The pressed juice had LD50, orally in rats, more than 120 g/kg body weight, and did not damage the growth, the blood chemistry and the pathology of organs. It was safe according to World Health Organization (Tappayuthpijarn et al., 1991; Intayot et al., 2002). The subchronic toxicity study of *M. loriformis* in rats for 3 months did not damage the growth, the blood chemistry and the pathology of organs (Tappayuthpijarn et al., 1991). The ethanol extract of this plant induced in vitro DT-diaphorase, a detoxifying enzyme (Vinitketkumnuen, 1996), and decreased in vivo the formation of aberrant crypt foci (ACF), which was related to the colon cancer (Intayot et al., 2002).

## **2.1.3** *Ficus hispida* **L. f.**

*F. hispida* is a traditional Thai plant from family Moraceae. It is a moderate sized tree and grows well in damp and shady place (Fig. 5). Almost all parts of this plant can be used in traditional medicine for the treatment of various ailments, for example, an anti-diarrhea, emetic, astringent, hepatoprotective, antitussive, antipyretic, antiinflammatory, depurative, vulnerary, hemostatic, antiulcer as well as in the treatment of anemia (Wuttidhammaved, 1997; Mandal and Ashok Kumar, 2002; Peraza-Sanchez et al., 2002).

Fig. 5. *Ficus hispida* L. f.

*F. hispida*, was investigated for antiproliferative activity on human breast cancer cell lines: SKBR3, MDA-MB435, T47D, and MCF7. Based on the results, the methanolic extract exhibited the most powerful activity on T47D breast cancer cell line with ED50 of 110.3±9.63 g/mL as compared to DMSO, the negative control. Confirmation of the result using colony-forming assay (clonogenic assay) yielded the effect in a dose - dependent manner. The cell cycle analysis of this plant extract revealed non-specificity with prominent in apoptosis. Therefore, *F. hispida* might provide some benefits in the treatment of breast cancer. (Pratumvinit et al., 2009)

Experimental Therapeutics in Breast Cancer Cells 249

The usage of herbs nowadays has been developed according to the laboratory evidence. Herbs are prepared as drug materials, which are conformed to the following pattern

The nature (crude drug, tincture, pressed juice, fluidextract, dry extract, etc.) of the drug

The quantity of the drug material in each dose of the solid dosage form (tablets) or in

The drug extract ratio (DER), the amount (g) of crude drug to produce one g extract or

The indication must be conformed to the monograph established by the Ministry of the

labeled DER = 4-6.7 : 1 recommended daily dose = 500 mg extract thus, the daily dose = 2-3.35 g crude drug.

The drug material comprises pressed juice from *T. cucumerina* fruit, which

The drug material comprises pressed juice from *M. loriformis* herb, which is standardized with glycosphingolipid namely 1-*O*-D-glucopyranosyl-2- (2-hydroxy-6-ene-cosamide)-sphingosine, a specific marker compound.

is standardized with cucurbitacin B, a specific marker compound.

One g pressed juice is prepared from 10.5 g fresh fruit. According to the preparation which is made from the pressed juice it is non-drug and can be considered as traditional remedy (Haensel Hoelzl, 1996). The indication is supported by the laboratory evidence and the traditional use. It is supposed to be used

One g pressed juice is prepared from 1.7 g fresh herb.

**3. The herbal preparation**

material (drug composition) must be specified.

each package of the liquid dosage form must be specified.

100 mL fluidextract

drug material, and the extraction method must be specified.

The daily dose must be specified as the weight of the crude drug.

Public Health, or supported by the clinical evidence.

For example :

**3.1 The preparation of** *Trichosanthes cucumerina*

10.5 g fresh fruit

**3.2 The preparation of** *Murdannia loriformis*

1.7:1

10.5:1

as natural cytostatic agent.

For example: 200 mg dry extract / tablet or

The solvent or solvent mixture used for the extraction must be specified.

(Dingermann, 2000).

Declaration

The daily dose

Declaration

DER

DER

## **2.1.4** *Erycibe elliptilimba* **Merr. & Chun.**

*Erycibe elliptilimba*, belonging to the Convolvulaceae family, is a Thai medicinal plant that has long been used for many decades to relieve symptoms from fever caused by infection, inflammation and prescribed in the mixture of traditional medicine for treating of various malignancies (Wuttidhammaved, 1997; Sintusarn, 2002).

Fig. 6. *Erycibe elliptilimba* Merr. & Chun.

The ED50 values of the methanol fraction against SKBR3 and MDA-MB435 were 56.07 and 30.61 g/mL, respectively. Doxorubicin as a positive control. This inhibitory growth activity of the treated cells acted in a dose-dependent manner which was also confirmed by the cell viability with the trypan blue exclusion assay. (Kummalue et al., 2007).

## **2.2 Herbal mixture**

Two Thai remedies (TR1 and TR2) were tested for antiproliferative effect. TR1 was composed of following ingredients : *Albizia procera* (stem), *Diospyros mollis* (stem), *Ficus hispida* (stem), *Smilax glabra* (stem), *Smilax china* (stem) and cobra bone. TR2 was composed of *Gelonium multiflorum* (stem), *Erycibe elliptilimba* (stem), *Balanophora abbreviate* (stem), *Smilax glabra* (stem), *Smilax china* (stem) and *Millingtonia hortensis* (stem). TR1 and TR2 had no antiproliferative effect on breast, lung and colon cancer cell lines. However the combination of the remedies (either TR1 or TR2, 30 g/mL) with doxorubicin (0.5 g/mL) could significantly inhibit growth of lung cancer cell line (A549) at G2/M phase stronger than doxorubicin alone (Srisapoomi et al., 2008).

In addition two Thai medicinal plants, they were *P. indica and S. rarax*, were tested for antiproliferative effect.  Both did not inhibit the breast cancer cell.

## **3. The herbal preparation**

248 Breast Cancer – Current and Alternative Therapeutic Modalities

*Erycibe elliptilimba*, belonging to the Convolvulaceae family, is a Thai medicinal plant that has long been used for many decades to relieve symptoms from fever caused by infection, inflammation and prescribed in the mixture of traditional medicine for treating of various

The ED50 values of the methanol fraction against SKBR3 and MDA-MB435 were 56.07 and 30.61 g/mL, respectively. Doxorubicin as a positive control. This inhibitory growth activity of the treated cells acted in a dose-dependent manner which was also confirmed by the cell

Two Thai remedies (TR1 and TR2) were tested for antiproliferative effect. TR1 was composed of following ingredients : *Albizia procera* (stem), *Diospyros mollis* (stem), *Ficus hispida* (stem), *Smilax glabra* (stem), *Smilax china* (stem) and cobra bone. TR2 was composed of *Gelonium multiflorum* (stem), *Erycibe elliptilimba* (stem), *Balanophora abbreviate* (stem), *Smilax glabra* (stem), *Smilax china* (stem) and *Millingtonia hortensis* (stem). TR1 and TR2 had no antiproliferative effect on breast, lung and colon cancer cell lines. However the combination of the remedies (either TR1 or TR2, 30 g/mL) with doxorubicin (0.5 g/mL) could significantly inhibit growth of lung cancer cell line (A549) at G2/M phase stronger

In addition two Thai medicinal plants, they were *P. indica and S. rarax*, were tested for antiproliferative effect.  Both

viability with the trypan blue exclusion assay. (Kummalue et al., 2007).

**2.1.4** *Erycibe elliptilimba* **Merr. & Chun.**

Fig. 6. *Erycibe elliptilimba* Merr. & Chun.

than doxorubicin alone (Srisapoomi et al., 2008).

did not inhibit the breast cancer cell.

**2.2 Herbal mixture**

malignancies (Wuttidhammaved, 1997; Sintusarn, 2002).

The usage of herbs nowadays has been developed according to the laboratory evidence. Herbs are prepared as drug materials, which are conformed to the following pattern (Dingermann, 2000).

For example: 200 mg dry extract / tablet or

100 mL fluidextract

	- For example :

## **3.1 The preparation of** *Trichosanthes cucumerina*

Declaration

The drug material comprises pressed juice from *T. cucumerina* fruit, which is standardized with cucurbitacin B, a specific marker compound.

DER

10.5:1

One g pressed juice is prepared from 10.5 g fresh fruit.

10.5 g fresh fruit

## **3.2 The preparation of** *Murdannia loriformis*

Declaration

The drug material comprises pressed juice from *M. loriformis* herb, which is standardized with glycosphingolipid namely 1-*O*-D-glucopyranosyl-2- (2-hydroxy-6-ene-cosamide)-sphingosine, a specific marker compound.

DER

1.7:1 One g pressed juice is prepared from 1.7 g fresh herb.

Experimental Therapeutics in Breast Cancer Cells 251

Cells are weakly invasive in vitro. In Matrigel, most of these cells grow as non-fused spheres. Moreover, on plastic, they will accumulate in clusters of loosely attached cells and rarely reach full confluency. Breast cancer cell lines in this group are BT-474, CAMA-1,

The third group is mesenchymal-like or stromal-like cells. It does not express the markers found in the first and second group. In contrast, it exhibits a high level of vimentin (gene VIM) which is the marker of mesenchymal cells. These cells have fibroblastoid phenotype on plastic and grow as colonies with large stellate projections in Matrigel. They are highly invasive in

Herein, details of breast cancer cell lines which have been employed in our medicinal plant

SKBR3 is a human breast carcinoma cell line established in late 1970s and derived from pleural effusion of Caucasian patient diagnosed as invasive ductal carcinoma (CLS, Germany). It is an adherent cell which is the usual character found in most breast cancer cell lines. Ultra structures of cells show microvilli and desmosomes, large lysosomes, and bundles of cytoplasmic fibrils. In nude mice, these cells can form poorly differentiated adenocarcinoma (http://www.cell-lines-service). In addition in nude mice, c-Jun, the ending molecule of signal transduction pathways, is demonstrated to have the critical role in

Estrogen receptor (ER) is found to be absent or expressed at the very low level in SKBR3 cells and progesterone receptor is also absent in this type of cell (Lacroix and Leclercq, 2004). In contrast, Her2/neu receptor including leptin receptor are overexpressed in this cell line whereas insulin- like growth factor receptor- I is expressed at the lower level when compared with those in MCF7 cells (Ozbay and Nahta, 2008). Interestingly, recent report revealed that SKBR3 expressed NMDAR1 and NMDAR2 receptors, both of which are important calcium channels. These receptors are reported to be essential for the growth of

This SKBR3 cell line with high expression of HER2 protein has long been used in various research. Recently, it has been shown to be the suitable candidate for reference materials in quality control of HER2 testing together with MCF7 cell line. Based on this report, SKBR3 and MCF7 breast cancer cells provide valuable controls for quantitative measurement of HER2 amplification and production (Xiao et al, 2009). The photograph of SKRB3 cultured in

MCF7 is a human breast carcinoma cell line derived from pleural effusion of Caucasian patient diagnosed with breast adenocarcinoma (CLS, Germany). It is a relatively resistant to cisplatin treatment (Westmose Yde and Issinger, 2006). Morphology of this cell line exhibits epithelial-like cell including the ability to process estradiol via cytoplasmic estrogen receptors and domes formation. This cell line has oncogene, wnt7h and can induce tumor in

This MCF7 cell line expresses both estrogen and progesterone receptors whereas the expression of Her2/neu is absent (Lacroix and Leclercq, 2004). The cells can be suppressed by catechin hydrate, product from plant sources such as green tea, through TP53/caspase-

vitro. MDA-MB435S, MDA-MB231, Hs578T, and BT-549 are classified in this group.

the tumorigenesis and metastasis in SKBR3 breast cancer cells (Zhang et al, 2007).

human breast cancer xenografts in mice (North et al, 2010).

MDA-MB134, MDA-MB361, MDA-MB453, MDA-MB468, and SKBR3.

research are demonstrated.

the flask is shown in Figure 7.

**4.1.2 MCF7 breast cancer cell line**

nude mice (http://www.cell-lines-service).

**4.1.1 SKBR3 breast cancer cell line**

100 g fresh herb (aerial plant parts)

## **4. Breast cancer cells and experimental procedure in antiproliferative activity background**

Breast cancer is one of the leading causes of cancer death around the world. In Thailand, the incidence of breast cancer disease accounts for 20.5 per 100,000 female population (Chaiwerawatana, 2007). Though breast cancer treatment nowadays has dramatically improved due to new drug emerging such as monoclonal antibody, and tyrosine kinase inhibitor, the resistance to the drug itself, unfortunately, has significantly increased. Therefore, searching for the new drug to treat breast cancer is very essential. Based on this important issue, four specific breast cancer cell lines, i.e., SKBR3, MCF7, T47D, and MDA-MB435 have been used to evaluate the potential and promising chemotherapeutic agents from medicinal plants sources.

These four breast cancer cell lines, SKBR3, MCF7, T47D, and MDA-MB435, have different origins and properties which are considered as the very useful tools for investigating the medicinal plants effects. Actually, medicinal plants have long been prescribed for over centuries to treat various diseases including infections and malignancies such as breast cancer in many countries, for example, China, and Thailand. This traditional medicine nowadays plays an important role in healthcare system with approximately 80% of people around the world using it (Itharat and Ooraikul, 2007). Determination of the plants activities especially growth inhibition and their mechanisms for cancer therapy is, indeed, undergoing in much effort.

In this section, four breast cancer cell lines and their experiments on medicinal plants are demonstrated. These include antiproliferative activity and cytotoxicity, cell cycle study, apoptotic study, mechanisms of action in plants, and future direction for testing the drugs from plant sources. Most of the experiments are performed in the biosafety level 1 which is suitable enough for manipulating these breast cancer cell lines in vitro.

## **4.1 Breast cancer cell lines: Their origins and properties**

Breast cancer cell lines can be categorized into three groups according to their phenotypes and invasiveness (Lacroix and Leclercq, 2004).

The first group is luminal epithelial-like cells. This group expresses high amount of typical luminal epithelial phenotype of breast cells such as estrogen receptor (ER), E-cadherin (gene CDH1), zonula occludens-1(TJP1), desmoplakin I/II (DSP), and desmosomal junctions. This group of breast cancer cells will grow as interconnected colonies of polygonal cells on plastic and as fused colonies in Matrigel, which is semisolid medium. This kind of cells are weakly invasive. BT-483, MCF7, T47D, and ZR-75 are all in this group.

The second group is called weakly luminal epithelial-like cells. This group of cells shows the expression closely to the first group with a reduced extent or at least some of those markers.

 According to the preparation made from the pressed juice it is non-drug and can be considered as traditional remedy (Haensel Hoelzl, 1996). The indication can be supported by the traditional use over 30 years and the case reported by the physicians. To use as an adjunct to anticancer therapy in order to reduce the side effects and

**4. Breast cancer cells and experimental procedure in antiproliferative activity**

Breast cancer is one of the leading causes of cancer death around the world. In Thailand, the incidence of breast cancer disease accounts for 20.5 per 100,000 female population (Chaiwerawatana, 2007). Though breast cancer treatment nowadays has dramatically improved due to new drug emerging such as monoclonal antibody, and tyrosine kinase inhibitor, the resistance to the drug itself, unfortunately, has significantly increased. Therefore, searching for the new drug to treat breast cancer is very essential. Based on this important issue, four specific breast cancer cell lines, i.e., SKBR3, MCF7, T47D, and MDA-MB435 have been used to evaluate the potential and promising chemotherapeutic agents

These four breast cancer cell lines, SKBR3, MCF7, T47D, and MDA-MB435, have different origins and properties which are considered as the very useful tools for investigating the medicinal plants effects. Actually, medicinal plants have long been prescribed for over centuries to treat various diseases including infections and malignancies such as breast cancer in many countries, for example, China, and Thailand. This traditional medicine nowadays plays an important role in healthcare system with approximately 80% of people around the world using it (Itharat and Ooraikul, 2007). Determination of the plants activities especially growth inhibition and their mechanisms for cancer therapy is, indeed,

In this section, four breast cancer cell lines and their experiments on medicinal plants are demonstrated. These include antiproliferative activity and cytotoxicity, cell cycle study, apoptotic study, mechanisms of action in plants, and future direction for testing the drugs from plant sources. Most of the experiments are performed in the biosafety level 1 which is

Breast cancer cell lines can be categorized into three groups according to their phenotypes

The first group is luminal epithelial-like cells. This group expresses high amount of typical luminal epithelial phenotype of breast cells such as estrogen receptor (ER), E-cadherin (gene CDH1), zonula occludens-1(TJP1), desmoplakin I/II (DSP), and desmosomal junctions. This group of breast cancer cells will grow as interconnected colonies of polygonal cells on plastic and as fused colonies in Matrigel, which is semisolid medium. This kind of cells are weakly

The second group is called weakly luminal epithelial-like cells. This group of cells shows the expression closely to the first group with a reduced extent or at least some of those markers.

suitable enough for manipulating these breast cancer cell lines in vitro.

**4.1 Breast cancer cell lines: Their origins and properties**

invasive. BT-483, MCF7, T47D, and ZR-75 are all in this group.

and invasiveness (Lacroix and Leclercq, 2004).

100 g fresh herb (aerial plant parts)

prevent the cancer metastasis.

from medicinal plants sources.

undergoing in much effort.

The daily dose

**background**

Cells are weakly invasive in vitro. In Matrigel, most of these cells grow as non-fused spheres. Moreover, on plastic, they will accumulate in clusters of loosely attached cells and rarely reach full confluency. Breast cancer cell lines in this group are BT-474, CAMA-1, MDA-MB134, MDA-MB361, MDA-MB453, MDA-MB468, and SKBR3.

The third group is mesenchymal-like or stromal-like cells. It does not express the markers found in the first and second group. In contrast, it exhibits a high level of vimentin (gene VIM) which is the marker of mesenchymal cells. These cells have fibroblastoid phenotype on plastic and grow as colonies with large stellate projections in Matrigel. They are highly invasive in vitro. MDA-MB435S, MDA-MB231, Hs578T, and BT-549 are classified in this group.

Herein, details of breast cancer cell lines which have been employed in our medicinal plant research are demonstrated.

#### **4.1.1 SKBR3 breast cancer cell line**

SKBR3 is a human breast carcinoma cell line established in late 1970s and derived from pleural effusion of Caucasian patient diagnosed as invasive ductal carcinoma (CLS, Germany). It is an adherent cell which is the usual character found in most breast cancer cell lines. Ultra structures of cells show microvilli and desmosomes, large lysosomes, and bundles of cytoplasmic fibrils. In nude mice, these cells can form poorly differentiated adenocarcinoma (http://www.cell-lines-service). In addition in nude mice, c-Jun, the ending molecule of signal transduction pathways, is demonstrated to have the critical role in the tumorigenesis and metastasis in SKBR3 breast cancer cells (Zhang et al, 2007).

Estrogen receptor (ER) is found to be absent or expressed at the very low level in SKBR3 cells and progesterone receptor is also absent in this type of cell (Lacroix and Leclercq, 2004). In contrast, Her2/neu receptor including leptin receptor are overexpressed in this cell line whereas insulin- like growth factor receptor- I is expressed at the lower level when compared with those in MCF7 cells (Ozbay and Nahta, 2008). Interestingly, recent report revealed that SKBR3 expressed NMDAR1 and NMDAR2 receptors, both of which are important calcium channels. These receptors are reported to be essential for the growth of human breast cancer xenografts in mice (North et al, 2010).

This SKBR3 cell line with high expression of HER2 protein has long been used in various research. Recently, it has been shown to be the suitable candidate for reference materials in quality control of HER2 testing together with MCF7 cell line. Based on this report, SKBR3 and MCF7 breast cancer cells provide valuable controls for quantitative measurement of HER2 amplification and production (Xiao et al, 2009). The photograph of SKRB3 cultured in the flask is shown in Figure 7.

#### **4.1.2 MCF7 breast cancer cell line**

MCF7 is a human breast carcinoma cell line derived from pleural effusion of Caucasian patient diagnosed with breast adenocarcinoma (CLS, Germany). It is a relatively resistant to cisplatin treatment (Westmose Yde and Issinger, 2006). Morphology of this cell line exhibits epithelial-like cell including the ability to process estradiol via cytoplasmic estrogen receptors and domes formation. This cell line has oncogene, wnt7h and can induce tumor in nude mice (http://www.cell-lines-service).

This MCF7 cell line expresses both estrogen and progesterone receptors whereas the expression of Her2/neu is absent (Lacroix and Leclercq, 2004). The cells can be suppressed by catechin hydrate, product from plant sources such as green tea, through TP53/caspase-

Experimental Therapeutics in Breast Cancer Cells 253

(Lacroix, 2009; Rae et al, 2004). This cell line is found to express certain genes commonly transcribed in melanocyte which are not seen in breast cancer cell lines such as DCP gene. Moreover, expression of melanocyte proteins tyrosinase and melan-A has also been demonstrated in MDA-MB435. However, some evidences have been presented that MDA-MB435 might be breast cancer cell line expressing a poorly differentiated aggressive breast tumor together with epithelial and melanocyte markers (Chambers, 2009; http://www.cell-

Investigation on protein identification in MDA-MB435 cells was performed and found 30 important proteins expressing in this cell line (Chandramouli et al, 2009). For example, caspase 14 precursor, heat shock protein 60, cell adhesion proteins, and some immune

Antiproliferative and cytotoxic activity are the basic and very common investigations used for determination in the efficacy of plant extracts for searching the novel drugs. The measurement of herb treated mammalian cells either surviving or proliferating cells could be achieved by several techniques. Counting cells that include or exclude a dye such as trypan blue dye with the use of a hemocytometer under a microscope or in an automated cell counter is one assay. Quantitation of suspension cells could be performed by counting them directly whereas adherent cells which grow in monolayer culture require proteolytic agents such as trypsin prior to quantitation. This method is, therefore, not only time

A number of various indirect methods to quantitate living or dead cell number has been developed. Based on the fact that active or living cells will increase or decrease some specific proteins or even nucleic acids that can indicate cellular proliferation or cytotoxicity. Measuring released 51Cr-labeled protein after cell lysis, and measuring incorporation of 3Hthymidine during cell proliferation have been employed (Mosmann, 1983). These traceable radioactive moieties are very efficient and specific. However, the radioactive methods could handle very limited numbers of samples at each time and also require long sample preparation procedure with inherent dangers and high cost (Haslam et al, 2000). Therefore, several non-radioactive methods have been developed instead of an old traditional method,

In this section, breast cancer cells and experimental procedures in antiproliferative activity, three non-radioactive assays, i.e., MTT assay, Lactate dehydrogenase (LDH) cytotoxicity

This colorimetric assay was first developed and reported by Mosmann (Mosmann, 1983). Tetrazolium salt MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide) is tested and used in this assay. These tetrazolium salts can measure the activity of several dehydrogenase enzymes especially mitochondrial enzyme succinate-dehydrogenase (Slater et al., 1963). The reaction is known to be occurred in the living cells by cleaving the tetrazolium ring in the metabolically active mitochondria, not in dead cells or erythrocytes. From the MTT pale yellow substrate, the dark blue (purple) formazan products are generated in the direct proportion to the living cell numbers. Notably, this MTT assay

assay, and clonogenic assay, commonly used have been demonstrated in details.

**4.2 Breast cancer cells: Experimental procedures in antiproliferative activity**

response proteins such as complement C3 were also demonstrated.

consuming, labor intensive, but also dependent upon individual skills.

lines-service).

the cell counting.

**4.2.1 MTT assay**

**Principle & mechanism of action**

Fig. 7. SKBR3 cells grow in DMEM supplemented with 10% fetal bovine serum and 1% penicillin+ streptomycin. Cells show monolayer when grow in plastic with polygonal shaped. This cell line is categorized as weakly luminal epithelial-like cell.

mediated apoptosis (Alshatwi, 2010). Some vasoactive peptides such as endothelin 1 is found at low level in MCF7 while in SKBR3 is expressed at the higher level. This expression might correlate with high invasiveness phenotype in breast cancer (Hagemann et al, 2005). MCF7 has been extensively used as the model for breast cancer and breast cancer therapy. However, different sources of MCF7 show the differences in response to 17beta-estradiol resulting from activation or inhibition of insulin-like growth factor I (IGF-1) (Hamelers et al, 2003). Therefore, the different responses of MCF7 should be realized due to the expression of IGF-1. As mentioned above, MCF7 is suitable to be candidate of reference materials in quality control for HER2 testing (Xiao et al, 2009).

## **4.1.3 T47D breast cancer cell line**

T47D is a human breast carcinoma cell line derived from pleural effusion of an infiltrating ductal carcinoma of the breast (CLS, Germany). It contains cytoplasmic junctions, receptors to 17 beta estradiol including steroids and calcitonin ( Moseley et al, 1983). Receptors for estrogen and progesterone are also present (Hevir et al, 2011). This cell line has tumorigenicity in nude mice and has three oncogenes, i.e., wnt3, wnt7h, and wnt7b (http://www.cell-lines-service).

By activation Janus kinase2/signal transducer and transcription 5 pathway, this cell line expresses ample growth hormone receptor and prolactin receptor (Xu et al, 2011). Both receptors are structurally similar cytokine receptor superfamily members. Direct protein identification by MALDI post-source decay (PSD) or MALDI collision-induced dissociation (CID) in T47D cells showed only histone H2B (Pevsner et al, 2007). Chemokine CXCL12 and its promoter activity are found to be increase in human T47D breast cancer cells (Chen et al, 2010). Indeed, T47D cell line, as well as MCF7, represents the good model of estrogen-dependent breast cancer with capacity of 17beta-estradiol local production (Hevir et al, 2011).

#### **4.1.4 MDA-MB435 breast cancer cell line**

MDA-MB435 is derived from the pleural effusion of metastatic ductal adenocarcinoma of the breast. Recent data have pointed out that MDA-MB435 cells are from melanoma

Fig. 7. SKBR3 cells grow in DMEM supplemented with 10% fetal bovine serum and 1% penicillin+ streptomycin. Cells show monolayer when grow in plastic with polygonal

mediated apoptosis (Alshatwi, 2010). Some vasoactive peptides such as endothelin 1 is found at low level in MCF7 while in SKBR3 is expressed at the higher level. This expression might correlate with high invasiveness phenotype in breast cancer (Hagemann et al, 2005). MCF7 has been extensively used as the model for breast cancer and breast cancer therapy. However, different sources of MCF7 show the differences in response to 17beta-estradiol resulting from activation or inhibition of insulin-like growth factor I (IGF-1) (Hamelers et al, 2003). Therefore, the different responses of MCF7 should be realized due to the expression of IGF-1. As mentioned above, MCF7 is suitable to be candidate of reference materials in

T47D is a human breast carcinoma cell line derived from pleural effusion of an infiltrating ductal carcinoma of the breast (CLS, Germany). It contains cytoplasmic junctions, receptors to 17 beta estradiol including steroids and calcitonin ( Moseley et al, 1983). Receptors for estrogen and progesterone are also present (Hevir et al, 2011). This cell line has tumorigenicity in nude mice and has three oncogenes, i.e., wnt3, wnt7h, and wnt7b

By activation Janus kinase2/signal transducer and transcription 5 pathway, this cell line expresses ample growth hormone receptor and prolactin receptor (Xu et al, 2011). Both receptors are structurally similar cytokine receptor superfamily members. Direct protein identification by MALDI post-source decay (PSD) or MALDI collision-induced dissociation (CID) in T47D cells showed only histone H2B (Pevsner et al, 2007). Chemokine CXCL12 and its promoter activity are found to be increase in human T47D breast cancer cells (Chen et al, 2010). Indeed, T47D cell line, as well as MCF7, represents the good model of estrogen-dependent

MDA-MB435 is derived from the pleural effusion of metastatic ductal adenocarcinoma of the breast. Recent data have pointed out that MDA-MB435 cells are from melanoma

breast cancer with capacity of 17beta-estradiol local production (Hevir et al, 2011).

shaped. This cell line is categorized as weakly luminal epithelial-like cell.

quality control for HER2 testing (Xiao et al, 2009).

**4.1.3 T47D breast cancer cell line**

(http://www.cell-lines-service).

**4.1.4 MDA-MB435 breast cancer cell line**

(Lacroix, 2009; Rae et al, 2004). This cell line is found to express certain genes commonly transcribed in melanocyte which are not seen in breast cancer cell lines such as DCP gene. Moreover, expression of melanocyte proteins tyrosinase and melan-A has also been demonstrated in MDA-MB435. However, some evidences have been presented that MDA-MB435 might be breast cancer cell line expressing a poorly differentiated aggressive breast tumor together with epithelial and melanocyte markers (Chambers, 2009; http://www.celllines-service).

Investigation on protein identification in MDA-MB435 cells was performed and found 30 important proteins expressing in this cell line (Chandramouli et al, 2009). For example, caspase 14 precursor, heat shock protein 60, cell adhesion proteins, and some immune response proteins such as complement C3 were also demonstrated.

## **4.2 Breast cancer cells: Experimental procedures in antiproliferative activity**

Antiproliferative and cytotoxic activity are the basic and very common investigations used for determination in the efficacy of plant extracts for searching the novel drugs. The measurement of herb treated mammalian cells either surviving or proliferating cells could be achieved by several techniques. Counting cells that include or exclude a dye such as trypan blue dye with the use of a hemocytometer under a microscope or in an automated cell counter is one assay. Quantitation of suspension cells could be performed by counting them directly whereas adherent cells which grow in monolayer culture require proteolytic agents such as trypsin prior to quantitation. This method is, therefore, not only time consuming, labor intensive, but also dependent upon individual skills.

A number of various indirect methods to quantitate living or dead cell number has been developed. Based on the fact that active or living cells will increase or decrease some specific proteins or even nucleic acids that can indicate cellular proliferation or cytotoxicity. Measuring released 51Cr-labeled protein after cell lysis, and measuring incorporation of 3Hthymidine during cell proliferation have been employed (Mosmann, 1983). These traceable radioactive moieties are very efficient and specific. However, the radioactive methods could handle very limited numbers of samples at each time and also require long sample preparation procedure with inherent dangers and high cost (Haslam et al, 2000). Therefore, several non-radioactive methods have been developed instead of an old traditional method, the cell counting.

In this section, breast cancer cells and experimental procedures in antiproliferative activity, three non-radioactive assays, i.e., MTT assay, Lactate dehydrogenase (LDH) cytotoxicity assay, and clonogenic assay, commonly used have been demonstrated in details.

#### **4.2.1 MTT assay**

#### **Principle & mechanism of action**

This colorimetric assay was first developed and reported by Mosmann (Mosmann, 1983). Tetrazolium salt MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide) is tested and used in this assay. These tetrazolium salts can measure the activity of several dehydrogenase enzymes especially mitochondrial enzyme succinate-dehydrogenase (Slater et al., 1963). The reaction is known to be occurred in the living cells by cleaving the tetrazolium ring in the metabolically active mitochondria, not in dead cells or erythrocytes. From the MTT pale yellow substrate, the dark blue (purple) formazan products are generated in the direct proportion to the living cell numbers. Notably, this MTT assay

Experimental Therapeutics in Breast Cancer Cells 255

 For pure compounds, serial concentrations can be diluted to the final concentrations as low as 0.1 µg/mL. This is because pure compounds are active compounds which are

After adding MTT solution, remember to wrap plate with aluminium foil because MTT

We usually perform the O.D. measurement at 595 nm. because of the limitation of our

 Rotate the cell culture plate at the rotator platform for 10 minutes is important to induce the homogeneous solution that will be easy for the O.D. measurement and to achieve

 The rapid flick-off of the medium after the MTT incubation will be done only in adherent cell lines such as breast cancer cell lines. For suspension cells such as leukemic cell lines, K562, the 100 µl of the stop solution (10% SDS dissolved in 0.01 N HCl) will be

 If dilute the herbal extract with DMSO, it is better to keep the percentage of DMSO in the final concentration not more than 0.5% to avoid cell damage and false positive

The number of the seeding cells used in our experiment is from the standard curve at

result. In our experience, 1% DMSO still can cause some effect on cell viability. Please remember to set the control wells (both positive and negative control). We usually use doxorubicin or vinca alkaloid for positive control. Only cells in the medium

We always perform the MTT assay with three independent experiments.

Fig. 8. The MTT assay showed the dark purple formazan products in the wells after

For MTT tetrazolium salt assay, the method measures the surviving cells after treatment with chemical substances. However, for the very low toxic reagents, calculation from the

**Special comments:**

instrument.

the correct data.

the O.D 0.7-1.2.

purified from crude extracts.

added into each well and incubated overnight.

will be used as negative control.

dissolving the crystal with 100 µl of 100% DMSO.

**Principle & mechanism of action**

**4.2.2 Lactate dehydrogenase (LDH) cytotoxicity assay**

substrate is light sensitive.

cannot distinquish between the cytotoxic and cytostatic effect of the tested drug or herb (Plumb, 2004).

Interestingly, MTT solution at 1 mg/mL increases the formazan product whereas MTT solution at 1-2 mg/mL will slow down the production (Sylvester, 2011). This is the reason why MTT solution is usually used at the concentration of 1 mg/mL. From the kinetics of formazan production, incubation time over 4 hours will get the plateau production curve. Therefore, standard incubation time at 3-4 hours is usually enough for the production time (Denizot and Lang, 1986).

As reported, the blue formazan products are partially dissolved in the medium. To induce complete solubilization of these MTT formazan products, several organic solvents have been tested and used such as ethanol, dimethylsulfoxide (DMSO), and isopropanol which is reported to be the most suitable solvent. After getting the homogeneous solution, optical density (OD) will be measured by reading on a scanning multiwell spectrophotometer (ELISA reader). The absorption spectrum of MTT formazan crystal is pH dependent. At pH 10.5, the maximum absorption is 570 nm. whereas, at pH 7.0, the absorption peak will show at 500 and 570 nm. (Plumb, 2004). The use of flat-bottomed well will help in increasing the sensitivity due to the doubled light-path length in the final optical density reading (Denizot and Lang, 1986).

In general, the accurate and reliable results from the MTT assay depend upon various important parameters which need to be optimized first. The proper cell number for seeding should be characterized by using standard curve. Changing into fresh medium prior to adding MTT solution is also critical in getting the good yield because of reduction of MTT activity in nutrient-depleted medium. Time and dose of MTT solution in each experiment may be different for each cell or cell lines used (Sylvester, 2011). Importantly, chemotherapeutic agents and some drugs such as valproic acid can yield the lower results from the MTT assay (Ari et al, 2010; Ulukaya et al, 2008). The experimental procedure is as followed (Kummalue et al, 2005).

## **Experimental procedure:**

percentage of viability = OD sample/OD control 100 (1)

percentage of inhibition = 100 - % viability

## **Special comments:**

254 Breast Cancer – Current and Alternative Therapeutic Modalities

cannot distinquish between the cytotoxic and cytostatic effect of the tested drug or herb

Interestingly, MTT solution at 1 mg/mL increases the formazan product whereas MTT solution at 1-2 mg/mL will slow down the production (Sylvester, 2011). This is the reason why MTT solution is usually used at the concentration of 1 mg/mL. From the kinetics of formazan production, incubation time over 4 hours will get the plateau production curve. Therefore, standard incubation time at 3-4 hours is usually enough for the production time

As reported, the blue formazan products are partially dissolved in the medium. To induce complete solubilization of these MTT formazan products, several organic solvents have been tested and used such as ethanol, dimethylsulfoxide (DMSO), and isopropanol which is reported to be the most suitable solvent. After getting the homogeneous solution, optical density (OD) will be measured by reading on a scanning multiwell spectrophotometer (ELISA reader). The absorption spectrum of MTT formazan crystal is pH dependent. At pH 10.5, the maximum absorption is 570 nm. whereas, at pH 7.0, the absorption peak will show at 500 and 570 nm. (Plumb, 2004). The use of flat-bottomed well will help in increasing the sensitivity due to the doubled light-path length in the final optical density reading (Denizot and Lang, 1986). In general, the accurate and reliable results from the MTT assay depend upon various important parameters which need to be optimized first. The proper cell number for seeding should be characterized by using standard curve. Changing into fresh medium prior to adding MTT solution is also critical in getting the good yield because of reduction of MTT activity in nutrient-depleted medium. Time and dose of MTT solution in each experiment may be different for each cell or cell lines used (Sylvester, 2011). Importantly, chemotherapeutic agents and some drugs such as valproic acid can yield the lower results from the MTT assay (Ari et al, 2010; Ulukaya et al, 2008). The experimental procedure is as

 Breast cancer cells will be seeded at 1x104 cells per well in 96 flat-bottomed well plate containing 100µl of the culture medium DMEM supplemented with 10% heat

 After 24 hours, the cells will be treated with herbal extract at final concentrations from 1, 10, 100, and 500 µg/mL for 48 hours. These serial concentrations are used for

After 48 hours incubation, medium will be changed into fresh medium, and MTT 50 µl

 After 4 hours incubation, the MTT solution will then be removed by rapid flick-off of the medium and 100 µl of 100% DMSO will be added to solubilize the formazan products. The dark purple solution from formazan products can be seen as shown in Figure 8. Plate will be rotated on the platform rotator for 10 minutes before further measurement

Calculation of ED50 (50% of inhibition of cancer cell growth) will be performed by using

percentage of inhibition = 100 - % viability

percentage of viability = OD sample/OD control 100 (1)

(stock concentration at 1mg/mL in PBS) will be added to each well.

(Plumb, 2004).

(Denizot and Lang, 1986).

followed (Kummalue et al, 2005).

inactivated fetal bovine serum and 1% antibiotic.

determining the activity in crude extracts only.

the O.D. by a microplate Elisa reader at 595 nm.

**Experimental procedure:**

the formula:

Fig. 8. The MTT assay showed the dark purple formazan products in the wells after dissolving the crystal with 100 µl of 100% DMSO.

## **4.2.2 Lactate dehydrogenase (LDH) cytotoxicity assay**

## **Principle & mechanism of action**

For MTT tetrazolium salt assay, the method measures the surviving cells after treatment with chemical substances. However, for the very low toxic reagents, calculation from the

Experimental Therapeutics in Breast Cancer Cells 257

solutions such as methylene blue, ethidium bromide, and crystal violet (Guda et al, 2007). Counting colony can be done by using manual counting with microscope or using the specific software (Niyazi et al, 2007). The experimental procedure is demonstrated belowed

 Breast cancer cell lines will be plated in 12 well plate at a density of 7x102 cells per well. The next morning, cells will be treated with the herbal extract at the concentrations of 0,

Colonies will be counted by using ChemiDocXRS with specific software. Figure 9

 The number of cells seeded in each well will be vary depending on the area of the well. The concentrations used in the experiment depend upon the ED50 value of each

Changing into fresh medium (after treated cells) before continue keeping cells in culture

 The time for culturing after exposure to the herbal extract depend upon the cell growth curve. For breast cancer cell lines, small colony can be detected after culturing cells for

Counting the colonies can be achieved by using only light microscope, no software use

We always perform the clonogenic assay with three independent experiments.

A B C

Fig. 9.  B. T47D treated with plant extract at 0.5 times ED50. No colony was detected which was due to the cytotoxicity of the extract. C. T47D treated with 0.5% DMSO as a control because the extract was diluted with 0.5% DMSO. Colonies could be seen as dots at the bottom of the wells after staining with crystal violet in this experiment and

After 48 hours incubation, culture medium will be changed into fresh medium.

Plates will be stained with 1 gm of crystal violet in 50% methanol for 30 minutes.

Cells will be further cultured for 7-14 days until colonies are observed.

Pour off the stain, carefully rinse with running water.

showed the colonies detected in our experiment.

is very important for cell growth and colony formation.

(but may be too difficult to count in some cases).

(Pratumvinit et al, 2009). **Experimental procedure:**

**Special comments:**

medicinal plant.

at least 7 days.

counted by using the software.

50, 100, and 150 µg/mL.

data of control cells and treated cells from MTT assay results in low sensitivity. In this case, direct measurement of dead cells will give more accurate information (Sasaki et al, 1992). Therefore, development of assay using cytolytic enzymes releasing from dead cells has been initiated which can represent proportionally to the dead cell number (Sasaki et al, 1992). Detection of lactate dehydrogenase enzyme, one of the stable cytolytic enzyme found in animal cells, has been established for this purpose (Decker and Lohmann-Matthes, 1988). Lactate dehydrogenase (LDH) enzyme has the function to catalyze lactate to pyruvate under anaerobic condition. It is a soluble enzyme in the cytoplasm. This enzyme will be released to the culture medium when the cell is ruptured or dead. Usually the assay will contain two steps. First, LDH releasing from the cells will oxidize lactate to generate NADH and H+. The second step, diaphorase in the reaction will use NADH and H+ to catalyze the tetrazolium salt such as WST-8, or INT to generate colored formazan which will absorbs strongly at wavelength 490-520 nm. The amount of the formazan production will correlate in the proportion to the damaged cells in the reaction (Haslam et al, 2000; Rodney et al, 1966). The experimental procedure is shown belowed (Kummalue et al, 2009; LDH-cytotoxicity assay kit with WST-8, MBL international corporation).

## **Experimental procedure:**

#### **Special comments:**

All MTT, WST-8, XTT, and INT are commonly used tetrazolium salts. They have different chemical structures and properties such as solubility. These properties depend on the substituents on the tetrazole rings in the compounds (Zhivich et al, 1990).

#### **4.2.3 Clonogenic assay**

#### **Principle & mechanism of action**

This method for cell sensitivity assay is considered as the gold standard technique and has been used for a long time (Plumb, 2004). It is a well known method for testing the effects of drugs on the proliferation of treated cell lines. This assay can measure the ability of cells to proliferate and form colonies after treating cells with chemical substances or herbal extracts (cytostatic effect of drug) whereas cells that are killed by the drugs cannot grow and form colonies (cytotoxic effect of drug). In addition, clonogenic assay is reported to be able to detect cell survival as low as 1% (Plumb, 2004).

Staining technique is a very important step because the accuracy of counting colonies depends on the good visualization. Colony staining has been performed with several dye solutions such as methylene blue, ethidium bromide, and crystal violet (Guda et al, 2007). Counting colony can be done by using manual counting with microscope or using the specific software (Niyazi et al, 2007). The experimental procedure is demonstrated belowed (Pratumvinit et al, 2009).

## **Experimental procedure:**

256 Breast Cancer – Current and Alternative Therapeutic Modalities

data of control cells and treated cells from MTT assay results in low sensitivity. In this case, direct measurement of dead cells will give more accurate information (Sasaki et al, 1992). Therefore, development of assay using cytolytic enzymes releasing from dead cells has been initiated which can represent proportionally to the dead cell number (Sasaki et al, 1992). Detection of lactate dehydrogenase enzyme, one of the stable cytolytic enzyme found in animal cells, has been established for this purpose (Decker and Lohmann-Matthes, 1988). Lactate dehydrogenase (LDH) enzyme has the function to catalyze lactate to pyruvate under anaerobic condition. It is a soluble enzyme in the cytoplasm. This enzyme will be released to the culture medium when the cell is ruptured or dead. Usually the assay will contain two steps. First, LDH releasing from the cells will oxidize lactate to generate NADH and H+. The second step, diaphorase in the reaction will use NADH and H+ to catalyze the tetrazolium salt such as WST-8, or INT to generate colored formazan which will absorbs strongly at wavelength 490-520 nm. The amount of the formazan production will correlate in the proportion to the damaged cells in the reaction (Haslam et al, 2000; Rodney et al, 1966). The experimental procedure is shown belowed (Kummalue et al, 2009; LDH-cytotoxicity assay

 Breast cancer cells will be seeded at a density of 1 x 104 cells per well in 96 well plates flat-bottom in 100 µl of culture medium DMEM supplemented with 10% heat

 The next morning, cells will be treated with the herbal extract at various final concentrations from 1 to 500 µg/mL for 48 hours in the incubator. These concentrations

 Transfer 10 µl of culture medium into the new 96 well plate with corresponding well. Add 100 µl of LDH reaction mix (containing LDH and WST-8) to 10 µl of tested culture medium and incubated at 37 ºC with a 5% CO2 incubator for an additional 30 minutes. The absorbance at 450 nm of the dissolved solution will be measured by using an Elisa

All MTT, WST-8, XTT, and INT are commonly used tetrazolium salts. They have different chemical structures and properties such as solubility. These properties depend on the

This method for cell sensitivity assay is considered as the gold standard technique and has been used for a long time (Plumb, 2004). It is a well known method for testing the effects of drugs on the proliferation of treated cell lines. This assay can measure the ability of cells to proliferate and form colonies after treating cells with chemical substances or herbal extracts (cytostatic effect of drug) whereas cells that are killed by the drugs cannot grow and form colonies (cytotoxic effect of drug). In addition, clonogenic assay is reported to be able to

Staining technique is a very important step because the accuracy of counting colonies depends on the good visualization. Colony staining has been performed with several dye

substituents on the tetrazole rings in the compounds (Zhivich et al, 1990).

kit with WST-8, MBL international corporation).

inactivated fetal bovine serum and 1% antibiotic.

are used to evaluate the ED50 for crude extracts only. Centrifuge the cells at 1500 rpm or 250 g for 10 minutes.

**Experimental procedure:**

plate reader. **Special comments:**

**4.2.3 Clonogenic assay**

**Principle & mechanism of action**

detect cell survival as low as 1% (Plumb, 2004).

## **Special comments:**

Fig. 9.  B. T47D treated with plant extract at 0.5 times ED50. No colony was detected which was due to the cytotoxicity of the extract. C. T47D treated with 0.5% DMSO as a control because the extract was diluted with 0.5% DMSO. Colonies could be seen as dots at the bottom of the wells after staining with crystal violet in this experiment and counted by using the software.

Experimental Therapeutics in Breast Cancer Cells 259

The incubation time for cell cycle study depends on the time that is used for cell

Dosage used in the experiment to investigate the effect of plant on cell cycle depends

Apoptosis or programmed cell death is a very interesting investigation in drug discovery and development especially for cancer therapies. Searching for the anticancer agents that can induce apoptosis is necessary and needed for the effective treatment. For example, tamoxifen, which is prescribed as an adjuvant therapy in breast cancer, has been shown to induce apoptosis by down regulation of bcl-2 (Zhang et al, 1999). Therefore, most of medicinal plant researchers keep looking on finding these properties of plants. By the way,

Apoptosis is a process that organisms, including human beings, use to tightly regulate the cell numbers and tissue size (Hengartner, 2000). It is caused by a group of cysteine proteases known as caspases. There are 2 major apoptotic pathways. One is via death receptor pathway which is triggered by death receptor superfamily such as CD95 and also tumor necrosis factor receptor I. This results in activation of caspase 8. The other pathway is mitochondrial pathway. This activates pro-apoptotic member of the Bcl-2 family and then pro caspase 9. Both pathways converge at the caspase 3 activation (Hengartner, 2000). To evaluate the apoptotic activity of plant extracts, caspase activity is usually investigated either by detection at the level of the enzyme caspase itself or by the level of its cleavage product, poly (ADP-ribose) polymerase-1 (PARP-1) (Los et al, 2002;

In summary, characterization of apoptosis is chromatin condensation, fragmentation of DNA (seen as ladder pattern in gel electrophoresis), and reduction of cell volume. Externalization of phosphatidylserine at the cell surface also plays an important role in macrophages recognition and consequently eradication of these apoptotic cells (Koopman et al, 1994). With these specific expression of phosphatidylserine, the new method was developed for detection of apoptotic cells by flow cytometry and has been frequently used nowadays. (Koopman et al, 1994). The experimental procedure is shown belowed.

Cells will be seeded on 100 cm tissue culture dish at the density of 1x106 cells.

concentrations ranging from 0.5 times ED50, to 2 times ED50.

used to treat cells depend on the efficacy of the plant extract itself.

After 24 hours, cells will be treated with plant extracts at the various final

After incubating cells for various time points, cells will be harvested. The time points

 Cells will be labeled with Annexin V-PI following the protocol of Annexin V-FITC Apoptosis Detection Kit from the manufacture and analysis by flow cytometry.

Detection of early and late apoptosis using annexin V depends upon the doses and time

**4.3.2 Breast cancer cells and experimental procedure in apoptotic study**

**Special comments:**

sensitivity assay.

Kummalue et al, 2011).

(Kummalue et al, 2011) **Experimental procedure:**

**Special comments:**

that treating the cells.

upon the ED50 of the plant extract.

high cost and time consumption are unavoidable to face.

## **4.3 Breast cancer cells and experimental procedures in mechanism of actions**

This is usually the next step of investigation in searching for the new drug from plant sources. Plant extracts that exhibit the promising ED50 values, i.e., the plant extracts show some antiproliferative or cytotoxic activities on specific breast cancer cell lines, the extracts will be further studied to find the mechanism of actions in view of inhibition of cancer cells growth or even killing cells. Cell cycle analysis and apoptotic study are the most two common methods used in the field for searching the mechanism of actions in plants.

## **4.3.1 Breast cancer cells and experimental procedure in cell cycle study**

Based on the knowledge of normal cell cycle, breast cancer cells actively synthesize DNA in the same process (Ross et al, 2003). Normally, the cell cycle phase contains G0, G1, S, G2, and M phase in order. Cells in the G0 phase or resting state contain diploid DNA which will enter the second step G1 (Gap 1). After this step, cells increase their DNA content to two folds with twice the diploid DNA content at the end. This accumulation state of DNA is S phase. Cells then enter the G2 phase (Gap 2) and finally go into the M phase, while M means the mitotic state. Finally, each mitotic cell will end up with 2 daughter cells.

In breast cancer cells, DNA ploidy has been reported to correlate with the prognosis in patients. The first retrospective report using flow cytometry to study DNA content as a predictor in breast cancer was done by Auer et al with pure diploid showing an excellent prognosis (Auer et al, 1980). However, the use of DNA ploidy as an prognostic indicator in clinical practice still remains in controversy (Ross et al, 2003). Estrogen has also been reported to induce breast cancer which might due to the inappropriate activation of cyclin dependent kinase and consequently abnormal transition through G1 phase (Foster et al, 2001).

The most popular technique used for demonstration DNA content in the cells is by flow cytometry (Nunez, 2001). Four distinct phases could be categorized, i.e., G1, G2, S, and M phases with G2 and M phase will represent at the same DNA content (so called G2/M phase). The staining techniques to distinquish these cell cycle phases are the measurement of the incorporation of bromodeoxyuridine (BrdU) and propidium iodide (Kummalue et al, 2002; Pratumvinit et al, 2009). Interference from RNA may result in misinterpretation as false positive. Therefore, the use of RNase should be added in the protocol. The experiment in breast cancer demonstrated here will share the experience in using the cell cycle kit which is much easier than the traditional technique. (The details of cell cycle study using BrdU incorporation and propidium iodide can be found in the references as mentioned in the text.) Belowed is the experimental procedure (Kummalue et al, 2007).

#### **Experimental procedure:**

## **Special comments:**

258 Breast Cancer – Current and Alternative Therapeutic Modalities

This is usually the next step of investigation in searching for the new drug from plant sources. Plant extracts that exhibit the promising ED50 values, i.e., the plant extracts show some antiproliferative or cytotoxic activities on specific breast cancer cell lines, the extracts will be further studied to find the mechanism of actions in view of inhibition of cancer cells growth or even killing cells. Cell cycle analysis and apoptotic study are the most two

Based on the knowledge of normal cell cycle, breast cancer cells actively synthesize DNA in the same process (Ross et al, 2003). Normally, the cell cycle phase contains G0, G1, S, G2, and M phase in order. Cells in the G0 phase or resting state contain diploid DNA which will enter the second step G1 (Gap 1). After this step, cells increase their DNA content to two folds with twice the diploid DNA content at the end. This accumulation state of DNA is S phase. Cells then enter the G2 phase (Gap 2) and finally go into the M phase, while M means

In breast cancer cells, DNA ploidy has been reported to correlate with the prognosis in patients. The first retrospective report using flow cytometry to study DNA content as a predictor in breast cancer was done by Auer et al with pure diploid showing an excellent prognosis (Auer et al, 1980). However, the use of DNA ploidy as an prognostic indicator in clinical practice still remains in controversy (Ross et al, 2003). Estrogen has also been reported to induce breast cancer which might due to the inappropriate activation of cyclin dependent kinase and consequently abnormal transition through G1 phase (Foster et al,

The most popular technique used for demonstration DNA content in the cells is by flow cytometry (Nunez, 2001). Four distinct phases could be categorized, i.e., G1, G2, S, and M phases with G2 and M phase will represent at the same DNA content (so called G2/M phase). The staining techniques to distinquish these cell cycle phases are the measurement of the incorporation of bromodeoxyuridine (BrdU) and propidium iodide (Kummalue et al, 2002; Pratumvinit et al, 2009). Interference from RNA may result in misinterpretation as false positive. Therefore, the use of RNase should be added in the protocol. The experiment in breast cancer demonstrated here will share the experience in using the cell cycle kit which is much easier than the traditional technique. (The details of cell cycle study using BrdU incorporation and propidium iodide can be found in the references as mentioned in the

Breast cancer cells will be seeded at 1x106 cells in 100 mm tissue culture dish and

The next morning, cells will be treated with the plant extract at the dosage ranging from

 After 48 hours incubation, cells will be harvested and incubated with reagents as described in the protocol of the CycleTEST™PLUS DNA reagent kit (Becton Dickinson,

Measurement of DNA content of cells will be performed within 3 hours by flow

**4.3 Breast cancer cells and experimental procedures in mechanism of actions**

common methods used in the field for searching the mechanism of actions in plants.

**4.3.1 Breast cancer cells and experimental procedure in cell cycle study**

the mitotic state. Finally, each mitotic cell will end up with 2 daughter cells.

text.) Belowed is the experimental procedure (Kummalue et al, 2007).

2001).

**Experimental procedure:**

USA).

incubated overnight.

0.5 times ED50 to 2 times ED50.

cytometry and analysed by CellQuest Software.

## **4.3.2 Breast cancer cells and experimental procedure in apoptotic study**

Apoptosis or programmed cell death is a very interesting investigation in drug discovery and development especially for cancer therapies. Searching for the anticancer agents that can induce apoptosis is necessary and needed for the effective treatment. For example, tamoxifen, which is prescribed as an adjuvant therapy in breast cancer, has been shown to induce apoptosis by down regulation of bcl-2 (Zhang et al, 1999). Therefore, most of medicinal plant researchers keep looking on finding these properties of plants. By the way, high cost and time consumption are unavoidable to face.

Apoptosis is a process that organisms, including human beings, use to tightly regulate the cell numbers and tissue size (Hengartner, 2000). It is caused by a group of cysteine proteases known as caspases. There are 2 major apoptotic pathways. One is via death receptor pathway which is triggered by death receptor superfamily such as CD95 and also tumor necrosis factor receptor I. This results in activation of caspase 8. The other pathway is mitochondrial pathway. This activates pro-apoptotic member of the Bcl-2 family and then pro caspase 9. Both pathways converge at the caspase 3 activation (Hengartner, 2000). To evaluate the apoptotic activity of plant extracts, caspase activity is usually investigated either by detection at the level of the enzyme caspase itself or by the level of its cleavage product, poly (ADP-ribose) polymerase-1 (PARP-1) (Los et al, 2002; Kummalue et al, 2011).

In summary, characterization of apoptosis is chromatin condensation, fragmentation of DNA (seen as ladder pattern in gel electrophoresis), and reduction of cell volume. Externalization of phosphatidylserine at the cell surface also plays an important role in macrophages recognition and consequently eradication of these apoptotic cells (Koopman et al, 1994). With these specific expression of phosphatidylserine, the new method was developed for detection of apoptotic cells by flow cytometry and has been frequently used nowadays. (Koopman et al, 1994). The experimental procedure is shown belowed. (Kummalue et al, 2011)

#### **Experimental procedure:**

#### **Special comments:**

Detection of early and late apoptosis using annexin V depends upon the doses and time that treating the cells.

Experimental Therapeutics in Breast Cancer Cells 261

the library for human cancer that will represent the genetic and epigenetic variation of the population (Chun et al, 2010). In addition, since iPSs grow in culture like cell lines, therefore,

The investigation of the traditional used and evidence-based medicinal plants provides the chance to discover natural anticancer agents. The breast cancer cell lines with specific properties and the progress in antiproliferative and cytotoxic experiments are the important tools for the investigation of the medicinal plants. Clear differences in specific properties including receptors of these 4 breast cancer cell lines have been demonstrated so far. Comparing specific receptors, i.e., estrogen receptor, progesterone receptor, and Her2/neu in these 4 breast cancer cell lines: SKBR3, MCF7, T47D, and MDA-MB435 are demonstrated in Table 2. These 4 breast cancer cell lines are classified in different groups and express different receptors. Therefore, treatment of plant extracts on these 4 breast cancer cell lines can help, at least, in the decision whether or not the extracts will exert activities in breast cancer with different subtypes. However, the established cell lines from patients do not always represent the genotypes of parental tumor tissues (Tsuji et al, 2010). Therefore, testing in cell lines may not yield the same results when performing in vivo though most of these cells deregulate in signaling along the EgfR-MAPK pathway (Heiser et al, 2009).

**Characters SKBR3 MCF7 T47D MDA-MB435**  Category Second group First group First group Third group Estrogen receptor - + + -

Her-2/neu overexpression - - -

Table 2. Characters of receptors on four breast cancer cell lines: SKBR3, MCF7, T47D, and MDA-MB435 (http://www.cell-lines-service; Lacroix and Leclercq, 2004; Hevir et al, 2011). Searching for the novel chemotherapeutic agents from plant sources usually involves with the initial process of antiproliferative activity. These three non-radioactive assays are commonly used nowadays in the determination of antiproliferative and cytotoxic activities. Several aspects from them are summarized in Table 3, i.e., sensitivity, cost and time consuming for each assay. Notably, each assay has its own advantages and disadvantages

Prediction of toxicology and therapeutic responses induced by the novel drugs can be approached by iPS technology as mentioned aboved. Lots of current disease specific based patients iPS have been reported since then (Chun et al, 2010). This application as a personalized approach leads to the specialized model in vitro. However, the safety and risk in applying to the clinical trials should be aggressively considered because of the techniques

The elucidation of the action mechanism provides the concrete evidence of the study plant. The utilization of herbs as natural anticancer agents can solve the problem of the

this technology can provide the unlimited sources of cells for desired situation.

**6. Discussion and conclusion**

Progesterone receptor

(-) = not expressed; (+) = expressed

which should be carefully considered.

in generation of iPS with viral gene transfer.

## **4.3.3 Breast cancer cells and specific mechanism of action of the plant extracts**

Elucidation of the mechanism of action will greatly help in understanding the activity from plant sources on these breast cancer cell lines. Following the cell cycle and apoptotic studies as the basis, the mechanisms of these activities such as telomerase activity, antioxidant activity, and details of special signaling pathways are always considered for studying.

Telomerase is a eukaryotic ribonucleoprotein complex that helps in stabilizing telomere length in human stem cells, reproductive cells, and cancer cells (Shay and Bacchetti, 1997; Shay et al, 2001). The activity of telomerase increases in almost all human cancers except for the normal cells (Shay et al, 2001;Kim et al, 1994). Based on several studies, there are correlations between telomere shortening and growth failure of human cells (Harley et al, 1990; Shay et al, 2001). Therefore, human cancer cells show to have short telomeres with high telomerase, which is in contrast to normal human cells. Notably, cancer cells need to maintain telomeres for their immortalization. Based on this correlation, investigations of human cancer on telomerase activity have been done for over decades due to the potential development in drug targeting cancers.

Human telomerase consists of a catalytic protein component (hTERT) and integral RNA which are essential for the function of telomerase (Shay et al, 2001). hTERT is reported to be the critical for the production of telomerase (Nakamura et al, 1997). Investigation on telomerase activity can usually perform by detection the hTERT expression using RT-PCR. RNA extraction from treated breast cancer cells will be done before further processing to quantitate the level of transcripts. The method can be undertaken by real time RT-PCR and specific software for interpretation (Duangmano et al, 2010).

## **5. Breast cancer cells in medicinal plants research: Future direction in drug testing**

Generating of induced pluripotent stem cells (iPS) for the first time in the world by Professor Dr. Shinya Yamanaka in Year 2006 has very high impact on several fields such as stem cell therapies, regenerative medicine, replacement therapy, and also drug screening and toxicity (Inoue and Yamanaka, 2011). Researchers worldwide are very interested in this iPS technology because of the high possibility in its usefulness and application to human diseases.

Induced pluripotent stem cells or iPS has first been generated from mouse tip tail fibroblasts which were somatic cells (Takahashi and Yamanaka, 2006). The process involved in transduction of fibroblasts with retrovirus carrying 4 transcription factors, i.e., Sox2, Klf4, c-Myc, and Oct3/4. These four factors are now known as Yamanaka's four factors. iPS cells exhibit several features characteristics of embryonic stem cells such as positive for alkaline phosphatase, forming teratoma in mice, and expression of specific embryonic antigen (Park et al, 2008). Nowadays, iPSs have been generated from various kinds of animals cells such as monkey, mouse, etc., including normal human cells, human cancer cell lines, and patients' cells to generate the patient-specific iPSs (Chun et al, 2010; Raya et al, 2009).

In drug screening and toxicity testing, human iPSs offer the high values in this field. For example, breast cancer patients' fibroblasts can be induced with transcription factors either 2 or 4 factors to create patient-specific iPS. Based on the fact that the iPS cells are generated from patients' own somatic cells, therefore, these specific iPS cells can be used to identify and test with the novel drug developing for cancer patients including the toxic effects of the drugs themselves (Chun et al, 2010). Interestingly, the advantage of iPS is the generation of the library for human cancer that will represent the genetic and epigenetic variation of the population (Chun et al, 2010). In addition, since iPSs grow in culture like cell lines, therefore, this technology can provide the unlimited sources of cells for desired situation.

## **6. Discussion and conclusion**

260 Breast Cancer – Current and Alternative Therapeutic Modalities

Human telomerase consists of a catalytic protein component (hTERT) and integral RNA which are essential for the function of telomerase (Shay et al, 2001). hTERT is reported to be the critical for the production of telomerase (Nakamura et al, 1997). Investigation on telomerase activity can usually perform by detection the hTERT expression using RT-PCR. RNA extraction from treated breast cancer cells will be done before further processing to quantitate the level of transcripts. The method can be undertaken by real time RT-PCR and

**5. Breast cancer cells in medicinal plants research: Future direction in drug**

Generating of induced pluripotent stem cells (iPS) for the first time in the world by Professor Dr. Shinya Yamanaka in Year 2006 has very high impact on several fields such as stem cell therapies, regenerative medicine, replacement therapy, and also drug screening and toxicity (Inoue and Yamanaka, 2011). Researchers worldwide are very interested in this iPS technology because of the high possibility in its usefulness and application to human

Induced pluripotent stem cells or iPS has first been generated from mouse tip tail fibroblasts which were somatic cells (Takahashi and Yamanaka, 2006). The process involved in transduction of fibroblasts with retrovirus carrying 4 transcription factors, i.e., Sox2, Klf4, c-Myc, and Oct3/4. These four factors are now known as Yamanaka's four factors. iPS cells exhibit several features characteristics of embryonic stem cells such as positive for alkaline phosphatase, forming teratoma in mice, and expression of specific embryonic antigen (Park et al, 2008). Nowadays, iPSs have been generated from various kinds of animals cells such as monkey, mouse, etc., including normal human cells, human cancer cell lines, and patients'

In drug screening and toxicity testing, human iPSs offer the high values in this field. For example, breast cancer patients' fibroblasts can be induced with transcription factors either 2 or 4 factors to create patient-specific iPS. Based on the fact that the iPS cells are generated from patients' own somatic cells, therefore, these specific iPS cells can be used to identify and test with the novel drug developing for cancer patients including the toxic effects of the drugs themselves (Chun et al, 2010). Interestingly, the advantage of iPS is the generation of

cells to generate the patient-specific iPSs (Chun et al, 2010; Raya et al, 2009).

**4.3.3 Breast cancer cells and specific mechanism of action of the plant extracts**  Elucidation of the mechanism of action will greatly help in understanding the activity from plant sources on these breast cancer cell lines. Following the cell cycle and apoptotic studies as the basis, the mechanisms of these activities such as telomerase activity, antioxidant activity, and details of special signaling pathways are always considered for studying. Telomerase is a eukaryotic ribonucleoprotein complex that helps in stabilizing telomere length in human stem cells, reproductive cells, and cancer cells (Shay and Bacchetti, 1997; Shay et al, 2001). The activity of telomerase increases in almost all human cancers except for the normal cells (Shay et al, 2001;Kim et al, 1994). Based on several studies, there are correlations between telomere shortening and growth failure of human cells (Harley et al, 1990; Shay et al, 2001). Therefore, human cancer cells show to have short telomeres with high telomerase, which is in contrast to normal human cells. Notably, cancer cells need to maintain telomeres for their immortalization. Based on this correlation, investigations of human cancer on telomerase activity have been done for over decades due to the potential

development in drug targeting cancers.

**testing**

diseases.

specific software for interpretation (Duangmano et al, 2010).

The investigation of the traditional used and evidence-based medicinal plants provides the chance to discover natural anticancer agents. The breast cancer cell lines with specific properties and the progress in antiproliferative and cytotoxic experiments are the important tools for the investigation of the medicinal plants. Clear differences in specific properties including receptors of these 4 breast cancer cell lines have been demonstrated so far. Comparing specific receptors, i.e., estrogen receptor, progesterone receptor, and Her2/neu in these 4 breast cancer cell lines: SKBR3, MCF7, T47D, and MDA-MB435 are demonstrated in Table 2. These 4 breast cancer cell lines are classified in different groups and express different receptors. Therefore, treatment of plant extracts on these 4 breast cancer cell lines can help, at least, in the decision whether or not the extracts will exert activities in breast cancer with different subtypes. However, the established cell lines from patients do not always represent the genotypes of parental tumor tissues (Tsuji et al, 2010). Therefore, testing in cell lines may not yield the same results when performing in vivo though most of these cells deregulate in signaling along the EgfR-MAPK pathway (Heiser et al, 2009).

(-) = not expressed; (+) = expressed

Table 2. Characters of receptors on four breast cancer cell lines: SKBR3, MCF7, T47D, and MDA-MB435 (http://www.cell-lines-service; Lacroix and Leclercq, 2004; Hevir et al, 2011).

Searching for the novel chemotherapeutic agents from plant sources usually involves with the initial process of antiproliferative activity. These three non-radioactive assays are commonly used nowadays in the determination of antiproliferative and cytotoxic activities. Several aspects from them are summarized in Table 3, i.e., sensitivity, cost and time consuming for each assay. Notably, each assay has its own advantages and disadvantages which should be carefully considered.

Prediction of toxicology and therapeutic responses induced by the novel drugs can be approached by iPS technology as mentioned aboved. Lots of current disease specific based patients iPS have been reported since then (Chun et al, 2010). This application as a personalized approach leads to the specialized model in vitro. However, the safety and risk in applying to the clinical trials should be aggressively considered because of the techniques in generation of iPS with viral gene transfer.

The elucidation of the action mechanism provides the concrete evidence of the study plant. The utilization of herbs as natural anticancer agents can solve the problem of the

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*F. hispida* and *E. elliptilimba* are interesting herbs for further investigation. It was also noticeable that herbal remedy could potentiate the effect of anticancer drug.

## **7. References**

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+

+

+++

**WST-8**

+++

++

+

Table 3. Comparison of three non-radioactive methods for assaying the antiproliferative activities (Kosaka et al, 1996; Kawada et al, 2002; Fotakis and Timbrell, 2006; Miyamoto et al,

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**13**

*U.S.A.*

**Red American Ginseng and Breast Cancer**

*Tang Center for Herbal Medicine Research and Department of Anesthesia & Critical Care,*

Breast cancer is the most frequently diagnosed form of cancer among women. Breast cancer is the leading cause of cancer death in nearly all developed countries, showing an increased incidence over the last decades, and the expected number of new U.S. patients in 2010 would be 209,060, and the death number will be 40,230 1. The clinical management of breast cancer invariably involves diverse conventional modalities, including surgery, chemotherapy, and radiation 2. The complex characteristics of breast cancer may also require some alternative management to improve the therapeutic efficacy of conventional treatment

American ginseng (*Panax quinquefolius* L.) is a commonly used herb in the United States which belongs to the genus *Panax* L. in family Araliaceae 4. *Panax* is a small genus, and nearly all species in this genus are important herbal medicines, especially Asian ginseng (*Panax ginseng* C. A. Meyer) 5. Since Asian ginseng has been used as herbal medicine for a long history in oriental countries, many studies have been conducted on its constituents and its pharmacological effects 6. Asian ginseng has many reported health benefits including anticancer activities 7-9. In the 1990s, a case-control study on over a thousand Korean subjects showed that long-term ginseng consumption was associated with a decreased risk for many different cancers compared with those who did not consume ginseng 10,11. In contrast to many studies on Asian ginseng's anticancer effects, investigation of American ginseng is

American ginseng extracts were found to inhibit the growth of breast cancer cells12. We previously investigated the effects of several herbal extracts on reducing chemotherapeutic side effects and found that American ginseng can attenuate cisplatin-induced nausea and vomiting in a rat model, while not affecting its anticancer properties in human cancer cells13. In addition, the extract from American ginseng enhanced the antiproliferation effect of cisplatin

In Asia, ginseng root is air-dried into white ginseng or steamed at 100 °C to give red ginseng. It is believed that red ginseng is more pharmacologically effective than white ginseng 14. The differences in the biological effects of white and red Asian ginseng are attributed to the significant changes in ginsenosides from the steaming treatment 15. In this study, we treated American ginseng root at various temperatures and heating times. After heat-processing, the root of American ginseng, like Asian ginseng, changes from white to

on human breast cancer cells, suggesting that it possesses its own anticancer activity13.

red; steamed *P. quinquefolius* root is therefore referred to as red American ginseng.

**1. Introduction**

and the quality of life for cancer patients 3.

limited 7, and its mechanisms of action are largely unknown.

Chong-Zhi Wang, Guang-Jian Du and Chun-Su Yuan

*The Pritzker School of Medicine, University of Chicago, Chicago,*

## **Red American Ginseng and Breast Cancer**

Chong-Zhi Wang, Guang-Jian Du and Chun-Su Yuan

*Tang Center for Herbal Medicine Research and Department of Anesthesia & Critical Care, The Pritzker School of Medicine, University of Chicago, Chicago, U.S.A.*

## **1. Introduction**

268 Breast Cancer – Current and Alternative Therapeutic Modalities

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Breast cancer is the most frequently diagnosed form of cancer among women. Breast cancer is the leading cause of cancer death in nearly all developed countries, showing an increased incidence over the last decades, and the expected number of new U.S. patients in 2010 would be 209,060, and the death number will be 40,230 1. The clinical management of breast cancer invariably involves diverse conventional modalities, including surgery, chemotherapy, and radiation 2. The complex characteristics of breast cancer may also require some alternative management to improve the therapeutic efficacy of conventional treatment and the quality of life for cancer patients 3.

American ginseng (*Panax quinquefolius* L.) is a commonly used herb in the United States which belongs to the genus *Panax* L. in family Araliaceae 4. *Panax* is a small genus, and nearly all species in this genus are important herbal medicines, especially Asian ginseng (*Panax ginseng* C. A. Meyer) 5. Since Asian ginseng has been used as herbal medicine for a long history in oriental countries, many studies have been conducted on its constituents and its pharmacological effects 6. Asian ginseng has many reported health benefits including anticancer activities 7-9. In the 1990s, a case-control study on over a thousand Korean subjects showed that long-term ginseng consumption was associated with a decreased risk for many different cancers compared with those who did not consume ginseng 10,11. In contrast to many studies on Asian ginseng's anticancer effects, investigation of American ginseng is limited 7, and its mechanisms of action are largely unknown.

American ginseng extracts were found to inhibit the growth of breast cancer cells12. We previously investigated the effects of several herbal extracts on reducing chemotherapeutic side effects and found that American ginseng can attenuate cisplatin-induced nausea and vomiting in a rat model, while not affecting its anticancer properties in human cancer cells13. In addition, the extract from American ginseng enhanced the antiproliferation effect of cisplatin on human breast cancer cells, suggesting that it possesses its own anticancer activity13.

In Asia, ginseng root is air-dried into white ginseng or steamed at 100 °C to give red ginseng. It is believed that red ginseng is more pharmacologically effective than white ginseng 14. The differences in the biological effects of white and red Asian ginseng are attributed to the significant changes in ginsenosides from the steaming treatment 15. In this study, we treated American ginseng root at various temperatures and heating times. After heat-processing, the root of American ginseng, like Asian ginseng, changes from white to red; steamed *P. quinquefolius* root is therefore referred to as red American ginseng.

Red American Ginseng and Breast Cancer 271

The HPLC system was a Waters 2960 instrument (Milford, MA) with a quaternary pump, an automatic injector, a photodiode array detector (Model 996), and Waters Millennium 32 software for peak identification and integration. The separation was carried out on a 250×3.2 mm i.d., 5μ, Ultrasphere C18 column (Alltech, Deerfield, IL) with a 7.5×3.2 mm i.d. guard column. For HPLC analysis, a 20-μL sample was injected into the column and eluted at room temperature with a constant flow rate of 1.0 ml/min. For the mobile phase, acetonitrile (solvent A) and water (solvent B) were used. Gradient elution started with 18% solvent A and 82% solvent B. Elution was changed to 21% A for 20 min, then to 26% A for 3 min and held for 19 min. It was then changed to 36% A for 13 min, to 50% A for 9 min, to 95% A for 2 min, and held for 3 min. Lastly elution was changed to 18% A for 3 min and held for 8 min. The detection wavelength was set to 202 nm. All tested solutions were filtered through Millex 0.2-μm nylon membrane syringe filters (Millipore Co., Bedford, MA) before use. The linearity of this method was assayed by analyzing standard solutions in the range of 2-400 μg/ml for the 12 ginsenosides. Calibration curves were constructed from the measured peak areas and the related amount of ginsenosides. Ginsenosides Rb1, Rb2, Rb3, Rc, Rd, Re, Rg1, Rg2, 20*R*-Rg2, Rg3, Rh1 and Rh2 (Figure **1**) in extract samples were identified by comparison of their retention times with those obtained from the chromatograms of mixed ginsenoside standards. The contents of saponins in each sample were calculated using standard curves

**OH**

**R2**

Fig. 1. Chemical structures of assayed ginsenosides in American ginseng.

**R3O**

**20(S)**

**Name R1 R2 R3**  Ginsenoside Rb1 -glc2- 1glc -H 20(S), -glc6- 1glc Ginsenoside Rb2 -glc2- 1glc -H 20(S), -glc6- 1ara(pyr) Ginsenoside Rb3 -glc2- 1glc -H 20(S), -glc6- 1xyl Ginsenoside Rc -glc2- 1glc -H 20(S), -glc6- 1ara(fur) Ginsenoside Rd -glc2- 1glc -H 20(S), -glc Ginsenoside Re -H -O-glc2- 1rha 20(S), -glc Ginsenoside Rg1 -H -O-glc 20(S), -glc Ginsenoside Rg2 -H -O-glc2- 1rha 20(S), -H 20R-ginsenoside Rg2 -H -O-glc2- 1rha 20(R), -H Ginsenoside Rg3 -glc2- 1glc -H 20(S), -H Ginsenoside Rh1 -H -O-glc 20(S), -H Ginsenoside Rh2 -glc -H 20(S), -H

**OR3 20(R)**

**2.3 HPLC analysis**

of ginsenosides.

**R1O**

Currently, the most popular herbal products are in extract forms. New regulations released by FDA on alternative complementary supplements require the botanical extracts to be standardized 16. The contents of ginsenosides in the extracts were not assayed before. We treated American ginseng root at various temperatures and heating times. The contents of representative ginsenosides in unsteamed and steamed American ginseng root extracts were determined using HPLC. In addition to the cell line MCF-7, we also used the MDA-MB-230 human breast cancer cells to evaluate the chemopreventive activities of unsteamed and steamed American ginseng root extracts and four ginsenosides. To observe the anticancer mechanisms, the effects of American ginseng extracts and single compounds on apoptosis and cell cycle were evaluated.

## **2. Materials and methods**

## **2.1 Chemicals**

All solvents were of high-performance liquid chromatography (HPLC) grade from Fisher Scientific (Norcross, GA). Milli Q water was supplied by a water purification system (US Filter, Palm Desert, CA). Standards for ginsenosides Rb1, Rb2, Rc, Rd, Re, and Rg1 were obtained from Indofine Chemical Company (Somerville, NJ); ginsenosides Rb3, Rg2, Rg3, Rh1, Rh2 and 20*(R)*-ginsenoside Rg2 (20*R*-Rg2) were obtained from the Delta Information Center for Natural Organic Compounds (Xuancheng, Anhui, China). All standards were of biochemical-reagent grade and at least 95% pure as confirmed by HPLC. All the plastic materials were purchased from Falcon Labware (Franklin Lakes, NJ). Trypsin, RPMI 1640 medium, fetal bovine serum (FBS), and penicillin/streptomycin solution (200×) were obtained from Mediatech, Inc. (Herndon, VA). A CellTiter 96 Aqueous One Solution Cell Proliferation Assay kit was obtained from Promega (Madison, WI). Annexin V-FITC, cyclin A-FITC and cyclin D1-FITC were obtained from BD Biosciences (San Diego, CA).

#### **2.2 Plant materials, processing and extraction**

American ginseng roots (*Panax quinquefolius* L.) were obtained from Roland Ginseng, LLC (Wausau, WI). The voucher samples were deposited at the Tang Center for Herbal Medicine Research at University of Chicago (Chicago, IL). For the heat-processing of American ginseng, the roots were steamed at 100 °C and 120 °C for 1 h or at 120 °C for 0.5, 1, 2, 3, and 4 h.

For the HPLC analysis, dried American ginseng sample (0.5 g) was extracted with methanol in a Soxhlet extractor for 8 h. The extract was concentrated, transferred into a 25-ml volumetric flask and diluted to the desired volume with methanol. One millimeter of the solution was purified by solid-phase extraction 17. Purified solutions were stored at 4 °C until HPLC analysis.

For the *in vitro* anti-cancer studies, the process for the extraction of American ginseng samples was as follows: A root sample, unsteamed or steamed at various temperatures and times, was ground to powder and passed through a 40 mesh screen. Then 25 g of powder was extracted with 500 ml of 70% ethanol for 4 h; the water bath was maintained at 90 °C. When cooled, the solution was filtered with P8 filter paper (Fisher Scientific, Pittsburgh, PA) and the filtrate was collected. The residue was extracted with 500 ml of 70% ethanol once more and then filtered while the solution was cooled. The filtrates were combined and the solvent was evaporated under vacuum. The dried extract was dissolved in 100 ml water and then extracted with water-saturated *n*-butanol. The *n*-butanol phase was evaporated under vacuum and then lyophilized.

The root of *P. quinquefolius* L. was collected from Roland Ginseng, LLC (Wausau, WI, USA).

#### **2.3 HPLC analysis**

270 Breast Cancer – Current and Alternative Therapeutic Modalities

Currently, the most popular herbal products are in extract forms. New regulations released by FDA on alternative complementary supplements require the botanical extracts to be standardized 16. The contents of ginsenosides in the extracts were not assayed before. We treated American ginseng root at various temperatures and heating times. The contents of representative ginsenosides in unsteamed and steamed American ginseng root extracts were determined using HPLC. In addition to the cell line MCF-7, we also used the MDA-MB-230 human breast cancer cells to evaluate the chemopreventive activities of unsteamed and steamed American ginseng root extracts and four ginsenosides. To observe the anticancer mechanisms, the effects of American ginseng extracts and single compounds on apoptosis

All solvents were of high-performance liquid chromatography (HPLC) grade from Fisher Scientific (Norcross, GA). Milli Q water was supplied by a water purification system (US Filter, Palm Desert, CA). Standards for ginsenosides Rb1, Rb2, Rc, Rd, Re, and Rg1 were obtained from Indofine Chemical Company (Somerville, NJ); ginsenosides Rb3, Rg2, Rg3, Rh1, Rh2 and 20*(R)*-ginsenoside Rg2 (20*R*-Rg2) were obtained from the Delta Information Center for Natural Organic Compounds (Xuancheng, Anhui, China). All standards were of biochemical-reagent grade and at least 95% pure as confirmed by HPLC. All the plastic materials were purchased from Falcon Labware (Franklin Lakes, NJ). Trypsin, RPMI 1640 medium, fetal bovine serum (FBS), and penicillin/streptomycin solution (200×) were obtained from Mediatech, Inc. (Herndon, VA). A CellTiter 96 Aqueous One Solution Cell Proliferation Assay kit was obtained from Promega (Madison, WI). Annexin V-FITC, cyclin

A-FITC and cyclin D1-FITC were obtained from BD Biosciences (San Diego, CA).

the roots were steamed at 100 °C and 120 °C for 1 h or at 120 °C for 0.5, 1, 2, 3, and 4 h.

American ginseng roots (*Panax quinquefolius* L.) were obtained from Roland Ginseng, LLC (Wausau, WI). The voucher samples were deposited at the Tang Center for Herbal Medicine Research at University of Chicago (Chicago, IL). For the heat-processing of American ginseng,

For the HPLC analysis, dried American ginseng sample (0.5 g) was extracted with methanol in a Soxhlet extractor for 8 h. The extract was concentrated, transferred into a 25-ml volumetric flask and diluted to the desired volume with methanol. One millimeter of the solution was purified by solid-phase extraction 17. Purified solutions were stored at 4 °C

For the *in vitro* anti-cancer studies, the process for the extraction of American ginseng samples was as follows: A root sample, unsteamed or steamed at various temperatures and times, was ground to powder and passed through a 40 mesh screen. Then 25 g of powder was extracted with 500 ml of 70% ethanol for 4 h; the water bath was maintained at 90 °C. When cooled, the solution was filtered with P8 filter paper (Fisher Scientific, Pittsburgh, PA) and the filtrate was collected. The residue was extracted with 500 ml of 70% ethanol once more and then filtered while the solution was cooled. The filtrates were combined and the solvent was evaporated under vacuum. The dried extract was dissolved in 100 ml water and then extracted with water-saturated *n*-butanol. The *n*-butanol phase was evaporated under

The root of *P. quinquefolius* L. was collected from Roland Ginseng, LLC (Wausau, WI, USA).

**2.2 Plant materials, processing and extraction**

and cell cycle were evaluated.

**2. Materials and methods**

**2.1 Chemicals**

until HPLC analysis.

vacuum and then lyophilized.

The HPLC system was a Waters 2960 instrument (Milford, MA) with a quaternary pump, an automatic injector, a photodiode array detector (Model 996), and Waters Millennium 32 software for peak identification and integration. The separation was carried out on a 250×3.2 mm i.d., 5μ, Ultrasphere C18 column (Alltech, Deerfield, IL) with a 7.5×3.2 mm i.d. guard column. For HPLC analysis, a 20-μL sample was injected into the column and eluted at room temperature with a constant flow rate of 1.0 ml/min. For the mobile phase, acetonitrile (solvent A) and water (solvent B) were used. Gradient elution started with 18% solvent A and 82% solvent B. Elution was changed to 21% A for 20 min, then to 26% A for 3 min and held for 19 min. It was then changed to 36% A for 13 min, to 50% A for 9 min, to 95% A for 2 min, and held for 3 min. Lastly elution was changed to 18% A for 3 min and held for 8 min. The detection wavelength was set to 202 nm. All tested solutions were filtered through Millex 0.2-μm nylon membrane syringe filters (Millipore Co., Bedford, MA) before use. The linearity of this method was assayed by analyzing standard solutions in the range of 2-400 μg/ml for the 12 ginsenosides. Calibration curves were constructed from the measured peak areas and the related amount of ginsenosides. Ginsenosides Rb1, Rb2, Rb3, Rc, Rd, Re, Rg1, Rg2, 20*R*-Rg2, Rg3, Rh1 and Rh2 (Figure **1**) in extract samples were identified by comparison of their retention times with those obtained from the chromatograms of mixed ginsenoside standards. The contents of saponins in each sample were calculated using standard curves of ginsenosides.

Fig. 1. Chemical structures of assayed ginsenosides in American ginseng.

Red American Ginseng and Breast Cancer 273

Cells were seeded in 24-well tissue culture plates. After culturing for 1 day, the medium was changed and extracts/ginsenosides were added. After treated for 48 h, cells floating in the medium were collected. The adherent cells were detached with 0.05% trypsin. Then culture medium containing 10% FBS (and floating cells) was added to inactivate trypsin. After being pipetted gently, the cells were centrifuged for 5 min at 1500 g. The supernatant was removed and cells were stained with annexin V-FITC and propidium iodide according to the manufacturer's instructions. Untreated cells were used as control for double staining. Cells were analyzed immediately after staining using a FACScan flow cytometer (Becton Dickinson, Mountain View, CA) and FlowJo 7.1.0 software (Tree Star, Ashland, OR). For

Cells were seeded in 24-well tissue culture plates. On the second day, the medium was changed and cells were treated with extracts. Cells were incubated for 48 h before the cells were harvested. The cells were fixed gently by putting 80% ethanol in freezer for 2 h and were then treated with 0.25% Triton X-100 for 5 min on ice bath. Cells were resuspended in 300 ul of PBS containing 40 µg/ml propidium iodide and 0.1 mg/ml RNase. Then, 20 µl of cyclin A-FITC or cyclin D1-FITC was added to the cell suspension. Then the cells were incubated in a dark room for 20 min at room temperature, and analyzed with a FACScan

After cells were treated with extracts/ginsenosides, the cells were harvested in similar manner to the method used in the cyclins assay. Then, the cells were stained with propidium iodide, and analyzed with a FACScan flow cytometer. For each measurement, at

Data are presented as mean ± standard deviation (SD) with n = 3. A one-way ANOVA determined whether the results had statistical significance. In some cases, Student's t-test was used for comparing two groups. The level of statistical significance was set at P<0.05.

Twelve ginsenosides (Figure **1**) were determined in American ginseng using high performance liquid chromatography (HPLC). All the assayed ginsenosides were dammarane glycosides 17,19. The influence of steaming at 100°C and 120°C on the ginsenoside content of American ginseng was tested. Compared with unsteamed American ginseng, the roots treated at 100°C for 1 h decreased total ginsenoside content slightly, from 7.95% to 7.32%. For the main saponin contents, ginsenoside Rb1 was changed from 4.940% to 4.463%, and Re was from 1.756% to 1.630%; ginsenoside Rg3 increased from 0.003% to 0.048%. Steaming at 120°C for 1 h decreased the total ginsenoside content to 5.85%, as follows: Rb1, 3.252%; Re, 0.968%; and Rg3, 0.271%. Ginsenoside Rg3 increased significantly

flow cytometer. For each measurement, at least 20,000 cells were counted.

**3.1 Saponin composition changes in extracts during heat-processing**

**2.6 Apoptosis assay**

each measurement, at least 20,000 cells were counted.

**2.7 Cyclin A and cyclin D1 assay**

**2.8 Cell cycle assay**

least 20,000 cells were counted.

**2.9 Statistical analysis**

**3. Results**

at 120°C.

Ginsenoside Rg3 increased significantly from 0.5 to 3 h of steaming. Ginsenoside Rh2 is also an active anticancer saponin, however, after steaming treatment, the content of Rh2 was very low (Table **1**).

n = 3; ND, not detected; values are expressed as percentage of dry weight; RSD (relative standard derivation) are less than 15%.

Table 1. Ginsenoside contents in Asian ginseng, red Asian ginseng and American ginseng which were unsteamed or steamed at 120°C for 0.5-4 h.

## **2.4 Cell culture**

The human breast cancer cell lines MCF-7 and MDA-MB-231 were purchased from American Type Culture Collection, ATCC (Manassas, VA, USA) and grown in RPMI 1640 medium supplemented with 10% FBS and 50 IU penicillin/streptomycin in a humidified atmosphere of 5% CO2 at 37°C.

## **2.5 Cell proliferation analysis**

Cells were seeded in 96-well plates. After 1 d, various concentrations of extracts/ginsenosides were added to the wells. The final concentration of ethanol was 0.5%. Controls were exposed to culture medium containing 0.5% ethanol without drugs. All experiments were performed in triplicate and repeated 3 times. Cell proliferation was evaluated using an MTS assay according to the manufacturer's instructions. Briefly, at the end of the drug exposure period, the medium was replaced with 100 μl of fresh medium, 20 μl of MTS reagent (CellTiter 96 Aqueous Solution) in each well, and the plate was returned to the incubator for 1-2 h. A 60-μl aliquot of medium from each well was transferred to an ELISA 96-well plate and its absorbance at 490 nm was recorded 4,18. Results were expressed as percentage of control (ethanol vehicles set at 100%).

## **2.6 Apoptosis assay**

272 Breast Cancer – Current and Alternative Therapeutic Modalities

Ginsenoside Rg3 increased significantly from 0.5 to 3 h of steaming. Ginsenoside Rh2 is also an active anticancer saponin, however, after steaming treatment, the content of Rh2 was

Rg1 0.279 0.276 0.133 0.098 0.075 0.017 ND ND Re 0.234 0.188 1.750 1.347 0.968 0.221 0.032 ND Rh1 ND 0.015 ND 0.008 0.017 0.040 0.042 0.049 Rg2 0.031 0.034 0.015 0.154 0.266 0.405 0.444 0.363 20*R*-Rg2 ND 0.022 ND 0.086 0.155 0.388 0.425 0.422 Rb1 0.545 0.462 4.940 3.918 3.252 1.743 0.735 0.177 Rc 0.523 0.294 0.387 0.325 0.278 0.141 0.087 0.018 Rb2 0.443 0.200 0.043 0.042 0.036 0.026 0.018 ND Rb3 0.067 0.032 0.063 0.064 0.050 0.035 0.017 ND Rd 0.257 0.073 0.604 0.510 0.460 0.335 0.206 0.097 Rg3 0.004 0.033 0.003 0.143 0.271 0.664 1.053 1.225 Rh2 ND 0.009 0.007 0.007 0.018 0.047 0.057 0.066 **Total 2.38 1.64 7.95 6.70 5.85 4.05 3.11 2.42**

**Steaming Time (Red American Ginseng) 0.5 h 1 h 2 h 3 h 4 h**

**American Ginseng**

n = 3; ND, not detected; values are expressed as percentage of dry weight; RSD (relative standard

Table 1. Ginsenoside contents in Asian ginseng, red Asian ginseng and American ginseng

The human breast cancer cell lines MCF-7 and MDA-MB-231 were purchased from American Type Culture Collection, ATCC (Manassas, VA, USA) and grown in RPMI 1640 medium supplemented with 10% FBS and 50 IU penicillin/streptomycin in a humidified

Cells were seeded in 96-well plates. After 1 d, various concentrations of extracts/ginsenosides were added to the wells. The final concentration of ethanol was 0.5%. Controls were exposed to culture medium containing 0.5% ethanol without drugs. All experiments were performed in triplicate and repeated 3 times. Cell proliferation was evaluated using an MTS assay according to the manufacturer's instructions. Briefly, at the end of the drug exposure period, the medium was replaced with 100 μl of fresh medium, 20 μl of MTS reagent (CellTiter 96 Aqueous Solution) in each well, and the plate was returned to the incubator for 1-2 h. A 60-μl aliquot of medium from each well was transferred to an ELISA 96-well plate and its absorbance at 490 nm was recorded 4,18. Results were expressed

very low (Table **1**).

**Ginsenoside Asian**

derivation) are less than 15%.

atmosphere of 5% CO2 at 37°C.

**2.5 Cell proliferation analysis**

**2.4 Cell culture**

which were unsteamed or steamed at 120°C for 0.5-4 h.

as percentage of control (ethanol vehicles set at 100%).

**Ginseng**

**Red Asian Ginseng**

Cells were seeded in 24-well tissue culture plates. After culturing for 1 day, the medium was changed and extracts/ginsenosides were added. After treated for 48 h, cells floating in the medium were collected. The adherent cells were detached with 0.05% trypsin. Then culture medium containing 10% FBS (and floating cells) was added to inactivate trypsin. After being pipetted gently, the cells were centrifuged for 5 min at 1500 g. The supernatant was removed and cells were stained with annexin V-FITC and propidium iodide according to the manufacturer's instructions. Untreated cells were used as control for double staining. Cells were analyzed immediately after staining using a FACScan flow cytometer (Becton Dickinson, Mountain View, CA) and FlowJo 7.1.0 software (Tree Star, Ashland, OR). For each measurement, at least 20,000 cells were counted.

## **2.7 Cyclin A and cyclin D1 assay**

Cells were seeded in 24-well tissue culture plates. On the second day, the medium was changed and cells were treated with extracts. Cells were incubated for 48 h before the cells were harvested. The cells were fixed gently by putting 80% ethanol in freezer for 2 h and were then treated with 0.25% Triton X-100 for 5 min on ice bath. Cells were resuspended in 300 ul of PBS containing 40 µg/ml propidium iodide and 0.1 mg/ml RNase. Then, 20 µl of cyclin A-FITC or cyclin D1-FITC was added to the cell suspension. Then the cells were incubated in a dark room for 20 min at room temperature, and analyzed with a FACScan flow cytometer. For each measurement, at least 20,000 cells were counted.

## **2.8 Cell cycle assay**

After cells were treated with extracts/ginsenosides, the cells were harvested in similar manner to the method used in the cyclins assay. Then, the cells were stained with propidium iodide, and analyzed with a FACScan flow cytometer. For each measurement, at least 20,000 cells were counted.

## **2.9 Statistical analysis**

Data are presented as mean ± standard deviation (SD) with n = 3. A one-way ANOVA determined whether the results had statistical significance. In some cases, Student's t-test was used for comparing two groups. The level of statistical significance was set at P<0.05.

## **3. Results**

## **3.1 Saponin composition changes in extracts during heat-processing**

Twelve ginsenosides (Figure **1**) were determined in American ginseng using high performance liquid chromatography (HPLC). All the assayed ginsenosides were dammarane glycosides 17,19. The influence of steaming at 100°C and 120°C on the ginsenoside content of American ginseng was tested. Compared with unsteamed American ginseng, the roots treated at 100°C for 1 h decreased total ginsenoside content slightly, from 7.95% to 7.32%. For the main saponin contents, ginsenoside Rb1 was changed from 4.940% to 4.463%, and Re was from 1.756% to 1.630%; ginsenoside Rg3 increased from 0.003% to 0.048%. Steaming at 120°C for 1 h decreased the total ginsenoside content to 5.85%, as follows: Rb1, 3.252%; Re, 0.968%; and Rg3, 0.271%. Ginsenoside Rg3 increased significantly at 120°C.

Red American Ginseng and Breast Cancer 275

Fig. 3. Saponin content in American ginseng roots steamed at 120 °C for 0-4 h. The content of