Anti-cancer effect of Cassia auriculata leaf extract in vitro through cell cycle arrest and induction of apoptosis in human breast and larynx cancer cell lines

Cell Biology International 33 (2009) 127e134 www.elsevier.com/locate/cellbi

Anti-cancer effect of Cassia auriculata leaf extract in vitro through cell cycle arrest and induction of apoptosis in human breast and larynx cancer cell lines R. Prasanna a,*, C.C. Harish b, R. Pichai a, D. Sakthisekaran c, P. Gunasekaran b a Department of Chemistry, Presidency College, Chennai 600 005, Tamil Nadu, India Department of Virology, King Institute of Preventive Medicine, Gundy, Chennai 600 032, Tamil Nadu, India c Department of Medical Biochemistry, University of Madras, Tharamani Campus, Chennai, Tamil Nadu, India b

Received 4 February 2008; revised 21 August 2008; accepted 13 October 2008

Abstract The in vitro anti-cancer effect of Cassia auriculata leaf extract (CALE) was evaluated in human breast adenocarcinoma MCF-7 and human larynx carcinoma Hep-2 cell lines. CALE preferentially inhibited the growth of both the cell lines in a dose-dependent manner with IC50 values of 400 and 500 mg for MCF-7 and Hep-2 cells, respectively. The results showed the anti-cancer action is due to nuclear fragmentation and condensation, associated with the appearance of A0 peak in cell cycle analysis that is indicative of apoptosis. In addition, CALE treated MCF-7 and Hep-2 cells had decreased expression of anti-apoptotic Bcl-2 protein and increased expression of pro-apoptotic Bax protein, eventually leading a decrease in the Bcl-2/Bax ratio. These results demonstrated that CALE inhibits the proliferation of MCF-7 and Hep-2 cells through induction of apoptosis, making CALE a candidate as new anti-cancer drug. Ó 2008 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. Keywords: Cassia auriculata; MCF-7; Hep-2; Cell cycle; Apoptosis; Bax/Bcl-2

1. Introduction Plants and plant products both as extracts and derived compounds are known to be effective and versatile chemopreventive agents against various types of cancers (Aruna and Sivarama Krishnan, 1990; Graham et al., 2000; Moongkarndi et al., 2004). Traditional background of Indian medicine shows extensive use of plant products in cancer (Cha, 1977; Gupta, 1979; Rabi and Gupta, 1995; Hussian et al., 1993). A remarkable surge of interest in chemoprevention research has thus led to the identification of many phytochemicals as effective chemopreventive agents (Cordell et al., 1999). Today there are at

* Corresponding author. Department of Chemistry, Presidency College, Kamarajar Salai, Chepauk, Chennai 600 005, Tamil Nadu, India. Tel.: þ91 9701447730. E-mail address: [email protected] (R. Prasanna).

least 120 distinct chemical substances derived from plants that are considered as important drugs and active ingredients. Dried flower and leaf extracts of Cassia auriculata (Cesalpiniaceae) are widely used in Indian traditional medicine (Shawney et al., 1978). The flower and seed extracts of the plant have anti-diabetic activity (Jain and Sharma, 1967) and they have an emollient effect (Dhar et al., 1968). The alcoholic leaf extract of Cassia auriculata is effective in alcoholic liver injury (Rajagopala et al., 2003). There are a few experimental studies to show the anti-viral activity of the plant (Dhar et al., 1968). Natural compounds are perfectly suited to the current molecular-target approach of drug development, as well as the use of combinations. They produce few adverse effects, many act as tonics and stimulators that inhibit multiple aspects of disease progression (anti-proliferative, anti-adhesive; as a group they show a propensity for synergistic interactions). Although this plant has been widely studied, biochemical studies on the anti-carcinogenic effects of Cassia auriculata

1065-6995/$ - see front matter Ó 2008 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.cellbi.2008.10.006

128

R. Prasanna et al. / Cell Biology International 33 (2009) 127e134

has not been reported. Many chemotherapeutic drugs eliminate cancer cells by inducing, a genetically programmed form of cell death (Nanba et al., 1994). It is therefore important to establish the chemopreventive efficacy of the plant by evaluating cytotoxicity and apoptosis induction in cancer cell lines before whole animal studies or clinical trials begin. We therefore decided to screen the leaf extract for its anti-cancer activity against the human breast carcinoma MCF-7 and human larynx carcinoma Hep-2 cells. 2. Materials and methods 2.1. Leaf extract Freshly collected leaves of Cassia auriculata were cleaned, shade dried. These leaves were coarse powdered in a low speed blender and stored in an airtight container. One gram of the powdered leaves was soaked in 100 ml of absolute ethanol. The mixture was kept in the rotary shaker for 48 h. The contents were filtered through muslin cloth and the filter was dried at 55  C. The sediments were re-extracted as mentioned above. The dried extract was scrapped and stored at 4  C in airtight vials. 2.2. Chemicals

the stringent literature and previous studies we had conducted. HBL-100 and Vero cells are immortal, but non-tumorogenic and do not express most specific tumorogenic markers. Both cell types were seeded at 1  105 cells per well in 24-well plates. After overnight growth, the medium was replaced with maintenance medium (SMEM without FBS) containing various concentrations the CALE and incubated for 24 h. The plates were microscopically examined for cytotoxicity. MTT assay was used to assess the cell viability based on its reduction by mitochondrial dehydrogenase enzyme of the viable cells to purple formazan product (Mosmann, 1983). Briefly, cells were diluted in the growth medium and seeded in 24-well plates at 5  104 cells/well. After overnight incubation, growth medium was replaced with exposure medium (SMEM without FBS) containing the desired concentrations of CALE. After 24 h, the cells were washed with 200 ml of PBS, and incubated with 100 ml of 500 mg/ml MTT in PBS at 37  C for 3 h. The formazan product was dissolved in 200 ml of DMSO and estimated by measuring the absorbance at 570 nm in an ELISA plate reader. Cell survival was expressed as a percentage of viable cells of treated samples to control samples. The test was performed in triplicate and each experiment was repeated three times. The same protocol was repeated to check the anti-cancer activity of CALE using the known tumorogenic MCF-7 and Hep-2 cell lines.

All reagents used in the study were of analytical grade. Stock solution of the leaf extract was dissolved in PBS containing 0.5% dimethyl sulfoxide (DMSO). The stock solutions were diluted with the medium to the desired concentration (the final concentration of DMSO on the medium was <0.01%, which had no detectable effect on cell growth). Eagle’s minimal essential media (SMEM) and fetal bovine serum (FBS) were purchased from GIBCO. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), propidium iodide, proteinase K, phenylmethanesulfonyl fluoride (PMSF), rhodamine 123, and RNAse A were purchased from Sigma Chemical Company (St. Louis, MO, USA).

Both MCF-7 and Hep-2 cells were plated at 5  104 cells/ well into a six-well chamber plate. At >80% confluence, the cells were treated with CALE for 24 h. The cells were washed with PBS fixed in methanol:acetic acid (3:1 v/v) for 10 min and stained with 50 mg/ml of propidium iodide for 20 min. Nuclear morphology of apoptotic cells with condensed/fragmented nuclei was examined under a fluorescent microscope and at least 1  103 cells were counted to assess apoptotic cell death (Keum et al., 2002).

2.3. Cell lines and cell cultures

2.6. Cell cycle analysis

MCF-7 (human breast adenocarcinoma cell line), HBL-100 (normal breast cell line), Hep-2, (human epithelial larynx cancer cell line) and Vero (a non-tumorogenic cell line) were obtained from the National Centre for Cell Sciences, Pune, India, and were grown in SMEM media supplemented with 10% FBS 100 IU/ml, penicillin 100 mg/ml, streptomycin 20 mg/ml, kanamycin acid sulphate 20 mg/ml, amphotericin-B, 3% L-glutamine and 7.5% sodium bi-carbonate solution. The cells were maintained as monolayers in 25 cm2 plastic tissue culture flasks at 37  C in a humidified atmosphere containing 5% CO2 in air. Exponentially growing cells were used in all the experiments.

Cell cycle distribution and measurement of the percentage of apoptotic cells were performed by flow cytometry (Tai et al., 2000). After treatment, floating cells in the medium were combined with attached cells collected by trypsinization. Cells were washed with cold PBS and fixed in 80% ethanol in PBS at 20  C. After 12 h, fixed cells were pelleted and stained with propidium iodide (50 mg/ml) in the presence of RNase A (20 mg/ ml) for 30 min at 37  C. About 104 cells were analysed in a Becton Dickinson FACScan flow cytometer. Cell cycle histograms were analysed using Cell Quest software. Apoptotic cells were distinguished by their decreased DNA content, as shown by their weaker staining intensity in the area of the sub-G0/G1 phase.

2.5. Nuclear morphology assay

2.4. Cytotoxicity and anti-cancer assay 2.7. Western blotting Initially the cytotoxicity of CALE was followed by the nontumorogenic breast (HBL-100) and the African green monkey epithelial (Vero) cells. These cell lines were selected based on

MCF-7 and Hep-2 cells were treated with their respective concentrations of CALE, washed twice with ice-cold PBS and

R. Prasanna et al. / Cell Biology International 33 (2009) 127e134

lysed in lysis buffer (50 mM TriseHCl, pH 8.0, 5 mM EDTA, 150 mM sodium chloride, 0.5% Nonidet P-40, 0.5 mM PMSF and 0.5 mM DTT) for 30 min at 4  C. The supernatant was collected by centrifugation at 12,500  g for 20 min. About 50 mg of total protein from the supernatant, as determined by Bradford’s protein estimation kit, was separated on 10% SDSPAGE before being transferred to a nitrocellulose membrane, using a semi-dry system (BIORAD; Towbin et al., 1979). The membranes were incubated with TBS (Tris buffered

129

saline e 150 mM/L sodium chloride, 50 mM/LTris, and pH 7.4) containing 5% non-fat dried milk for >1 h to block the nonspecific binding sites. The blocks were incubated with 1:1000 dilution of anti Bcl-2, Bax primary antibodies (NeoMarkers USA) and b-actin (Santa Cruz, CA, USA) overnight. The blocks were washed with TBS containing 0.1% Tween-20 and the proteins were detected by incubating with the corresponding horse-radish peroxidase-conjugated secondary antibodies at 1:2000 dilution for 60e90 min at room temperature. After

A

HBL-100 Control

B

HBL-100 CALE toxicity

C

MCF-7 Control

D

MCF-7 CALE toxicity

E

Vero Control

F

G

Hep-2 Control

H

Vero CALE toxicity

Hep-2 CALE toxicity

Fig. 1. Light microscopy photographs of untreated control and CALE treated cells for 24 h.

130

R. Prasanna et al. / Cell Biology International 33 (2009) 127e134

extensive washes in TBS containing 0.1% Tween-20, the transferred proteins were visualized using DAB. Densitometry was performed on an IISP flat-bed scanner and quantified with total lab 1.11 software.

A 120

2.8. Statistics

3. Results 3.1. Cytotoxicity assay

100

Percentage cell viability

The cytotoxicity data are presented as mean percentages of control  SD and linear regression analysis was used to calculate the IC50 values for the cytotoxicity assays. Other datas were analysed using analysis of variance (ANOVA) followed by Tukey-HSD test and the results were considered statistically significant if p < 0.05.

We first examined the cytotoxic effects of CALE on the growth of the cancerous MCF-7 and Hep-2 cells with the normal HBL-100 and Vero cells as respective controls (Fig. 1AeH). CALE showed a dose-dependent cytotoxic effects on both MCF-7 and Hep-2 cells with IC50 values of 400 and 500 mg, respectively. Although not significant, these concentrations of CALE had their effects on normal cell lines (Fig. 2A,B). For all further experiments, IC50 values were used.

The morphological and biochemical features of apoptosis were distinct from that of necrosis (Paneerselvam, 1998). The morphology of apoptotic cell showed highly condensed chromatin in a fragmented nucleus (Darzynkiewicz et al., 1992). Incubation of the MCF-7 and Hep-2 cells with CALE for 24 h significantly increased the number of apoptotic cells compared to control, as evidenced by nuclear fragmentation and condensation (Fig. 3A,B). 3.3. Effect of CALE on cell cycle control of MCF-7 and Hep-2 cells To investigate whether the CALE affected cell cycle regulation, flow cytometry was performed. Fig. 4 shows incubation of CALE with MCF-7 and Hep-2 cells for 24 h significantly reduced the DNA content, making them appear in the sub-G0/G1 or A0 region indicative of apoptosis, with consequent loss of cells in the G1 phase. 56.1% of cells were in sub G0/G1 phase, with 9.8% cells in the same phase in the respective control. In the CALE treated Hep-2 cells, the proportions of cells with reduced DNA content in G0/G1 phase of control increased from 7.8% to 53.1%. 3.4. Effect of CALE on Bcl-2/Bax ratio We examined the effects of CALE on the expression of Bcl2 and Bax that play a key role in regulating the apoptosis process. Incubation of MCF-7 and Hep-2 cells with CALE

60

40

20 HBL-100 MCF-7 0 cc

100

200

300

400

500

600

CALE Treatment µg/ml, 24 h

B

120

100

Percentage cell viability

3.2. Induction of apoptosis by CALE

80

80

60

40

20 Vero Hep-2 0 cc

100

200

300

400

500

600

CALE Treatment µg/ml, 24 h Fig. 2. Effects of CALE on MCF-7 and Hep-2 cancerous cells at 24 h incubation time along with HBL-100 and Vero as normal cell lines. Cell survival was measured using MTT assay and expressed as percentage of viable cells of treated samples to untreated control samples. Data are represented by mean  SD of two independent experiments each performed in tetrads. IC50 value was calculated using linear regression analysis.

R. Prasanna et al. / Cell Biology International 33 (2009) 127e134

131

Fig. 3. Morphological changes (A) and the number of apoptotic nuclei (B) in MCF-7 and Hep-2 cells after 400 and 500 mg of CALE treatment respectively for 24 h. Error bar represents SD between counts of three independent experiments. *** e Significantly increased compared to control ( p < 0.001) by ANOVA followed by Tukey-HSD test.

significantly decreased the Bcl-2/Bax ratio compared to the untreated control cells (Fig. 5). 4. Discussion The results demonstrate that CALE preferentially are cytotoxic to human breast adenocarcinoma (MCF-7) and human larynx carcinoma (Hep-2) cells in a dose-dependent manner. Wyllie (1980) described how DNA fragmentation is a component of apoptosis. The morphology of an apoptotic cell shows a highly condensed chromatin in a fragmented

nucleus (Darzynkiewicz et al., 1992; Wyllie et al., 1980). The nuclear morphology and appearance of A0 peak in the flow cytometry histograms both demonstrate that CALE induces apoptosis in MCF-7 and Hep-2 cells. Both DNA loss and a decrease of DNA accessibility to the dye are responsible for the lower fluorescence of apoptotic cells (Zamai et al., 1993), making them appearing in the sub-G0/G1 region of the histogram. Incubation of MCF-7 and Hep-2 cells with CALE for 24 h resulted in the loss of cells in G0/G1 phase with concomitant increase in appearance of apoptotic cells (A0 peak). The occurrence of such hypo-diploid peak after staining

132

R. Prasanna et al. / Cell Biology International 33 (2009) 127e134

Fig. 4. Cell cycle analysis of MCF-7 and Hep-2 treated cells. (A) Representative histograms demonstrate the cell population according to the DNA content determined by propidium iodide staining. (B) Quantitative analysis of apoptotic cell population in MCF-7 and Hep-2 cells Error bar represents SD between counts of three independent experiments. *** e Significantly increased compared to control ( p < 0.001) by ANOVA followed by Tukey-HSD test.

with DNA specific fluorescent dyes (Fig. 4) helps in identifying the apoptotic mode of cell death (Nicoletti et al., 1991; Swat et al., 1991; Telford et al., 1992). In the apoptotic mode of cell death, there is tight regulation by a number of gene products that either promote or block cell

death in different stages of the cell cycle. The Bcl-2 gene family in mammals consists of a number of anti-apoptotic proteins, including the Bcl-2 protein (Hong et al., 2002), which is located primarily in the outer mitochondrial membrane and blocks apoptosis by preventing cytochrome c

R. Prasanna et al. / Cell Biology International 33 (2009) 127e134

Fig. 5. The effect of CALE on Bcl-2/Bax ratio. Error bar represents SD of three independent experiments. *** e Significantly increased compared to control ( p < 0.001) by ANOVA followed by Tukey-HSD test. b actin was used as loading control.

release from the mitochondria as well as inhibiting caspase-3 activity. Bax and BH-3 gene families consists of pro-apoptotic proteins, including Bax that translocates from the cytosol to the mitochondria and interacts with outer membrane proteins to form pores, thereby releasing cytochrome c and triggering caspase-mediated apoptotic cell death. The relative levels of Bcl-2 and Bax proteins were the most highly predictive, and most sensitivity to apoptotic agents in breast cancer cells, compared to other members of Bcl-2 family (Hong et al., 2002). Bcl-2 and Bax are therefore attractive targets for designing new anti-cancer drugs (Mackey et al., 1998; Lohmann et al., 2000). From Fig. 5, it is evident that downregulation of Bcl-2 in both MCF-7 and Hep-2 cells after CALE treatment lowered the Bcl-2/Bax ratio, suggesting that Bcl-2 participated in the CALE induced apoptosis in both breast and larynx cancer cells. Bcl-2 can control calcium ion release from mitochondria and endoplasmic reticulum. Calcium ions activate nucleases and proteases that mediate cellular destruction during apoptosis (Hong et al., 2002). Bcl-2 can also activate apoptosis by controlling the cellular redox reactions (Hong et al., 2002). The translocation of Bax to mitochondria from the cytosol, results in the depolarization of mitochondria and release of apoptotic factors trough outer membrane formed by Bax oligomers (Hong et al., 2002). Bax affects the mitochondrial membrane integrity and influences the mitochondrial release of apoptosis associated factors like Apaf-1 (apoptosis protease activated factor), Aif (apoptosis inducing factor) and cytochrome

133

c into the cytoplasm (Zhang et al., 2006). Cytochrome c interacts with procaspase-9 and switches on the caspase-3, caspase-6 and caspase-7, leading to apoptosis (Hsu et al., 2006). The downregulation of Bcl-2 protein leads to the formation of Bax homodimers that increase the rate of apoptosis (Zhang et al., 2006). The upregulation of Bax proteins may thus be a reason for apoptosis in CALE treated MCF-7 and Hep-2 cells. Although we have not done caspase assay, it is evident from other studies that caspase can be activated, paving the way for apoptosis (Shim et al., 2007). The ratio of Bcl-2/Bax has been shown to be critical in determining the susceptibility of cells to induce apoptosis (Rasiova, 2001). Thus the relative ratio of these proteins determines the fate of the cell. There are studies to show apoptosis is readily triggered in MCF-7 cells by downregulation of Bcl-2 and upregulation of Bax, thereby reducing their relative ratio (Hong et al., 2002; Zhang et al., 2006; Hsu et al., 2006). Further CALE may also activate caspase suggesting a new group of lead molecules to fight against chemo resistance. The data in Fig. 5 shows a positive association between Bcl-2/Bax expression and mitochondrial events in apoptosis induced by CALE. This clearly demonstrates that CALE induces apoptosis in both MCF-7 and Hep-2 cells, which might account for its anti-cancer activities. Flavanoids and procyanidins were reported to be present in Cassia auriculata (Kumaran and Joel Karunakaran, 2007). Epigallocatechin-3-gallate, a flavonoid rapidly inhibited proliferation of HL-60 cells, which correlated with the formation of apoptotic DNA fragments (Nakamura et al., 2003). Kaur et al. (1992) showed the anti-proliferative and cytostatic effect of a polyhydroxylated flavonoid in vitro, which exhibited strong inhibition of DNA, RNA and protein synthesis and selectively blocked cell cycle progression in vitro. Proanthocyanidins was found to exhibit a concentration and time-dependent cytotoxic effects on MCF-7 cells, A-427 lung cancer cells and gastric adenocarcinoma cells while the viability of normal human gastric mucosal cells and murine macrophage cells were enhanced (Ye et al., 1999). Thus these properties of flavonoids and procyanidins could have been responsible for the anti-cancer activity of CALE exhibited in cancerous cell lines. 3-O-beta-D-xylopyranosides, a class of triterpene glycosides, are present in Cassia auriculata (Sanghi et al., 2000). The methanol extract of Tribulus macropterus and its isolated compound b-D-xylopyranoside are cytotoxic to human tumor cell lines (Abdel-Hameed el et al., 2007). 3-O-beta-D-xylopyranoside isolated from C. dahurica is effective against HepG2 cell line by regulating protein expression of bcl-2 family (Tian et al., 2006). The same class of compound from Actaea asiatica was effective against MCF-7 cell line (Gao et al., 2006), and also to hepatoma cells, inducing apoptosis by G0/G1 cell cycle arrest, upregulating Bax and downregulating Bcl-2 levels (Tian et al., 2006). Thus these compounds present in CALE would have been responsible for the anti-cancer activity against MCF-7 and Hep-2 cells. In conclusion CALE offers a valuable candidate lead compound to counter growing drug resistance in breast and larynx cancers.

134

R. Prasanna et al. / Cell Biology International 33 (2009) 127e134

References Abdel-Hameed el SS, El-Nahas HA, El-Wakil EA, Ahmed WS. Cytotoxic cholestane and pregnane glycosides from Tribulus macropterus. Zeitschrift fu¨r Naturforschung 2007;62(5-6):319e25. Aruna K, Sivarama Krishnan VM. Plant products as protective agents against cancer. Indian Journal of Experimental Biology 1990;28:1008e11. Cha S. Potential anticancer medicinal plants. A statistical evaluation of their frequencies of appearance in oriental medicine formularies. Korean Journal of Pharmacognosy 1977;8:1e14. Cordell GA, Farnvorth NR, King Horn CWW. Plant derived anti cancer agents for therapy (abstr), 4th International Congresson Phytotherapy, PLI, Munich, Germany, 1999. Darzynkiewicz Z, Bruno S, Del Bino G, Gorczyca W, Hots MA, Lassota P, et al. Features of apoptotic cells measured by flow cytometry. Cytometry 1992;14:795e808. Dhar ML, Dhawan BN, Mehrotra BN, Ray C. Screening of Indian plants for biological activity. Indian Journal of Experimental Biology 1968;6: 232e47. Gao J, Huang F, Zhang J, Zhu G, Yang M, Xiao P. Cytotoxic cycloartane triterpene saponins from Actaea asiatica. Journal of Natural Products 2006;69(10):1500e2. Graham JG, Quinn ML, Fabricant DS, Fransworth NR. Plants used against cancer-an extension of the work of Jonathan Hartwell. Journal of Ethanopharmacology 2000;73:347e77. Gupta SK. Apocynaceous plants of Varanasi with notes on their medicinal importance. Journal of Research Indian Medicine Yoga and Homeopathy 1979;14:140e2. Hong C, Firestone GL, Boeldanes LF. Bcl-2 family-mediated apoptotic effects of 3,30 -diionoilmethane (DIM) in human breast cancer cells. Biochemical Pharmacology 2002;63:1085e97. Hsu YL, Kuo PL, Zeng WS, Lin CC. Chalcone inhibits the proliferation of human breast cancer cell by blocking cell cycle progression and inducing apoptosis. Food and Chemical Toxicology 2006;44:704e13. Hussian SJ, Alvi AB, Jahan MA. Study on Unani medicinal plants, Asteratiqus. Journal of Research and Education in Indian Medicine 1993;2: 35e9. Jain SR, Sharma SN. Hypoglycaemic drugs of Indian indigenous origin. Planta Medica 1967;15:439e42. Kaur G, Stetler-Stevenson M, Sebers S, Worland P, Sedlacek H, Myers C, et al. Growth inhibition with reversible cell cycle arrest of carcinoma cells by flavone L86-8275. Journal of the National Cancer Institute 1992;84(22): 1736e40. Keum YS, Kim J, Lee KH, Park KK, Surh YJ, Lee JM. Induction of apoptosis and caspase-3 activation by chemopreventive [6]-paradol and structurally related compounds in KB cells. Cancer Letters 2002;177:41e7. Kumaran A, Joel Karunakaran R. Antioxidant activity of Cassia auriculata flowers. Fitoterapia 2007;78(1):46e7. Lohmann CM, League AA, Clark WS, Lawson D, DeRose PB, Cohen C. Bcl2:Bax and Bcl-2:Bcl-x ratios by image cytometric quantitation of immunohistochemical expression on ovarian carcinoma: correlation with prognosis. Cytometry 2000;42:61e6. Mackey TJ, Borkowski A, Amin P, Jacobs SC, Kyprianou N. Bcl-2/Bax ratio as a preventive marker for therapeutic responses to radiotherapy in patients with prostrate cancer. Urology 1998;52:85e90. Moongkarndi P, Kosem N, Luanratana O, Jongsomboonkusol S, Pongpan N. Antiproliferative activity of Thai medicinal plant extracts on human breast adenocarcinoma cell line. Fitoterapia 2004;75:375e7. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. Journal of Immunological Methods 1983;65:55e63.

Nakamura C, Yasumoto E, Nakano K, Nakayachi T, Hashimoto K, Kusama K, et al. Changes in intracellular concentrations of polyamines during apoptosis of HL-60 cells. Anticancer Research 2003;23(6C):4797e803. Nanba TK, Adota S, Shimomura K, Iida K. Skin-lightning cosmetic containing hyaluronidase and collagenase inhibiting Cassia auriculata extracts. Japan Kokai To Kkya Koho 1994;26:960. Nicoletti I, Migliorati G, Pagliacci MC, Grignani F. Riccardic. A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. Journal of Immunological Methods 1991;139:271e9. Paneerselvam N. Apoptosis and gene regulation. Current Science 1998;75: 829e39. Rabi T, Gupta RC. Antitumor and cytotoxic investigation of Amoora rohituka. International Journal of Pharmacognogy 1995;33:359e61. Rajagopala SK, Manikama P, Periyasamyb V, Namasivayam N. Activity of Cassia auriculata leaf extract in rats with alcoholic liver injury. Journal of Nutritional Biochemistry 2003;14:452e8. Rasiova M. The Bax/Bcl-2 ratio determines the susceptibility of human melanoma cells to CD95/Fas-mediated apoptosis. Journal of Investigation of Dermatology 2001;117:333e40. Sanghi R, Tripathi K, Sharma JP. New triterpenoid, O-beta-D-xylopyranosides from Cassia auriculata. Indian Journal of Chemistry Section B, Organic Including Medicinal 2000;39(6):477e9. Shawney AN, Khan MR, Ndaalio G, Nkunya MHH, Wavers H. Studies on the rational of African traditional medicine, part II, Preliminary screening of medicinal plants for anti-Gonococci activity. Pakistan Journal of Science Indigenous Research 1978;21:189e92. Shim HY, Park JH, Paik HD, Nah SY, Kim DSHL, Han YS. Acacetin-induced apoptosis of human breast cancer MCF-7 cells involves caspase cascade, mitochondria-mediated death signaling and SAPK/JNK1/2-c-Jun activation. Molecules and Cells 2007;24:95e104. Swat W, Ignatowicz L, Kisielow P. Detection of apoptosis of immature CD4þ8þ thymocytes by flow cytometry. Journal of Immunological Methods 1991;37:79e87. Tai KW, Chou MY, Hu CC, Yang JJ, Chang YC. Induction of apoptosis in KB cells by pingyangmucin. Oral Oncology 2000;36Z:242e7. Telford WG, King LE, Fraker PJ. Comparative evaluation of several DNA binding dyes in the detection of apoptosis-associated chromatin degradation by flow cytometry. Cytometry 1992;13:137e43. Tian Z, Si JY, Chen SB, Yang MS, Xiao PG, Wu EX. Cytotoxicity and mechanism of 23-O-acetylcimigenol-3-O-beta-D-xylopyranoside on HepG2 cells. Zhongguo Zhong Yao Za Zhi 2006;31(21):1818e21. Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proceedings of the National Academy of Sciences USA 1979;76: 4350e5354. Wyllie AH. Glucocorticoid-induced apoptosis is associated with endogenous, endonuclease activation. Nature 1980;284:555e6. Wyllie AH, Kerr JFR, Currie AR. Cell death: the significance of apoptosis. In: Bourne GH, Danielli FJ, Jeon KW, editors. International review of cytology, Vol. 68, Academic Press, New York 1980, pp. 251e306. Ye X, Krohn RL, Liu W, Joshi SS, Kuszynski CA, McGinn TR, et al. The cytotoxic effects of a novel IH636 grape seed proanthocyanidin extract on cultured human cancer cells. Molecular and Cellular Biochemistry 1999; 196(1-2):99e108. Zamai L, Falcieri E, Zauli G, Cataldi A, Vitale M. Optimal detection of apoptosis by flow cytometry depends on cell morphology. Cytometry 1993;14:891e7. Zhang M, Peter C, Cheng K, Lawrence C, Chium M, Elaine Y, et al. Cell cycle arrest and apoptosis induction in human breast carcinoma MCF-7 cells by carboxymethylated b-glucan from the mushroom Sclertia of Pleurotus tuber-regium. Carbohydrate Polymers 2006;66:455e62.