Genistein‐induced G2‐m arrest,P21WAF1 upregulation,and apoptosis in a non‐small‐cell lung cancer cell line

Lung cancer is the leading cause of cancer‐related death in the world, with increasing incidence in many developed countries. Epidemiological data suggest that consumption of soy products (the isoflavone genistein) may be associated with a decreased risk of breast and prostate cancer; however, such studies are not available for lung cancer. We investigated cell growth inhibition, modulation in gene expression, and induction of apoptosis by genistein in H460 non‐small lung cancer cells. Genistein inhibited H460 cell growth in a dose‐dependent manner. Flow‐cytometric analysis showed that 30 μM genistein arrested cell cycle progression at the G2‐M phase. 4,6‐Diamidino‐2‐phenylindole staining, flow‐cytometric analysis, and DNA laddering were used to investigate apoptotic cell death, and the results show that 30 μM genistein can cause typical DNA laddering, a hallmark for apoptosis. In addition, flow cytometry and 4,6‐diamidino‐2‐phenylindole staining showed induction of apoptosis by genistein. Our investigation also demonstrated the modulation of p21^WAF1 by Western blot analysis of cell lysates obtained from cultured cells treated with 30 and 50 μM genistein for 24, 48, and 72 hours. Simultaneously, im‐munocytochemical staining was conducted for the expression of p21^WAF1 protein. Our results showed that genistein can upregulate p21^WAFI expression in genistein‐treated cells. From these results, we conclude that genistein may act as an anticancer agent, and further studies may prove its efficacy in non‐small lung cancer cells. Thus the biological effects of genistein may, indeed, be due to the modulation of cell growth, cell death, and cell cycle regulatory molecules.     

Differential Effect of Grape Seed Extract against Human Non-small-Cell Lung Cancer Cells: The Role of Reactive Oxygen Species and Apoptosis Induction

The present study examines grape seed extract (GSE) efficacy against a series of non-small-cell lung cancer (NSCLC) cell lines that differ in their Kras and p53 status to establish GSE potential as a cytotoxic agent against a wide range of lung cancer cells. GSE suppressed growth and induced apoptotic death in NSCLC cells irrespective of their k-Ras status, with more sensitivity toward H460 and H322 (wt k-Ras) than A549 and H1299 cells (mutated k-Ras). Mechanistic studies in A549 and H460 cells, selected, based on comparative efficacy of GSE at higher and lower doses, respectively, showed that apoptotic death involves cytochrome c release associated caspases 9 and 3 activation, and poly (ADP-ribosyl) polymerase cleavage, strong phosphorylation of ERK1/2 and JNK1/2, downregulation of cell survival proteins, and upregulated proapoptotic Bak expression. Importantly, GSE treatment caused a strong superoxide radical-associated oxidative stress, significantly decreased intracellular reduced glutathione levels, suggesting, for the first time, the involvement of GSE-caused oxidative stress in its apoptotic inducing activity in these cells. Because GSE is a widely-consumed dietary agent with no known untoward effects, our results support future studies to establish GSE efficacy and usefulness against NSCLC control.     

Genistein-induced upregulation of p21WAF1, downregulation of cyclin B, and induction of apoptosis in prostate cancer cells

Increased soy consumption in Asian diets, resulting in increased serum isoflavone levels, has been associated with a decreased risk for prostate adenocarcinoma (PCa). The isoflavone genistein is believed to be the anticancer agent found in soy, and significant levels of genistein have been detected in human prostatic fluid, implicating the role of genistein in PCa prevention. Recent studies have demonstrated genistein's ability to inhibit cell growth and induce apoptosis in several cell lines; however, the molecular mechanisms of genistein's effect are not known. We have evaluated the mechanism by which genistein may inhibit PCa cell growth. Here we report that genistein inhibits PCa cell growth in culture in a dose-dependent manner, which is accompanied by a G2/M cell cycle arrest. Cell growth inhibition was observed with concomitant downregulation of cyclin B, upregulation of the p21WAF1 growth-inhibitory protein, and induction of apoptosis. Collectively, these results provide experimental evidence for a novel effect of genistein on cell cycle gene regulation, resulting in the inhibition of cell growth and ultimate demise of tumor cells.     

Role of wild-type p53 in apoptotic and non-apoptotic cell death induced by X-irradiation and heat treatment in p53-mutated mouse M10 cells

The sensitizing effects of wild-type p53 on X-ray-induced cell death and on heat-induced apoptosis in M10, a radiosensitive and Trp53 (mouse p53 gene)-mutated lymphoma cell line which dies through necrosis by X-irradiation, were investigated using three M10 derived transfectants with wild-type TP53 (human p53 gene). Cell death was determined by colony formation and/or dye exclusion test, and apoptosis was detected as the changes in nuclear morphology by Giemsa staining. Expression of wild-type p53 protein increased radiosensitivity of cell death as determined by both clonogenic and dye exclusion assays. This increase in radiosensitivity was attributable largely to apoptosis induction in addition to a small enhancement of necrosis. Interestingly neither pathway to cell death was accompanied by caspase-3 activation. On the other hand, heat-induced caspase-3 dependent apoptotic cell death without transfection was further increased by the transfection of wild-type p53. In conclusion, the introduction of wild-type p53 enhanced apoptotic cell death by X-rays or heat via different mechanisms that do or do not activate caspase-3, respectively. In addition, p53 also enhanced the X-ray-induced necrosis in M10 cells.     

Genistein‐induced upregulation of p21 WAF1 , downregulation of cyclin B,and induction of apoptosis in prostate cancer cells

Increased soy consumption in Asian diets, resulting in increased serum isoflavone levels, has been associated with a decreased risk for prostate adenocarcinoma (PCa). The isoflavone genistein is believed to be the anticancer agent found in soy, and significant levels of genistein have been detected in human prostatic fluid, implicating the role of genistein in PCa prevention. Recent studies have demonstrated genistein's ability to inhibit cell growth and induce apoptosis in several cell lines; however, the molecular mechanisms of genistein's effect are not known. We have evaluated the mechanism by which genistein may inhibit PCa cell growth. Here we report that genistein inhibits PCa cell growth in culture in a dose‐dependent manner, which is accompanied by a G₂/M cell cycle arrest. Cell growth inhibition was observed with concomitant downregulation of cyclin B, upregulation of the p21^WAF1 growth‐inhibitory protein, and induction of apoptosis. Collectively, these results provide experimental evidence for a novel effect of genistein on cell cycle gene regulation, resulting in the inhibition of cell growth and ultimate demise of tumor cells.     

Diallyl disulfide (DADS) induces the antitumorigenic NSAID-activated gene (NAG-1) by a p53-dependent mechanism in human colorectal HCT 116 cells

Garlic is appealing as an anti-carcinogenic agent due to its ability to induce apoptosis in vitro and inhibit the formation and growth of tumors in animals in vivo. Diallyl disulfide (DADS) is a constituent of garlic that suppresses neoplastic cell growth and induces apoptosis. We examined the effects of DADS on various cancer cell lines to better understand its effect on apoptosis and apoptosis-related genes. The nonsteroidal anti-inflammatory drug (NSAID)-activated gene (NAG-1) has proapoptotic and antitumorigenic activities and is upregulated by anticancer agents such as NSAIDs. In this study, human colorectal HCT-116 (wild-type p53), HCT-15 (p53 mutant) and human prostate PC-3 (p53 mutant) cells were exposed to DADS. DADS inhibited cell proliferation in all cell lines albeit to a lesser extent in HCT-15 and PC-3 cells at 11.5 and 23 micromol/L. In HCT-116 cells, DADS induced p53 and NAG-1 in a dose-dependent manner and the induction of p53 preceded that of NAG-1. In HCT-116 cells, NAG-1 protein expression was increased 2.4-fold +/- 0.6 at 4.6 micromol/L and 6.1-fold +/- 1.7 at 23 micromol/L DADS, whereas p53 was induced 1.5-fold +/- 0.1 and 2.3-fold +/- 0.4. DADS did not induce NAG-1 or p53 in p53 mutant cell lines; however, NAG-1 expression was induced by sulindac sulfide. HCT-116 cells treated with 4.6 and 23 micromol/L DADS resulted in a 1.9- and 2.9-fold increase in apoptosis, respectively. In contrast, 23 micromol/L DADS induced apoptosis only 1.8-fold in HCT-15 cells and not at all in PC-3 cells. Thus, DADS-induced apoptosis and NAG-1 protein expression appear to occur via p53.     

对曲古抑菌素A敏感的人肺腺癌细胞株的初步筛选

目的初步筛选不同p53表达型肺癌细胞株对曲古抑菌素A（TSA）敏感性不同的影响因素。方法分别以体外药物敏感试验（MTT法）、流式细胞术观察TSA处理3种肺癌细胞株（H1299为p53缺失型,H322为p53变异型,A549为p53野生型）后,其生长抑制情况以及细胞周期、细胞凋亡等指标的变化;用Western blot法检测3种细胞的人类表皮生长因子受体2（HER2）表达水平。结果TSA对3种细胞均有抑制作用,在96h时对A549的IC50抑制作用明显低于对H1299和H322的IC50（P〈0.05）,A549细胞凋亡也明显多于H1299和H322的细胞凋亡（P〈0.05）。3种细胞株HER2蛋白表达水平由高到低依次为H322、A549和H1299,与TSA的IC50值之间无相关性。结论TSA对p53不同表达型人肺腺癌细胞株均有生长抑制作用,肺腺癌细胞株p53的不同表达型可能对TSA的敏感性产生影响,而HER2的表达强度不影响TSA的敏感性。     

Delayed expression of apoptosis in X-irradiated human leukemic MOLT-4 cells transfected with mutant p53

The effects of X-rays on cell survival, apoptosis, and long-term response in the development of cell death as measured by the dye exclusion test were studied in human leukemic MOLT-4 cells (p53 wild-type) stably transfected with a mutant p53 cDNA expression vector. Cell survival, as determined from colony-forming ability, was increased in an expression level dependent manner, but the increase was partial even with the highest-expressing clone (B3). This contrasts with the prior observation that cell death and apoptosis in B3 are completely inhibited at 24 h after irradiation with 1.8 Gy of X-rays. The examination of B3 cells incubated for longer than 24 h after X-irradiation showed a delay in the induction of cell death and apoptosis. Western blot analysis revealed that the time required to reach the highest level of wild-type p53 protein in B3 was longer than the time in MOLT-4 and that the p53 may be stabilized by the phosphorylation at Ser-15. These results suggest that the introduction of mutant p53 into MOLT-4 merely delays the development of apoptosis, during which the cells could repair the damage induced by X-rays, and results in the partial increase in cell survival.     

金雀异黄素诱导人胃癌细胞凋亡的机制研究

采用 4’ ,6 二乙酰基 2 苯基吲哚染色、琼脂糖凝胶电泳实验观察了金雀异黄素 (Gen)对体外培养的人胃癌SGC 790 1细胞的凋亡诱导作用 ,并采用WesternBlot法检测Gen对人胃癌细胞p5 3、Caspase 3蛋白的表达情况。结果显示 ,Gen诱导了胃癌细胞凋亡 ,并抑制突变型p5 3蛋白表达 ,增加Caspase 3蛋白表达。结果提示 ,Gen抑制突变型p5 3蛋白表达 ,增加Caspase 3蛋白表达 ,可能是其诱导胃癌细胞凋亡的机制之一     

Effects of Selenite and Genistein on G2/M Cell Cycle Arrest and Apoptosis in Human Prostate Cancer Cells

Combination of chemopreventive agents with distinct molecular mechanisms is considered to offer a potential for enhancing cancer prevention efficacy while minimizing toxicity. Here we report two chemopreventive agents, selenite and genistein, that have synergistic effects on apoptosis, cell cycle arrest, and associated signaling pathways in p53-expressing LNCaP and p53-null PC3 prostate cancer cells. We show that selenite induced apoptosis only, whereas genistein induced both apoptosis and G ₂ /M cell cycle arrest. Combination of these two agents exhibited enhanced effects, which were slightly greater in LNCaP than PC3 cells. Selenite or genistein alone upregulated protein levels of p53 in LNCaP cells only and p21 ^waf1 and Bax in both cell lines. Additionally, genistein inhibited AKT phosphorylation. Downregulation of AKT by siRNA caused apoptosis and G ₂ /M cell cycle arrest and masked the effects of genistein. Treatment with insulin-like growth factor I (IGF-I) elevated levels of total and phosphorylated AKT and suppressed the effects of genistein. Neither downregulation of AKT nor IGF-I treatment altered the cellular effects of selenite. Our study demonstrates that selenium and genistein act via different molecular mechanisms and exhibit enhanced anticancer effects, suggesting that a combination of selenium and genistein may offer better efficacy and reduction of toxicity in prostate cancer prevention.     

Radiation-induced apoptosis in peritoneal resident macrophages of C3H mice

Gamma ray-radiation induced significant apoptosis in peritoneal resident macrophages (PRMs) of C3H/HeJ (C3H) mice, but not in other strains of mice. To investigate the role of DNA damage in the apoptosis, DNA damage was quantified in PRMs by use of the alkaline single-cell gel electrophoresis (Comet) assay. No significant difference was found between C3H and C57Black/6 mice in either radiation-induced DNA damage or repair. Radiation induced apoptosis at the same levels in PRMs of p53 knockout mice and atm knockout mice as those of wild-type C3H mice; however radiation-induced apoptosis was significantly less extensive in the thymocytes of these mutant mice than in those of wild-type mice. Apoptosis was also induced at the same level by an irradiation in PRMs of C3H scid mice as in those of wild-type C3H mice. Therefore it was suggested that radiation-induced DNA damage and TP53, ATM, or DNA-PK-mediated cellular responses occurring downstream thereof were not involved in the radiation-induced apoptotic cell death in C3H mouse PRMs.     

Influence of genistein isoflavone on matrix metalloproteinase-2 expression in prostate cancer cells

We investigated the expression of matrix metalloproteinase (MMP)-2 in human LNCaP and PC3 prostate cancer cell lines in response to genistein exposure. Initially we studied the phytosensitivity of the cells to genistein using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay to determine percentage cell viability/inhibition and the terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick end-labeling apoptosis assay to assess the type of cell death. The results revealed that genistein inhibited growth and proliferation in both PC3 (hormone-dependent) and LNCaP (hormone-independent) prostate cancer cell lines, that there was no significant difference in sensitivity to genistein between PC3 and LNCaP cells, and that the effect of genistein on the cells was dose- and time-dependent. The results also revealed that inhibition of cell growth in both PC3 and LNCaP cells was predominantly due to apoptotic cell death. These results were consistent with data in previous studies. This was followed by determination of the MMP-2 profile in response to genistein treatment. The results indicated a significant dose- and time-dependent inhibition of MMP-2 expression levels in both cells, with a highly significant negative correlation between MMP-2 levels and concentration of genistein. This is of phytotherapeutic significance in view of the pivotal role of MMP-2 expression in the pathogenesis of prostate cancer. Increasing expression of MMPs has been identified in many human cancers, including prostate cancer. Our findings indicate that genistein could be a potent therapeutic inhibitor of MMP-2 in line with current concepts of targeted treatment.     

Genistein-induced apoptosis of p815 mastocytoma cells is mediated by Bax and augmented by a proteasome inhibitor,lactacystin

Although genistein has been demonstrated to induce apoptosis of various cells, there is no report of its effect on mast cell proliferation. Here we show that genistein reduced the viability of mast cell tumor cell lines, p815 and RBL-2H, but not of a human mast cell line, HMC-1. Further investigation on its growth-inhibitory mechanism was undertaken on p815 mastocytoma cells. Genistein induced G2/M arrest and subsequent apoptotic death. p815 cells undergoing apoptosis showed many apoptotic manifestations, such as reduction of mitochondrial membrane potential, release of cytochrome c to cytosol, translocation of apoptosis-inducing factor to nucleus, activation of caspase-3, nuclear condensation, and generation of DNA fragmentation. Genistein treatment resulted in the increase of Bax expression and its translocation into mitochondria, whereas expression levels of Bcl-2 remained unchanged. Proteasome activity decreased at the early time points after genistein treatment, but thereafter it fluctuated at increased levels. A proteasome inhibitor, lactacystin, potentiated the induction of apoptosis. Taken together, genistein-induced apoptosis of p815 mastocytoma cells is at least in part mediated by proteasome, Bax, apoptosis-inducing factor, and caspase and augmented by cotreatment with a proteasome inhibitor, lactacystin.     

Growth inhibition and induction of apoptosis in MCF-7 breast cancer cells by fermented soy milk

The effect of a fermented soy milk product (FSP) on various human breast carcinoma cell lines was investigated, and it was shown to have a growth-inhibitory effect, especially on MCF-7 cells. Thus the MCF-7 cell line was used to study the mechanism of action. In female severe combined immune deficiency mice implanted with MCF-7 cells, pretreatment with FSP significantly inhibited tumor growth. The inhibitory effect of FSP on MCF-7 cells seemed to be caused by the additive effects of a wide variety of constituents. The active components of FSP are mainly in the water phase, and the lipid-soluble fraction, which includes the soy isoflavones such as genistein and daidzein, is relatively ineffective. A variety of methods were used to demonstrate that FSP caused apoptotic cell death in MCF-7 cells. FSP induced generation of reactive oxygen species (ROS). Growth inhibition and ROS generation induced by FSP could be inhibited by catalase and deferoxamine, indicating that the ROS production probably was the cause of this apoptotic cell death. This study suggests that FSP retards tumor growth in vivo and can trigger apoptosis in vitro. It may, therefore, be a potential nutritional supplement in chemotherapy.     

Induction of apoptotic cell death by phytoestrogens by up-regulating the levels of phospho-p53 and p21 in normal and malignant estrogen receptor α-negative breast cells

In this study, we investigated the underlying mechanism by which phytoestrogens suppress the growth of normal (MCF-10A) and malignant (MDA-MB-231) estrogen receptor α (ERα)-negative breast cells. We hypothesized that phytoestrogen inhibits the proliferation of ERα-negative breast cancer cells. We found that all tested phytoestrogens (genistein, apigenin, and quercetin) suppressed the growth of both MCF-10A and MDA-MB-231 cells, as revealed by proliferation assays. These results were accompanied by an increase in the sub-G0/G1 apoptotic fractions as well as an increase in the cell population in the G2/M phase in both cell types, as revealed by cell cycle analysis. When we assessed the effect of phytoestrogens on the level of intracellular signaling molecules by Western blot analysis, we found that phytoestrogens increased the level of active p53 (phospho-p53) without changing the p53 level in both MCF-10A and MDA-MB-231 cells. Phytoestrogens also induced an increase in p21, a p53 target gene, and a decrease in either Bcl-xL or cyclin B1 in both cell types. In contrast, the protein levels of phosphatase and tensin homolog, cyclin D1, cell division control protein 2 homolog, phospho-cell division control protein 2 homolog, and p27 were not changed after phytoestrogen treatment. Our data indicate that phytoestrogens induce apoptotic cell death of ERα-negative breast cancer cells via p53-dependent pathway and suggest that phytoestrogens may be promising agents in the treatment and prevention of ERα-negative breast cancer.     

Genistein inhibits insulin-like growth factor-I receptor signaling in HT-29 human colon cancer cells: a possible mechanism of the growth inhibitory effect of Genistein

Genistein, a soy isoflavone, has attracted much attention for its chemopreventive properties. Overexpression and constitutive activation of receptor tyrosine kinases are frequent events in human cancer. Because genistein has previously been reported to decrease HT-29 cell growth, the present study compared the effects of genistein with daidzein on the protein levels of the members of the ErbB receptor family and insulin-like growth factor-I (IGF-I) receptor (IGF-IR). HT-29 cells were cultured in serum-free medium, with 0, 25, 50, or 100 micromol/L genistein, daidzein, and/or 10 nmol/L IGF-I. DNA synthesis was estimated by 5-bromo-2'-deoxyuridine incorporation. Apoptotic cells were analyzed by annexin-V staining followed by flow cytometry. Genistein inhibited viable HT-29 cell numbers, in a dose-dependent manner, whereas daidzein had no effect on cell growth. The decrease in cell growth caused by genistein was due to decreased DNA synthesis and apoptosis induction. Immunoblot analysis showed that neither genistein nor daidzein decreased the protein levels of either of the epidermal growth factor receptors, ErbB2 or ErbB3. Genistein did, however, decrease the IGF-IR protein levels, whereas daidzein had no effect. Genistein did not change the protein levels of insulin-receptor substrate-1 (IRS-1), the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI3K), or Akt. Immunoprecipitation/western blot analyses revealed that genistein decreased IGF-I-stimulated phosphorylation of IGF-IR and IRS-1, recruitment of p85 to IGF-IR, and phosphorylation of Akt. These results suggest that inhibition of cell proliferation and induction of apoptosis by genistein are mediated, at least in part, by its ability to inhibit IGF-IR signaling and the PI3K/Akt pathway.     

Soybean phytochemicals inhibit the growth of transplantable human prostate carcinoma and tumor angiogenesis in mice.

The objectives of our studies are to characterize the ability of dietary soybean components to inhibit the growth of prostate cancer in mice and alter tumor biomarkers associated with angiogenesis. Soy isoflavones (genistein or daidzein) or soy phytochemical concentrate inhibit the growth of prostate cancer cells LNCaP, DU 145 and PC-3 in vitro, but only at supraphysiologic concentrations, i.e., 50% inhibitory concentration (IC(50)) > 50 micromol/L. G2-M arrest and DNA fragmentation consistent with apoptosis of prostate cancer cells are also observed at concentrations causing growth inhibition. In contrast, the in vitro proliferation of vascular endothelial cells was inhibited by soy phytochemcials at much lower concentrations. We evaluated the ability of dietary soy phytochemical concentrate and soy protein isolate to inhibit the growth of the LNCaP human prostate cancer in severe combined immune-deficient mice. Mice inoculated subcutaneously with LNCaP cells (2 x 10(6)) were randomly assigned to one of the six dietary groups based on the AIN-76A formulation for 3 wk. A 2 x 3 factorial design was employed with two protein sources (20%, casein vs. soy protein) and three levels of soy phytochemical concentrate (0, 0.2 and 1.0% of the diet). Soy components did not alter body weight gain or food intake. Compared with casein-fed controls, the tumor volumes after 3 wk were reduced by 11% (P = 0.45) by soy protein, 19% (P = 0.17) by 0.2% soy phytochemical concentrate, 28% by soy protein with 0.2% soy phytochemical concentrate (P < 0.05), 30% by 1.0% soy phytochemical concentrate (P < 0.05) and 40% by soy protein with 1.0% soy phytochemical concentrate (P < 0.005). Histologic examination of tumor tissue showed that consumption of soy products significantly reduced tumor cell proliferation, increased apoptosis and reduced microvessel density. The angiogenic protein insulin-like growth factor-I was reduced in the circulation of mice fed soy protein and phytochemical concentrate. Our data suggest that dietary soy products may inhibit experimental prostate tumor growth through a combination of direct effects on tumor cells and indirect effects on tumor neovasculature.     

EGCG Enhances the Efficacy of Cisplatin by Downregulating hsa-miR-98-5p in NSCLC A549 Cells

In the current study, the enhanced efficacy of cisplatin caused by (-)-epigallocatechin-3-gallate (EGCG) in nonsmall cell lung cancer (NSCLC) A549 cells was observed. The tumor size was significantly smaller in vivo in the combination of cisplatin and EGCG group, as compared with cisplatin-only group. However, in NCI-H460 cells, another kind of NSCLC cells, the efficacy of cisplatin was antagonized by EGCG. MiRNA microarray showed that hsa-miR-98–5p and hsa-miR-125a-3p were differentially expressed after EGCG treatment in these 2 cell lines. After transfection of hsa-miR-98–5p inhibitor, the survival fraction of both A549 and NCI-H460 cells was decreased upon cisplatin treatment. Meanwhile, as a critical regulator in the cisplatin-induced apoptosis, p53 was elevated by silencing of hsa-miR-98–5p. These results suggested that EGCG inhibited the expression of hsa-miR-98–5p, followed by an increase of p53, thus the efficacy of cisplatin was enhanced. Bioinformatics analysis showed that hsa-miR-125a-3p might have a strong connection with classical MAPK pathway. Taken together, these findings indicate that hsa-miR-98–5p could be a potential target in clinical cisplatin treatment of NSCLC. The combination of EGCG and cisplatin might be an effective therapeutic strategy in treating some type of NSCLC, although the possibility of antagonistic interactions must also be taken into account.