prognosis; the FDA has always had concerns regarding patient safety during Phase I trials with unknown drugs that may cause CNS toxicity and finally once a new drug is identi‐ fied to have activity in non-CNS cancer involvement, few sponsors want to risk the poten‐ tial hazards of toxicity that could result from treating patients with CNS involvement – primary or metastatic.  The problem has been com‐ pounded by the lack of attention to CNS drug distribution and penetration of brain

Thus, there is a major need for the development of unique anticancer molecules that are able to cross the blood brain barrier (BBB), initiate anticancer activity in the CNS tumors and are

**• Temodar** (temozolamide, TMZ (Schering-Plough,) is currently approved and marketed by Schering-Plough for the treatment of high-grade glioblastoma multiforme and refrac‐

**• Gliadel** (BCNU wafer, Eisai) is an implantable wafer also used for the treatment of pri‐

**• Avastin** (bevacizumab, Genentech/Roche) is an anti-angiogenic inhibitor that is being combined with other agents that can penetrate the BBB. Combinations such as *Avastin*

**• Tarceva** (erlotinib, OSI Pharmaceuticals), was granted FDA orphan drug designation in August 2003 for patients with malignant gliomas, a recent study of Tarceva found 16% of patients with malignant gliomas who were given Tarciva alone or in combination with

**• Iressa** (gefitinib, AstraZeneca) and has been approved for relapsing non-small cell lung cancer. A recent phase II trial of Iressa in patients with relapsed glioblastoma resulted in one of 52 patients achieving a partial response and 22 patients achieving stable disease.

**• Zarnestra** (tipifarnib, Johnson & Johnson Pharmaceutical) is in early phase trials for brain tumors. The drug is also being studied in patients with leukemia and breast or

**• 81C6** is a tenascin radioactive monoclonal antibody that is injected into a cavity created by the neurosurgeon after removal of the tumor.

The product is under development at Duke Comprehensive Cancer Center.

**• BMS-247550** (Bristol Myers Squibb) is in Phase II/III @ Memorial Sloan-Kettering.

Temodar showed tumor shrinkage. The main side effect was an acne-like rash.

**2. Landscape analysis of drugs available for brain tumors**

436 Evolution of the Molecular Biology of Brain Tumors and the Therapeutic Implications

tory anaplastic astrocytoma in both in combination with radiation.

plus *Camptosar* (*irinotecan/CPT-11*) +/- *Temodar* are in trials.

Side effects were limited to rash and diarrhea.

tumors, with reduction in toxicities.

non-neurotoxicity.

mary brain tumors.

lung cancers.

#### **3. A new approach to new novel agents for – 1o and 2o brain tumors**

The current trend to develop 'personalized therapy' based on the presence of specific pheno‐ typic or genotypic pathways is an improved approach over the structure activity relation‐ ships (SAR) of the 20th century medicinal chemists [10]. However, the management of unresectable brain tumors has not improved. There is still a deficit in drugs that cross the blood brain barrier (BBB), are not recycled out of the brain, are effective anticancer agents in the CNS and do not require hepatic activation. Most of the 21st century '*personalized medicine'* drugs are large molecules that are given with other drugs and are ionizable chemicals that cannot penetrate the BBB and/or recycled out of the brain.

**A suggested platform for new novel agents** – that might be effective therapies for brain tu‐ mors comes from the earlier 20th century literature that focused on – '*the energy of cancer cells vs. normal cells'* [11-13].

Brain tumor biochemistry began in 1930 with Warburg's publication - *The Metabolism of Tu‐ mors* [12]. The fundamental finding of this publication was that embryonic and tumor tissues have in common a high glycolytic metabolism even under aerobic conditions; in contrast to most normal tissue where oxidative metabolism predominates. The oxidative metabolism in cells is generally 16 x more economical in respect to energy than the glycolytic pathway. The Embden-Meyerhof glycolytic pathway is phylogenetically the more primitive form [12]. *In rapidly growing de-differentiated tissue, this pathway prevails over oxidative metabolism.*

These concepts are of interest because the energy for a healthy cell is generated from glucose where 36 ATP molecules are produced from 1 glucose molecule. Hydrolysis of ATP results in a free energy change of ~ -16,000 cal/ATP molecule, depending on the conditions.

Classically cancer cells generate 2 ATP/1 glucose molecule *vs.* 36 ATP/1 glucose molecule (for healthy normal cells) – with the end product of pyruvate, rather than CO2 and water as occurs in healthy cells [12]. Otto Warburg explained the latter as a result of the cancer cell's energy-transfer conversion to aerobic glycolysis, a major shift in energy available for cellular work [12].

Albert Szent-Gyorgyi referred to the energy differences of cancer cells *vs.* normal cells as – the *alpha* (α) state with ↑∆S and ↓∆F *vs.* the *beta* state (β), with ↓∆S and ↑∆F), resp. [13].

with a +ΔF. When hydrolyzed in the presence of suitable transformers (transducing sys‐ tems), the energy liberated may be used for cellular or tissue processes – cellular metabo‐

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Energy rich phosphates usually possess either a second phosphoryl group or a resonating organic radical capable of entering into conjugation with the mobile electrons. This type of molecule usually contains a chain of at least three adjacent atoms, all bearing a net positive charge (deficient in π electrons), the chain sometimes extends over a five or even six atoms

Many of the small molecules used as anti-CNS cancer agents also possess 3-atom high ener‐

lism, nerve conduction, cell membrane transport, etc.

**Figure 1.** Adenosine triphosphate (ATP)

**Figure 2.** Examples of 3-atom high-energy moieties

gy moieties (in red) [3]

[Fig. 2], [16].

The α state is the ground state of life! The most stable state of any system is with minimum ∆F and maximum ∆S. If the cellular organization becomes unstable, cells will return to an α state and remain there. The more complete the return – the harder it will be to reverse.

The β state is where there is a high degree of differentiation and potential for release of F if the cells react and initiate differentiation. Here the energy present allow cellular regulation and multicellular organisms replication; not a mass of undifferentiated cells – cancer.

[Where, S = entropy or potential (stored capacity) for a reaction to occur and F (free energy), a state of function depending only on the initial and final states of a system [14].

The importance of ΔF is that the value immediately provides quantitative information about the potential ability of a substance to undergo a chemical or physical transformation. A reac‐ tion may proceed spontaneously without external energy only if the ΔF is negative (energy dissipated) [14]. Chemical thermodynamics tells us that "*F needs to be a released in order for the cell to be productive and do work, not just replicate with a low productive reactivity"* [13].

We should view entropy as an index of a condition or character. It is an index of the capacity for spontaneous changes. By historical accident, the index was actually defined so that it *increased* as the capacity of an isolated system for spontaneous change *decreased*.

*Hence, entropy is an index of exhaustion*. The more a system has lost its capacity for spontane‐ ous change, the greater is the entropy [14].

Yes, cancer cells represent a primitive, high random energy, low functional state with unbri‐ dled proliferation [13]. Cancer cells are not sick or weak, their vitality, as a wild type, may be even higher than that of a normal cell.

The cancer cell is unable to rebuild its beta state after it has completed its division and has to persist in the proliferative alpha state until death. In itself, the proliferative state would not be pathological – it is only its timing and irreversibility, which makes it so. *It is not pathologi‐ cal to be an infant, but it is pathological to be one and remain so x 50 years!*

Is cancer an outward manifestation resulting from depletion in cellular free energy within cells and increased entropy?

Although oxygen is considered essential for life, sulfur (S) and phosphorus (P) are the most common biological selections for energy transfer. The latter two elements are members of the Third Period of the Periodic Table and are capable of accepting an additional pair of electrons into unoccupied *3d* orbitals. This phenomenon is characteristic of the elements of the Third Period, which possess *d,* in addition to *s* and *p* orbitals, and thus have a place to hold electrons beyond the normal outer-shell octet. Molecules that possess P or S can store electrons (energy) and then release the energy during the transfer of shared electrons to oth‐ er electron deficient molecules [15].

ATP (Fig. 1) is a unique example of how a 3rd Period element - 'P' participates in a key natu‐ ral energy-rich compound, which upon hydrolysis generates ~ 8 kcal/mole upon hydrolysis with a +ΔF. When hydrolyzed in the presence of suitable transformers (transducing sys‐ tems), the energy liberated may be used for cellular or tissue processes – cellular metabo‐ lism, nerve conduction, cell membrane transport, etc.

**Figure 1.** Adenosine triphosphate (ATP)

Albert Szent-Gyorgyi referred to the energy differences of cancer cells *vs.* normal cells as – the *alpha* (α) state with ↑∆S and ↓∆F *vs.* the *beta* state (β), with ↓∆S and ↑∆F), resp. [13].

The α state is the ground state of life! The most stable state of any system is with minimum ∆F and maximum ∆S. If the cellular organization becomes unstable, cells will return to an α state and remain there. The more complete the return – the harder it will be to reverse.

The β state is where there is a high degree of differentiation and potential for release of F if the cells react and initiate differentiation. Here the energy present allow cellular regulation

[Where, S = entropy or potential (stored capacity) for a reaction to occur and F (free energy),

The importance of ΔF is that the value immediately provides quantitative information about the potential ability of a substance to undergo a chemical or physical transformation. A reac‐ tion may proceed spontaneously without external energy only if the ΔF is negative (energy dissipated) [14]. Chemical thermodynamics tells us that "*F needs to be a released in order for the*

We should view entropy as an index of a condition or character. It is an index of the capacity for spontaneous changes. By historical accident, the index was actually defined so that it

*Hence, entropy is an index of exhaustion*. The more a system has lost its capacity for spontane‐

Yes, cancer cells represent a primitive, high random energy, low functional state with unbri‐ dled proliferation [13]. Cancer cells are not sick or weak, their vitality, as a wild type, may

The cancer cell is unable to rebuild its beta state after it has completed its division and has to persist in the proliferative alpha state until death. In itself, the proliferative state would not be pathological – it is only its timing and irreversibility, which makes it so. *It is not pathologi‐*

Is cancer an outward manifestation resulting from depletion in cellular free energy within

Although oxygen is considered essential for life, sulfur (S) and phosphorus (P) are the most common biological selections for energy transfer. The latter two elements are members of the Third Period of the Periodic Table and are capable of accepting an additional pair of electrons into unoccupied *3d* orbitals. This phenomenon is characteristic of the elements of the Third Period, which possess *d,* in addition to *s* and *p* orbitals, and thus have a place to hold electrons beyond the normal outer-shell octet. Molecules that possess P or S can store electrons (energy) and then release the energy during the transfer of shared electrons to oth‐

ATP (Fig. 1) is a unique example of how a 3rd Period element - 'P' participates in a key natu‐ ral energy-rich compound, which upon hydrolysis generates ~ 8 kcal/mole upon hydrolysis

and multicellular organisms replication; not a mass of undifferentiated cells – cancer.

a state of function depending only on the initial and final states of a system [14].

438 Evolution of the Molecular Biology of Brain Tumors and the Therapeutic Implications

*cell to be productive and do work, not just replicate with a low productive reactivity"* [13].

*increased* as the capacity of an isolated system for spontaneous change *decreased*.

*cal to be an infant, but it is pathological to be one and remain so x 50 years!*

ous change, the greater is the entropy [14].

be even higher than that of a normal cell.

cells and increased entropy?

er electron deficient molecules [15].

Energy rich phosphates usually possess either a second phosphoryl group or a resonating organic radical capable of entering into conjugation with the mobile electrons. This type of molecule usually contains a chain of at least three adjacent atoms, all bearing a net positive charge (deficient in π electrons), the chain sometimes extends over a five or even six atoms [Fig. 2], [16].

$$\begin{array}{cccccccccccccccc} \circ & \bullet & \bullet & \bullet & \bullet & \bullet & \bullet & \bullet \\ \bullet & \bullet & \bullet & \bullet & \bullet & \bullet & \bullet & \bullet & \bullet \\ \bullet & \bullet & \bullet & \bullet & \bullet & \bullet & \bullet & \bullet & \bullet & \bullet \\ \bullet & \bullet & & & & & & \\ \bullet & \bullet & & & & & \\ \bullet & \bullet & \bullet & \bullet & \bullet & \bullet & \bullet & \bullet \\ \bullet & \bullet & \bullet & \bullet & \bullet & \bullet & \bullet & \bullet \\ \end{array}$$

**Figure 2.** Examples of 3-atom high-energy moieties

Many of the small molecules used as anti-CNS cancer agents also possess 3-atom high ener‐ gy moieties (in red) [3]

DEKK-TEC has designed a series of polychlorinated pyridyl carbonates (with 1 as a tem‐ plate, Fig. 3) that are linked with lipids and lipophilic moieties to promote CNS penetration [19]. DM-CHOC-PEN (4-demethyl-4-cholesteryloxycarbonylpenclomedine) is the most ac‐ tive and stable member of a large series of carbonates with improved activity vs. intracrani‐ ally (IC) implanted human xenograft models – U251 and D54 glioblastoma and MX-1 breast

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DM-CHOC-PEN is a unique molecule with several functional moieties that enable it to in‐

**Figure 4.** Lipophilic cholesterol link - blue. Carbonate linker that contains the properties seen in the elements of 2nd and 3rd period elements with higher energz storage potential - green. A pyridinium ring system that can transfer elec‐ trons via resonance into intermediary metabolism, ATP synthesis and substrate for the ABC cassette transport / red

The importance of the carbonate moiety is its internal energy and ability to transfer electrons

The additional oxygen in the carbonates [Fig. 5, lower structures] stabilizes the moiety and allows for distribution of charge through resonance (Fig. 5). This chemical property exists in

through multiple structures (resonance) (see Fig. 5):

**Figure 5.** Mesomeric forms of the carbonate; where R = pyridyl group & R'=cholesterol

the phosphates and other oxides of the 3rd period elements [see below] [15].

hibit cancer replication. The structure contains the following moieties of interest:

cancer [Table 1] [19].

**Figure 3.** Small Molecules that are used to treat CNS cancer

Where: **DM-PEN carbonate**, 1 is a carbonate analog of 4-demethyl penclomedine; **PRZ**, 2 is dacarbazine; **Temodar**®, 3 is temozolamide (TMZ); **BCNU**, 4 is bis-(chloroethyl)nitrosourea; **HOOI**, 5 is 4-hydroperoxyifosfamide; **Clomesone**, 6 is a chloroethylating agent similar to BCNU – alkylates guanine-O6 [17].

Compounds can be classified as "energy-rich" or "energy-poor" based on their ΔF. Energy poor compounds do not possess more than two adjacent atoms bearing a positive charge and poorly absorbed by the CNS.

Thus, the design of "high-energy molecules" that are lipophilic, energetic and absorbed into a low free energy (ΔF) and high entropy (ΔS) cancer environment are timely. The latter is a unique therapeutic target – *the result of inefficient energy of cancer cells* [14].

Life is controlled by the transfer of energy. All living systems obey the 1st law of thermo‐ dynamics – which states that the sum of all energies in an isolated system remains con‐ stant. Energy may be converted into another form but the quantity can be neither created nor destroyed [18].

A question to be answered – are high energy compounds really attracted to low energy highly utilized regions in the body?

#### **4. Polyhalogenated pyridyl carbonates [16, 19-21]**

The polyhalogenated pyridyl carbonates are neutral molecules that when coupled with lip‐ ids via high energy linkers – carbonates, phosphates, etc., fulfill the above requirements for novel high energy anticancer agents that could be attracted to and accumulate in CNS can‐ cer tissue [16, 19-21]. The carbonate moiety like phosphate has the properties (resonance) of the high energy 3-atom moieties described in Fig. 3.

DEKK-TEC has designed a series of polychlorinated pyridyl carbonates (with 1 as a tem‐ plate, Fig. 3) that are linked with lipids and lipophilic moieties to promote CNS penetration [19]. DM-CHOC-PEN (4-demethyl-4-cholesteryloxycarbonylpenclomedine) is the most ac‐ tive and stable member of a large series of carbonates with improved activity vs. intracrani‐ ally (IC) implanted human xenograft models – U251 and D54 glioblastoma and MX-1 breast cancer [Table 1] [19].

DM-CHOC-PEN is a unique molecule with several functional moieties that enable it to in‐ hibit cancer replication. The structure contains the following moieties of interest:

**Figure 3.** Small Molecules that are used to treat CNS cancer

[17].

440 Evolution of the Molecular Biology of Brain Tumors and the Therapeutic Implications

unique therapeutic target – *the result of inefficient energy of cancer cells* [14].

**4. Polyhalogenated pyridyl carbonates [16, 19-21]**

the high energy 3-atom moieties described in Fig. 3.

BCNU – alkylates guanine-O6

and poorly absorbed by the CNS.

highly utilized regions in the body?

nor destroyed [18].

Where: **DM-PEN carbonate**, 1 is a carbonate analog of 4-demethyl penclomedine; **PRZ**, 2 is dacarbazine; **Temodar**®, 3 is temozolamide (TMZ); **BCNU**, 4 is bis-(chloroethyl)nitrosourea; **HOOI**, 5 is 4-hydroperoxyifosfamide; **Clomesone**, 6 is a chloroethylating agent similar to

Compounds can be classified as "energy-rich" or "energy-poor" based on their ΔF. Energy poor compounds do not possess more than two adjacent atoms bearing a positive charge

Thus, the design of "high-energy molecules" that are lipophilic, energetic and absorbed into a low free energy (ΔF) and high entropy (ΔS) cancer environment are timely. The latter is a

Life is controlled by the transfer of energy. All living systems obey the 1st law of thermo‐ dynamics – which states that the sum of all energies in an isolated system remains con‐ stant. Energy may be converted into another form but the quantity can be neither created

A question to be answered – are high energy compounds really attracted to low energy

The polyhalogenated pyridyl carbonates are neutral molecules that when coupled with lip‐ ids via high energy linkers – carbonates, phosphates, etc., fulfill the above requirements for novel high energy anticancer agents that could be attracted to and accumulate in CNS can‐ cer tissue [16, 19-21]. The carbonate moiety like phosphate has the properties (resonance) of

**Figure 4.** Lipophilic cholesterol link - blue. Carbonate linker that contains the properties seen in the elements of 2nd and 3rd period elements with higher energz storage potential - green. A pyridinium ring system that can transfer elec‐ trons via resonance into intermediary metabolism, ATP synthesis and substrate for the ABC cassette transport / red

The importance of the carbonate moiety is its internal energy and ability to transfer electrons through multiple structures (resonance) (see Fig. 5):

**Figure 5.** Mesomeric forms of the carbonate; where R = pyridyl group & R'=cholesterol

The additional oxygen in the carbonates [Fig. 5, lower structures] stabilizes the moiety and allows for distribution of charge through resonance (Fig. 5). This chemical property exists in the phosphates and other oxides of the 3rd period elements [see below] [15].

[20]. The trichloromethylene group is required, since the dichloromethylene analog of PEN is not active [does not react with 4-(*p*-nitrobenzyl) pyridine (NBP] (Struck, person‐

**Figure 6.** Proposed mechanism of action of 4 - demethyl - 4 - cholesterolyl - penclomedine (DM - CHOC - PEN)

were assayed by HPLC and DM-CHOC-PEN, a guanine-N7

Thus, DM-CHOC-PEN alkylates DNA with adducts at guanine-N7

normal brain tissue.

[22].

guanine-O6

Mice with IC growing U251 gliomas were treated with DM-CHOC-PEN, 135 mg/kg daily x 5-days and after 3-days the animals were sacrificed and the brains with tumors removed. The tumors were easily separated from the normal brain tissue. Each was homogenized in phosphate buffer, extracted with dichloromethane, and evaporated to dryness. The residues

PEN involving the –CCl3 moiety and the DM-CHCO-PEN dimer-Cl2C-CCl2- were identified in the glioma tissue [16]. Neither DM-CHOC-PEN nor metabolites could be identified in the

in tumor tissue. The above MOA is in contrast to TMZ, BCNU and others, which alkylate

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that could be quantitated

nel communication).

**Scheme 1.**

In contrast, esters lack the degree of resonance/electron distribution [Fig. 5, upper structures].

As indicated in Fig. 5, a carbonate is not isosteric with an acyl or ester group nor is it a direct extension of an ester group. The carbonate group is unique and when combined with cho‐ lesterol its CNS penetration properties are enhanced [Table 1] [16].

**Table 1.** Activity of DM-CHOC-PEN vs. Intracerebrally Implanted Xenograft Tumors in Mice

Implant: 106 cells IC; Treatment Route: Intraperitoneal (- 5 days post IC implant); Schedule: \*IV q1d x 5d; + IV q1d x 3d; # po 1d x 5d; Species: Athymic NCr/nu mice – female, Charles River; Dose: mg/kg; %ILS = % increased life span; %LTS = long term survival; all studies were terminated at 54 days

Based on the preclinical antitumor activities in Table 1, DM-CHOC-PEN produced a moder‐ ate increase in life span (% ILS); however it was comparable to that seen with BCNU and TMZ. DM-CHOC-PEN did yield long-term survivors that were healthy with no weight loss. Even the highly resistant IC implanted D54 glioma was sensitive to DM-CHOC-PEN. The drug has minimal toxicity and has encouraging potential [16, 19, 21].

An *in vivo* activation mechanism scheme is shown in Fig. 6, where DM-CHOC-PEN is proposed to be acting as a non-classical alkylating agent. The scheme describes a dual carbonium ion - DNA cross-linking mechanism (in a G-X-C sequence) with tumor DNA in the major groove via N7 - guanine [20]. A bi-molecular coupled product (*via* the tri‐ chloromethylene group) has been identified as the major microsomal metabolite of PEN [20]. The trichloromethylene group is required, since the dichloromethylene analog of PEN is not active [does not react with 4-(*p*-nitrobenzyl) pyridine (NBP] (Struck, person‐ nel communication).

**Scheme 1.**

U251 Glioblasto ma

D54 Glioblasto ma

> MX-1 Breast Cancer

\*IV q1d x 5d; +

135 +54

200 +3

+20 Toxic

IV q1d x 3d; #

50 100

were terminated at 54 days

in the major groove via N7

In contrast, esters lack the degree of resonance/electron distribution [Fig. 5, upper structures].

lesterol its CNS penetration properties are enhanced [Table 1] [16].

442 Evolution of the Molecular Biology of Brain Tumors and the Therapeutic Implications

+20(1/5 CR)

+20(1/5 CR)

**Table 1.** Activity of DM-CHOC-PEN vs. Intracerebrally Implanted Xenograft Tumors in Mice

drug has minimal toxicity and has encouraging potential [16, 19, 21].

As indicated in Fig. 5, a carbonate is not isosteric with an acyl or ester group nor is it a direct extension of an ester group. The carbonate group is unique and when combined with cho‐

**DM-CHOC-PEN BCNU TMZ**

+20(1/5 CR)

17(1/6 CR) 60 12 0/5 NA NA NA

po 1d x 5d; Species: Athymic NCr/nu mice – female, Charles

NA NA NA NA NA NA

120 +54

+20(1/5 CR)

**Tumor \*Dose %ILS %LTS +Dose %ILS %LTS #Dos %ILS %LTS**

9 52

Implant: 106 cells IC; Treatment Route: Intraperitoneal (- 5 days post IC implant); Schedule:

River; Dose: mg/kg; %ILS = % increased life span; %LTS = long term survival; all studies

Based on the preclinical antitumor activities in Table 1, DM-CHOC-PEN produced a moder‐ ate increase in life span (% ILS); however it was comparable to that seen with BCNU and TMZ. DM-CHOC-PEN did yield long-term survivors that were healthy with no weight loss. Even the highly resistant IC implanted D54 glioma was sensitive to DM-CHOC-PEN. The

An *in vivo* activation mechanism scheme is shown in Fig. 6, where DM-CHOC-PEN is proposed to be acting as a non-classical alkylating agent. The scheme describes a dual carbonium ion - DNA cross-linking mechanism (in a G-X-C sequence) with tumor DNA

chloromethylene group) has been identified as the major microsomal metabolite of PEN

**Figure 6.** Proposed mechanism of action of 4 - demethyl - 4 - cholesterolyl - penclomedine (DM - CHOC - PEN)

Mice with IC growing U251 gliomas were treated with DM-CHOC-PEN, 135 mg/kg daily x 5-days and after 3-days the animals were sacrificed and the brains with tumors removed. The tumors were easily separated from the normal brain tissue. Each was homogenized in phosphate buffer, extracted with dichloromethane, and evaporated to dryness. The residues were assayed by HPLC and DM-CHOC-PEN, a guanine-N7 -CCl2-adduct with DM-CHOC-PEN involving the –CCl3 moiety and the DM-CHCO-PEN dimer-Cl2C-CCl2- were identified in the glioma tissue [16]. Neither DM-CHOC-PEN nor metabolites could be identified in the normal brain tissue.

Thus, DM-CHOC-PEN alkylates DNA with adducts at guanine-N7 that could be quantitated in tumor tissue. The above MOA is in contrast to TMZ, BCNU and others, which alkylate guanine-O6 [22].

The polyhalogenated pyridyl carbonates are unique structures that cross the BBB, accumu‐ late in CNS tumor tissue, not normal brain tissue and are not recycled out of the CNS are cytotoxic *vs*. intracranially (IC) implanted human tumor xenografts in mouse models; fulfill‐ ing all of the criteria of the preceding paragraphs [23].

To test the potential sensitivity of DM-CHOC-PEN as therapy for melanoma, the B-16 mouse melanoma was selected as a screening model and treated with DM-CHOC-PEN *in*

**In Vitro** – B-16 mouse melanoma cells [obtained from ATCC, Menassas, VA] were assayed

µg/mL) were added to the cells in a growth phase and removed after 8-12 h; cultures were washed with fresh medium. For florescent studies - cells in growth phase were incubated with dichlorofluorescein diacetate (DCFDA, 3 µg/mL RPMI) x 1-hr, washed and DM-CHOC-PEN added (1-5 µg/mL); incubation continued for an additional 5-hour. Cells were washed with fresh complete media and monitored with fluorescent microscopy. For the lat‐

C57BL mice (age – 7-9 weeks, Harlan Labs.) and treatments with DM-CHOC-PEN, HOOI, and other drugs began were initiated when lesions were palpable - 3-5 days post implant.

**Drugs** – were dissolved in saline or in a soybean oil/egg yolk lecithin emulsion (2 mg/mL)

**Results** – B-16 cells were incubated with DM-CHOC-PEN and after ~ 6-8 hours became heavily melanotic, lost adherence and floated to the top of the wells. Isolation of the floating heavily pigmented melanotic cells that formed (Fig. 7b), followed by washing, extraction with dichloromethane, and HPLC assays revealed that the cells contained DM-CHOC-PEN in concentrations of 0.003-0.09 µg/mL of packed B-16 cells. Attached amelanotic cells con‐

and administer by IP injection. HOOI was administered as the L-lysine salt [27].

/mL) using a complete RPMI media containing 10% FBS with pen/strep

C in a CO2 incubator. Drugs (in 1 mL volumes; in conc. - 0.1-5

) were implanted SC into the flank of adult female

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*vitro* and *in vivo* [28].

in culture wells (104

**7. Experimental methods**

and maintaining cells @ 36o

ter – RPMI without phenol red was used.

**In Vivo** – B-16 mouse melanoma cells (106

tained 0-20 ng/mL of DM-CHOC-PEN (significant) [28].

(a) (b)

**Figure 7.** a) B-16 melanoma cells – untreated; b) After treatment with DM-CHOC-PEN

The combinations of responses in mouse xenograft tumor models, and a lack of neurotoxici‐ ty in rat and dog biopsychology studies, have allowed a Phase I trial to be conducted with DM- CHOC-PEN in patients with advanced cancer, including CNS involvement - IND 68,876 [24].

DEKK-TEC and its clinical investigators have satisfied the FDA that they are capable of treating patients with Phase I drugs and diagnosing physical and mental/cognitive altera‐ tions that may be associated with CNS tumor growth vs. toxicity secondary to drugs.

#### **5. Phase I clinical trial with a high energy drug – DM-CHOC-PEN**

Weiner, Ware, Friedlander, et al. reported preliminary data from a Phase I trial involving 18 patients treated with DM-CHOC-PEN in single IV doses of 39-87.5 mg/m2 once every 21 days [25]. Six [6] patients had either CNS or spinal nervous system (SNS) involvement. Most patients had multiple treatments. The longest survivor (a sarcoma w/ SNS involvement) is 19+ months with NED [who also received doxorubicin at a later time] [25].

Support for DM-CHOC-PEN crossing the blood brain barrier in humans is provided from the above trial with reported observations for – GBM, CNS melanoma and breast cancer (BC) having PFS 4-13 mos., objective reductions in tumor sizes and no CNS toxicity.

One patient with pelvic sarcoma spread to the spinal cord required debulking surgery 21 days post treatment with DM-CHOC-PEN (39 mg/m2 ). Tumor tissue was removed, assayed and DM-CHOC-PEN was quantitated in the spinal sarcoma tissue – 92 ng/g of tumor [25].

The only SLTs noted to date are hyperbilirubinemia in 2-patients with liver metastasis; pa‐ tients with no liver pathology have not demonstrated SLTs. CNS toxicity has not been noted in any of the patients. Overall, the drug was reported as being tolerated very well [25].

#### **6. CNS melanoma**

Melanoma is the third most frequent cancer to metastasize to the brain. It is anticipated that there will be ~ 20,000 new cases of CNS stage melanoma diagnosed in 2012 [9]. Enrollment of these patients with CNS melanoma would be critical to appreciating the activity of the demethylpenclomedine carbonates in this stage of disease [9, 26]. Two of the patients enrol‐ led and treated in the DM-CHOC-PEN Phase I trial had CNS melanoma; one patient had significant shrinkage of CNS lesions, the other stable (NC) disease [25].

To test the potential sensitivity of DM-CHOC-PEN as therapy for melanoma, the B-16 mouse melanoma was selected as a screening model and treated with DM-CHOC-PEN *in vitro* and *in vivo* [28].

### **7. Experimental methods**

The polyhalogenated pyridyl carbonates are unique structures that cross the BBB, accumu‐ late in CNS tumor tissue, not normal brain tissue and are not recycled out of the CNS are cytotoxic *vs*. intracranially (IC) implanted human tumor xenografts in mouse models; fulfill‐

The combinations of responses in mouse xenograft tumor models, and a lack of neurotoxici‐ ty in rat and dog biopsychology studies, have allowed a Phase I trial to be conducted with DM- CHOC-PEN in patients with advanced cancer, including CNS involvement - IND

DEKK-TEC and its clinical investigators have satisfied the FDA that they are capable of treating patients with Phase I drugs and diagnosing physical and mental/cognitive altera‐

Weiner, Ware, Friedlander, et al. reported preliminary data from a Phase I trial involving 18-

days [25]. Six [6] patients had either CNS or spinal nervous system (SNS) involvement. Most patients had multiple treatments. The longest survivor (a sarcoma w/ SNS involvement) is

Support for DM-CHOC-PEN crossing the blood brain barrier in humans is provided from the above trial with reported observations for – GBM, CNS melanoma and breast cancer

One patient with pelvic sarcoma spread to the spinal cord required debulking surgery 21-

The only SLTs noted to date are hyperbilirubinemia in 2-patients with liver metastasis; pa‐ tients with no liver pathology have not demonstrated SLTs. CNS toxicity has not been noted in any of the patients. Overall, the drug was reported as being tolerated very well [25].

Melanoma is the third most frequent cancer to metastasize to the brain. It is anticipated that there will be ~ 20,000 new cases of CNS stage melanoma diagnosed in 2012 [9]. Enrollment of these patients with CNS melanoma would be critical to appreciating the activity of the demethylpenclomedine carbonates in this stage of disease [9, 26]. Two of the patients enrol‐ led and treated in the DM-CHOC-PEN Phase I trial had CNS melanoma; one patient had

significant shrinkage of CNS lesions, the other stable (NC) disease [25].

and DM-CHOC-PEN was quantitated in the spinal sarcoma tissue – 92 ng/g of tumor [25].

(BC) having PFS 4-13 mos., objective reductions in tumor sizes and no CNS toxicity.

once every 21-

). Tumor tissue was removed, assayed

tions that may be associated with CNS tumor growth vs. toxicity secondary to drugs.

**5. Phase I clinical trial with a high energy drug – DM-CHOC-PEN**

patients treated with DM-CHOC-PEN in single IV doses of 39-87.5 mg/m2

19+ months with NED [who also received doxorubicin at a later time] [25].

days post treatment with DM-CHOC-PEN (39 mg/m2

**6. CNS melanoma**

ing all of the criteria of the preceding paragraphs [23].

444 Evolution of the Molecular Biology of Brain Tumors and the Therapeutic Implications

68,876 [24].

**In Vitro** – B-16 mouse melanoma cells [obtained from ATCC, Menassas, VA] were assayed in culture wells (104 /mL) using a complete RPMI media containing 10% FBS with pen/strep and maintaining cells @ 36o C in a CO2 incubator. Drugs (in 1 mL volumes; in conc. - 0.1-5 µg/mL) were added to the cells in a growth phase and removed after 8-12 h; cultures were washed with fresh medium. For florescent studies - cells in growth phase were incubated with dichlorofluorescein diacetate (DCFDA, 3 µg/mL RPMI) x 1-hr, washed and DM-CHOC-PEN added (1-5 µg/mL); incubation continued for an additional 5-hour. Cells were washed with fresh complete media and monitored with fluorescent microscopy. For the lat‐ ter – RPMI without phenol red was used.

**In Vivo** – B-16 mouse melanoma cells (106 ) were implanted SC into the flank of adult female C57BL mice (age – 7-9 weeks, Harlan Labs.) and treatments with DM-CHOC-PEN, HOOI, and other drugs began were initiated when lesions were palpable - 3-5 days post implant.

**Drugs** – were dissolved in saline or in a soybean oil/egg yolk lecithin emulsion (2 mg/mL) and administer by IP injection. HOOI was administered as the L-lysine salt [27].

**Results** – B-16 cells were incubated with DM-CHOC-PEN and after ~ 6-8 hours became heavily melanotic, lost adherence and floated to the top of the wells. Isolation of the floating heavily pigmented melanotic cells that formed (Fig. 7b), followed by washing, extraction with dichloromethane, and HPLC assays revealed that the cells contained DM-CHOC-PEN in concentrations of 0.003-0.09 µg/mL of packed B-16 cells. Attached amelanotic cells con‐ tained 0-20 ng/mL of DM-CHOC-PEN (significant) [28].

**Figure 7.** a) B-16 melanoma cells – untreated; b) After treatment with DM-CHOC-PEN

In comparison with other agents – DM-CHOC-PEN had an IC50 of 0.4 µg/mL and TMZ, BCNU, DOX and actinomycin D (Act D), HOOI – 0.5-0.9 µg/mL; *cis*-platinum (*cis*-platin) – 1.5 µg/mL resulted in IC50 cell death @ 36 hour post-intial exposure. With the excep‐ tion of DM-CHOC-PEN, all treated cells died with ghosts remaining; excesive melanin formation only occurred with DM-CHOC-PEN; the terminal pathways were quite differ‐ ent for the other drugs.

Melanoma cell death due to DM-CHOC-PEN could be a result of increased intracellular melanin, as well as DNA alkylation. Addition of DCF-DA (dichlorofluorescein diacetate) to the culture medium (10 µg/mL) documented the formation of radical oxygen species (ROS) in the DM- CHOC-PEN treated cultures; green labeled cells (Fig. 8b). ROS release was documented with DCF, which is due to the hydrolysis and oxidized of DCF-DA by ROS with the release of fluorescent DCF. The generation of ROS was not observed with B-16 melanoma cells treated with *cis*-platin or actinomycin D and cultured under similar conditions. There is some back ground florescence and secondary to melanin-associated

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**In Vivo Studies** – Adult female C57BL mice in groups of 5-6 mice with palpable SC nodules were dosed IP daily (175 or 200 mg/kg) for 5-days with DM-CHOC-PEN and monitored dai‐

Mice treated with DM-CHOC-PEN (200 mg/kg/d x 5 days, IP) alone demonstrated an ILS of 142% (Fig. 9a). No drug related toxicity was noted – weight gain occurred until tumor

Cis-platin was used as a drug control and administered (IP) once weekly x 3 (Fig. 9b); ani‐ mals did not tolerate 3- or 5-day dosing schedules [28, 29]. Although active *in vitro, cis*-platin does not improve *in vivo* survival – as seen in Fig. 9b, although it is used in many melanoma

Tumor histology of the DM-CHOC-PEN treated B-16 melanomas are reviewed in Fig. 10. On the left (a) is the saline control and right (b) the DM-CHOC-PEN treated – 200 mg/kg IP dai‐ ly x 5 days. *Note* - the melanin deposits in the cells with vacuoles, similar to what was seen

ROS formation [16].

ly until death (Fig. 9a).

growth was obvious.

clinical protocols [3].

in culture [29].

**Figure 9.** a)DM-CHOC-PEN treated; b)Cis-platin treated

**Table 2.** Drugs Responses vs. B-16 Melanoma (IC50)

**Figure 8.** a)B-16 cells + DCF-DA; b)B-26 cells plus DM-CHOC-PEN + DCF-DA

Viewed with florescent microscopy – green florescent cells are generating ROS and the DCF is florescent.

Melanoma cell death due to DM-CHOC-PEN could be a result of increased intracellular melanin, as well as DNA alkylation. Addition of DCF-DA (dichlorofluorescein diacetate) to the culture medium (10 µg/mL) documented the formation of radical oxygen species (ROS) in the DM- CHOC-PEN treated cultures; green labeled cells (Fig. 8b). ROS release was documented with DCF, which is due to the hydrolysis and oxidized of DCF-DA by ROS with the release of fluorescent DCF. The generation of ROS was not observed with B-16 melanoma cells treated with *cis*-platin or actinomycin D and cultured under similar conditions. There is some back ground florescence and secondary to melanin-associated ROS formation [16].

**In Vivo Studies** – Adult female C57BL mice in groups of 5-6 mice with palpable SC nodules were dosed IP daily (175 or 200 mg/kg) for 5-days with DM-CHOC-PEN and monitored dai‐ ly until death (Fig. 9a).

Mice treated with DM-CHOC-PEN (200 mg/kg/d x 5 days, IP) alone demonstrated an ILS of 142% (Fig. 9a). No drug related toxicity was noted – weight gain occurred until tumor growth was obvious.

**Figure 9.** a)DM-CHOC-PEN treated; b)Cis-platin treated

In comparison with other agents – DM-CHOC-PEN had an IC50 of 0.4 µg/mL and TMZ, BCNU, DOX and actinomycin D (Act D), HOOI – 0.5-0.9 µg/mL; *cis*-platinum (*cis*-platin) – 1.5 µg/mL resulted in IC50 cell death @ 36 hour post-intial exposure. With the excep‐ tion of DM-CHOC-PEN, all treated cells died with ghosts remaining; excesive melanin formation only occurred with DM-CHOC-PEN; the terminal pathways were quite differ‐

ent for the other drugs.

**Drug IC50 (µg/mL)** DM-CHOC-PEN 0.4 +/- 0.01

446 Evolution of the Molecular Biology of Brain Tumors and the Therapeutic Implications

Actinomycin D 0.5 +/- 0.02

BCNU 0.9 +/- 0.1

*cis*-Platin 1.5 +/- 0.1

DOX 0.7 +/- 0.1

HOOI 0.8 +/- 0.2

**Figure 8.** a)B-16 cells + DCF-DA; b)B-26 cells plus DM-CHOC-PEN + DCF-DA

is florescent.

Viewed with florescent microscopy – green florescent cells are generating ROS and the DCF

TMZ "/>3.0

**Table 2.** Drugs Responses vs. B-16 Melanoma (IC50)

Cis-platin was used as a drug control and administered (IP) once weekly x 3 (Fig. 9b); ani‐ mals did not tolerate 3- or 5-day dosing schedules [28, 29]. Although active *in vitro, cis*-platin does not improve *in vivo* survival – as seen in Fig. 9b, although it is used in many melanoma clinical protocols [3].

Tumor histology of the DM-CHOC-PEN treated B-16 melanomas are reviewed in Fig. 10. On the left (a) is the saline control and right (b) the DM-CHOC-PEN treated – 200 mg/kg IP dai‐ ly x 5 days. *Note* - the melanin deposits in the cells with vacuoles, similar to what was seen in culture [29].

There were less viable cells available in the treated animals. DM-CHOC-PEN reduced the cellular concentration of the tumors and the cells accumulated in G1 phase, with an inhibi‐ tion in S-phase concentration of cells (Fig. 11b). Thus, more support for DM-CHOCPEN's potential use in melanoma. Unfortunately, there was a significant amount of debris present.

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**Discussion** – Mice bearing B-16 melanoma, tolerated DM-CHOC-PEN well when adminis‐ tered IP daily x 5 days or days 1 & 5 with improved overall survival (OAS) for both sched‐

Histological examinations of the melanoma tissue from animals that had been treated with DM-CHOC-PEN revealed tumor cells encompassed in extracellular melanin, very similar to what was seen in tissue culture (Fig. 7b). DM-CHOC-PEN was extracted in up to 92 ng/g

DM-CHOC-PEN is a pseudo-alkylator that binds to DNA's guanine-N7 via the trichloro‐ methyl moiety → dichloromethylene carbonium ion and can form DNA-guanine adducts and cell death [Fig. 6] [9]. However, the current observations in a melanoma model support DM-CHOC-PEN's additional ability to disrupt cellular metabolism with death via autooxi‐ dation of DOPA to melanin and superoxide formation – an additional MOA for considera‐

Electronic modeling studies support DM-CHOC-PEN's ability to act as a pyridinium co-fac‐ tor oxidized by the pyridine nucleotides (NAD, etc.) with red-ox transfer of electrons from 3,4-dihydroxyphenylalanine (DOPA) to the mitochondria and cytochrome C/cytochrome ox‐ idase transport system (Fig. 13). This result is the formation of 5, 6-indolequinone and mela‐ nin with a trail of electrons that can enter the mitochondrion intermediary pool resulting in all the same type of lethal changes seen with electron beam therapy for cancer [30, 31].

The 'melanin balls' that were seen *in vitro* are spheres of extracellular melanin polymer en‐ capsulating colonies of melanoma cells that could induce a hostile microenvironment through ROS formation with death via oxidative stress and apoptosis (Figs. 10) [30]. Clus‐ ters of melanin laden cells contained significantly elevated concentrations of DM-CHOC-PEN, as compared to the amelanotic variant; a possible storage site for the drug [31]. The initial DOPA → DOPA quinone transformation is catalyzed by tyrosinase (DOPA oxidase) intracellular [32-35]. However, the red-ox potential for DM-CHOC-PEN is sufficient to cata‐

lyze the conversion with excess melanin formation per the Rapier Scheme below [32].

chemical reactions as depicted in the classical Rapier Scheme in Fig.12 [32].

ducing electron traps in the system [36].

Both malignant and benign melanocytes generate melanin pigment via the sequence of

Melanin is far from being an end-product of oxidation [34-37]. At a glance the existence of energy bands are obvious from the highly conjugated heterocyclic indole quinone structure for melanin (Fig. 12). The hypothesis has been proposed that non-localized empty molecular orbitals are associated with the copolymer chain of the indole quinoid units and that the melanin polymer acts as a one-dimensional semi-conductor/trap with bound protons pro‐

tissue quantities from the melanoma tissue removed in Fig. 11b [16].

ules; the former was the superior.

tion – Fig. 12 [28, 29].

**Figure 11.** a)Histology of saline controls; b)Histology of DM-CHOC-PEN treated

Tumor tissue from the above DM-CHOC-PEN treated mice were assayed per cytoflorimetry using a BD FAC Scanner.

**Figure 12.** a) Mice bearing SC nodules – control animals. b) Mice bearing SC nodules dosed - 200 mg/kg; days 1-5 and sacrificed 3-days after the last treatment.

There were less viable cells available in the treated animals. DM-CHOC-PEN reduced the cellular concentration of the tumors and the cells accumulated in G1 phase, with an inhibi‐ tion in S-phase concentration of cells (Fig. 11b). Thus, more support for DM-CHOCPEN's potential use in melanoma. Unfortunately, there was a significant amount of debris present.

(a) (b)

448 Evolution of the Molecular Biology of Brain Tumors and the Therapeutic Implications

**Figure 11.** a)Histology of saline controls; b)Histology of DM-CHOC-PEN treated

using a BD FAC Scanner.

(a)

(b)

sacrificed 3-days after the last treatment.

Tumor tissue from the above DM-CHOC-PEN treated mice were assayed per cytoflorimetry

**Figure 12.** a) Mice bearing SC nodules – control animals. b) Mice bearing SC nodules dosed - 200 mg/kg; days 1-5 and

**Discussion** – Mice bearing B-16 melanoma, tolerated DM-CHOC-PEN well when adminis‐ tered IP daily x 5 days or days 1 & 5 with improved overall survival (OAS) for both sched‐ ules; the former was the superior.

Histological examinations of the melanoma tissue from animals that had been treated with DM-CHOC-PEN revealed tumor cells encompassed in extracellular melanin, very similar to what was seen in tissue culture (Fig. 7b). DM-CHOC-PEN was extracted in up to 92 ng/g tissue quantities from the melanoma tissue removed in Fig. 11b [16].

DM-CHOC-PEN is a pseudo-alkylator that binds to DNA's guanine-N7 via the trichloro‐ methyl moiety → dichloromethylene carbonium ion and can form DNA-guanine adducts and cell death [Fig. 6] [9]. However, the current observations in a melanoma model support DM-CHOC-PEN's additional ability to disrupt cellular metabolism with death via autooxi‐ dation of DOPA to melanin and superoxide formation – an additional MOA for considera‐ tion – Fig. 12 [28, 29].

Electronic modeling studies support DM-CHOC-PEN's ability to act as a pyridinium co-fac‐ tor oxidized by the pyridine nucleotides (NAD, etc.) with red-ox transfer of electrons from 3,4-dihydroxyphenylalanine (DOPA) to the mitochondria and cytochrome C/cytochrome ox‐ idase transport system (Fig. 13). This result is the formation of 5, 6-indolequinone and mela‐ nin with a trail of electrons that can enter the mitochondrion intermediary pool resulting in all the same type of lethal changes seen with electron beam therapy for cancer [30, 31].

The 'melanin balls' that were seen *in vitro* are spheres of extracellular melanin polymer en‐ capsulating colonies of melanoma cells that could induce a hostile microenvironment through ROS formation with death via oxidative stress and apoptosis (Figs. 10) [30]. Clus‐ ters of melanin laden cells contained significantly elevated concentrations of DM-CHOC-PEN, as compared to the amelanotic variant; a possible storage site for the drug [31]. The initial DOPA → DOPA quinone transformation is catalyzed by tyrosinase (DOPA oxidase) intracellular [32-35]. However, the red-ox potential for DM-CHOC-PEN is sufficient to cata‐ lyze the conversion with excess melanin formation per the Rapier Scheme below [32].

Both malignant and benign melanocytes generate melanin pigment via the sequence of chemical reactions as depicted in the classical Rapier Scheme in Fig.12 [32].

Melanin is far from being an end-product of oxidation [34-37]. At a glance the existence of energy bands are obvious from the highly conjugated heterocyclic indole quinone structure for melanin (Fig. 12). The hypothesis has been proposed that non-localized empty molecular orbitals are associated with the copolymer chain of the indole quinoid units and that the melanin polymer acts as a one-dimensional semi-conductor/trap with bound protons pro‐ ducing electron traps in the system [36].

Thus, melanin is a polymer of indole-5, 6-quinone which is highly electrophilic due to its conjugated structure and capable of attracting, storing and/or transferring electrons as elec‐ trical energy [30, 31, 34]. DM-CHOC-PEN treatment induced ROS formation *via* the melanin system resulting in cancer cell death – an end point.

ronmental ΔF and a decrease in ΔS; a new resting high energy state – the *beta state*.  DM-CHOC-PEN is a potential triple treat for CNS melanoma, the 3rd most common

Another high energy drug, 4-hydroperoxyifosfamide (HOOI) [Fig. 3, 14] and is a pre-activat‐ ed form of ifosfamide (IFOS) which possesses the criteria for discussion [Fig. 14]. HOOI also

IFOS is a well-known anticancer agent that requires hepatic activation to 4-HO-IFOS which spontaneously undergoes hydrolysis with ring opening resulting in isophosphoramide mus‐ tard (IPM) [Fig. 14], the latter is the active cytotoxic form of IFOS [37-39]. As part of the IFOS activation process, hepatic metabolic dechloroethylation releases chloroacetaldehyde, which has been proposed to be the major cause of IFOS associated neurotoxicity [40]. Acrolein is also released and has been implicated in dose limiting toxicities - hemorrhagic cystitis and 2<sup>o</sup>

In contrast, HOOI does not require hepatic microsomal activation, crosses the BBB and is readily absorbed by cancer cells where it releases IPM *in situ* [46, 47]. The former does not generate chloroacetaldehyde (neurotoxic) during its conversion to IPM, as does IFOS. Nei‐ ther hemorrhagic cystitis nor renal toxicity has been observed with HOOI in animal toxicol‐ ogy models. Pulmonary damage that could occur with peroxides – pulmonary air emboli was not observed in the dog HOOI study [46]. Hematological toxicity (bone marrow deple‐

The potential therapeutic usefulness of HOOI is supported by in vivo activity in human tu‐ mor xenografts and cyclophosphamide (CPA)-resistant murine tumor models plus reduced

HO P HN O

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CH2CH2Cl

Isophosphoramide Mustard (IPM)

NHCH2CH2Cl

contains a 3rd Period atom – 'P'; thus it has two centers of high energy – Fig 3, 14.

[28, 30, 31].

cancer that spreads to the CNS.

**8. A 2nd High energy drug**

R

Where: R = H; Ifosfamide (IFOS)

**Figure 14.** Ifosfamide and analogs

tumor promotion [41-45].

toxicities.

**Figure 13.** Interaction of DM-CHOC-PEN with Rapier melanin cell cycle and mitochondrion [32]

Melanin is potentially a storage deposit of electrons and/or for electron-rich molecules (re‐ ported by our group years ago) [31]. Due to the high redox potential between DOPA and DOPA quinone, +0.37v, a possible yield of -19.8 kcal/mole occurs upon the oxidation of 1 mole DOPA. A total of 12 e are generated per mole of DOPA oxidized to indole quinone which polymerize to melanin [Fig. 13 [31]. A field of melanin encompassing a colony of can‐ cer cells could generate a hostile electrical microenvironment and inhibit cellular metabo‐ lism and replication [33].

Melanoma cells are a classical representation of cancer cells with a resting low free energy (ΔF) and high entropy (ΔS) – the *alpha state* [14]. The interactions of DM-CHOC-PEN and DOPA induced the formation of melanin and ROS which resulted in an increased envi‐ ronmental ΔF and a decrease in ΔS; a new resting high energy state – the *beta state*.

Thus, DM-CHOC-PEN is a new drug entity that crosses the BBB and couples two new tar‐ gets – the melanin cycle and superoxide formation with apoptosis plus DNA guanine – *N7* adducts. DM-CHOC-PEN is a potential triple treat for CNS melanoma, the 3rd most common cancer that spreads to the CNS.

#### **8. A 2nd High energy drug**

Thus, melanin is a polymer of indole-5, 6-quinone which is highly electrophilic due to its conjugated structure and capable of attracting, storing and/or transferring electrons as elec‐ trical energy [30, 31, 34]. DM-CHOC-PEN treatment induced ROS formation *via* the melanin

**Figure 13.** Interaction of DM-CHOC-PEN with Rapier melanin cell cycle and mitochondrion [32]

mole DOPA. A total of 12 e-

lism and replication [33].

Melanin is potentially a storage deposit of electrons and/or for electron-rich molecules (re‐ ported by our group years ago) [31]. Due to the high redox potential between DOPA and DOPA quinone, +0.37v, a possible yield of -19.8 kcal/mole occurs upon the oxidation of 1

which polymerize to melanin [Fig. 13 [31]. A field of melanin encompassing a colony of can‐ cer cells could generate a hostile electrical microenvironment and inhibit cellular metabo‐

Melanoma cells are a classical representation of cancer cells with a resting low free energy (ΔF) and high entropy (ΔS) – the *alpha state* [14]. The interactions of DM-CHOC-PEN and DOPA induced the formation of melanin and ROS which resulted in an increased envi‐

are generated per mole of DOPA oxidized to indole quinone

system resulting in cancer cell death – an end point.

450 Evolution of the Molecular Biology of Brain Tumors and the Therapeutic Implications

Another high energy drug, 4-hydroperoxyifosfamide (HOOI) [Fig. 3, 14] and is a pre-activat‐ ed form of ifosfamide (IFOS) which possesses the criteria for discussion [Fig. 14]. HOOI also contains a 3rd Period atom – 'P'; thus it has two centers of high energy – Fig 3, 14.

**Figure 14.** Ifosfamide and analogs

IFOS is a well-known anticancer agent that requires hepatic activation to 4-HO-IFOS which spontaneously undergoes hydrolysis with ring opening resulting in isophosphoramide mus‐ tard (IPM) [Fig. 14], the latter is the active cytotoxic form of IFOS [37-39]. As part of the IFOS activation process, hepatic metabolic dechloroethylation releases chloroacetaldehyde, which has been proposed to be the major cause of IFOS associated neurotoxicity [40]. Acrolein is also released and has been implicated in dose limiting toxicities - hemorrhagic cystitis and 2<sup>o</sup> tumor promotion [41-45].

In contrast, HOOI does not require hepatic microsomal activation, crosses the BBB and is readily absorbed by cancer cells where it releases IPM *in situ* [46, 47]. The former does not generate chloroacetaldehyde (neurotoxic) during its conversion to IPM, as does IFOS. Nei‐ ther hemorrhagic cystitis nor renal toxicity has been observed with HOOI in animal toxicol‐ ogy models. Pulmonary damage that could occur with peroxides – pulmonary air emboli was not observed in the dog HOOI study [46]. Hematological toxicity (bone marrow deple‐ tion) was the dose limiting toxicity in the dog study [47].

The potential therapeutic usefulness of HOOI is supported by in vivo activity in human tu‐ mor xenografts and cyclophosphamide (CPA)-resistant murine tumor models plus reduced toxicities.

*In vivo* in comparison to CPA and IFOS in rodent and dog species [Table 3] [47], a potential‐ ly improved safety profile is anticipated – with reduced systemic formation of acrolein and the absence of chloroacetaldehyde during the conversion of HOOI to IPM – *in situ* [47].

An early study reported that SK-MEL-31 human melanoma cells were sensitive to HOOI, which prompted the comparison of the drug with DM-CHOC-PEN in the B-16 melanoma

In 2012, the incidence of new cases of melanoma in the US alone is estimated at 76,250, an increase of ~ 20% during the past 10 years [9]. Of these cases, ~ 25% will metastasize to the

Fig. 15 describes the impact on survival of B-16 mouse melanoma when HOOI is adminis‐

DM-CHOC-PEN (200 mg/kg/d) was injected IP daily x 5-days followed by HOOI (90 mg/m2

IP daily x 3 days (Day 6-8). The latter is a more classical mustard type agent with activity in the G1 phase, as compared to DM-CHOC-PEN, which inhibited cell replication at the S

The %LTS for the 2-drug combination was 173% vs. HOOI alone - 78%; for DM-CHOC-PEN

No drug related toxicity was noted – weight gain continued until tumor growth was a burden and animals sacrificed. The use of 5-day dosing for HOOI was toxic and not considered [48].

Combination of the two -'high energy' agents selected for discussion as binary therapy has po‐ tential. It will be a while before these 2-drugs are administered together; however, other binary

selections that incorporate DM-CHOC-PEN are in the planning stage for Phase II trials.

**Figure 15.** DM-CHOC-PEN plus HOOI in the Treatment of B-16 Mouse Melanoma

)

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model [47].

phase – Fig. 12b [49].

alone - 142% (see Fig. 14).

CNS; thus our interest in the drug [26].

tered alone and with DM-CHOC-PEN [49].

Dose – qd x 5d (IP) – except TMZ – PO Q4D x 3; \*human breast cancer – SC implanted, \*\*human glioblastoma – IC implanted; \*\*\*CPA (cyclophosphamide) – resistant murine leukemia. For MX-1 – T-C (days) = difference in median times post implant for tumors of treated groups to attain an evaluation size compared to median of control group; +6 log cell kill; ++>40 mg/kg was too toxic; \*\*\*% ILS (increased length of survival – study terminated @ 54 days.

**Table 3.** Human Xenografts and Murine Tumors Growing in Mouse Models

HOOI possesses two [2] 3-high energy atom chains, thus the drug more than fits the criteria proposed earlier in this chapter (3). In addition, HOOI does not require activation by the liv‐ er and spontaneously undergoes conversion to IPM and cross-links with nuclear DNA via major groove alkylation – *N7* -guanine, forming G-X-C adducts [39].

Of significance is that HOOI was curative (54% LTS, with 20% CR) *vs.* the human U251 glio‐ blastoma implanted IC as well as the MX-1 breast cancer IC model (Table 3) [47]. It should also be noted that BCNU – the gold standard for years in the treatment of gliomas produced no CRs and TMZ the current standard produced identical responses to HOOI.

Like DM-CHOC-PEN, HOOI accumulates in U-251 glioma tissue in 50 ng/g tissue content when the drug is administered IP to animals bearing IC implanted tumors [47]. No CNS/ behavioral alterations or toxicity have been noted for HOOI [48].

Unlike ifosfamide (IFOS), HOOI is more lipophilic, activated *in situ*, with less extracellular acrolein, no chloroacetaldehyde released and no IFOS associated CNS or GU toxicity [48]. In dogs the principal toxicity with HOOI was bone marrow suppression that reversed with time [47, 48].

An early study reported that SK-MEL-31 human melanoma cells were sensitive to HOOI, which prompted the comparison of the drug with DM-CHOC-PEN in the B-16 melanoma model [47].

*In vivo* in comparison to CPA and IFOS in rodent and dog species [Table 3] [47], a potential‐ ly improved safety profile is anticipated – with reduced systemic formation of acrolein and the absence of chloroacetaldehyde during the conversion of HOOI to IPM – *in situ* [47].

> **MX-1\* T- C (Days)**

>28.8 9

>54 days (20% LTS) [Dose - 50]

BCNU 9-15 (MTD) 5 12% LTS; 0 CR NA +52 (1/5 CR) NA

Dose – qd x 5d (IP) – except TMZ – PO Q4D x 3; \*human breast cancer – SC implanted, \*\*human glioblastoma – IC implanted; \*\*\*CPA (cyclophosphamide) – resistant murine leukemia. For MX-1 – T-C (days) = difference in median times post implant for tumors of treated groups to attain an evaluation size compared to median of control group; +6-

HOOI possesses two [2] 3-high energy atom chains, thus the drug more than fits the criteria proposed earlier in this chapter (3). In addition, HOOI does not require activation by the liv‐ er and spontaneously undergoes conversion to IPM and cross-links with nuclear DNA via

Of significance is that HOOI was curative (54% LTS, with 20% CR) *vs.* the human U251 glio‐ blastoma implanted IC as well as the MX-1 breast cancer IC model (Table 3) [47]. It should also be noted that BCNU – the gold standard for years in the treatment of gliomas produced

Like DM-CHOC-PEN, HOOI accumulates in U-251 glioma tissue in 50 ng/g tissue content when the drug is administered IP to animals bearing IC implanted tumors [47]. No CNS/

Unlike ifosfamide (IFOS), HOOI is more lipophilic, activated *in situ*, with less extracellular acrolein, no chloroacetaldehyde released and no IFOS associated CNS or GU toxicity [48]. In dogs the principal toxicity with HOOI was bone marrow suppression that reversed with

no CRs and TMZ the current standard produced identical responses to HOOI.

behavioral alterations or toxicity have been noted for HOOI [48].

log cell kill; ++>40 mg/kg was too toxic; \*\*\*% ILS (increased length of survival – study terminated @ 54 days.

**ZR-75-1\* T- C (Days)**

(33% LTS) >46.1 (83% CR) +54 (1/5 CR) +209+

10 8.6 (7% LTS) >43.8 (17% CR) 50 (0/5 CR) 42

10 2.1 NA NA +85

5 NA NA +54 (1/5 CR) NA

NA +54 (1/5 CR) NA

**U251 (%ILS)\*\*** **P388/CPA (% ILS)\*\***

**Drug**

HOOI

IFOS

IPM

**Dose (mg/kg/day)**

90 (<LD10)

DM-CHOC-PEN 135 10

40 (MTD)++

40 (MTD)++

TMZ 120 mg (MTD)

major groove alkylation – *N7*

time [47, 48].

**No of Mice**

452 Evolution of the Molecular Biology of Brain Tumors and the Therapeutic Implications

10

**Table 3.** Human Xenografts and Murine Tumors Growing in Mouse Models

In 2012, the incidence of new cases of melanoma in the US alone is estimated at 76,250, an increase of ~ 20% during the past 10 years [9]. Of these cases, ~ 25% will metastasize to the CNS; thus our interest in the drug [26].

Fig. 15 describes the impact on survival of B-16 mouse melanoma when HOOI is adminis‐ tered alone and with DM-CHOC-PEN [49].

DM-CHOC-PEN (200 mg/kg/d) was injected IP daily x 5-days followed by HOOI (90 mg/m2 ) IP daily x 3 days (Day 6-8). The latter is a more classical mustard type agent with activity in the G1 phase, as compared to DM-CHOC-PEN, which inhibited cell replication at the S phase – Fig. 12b [49].

The %LTS for the 2-drug combination was 173% vs. HOOI alone - 78%; for DM-CHOC-PEN alone - 142% (see Fig. 14).

No drug related toxicity was noted – weight gain continued until tumor growth was a burden and animals sacrificed. The use of 5-day dosing for HOOI was toxic and not considered [48].

Combination of the two -'high energy' agents selected for discussion as binary therapy has po‐ tential. It will be a while before these 2-drugs are administered together; however, other binary selections that incorporate DM-CHOC-PEN are in the planning stage for Phase II trials.

**Figure 15.** DM-CHOC-PEN plus HOOI in the Treatment of B-16 Mouse Melanoma

#### **9. Conclusion**

Melanoma cells, as seen in Fig. 7a represent classical cancer cells with a resting low free en‐ ergy (ΔF) and high entropy (ΔS) – the *alpha state* [14]. The interaction of DM-CHOC-PEN and DOPA induced the formation of melanin – a high energy component/storage that re‐ sulted in an increase in environmental ΔF and a decrease in ΔS; a new resting high energy state – the *beta state* [14]. Cells that are encompassed in the 'melanin balls' (Fig. 7b) were con‐ verted to a well differentiated state and die. The addition of HOOI to DM-CHOC-PEN in‐ hibited those cells that reversed to the *alpha state* – survive and continue to replicate. Thus, the binary combination improved %LTS vs. either drug alone; supporting the *in vitro* obser‐ vations.

**Acknowledgement**

**Author details**

Lee Roy Morgan\*

**References**

7: 337-44, 1996.

surg., 88: 11-20, 1998.

gist 12: 884-98, 2007.

Address all correspondence to<EMAIL_ADDRESS>
Grant support from NCI/SBIR R43/44CA85021 and R43CA132257 is appreciated.

discussions, review and comments during the preparation of this manuscript.

CEO DEKK-TEC, Inc University of New Orleans New Orleans, LA, USA

ology of a grave matter. Genes Dev., 15:1311-33, 2001.

The author wishes to thank Drs. Robert F. Struck, Branko S. Jursic, Roy S. Weiner, Marcus L. Ware, Barry Sartin, Philip Friedlander, Andrew Rodgers and Mr. Edmund Benes for their

A Rational for Novel Anti-NeuroOncology Drugs

http://dx.doi.org/10.5772/53889

455

[1] Maher, EA, Furnari, FB, Bachoo, RM, et al. Malignant glioblastoma: genetics and bi‐

[2] Van Brachlyn, JR, Jackson, CA, Pearl, DK, et al. Spingosine kinase -1 expression cor‐ relates with poor survival of patients with GBM: roles of spingosine kinase isoforms

[3] Patchell, RA. The management of brain metastasis. Cancer Treatment Rev. 29: 533-40. [4] Johnson, JD, Young, B, Demographics of brain metastasis. Neurosurg. Clin. N. Am.,

[5] Sampson, JH, Carter, JH, Friedman, AH, Seiger, HF. Demographics, prognosis and therapy in 702 patients with brain metastasis from malignant melanoma. J. Neuro‐

[6] Eichler, AF, Loeffler, JS. Multidisciplinary management of brain metastasis. Oncolo‐

[8] Villano, JL, Seery, TE, Bressler, LR. Temozolomide in malignant gliomas: current use and future targets. Cancer Chemotherapy and Pharmacology, 64: 647-655, 2009.

[9] Thompson, JA. Ten years of progress in melanoma. JNCCN, 10: 931-35, 2012.

[10] Genomics and Personalized Medicine Act of 2006; Bill of Congress 1093822.

[7] Smit and Marshall. Editorial, Community Oncology, 9: 250-258, 2012.

in glioblastoma cell lines. J. Neuropatol. Exp. Neurol, 64: 695-705, 2005.

Although, only two patients with CNS melanoma have been treated with DM-CHOC-PEN in the Phase I trial, the report that one patient did demonstrate an interval response and one patent had a no change response, both with no neurotoxicity is encouragement for the po‐ tential use of the drug in the treatment of CNS melanoma [25]. HOOI has not been used in clinical trials as yet.

There are many novel drugs that fall in to the 'high-energy' category that should be evaluat‐ ed or re-visited. In particular – clomesone [Fig. 3], a drug developed at Southern Research Institute, US had potential in animal studies, but produced hematological toxicity in Phase I trials and discontinued from studies [17, 50]. The drug has novel chloroethylating properties and like BCNU forms adducts with DNA guanine-O6 [50]. In addition, the drug contains two - S - atoms which are a 3rd Period element and able to expand its 3d orbital – a real 'high-energy' molecule. However, only one of the latter groups has the ability to resonant with energy storage.

With the two sets of 3-atom chain as a point of interest (Fig. 3, 6), clomesone should be revis‐ ited with dose modifications; it has potential [52].

In summary, three 'high energy' drugs have been reviewed as support that a novel ap‐ proach to the treatment of CNS malignancies could be through the inefficient energy system of brain tumors [11, 12]. Anticancer agents that have the appropriate structure, can penetrate the CNS, not be recycled out and inhibit cancer growth.

Over all, DM-CHOC-PEN has been well tolerated in Phase I studies with advanced –breast cancer, sarcoma, glioblastoma multiforme, melanoma, pancreas, esophageal, non-small cell lung cancer colorectal cancer and cervical cancer and will obviously enter Phase II trials alone or as binary therapy [25].

Furthermore, HOOI is being readied for Phase I clinical trials and the results should be equally interesting.

#### **Acknowledgement**

**9. Conclusion**

vations.

clinical trials as yet.

with energy storage.

alone or as binary therapy [25].

equally interesting.

ited with dose modifications; it has potential [52].

the CNS, not be recycled out and inhibit cancer growth.

Melanoma cells, as seen in Fig. 7a represent classical cancer cells with a resting low free en‐ ergy (ΔF) and high entropy (ΔS) – the *alpha state* [14]. The interaction of DM-CHOC-PEN and DOPA induced the formation of melanin – a high energy component/storage that re‐ sulted in an increase in environmental ΔF and a decrease in ΔS; a new resting high energy state – the *beta state* [14]. Cells that are encompassed in the 'melanin balls' (Fig. 7b) were con‐ verted to a well differentiated state and die. The addition of HOOI to DM-CHOC-PEN in‐ hibited those cells that reversed to the *alpha state* – survive and continue to replicate. Thus, the binary combination improved %LTS vs. either drug alone; supporting the *in vitro* obser‐

454 Evolution of the Molecular Biology of Brain Tumors and the Therapeutic Implications

Although, only two patients with CNS melanoma have been treated with DM-CHOC-PEN in the Phase I trial, the report that one patient did demonstrate an interval response and one patent had a no change response, both with no neurotoxicity is encouragement for the po‐ tential use of the drug in the treatment of CNS melanoma [25]. HOOI has not been used in

There are many novel drugs that fall in to the 'high-energy' category that should be evaluat‐ ed or re-visited. In particular – clomesone [Fig. 3], a drug developed at Southern Research Institute, US had potential in animal studies, but produced hematological toxicity in Phase I trials and discontinued from studies [17, 50]. The drug has novel chloroethylating properties and like BCNU forms adducts with DNA guanine-O6 [50]. In addition, the drug contains two - S - atoms which are a 3rd Period element and able to expand its 3d orbital – a real 'high-energy' molecule. However, only one of the latter groups has the ability to resonant

With the two sets of 3-atom chain as a point of interest (Fig. 3, 6), clomesone should be revis‐

In summary, three 'high energy' drugs have been reviewed as support that a novel ap‐ proach to the treatment of CNS malignancies could be through the inefficient energy system of brain tumors [11, 12]. Anticancer agents that have the appropriate structure, can penetrate

Over all, DM-CHOC-PEN has been well tolerated in Phase I studies with advanced –breast cancer, sarcoma, glioblastoma multiforme, melanoma, pancreas, esophageal, non-small cell lung cancer colorectal cancer and cervical cancer and will obviously enter Phase II trials

Furthermore, HOOI is being readied for Phase I clinical trials and the results should be

Grant support from NCI/SBIR R43/44CA85021 and R43CA132257 is appreciated.

The author wishes to thank Drs. Robert F. Struck, Branko S. Jursic, Roy S. Weiner, Marcus L. Ware, Barry Sartin, Philip Friedlander, Andrew Rodgers and Mr. Edmund Benes for their discussions, review and comments during the preparation of this manuscript.

#### **Author details**

Lee Roy Morgan\*

Address all correspondence to<EMAIL_ADDRESS>
CEO DEKK-TEC, Inc University of New Orleans New Orleans, LA, USA

#### **References**

[11] Wollemann, M. Biochemistry of brain tumours, University Park Press, Baltimore, 1974.

[24] Morgan, LR IND 0688876 DM-CHOC-PEN- Study May Proceed, FDA September 24,

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[25] Weiner, RS, Friedlander, P, Gordon, C, Ware, ML, Bastian, G, Rodgers, AH, Urien, S, Morgan, LR. Comparative Pharmacokinetics of 4-Demethyl-4-cholesteryloxycarbo‐ nylpenclomedine (DM-CHOC-PEN) in Humans, Proc. Amer. Assoc. Cancer Res., 53:

[27] Morgan, LR. Complexes of 4-hydroperoxyifosfamide as antitumor agents, EU Patent

[28] Morgan, LR, Benes, E, Rodgers, AH, Jursic, BS, Struck, BF, Waud, WR, Weiner, RS, Ware, M, Friedlander, P. Interaction of 4-Demethyl-4-cholesteryloxycarbonyl penclo‐ medine (DM-CHOC-PEN) with Melanoma Melanin Metabolism and Cell Death, EC‐

[29] Morgan, LR, Rodgers, AH, Bastian, G, Papagiannis, C, Krietlow, D, Struck, RF, Waud, WR. Comparative pharmacokinetics and intermediary metabolism of 4-de‐ methyl-4-cholesteryloxycarbonylpenclomedine (DM-CHOC-PEN), EORTC/AACR/

[30] Morgan, LR, Singh, R. Cytochrome oxidase-succinic dehydrogenase activities and the melanin pigment cycle in poikilothermic vertebrates. Comp. Biochem. Physiol.,

[31] Morgan, LR, Singh, R, Sylvest, V, Weimort, D. Oxidation of o-phenols by mouse and human melanoma dihydroxyphenyl alanine oxidase and dihydroxyphenyl alanine.

[33] Mason, HS. Structure of Melanins, In: Pigment Cell Biology, Ed. Gordon, M. Aca‐

[34] Van Woert, HH, Nicholson, A, Cotzias, GC. Functional similarities between the cyto‐ plasmic organelles of melanocytes and mitochondria of hepatocytes. Nature, 208,

[35] Traub, EF, Spoor, HJ. Melanin and tyrosinase in skin pigmentation. In: Pigment Cell Growth, 3rd Conference on Biology of Normal and Atypical Pigment Cell Growth.

[36] Pullmans, A and Pullman, B. The band structure of melanin. Biochem. Biophys. Acta,

[38] Norpoth, K. Studies on the metabolism of isophosphamide in man. Cancer Treat.

[37] Zolwyski, M., and Baker, L.H. Ifosfamide. J. Natl. Cancer Inst. 80:556-566, 1988.

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[15] Wald, G. Life in the second and third periods; or why phosphorus and sulfur for high energy bonds? In: Horizons in Biochemistry, Ed. Kasha, M, Pullman, Academic

[16] Morgan, LR, Struck, RF, Rodgers, AH, Bastian, G, Jursic, BS, Papaginnis, C, Waud, W. Intracerebral metabolism and pharmacokinetics of 4-demethyl-4-cholesteryl-oxy‐ carbonylpenclomedine (DM-CHOC-PEN). Proc. Amer. Assoc. Res., 49: Abst. 3745,

[17] Shealy YF, Krauth CA, Laster WR. 2-Chloroethylmethylsulfonyl)methanesulfonate and related (methylsulfonyl)methanesulfonates. Antineoplastic activity in vivo. J. Med. Chem. 27: 664-670, 1984; Dykes DJ, Waud WR, Harrison SD, Griswold DP, Shealy YF, Montgomery JA. Antitumor activity of 2-chloroethyl (ethylsulfonyl)meth‐ ane-sulfonate (clomesone, NSC 33847) against selected tumor systems in mice. Can‐

[18] Klotz, IM. Energy changes in biochemical reactions, Academic Press, New York,

[19] Morgan, LR, Struck, RF, Waud, WR, LeBlanc, B, Rodgers, AH, Jursic, BS. Carbonate and carbamate derivatives of 4-demethylpenclomedine as novel anticancer agents.

[20] Morgan, LR, Rodgers, AH, Bastian, G, Struck, RF, Waud, WR. Comparative pharma‐ cokinetics and intermediary metabolism of 4-demethyl-4-cholesteryl- oxycarbonyl‐

[21] Morgan, LR, Struck, RF, Rodgers, AH, Serota, DG. Preclinical Toxicity of 4- Demeth‐ yl-4-cholestryloxyl-carbonylpenclomedine (DM-CHOC-PEN). Proc. Amer. Assoc.

[22] Pletsas, D, Wheelhouse, RT, Pletsa, V, Nicolaou, A, Jenkins, TC, Bibby, MC, Kyto‐

[23] Morgan, LR. Demethylpenclomedine analogs and their use as anti-cancer agents. US

kylguanine-DNA alkyltransferase: implications for the design of DNA-modifying

derivatives resistant to repair by O6

Cancer Chemotherapy and Pharmacology, 64: 829-836, 2009.

penclomedine (DM-CHOC-PEN), EORTC/AACR/NCI, 567, 2010.

[12] Warburg, O. The Metabolism of tumours, Constable, London, 1930.

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1974.

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[39] Struck, RF, Dykes, DJ, Corbett, TH, Suling, WJ, MW. Isophosphoroamide mustard, a metabolite of ifosfamide with activity against murine tumors comparable to cyclo‐ phosphamide. Brit. J. Cancer 47:15-26, 1983.

**Chapter 16**

**DNA-PK is a Potential Molecular**

**Therapeutic Target for Glioblastoma**

P. O. Carminati, F. S. Donaires, P. R. D. V. Godoy,

A. P. Montaldi, J. A. Meador, A.S. Balajee,

Additional information is available at the end of the chapter

Glioblastoma multiforme is one of the deadliest forms of brain tumor with a median survival of <12 months with a high rate of recurrence. Glioblastoma cells respond poorly to radiother‐ apy and chemotherapy due to elevated DNA repair efficiency coupled with anti-apoptotic mechanisms [1, 2]. Therefore, development of new therapeutic strategies is absolutely critical either to control or cure the disease. Since glioblastoma cells have inherently elevated levels of DNA damage response (DDR) and DNA repair efficiency, we sought to determine whether or not suppression of key DNA repair factor(s) might be effective in sensitizing the brain tumor cells to chemotherapy. To test our objective, a pair of isogenic glioblastoma cell lines that differ in the functional status of DNA dependent protein kinase (DNA-PK) has been chosen. DNA dependent protein kinase (DNA-PK) belongs to a super family of phosphatidylinositol-3 kinase like kinases (PIKK) which also includes the gene products of ataxia telangiectasia mutated (ATM) and ATM and Rad 3 related (ATR) kinases [3, 4]. DNA-PK is the main component of mammalian NHEJ (non-homologous end joining), and also plays a role as a sensor of DNA damage by phosphorylating several downstream targets [5]. Further, DNA-PK has been demonstrated to be an important component of nucleotide excision repair (NER) pathway, which is chiefly responsible for the repair of bulky and helix distorting lesions induced by ultraviolet radiation (UV) and chemotherapeutic drugs such as cisplatin. To determine whether or not DNA-PK is a potential molecular therapeutic target for glioblastoma, two agents with different modes of action have been chosen for our study: (1) Zebularine and

> © 2013 Carminati et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

G. A. Passos and E. T. Sakamoto-Hojo

http://dx.doi.org/10.5772/53181

**1. Introduction**

(2) Cisplatin.

**Chapter 16**

## **DNA-PK is a Potential Molecular Therapeutic Target for Glioblastoma**

P. O. Carminati, F. S. Donaires, P. R. D. V. Godoy,

A. P. Montaldi, J. A. Meador, A.S. Balajee,

G. A. Passos and E. T. Sakamoto-Hojo

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53181

#### **1. Introduction**

[39] Struck, RF, Dykes, DJ, Corbett, TH, Suling, WJ, MW. Isophosphoroamide mustard, a metabolite of ifosfamide with activity against murine tumors comparable to cyclo‐

[40] Goren, MP, Wright, RK, Pratt, CB, Pell, FE. Dechloroethylation of ifosfamide and

[41] Cox, PJ. Cyclophosphamide cystitis – Identification of acrolein as the causativet. Bio‐

[42] Seo, IS, Clark, SA, McGovern, FD, Clark, DL, Johnson, EH. Leiomyosarcoma of the urinary bladder 13 years after cyclophosphamide therapy for Hodgkin's disease.

[43] Colburn, KK, Cao, JD, Krick, EH, Mortensen, SE, Wong, LG. Hodgkins lymphoma in a patient treated for Wegeners granulomatosis with cyclophosphamide and azathio‐

[44] Cuzick, J, Erskine, S, Edelman, D, Gelton, DAG. A comparison of the incidence of myelodyplastic syndrome and acute myeloid leukemia following melphalan and cy‐

[45] Durst, J., Ahrens, S., Paulussen, M., Rube, C., Winkelmann, W., Zoubek, A., Harms, D. and Jurgens, H. Second malignancies after treatment for Ewing's sarcoma: report

[46] Narayanan, V. In vivo evaluation of 4-hydroperoxyifosfamide (NSC 207117 and 227114). Screening Data Summary, Dev. Ther. Program, Dir. Cancer Treat. NCI, Be‐

[47] Morgan, L.R, Struck, R.F. Rodgers, A.H., Jursic, B.S., Waud, W.S., Butera, D., Devel‐ opment of Clinical Products. NCI Translational Science Meeting, Washington, DC,

[48] Morgan, L.R., Struck, R.F., Rodgers, A.H., Jursic, B.S. Waud, W. R. Pre- clinical Phar‐ macology and Toxicology for 4-Hydeoperoxyifosfamide (HOOI) and its L-lysine Salt – A Novel Anticancer Agent. Amer. Assoc Cancer Res, 42, 3222, 2011; Cancer Chemo‐

[49] Morgan, LR, Benes, E, Rodgers, AH, Jursic, BS, Friedlander, P, Weiner, RS, Ware, ML, Struck, RF. 4-Demethyl-4-cholesterylcarbonylpenclomedine (DM-CHOC-PEN) and 4-Hydroperoxyifosfamide (HOOI) as Binary Therapy for Melanoma, EORTC/

clophosphamide treatment for myelomatosis. Brit. J. Cancer 55:523-530, 1987.

of the CESS-studies. Int. J. Radiat. Oncol. Biol. Phys. 42:379-384, 1998.

phosphamide. Brit. J. Cancer 47:15-26, 1983.

458 Evolution of the Molecular Biology of Brain Tumors and the Therapeutic Implications

neurotoxicity. Lancet 2:1219-1220, 1986.

chem. Pharmacol. 28:2045-2049, 1979.

prine. J. Rheumatol. 12:599-602, 1985.

therapy Pharmacology, submitted.

[50] Robert F. Struck, Personnel communication.

AACR/NCI, Abst, 703, 2011.

Cancer 55:1597-1603, 1985.

thesda, MD 20205.

2008.

Glioblastoma multiforme is one of the deadliest forms of brain tumor with a median survival of <12 months with a high rate of recurrence. Glioblastoma cells respond poorly to radiother‐ apy and chemotherapy due to elevated DNA repair efficiency coupled with anti-apoptotic mechanisms [1, 2]. Therefore, development of new therapeutic strategies is absolutely critical either to control or cure the disease. Since glioblastoma cells have inherently elevated levels of DNA damage response (DDR) and DNA repair efficiency, we sought to determine whether or not suppression of key DNA repair factor(s) might be effective in sensitizing the brain tumor cells to chemotherapy. To test our objective, a pair of isogenic glioblastoma cell lines that differ in the functional status of DNA dependent protein kinase (DNA-PK) has been chosen. DNA dependent protein kinase (DNA-PK) belongs to a super family of phosphatidylinositol-3 kinase like kinases (PIKK) which also includes the gene products of ataxia telangiectasia mutated (ATM) and ATM and Rad 3 related (ATR) kinases [3, 4]. DNA-PK is the main component of mammalian NHEJ (non-homologous end joining), and also plays a role as a sensor of DNA damage by phosphorylating several downstream targets [5]. Further, DNA-PK has been demonstrated to be an important component of nucleotide excision repair (NER) pathway, which is chiefly responsible for the repair of bulky and helix distorting lesions induced by ultraviolet radiation (UV) and chemotherapeutic drugs such as cisplatin. To determine whether or not DNA-PK is a potential molecular therapeutic target for glioblastoma, two agents with different modes of action have been chosen for our study: (1) Zebularine and (2) Cisplatin.

© 2013 Carminati et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Many tumor cells have characteristic epigenetic alterations involving genome wide DNA hypo-methylation and gene specific hyper-methylation at CpG dinucleotide sequences leading to aberrant silencing of critical genes involved in DNA damage response, cell cycle and DNA metabolic processes. Recent studies [6-8] have demonstrated the usefulness of epigenetic targeting in treating many cancer cell types through inhibition of histone deacety‐ lases (HDAC) or DNA methyl transferases (DNMTs). Zebularine is a potent inhibitor of DNMTs and is superior to 5-AzaCR in terms of lower cytotoxicity and increased stability in aqueous solutions [9]. While Zebularine is a DNA-demethylating agent, cisplatin is a DNA cross-linking agent that induces the formation of DNA inter- and intra-strand cross-links. These cross-links are removed preferentially by NER pathway and NER deficient tumor cells are often sensitized to cisplatin treatment. Since DNA-PK plays a critical role in genome surveillance and DNA repair mechanisms, we wished to evaluate the potential of DNA-PK as a molecular therapeutic target for glioblastoma.

**2.2. DNA–PK deficient cells are sensitive to cisplatin treatment**

The relationship between DNA-PK/Ku activity and cisplatin sensitivity has been observed earlier in gliomas. Gliomas with high expression level of DNA-PK are resistant to cisplatin [11]. In an earlier review, we suggested that DNA-PK inhibition may be a promising strategy for treating glioblastomas [for review see 12]. To determine the role of DNA-PK in conferring cisplatin resistance, MO59J and MO59K cells were treated with cisplatin (5-75 µM; Sigma Aldrich, USA) for 72 h and assayed for proliferation using a fluorescence based CyQuant assay (Invitrogen, USA). MO59J showed a greater sensitivity to cisplatin compared to MO59K, and displayed a dose dependent decrease in cell proliferation, being reduced to 20% relative to the vehicle (DMSO) treated control cells for the highest drug concentration (75 µM). In contrast, MO59K was sensitive to cisplatin only at high doses (>50 µM). These results clearly indicate

DNA-PK is a Potential Molecular Therapeutic Target for Glioblastoma

http://dx.doi.org/10.5772/53181

461

that DNA-PK is a critical factor for cellular protection against cisplatin (Figure 1).

**Figure 1.** Cell proliferation assay using CyQuant (Invitrogen, USA) in MO59K and MO59J cells treated with cisplatin (5,

Cell cycle analysis was next performed to verify whether the enhanced cisplatin cytotoxicity of MO59J cells was due to defective cell cycle checkpoints. MO59K and MO59J cells in exponential growth phase were exposed to different concentrations (5 to 25 µM) of cisplatin and the cell cycle profile was analyzed by flow cytometry (24, 48 and 72 h after treatment). While DNA-PK proficient MO59K cells displayed an efficient accumulation of S-phase cells at all the concentrations of cisplatin exposure up to 72 h (Figure 2A), DNA-PK mutant MO59J cells exhibited only a modest S-phase accumulation after 24 h of drug treatment (25 µM

cisplatin) (Figure 2B), which was alleviated at later time points (48 and 72 h).

10, 25, 50, and 75 µM) for 72 h.

### **2. DNA-PK deficient cells are sensitive to zebularine and cisplatin treatments**

#### **2.1. Zebularine sensitizes DNA–PK deficient glioblastoma cells**

DNA-PK deficient (MO59J) and proficient (MO59K) glioblastoma cells were purchased from American Type Culture Collection (Rockville, MD, USA). Cells were routinely cultured in OPTI-MEM supplemented with 5% fetal bovine serum, vitamins and antibiotics. Zebularine [1-beta-D-ribofuranosyl)-1, 2-dihydropyrimidin-2-one] was purchased from EMD Biosciences (San Diego, CA, USA). Zebularine was dissolved in dimethyl sulfoxide (DMSO) at a stock concentration of 100 mM and stored at 4ºC. Clonogenic survival and proliferations assays were carried to determine the sensitivity of human gliomas (MO59J and MO59K) to different Zebularine concentrations (0-300 µM). The LC50 value for Zebularine was found to be 245 µM for DNA-PK deficient MO59J cells and at this concentration the survival of MO59K cells was reduced only by 20% [10]. This finding indicates that lack of DNA-PK potentiates the sensiti‐ zation of gliomas to Zebularine. In contrast to MO59K cells, DNA-PK deficient cells (MO59J) showed a reduction in survival (20%) even at the lowest Zebularine concentration (10 µM). Further, an elevated frequency of polyploid cells observed specifically in MO59J cells after Zebularine treatment pointed out a mitotic checkpoint deficiency. Although depletion of DNMT1 by Zebularine occurred at similar levels in both glioblastoma cell lines, DNA-PK deficient MO59J cells displayed elevated DNA hypo-methylation detected both at genome overall and gene promoter levels. Consistent with increased sensitivity, deoxy-Zebularine adduct level, measured for the first time by us in the genomic DNA, was 3-6 fold higher in DNA-PK deficient MO59J cells relative MO59K cells. Elevated micronuclei frequency was also observed in MO59J cells indicating the impairment of DNA damage response and repair. Collectively, our study suggests that DNA-PK is a potentially useful molecular therapeutic target for gliomas.

#### **2.2. DNA–PK deficient cells are sensitive to cisplatin treatment**

Many tumor cells have characteristic epigenetic alterations involving genome wide DNA hypo-methylation and gene specific hyper-methylation at CpG dinucleotide sequences leading to aberrant silencing of critical genes involved in DNA damage response, cell cycle and DNA metabolic processes. Recent studies [6-8] have demonstrated the usefulness of epigenetic targeting in treating many cancer cell types through inhibition of histone deacety‐ lases (HDAC) or DNA methyl transferases (DNMTs). Zebularine is a potent inhibitor of DNMTs and is superior to 5-AzaCR in terms of lower cytotoxicity and increased stability in aqueous solutions [9]. While Zebularine is a DNA-demethylating agent, cisplatin is a DNA cross-linking agent that induces the formation of DNA inter- and intra-strand cross-links. These cross-links are removed preferentially by NER pathway and NER deficient tumor cells are often sensitized to cisplatin treatment. Since DNA-PK plays a critical role in genome surveillance and DNA repair mechanisms, we wished to evaluate the potential of DNA-PK as

**2. DNA-PK deficient cells are sensitive to zebularine and cisplatin**

DNA-PK deficient (MO59J) and proficient (MO59K) glioblastoma cells were purchased from American Type Culture Collection (Rockville, MD, USA). Cells were routinely cultured in OPTI-MEM supplemented with 5% fetal bovine serum, vitamins and antibiotics. Zebularine [1-beta-D-ribofuranosyl)-1, 2-dihydropyrimidin-2-one] was purchased from EMD Biosciences (San Diego, CA, USA). Zebularine was dissolved in dimethyl sulfoxide (DMSO) at a stock concentration of 100 mM and stored at 4ºC. Clonogenic survival and proliferations assays were carried to determine the sensitivity of human gliomas (MO59J and MO59K) to different Zebularine concentrations (0-300 µM). The LC50 value for Zebularine was found to be 245 µM for DNA-PK deficient MO59J cells and at this concentration the survival of MO59K cells was reduced only by 20% [10]. This finding indicates that lack of DNA-PK potentiates the sensiti‐ zation of gliomas to Zebularine. In contrast to MO59K cells, DNA-PK deficient cells (MO59J) showed a reduction in survival (20%) even at the lowest Zebularine concentration (10 µM). Further, an elevated frequency of polyploid cells observed specifically in MO59J cells after Zebularine treatment pointed out a mitotic checkpoint deficiency. Although depletion of DNMT1 by Zebularine occurred at similar levels in both glioblastoma cell lines, DNA-PK deficient MO59J cells displayed elevated DNA hypo-methylation detected both at genome overall and gene promoter levels. Consistent with increased sensitivity, deoxy-Zebularine adduct level, measured for the first time by us in the genomic DNA, was 3-6 fold higher in DNA-PK deficient MO59J cells relative MO59K cells. Elevated micronuclei frequency was also observed in MO59J cells indicating the impairment of DNA damage response and repair. Collectively, our study suggests that DNA-PK is a potentially useful molecular therapeutic

**2.1. Zebularine sensitizes DNA–PK deficient glioblastoma cells**

460 Evolution of the Molecular Biology of Brain Tumors and the Therapeutic Implications

a molecular therapeutic target for glioblastoma.

**treatments**

target for gliomas.

The relationship between DNA-PK/Ku activity and cisplatin sensitivity has been observed earlier in gliomas. Gliomas with high expression level of DNA-PK are resistant to cisplatin [11]. In an earlier review, we suggested that DNA-PK inhibition may be a promising strategy for treating glioblastomas [for review see 12]. To determine the role of DNA-PK in conferring cisplatin resistance, MO59J and MO59K cells were treated with cisplatin (5-75 µM; Sigma Aldrich, USA) for 72 h and assayed for proliferation using a fluorescence based CyQuant assay (Invitrogen, USA). MO59J showed a greater sensitivity to cisplatin compared to MO59K, and displayed a dose dependent decrease in cell proliferation, being reduced to 20% relative to the vehicle (DMSO) treated control cells for the highest drug concentration (75 µM). In contrast, MO59K was sensitive to cisplatin only at high doses (>50 µM). These results clearly indicate that DNA-PK is a critical factor for cellular protection against cisplatin (Figure 1).

**Figure 1.** Cell proliferation assay using CyQuant (Invitrogen, USA) in MO59K and MO59J cells treated with cisplatin (5, 10, 25, 50, and 75 µM) for 72 h.

Cell cycle analysis was next performed to verify whether the enhanced cisplatin cytotoxicity of MO59J cells was due to defective cell cycle checkpoints. MO59K and MO59J cells in exponential growth phase were exposed to different concentrations (5 to 25 µM) of cisplatin and the cell cycle profile was analyzed by flow cytometry (24, 48 and 72 h after treatment). While DNA-PK proficient MO59K cells displayed an efficient accumulation of S-phase cells at all the concentrations of cisplatin exposure up to 72 h (Figure 2A), DNA-PK mutant MO59J cells exhibited only a modest S-phase accumulation after 24 h of drug treatment (25 µM cisplatin) (Figure 2B), which was alleviated at later time points (48 and 72 h).

Proliferating cell nuclear antigen (PCNA) is recruited to chromatin rapidly after DNA damage and the kinetics of disassembly from the chromatin is considered to reflect the DNA repair efficiency [13, 14]. PCNA expression was evaluated by Western blot in cisplatin- treated MO59K and MO59J cells. The expression of PCNA was increased in a dose-dependent manner in MO59K cells after 24 h of drug treatment, while a constant level of PCNA expression was observed in MO59J cells indicating the lack of S-phase specific PCNA accumulation (Figure 4). Our results from MO59K and MO59J cell lines indicate that DNA-PK is a critical determi‐ nant of cell survival after cisplatin treatment and the lack of S-phase arrest (as seen in MO59J

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463

**Figure 4.** Expression of PCNA protein (30 KDa) analyzed by Western blot using specific antibody. MO59K and MO59J

Therefore, DNA-PK may be an important molecule conferring cisplatin resistance in glioblas‐ toma cells, with potential use as a molecular target, and we also observed that the combined effect of cisplatin and LY294002 (an inhibitor of DNA-PK) caused a significant decrease in cell proliferation and an increase in apoptotic cell death in glioma cell lines [15]. Our studies thus far performed indicate that the specific targeting of DNA-PK by small molecule inhibitors may

**3.** *In silico* **analysis of transcription factors associated with differentially**

Transcription factors (TFs) regulate gene expression by binding to specific DNA sequences of gene promoter regions. Their expression can be deregulated under certain pathological conditions, such as cancer. Therefore, studies on regulatory elements of the transcription machinery are essential for understanding the mechanisms involved in the regulation of oncogenes and tumor suppressor genes. Additionally, knowledge of the essential functions of TFs, may lead to strategic development of novel chemotherapy regimen for selectively killing

cells were treated with 5, 10 and 25 µM of cisplatin for 3 and 24 h. β-actin was used as control.

be an effective strategy for the treatment of gliomas.

**expressed genes in GBM cells**

**3.1. Introduction**

cancer cells.

cells) is presumably responsible for the increased cell death after drug treatment.

**Figure 2.** Cell cycle analysis for MO59K and MO59J cells treated with 5, 10 and 25 µM of cisplatin for 24, 48 and 72 h. DMSO was used as control.

Consistent with impaired cell cycle regulation, an increased frequency of apoptosis (43% for 25 µM cisplatin) was observed after cisplatin treatment in MO59J cells; in contrast to MO59K, DNA-PK deficient MO59J cells showed apoptotic death even at 5 µM of cisplatin (Figure 3A,B).

**Figure 3.** Frequency of apoptotic MO59K (A) and MO59J (B) cells detected by flow cytometry as a subG1 population after exposure to different concentrations of cisplatin (5, 10 and 25 μM). The results were obtained at 24, 48 and 72 h after treatment. 10,000 events were analyzed for each experiment.

Proliferating cell nuclear antigen (PCNA) is recruited to chromatin rapidly after DNA damage and the kinetics of disassembly from the chromatin is considered to reflect the DNA repair efficiency [13, 14]. PCNA expression was evaluated by Western blot in cisplatin- treated MO59K and MO59J cells. The expression of PCNA was increased in a dose-dependent manner in MO59K cells after 24 h of drug treatment, while a constant level of PCNA expression was observed in MO59J cells indicating the lack of S-phase specific PCNA accumulation (Figure 4). Our results from MO59K and MO59J cell lines indicate that DNA-PK is a critical determi‐ nant of cell survival after cisplatin treatment and the lack of S-phase arrest (as seen in MO59J cells) is presumably responsible for the increased cell death after drug treatment.

**Figure 4.** Expression of PCNA protein (30 KDa) analyzed by Western blot using specific antibody. MO59K and MO59J cells were treated with 5, 10 and 25 µM of cisplatin for 3 and 24 h. β-actin was used as control.

Therefore, DNA-PK may be an important molecule conferring cisplatin resistance in glioblas‐ toma cells, with potential use as a molecular target, and we also observed that the combined effect of cisplatin and LY294002 (an inhibitor of DNA-PK) caused a significant decrease in cell proliferation and an increase in apoptotic cell death in glioma cell lines [15]. Our studies thus far performed indicate that the specific targeting of DNA-PK by small molecule inhibitors may be an effective strategy for the treatment of gliomas.

### **3.** *In silico* **analysis of transcription factors associated with differentially expressed genes in GBM cells**

#### **3.1. Introduction**

**Figure 2.** Cell cycle analysis for MO59K and MO59J cells treated with 5, 10 and 25 µM of cisplatin for 24, 48 and 72 h.

Consistent with impaired cell cycle regulation, an increased frequency of apoptosis (43% for 25 µM cisplatin) was observed after cisplatin treatment in MO59J cells; in contrast to MO59K, DNA-PK deficient MO59J cells showed apoptotic death even at 5 µM of cisplatin (Figure 3A,B).

**Figure 3.** Frequency of apoptotic MO59K (A) and MO59J (B) cells detected by flow cytometry as a subG1 population after exposure to different concentrations of cisplatin (5, 10 and 25 μM). The results were obtained at 24, 48 and 72 h

after treatment. 10,000 events were analyzed for each experiment.

462 Evolution of the Molecular Biology of Brain Tumors and the Therapeutic Implications

DMSO was used as control.

Transcription factors (TFs) regulate gene expression by binding to specific DNA sequences of gene promoter regions. Their expression can be deregulated under certain pathological conditions, such as cancer. Therefore, studies on regulatory elements of the transcription machinery are essential for understanding the mechanisms involved in the regulation of oncogenes and tumor suppressor genes. Additionally, knowledge of the essential functions of TFs, may lead to strategic development of novel chemotherapy regimen for selectively killing cancer cells.

TFs play a critical role in various cellular processes through regulation of genes involved in cell cycle, proliferation, cell differentiation, and apoptosis. It has been suggested that TFs can be potential therapeutic targets for treating the patients with prostate [16] and breast cancer [17]. Recently, *in silico* prediction for regulation of TFs has been effectively utilized for diagnostics and therapeutics of breast cancer [17]. Identification and characterization of activated transcription regulatory elements hold a great promise not only in providing information on neoplastic mechanisms, but also in predicting targets for therapeutic interven‐ tion [18]. Although enormous amount of data exist for genes that are differentially expressed in tumor cells, knowledge of TFs associated with these genes is rather limited. Information on TFs may enable us to understand how differentially expressed genes in tumor cells respond to anticancer drugs, as a single or combined treatment with DNA repair inhibitors, which targets DNA repair proteins.

through the FatiGO tool, Babelomics v3.2 [28]. For comparisons, the entire list of genes present in the microarray was subjected to analysis (4,300 transcripts).The data were analyzed by the two-tailed Fisher's exact test, with significance threshold for the p-values set at 0.05. Similar procedures were performed for the list of 108 genes (set of differentially expressed genes under 25µM cisplatin treatment). Information regarding the biological functions was obtained at SOURCE (http://smd.stanford.edu/cgi-bin/source/sourceSearch), and NCBI AceView (http://

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465

Among the predicted TFs associated with up-regulated genes after treatment with cisplatin alone, the most significant ones are AHR (p = 9.9E–3, 7.7% of associated genes), HNF4 (p = 9.6E–3, 81% of associated genes), NFkappaB (p = 5.8E–3, 11.5% of associated genes), MRF2 (p = 1.9E–3, 53.8%), and SOX5 (p = 1.6E–2, 7.7% of associated genes). These TFs have been linked with distinct biological functions, including differentiation, homeostasis, cell growth, senes‐ cence and apoptosis. AHR regulates an array of physiological responses including xenobiotic metabolism, vasculature development, immunosuppression, T-cell differentiation, reproduc‐ tion, and cell cycle progression [29]. Most genes known to be controlled by HNF4α are involved in lipid, carbohydrate or amino acid transport and metabolism, indicating a central role in energy homeostasis [30]. MRF2 (ARID5B) plays a vital role in regulating embryonic develop‐ ment, cell growth and differentiation through tissue-specific repression of differentiationspecific gene expression [31, 32]. Aberrant *ARID5B* expression in developing fetus could halt B-lymphocyte maturation and contribute to leukemogenesis [33]. SOX5 is involved in gene regulation and maintenance of chromatin structure during diverse developmental processes. Recently, SOX5 has been found associated with induced acute cellular senescence [34], while

www.ncbi.nlm.nih.gov/IEB/Research/Acembly/).

NFkappaB is related to apoptosis evasion in GBM cells [35, 36].

**Percentage with term**

**List #1 List #2**

*GATA-3* 30.1 16.3 1.85 1.7E–3 *FOXP3* 60.2 46.0 1.31 8.0E–3 *FOXJ2* 46.2 32.6 1.42 9.5E–3 *C/EBP* 18.3 9.8 1.87 1.5E–2

**Table 1.** Transcription factors (Term) associated with up-regulated genes in cisplatin and LY294002 treated U343 cells. "Percentage with term" corresponds to the amount of genes that was associated to each TF in each list: List #1 represents up-regulated genes obtained by statistical analysis, and List #2 represents all the genes present in the microarray slide. "Enrichment index" was calculated for each Term by dividing the percentage in the List #1 by that in

62.4 54.6 1.14 1.6E–2

**Enrichment index p-value**

**Term**

*LXR, PXR, CAR COUP, RAR*

the List #2.

**3.2. TFs associated with up–regulated genes**

DNA-PK, one of the key components of mammalian NHEJ (non-homologous end joining) repair process, belongs to PI3K-related protein kinase (PIKK) family [19]. Recent studies have projected DNA-PK as a potential molecular target for cancer treatment. Many known PI3K inhibitors such as LY294002, wortmannin and PI-103, inhibit DNA-PK activity with a compa‐ rable potency to that observed for PI3K inhibition [20]. LY294002 is a PI3K (phosphatidylino‐ sitol-3 kinase) inhibitor, well recognized by its antitumor and pro-apoptotic properties in several cancer cell lines [21]. Although LY294002 alone can inhibit cell proliferation or induce apoptosis in cancer cells, its cytotoxic effects can be further enhanced when combined with radio- or chemotherapy [12, 22]. Consequently, PI3K inhibitors including LY294002 have been investigated as a possible adjuvant for cancer therapy in many tumor models [23-25], and its synergistic effects with other anti-tumoral drugs deserve to be investigated in cancer cells.

Our recent study demonstrated that a combined treatment of cisplatin (5-50µM) and LY294002 (50µM) for 24 h reduced the survival of U343 GBM cells with elevated apoptotic death at 72 h [15]. In this study, gene expression analysis was also performed using a glass slide microarray containing 4,300 cDNA clones from the human IMAGE Consortium cDNA library [http:// image.llnl.gov/image/] essentially following the procedures published earlier [26]. Statistical analysis of the results [27] showed that 25µM cisplatin alone caused significant changes in the expression of 108 genes (28 up- and 80 down-regulated), while LY294002 alone altered the expression of 33 genes (6 up- and 27 down-regulated); additionally, 274 genes (97 up- and 177 down-regulated) were modulated by the combined treatment of cisplatin and LY294002. The complete gene lists are available at http//www.rge.fmrp.usp.br/passos/cislygbm/. The drug combination induced 2.6-fold higher numbers of modulated genes, relative to cisplatin alone, and these genes participate in several biological processes, such as DNA repair, cell death, and cell cycle control/proliferation, metabolism, transcription regulation, and cellular adhesion.

Using the gene sets obtained in the above-mentioned study, we performed an *in silico* analysis of TFs for the differentially expressed genes obtained in the following comparisons: 25µM cisplatin *versus* control, and 25µM cisplatin plus 50µM LY294002 *versus* control; these lists contained 108 and 274 differentially expressed genes (SAM analysis, FDR ≤ 0.05), respectively. Among the 274 genes, 97 (35.4%) genes were up-regulated and 177 (64.6%) genes were downregulated. Both up- and down-regulated genes were submitted separately to *in silico* analysis through the FatiGO tool, Babelomics v3.2 [28]. For comparisons, the entire list of genes present in the microarray was subjected to analysis (4,300 transcripts).The data were analyzed by the two-tailed Fisher's exact test, with significance threshold for the p-values set at 0.05. Similar procedures were performed for the list of 108 genes (set of differentially expressed genes under 25µM cisplatin treatment). Information regarding the biological functions was obtained at SOURCE (http://smd.stanford.edu/cgi-bin/source/sourceSearch), and NCBI AceView (http:// www.ncbi.nlm.nih.gov/IEB/Research/Acembly/).

#### **3.2. TFs associated with up–regulated genes**

TFs play a critical role in various cellular processes through regulation of genes involved in cell cycle, proliferation, cell differentiation, and apoptosis. It has been suggested that TFs can be potential therapeutic targets for treating the patients with prostate [16] and breast cancer [17]. Recently, *in silico* prediction for regulation of TFs has been effectively utilized for diagnostics and therapeutics of breast cancer [17]. Identification and characterization of activated transcription regulatory elements hold a great promise not only in providing information on neoplastic mechanisms, but also in predicting targets for therapeutic interven‐ tion [18]. Although enormous amount of data exist for genes that are differentially expressed in tumor cells, knowledge of TFs associated with these genes is rather limited. Information on TFs may enable us to understand how differentially expressed genes in tumor cells respond to anticancer drugs, as a single or combined treatment with DNA repair inhibitors, which

464 Evolution of the Molecular Biology of Brain Tumors and the Therapeutic Implications

DNA-PK, one of the key components of mammalian NHEJ (non-homologous end joining) repair process, belongs to PI3K-related protein kinase (PIKK) family [19]. Recent studies have projected DNA-PK as a potential molecular target for cancer treatment. Many known PI3K inhibitors such as LY294002, wortmannin and PI-103, inhibit DNA-PK activity with a compa‐ rable potency to that observed for PI3K inhibition [20]. LY294002 is a PI3K (phosphatidylino‐ sitol-3 kinase) inhibitor, well recognized by its antitumor and pro-apoptotic properties in several cancer cell lines [21]. Although LY294002 alone can inhibit cell proliferation or induce apoptosis in cancer cells, its cytotoxic effects can be further enhanced when combined with radio- or chemotherapy [12, 22]. Consequently, PI3K inhibitors including LY294002 have been investigated as a possible adjuvant for cancer therapy in many tumor models [23-25], and its synergistic effects with other anti-tumoral drugs deserve to be investigated in cancer cells.

Our recent study demonstrated that a combined treatment of cisplatin (5-50µM) and LY294002 (50µM) for 24 h reduced the survival of U343 GBM cells with elevated apoptotic death at 72 h [15]. In this study, gene expression analysis was also performed using a glass slide microarray containing 4,300 cDNA clones from the human IMAGE Consortium cDNA library [http:// image.llnl.gov/image/] essentially following the procedures published earlier [26]. Statistical analysis of the results [27] showed that 25µM cisplatin alone caused significant changes in the expression of 108 genes (28 up- and 80 down-regulated), while LY294002 alone altered the expression of 33 genes (6 up- and 27 down-regulated); additionally, 274 genes (97 up- and 177 down-regulated) were modulated by the combined treatment of cisplatin and LY294002. The complete gene lists are available at http//www.rge.fmrp.usp.br/passos/cislygbm/. The drug combination induced 2.6-fold higher numbers of modulated genes, relative to cisplatin alone, and these genes participate in several biological processes, such as DNA repair, cell death, and cell cycle control/proliferation, metabolism, transcription regulation, and cellular adhesion.

Using the gene sets obtained in the above-mentioned study, we performed an *in silico* analysis of TFs for the differentially expressed genes obtained in the following comparisons: 25µM cisplatin *versus* control, and 25µM cisplatin plus 50µM LY294002 *versus* control; these lists contained 108 and 274 differentially expressed genes (SAM analysis, FDR ≤ 0.05), respectively. Among the 274 genes, 97 (35.4%) genes were up-regulated and 177 (64.6%) genes were downregulated. Both up- and down-regulated genes were submitted separately to *in silico* analysis

targets DNA repair proteins.

Among the predicted TFs associated with up-regulated genes after treatment with cisplatin alone, the most significant ones are AHR (p = 9.9E–3, 7.7% of associated genes), HNF4 (p = 9.6E–3, 81% of associated genes), NFkappaB (p = 5.8E–3, 11.5% of associated genes), MRF2 (p = 1.9E–3, 53.8%), and SOX5 (p = 1.6E–2, 7.7% of associated genes). These TFs have been linked with distinct biological functions, including differentiation, homeostasis, cell growth, senes‐ cence and apoptosis. AHR regulates an array of physiological responses including xenobiotic metabolism, vasculature development, immunosuppression, T-cell differentiation, reproduc‐ tion, and cell cycle progression [29]. Most genes known to be controlled by HNF4α are involved in lipid, carbohydrate or amino acid transport and metabolism, indicating a central role in energy homeostasis [30]. MRF2 (ARID5B) plays a vital role in regulating embryonic develop‐ ment, cell growth and differentiation through tissue-specific repression of differentiationspecific gene expression [31, 32]. Aberrant *ARID5B* expression in developing fetus could halt B-lymphocyte maturation and contribute to leukemogenesis [33]. SOX5 is involved in gene regulation and maintenance of chromatin structure during diverse developmental processes. Recently, SOX5 has been found associated with induced acute cellular senescence [34], while NFkappaB is related to apoptosis evasion in GBM cells [35, 36].

**Table 1.** Transcription factors (Term) associated with up-regulated genes in cisplatin and LY294002 treated U343 cells. "Percentage with term" corresponds to the amount of genes that was associated to each TF in each list: List #1 represents up-regulated genes obtained by statistical analysis, and List #2 represents all the genes present in the microarray slide. "Enrichment index" was calculated for each Term by dividing the percentage in the List #1 by that in the List #2.

For the combined treatment (cisplatin and LY294002), the predicted TFs associated with upregulated genes showed different TFs (Table 1), indicating that the drug combination in‐ duced quantitative and qualitative differences in transcript expression profiles displayed by GBM treated cells.

binding protein alpha, involved in cell cycle arrest [51], and also plays a role in DNA damage response dependent on TP53 [52]. The protein acts as a dominant-negative inhibitor by forming hetero-dimers with other C/EBP members, such as C/EBP and LAP (*liver activator protein*), and preventing their DNA binding activity. Another member, GADD153 (CHOP), is a critical initiator of ER stress-induced apoptosis, and its knockdown prevents perturbations in the *AKT* (*protein kinase B*)/*FOXO3a* (*forkhead box, class O, 3a*) pathway in response to ER stress [53]. C/EBP β, a member of a subfamily of basic leucine zipper (bZIP) protein family, play a potential role in the proliferation and invasion of glioma, being considered as a novel molecular target for therapy, as well as a predictive marker for survival of patients with glioma [54]. In the present study, C/EBP showed an association with up-regulated genes (EI = 1.87), suggesting a possible role in DNA damage responses under drug treatment, and most probably, via AKT

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Three TFs (*LXR*, *PXR* and *CAR*) were related to the same percentage (62.4%) of up-regulated genes, presenting EI = 1.14. The *liver X receptors* (*LXR*), *LXRA* and *LXRB*, comprise a subfamily of the nuclear receptor superfamily and are key regulators of macrophage function, controlling transcriptional programs involved in lipid homeostasis and inflammation. Polymorphism of *LXR* gene is associated with obesity since this gene is involved in cellular lipid metabolic process [55]. *PXR*, also named *NR1I2* (*Nuclear receptor subfamily 1, group I, member 2*) is a TF that acts in both positive and negative regulation of transcription, as well as in xenobiotic transport and metabolic process, in addition to exogenous drug catabolic process; this gene seems to be also implicated in breast cancer development [56, 57]. Similarly to *PXR*, *CAR* (*NR1I3*, *Nuclear receptor subfamily 1, group I, member 3*) is also a key regulator of xenobiotic and endobiotic metabolism [58]. Identification of TFs related to liver and xenobiotic metabolism regulating the over-expressed genes suggests that these genes may play a role in drug

The foregoing account demonstrates that all the TFs that are associated with up-regulated genes after combined treatment (cisplatin and LY294002) are regulators of important biological processes, such as cell differentiation, metabolism, development, and immune response, but the literature is still scarce regarding the role of above mentioned TFs in cellular response mechanisms to chemotherapeutic drugs. In spite of this, the increment observed in the number of associated genes (in terms of EI index, which ranged from 1.14 to 1.87) tends to suggest the

Similar to the prediction of TFs associated with up-regulated genes, we performed the same kind of analysis for determining the TFs associated with down-regulated genes. None of the TF was found associated with down-regulated genes after treatment with cisplatin alone. However, for the combined treatment (cisplatin and LY294002), six TFs were predicted (Table 2). All TFs displayed enrichment indexes greater than 1 (ranging from 1.10 to 1.80), indicating that the enrichment was greater in the list of down-regulated genes (list #1) in comparison to

involvement of these TFs in the outcome of cellular response to combined treatment.

responses. However, this interesting possibility needs further validation.

pathway, which is a target for LY294002.

**3.3. TFs associated with down–regulated genes**

all the genes (list #2) included in the array.

All the TFs that were associated with up-regulated genes showed enrichment indexes greater than 1 (in the range of 1.14 to 1.87), indicating that these TFs were more specifically associated with the up-regulated genes as compared to the entire array of genes (Table 1). Biological functions of the predicted TFs associated with up- and down-regulated genes after the combined treatment (cisplatin and LY294002) in U343 cells are described below:

*GATA-3* (*Trans-acting T-cell-specific transcription factor*) gene encodes a protein, which be‐ longs to the GATA family of transcription factors. The protein contains two GATA-type zinc fingers and is an important regulator of T-cell development and plays an important role in endothelial cell biology; *GATA-3* is essential in the embryonic development of the parathyroid, auditory system and kidneys, and defects in this gene cause HDR syndrome (hypoparathyroidism with sensorineural deafness and renal dysplasia) [37]. *GATA-3* anal‐ ysis and the phenotypic spectrum obtained for nine Japanese families with the HDR syn‐ drome suggest that this syndrome is primarily caused by *GATA-3* haploinsufficiency [38]. This TF plays a dual role in transcription regulation in a positive and negative manner. In accordance with this, we found the association of GATA-3 with up-regulated genes (EI = 1.85) after drug treatment in U343 cells.

Another TF, FOXP3 (*Forkhead box P3*), a positive regulator of transcription, plays a critical role in the control of immune response. This TF was associated with up-regulated genes (EI = 1.31) in drug-treated U343 cells. Defects in this gene lead to X-linked autoimmunity-immunodefi‐ ciency syndrome [39]. In several neoplasias, such as breast [40], ovarian [41], skin [42] and stomach, FOXP3 was found over-expressed [43], or mutated in prostate cancer [44]. It is well established that tumor cells acquire molecular and biochemical changes which make them potentially vulnerable to the immune system [45]. Alterations in the immune system can be applied for cancer detection and therapies [46]. Interestingly, [47] demonstrated a dependency on functional TP53 for DNA damage induced activation of *FOXP3* in human breast and colon carcinoma cells. However, the precise role of *FOXP3* in drug responses is still unknown.

*FOXJ2* (*Forkhead box J2*) was also associated with up-regulated genes (EI = 1.42); this TF is often described as a positive regulator of transcription [48] and is a member of Forkhead Box TFs, many of which have been reported to participate in tumor migration and invasion. Wang et al. [49] showed that the expression of *FOXJ2* was high in primary breast cancer tissues without lymph nodes metastases; moreover, *FOXJ2* may play a role in maintenance and survival of developing and adult neurons, but its function in the central nervous system is still unclear. Knockdown of this TF in cultured primary astrocytes by siRNA showed that *FOXJ2* played an important role in lipopolysaccharide-induced inflammatory responses [50]. Nonetheless, there is no information in the literature about its function in relation to chemotherapeutic drugs.

*C/EBP* (*CCAAT/enhancer-binding protein*) family plays important roles in many processes such as cell differentiation, metabolism, and development. *C/EBP* (CEBPA) gene, CCAAT/enhancer binding protein alpha, involved in cell cycle arrest [51], and also plays a role in DNA damage response dependent on TP53 [52]. The protein acts as a dominant-negative inhibitor by forming hetero-dimers with other C/EBP members, such as C/EBP and LAP (*liver activator protein*), and preventing their DNA binding activity. Another member, GADD153 (CHOP), is a critical initiator of ER stress-induced apoptosis, and its knockdown prevents perturbations in the *AKT* (*protein kinase B*)/*FOXO3a* (*forkhead box, class O, 3a*) pathway in response to ER stress [53]. C/EBP β, a member of a subfamily of basic leucine zipper (bZIP) protein family, play a potential role in the proliferation and invasion of glioma, being considered as a novel molecular target for therapy, as well as a predictive marker for survival of patients with glioma [54]. In the present study, C/EBP showed an association with up-regulated genes (EI = 1.87), suggesting a possible role in DNA damage responses under drug treatment, and most probably, via AKT pathway, which is a target for LY294002.

Three TFs (*LXR*, *PXR* and *CAR*) were related to the same percentage (62.4%) of up-regulated genes, presenting EI = 1.14. The *liver X receptors* (*LXR*), *LXRA* and *LXRB*, comprise a subfamily of the nuclear receptor superfamily and are key regulators of macrophage function, controlling transcriptional programs involved in lipid homeostasis and inflammation. Polymorphism of *LXR* gene is associated with obesity since this gene is involved in cellular lipid metabolic process [55]. *PXR*, also named *NR1I2* (*Nuclear receptor subfamily 1, group I, member 2*) is a TF that acts in both positive and negative regulation of transcription, as well as in xenobiotic transport and metabolic process, in addition to exogenous drug catabolic process; this gene seems to be also implicated in breast cancer development [56, 57]. Similarly to *PXR*, *CAR* (*NR1I3*, *Nuclear receptor subfamily 1, group I, member 3*) is also a key regulator of xenobiotic and endobiotic metabolism [58]. Identification of TFs related to liver and xenobiotic metabolism regulating the over-expressed genes suggests that these genes may play a role in drug responses. However, this interesting possibility needs further validation.

The foregoing account demonstrates that all the TFs that are associated with up-regulated genes after combined treatment (cisplatin and LY294002) are regulators of important biological processes, such as cell differentiation, metabolism, development, and immune response, but the literature is still scarce regarding the role of above mentioned TFs in cellular response mechanisms to chemotherapeutic drugs. In spite of this, the increment observed in the number of associated genes (in terms of EI index, which ranged from 1.14 to 1.87) tends to suggest the involvement of these TFs in the outcome of cellular response to combined treatment.

#### **3.3. TFs associated with down–regulated genes**

For the combined treatment (cisplatin and LY294002), the predicted TFs associated with upregulated genes showed different TFs (Table 1), indicating that the drug combination in‐ duced quantitative and qualitative differences in transcript expression profiles displayed by

All the TFs that were associated with up-regulated genes showed enrichment indexes greater than 1 (in the range of 1.14 to 1.87), indicating that these TFs were more specifically associated with the up-regulated genes as compared to the entire array of genes (Table 1). Biological functions of the predicted TFs associated with up- and down-regulated genes after the

*GATA-3* (*Trans-acting T-cell-specific transcription factor*) gene encodes a protein, which be‐ longs to the GATA family of transcription factors. The protein contains two GATA-type zinc fingers and is an important regulator of T-cell development and plays an important role in endothelial cell biology; *GATA-3* is essential in the embryonic development of the parathyroid, auditory system and kidneys, and defects in this gene cause HDR syndrome (hypoparathyroidism with sensorineural deafness and renal dysplasia) [37]. *GATA-3* anal‐ ysis and the phenotypic spectrum obtained for nine Japanese families with the HDR syn‐ drome suggest that this syndrome is primarily caused by *GATA-3* haploinsufficiency [38]. This TF plays a dual role in transcription regulation in a positive and negative manner. In accordance with this, we found the association of GATA-3 with up-regulated genes (EI =

Another TF, FOXP3 (*Forkhead box P3*), a positive regulator of transcription, plays a critical role in the control of immune response. This TF was associated with up-regulated genes (EI = 1.31) in drug-treated U343 cells. Defects in this gene lead to X-linked autoimmunity-immunodefi‐ ciency syndrome [39]. In several neoplasias, such as breast [40], ovarian [41], skin [42] and stomach, FOXP3 was found over-expressed [43], or mutated in prostate cancer [44]. It is well established that tumor cells acquire molecular and biochemical changes which make them potentially vulnerable to the immune system [45]. Alterations in the immune system can be applied for cancer detection and therapies [46]. Interestingly, [47] demonstrated a dependency on functional TP53 for DNA damage induced activation of *FOXP3* in human breast and colon carcinoma cells. However, the precise role of *FOXP3* in drug responses is still unknown.