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#### **15. References**

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**7**

*Spain*

**V-ATPase Inhibitors in Cancer Treatment and**

ATPases are enzyme systems that originated in a common ancestor and are distributed universally among all organisms. There are three types of ATPases: those found in archaea (A-ATPases), synthases (F-APTases), and vacuole or vacuolar ATPases (V-ATPases) (Nelson, Nelson 1989). They are essential for life and have in common the fact that they create an electrochemical ion gradient across the membrane to hydrolyze or synthesize ATP. Structurally, they are enzymatic complexes that work as molecular rotary motors. ATPases are formed by two domains, a hydrophobic domain (A0, V0, and F0) and a hydrophilic domain (A1, V1, and F1) connected by a central axis and either one or two lateral axes. In

Unlike F-ATPases, whose primary function in eukaryotic cells is to generate ATP from proton motive force, V-ATPases function exclusively as ATP-dependent proton pumps, performing diverse biological functions within cells (Nelson 1992; Kane 1999; Saroussi &

Regarding to the membrane transport, V-ATPases play an important role in receptormediated endocytosis (Forgac 1998), intracellular transport, and the acidification of late endosomes (Kane 1999; Stevens & Forgac 1997; Nishi & Forgac 2002; Kawasaki-Nishi & Forgac 2003; Finbow, Harrison 1997). Vacuolar acidification has also been reported to be involved in the transport of lysosomal enzymes from the Golgi apparatus to the lysosomes (Stevens, Forgac 1997; Moriyama, Nelson 1989). V-ATPases appear to play an important role in the creation of the microenvironment needed for correct protein transport, exchange, and

Although V-ATPases were initially identified in intracellular compartments, knowledge on the roles they play in the plasma membrane has increased enormously. V-ATPases located at the apical membrane of type A intercalated cells are involved in the secretion of protons in renal fluid (Smith et al. 2005; van Hille et al. 1993). Type B intercalated cells, whose function is to secrete bicarbonate, also contain V-ATPases, but they are located between the apical and basolateral membranes (Nishi & Forgac 2002, van Hille et al. 1993). In macrophages and neutrophils, plasma membrane V-ATPases (pmV-ATPases) are involved

**1. Introduction**

**1.1 Biological functions**

Nelson 2008, Stevens & Forgac 1997).

secretion (Schoonderwoert et al. 2000).

this chapter, we are going to discuss V-ATPases.

**Their Implication in Multidrug Resistance**

 **in Oral Squamous Cell Carcinoma**

Mario Pérez-Sayáns and Abel García García

*University of Santiago de Compostela*

## **V-ATPase Inhibitors in Cancer Treatment and Their Implication in Multidrug Resistance in Oral Squamous Cell Carcinoma**

Mario Pérez-Sayáns and Abel García García *University of Santiago de Compostela Spain*

### **1. Introduction**

128 Current Cancer Treatment – Novel Beyond Conventional Approaches

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#### **1.1 Biological functions**

Unlike F-ATPases, whose primary function in eukaryotic cells is to generate ATP from proton motive force, V-ATPases function exclusively as ATP-dependent proton pumps, performing diverse biological functions within cells (Nelson 1992; Kane 1999; Saroussi & Nelson 2008, Stevens & Forgac 1997).

Regarding to the membrane transport, V-ATPases play an important role in receptormediated endocytosis (Forgac 1998), intracellular transport, and the acidification of late endosomes (Kane 1999; Stevens & Forgac 1997; Nishi & Forgac 2002; Kawasaki-Nishi & Forgac 2003; Finbow, Harrison 1997). Vacuolar acidification has also been reported to be involved in the transport of lysosomal enzymes from the Golgi apparatus to the lysosomes (Stevens, Forgac 1997; Moriyama, Nelson 1989). V-ATPases appear to play an important role in the creation of the microenvironment needed for correct protein transport, exchange, and secretion (Schoonderwoert et al. 2000).

Although V-ATPases were initially identified in intracellular compartments, knowledge on the roles they play in the plasma membrane has increased enormously. V-ATPases located at the apical membrane of type A intercalated cells are involved in the secretion of protons in renal fluid (Smith et al. 2005; van Hille et al. 1993). Type B intercalated cells, whose function is to secrete bicarbonate, also contain V-ATPases, but they are located between the apical and basolateral membranes (Nishi & Forgac 2002, van Hille et al. 1993). In macrophages and neutrophils, plasma membrane V-ATPases (pmV-ATPases) are involved

V-ATPase Inhibitors in Cancer Treatment and Their

Wang & Forgac 2007, Inoue et al. 2005) (Figure 1).

contains the catalytic unit (Nishi & Forgac 2002).

fusion of the membrane (Muller et al. 2003).

**1.3 V-ATPase regulation**

Implication in Multidrug Resistance in Oral Squamous Cell Carcinoma 131

sector (V1) and a hydrophobic membrane sector (V0) responsible for driving protons (Gruber 2005). The catalytic sector is composed of five polypeptides known as subunits A, B, C, D, and E, with a molecular weight, in decreasing order, ranging from 72 to 33 kDa. Recent advances in knowledge of the mechanism of action of F-ATPases have clarified the relationship between function and structure for each of the subunits of these enzymes (Qi,

Fig. 1. Diagram of V-ATPase. The cytosolic domain (in yellow) is formed by three A subunits, three B subunits, three G subunits, and one C, D, E, F, and H subunit. The V0 transmembrane domain is formed by five subunits: a, c, c', c'' and d. The V1 domain

Three major regulatory mechanisms have been described for V-ATPase: *1)* the regulation of pump density, which allows different cells to maintain their cytoplasmic and vacuolar pH stable; *2)* the regulation of V1 and V0 domain association/dissociation, for example, a decrease in glucose levels can cause a 70% dissociation of the V1 domains of the membrane; and *3)* the regulation of secretory activity, via the maintenance of balance in the formation of bisulfite and binding efficiency between H+ and the pump. Other mechanisms include the necessary modifications in the membrane potential for the generation of electrogenic force (Forgac 1998; Peng, Stone & Xie 1993) and alterations in the vacuolar transporter chaperone (Vtc) complex, which affect the conformation of the V0 domain and its function in vacuole

in the homeostasis of cytoplasmic pH (Stevens & Forgac 1997, Nishi & Forgac 2002, Nanda et al. 1996). These ATPases also play an important role in bone reabsorption (Marshansky, Futai , Stevens, Forgac 1997, Nishi, Forgac 2002, Smith et al. 2005, van Hille et al. 1993). Another of their functions is to regulate sperm motility and maturation on the apical membrane of epididymal cells and vas deferens by stabilizing the sperm medium (Nishi, Forgac 2002). The role of V-ATPases in cancer cells will be discussed in a specific place.

Other additional functions of V-ATPases involves the low pH maintained by them in lysosomes and phagosomes, which is necessary for the activity of the degradative enzymes in these compartments (Sun-Wada, Wada & Futai 2003, Sun-Wada, Wada & Futai 2004, Kurashima et al. 1996) and the transport of small molecules and ions (Nishi, Forgac 2002, Kurashima et al. 1996). The driving force necessary for the accumulation of neurotransmitters in synaptic vesicles is proton motive force, which is generated by V-ATPases (Nelson, Harvey 1999). The fusion-fission balance of the vacuolar system of eukaryotic cells is also controlled by V-ATPases, i.e. via the interaction between vacuolar SNARE proteins and GTPase Vps1p (Baars et al. 2007, Muller et al. 2003). Exocytosis in eosinophils and binding to actin cytoskeleton is also regulated by V-ATPases (Kurashima et al. 1996). The association between V-ATPase subunits and other cellular proteins, for example, that which occurs between the C subunit of the V0 domain and the E5 oncoprotein, or between platelet-derived growth factor (PDGF) and b1 integrin, indicate that these subunits play a role in cell growth and transformation. V-ATPases also allow the entry of certain viruses (e.g. influenza) and toxins (e.g. diphtheria) into the intracellular space via the binding of these pathogens to the endosomal membrane (Stevens, Forgac 1997). In the case of the human immunodeficiency virus (HIV), the association between the V-ATPase H subunit and the HIV-1 Nef protein, which controls the expression of CD4 (the main HIV receptor), facilitates endocytosis of Nef and/or alterations in the acidification of the endosomal pathway by this protein (Nishi, Forgac 2002)(Marshansky, Futai ). The most recent function attributed to V-ATPases is their involvement in the regulation of cell-cell fusion to form larger cells, as is the case with osteoclasts and macrophages (Wada et al. 2008).

#### **1.2 V-atpase structure**

The V-ATPase proton pump has multiple subunits, each with multiple isoforms, hence the need for a clear, standardized nomenclature system. Initially, the HUGO Gene Nomenclature Committee agreed to use the ATP as the stem, or root, symbol. ATP6, for example, indicated ATPase, H+ transport, lysosomal (vacuolar proton pump). In 2003, the nomenclature system for genes encoding V-ATPase subunits was revised and it was decided to maintain the root ATP6 and add the domain to which a particular subunit belonged, followed by the letter of the subunit, and finally the number of the isoform, where relevant, (e.g. *ATP6V1C1*, *ATP6V1E,* etc.) (Smith A.N. et al. 2003).

V-ATPase structure, function, biogenesis, and regulation was widely revised by Stevens and Forgac (Stevens & Forgac 1997). We will use the nomenclature system proposed by these authors to explain the structural subunits of V-ATPase together with relevant modifications based on recent research using transmission electron microscopy (Wilkens, Zhang & Zheng 2005).

V-ATPases have been found to be practically identical in terms of the composition of their subunits in all eukaryotic cells. They have two distinct structures: a peripheral catalytic

in the homeostasis of cytoplasmic pH (Stevens & Forgac 1997, Nishi & Forgac 2002, Nanda et al. 1996). These ATPases also play an important role in bone reabsorption (Marshansky, Futai , Stevens, Forgac 1997, Nishi, Forgac 2002, Smith et al. 2005, van Hille et al. 1993). Another of their functions is to regulate sperm motility and maturation on the apical membrane of epididymal cells and vas deferens by stabilizing the sperm medium (Nishi, Forgac 2002). The role of V-ATPases in cancer cells will be discussed in a specific place. Other additional functions of V-ATPases involves the low pH maintained by them in lysosomes and phagosomes, which is necessary for the activity of the degradative enzymes in these compartments (Sun-Wada, Wada & Futai 2003, Sun-Wada, Wada & Futai 2004, Kurashima et al. 1996) and the transport of small molecules and ions (Nishi, Forgac 2002, Kurashima et al. 1996). The driving force necessary for the accumulation of neurotransmitters in synaptic vesicles is proton motive force, which is generated by V-ATPases (Nelson, Harvey 1999). The fusion-fission balance of the vacuolar system of eukaryotic cells is also controlled by V-ATPases, i.e. via the interaction between vacuolar SNARE proteins and GTPase Vps1p (Baars et al. 2007, Muller et al. 2003). Exocytosis in eosinophils and binding to actin cytoskeleton is also regulated by V-ATPases (Kurashima et al. 1996). The association between V-ATPase subunits and other cellular proteins, for example, that which occurs between the C subunit of the V0 domain and the E5 oncoprotein, or between platelet-derived growth factor (PDGF) and b1 integrin, indicate that these subunits play a role in cell growth and transformation. V-ATPases also allow the entry of certain viruses (e.g. influenza) and toxins (e.g. diphtheria) into the intracellular space via the binding of these pathogens to the endosomal membrane (Stevens, Forgac 1997). In the case of the human immunodeficiency virus (HIV), the association between the V-ATPase H subunit and the HIV-1 Nef protein, which controls the expression of CD4 (the main HIV receptor), facilitates endocytosis of Nef and/or alterations in the acidification of the endosomal pathway by this protein (Nishi, Forgac 2002)(Marshansky, Futai ). The most recent function attributed to V-ATPases is their involvement in the regulation of cell-cell fusion to form larger cells, as is the case with osteoclasts and macrophages (Wada et al.

The V-ATPase proton pump has multiple subunits, each with multiple isoforms, hence the need for a clear, standardized nomenclature system. Initially, the HUGO Gene Nomenclature Committee agreed to use the ATP as the stem, or root, symbol. ATP6, for example, indicated ATPase, H+ transport, lysosomal (vacuolar proton pump). In 2003, the nomenclature system for genes encoding V-ATPase subunits was revised and it was decided to maintain the root ATP6 and add the domain to which a particular subunit belonged, followed by the letter of the subunit, and finally the number of the isoform, where relevant,

V-ATPase structure, function, biogenesis, and regulation was widely revised by Stevens and Forgac (Stevens & Forgac 1997). We will use the nomenclature system proposed by these authors to explain the structural subunits of V-ATPase together with relevant modifications based on recent research using transmission electron microscopy (Wilkens, Zhang & Zheng

V-ATPases have been found to be practically identical in terms of the composition of their subunits in all eukaryotic cells. They have two distinct structures: a peripheral catalytic

2008).

2005).

**1.2 V-atpase structure**

(e.g. *ATP6V1C1*, *ATP6V1E,* etc.) (Smith A.N. et al. 2003).

sector (V1) and a hydrophobic membrane sector (V0) responsible for driving protons (Gruber 2005). The catalytic sector is composed of five polypeptides known as subunits A, B, C, D, and E, with a molecular weight, in decreasing order, ranging from 72 to 33 kDa. Recent advances in knowledge of the mechanism of action of F-ATPases have clarified the relationship between function and structure for each of the subunits of these enzymes (Qi, Wang & Forgac 2007, Inoue et al. 2005) (Figure 1).

Fig. 1. Diagram of V-ATPase. The cytosolic domain (in yellow) is formed by three A subunits, three B subunits, three G subunits, and one C, D, E, F, and H subunit. The V0 transmembrane domain is formed by five subunits: a, c, c', c'' and d. The V1 domain contains the catalytic unit (Nishi & Forgac 2002).

## **1.3 V-ATPase regulation**

Three major regulatory mechanisms have been described for V-ATPase: *1)* the regulation of pump density, which allows different cells to maintain their cytoplasmic and vacuolar pH stable; *2)* the regulation of V1 and V0 domain association/dissociation, for example, a decrease in glucose levels can cause a 70% dissociation of the V1 domains of the membrane; and *3)* the regulation of secretory activity, via the maintenance of balance in the formation of bisulfite and binding efficiency between H+ and the pump. Other mechanisms include the necessary modifications in the membrane potential for the generation of electrogenic force (Forgac 1998; Peng, Stone & Xie 1993) and alterations in the vacuolar transporter chaperone (Vtc) complex, which affect the conformation of the V0 domain and its function in vacuole fusion of the membrane (Muller et al. 2003).

V-ATPase Inhibitors in Cancer Treatment and Their

Macrolide antibiotics with 18 membered lactone rings

Macrocyclic salicylate

Substitution of enamide NH, salicylate, and phenyl salicylate

lactone ring with a thiazole

side

based

**3. Role of v-ATPases inhibitors in cancer**

CLASSES OF V-ATPase INHIBITORS

Concanamycin

Salicylihamide

Apicularens

Lobatamides

Oximidines

Cruentaren

Late-generation V-ATPase inhibitors

**3.1 Tumor metastasis**

Archazolid Macrocyclic

Indolyls Bafilomycin-

Table 1. Classes of V-ATPase inhibitors

Bafilomycin

Plecomacrolide

Benzolactone enamides

&

A

Implication in Multidrug Resistance in Oral Squamous Cell Carcinoma 133

Sponge *Haliclona*

*Tunicate Aplidium*

*lobatum*

*Archangium gephyra Cystobacter violaceus*

Tributyltin chloride (TBTCl), FR177995, FR167356

The development and maintenance of the proton gradient present in tumors is due directly to the ability of tumor cells to secrete protons (H+) (Martinez-Zaguilan et al. 1993, McLean et al. 2000), acidify the extracellular medium (Cardone, Casavola & Reshkin 2005, Perona, Serrano 1988), and keep the cytosolic pH alkaline (Sennoune, Martinez-Zaguilan 2007). This ability also

sp.

Chemistry Provenience Binding site Action

*Streptomyces* Unknown

Lactone ring *Chondromyces* VO complex Highly toxic for

Lactone ring *Pseudomonas* sp. VO complex Animal and

Lactone ring *Byssovorax cruenta* VO complex Cytotoxicity on

NiK12192, SB 242784, FR202126, 3-bromopyruvate (3-Br PA),

V-ATPases inhibition Ionophoric properties

human and animal

mammalian V-ATPases inhibition

mammalian V-ATPases inhibition

mammalian and fungal cells at mitochondrial F-

mammalian cell line

ATPases

VO subunit c Cytotoxicity on

Synthesis VO subunit c V-ATPase inhibitor

VO complex Animal V-ATPases inhibition Cytotoxin

cell

VO complex Animal and

## **2. V-ATPase inhibitors**

Scientific evidence suggests that the acidic tumor microenvironment is key to managing cancer progression and metastasis. In particular, V-ATPases play a major role in metastasis tumor development because many tumor cells secrete lysosomal enzymes that participate in the extracellular matrix degradation necessary for metastatic invasion. These enzymes are most active at low optimal pH; moreover, V-ATPases are responsible for microenvironment acidification (Nishi, Forgac 2002, Martinez-Zaguilan et al. 1993). Among the many mechanisms that regulate the tumor microenvironment, V-ATPases are especially significant because they can be inhibited by proton pump inhibitors. (Fais et al. 2007).

#### **2.1 Classes of V-ATPase inhibitors**

Initial attempts to block V-ATPases were made after bafilomycin and concanamycin were discovered in 1988 (Bowman, Siebers & Altendorf 1988). New molecules capable of inhibiting V-ATPase to a greater or lesser extent via different mechanisms of action were later discovered. Such molecules include benzolactone enamides salicylihalamide (Erickson et al. 1997), lobatamide A and B (Galinis et al. 1997), apicularen (Kunze B., Janse R., Sasse F., Höfle G. and Reichenbach H. 1998), indolyls (Gagliardi et al. 1998, Nadler et al. 1998), oximidine (Kim et al. 1999), macrolactone archazolid (Sasse et al. 2003), lobatamide C (Shen et al. 2003), and cruentaren(Kunze et al. 2006). The latest generation of inhibitors include NiK12192 (Saroussi, Nelson 2008, Petrangolini et al. 2006), FR202126 (Niikura 2007), and PPI SB 242784 (Hesselink et al. 2008). We can see the differences and similarities of V-ATPase inhibitors in Table 1:

The V-ATPase inhibitors studied most thoroughly and used most often are macrolide antibiotics with 18-membered lactone rings, namely, bafilomycins and concanamycins. Bafilomycin and concanamycin are commercially available, and various laboratories have developed in vitro synthesis processes for experimental purposes (Scheidt et al. 2002). The remaining V-ATPase inhibitors are still in experimental phase, due to possible side effects that may occur in humans. However, PPIs are the treatment of choice for peptic diseases such as gastroesophageal reflux (Larsson et al. 1985). While these pumps block the secretion of gastric acid, they also directly inhibit V-ATPase activity. Examples of PPIs include omeprazole, esomeprazole, lansoprazole, pantoprazole, and rabeprazole (Horn 2000), all of which accumulate in acidic compartments (De Milito, Fais 2005a). PPI treatment has been associated with V-ATPase activity inhibition and an increase in both extracellular pH and pH in lysosomal organelles. In vivo experiments using mice/human xenografts have shown that pretreatment with PPIs can sensitize solid human tumors to chemotherapy drugs (De Milito, Fais 2005a).

Treatment with PPIs has also been found to sensitize tumor cells to cisplatin, 5-fluoracil, and vinblastine through changes in cellular pH gradients, with retention of the drugs in the cytoplasm, and in the nucleus in the case of doxorubicin (De Milito, Fais 2005a, Luciani et al. 2004, Luciani et al. 2004, Cianfriglia et al. 1990).

It is also known that low pH levels are suitable for the complete activation of PPIs (De Milito et al. 2007), suggesting that tumor alkalinization may be an extremely interesting target for future anticancer treatments (De Milito, Fais 2005a, Luciani et al. 2004, De Milito, Fais 2005b). Specific V-ATPase inhibitors such as concanamycin and bafilomycins are other candidates for investigation, not only to treat cancer but also to reduce MDR in tumors (Perez-Sayans et al. 2009, Sasazawa et al. 2009).

Scientific evidence suggests that the acidic tumor microenvironment is key to managing cancer progression and metastasis. In particular, V-ATPases play a major role in metastasis tumor development because many tumor cells secrete lysosomal enzymes that participate in the extracellular matrix degradation necessary for metastatic invasion. These enzymes are most active at low optimal pH; moreover, V-ATPases are responsible for microenvironment acidification (Nishi, Forgac 2002, Martinez-Zaguilan et al. 1993). Among the many mechanisms that regulate the tumor microenvironment, V-ATPases are especially significant because they can be inhibited by proton pump inhibitors. (Fais et al. 2007).

Initial attempts to block V-ATPases were made after bafilomycin and concanamycin were discovered in 1988 (Bowman, Siebers & Altendorf 1988). New molecules capable of inhibiting V-ATPase to a greater or lesser extent via different mechanisms of action were later discovered. Such molecules include benzolactone enamides salicylihalamide (Erickson et al. 1997), lobatamide A and B (Galinis et al. 1997), apicularen (Kunze B., Janse R., Sasse F., Höfle G. and Reichenbach H. 1998), indolyls (Gagliardi et al. 1998, Nadler et al. 1998), oximidine (Kim et al. 1999), macrolactone archazolid (Sasse et al. 2003), lobatamide C (Shen et al. 2003), and cruentaren(Kunze et al. 2006). The latest generation of inhibitors include NiK12192 (Saroussi, Nelson 2008, Petrangolini et al. 2006), FR202126 (Niikura 2007), and PPI SB 242784 (Hesselink et al. 2008). We can see the differences and similarities of V-ATPase

The V-ATPase inhibitors studied most thoroughly and used most often are macrolide antibiotics with 18-membered lactone rings, namely, bafilomycins and concanamycins. Bafilomycin and concanamycin are commercially available, and various laboratories have developed in vitro synthesis processes for experimental purposes (Scheidt et al. 2002). The remaining V-ATPase inhibitors are still in experimental phase, due to possible side effects that may occur in humans. However, PPIs are the treatment of choice for peptic diseases such as gastroesophageal reflux (Larsson et al. 1985). While these pumps block the secretion of gastric acid, they also directly inhibit V-ATPase activity. Examples of PPIs include omeprazole, esomeprazole, lansoprazole, pantoprazole, and rabeprazole (Horn 2000), all of which accumulate in acidic compartments (De Milito, Fais 2005a). PPI treatment has been associated with V-ATPase activity inhibition and an increase in both extracellular pH and pH in lysosomal organelles. In vivo experiments using mice/human xenografts have shown that pretreatment with PPIs can sensitize solid human tumors to chemotherapy drugs (De

Treatment with PPIs has also been found to sensitize tumor cells to cisplatin, 5-fluoracil, and vinblastine through changes in cellular pH gradients, with retention of the drugs in the cytoplasm, and in the nucleus in the case of doxorubicin (De Milito, Fais 2005a, Luciani et al.

It is also known that low pH levels are suitable for the complete activation of PPIs (De Milito et al. 2007), suggesting that tumor alkalinization may be an extremely interesting target for future anticancer treatments (De Milito, Fais 2005a, Luciani et al. 2004, De Milito, Fais 2005b). Specific V-ATPase inhibitors such as concanamycin and bafilomycins are other candidates for investigation, not only to treat cancer but also to reduce MDR in tumors

**2. V-ATPase inhibitors**

**2.1 Classes of V-ATPase inhibitors**

inhibitors in Table 1:

Milito, Fais 2005a).

2004, Luciani et al. 2004, Cianfriglia et al. 1990).

(Perez-Sayans et al. 2009, Sasazawa et al. 2009).

Table 1. Classes of V-ATPase inhibitors

### **3. Role of v-ATPases inhibitors in cancer**

#### **3.1 Tumor metastasis**

The development and maintenance of the proton gradient present in tumors is due directly to the ability of tumor cells to secrete protons (H+) (Martinez-Zaguilan et al. 1993, McLean et al. 2000), acidify the extracellular medium (Cardone, Casavola & Reshkin 2005, Perona, Serrano 1988), and keep the cytosolic pH alkaline (Sennoune, Martinez-Zaguilan 2007). This ability also

V-ATPase Inhibitors in Cancer Treatment and Their

favor metastatic dissemination (Halaban et al. 2002).

preventing ECM protease activation (Fais et al. 2007).

*vivo* (Volk, Albert & Kempski 1998, McSheehy et al. 2003).

neutrophils (Gottlieb et al. 1995)and osteoclasts (Xu et al. 2003).

**3.2 Tumor cell growth and survival**

detection in cancer cells (Lim et al. 2006).

al. 2008).

Hishita et al. 2001).

Implication in Multidrug Resistance in Oral Squamous Cell Carcinoma 135

Using RNA interference techniques, Lu et al. found that distant metastasis could be delayed and suppressed in human hepatocellular carcinoma *in vitro* by reducing proton extrusion and gelatinase activity through the inhibition of V-ATPase subunit c (ATP6L) (Lu et al. 2005). This fact is consistent with subunit c block by bafilomycin and concanamycin, as this is their main binding site to V-ATPase (Bowman et al. 2004). In tyrosinase-positive amelanotic melanoma cells, inactive tyrosinase accumulates in the endoplasmic reticulum because the presence of aberrant V-ATPases blocks trafficking through secretory pathways. The use of V-ATPase inhibitors, such as bafilomycin A1 or concanamycin A, improves transport, demonstrating the involvement of this enzyme and preventing conditions that

Hence, both *in vitro* or *in vivo*, V-ATPases are a target for anticancer therapeutic agents, either directly by regulating the pH gradient in the tumor environment or indirectly by

V-ATPases may also play a significant role in tumor cell survival by regulating pH and preventing apoptosis. As previously reported, plasma membrane V-ATPases help regulate cytosolic pH in macrophages and neutrophils (Nanda et al. 1996). This mechanism may also be used by tumor cells, which produce more H+ due to high glycolytic activity (Gatenby, Gillies 2004). Treatment with V-ATPase inhibitors lowers H+ extrusion, both *in vitro* and *in*

Bafilomycin A1 was assessed as a potential anticancer agent because it inhibits cell proliferation and tumor growth. Although this effect has been attributed to the inhibition of intracellular acidosis by blocking V-ATPases, the precise mechanism remains unknown (Bowman et al. 2004). A study conducted by Lim et al., hypothesized that bafilomycin A1 and its analogue, concanamycin A, stimulate a tumor growth factor, hypoxia-inducible 1α (HIF-1α) (Zhong et al. 1999). The interaction of bafilomycin with HIF-1α increases with hypoxia, causing strong induction of the p21 gene which, in turn, leads to cell cycle

V-ATPase inhibition has also been shown to trigger apoptosis through caspase-dependent and caspase-independent mechanisms (De Milito et al. 2007, Aiko et al. 2002), and bafilomycin and concanamycin induce apoptosis in other types of cells, including

Morimura et al. described the growth-inhibiting effect of apoptosis stimulation in human hepatoblastomas using bafilomycin A1. In particular, electron microscopy, morphological observations, and flow cytometry showed higher apoptotic cell ratios and diminished cell reproduction. Cell growth inhibition in normal liver cells was insignificant (Morimura et

In the case of human gastric cancer cells, Nakashima et al. investigated the mechanism of apoptosis induced by bafilomycin A1. Bafilomycin inhibits the growth of MKN-1 cancer cells through apoptosis. Flow cytometry was used to measure alterations in lysosomal pH, which increased in the presence of bafilomycin. Caspase-3 activity was also increased by bafilomycin; such findings suggest that bafilomycin A1 induces apoptosis in MKN-1 cells mediated by proteases released after lysosomal dysfunction, followed by caspase-3 activation of the cytochrome c-independent manner (Nakashima et al. 2003,

A study conducted by Wu et al. has shown that bafilomycin A1 suppresses macroautophagy by preventing lysosome acidification (Wu et al. 2009). Macroautophagy is a protein

increases with tumor aggressiveness (Montcourrier et al. 1997, Parkins et al. 1997). In addition, low pH may cause extracellular matrix (ECM) degradation and remodeling through activation of proteolytic enzymes which contribute to invasion and cancer metastasis (Martinez-Zaguilan et al. 1996, Rofstad et al. 2006). Proteases need low extracellular pH to optimize their activation, including metalloproteinases (MMP), morphogenetic bone metalloproteinases (protein type 1), tissue serine proteases, and adamalysin-related proteinases. Among them, MMPs are the proteases basically involved in degradation and remodelling of all extracellular matrix (ECM) structural components (Montcourrier et al. 1994, Rozhin et al. 1994, Johnson et al. 2000, Kato et al. 2005, Gocheva, Joyce 2007).

Sennoune et al. assessed the effect of bafilomycin A1 in breast tumor cells and found that cytoplasmic pH recovery was inhibited in response to acid load, in both highly and lowly metastatic cells, although to a greater extent in highly metastatic cells (Sennoune et al. 2004). This suggests that V-ATPases in the plasma membrane are involved in the acquisition of a more metastatic phenotype and that the use of V-ATPase inhibitors allows distant metastasis to be minimized (Figure 2).

Fig. 2. Proposed mechanism by which overexpression of pmVATPase at the leading edge of the cell modulates cell migration/invasion. The proposed model should be viewed as a framework to explain how pmV-ATPases determine the acquisition of an invasive phenotype needed for angiogenesis and metastasis. Changes in pHcyt are critical for establishing cell polarity needed for cell movement. A critical step in directed motility and migration is the asymmetric actin polymerization at the leading edge (Sennoune, Martinez-Zaguilan 2007).

Using RNA interference techniques, Lu et al. found that distant metastasis could be delayed and suppressed in human hepatocellular carcinoma *in vitro* by reducing proton extrusion and gelatinase activity through the inhibition of V-ATPase subunit c (ATP6L) (Lu et al. 2005). This fact is consistent with subunit c block by bafilomycin and concanamycin, as this is their main binding site to V-ATPase (Bowman et al. 2004). In tyrosinase-positive amelanotic melanoma cells, inactive tyrosinase accumulates in the endoplasmic reticulum because the presence of aberrant V-ATPases blocks trafficking through secretory pathways. The use of V-ATPase inhibitors, such as bafilomycin A1 or concanamycin A, improves transport, demonstrating the involvement of this enzyme and preventing conditions that favor metastatic dissemination (Halaban et al. 2002).

Hence, both *in vitro* or *in vivo*, V-ATPases are a target for anticancer therapeutic agents, either directly by regulating the pH gradient in the tumor environment or indirectly by preventing ECM protease activation (Fais et al. 2007).

## **3.2 Tumor cell growth and survival**

134 Current Cancer Treatment – Novel Beyond Conventional Approaches

increases with tumor aggressiveness (Montcourrier et al. 1997, Parkins et al. 1997). In addition, low pH may cause extracellular matrix (ECM) degradation and remodeling through activation of proteolytic enzymes which contribute to invasion and cancer metastasis (Martinez-Zaguilan et al. 1996, Rofstad et al. 2006). Proteases need low extracellular pH to optimize their activation, including metalloproteinases (MMP), morphogenetic bone metalloproteinases (protein type 1), tissue serine proteases, and adamalysin-related proteinases. Among them, MMPs are the proteases basically involved in degradation and remodelling of all extracellular matrix (ECM) structural components (Montcourrier et al. 1994, Rozhin et al. 1994, Johnson et

Sennoune et al. assessed the effect of bafilomycin A1 in breast tumor cells and found that cytoplasmic pH recovery was inhibited in response to acid load, in both highly and lowly metastatic cells, although to a greater extent in highly metastatic cells (Sennoune et al. 2004). This suggests that V-ATPases in the plasma membrane are involved in the acquisition of a more metastatic phenotype and that the use of V-ATPase inhibitors allows distant

Fig. 2. Proposed mechanism by which overexpression of pmVATPase at the leading edge of the cell modulates cell migration/invasion. The proposed model should be viewed as a framework to explain how pmV-ATPases determine the acquisition of an invasive phenotype needed for angiogenesis and metastasis. Changes in pHcyt are critical for establishing cell polarity needed for cell movement. A critical step in directed motility and migration is the asymmetric actin polymerization at the leading edge (Sennoune, Martinez-

al. 2000, Kato et al. 2005, Gocheva, Joyce 2007).

metastasis to be minimized (Figure 2).

Zaguilan 2007).

V-ATPases may also play a significant role in tumor cell survival by regulating pH and preventing apoptosis. As previously reported, plasma membrane V-ATPases help regulate cytosolic pH in macrophages and neutrophils (Nanda et al. 1996). This mechanism may also be used by tumor cells, which produce more H+ due to high glycolytic activity (Gatenby, Gillies 2004). Treatment with V-ATPase inhibitors lowers H+ extrusion, both *in vitro* and *in vivo* (Volk, Albert & Kempski 1998, McSheehy et al. 2003).

Bafilomycin A1 was assessed as a potential anticancer agent because it inhibits cell proliferation and tumor growth. Although this effect has been attributed to the inhibition of intracellular acidosis by blocking V-ATPases, the precise mechanism remains unknown (Bowman et al. 2004). A study conducted by Lim et al., hypothesized that bafilomycin A1 and its analogue, concanamycin A, stimulate a tumor growth factor, hypoxia-inducible 1α (HIF-1α) (Zhong et al. 1999). The interaction of bafilomycin with HIF-1α increases with hypoxia, causing strong induction of the p21 gene which, in turn, leads to cell cycle detection in cancer cells (Lim et al. 2006).

V-ATPase inhibition has also been shown to trigger apoptosis through caspase-dependent and caspase-independent mechanisms (De Milito et al. 2007, Aiko et al. 2002), and bafilomycin and concanamycin induce apoptosis in other types of cells, including neutrophils (Gottlieb et al. 1995)and osteoclasts (Xu et al. 2003).

Morimura et al. described the growth-inhibiting effect of apoptosis stimulation in human hepatoblastomas using bafilomycin A1. In particular, electron microscopy, morphological observations, and flow cytometry showed higher apoptotic cell ratios and diminished cell reproduction. Cell growth inhibition in normal liver cells was insignificant (Morimura et al. 2008).

In the case of human gastric cancer cells, Nakashima et al. investigated the mechanism of apoptosis induced by bafilomycin A1. Bafilomycin inhibits the growth of MKN-1 cancer cells through apoptosis. Flow cytometry was used to measure alterations in lysosomal pH, which increased in the presence of bafilomycin. Caspase-3 activity was also increased by bafilomycin; such findings suggest that bafilomycin A1 induces apoptosis in MKN-1 cells mediated by proteases released after lysosomal dysfunction, followed by caspase-3 activation of the cytochrome c-independent manner (Nakashima et al. 2003, Hishita et al. 2001).

A study conducted by Wu et al. has shown that bafilomycin A1 suppresses macroautophagy by preventing lysosome acidification (Wu et al. 2009). Macroautophagy is a protein

V-ATPase Inhibitors in Cancer Treatment and Their

play a key role in this resistance (Perez-Sayans et al. 2009).

**3.3.1 Multidrug resistance in OSCC**

*GST-π*, and *DNA* topoisomerase *II*.

Ling 1976).

(Uematsu et al. 2001).

MDR in OSCC.

Implication in Multidrug Resistance in Oral Squamous Cell Carcinoma 137

Extracellular pH is considerably more acidic in oral squamous cell carcinoma (OSCC), a solid tumor, than in normal tissue. This increased acidity interferes with the absorption of standard chemotherapy drugs, reducing their effect on tumors (Griffiths 1991, Negendank 1992). Vacuolar ATPases (V-ATPases) have been reported to be largely responsible for this acidic environment (Newell et al. 1993, Yamagata et al. 1998). While a clear association has been established between MDR and Pgp expression in some tumors, the mechanism by which drug resistance occurs within the multistep process of OSCC has not yet been fully elucidated (Tanigawara 2000). OSCC is highly resistant to a wide range of structurally different drugs with diverse cytotoxic mechanisms of action (McLeod, Evans 1999). This suggests that OSCC may be intrinsically chemoresistant and it is possible that V-ATPases

As already mentioned, while a clear association has been established between MDR and Pgp expression in certain tumors, the mechanism by which drug resistance occurs within the multistep process of OSCC has not yet been fully elucidated (Ling 1997). Several genes have been implicated in MDR, including *MDR1*, *MRP* (multidrug resistance*-*associated protein)*,*

Pgp is encoded by *MDR1* and flow cytometry studies have shown increased Pgp levels in recurrent OSCCs compared to normal mucosa with oral lesions at different stages of tumorigenesis (Jain et al. 1997). These findings were confirmed by immunohistochemical staining in a study that compared recurrent tumors with untreated primary oral tumors (Chomczynski, Sacchi 2006, Xie et al. 2000). The best known *MDR1* gene product is Pgp/P-170, which has been implicated in resistance to chemotherapy agents such as taxanes, anthracyclines, vinca alkaloids, podophyllotoxins, and camptothecins (Juliano,

The mechanism by which Pgp-mediated MDR is acquired in head and neck tumors, however, is different. Immunohistochemical studies, for example, have revealed high levels of Pgp in salivary gland adenocarcinoma (SGA) cell lines but insignificant levels in OSCC cell lines (Naramoto et al. 2007). Reverse transcription quantitative polymerase chain reaction analysis of *MDR1* expression has also revealed increased Pgp levels in different cell lines treated with vincristine (alkaloid cancer drug). These results suggest that Pgp-induced MDR in OSCC is essentially an acquired phenotype caused by the genetic induction of Pgp

MRP has been linked to MDR in multidrug-resistant Pgp-negative cells lines in small cell lung cancer, cancer of the stomach, bladder, cervix, and prostate, and leukemia (Kim et al. 1996, Kim et al. 1995, Endo et al. 1996b, Endo et al. 1996a). In head and neck tumors, overexpression of MRP1 mRNA has been found in human and murine OSCC and SGA cell lines mice treated with vincristine. sugesting the theory of Pgp- and MRP-independent

Overexpression of the isozyme GST-π is often associated with malignant transformation and/or MDR (Ruzza et al. 2009). GST-π is responsible for detoxifying xenobiotics such as carboplatin (used in chemotherapy) and elevated levels of this enzyme cause treatment resistance (Engel et al. 2005, Koshiyama et al. 2001). Whether or not this is also the case in OSCC, however, is a matter of debate. In a study by Chen et al (Chen, Lin 1997), GST-π levels increased with increased severity of oral epithelial dysplasia in line with the development of OSCC. The immunohistochemical expression of placental GST-π has been

degradation pathway that allows increased cell survival under stress and in cancer (Meijer, Codogno 2004, Mortimore, Hutson & Surmacz 1983). Macroautophagic inhibition in HT-29, HCT-116, and SW1116 colon cancer cells is accompanied by down-regulation of cyclin D and E and up-regulation of p21Cip1 and various caspases, causing an antiproliferative effect (Wu et al. 2009).

ancer cells are more likely to express V-ATPase than normal cells, causing abnormalities in the acidic microenvironment and affecting cancer cell growth and infiltration significantly (Saroussi, Nelson 2008, Cardone, Casavola & Reshkin 2005, Perona, Serrano 1988). Moreover, neoplastic cells are more sensitive to bafilomycin A1 than normal cells, a fact that may be used in anticancer therapy (Ohta et al. 1996).

#### **3.3 Chemoresistance**

Resistance to chemotherapy agents is the main reason for treatment failure in patients with cancer, and multidrug resistance (MDR) occurs in many types of tumors. The main mechanism that gives rise to the MDR phenotype is the overexpression of drug efflux transporters such as the P glycoprotein (Pgp) in the plasma membrane (Nielsen, Skovsgaard 1992) (Figure 3).

Fig. 3. Pgp resides in the cell membrane, where it may act to pump toxins out of the cell. The painting shows a model of the protein's structure that is based on its known sequence of amino acids. The protein chain is thought to snake back and forth 12 times across the lipid bilayer of the membrane forming a 12-sided pore. The pan of the protein outside the cell bears sugar chains (purple); two large and nearly identical domains protrude into the cell. They include regions (green) that bind the cellular energy-carrying compound ATP, which probably provides the energy that drives the efflux (arrows) (Kartner, Ling 1989).

Extracellular pH is considerably more acidic in oral squamous cell carcinoma (OSCC), a solid tumor, than in normal tissue. This increased acidity interferes with the absorption of standard chemotherapy drugs, reducing their effect on tumors (Griffiths 1991, Negendank 1992). Vacuolar ATPases (V-ATPases) have been reported to be largely responsible for this acidic environment (Newell et al. 1993, Yamagata et al. 1998). While a clear association has been established between MDR and Pgp expression in some tumors, the mechanism by which drug resistance occurs within the multistep process of OSCC has not yet been fully elucidated (Tanigawara 2000). OSCC is highly resistant to a wide range of structurally different drugs with diverse cytotoxic mechanisms of action (McLeod, Evans 1999). This suggests that OSCC may be intrinsically chemoresistant and it is possible that V-ATPases play a key role in this resistance (Perez-Sayans et al. 2009).

## **3.3.1 Multidrug resistance in OSCC**

136 Current Cancer Treatment – Novel Beyond Conventional Approaches

degradation pathway that allows increased cell survival under stress and in cancer (Meijer, Codogno 2004, Mortimore, Hutson & Surmacz 1983). Macroautophagic inhibition in HT-29, HCT-116, and SW1116 colon cancer cells is accompanied by down-regulation of cyclin D and E and up-regulation of p21Cip1 and various caspases, causing an antiproliferative effect

ancer cells are more likely to express V-ATPase than normal cells, causing abnormalities in the acidic microenvironment and affecting cancer cell growth and infiltration significantly (Saroussi, Nelson 2008, Cardone, Casavola & Reshkin 2005, Perona, Serrano 1988). Moreover, neoplastic cells are more sensitive to bafilomycin A1 than normal cells, a fact that

Resistance to chemotherapy agents is the main reason for treatment failure in patients with cancer, and multidrug resistance (MDR) occurs in many types of tumors. The main mechanism that gives rise to the MDR phenotype is the overexpression of drug efflux transporters such as the P glycoprotein (Pgp) in the plasma membrane (Nielsen, Skovsgaard

Fig. 3. Pgp resides in the cell membrane, where it may act to pump toxins out of the cell. The painting shows a model of the protein's structure that is based on its known sequence of amino acids. The protein chain is thought to snake back and forth 12 times across the lipid bilayer of the membrane forming a 12-sided pore. The pan of the protein outside the cell bears sugar chains (purple); two large and nearly identical domains protrude into the cell. They include regions (green) that bind the cellular energy-carrying compound ATP, which

probably provides the energy that drives the efflux (arrows) (Kartner, Ling 1989).

(Wu et al. 2009).

**3.3 Chemoresistance**

1992) (Figure 3).

may be used in anticancer therapy (Ohta et al. 1996).

As already mentioned, while a clear association has been established between MDR and Pgp expression in certain tumors, the mechanism by which drug resistance occurs within the multistep process of OSCC has not yet been fully elucidated (Ling 1997). Several genes have been implicated in MDR, including *MDR1*, *MRP* (multidrug resistance*-*associated protein)*, GST-π*, and *DNA* topoisomerase *II*.

Pgp is encoded by *MDR1* and flow cytometry studies have shown increased Pgp levels in recurrent OSCCs compared to normal mucosa with oral lesions at different stages of tumorigenesis (Jain et al. 1997). These findings were confirmed by immunohistochemical staining in a study that compared recurrent tumors with untreated primary oral tumors (Chomczynski, Sacchi 2006, Xie et al. 2000). The best known *MDR1* gene product is Pgp/P-170, which has been implicated in resistance to chemotherapy agents such as taxanes, anthracyclines, vinca alkaloids, podophyllotoxins, and camptothecins (Juliano, Ling 1976).

The mechanism by which Pgp-mediated MDR is acquired in head and neck tumors, however, is different. Immunohistochemical studies, for example, have revealed high levels of Pgp in salivary gland adenocarcinoma (SGA) cell lines but insignificant levels in OSCC cell lines (Naramoto et al. 2007). Reverse transcription quantitative polymerase chain reaction analysis of *MDR1* expression has also revealed increased Pgp levels in different cell lines treated with vincristine (alkaloid cancer drug). These results suggest that Pgp-induced MDR in OSCC is essentially an acquired phenotype caused by the genetic induction of Pgp (Uematsu et al. 2001).

MRP has been linked to MDR in multidrug-resistant Pgp-negative cells lines in small cell lung cancer, cancer of the stomach, bladder, cervix, and prostate, and leukemia (Kim et al. 1996, Kim et al. 1995, Endo et al. 1996b, Endo et al. 1996a). In head and neck tumors, overexpression of MRP1 mRNA has been found in human and murine OSCC and SGA cell lines mice treated with vincristine. sugesting the theory of Pgp- and MRP-independent MDR in OSCC.

Overexpression of the isozyme GST-π is often associated with malignant transformation and/or MDR (Ruzza et al. 2009). GST-π is responsible for detoxifying xenobiotics such as carboplatin (used in chemotherapy) and elevated levels of this enzyme cause treatment resistance (Engel et al. 2005, Koshiyama et al. 2001). Whether or not this is also the case in OSCC, however, is a matter of debate. In a study by Chen et al (Chen, Lin 1997), GST-π levels increased with increased severity of oral epithelial dysplasia in line with the development of OSCC. The immunohistochemical expression of placental GST-π has been

V-ATPase Inhibitors in Cancer Treatment and Their

the plasma membrane (Raghunand et al. 1999).

**4. Conclusions**

minimizing adverse effects.

pp. 3873-3882.

7972-7976.

**5. References**

improve drug resistance in breast cancer cells (You et al. 2009).

*signalling,* vol. 14, no. 8, pp. 717-722.

*surgery,* vol. 18, no. 5, pp. 1051-1054.

vol. 279, no. 32, pp. 33131-33138.

Implication in Multidrug Resistance in Oral Squamous Cell Carcinoma 139

has been found to sensitize tumor cell lines to the effect of different chemotherapy drugs (De Milito, Fais 2005a, Luciani et al. 2004, De Milito, Fais 2005b, Sennoune, Luo & Martinez-Zaguilan 2004). The effectiveness of the drug transport mechanism appears to be comparable to that of drug efflux pumps such as Pgp, although vesicle acid exchange (above all in vesicles that have an active H+/cation exchange system) may be an important factor in drug resistance, and particularly in cells that do not overexpress Pgp-type efflux pumps in

Murakami et al (Murakami et al. 2001) found cisplatin-resistant tumors to contain elevated levels of all V-ATPase subunits but levels of *ATP6C* were particularly. They also found significantly higher levels of cellular pH in cisplatin-resistant tumor cells than in cells sensitive to vincristine and etoposide. In a later study, however, Zhang et al (Zhang et al. 2006) identified 38 overexpressed genes and 25 underexpressed genes in cisplatin-resistant OSCC. Torigoe et al (Torigoe et al. 2002) showed that treatment with anticancer agents increased *ATP6L* (c subunit). Interfering RNA targeting the c subunit has also been found to

The above findings suggest that the induced expression of V-ATPases in MDR is an antiapoptotic defence and that the combined use of PPIs or V-ATPase inhibitors and low chemotherapy doses might be a possible treatment target (Torigoe et al. 2002). We believe that the future of these molecules in cancer treatment involves measuring the overexpression of specific V-ATPase subunits in tumors to be treated and then using inhibitors specific for the subunits being expressed (Perez-Sayans et al. 2009, Perez-Sayans et al. 2010). This will allow clinicians to provide more specific treatment, while also

Aiko, K., Tsujisawa, T., Koseki, T., Hashimoto, S., Morimoto, Y., Amagasa, T. & Nishihara,

Baars, T.L., Petri, S., Peters, C. & Mayer, A. 2007, "Role of the V-ATPase in Regulation of the

Becelli, R., Renzi, G., Morello, R. & Altieri, F. 2007, "Intracellular and extracellular tumor pH

Bowman, E.J., Graham, L.A., Stevens, T.H. & Bowman, B.J. 2004, "The

Bowman, E.J., Siebers, A. & Altendorf, K. 1988, "Bafilomycins: a class of inhibitors of

T. 2002, "Involvement of cytochrome c and caspases in apoptotic cell death of human submandibular gland ductal cells induced by concanamycin A", *Cellular*

Vacuolar Fission Fusion Equilibrium", *Molecular Biology of the Cell,* vol. 18, no. 10,

measurement in a series of patients with oral cancer", *The Journal of craniofacial*

bafilomycin/concanamycin binding site in subunit c of the V-ATPases from Neurospora crassa and Saccharomyces cerevisiae", *The Journal of biological chemistry,*

membrane ATPases from microorganisms, animal cells, and plant cells", *Proceedings of the National Academy of Sciences of the United States of America,* vol. 85, no. 21, pp.

studied in the oral epithelium of premalignant and malignant oral lesions, and has indeed been proposed as a good marker for premalignant lesions and tumors (Zhang, Xiao & Priddy 1994). Another study, however, that analyzed GST-π levels using enzyme-linked immunoassay failed to find a significant relationship between GST-π and TNM stage (Oude Ophuis et al. 1998). Finally, in a study that analyzed GST-π expression using Northern blot analysis and gene amplification with Southern blot analysis, Wang et al (Wang et al. 1997) concluded that the amplification of the *GST-π* gene was not critically related to the overexpression of GST-π mRNA. Furthermore, they found no relationship between GST-π mRNA overexpression and tumor size, neck nodal status, or patient survival.

The downregulation of topoisomerase II—an enzyme that breaks and rejoins doublestranded DNA in the interconversion of different topological forms of DNA—has also been associated with MDR (Deffie, Batra & Goldenberg 1989, Shi et al. 2008).

The mechanisms underlying MDR response are less clear in OSCC than in other types of tumors (Yajima et al. 2009). MDR holds challenges for both researchers and the pharmaceutical industry. Accordingly, efforts are being made on all sides to find anticancer compounds characterized not only by high tolerability and oral bioavailability but also by the ability to overcome the problem of drug resistance. OSCC is highly resistant to a wide range of structurally different drugs with different cytotoxic mechanisms of action (McLeod, Evans 1999). This suggests that OSCC may be intrinsically chemoresistant, with other molecules, including V-ATPases, playing an important role (Perez-Sayans et al. 2009).

#### **3.3.2 The role of v-ATPases in multidrug resistance**

It has been demonstrated that hypoxia and acidity contribute to the transition from benign to malignant growth via the selection of tumor cells capable of surviving in an acidic, oxygen-deprived environment. Acidity, for example, has been associated with chemotherapy resistance (Raghunand et al. 2001), proliferation (Morita et al. 1992), and metastatic behavior (Martinez-Zaguilan et al. 1996). Indeed, alteration of the pH gradient between the extracellular environment and the cell cytoplasm has been suggested as a possible mechanism of resistance to cytotoxic drugs (De Milito, Fais 2005a) (Figure 4).

The alteration of cytosolic pH also plays an important role in drug resistance in chemotherapy. Extracellular pH in solid tumors is significantly more acidic than in normal tissue. This increased acidity interferes with the absorption of basic chemotherapy drugs, reducing their effect on tumors (Griffiths 1991, Negendank 1992). Martínez-Zaguilán et al., found that unlike chemoresistant cells, chemosensitive cells did not recover from acid load (Martínez-Zaguilán et al. 1999). Becellli et al (Becelli et al. 2007) found that reversed pH gradient was directly related to drug resistance.

Anaerobic metabolism is an important determinant of tumor acidity that allows the selection of cells capable of surviving in a hypoxic-anaerobic environment via the synthesis of lactate. A complex system appears to regulate pH homeostasis in mammal cells, and it seems as if malignant tumor cells are capable of exploiting some of these mechanisms to protect themselves from the acidic environment, while maintaining levels of acidity that are poorly tolerated by normal or more differentiated cells (De Milito, Fais 2005b).

Recent studies have suggested that V-ATPases, which secrete protons through the plasma membrane, may play a key role in the acidification of the tumor environment (Perez-Sayans et al. 2009). Several human tumor cells are characterized by increased V-ATPase expression and activity (Perez-Sayans et al. 2010), and pretreatment with proton pump inhibitors (PPIs) has been found to sensitize tumor cell lines to the effect of different chemotherapy drugs (De Milito, Fais 2005a, Luciani et al. 2004, De Milito, Fais 2005b, Sennoune, Luo & Martinez-Zaguilan 2004). The effectiveness of the drug transport mechanism appears to be comparable to that of drug efflux pumps such as Pgp, although vesicle acid exchange (above all in vesicles that have an active H+/cation exchange system) may be an important factor in drug resistance, and particularly in cells that do not overexpress Pgp-type efflux pumps in the plasma membrane (Raghunand et al. 1999).

Murakami et al (Murakami et al. 2001) found cisplatin-resistant tumors to contain elevated levels of all V-ATPase subunits but levels of *ATP6C* were particularly. They also found significantly higher levels of cellular pH in cisplatin-resistant tumor cells than in cells sensitive to vincristine and etoposide. In a later study, however, Zhang et al (Zhang et al. 2006) identified 38 overexpressed genes and 25 underexpressed genes in cisplatin-resistant OSCC. Torigoe et al (Torigoe et al. 2002) showed that treatment with anticancer agents increased *ATP6L* (c subunit). Interfering RNA targeting the c subunit has also been found to improve drug resistance in breast cancer cells (You et al. 2009).

## **4. Conclusions**

138 Current Cancer Treatment – Novel Beyond Conventional Approaches

studied in the oral epithelium of premalignant and malignant oral lesions, and has indeed been proposed as a good marker for premalignant lesions and tumors (Zhang, Xiao & Priddy 1994). Another study, however, that analyzed GST-π levels using enzyme-linked immunoassay failed to find a significant relationship between GST-π and TNM stage (Oude Ophuis et al. 1998). Finally, in a study that analyzed GST-π expression using Northern blot analysis and gene amplification with Southern blot analysis, Wang et al (Wang et al. 1997) concluded that the amplification of the *GST-π* gene was not critically related to the overexpression of GST-π mRNA. Furthermore, they found no relationship between GST-π

The downregulation of topoisomerase II—an enzyme that breaks and rejoins doublestranded DNA in the interconversion of different topological forms of DNA—has also been

The mechanisms underlying MDR response are less clear in OSCC than in other types of tumors (Yajima et al. 2009). MDR holds challenges for both researchers and the pharmaceutical industry. Accordingly, efforts are being made on all sides to find anticancer compounds characterized not only by high tolerability and oral bioavailability but also by the ability to overcome the problem of drug resistance. OSCC is highly resistant to a wide range of structurally different drugs with different cytotoxic mechanisms of action (McLeod, Evans 1999). This suggests that OSCC may be intrinsically chemoresistant, with other molecules, including V-ATPases, playing an important role (Perez-Sayans et al. 2009).

It has been demonstrated that hypoxia and acidity contribute to the transition from benign to malignant growth via the selection of tumor cells capable of surviving in an acidic, oxygen-deprived environment. Acidity, for example, has been associated with chemotherapy resistance (Raghunand et al. 2001), proliferation (Morita et al. 1992), and metastatic behavior (Martinez-Zaguilan et al. 1996). Indeed, alteration of the pH gradient between the extracellular environment and the cell cytoplasm has been suggested as a possible mechanism of resistance to cytotoxic drugs (De Milito, Fais 2005a) (Figure 4). The alteration of cytosolic pH also plays an important role in drug resistance in chemotherapy. Extracellular pH in solid tumors is significantly more acidic than in normal tissue. This increased acidity interferes with the absorption of basic chemotherapy drugs, reducing their effect on tumors (Griffiths 1991, Negendank 1992). Martínez-Zaguilán et al., found that unlike chemoresistant cells, chemosensitive cells did not recover from acid load (Martínez-Zaguilán et al. 1999). Becellli et al (Becelli et al. 2007) found that reversed pH

Anaerobic metabolism is an important determinant of tumor acidity that allows the selection of cells capable of surviving in a hypoxic-anaerobic environment via the synthesis of lactate. A complex system appears to regulate pH homeostasis in mammal cells, and it seems as if malignant tumor cells are capable of exploiting some of these mechanisms to protect themselves from the acidic environment, while maintaining levels of acidity that are

Recent studies have suggested that V-ATPases, which secrete protons through the plasma membrane, may play a key role in the acidification of the tumor environment (Perez-Sayans et al. 2009). Several human tumor cells are characterized by increased V-ATPase expression and activity (Perez-Sayans et al. 2010), and pretreatment with proton pump inhibitors (PPIs)

poorly tolerated by normal or more differentiated cells (De Milito, Fais 2005b).

mRNA overexpression and tumor size, neck nodal status, or patient survival.

associated with MDR (Deffie, Batra & Goldenberg 1989, Shi et al. 2008).

**3.3.2 The role of v-ATPases in multidrug resistance**

gradient was directly related to drug resistance.

The above findings suggest that the induced expression of V-ATPases in MDR is an antiapoptotic defence and that the combined use of PPIs or V-ATPase inhibitors and low chemotherapy doses might be a possible treatment target (Torigoe et al. 2002). We believe that the future of these molecules in cancer treatment involves measuring the overexpression of specific V-ATPase subunits in tumors to be treated and then using inhibitors specific for the subunits being expressed (Perez-Sayans et al. 2009, Perez-Sayans et al. 2010). This will allow clinicians to provide more specific treatment, while also minimizing adverse effects.

## **5. References**

V-ATPase Inhibitors in Cancer Treatment and Their

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**8**

*India*

**Farnesyltransferase Inhibitor**

Anuj G. Agrawal1 and Rakesh R. Somani2

*RIICO Industrial Area, Bhiwadi 301019 (RJ), Near New Delhi,*

*Pharmacy, Behind Collector Colony, Chembur (E), Mumbai,*

*1Research and Development, Cachet Pharmaceuticals Pvt. Ltd., Alkem group, C-582,*

*2Department of Pharmaceutical Chemistry, Vivekanand Education Society's College of*

Cancer is a class of diseases characterized by uncontrolled growth of abnormal cells anywhere in the body and the ability of these cells to invade other locations in the body, either by direct growth into adjacent tissue or by migration of cells to distant sites. This unregulated growth is caused by damage to DNA, resulting in mutations to genes that encode proteins controlling cell division. To prevent this unregulated growth various anticancer drug (Chen et al., 2011; Kim & Dass 2011) have been developed. But these drugs have severe toxicity and are not well tolerated in the patient. Therefore, the major goal in anticancer drug discovery process is to discover and develop innovative therapies that exhibit a real improvement in effectiveness and/or tolerability. In cancer therapy, continuous effort has been made to explore the new targets. Cancer research is largely focused on prospective targets identified by basic science such as the oncogenic signal transduction pathway, oncogenes, tumor suppressor genes, and genes involved in the regulation of the cell cycle and apoptosis or programmed cell death (Gridelli et al., 2003; Hochhaus et al., 2004; Lau et al., 2011; Minna et al., 2004). Proteins mediating their effects are obvious targets for cancer therapy because, by definition, these proteins are involved in the primary transformation of normal cells. Proteins that transmit abnormal growth signals offer enticing points of intervention for the treatment of cancer. One potential target is the Ras family of proteins, which are mutationally activated in a wide range of human tumor types and are important contributors to the neoplastic phenotype (Barbacid et al., 1987; Biagi

Ras proteins have been the subject of intense research investigation by the biomedical research community since 1982 (James et al., 1996). Ras is the name of a protein, the gene that encodes it, and the family and superfamily of proteins to which it belongs. Ras proteins are guanine nucleotide–binding proteins that play pivotal roles in the control of normal and transformed cell growth. The Ras superfamily includes the Ras, Rho, and Rab families. There are three Ras proto-oncogenes: the H-*ras* gene (Harvey murine sarcoma viral

**1. Introduction**

**2. Ras protein**

et al., 2010; Bollag et al., 1991; Bos et al., 1989).

**in Cancer Treatment**

*research : an official journal of the American Association for Cancer Research,* vol. 3, no. 1, pp. 111-114.

## **Farnesyltransferase Inhibitor in Cancer Treatment**

Anuj G. Agrawal1 and Rakesh R. Somani2

*1Research and Development, Cachet Pharmaceuticals Pvt. Ltd., Alkem group, C-582, RIICO Industrial Area, Bhiwadi 301019 (RJ), Near New Delhi, 2Department of Pharmaceutical Chemistry, Vivekanand Education Society's College of Pharmacy, Behind Collector Colony, Chembur (E), Mumbai, India*

#### **1. Introduction**

148 Current Cancer Treatment – Novel Beyond Conventional Approaches

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#### **2. Ras protein**

Ras proteins have been the subject of intense research investigation by the biomedical research community since 1982 (James et al., 1996). Ras is the name of a protein, the gene that encodes it, and the family and superfamily of proteins to which it belongs. Ras proteins are guanine nucleotide–binding proteins that play pivotal roles in the control of normal and transformed cell growth. The Ras superfamily includes the Ras, Rho, and Rab families. There are three Ras proto-oncogenes: the H-*ras* gene (Harvey murine sarcoma viral

Farnesyltransferase Inhibitor in Cancer Treatment 151

variety of signal pathways that control such processes as cytoskeletal integrity, proliferation, cell adhesion, apoptosis, and cell migration (Zhao et al., 2011). The final four amino acids play an important role in specifying subcellular localization of the Ras protein. All Ras proteins have a specific amino acid sequence motif at the carboxyl (C) terminus, commonly referred to as the CAAX sequence (C, cysteine; A, aliphatic amino acid; X, any amino acid usually methionine or serine) which signals for posttranslational modifications (Cadinanos

Ras is a G protein and functions as a molecular switch cycling between GTP- bound "on" and GDP-bound "off" states (Seki et al., 1996). It is activated by guanine exchange factors which are themselves activated by mitogenic signals and through feedback from Ras itself. It is inactivated by GTPase-activating protein, which increases the rate of GTP hydrolysis, returning Ras to its GDP-bound form, simultaneously releasing an inorganic phosphate. Ras is synthesized in the cytoplasm as a biologically inactive cytosolic propeptide (Pro-Ras) and undergoes a series of closely linked posttranslational modifications by the covalent addition of a non-polar farnesyl group to the COOH-terminal, thereby increasing its hydrophobicity (Kyathanahalli & Kowluru, 2011). The C-termini triplet of amino acids is cleaved off, leaving a farnesylated, methylated cysteine residue at the carboxyterminus. Ras is then localized to the inner surface of the plasma membranes (Gibbs et al., 1993; Hancock et al., 1989, 1990; Jackson et al., 1990; Salaun et al., 1999), in which Ras cycles from an inactive GDP-bound state to an active GTP-bound state. Once in its GTP-bound form, Ras activates several downstream effector pathways that mediate increased gene transcription and rapid cell proliferation (Fig. 2). The most critical step, farnesylation, adds a 15-carbon farnesyl isoprenoid group to H-, K-, and N-Ras through a thioether bond and is catalyzed by

Fig. 2. Ras-dependent signal transduction with Farnesyltransferase inhibitor (FTI) target.

et al., 2003; Epifano et al., 2007; Roberts et al., 2008; Rowinsky et al., 2006).

 The *ras* oncogenes encode four low molecular weight (21 kDa) proteins, Ras (H-Ras, N-Ras, and K-Ras4A and K-Ras4B, resulting from two alternatively spliced K-*ras* gene products) (Morgillo et al., 2007), that, in normal untransformed cells, cycle between an inactive guanosine 5' diphosphate (GDP)-bound state and active guanosine 5'-triphosphate (GTP)-bound state at the inner surface of the plasma membrane in mammalian cells.

(b)

Fig. 1. a. Structure of the HRAS protein (Elaine 2009), b. Ribbon diagram of H-ras (Elaine 2010).

The highly conserved nature of the variable region across mammalian species indicates that Ras proteins serve specific functions. They are very important molecular switches for a wide

oncogene homolog, Fig. 1), the K-*ras* gene (Kirsten murine sarcoma viral oncogene homolog), and the N-*ras* gene (neuroblastoma oncogene homolog) (Boguski et al., 1993; Ellis et al., 1981; Marcos et al., 2003; Ruta et al., 1986; Shimizu et al., 1983). The *ras* oncogenes encode four low molecular weight (21 kDa) proteins, Ras (H-Ras, N-Ras, and K-Ras4A and K-Ras4B, resulting from two alternatively spliced K-*ras* gene products) (Morgillo et al., 2007), that, in normal untransformed cells, cycle between an inactive guanosine 5' diphosphate (GDP)-bound state and active guanosine 5'-triphosphate (GTP)-bound state at

(a)

(b)

The highly conserved nature of the variable region across mammalian species indicates that Ras proteins serve specific functions. They are very important molecular switches for a wide

Fig. 1. a. Structure of the HRAS protein (Elaine 2009), b. Ribbon diagram of H-ras

(Elaine 2010).

the inner surface of the plasma membrane in mammalian cells.

variety of signal pathways that control such processes as cytoskeletal integrity, proliferation, cell adhesion, apoptosis, and cell migration (Zhao et al., 2011). The final four amino acids play an important role in specifying subcellular localization of the Ras protein. All Ras proteins have a specific amino acid sequence motif at the carboxyl (C) terminus, commonly referred to as the CAAX sequence (C, cysteine; A, aliphatic amino acid; X, any amino acid usually methionine or serine) which signals for posttranslational modifications (Cadinanos et al., 2003; Epifano et al., 2007; Roberts et al., 2008; Rowinsky et al., 2006).

Ras is a G protein and functions as a molecular switch cycling between GTP- bound "on" and GDP-bound "off" states (Seki et al., 1996). It is activated by guanine exchange factors which are themselves activated by mitogenic signals and through feedback from Ras itself. It is inactivated by GTPase-activating protein, which increases the rate of GTP hydrolysis, returning Ras to its GDP-bound form, simultaneously releasing an inorganic phosphate. Ras is synthesized in the cytoplasm as a biologically inactive cytosolic propeptide (Pro-Ras) and undergoes a series of closely linked posttranslational modifications by the covalent addition of a non-polar farnesyl group to the COOH-terminal, thereby increasing its hydrophobicity (Kyathanahalli & Kowluru, 2011). The C-termini triplet of amino acids is cleaved off, leaving a farnesylated, methylated cysteine residue at the carboxyterminus. Ras is then localized to the inner surface of the plasma membranes (Gibbs et al., 1993; Hancock et al., 1989, 1990; Jackson et al., 1990; Salaun et al., 1999), in which Ras cycles from an inactive GDP-bound state to an active GTP-bound state. Once in its GTP-bound form, Ras activates several downstream effector pathways that mediate increased gene transcription and rapid cell proliferation (Fig. 2). The most critical step, farnesylation, adds a 15-carbon farnesyl isoprenoid group to H-, K-, and N-Ras through a thioether bond and is catalyzed by Farnesyl transferase (FTase) (Kho et al., 2004; Ljuca et al., 2011).

Fig. 2. Ras-dependent signal transduction with Farnesyltransferase inhibitor (FTI) target.

Farnesyltransferase Inhibitor in Cancer Treatment 153

In addition, there are other prenyltransferase enzymes, including geranylgeranyl transferases which transfer one or two 20-carbon geranylgeranyl isoprenoid lipid moieties to proteins, again facilitating membrane incorporation. Both farnesylation and geranylgeranylation result in more hydrophobic proteins. The potential for crossprenylation of proteins such as Ras suggests that geranylgeranyltransferase could restore the function of these proteins if FTase was inhibited (Kim et al., 2010; Marks et al., 2007). However, not all Ras proteins are prenylated by geranylgeranyltransferase, and it is not clear that the function of geranylgeranylated Ras is the same as that of farnesylated Ras, as suggested by the fact that geranylgeranylated normal Ras may be inhibitory. Strategies that are capable of blocking FTase and preventing farnesylation may be expected to inhibit the maturation of Ras into a biologically active molecule, thus turning off signal transduction

Farnesyl transferase is located in cell cytosol. FTase is one of the three enzymes in the prenyltransferase group that catalyzes most prenylation reactions and differs in their isoprenoid substrates and protein targets (Fig. 4). FTase adds a 15 carbon (Subramanian et al., 2008) isoprenoid lipid called a farnesyl group to proteins bearing a CAAX motif and its targets include members of the Ras superfamily of small GTP binding proteins critical to cell

FTase is a zinc metalloenzyme that exists as a heterodimer. This heterodimer has two distinct subunits denoted as α and β, having molecular weights of 48 kDa and 46 kDa respectively (Machida et al., 2011; Zhang & Casey, 1996). The X-ray crystal structure of FTase reveals that it has binding sites for both the CAAX peptide and the FDP (Kauh et al., 2011; Park et al., 1997; Wei et al., 2011). It has been shown that geranylgeranyltransferase can

(Appels et al., 2011; Geryk-Hall et al., 2010).

prenylate some of the substrates of FTase and vice versa.

Fig. 4. Structure of Farnesyltransferase (Berman et al., 2000)

**5. Farnesyl transferase**

cycle progression.

#### **3. Mutations of Ras in human cancers**

Ras is mutated to an oncogenic form in cancer, so the Ras and Ras-related proteins are often deregulated, leading to increased invasion and metastasis, and decreased apoptosis. In part of the human tumors, one of the three *ras* genes harbored a point mutation, they result in a permanently active GTP-bound form of Ras (Le Moulec et al., 2009; Lowry & Willumsen et al., 1993).

Mutant Ras proteins transform cells because they continuously activate the downstream effector pathways, including those involved in cell proliferation, in the absence of any upstream growth factor stimulation. Mutations of *ras* occur in approximately 30% of all human cancers, including a significant proportion of pancreatic and colorectal carcinomas (Clark et al., 1995; Khosravi-Far et al., 1994; Shimoyama, 2011; Widemann et al. 2006). With regard to the three *ras* genes, mutation of K*-ras* is most commonly found in human tumors, whereas N*-ras* mutations are encountered less often and H*-ras* mutations rarely. The type of *ras* mutation seems to correlate with tumor type. Although activating *ras* mutations are mainly involved with myeloid malignancies and carcinomas of the breast, colon, pancreas, lung, and thyroid, they have also been detected in many other types of cancer (Beaupre et al., 1999; Zheng et al., 2010).

#### **4. Post-translational modification of Ras**

Ras proteins are tethered to the inner face of the membrane by posttranslational modifications that make them more hydrophobic (Ageberg et al., 2011), which involve prenylation (addition of a lipid moiety) of the protein. Afterits synthesis as cytoplasmic Pro-Ras, Ras is sequentially modified by farnesylation of the cysteine residue, proteolytic cleavage of the AAX peptide by proteases, and carboxymethylation of the new C-terminal carboxylate by carboxymethyl transferase. As the first step in this sequence, farnesylation is the most critical part of the process (Casey et al., 1989; Cox & Der, 1997; Gibbs & Oliff, 1997; Gelb et al., 1997; Kato et al., 1992; McCormick et al., 1993; Omer et al., 1997; Schafer et al., 1989; Yamane et al., 1990), in which a 15-carbon farnesyl isoprenoid group is transferred from farnesyl diphosphate (FDP) to form a thioether bond with the cysteine moiety in the C terminal tetrapeptide sequence of the Ras protein (Fig. **3**).

Fig. 3. The first step in Ras posttranslational modification is mediated by FTase, which transfers a farnesyl moiety from FDP to the cysteine moiety in the CAAX motif at the carboxyl terminus of Ras.

Ras is mutated to an oncogenic form in cancer, so the Ras and Ras-related proteins are often deregulated, leading to increased invasion and metastasis, and decreased apoptosis. In part of the human tumors, one of the three *ras* genes harbored a point mutation, they result in a permanently active GTP-bound form of Ras (Le Moulec et al., 2009; Lowry & Willumsen et

Mutant Ras proteins transform cells because they continuously activate the downstream effector pathways, including those involved in cell proliferation, in the absence of any upstream growth factor stimulation. Mutations of *ras* occur in approximately 30% of all human cancers, including a significant proportion of pancreatic and colorectal carcinomas (Clark et al., 1995; Khosravi-Far et al., 1994; Shimoyama, 2011; Widemann et al. 2006). With regard to the three *ras* genes, mutation of K*-ras* is most commonly found in human tumors, whereas N*-ras* mutations are encountered less often and H*-ras* mutations rarely. The type of *ras* mutation seems to correlate with tumor type. Although activating *ras* mutations are mainly involved with myeloid malignancies and carcinomas of the breast, colon, pancreas, lung, and thyroid, they have also been detected in many other types of cancer (Beaupre et

Ras proteins are tethered to the inner face of the membrane by posttranslational modifications that make them more hydrophobic (Ageberg et al., 2011), which involve prenylation (addition of a lipid moiety) of the protein. Afterits synthesis as cytoplasmic Pro-Ras, Ras is sequentially modified by farnesylation of the cysteine residue, proteolytic cleavage of the AAX peptide by proteases, and carboxymethylation of the new C-terminal carboxylate by carboxymethyl transferase. As the first step in this sequence, farnesylation is the most critical part of the process (Casey et al., 1989; Cox & Der, 1997; Gibbs & Oliff, 1997; Gelb et al., 1997; Kato et al., 1992; McCormick et al., 1993; Omer et al., 1997; Schafer et al., 1989; Yamane et al., 1990), in which a 15-carbon farnesyl isoprenoid group is transferred from farnesyl diphosphate (FDP) to form a thioether bond with the cysteine moiety in the C

<sup>O</sup> <sup>P</sup> <sup>O</sup> <sup>P</sup> <sup>O</sup>

O O

O O

Ras-Cys-A1-A2-X

S

Fig. 3. The first step in Ras posttranslational modification is mediated by FTase, which transfers a farnesyl moiety from FDP to the cysteine moiety in the CAAX motif at the

**3. Mutations of Ras in human cancers**

al., 1993).

al., 1999; Zheng et al., 2010).

Ras-Cys-A1-A2-X

carboxyl terminus of Ras.

SH

**4. Post-translational modification of Ras**

terminal tetrapeptide sequence of the Ras protein (Fig. **3**).

Farnesyl diphosphate (FDP)

FTase