Caspase-3 protease activation during the process of genistein-induced apoptosis in TM4 testicular cells

Biology of the Cell 92 (2000) 115-124 © 2000 Editions scientifiques et medicales Elsevier SAS. All rights reserved

115

Original article

Caspase-3 protease activation during the process of genistein-induced apoptosis in TM4 testicular cells James Kumi-Diaka *, Andre Butler Florida Atlantic University, Department of Biology, College of Liberal Arts & Sciences, Davie, FL 33314, USA Received 18 December 1999; accepted 20 February 2000

The role of caspase-3 (CPP32) protease in the molecular pathways of genistein-induced cell death in TM4 cells was investigated. Fluorescence microscopy with Hoechst-33258-PI nuclear stain was used to distinguish between apoptosis and necrosis pathways of cell death. The viability of the test cells was assessed with both the trypan blue exclusion and MTT tetrazolium (3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyltetralzolium bromide, 2.5 m g / m L ) assays. Caspase-3 enzymatic activity was determined using CasPASE Apoptosis Assay Kit. The overall results from all the data demonstrated that: i) genistein exerts dose- and time-dependent effects on TM4 testis cells; ii) apoptosis is induced by lower concentrations of genistein and necrosis induced by higher concentrations of genistein; iii) genistein induced activation caspase-3 enzymatic activity; iv) genistein-induction of apoptosis and necrosis was significantly inhibited by the caspase-3 inhibitor, z-DEV-FMK; v) sodium azide induced necrosis without activation of CPP32 enzymatic activity, and induction of apoptosis; and vi) genistein-induced apoptosis was associated with activation of CPP32 enzymatic activity in the cells. The overall results indicate a strong evidence of caspase3 (CPP332) mediation in the molecular pathways of genistein-induced apoptosis in testicular cells. Apoptosis is the physiologically programmed cell death in which intrinsic mechanisms participate in the death of the cell, in contrast to necrosis, which induces inflammatory response in the affected cell. The fact that the chemopreventive role of several cancer drugs is due to induction of apoptosis augments the biotherapeutic potential of genistein for the treatment of malignant diseases including prostate and testicular cancers. It is therefore inevitable that identification of the apoptotic pathways and the points at which regulation occurs could be instrumental in the design of genistein biotherapy for such diseases. © 2000 Editions scientifiques et m6dicales Elsevier SAS

genistein/ caspase-3 protease/ apoptosis/ apoptosis/ TM4 cells

1. INTRODUCTION Epidemiological studies have demonstrated that soybased foods containing isoflavonoid could reduce the incidence and/or risk of chronic diseases, and certain

*C o r r e s p o n d e n c e and reprints

Caspase-3 pr0tease activation

hormone-dependent and hormone-independent cancers, including prostate and breast cancers (Tominaga, 1985; Severson et al., 1989; Lee et al., 1991). The chemopreventive-chemothrapeutic component of soybean products has been attributed to the phytochemical, genistein (Paterson and Barnes, 1991; Barnes and Patterson, 1995; Shao et al., 1998). Genistein (4',5',7-trihydroxyisoflavone) is the major isoflavonoid in soybeans. The phytochemical isoflavonoids are a group of

Kumi-Diaka and Butler

116 plant chemicals that resemble steroid estrogens and mimic their biological reactions (Whitten et al., 1992; Kim et al., 1998). Genistein isoflavone has several biological activities. The cellular mechanisms of the action of genistein are not completely elucidated. However, on a biochemical basis, genistein is a competitive inhibitor of protein tyrosine kinase and topoisomerase-II (Constantinou et al., 1990; Morris et al., 1998); induces apoptosis in certain cells (Bergamaschi et al., 1993; McCabe Jr., 1993; Morris et al., 1998; Kumi-Diaka et al., 1998); inhibits angiogenesis and modulates cell cycle activities by arresting cell cycle at G2-M stage (Matsukawa et al., 1993). Kim et al. (1998) indicated that in several cell systems in which genistein inhibits growth, genistein does not alter tyrosine phosphorylation of the EGF receptor thought to be involved in signal transduction pathways. This observation supports the concept of multi-mechanism of action of genistein Growth inhibition of cancer progression by genistein is associated with a specific G2/M arrest, induction of p21WAF/CIPI expression, and apoptosis (Shao et al., 1998). In general, the molecular mechanism of apoptosis induction has only been partially elucidated (Yiwei et al., 1999). Apoptosis in certain normal and cancer cells is partly mediated intracellularly by several genes, including ~53 tumor suppressor gene, Bcl-2, and p21WAFl(Chiarugi et al., 1994). P53 gene is a cell cycle regulator able to induce cell cycle arrest to allow DNA repair a n d / o r apoptosis (Vogelstein and Kinzler, 1992, 1993). The discovery of a family of cysteine proteases closely related to Ced-3 has greatly increased the understanding of apoptosis in many tissues and cells (Ellis and Horvitz, 1986; Higuchi et al., 1998), though the sequences of caspase protease activation in apoptosis signaling are still not completely understood (Chinnaiyan et al., 1996). Nicholson et al. (1995) have demonstrated that caspase-3 is one of the principal proteases in spontaneous and staurosporine-mediated apoptosis. Pharmacological studies indicated involvement of caspases in apoptosis in virus-infected cells without defining which caspase(s) is (are) directly involved (Chinnaiyan et al., 1997; Hoff and Donis., 1997). We have demonstrated in our laboratory that both genistein (a protein kinase inhibitor) and dexamethasone (antiinflammatory glucocorticoid) induce apoptosis in testicular (TM3, TM4, GC-lspg) (Kumi-Diaka et al., 1999) and prostate (DU-145, LNCaP) cell lines. However the potential mechanism of genistein- and dxm-induced apoptosis in these cell lines has not been elucidated. Apoptosis in many mammalian cells could be induced by cross-linking of the Fas/APO-1 (CD95) receptor through engagement of the receptor by its natural ligand (Kuwana et al., 1998) leading to activation of the caspases. The objective of the present study was to investigate the intracellular mechanisms of genistein-induced growthinhibition and apoptosis in a testis, by determining the

Caspase-3 protease activation

Biology of the Cell 92 (2000) 115-124 potential involvement of caspase-3 proteases in the common pathways of apoptosis.

2. MATERIALS AND METHODS 2.1. Reagents and cell lines Genistein (Indoline Chemical Co., Summerville, NJ, USA) was prepared by dissolving in dimethylsulfoxide (DMSO) to make 10 000 IJg/mL stock solution, from which pre-determined genistein concentrations of 15 (G15), 30 (G30), 45 (G45), and 60 (G~0) JJg/mL were made for this study. The purity of genistein as per manufacturer was 99%. Culture media, antibiotics, sera, trypsinEDTA, and other reagents were purchased from Sigma Scientific (St. Louis, MO, USA). Testicular cell line (Sertoli cell, TM4) was purchased from American tissue culture collection (ATCC) (Rockville, MD, USA), and maintained as monolayer in DMEM: HI2 (50:50), containing 15 mM HEPES, and supplemented with 0.45% glucose (w/w), 10% fetal bovine serum and 100 U / m L penicillin + 100 IJg/mL streptomycin. Caspase-3/ CPP32 and caspase-3 inhibitor (Z-VAD-FMK) was purchased from Geno-Tech lnc (St. Louis, MO, USA).

2.2. Cell growth and inhibition assays In order to determine the potential role of caspase-3 protease in the common pathways of genistein-induced growth inhibition and apoptosis, TM4 cells were exposed to varying concentrations of genistein (G0_60). 1.25 x 104 cells/500 [JL/well were seeded in: i) culture medium as control; and ii) medium + G0_60. Each treatment group (i & ii) was plated in replicates of six in a 24-well microtiter plates (MTP) (Costar, Fisher Scientific). Cells were incubated for up to 24 h in a humidified atmosphere of 5% CO 2 at 37°C. At 6, 12, and 24 h of incubation, 200 jJL of the supernatant from each well was gently aspirated into micro-centrifuge tube and stored at -70°C until assayed for lactate dehydrogenase (LDH) enzyme activity in an unrelated experiment. At each time point, the adherent cells in equal number of wells from each treatment group were trypsinized with trypsin-EDTA, and processed for post-incubation cell viability using trypan blue exclusion (TB) and MTT tetrazolium (3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyltetralzolium bromide, 2.5 m g / m L ) assays.

2.2.1. Trypan blue exclusion: growth and viability test To evaluate growth and viability of the genistein-exposed and non-exposed cells, the percentage of viablenon-viable cells was determined, using trypan blue exclusivity stain. Cell growth and viability was measured by adding 0.4% trypan blue in 0.9% saline to a 50% diKumi-Diaka and Butler

Biology of the Cell 92 (2000) 115-124 lution, and cells were counted, using the hemocytometer according to standard procedure (Pienata and Lehr, 1993). Briefly, 0.5 mL of the trypan blue solution was transferred to a test tube and 0.3 mL of phosphate buffered saline (PBS) plus 0.2 mL of the trypsinized cell suspension (dilution factor of 5) were added. The final solution was thoroughly and gently mixed and allowed standing for 5 min. Then a drop of this dye-cell suspension was loaded onto both chambers of the hemocytometer. Cells were examined and counted in duplicates under light microscope at 200 x (Olympus BH2). Concentration and total number of cells were determined, and percentage cell viability was calculated by the formula: No. of viable cells (unstained cells) Cell viability (%) = x 100 Total no. of cells Thus the percentage of viable and non-viable cells (spontaneous and treatment-induced death) was determined for the genistein-exposed and non-exposed controls.

2.2.2. MTT (tetrazolium} assay: growth and cellular metabolic activity At each time point of incubation, MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetral-zolium bromide, 2.5 m g / m L ) assay was performed on the adherent cells in selected wells. Briefly, 100 pL of the stock MTT solution was added to 0.5 mL of media + cells in each well, and the plate incubated for 60 min at 37°C. After the incubation the loosely attached cells were harvested and transferred into labeled microcentrifuge tubes. The purple color indicated a reduction of the MTT dye into insoluble formazan within the cells. The cell-dye solution was centrifuged at 1000 r p m for 10 rain and the supernatant was removed. The pelleted cells were lysed with 200 pL lysing solution (0.33 ml HC1 in 100 mL isopropanol). The resultant solution was transferred into correspondingly labeled wells in 96-well MTP, and absorbance read at 620 nm, using an ELISA plate reader (NX 1001 multi-font). The data obtained from the MTT cellular metabolic assay were correlated with the results of the trypan blue viability assay. The potential effect of DMSO solvent on the growth and viability of TM4 cells was tested in a preliminary study. DMSO solution was added to the culture media at the final concentration of 0.1% that was the highest concentration of DMSO used in the genistein-treated cell media. No significant differences in cell growth and viability were observed between the TM4 cells without DMSO and TM4-DMSO cells; which indicated that DMSO did not influence the outcome of the experiment.

Caspase-3 protease activation

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2.3. Genistein-induced apoptosis and caspase protease activation In this phase of the experiment, TM4 cells were plated at a density of 2.25 x 106 cells/500 pL/well in: i) vehicle (DMSO) alone as a negative control (G0); ii) G15_60, (15, 30, 45, and 60 btg/mL genistein); iii) G0_60 + Z-VAD-FMK (caspase-3 inhibitor); and iv) 1% sodium azide (naz), and all plates were incubated for up to 24 h at 37°C and 5% CO 2. Each treatment was plated in replicates of six wells per treatment in 24-well plates. At 6, 12 and 24 h post-incubation, cells were processed for determination of treatment-induced apoptosis and caspase-3 (CPP32) enzymatic expression, as described below (sections 2.3.1. and 2.3.2.).

2.3.1. Fluorescence microscopy: morphological assessment of apoptosis and necrosis After 6, 12 and 24 h of incubation, an equal number of wells in each treatment group was processed for assessment of apoptosis and / or necrosis. Adherent cells were trypsinized and added to the floating cells for centrifugation at 10 000 rpm for 10 min. Three wells of each treatment were used for CPP32 expression and three wells for apoptosis-necrosis assessment. Apoptotic cells with condensed a n d / o r fragmented nuclei were viewed by Hoechst 33342-Propidium iodide (PI) nuclear staining, using the Vybrant TM Apoptosis Assay Kit no. 5 (V-13244) (Molecular Probes, Eugene, USA). Briefly, cells were washed in cold phosphate-buffered saline (PBS) and the concentration adjusted to about 2 x 106cells/mL in PBS. To 0.6 mL of cell suspension, 1 btL of Hoechst 33342 + i blL of PI was added and the cell suspension incubated on ice for 30 min in the dark. Soon after the incubation, one to two drops of the stained suspension were put on a slide and covered with cover slip. The p r e p a r e d slides were examined for nuclear staining and morphologic characteristics of the cells using epifluorescent objective at 400 x. Morphologically, the nuclei of apoptotic cells became smaller, condensed and hyperfluorescent when labeled with Hoechst 33342 nuclear stains (Molecular Probes, Eugene, USA). Hoechst 33342 and PI vary in their spectral characteristics and in their ability to penetrate cells. The blue-fluorescent Hoechst 33342 stains the condensed chromatin of apoptotic cells more brightly than the looser chromatin of norreal cells. The red-fluorescent p r o p i d i u m iodide is permeant only to necrotic cells. Consequently, cell death was quantitated by counting 50 cells in separate fields of view, and noting the percentage of normal, apoptotic and necrotic cells based on the morphological and staining characteristics of the cells (Cotter and Martin, 1996). Fluoroscopic determination of apoptosis has been previously demonstrated and confirmed by the DNA fragmentation tunnel assay (Kumi-Diaka et al., 1998, 1999).

Kumi-Diaka and Butler

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Biology of the Cell 92 (2000) 115-124

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Genistein (gg/mL) Figure 1. The effect of genistein on growth and proliferation of Sertoli cells (TM4). Cells were seeded at 0.55 x 105 cells/well and co-incubated with varying concentrations of genistein at 37°C, 5% for up to 24 h. Cell viability was assessed by trypan blue exclusion. The percentage of live cells was calculated as a ratio of A62onm of treated and untreated control cells at 6, 12 and 24 h of incubation. Columns are means for two independent experiments; bars are S.E.

2.3.2. Caspase activity assay After centrifugation of the treated cells as previously described, caspase-3 enzymatic activity was determined using CasPASE Apoptosis Assay Kit (Cat. #-786-200/50)(Geno Technology Inc. St. Louis MO, USA). Prior to use, caspase kit reagents were first prepared, followed by lysis of the treated cells according to a modification of the manufacturer's protocol. In this study, cells were lysed with a sonicator (Misonix, Farmingdale, NY, USA), and caspase-3 enzymatic activity in the lysates determined as described. Briefly, microtiter wells were set up in duplicates for controls, blank, and test cells (lysates). Then 50 pL of 2 x CasPACE assay buffer was transferred into each well followed by addition of 50 ~tL of the cell lysate to the wells, and addition of 5 pL of the caspase substrate, Ac-DEV-AFC. A few minutes were allowed for reaction, and the plate was read (at a zero initial time) on ELIZA micro-plate reader (NX 1001 multi-font) at 405 nm. The plate was then incubated at 37°C for 2.5 h and the absorbance read again at 405 nrn wavelength. The level of caspase-3 (CPP32) enzymatic activity in the cell lysate was directly proportional to the color reaction. Therefore, to quantitate CPP32 expression in the lysates, the fold increase in caspase-3 (CPP32) protease activity was determined by comparing the absorbance from the treated samples with the non-treated controls. To further confirm, compare and establish non-specific protease activity, control experiments were repeated Caspase-3 protease activation

and run with or without caspase-3 specific inhibitor, ZVAD-FMK. Briefly, reaction wells of the MTP were prepared to contain the following: a) 5 pL lysate + 50 }.tLof 2 x assay buffer + 1 btL of Z-VAD-FMK + 5 gL Ac-DEVAFC conjugate; b) 50 pL of 2 x assay buffer + 5 btL AcDEV-AFC +1 pL distilled water; and c) 5 btL cell lysate + 50 btL of 2 x assay buffer + i pL distilled water. The plate was incubated at 37°C for 2.5 h and the absorbance read at 405 run as described above. The inter-treatment data were compared to ascertain and confirm the effect of ZVAD-FMK on caspase-3 enzymatic expression.

2.4. Statistical analysis Each experiment was performed in duplicate and repeated twice to confirm similar results. Significance of the differences between the mean values was determined using the Student's t-test (Wilkinson, 1990), and considering P < 0.05 to be statistically significant.

3. RESULTS 3.1. Cell viability assays. M'B" and TB exclusion In previous studies, we showed that genistein induced a concentration-dependent dual effect with regard to growth promotion and inhibition in TM3, TM4 and GCKumi-Diaka and Butler

Biology of the Cell 92 (2000) 115-124

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Effect of different concentrations of genistein on Leydig cells. Leydig cells were treated with genistein and incubated for 24 h. Cell viability was then determined by trypan blue (TB) stain and MTT assay for metabolic activity. The percentage of cell viability was calculated as a ratio of absorbance of treated and untreated cells. Data points are means for four wells from two independent experiments, expressed relative to untreated controls. The S.E. was consistently < 10% of the means in both the MTT and TB assays. The correlation between MTT and trypan blue results was highly significant (MTT versus TB, r = 0.98; P < 0.003).

lspg testis cells (Kumi-Diaka et al., 1999). The results from the present study are in conformity with the previous studies. As shown infigures 1 and 2, both the viability (trypan blue exclusion) and metabolic activity (MTF assay) of TM4 cells decreased with increasing doses of genistein above 15 ~ g / m L and exposure time of > 6 h. The differences in percentage of live cells between the treatment groups at > 15 btg/mL of genistein were significant (P < 0.05) at all time points (6, 12 and 24 h) (figure 1); thus demonstrating the dose- and time-dependent effects of genistein on viability and metabolic activities of Sertoli cells. The Sertoli (TM4) cells were exposed to increasing concentrations of genistein isoflavone at 37°C and 5% CO 2 for up to 24 h. The data obtained revealed that the correlation between the genistein concentration and the two viability-metabolic activity assays (TB, MTT) was significant (table !). The results demonstrated a significant inverse relationship between genistein concentration and cell viability; and a significantly high positive correlation between the MTT and TB assays (MT versus TB, r = 0.862, P < 0.003) (table/); this offers a strong support for the reliability of the data.

3.2. Genistein-induced apoptosis In this phase of the study, genistein-induced cell death and the type of cell death in TM4 was investigated. The Caspase-3 protease activation

Hoechst 33258-PI nuclear D N A stain provided both qualitative and quantitative data on the mode and degree of cell death. The fluorescent examination of the Hoechst 33258-PI stained TM4 cells revealed that genistein induced a concentration-dependent apoptosis and necrosis in the treated cells (figure 3). Apoptotic cells were identified (and quantitated) by the staining and morphologic characteristics of the cells; manifested by contraction of cell size and condensation of the DNA chromatin. At higher doses of genistein, necrotic cells were present in the cell population. With increasing dose of genistein, the percentage of cells with necrotic morphology increased progressively and significantly as the number of apoptotic cells levels up (figure 3). Pretreatment of cells (G15_60 + Z-VAD-FMK) with caspase3 inhibitor (Z-VAD-FMK) almost completely blocked genistein-induced apoptosis and necrosis at all levels of genistein dose. After 24 h of incubation, CPP32 enzymatic activity averaged 17.93% and 4.13% apoptosis respectively in G60-treated and G60 + Z-VAD-FMKtreated cells, with significant difference in caspase activity (P < 0.001) between the treatments. This observation demonstrates the significance of caspase-3 protease activity in the apoptotic pathways of genistein. At 1% concentration, sodium azide (naz) induced a time-dependent necrosis in TM4 with no indiKumi-Diaka and Butler

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Biology of the Cell 92 (2000) 115-124

Table I. Correlation coefficients between the tetrazolium assay (MTT), trypan blue exclusion (TB), and genistein concentration (Gn). Parameters

Gn versus MTT TB versus Gn MTT versusTB

r

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P-value *

-0.958 -0.912 0.982

0.918 0.831 0.964

0.010 0,031 0.003

• P < 0.01, 0.031 = significant; P < 0.003 = highly significant.

cation of significant apoptosis. At 24 h of incubation, naz-induced necrosis averaged 21.18 + 4.31 percent of the total cells counted. Previous studies demonstrated that both the fluorescence dye DNA staining and D N A fragmentation assay correlate significantly, thus confirming the reliability of the fluorescence stain in studying apoptosis.

3.3. Caspase enzymatic activity Following the induction of apoptosis by genistein, further studies were conducted to assess the involvement of caspase-3 protease activity in genistein-induced apoptosis in testis cells. The present data indicate that caspase-3 activation is significant in genistein-induced apoptosis. The results revealed that expression of CPP32 enzymatic activity in TM4 cells was dependent on the dose of genistein and time of exposure (figure 4); and that the level of caspase-3 activity was directly proportional to the color reaction and hence to the absorbance of the lysates (figure 4). After 24 h of incubation, CPP32 enzymatic activity in the genistein-exposed TM4 cells was 1.1-, 1.3-, 3.3- and 3.1-fold over non-treated controls at genistein doses of 15, 30, 45, and 60 p g / mL respectively. The inter-treatment differences in CPP32 expression were significant (P < 0.05 to P < 0.001). The pattern of CPP32 activity in the treated cells followed the dose- and time-dependent pattern of apoptotic cell death (figure 3). The data revealed a direct correlation between caspase-3 enzymatic activity and percentage apoptosis; percent of apoptotic cells increased correspondingly with increased caspase-3 expression (figures 3, 4). Furthermore, in the presence of caspase-3 inhibitor (pre-treatment of cells with z-VADFMK) genistein-induced expression of CPP32 protease activity was almost completely blocked (figure 5), thus demonstrating the specificity of CPP32 in genistein. Sodium azide exposure to TM4 cells induced necrosis in the cells, but no significant apoptosis, and only minimal expression of CPP32 enzymatic activity. At 24 h of incubation, percentage necrosis, apoptosis and CPP32 activation were 20.93 _+4.04 %, 0.07 +_0.01%, and Caspase-3 protease activation

1.01-fold over the control (1% increase over control), respectively. The overall data demonstrate that pre-treatment of TM4 cells with the caspase-3 specific inhibitor, ZVAD-FMK, significantly blocked genistein-induced expression of caspase-3 enzymatic activity and genistein-induced apoptosis, thus demonstrating the mediator role of the CPP32 in genistein-induced apoptosis.

4. DISCUSSION In this study the role of caspase-3 (CPP32) protease on genistein-induced cell death in Sertoli (TM4) cells was investigated. Fluorescence microscopy with Hoechst33258-PI nuclear stain was used to distinguish between apoptosis and necrosis pathways of cell death. The viability of the genistein-exposed cells relative to the nonexposed cells was assessed with both the trypan blue exclusion (TB) and MTT assays. Both assays demonstrated a significant inverse relationship between the dose of genistein and cell viability. Cell viability decreased with increasing genistein dose. The significantly high positive correlation between the two assays (MTT and TB) demonstrates the reliability of the data. In previous studies we demonstrated that the response of various testicular cells to genistein was both doseand time-dependent (Kumi-Diaka et al., 1999). As indicated infigure 1, genistein manifested a concentrationand time-dependent dual effect with regard to growth promotion and inhibition on Sertoli (TM4) cells. At lower concentration, up to 15 p g / m L , genistein stimulated TM4 cell growth; and at a dose of > 15 p g / m L , progressive inhibition of growth was manifested with increasing exposure time (figure 1). The decrease in percentage of live cells with increasing dose of genistein and exposure time was due to genistein-induced cell death, as revealed by the follow-up experiments. These dual biological activities of genistein in TM4 cells are in conformity with similar genistein action on a variety of cells, including cancer and non-cancerous cells (Akiyama et al., 1987; Okura et al., 1988; Murrill et al., 1996; Kyle et al., 1997). The results indicate that genistein manifests dual cell death pathways, apoptosis and necrosis, depending on the dose. At lower doses of genistein, cell death in TM4 cells was primarily through the apoptotic mechanism. This result was in conformity with that of previous studies (Kumi-Diaka et al., 1998). However, at higher genistein doses, cell death by apoptosis gradually declined in favor of increasing number of cells with necrotic morphology. This switch of apoptosis to necrosis at high concentrations of genistein is most likely due to toxic effects of genistein at higher doses. It has been hypothesized that a decrease in intracellular ATP might switch apoptosis to necrosis (Higuchi et al., 1998). However, it is yet to be determined if increasing Kumi-Diaka and Butler

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Genistein conc (pg/mL) F i g u r e 3. Genistein-induced apoptosis and necrosis in Sertoli (TM4) cells after 24 h of incubation.TM4 cells were seeded at a

density of 1.25 x 105cells/well and co-cultured with genistein (Go_6opg/mL) for 24 h at 37°C and 5% CO2. Both genisteininduced apoptosis and necrosis were dose-dependent. Percentages were calculated as a ratio of apoptotic cells to the total cell count a ratio of apoptotic ceils to the total cell count. Each data point represents the mean _+ standard error of the mean _+ standard error of the mean of two independent experiments. Bar = S.E.M.S.E.M. was consistently < 10% of the means in necrosis, too low to appear in the graph.

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exposed to varying concentrations of genistein and incubated for 6, 12 or 24 h at 37°C, 5% CO2 and assayed for CPP32 activity as described in Materials and methods. Caspase-3 enzymatic activity correlated with color intensity (Absorbance-A4o5). CPP32 expression in TM4 cells showed dependency on dose of genistein and exposure time. Each data point represents the mean -+ standard error of the mean of two independent experiments. Bar = S.E.M. Kumi-Diaka and Butler

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doses of genistein could decrease intracellular ATP in testis cell. Other agents could induce the same pattern of cell death in certain cells. The induction of apoptosis in HeLa cells at lower doses of isothiocyanates, and necrosis at higher doses has been reported (Yu et al., 1998). The induction of necrosis in TM4 cells by sodium azide (naz) as seen in this study, is most probably due to the cytotoxic effects of this potentially toxic environmental chemical. Sodium azide induces a non-apoptotic cell death on mouse thymocytes (Wyllie et al., 1984). Our results also showed that genistein activated caspase-3 (CPP32) enzymatic activity in a dose-dependent manner in TM4 cells; and that both genisteininduced CPP32 activity and genistein-induced apoptosis was inhibited by Z-VAD-FMK, a CPP32 specific inhibitor. These results indicate that caspase-3 protease activation is one of the principal pathways in genisteininduced apoptosis in TM4 cells. This observation is in agreement with the expression of multiple species of CPP32 enzymatic activity in other apoptotic cells (Miura et al., 1993; Faleim et al., 1997). Furthermore, treatment of TM4 cells with naz-induced necrosis on the cells without significant activation and expression of caspase-3 enzymatic activity and no significant apoptosis, further indicating that activation of CPP32 enzymatic activity could be a specific biochemical event in the molecular pathways of genistein-induced apoptosis in TM4 cells. Mediation of caspase-3 in the induction of apoptosis in other cells by other external stimuli has been reported (Liu and Zou, 1997; Higuchi et al., 1998; Caspase-3 protease activation

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CPP32 enzymatic activity in TM4 and TM4 + z-VAD-FMK (CPP32 inhibitor) after 24 h of incubation. The cells were exposed to varying concentrations of genistein and incubated for up to 24 h; caspase-3 enzymatic activity correlated with color intensity (absorbance); and showed dependency on genistein concentration and time of exposure. CPP32 inhibitor (z-VAD-FMK) significantly inhibited caspase-3 enzymatic expression in the treated cells at all levels of genistein concentration. Each data point represents the mean + standard error (bar) of the mean of two experimental data. In the + z-VAD-FMK data the S.E. was consistently < 10% of the means.

Yu et al., 1998; Ishisaki et al., 1999). The caspase family protease activation has been considered as a key step in the induction of cell death in some tissues a n d / o r cells (Higuchi et al., 1998). It is clear from these results that genistein could kill TM4 cells by two different mechanisms: induction of apoptosis and induction of necrosis, depending on the dosage. The mechanistic differences, if any, between the induction of apoptosis and necrosis were not investigated in this study. However, other investigators have indicated that the same signals that induce apoptosis in some cells could also induce necrosis in the same cells under certain conditions (Higuchi et al., 1998). The present results are in conformity with the reported observations. Furthermore, the involvement of caspases in necrotic cell death and apoptotic cell death in certain cells has been demonstrated (CasciolaRosen et al., 1995; Datta et al., 1997; Yu et al., 1998). Given its diversity of substrates, caspase-3 protease could serve as a general mediator of apoptosis induced by a variety of external stimuli in a variety of cells and tissues (Barry Behnke, 1990; Nagata and Golstein, 1995; Yu et al., 1998; Kumi-Diaka et al., 1999). The present study indicates that CPP32 is a major mediator in the molecular pathways of genistein-induced apoptosis in testicular cells. Figures 3 and 4 indicate induction of 2.5 to 10% apoptosis between G O and G30 without significant caspase-3 protease activation. This level of apoptosis could have been spontaneously induced by other stimuli in the microenvironment of the growth medium; most likely low levels of caspase-3 protease. Kumi-Diaka and Butler

Biology of the Cell 92 (2000) 115-124 Up to 30 ~tg/mL of genistein, induction of apoptosis increased as caspase-3 activity increased; and caspase3 activity optimized with significant outburst at > 30 p g / m L of genistein. In a parallel trial experiment using fluorometric assay, we showed that the irreversible caspase-3 inhibitor, z-VAD-FMK, significantly limited caspase-3 expression and spontaneous apoptosis induction in both genistein-treated and untreated TM4 and TM3 cells. The high sensitivity of fluorometric assay made possible, the detection and measurement of very low levels of genistein-induced caspase-3 protease activity. Caspase-3 protease has been demonstrated to play a critical role in spontaneous apoptosis (Nicholson et al., 1995). This report lends support to the results in the present studies. Several studies indicate that some potent chemopreventive drugs induce apoptosis in some tumors, resulting in the prevention of cancer (Tsujii and DuBois, 1995; Thompson et al., 1997). Thus induction of apoptosis may represent a major mechanism by which genistein isoflavone exerts its chemopreventive actions in some cancers and other chronic diseases. It is concluded that genistein exerts dose- and timedependent effects on TM4 testis cells; that low doses of genistein induced apoptosis and high doses of genistein induced necrotic cell death; that genisteininduced apoptosis in TM4 cells was associated with activation of caspase-3 enzymatic (CPP32) activity; that naz induced necrosis without activation of CPP32 activity and no induction of apoptosis in TM4 cells; that genistein-induction of apoptosis and necrosis in TM4 cells was inhibited by CPP32 specific inhibitor, zVAD-FMK. These results present evidence that the mechanism of genistein-induced apoptosis includes expression and amplification of caspase-3 protease; and strongly indicate caspase-3 (CPP332) mediation in the molecular pathways of genistein-induced apoptosis in TM4 testis cells. The fact that the chemopreventive role of several cancer drugs is due to induction of apoptosis justifies the inclusion of genistein in the therapeutic considerations and formulations for treatment of malignant diseases such as prostate and testicular cancers.

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