Synergistic inhibition of head and neck tumor growth by green tea (-)-epigallocatechin-3-gallate and EGFR tyrosine kinase inhibitor

One of the mechanisms of the antitumor activity of green tea (-)-epigallocatechin-3-gallate (EGCG) is associated with its effect on epidermal growth factor receptor (EGFR)-mediated signaling transduction pathways. We investigated whether combining EGCG with the EGFR-tyrosine kinase inhibitor (EGFR-TKI) erlotinib may augment erlotinib-induced cell growth inhibition of squamous cell carcinoma of the head and neck (SCCHN) in a mouse xenograft model. In vitro studies with 5 head and neck cancer cell lines revealed that synergistic cell growth inhibition by the combination of EGCG and erlotinib was associated with significantly greater inhibition of pEGFR and pAKT, increased activation of caspases 9, 3 and PARP compared to the inhibition induced by EGCG or erlotinib alone. Erlotinib inhibited phosphorylation of EGFR, stabilizing EGFR at the plasma membrane, whereas EGCG induced EGFR internalization and ubiquitin-degradation, ultimately undermining EGFR signaling. The efficacy of the combination treatment was investigated with nude mice (n = 25) orally gavaged with vehicle control, EGCG, erlotinib or the combination at the same doses for 7 days, followed by subcutaneous injection with Tu212 cells. Animals were continuously administered the agents 5 days weekly for 7 weeks. The combined treatment resulted in significantly greater inhibition of tumor growth and delayed tumor progression as a result of increased apoptosis, decreased cell proliferation and reduced pEGFR and pAKT compared to the single agent treatment groups. Our results suggest a synergistic antitumor effect of a combined treatment with EGCG and erlotinib, and provide a promising regimen for future chemoprevention and treatment of SCCHN.     

Prostate carcinoma and green tea: PSA-triggered basement membrane degradation and MMP-2 activation are inhibited by (-)epigallocatechin-3-gallate

Prostate-specific antigen (PSA) is a serine-protease that, in addition to cleaving semenogelins in the seminal coagulum, is able to cleave extracellular matrix glycoproteins, thereby affecting cell migration and metastasis. We here report some new activities of PSA that deserve careful consideration in the cancer context: degradation of gelatin, degradation of type IV collagen in reconstituted basement membrane (Matrigel) and activation of progelatinase A (MMP-2), but not pro-MMP-9, in a cell-free system. Since consumption of green tea has been reported to lower the risk of prostate cancer, we investigated the effects of the major flavanol of green tea, (-)epigallocatechin-3-gallate (EGCG), on expression and activity of PSA by prostate carcinoma cells. In addition to restraint of PSA expression, EGCG was found to inhibit in a dose-dependent manner all the above PSA activities, at concentrations lower than the cytotoxic serine-protease inhibitor PMSF and close to levels measured in the serum following ingestion of green tea. The activity of PSA was suppressed also by the elastase released by the inflammatory leukocytes. These results highlight new PSA activities, suggest gelatin zymography as a new convenient assay for PSA, propose EGCG as natural inhibitor of prostate carcinoma aggressiveness, but also stimulate further investigation on the role of prostatic inflammation.     

The green tea polyphenol,epigallocatechin-3-gallate,protects against the oxidative cellular and genotoxic damage of UVA radiation

A number of biological activities have been ascribed to the major green tea polyphenol epigallocatechin-3-gallate (EGCG) to explain its chemopreventive properties. Its antioxidant properties emerge as a potentially important mode of action. We have examined the effect of EGCG treatment on the damaging oxidative effects of UVA radiation in a human keratinocyte line (HaCaT). Using the ROS-sensitive probes dihydrorhodamine 123 (DHR) and 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA), we detected a reduction in fluorescence in UVA-irradiated (100 kJ/m(2)) cells in the case of the former but not the latter probe after a 24-hr treatment with EGCG (e.g., 14%, [p < 0.05] after 10 microM EGCG). In the absence of UVA, however, both DHR and DCFH detected a pro-oxidant effect of EGCG at the highest concentration used of 50 microM. Measurements of DNA damage in UVA-exposed cells using the single cell gel electrophoresis assay (comet assay) also showed the protective effects of EGCG. A concentration of 10 microM EGCG decreased the level of DNA single strand breaks and alkali-labile sites to 62% of the level observed in non-EGCG, irradiated cells (p < 0.001) with a 5-fold higher concentration producing little further effect. Correspondingly, EGCG ablated the mutagenic effects of UVA (500 kJ/m(2)) reducing an induced hypoxanthine-guanine phosphoribosyl transferase (HPRT) mutant frequency of (3.39 +/- 0.73) x 10(-6) to spontaneous levels (1.09 +/- 0.19) x 10(-6). Despite having an antiproliferative effect in the absence of UVA, EGCG also served to protect against the cytotoxic effects of UVA radiation. Our data demonstrate the ability of EGCG to modify endpoints directly relevant to the carcinogenic process in skin.     

Induction of apoptosis in human prostate cancer cell line, PC3, by 3,3'-diindolylmethane through the mitochondrial pathway

Prostate cancer is the most common malignancy and the second leading cause of male death in Western countries. Prostate cancer mortality results from metastases to the bones and lymph nodes and progression from androgen-dependent to androgen-independent disease. Although androgen ablation was found to be effective in treating androgen-dependent prostate cancer, no effective life-prolonging therapy is available for androgen-independent cancer. Epidemiological studies have shown a strong correlation between consumption of cruciferous vegetables and a lower risk of prostate cancer. These vegetables contain glucosinolates, which during metabolism give rise to several breakdown products, mainly indole-3-carbinol (I3C), which may be condensed to polymeric products, especially 3,3'-diindolylmethane (DIM). It was previously shown that these indole derivatives have significant inhibitory effects in several human cancer cell lines, which are exerted through induction of apoptosis. We have previously reported that I3C and DIM induce apoptosis in prostate cancer cell lines through p53-, bax-, bcl-2- and fasL-independent pathways. The objective of this study was examination of the apoptotic pathways that may be involved in the effect of DIM in the androgen-independent prostate cancer cell line, PC3, in vitro. Our results suggest that DIM induces apoptosis in PC3 cells, through the mitochondrial pathway, which involves the translocation of cytochrome c from the mitochondria to the cytosol and the activation of initiator caspase, 9, and effector caspases, 3 and 6, leading to poly ADP-ribose polymerase (PARP) cleavage and induction of apoptosis. Our findings may lead to the development of new therapeutic strategies for the treatment of androgen-independent prostate cancer.     

环王巴明诱导DU145细胞凋亡的机制研究

目的：研究环王巴明诱导DU145细胞凋亡作用及其对Gli-1和Bcl-2 mRNA表达的影响,探讨环王巴明诱导DU145细胞凋亡的机制。方法：不同浓度环王巴明处理DU145细胞后,吖啶橙染色,荧光显微镜下观察细胞形态变化。流式细胞术观察细胞凋亡率变化,RT—PCR检测5、10μmol／L环王巴明作用72h后的实验组和对照组Gli-1、Bcl-2mRNA表达水平差异。结果：当环王巴明浓度大于5μmol／L作用72h后,荧光显微镜下可见细胞核固缩或碎裂,核仁变形等典型的凋亡形态学改变。环王巴明5μmol／L组Gli-1、Bcl-2 mRNA表达减弱,但同对照组差异无统计学意义（P〉0．05）,与空白及DMSO对照组相比10μmol／L环王巴明可以显著下调Gli—1、Bcl-2 mRNA表达（P〈0．01）。结论：环王巴明可诱导人前列腺癌DU145细胞株凋亡,其作用机制可能与下调Gli-1和Bcl-2 mRNA表达,活化细胞凋亡的线粒体途径有关。     

Targeting multiple signaling pathways by green tea polyphenol (-)-epigallocatechin-3-gallate

Cell signaling pathways, responsible for maintaining a balance between cell proliferation and death, have emerged as rational targets for the management of cancer. Emerging data amassed from various laboratories around the world suggests that green tea, particularly its major polyphenolic constituent (-)-epigallocatechin-3-gallate (EGCG), possesses remarkable cancer chemopreventive and therapeutic potential against various cancer sites in animal tumor bioassay systems and in some human epidemiologic studies. EGCG has been shown to modulate multiple signal transduction pathways in a fashion that controls the unwanted proliferation of cells, thereby imparting strong cancer chemopreventive as well as therapeutic effects. This review discusses the modulations of important signaling events by EGCG and their implications in cancer management.     

Green tea constituent epigallocatechin-3-gallate selectively inhibits COX-2 without affecting COX-1 expression in human prostate carcinoma cells

Overexpression of cyclooxygenase (COX)-2 has been implicated in many pathologic conditions, including cancer. One practical inference of this finding is that sustained inhibition of COX-2 could serve as a promising target for prevention or therapy of cancer. Conventional nonsteroidal antiinflammatory drugs (NSAIDs) and recently developed COX-2-specific inhibitors have shown considerable promise in prevention of some forms of human cancer; however, its application is limited due to severe toxic side effects on normal cells. Therefore, there is a need to define novel, nontoxic dietary constituents with proven chemopreventive effects through other pathways that also possess COX-2 but not COX-1 inhibitory activity. Recent studies on green tea and its major polyphenolic constituent (-)epigallocatechin-3-gallate (EGCG) have established its remarkable cancer preventive and some cancer therapeutic effects. Here, we show that EGCG inhibits COX-2 without affecting COX-1 expression at both the mRNA and protein levels, in androgen-sensitive LNCaP and androgen-insensitive PC-3 human prostate carcinoma cells. Based on our study, it is tempting to suggest that a combination of EGCG with chemotherapeutic drugs could be an improved strategy for prevention and treatment of prostate cancer.     

Induction of apoptosis in prostate cancer cell lines by a flavonoid, baicalin

The flavonoid baicalin (baicalein 7-D-beta-glucuronate), isolated from the dried root of Scutellaria baicalensis Georgi (Huang Qin), is widely used in the traditional Chinese herbal medicine for its anti-inflammatory, anti-pyretic and anti-hypersensitivity effects. In the present study, we investigated the in vitro effects of baicalin on the growth, viability, and induction of apoptosis in several human prostate cancer cell lines, including DU145, PC-3, LNCaP and CA-HPV-10. The cell viability after treating with baicalin for 2-4 days was quantified by a colorimetric 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-s ulfophenyl)- 2H-tetrazolium (MTS) assay. The results showed that baicalin could inhibit the proliferation of prostate cancer cells. The responses to baicalin were different among different cell lines, with DU145 cells being the most sensitive and LNCaP cells the most resistant. Baicalin caused a 50% inhibition of DU145 cells at concentrations of 150 microM or above. The inhibition of proliferation of prostate cancer cells after a short period of exposure to baicalin was associated with induction by apoptosis, as evidenced by the typical nuclear fragmentation using Hoechst 33258 staining, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) labeling, DNA fragmentation, activation of caspase-3 and cleavage of poly-ADP-ribose polymerase (PARP). The results indicate that baicalin has direct anti-tumor effects on human prostate cancer cells.     

Regulation of apoptosis in prostate cancer

Transformation and malignant progression of prostate cancer is regulated by the inability of prostatic epithelial cells to undergo apoptosis rather than by increased cell proliferation. The basic apoptotic machinery of most prostate cancer cells is intact and the inability to undergo apoptosis is due to molecular alterations that result in failure to initiate or execute apoptotic pathways. This review discusses the role of anti-apoptotic proteins such as Bcl-2/Bcl_XL, NF-B, IGF, caveolin, and Akt, and pro-apoptotic molecules such as PTEN, p53, Bin1, TGF-, and Par-4 that can regulate progression of prostate cancer. In addition to highlighting the salient features of these molecules and their relevance in apoptosis, this review provides an appraisal of their therapeutic potential in prostate cancer. Molecular targeting of these proteins and/or their innate pro- or anti-apoptotic pathways, either singly or in combination, may be explored in conjunction with conventional and currently available experimental strategies for the treatment of both hormone-sensitive and hormone-resistant prostate cancer.     

Epigallocatechin-3-gallate is a potent natural inhibitor of fatty acid synthase in intact cells and selectively induces apoptosis in prostate cancer cells

Chemical inhibitors of fatty acid synthase (FAS) inhibit growth and induce apoptosis in several cancer cell lines in vitro and in tumor xenografts in vivo. Recently the green tea component epigallocatechin-3-gallate (EGCG) was shown to act as a natural inhibitor of FAS in chicken liver extracts. Here we investigated whether EGCG inhibits FAS activity in cultured prostate cancer cells and how this inhibition affects endogenous lipid synthesis, cell proliferation and cell viability. The high levels of FAS activity in LNCaP cells were dose-dependently inhibited by EGCG and this inhibition was paralleled by decreased endogenous lipid synthesis, inhibition of cell growth and induction of apoptosis. In contrast, epicatechin (EC), another closely related green tea polyphenolic compound, which does not inhibit FAS, had no effect on LNCaP cell growth or viability. Treatment of nonmalignant cells with low levels of FAS activity (fibroblasts) with EGCG led to a decrease in growth rate but not to induction of apoptosis. These data indicate that EGCG inhibits FAS activity as efficiently as presently known synthetic inhibitors and selectively causes apoptosis in LNCaP cells but not in nontumoral fibroblasts. These findings establish EGCG as a potent natural inhibitor of FAS in intact cells and strengthen the molecular basis for the use of EGCG as a chemopreventive and therapeutic antineoplastic agent.     

Biological significance of myeloperoxidase (MPO) on green tea component, (−)-epigallocatechin-3-gallate (EGCG)-induced apoptosis: its therapeutic potential for myeloid leukemia

The therapeutic approach to acute myeloid leukemia (AML) is usually chemotherapy, but severe side effects and complications induced by the anticancer drugs are sometimes fatal and are major problems in the clinical setting. Recently, more specifically targeted agents have been developed for the treatment of AML; however, most candidate agents for targeted therapy have yet to be translated into clinical application. Natural compounds appear to be safer than the current chemotherapeutic agents. Recently, it has been reported that reactive oxygen species (ROS) produced by natural compounds such as (−)-epigallocatechin-3-gallate (EGCG) induce apoptosis in myeloid leukemic cells. EGCG markedly induced apoptosis of myeloperoxidase (MPO)-positive myeloid leukemic cells. Treatment with EGCG caused a significant ROS production in MPO-positive leukemic cells. ROS are now thought of as signaling molecules in response to various extracellular stimuli. On the other hand, ROS may be the direct mediator of EGCG-induced apoptosis in myeloid leukemic cells. In particular, highly toxic ROS such as hydroxyl radical (·OH) generated via the H₂O₂/MPO halide system may directly mediate oxidative stress-induced apoptosis in myeloid leukemic cells.     

Isosilybin A induces apoptosis in human prostate cancer cells via targeting Akt,NF‐κB,and androgen receptor signaling

Prostate cancer (PCA) is the second most malignancy in American men. Advanced stage PCA cells possess unlimited replication potential as well as resistance to apoptosis. Therefore, targeting survival mechanisms and activating apoptotic machinery in PCA cells using nontoxic phytochemicals is suggested as an attractive strategy against this deadly malignancy. In the present study, we assessed the effect of one such botanical agent, namely isosilybin A, on apoptotic machinery and key members of cell survival signaling [Akt, NF‐κB, and androgen receptor (AR)] in different PCA cells. Results showed that isosilybin A (90–180 µM) treatment significantly induces apoptotic death by activating both extrinsic (increased level of DR5 and cleaved caspase 8) and intrinsic pathways (caspase 9 and 3 activation) of apoptosis in three different human PCA cell lines namely 22Rv1, LAPC4, and LNCaP. Further, isosilybin A treatment decreased the levels of phospho‐Akt (serine‐473), total Akt, and the nuclear levels of NF‐κB constituents (p50 and p65). Isosilybin A treatment also decreased the AR and PSA level in 22Rv1, LAPC4, and LNCaP cells. Employing pan‐caspase inhibitor (Z‐VAD.fmk), we confirmed that isosilybin A‐mediated decreased AR is independent of caspases activation. Temporal kinetics analysis showed that the primary effect of isosilybin A is on AR, as decrease in AR was evident much earlier (4 h) relative to caspase activation and apoptosis induction (12 h). Overall, our results demonstrated that isosilybin A activates apoptotic machinery in PCA cells via targeting Akt–NF‐κB–AR axis; thereby, indicating a promising role for this phytochemical in the management of clinical PCA. © 2010 Wiley‐Liss, Inc.     

Inhibition of intestinal tumorigenesis in Apcmin/+ mice by (-)-epigallocatechin-3-gallate, the major catechin in green tea

The present study was designed to investigate the effects of two main constituents of green tea, (-)-epigallocatechin-3-gallate (EGCG) and caffeine, on intestinal tumorigenesis in Apc(min/+) mice, a recognized mouse model for human intestinal cancer, and to elucidate possible mechanisms involved in the inhibitory action of the active constituent. We found that p.o. administration of EGCG at doses of 0.08% or 0.16% in drinking fluid significantly decreased small intestinal tumor formation by 37% or 47%, respectively, whereas caffeine at a dose of 0.044% in drinking fluid had no inhibitory activity against intestinal tumorigenesis. In another experiment, small intestinal tumorigenesis was inhibited in a dose-dependent manner by p.o. administration of EGCG in a dose range of 0.02% to 0.32%. P.o. administration of EGCG resulted in increased levels of E-cadherin and decreased levels of nuclear beta-catenin, c-Myc, phospho-Akt, and phospho-extracellular signal-regulated kinase 1/2 (ERK1/2) in small intestinal tumors. Treatment of HT29 human colon cancer cells with EGCG (12.5 or 20 micromol/L at different times) also increased protein levels of E-cadherin by 27% to 58%, induced the translocation of beta-catenin from nucleus to cytoplasm and plasma membrane, and decreased c-Myc and cyclin D1 (20 micromol/L EGCG for 24 hours). These results indicate that EGCG effectively inhibited intestinal tumorigenesis in Apc(min/+) mice, possibly through the attenuation of the carcinogenic events, which include aberrant nuclear beta-catenin and activated Akt and ERK signaling.     

Induction of prostate specific antigen production by steroids and tamoxifen in breast cancer cell lines

Summary We demonstrate that the steroid hormone receptor-positive breast carcinoma cell lines T-47D and MCF-7 can be induced by androgens, progestins, mineralocorticosteroids, glucocorticosteroids, and antiestrogens, to produce prostate specific antigen (PSA). Estrogens failed to induce such stimulation in both cell lines and, in addition, were able to block the induction by androgens in the cell line T-47D. These data support and extend our previous report on PSA production by breast tumors and describe anin vitro system which can be used to study the phenomenon for possible application in prognosis and design of new therapy.     

青蒿琥酯诱导人前列腺癌细胞凋亡的实验研究

目的:探讨青蒿琥酯(artesunate,Art)对人前列腺癌PC-3细胞株抑制作用及机制。方法:进行前列腺癌PC-3细胞培养,不同浓度Art干预,采用MTT法检测用药后细胞增殖率的改变,流式细胞术(FCM)检测细胞周期的改变和凋亡比。结果:与对照组比较,Art可显著抑制PC-3细胞增殖(P<0.01);PC-3细胞主要被阻滞在G0/G1期,FCM检测PC-3细胞凋亡比,各实验组均明显高于对照组(P<0.01),且呈时间、剂量依赖性。结论:Art可抑制PC-3细胞增殖,其作用机制可能与调控细胞周期、诱导细胞凋亡有关。     

Effects of interferons and double-stranded RNA on human prostate cancer cell apoptosis

Prostate cancer is the second most commonly diagnosed cancer among men in the United States. Prostate cancer therapy is severely hampered by lack of response and development of resistance to conventional chemotherapeutic drugs in patients. Therefore, the development and discovery of new drugs have become an urgent clinical need. Interferons (IFNs), a family of pleiotropic cytokines, exert antitumor activities due to their anti-proliferative, immunomodulatory and proapoptotic functions. Here, we report that pretreatment of prostate cancer PC-3 cells with IFNs sensitized these cells to double-stranded RNAs (dsRNAs)-induced apoptosis. The enhancement effect of IFN treatment was dependent on IFN subtypes, in particular, IFN γ. In comparison with IFN α or β, IFN γ treatment remarkably augmented apoptosis in PC-3 cells induced with polyinosinic:polycytidylic acid (poly I:C), a synthesized form of dsRNA. We demonstrated that IFN-signaling was necessary for these effects by using mutant cell lines. Transfection of 2–5A, the activator of RNase L, or silencing of dsRNA-dependent protein kinase R (PKR) by siRNA did not have any significant impact on this event, suggesting that neither RNase L nor PKR was involved in poly I:C/IFN γ-induced apoptosis in the cells. Further investigation of the apoptotic pathway revealed that Bak, a pro-apoptotic member of the Bcl-2family, was synergistically up-regulated by IFN γ and poly I:C, whereas other members of the family were not affected. Knocking down of Bak demonstrated its contribution to poly I:C/IFN γ-induced apoptosis in the cells. We believeour findings will precipitate the design of novel therapeutic strategies for prostate cancer.     

A novel prodrug of the green tea polyphenol (-)-epigallocatechin-3-gallate as a potential anticancer agent

The most abundant and biologically active green tea catechin, (-)-epigallocatechin-3-gallate or (-)-EGCG, has been shown to act as a proteasome inhibitor and tumor cell death inducer. However, (-)-EGCG is unstable under physiologic conditions and has poor bioavailability. Previously, in an attempt to increase the stability of (-)-EGCG, we introduced peracetate protections to its reactive hydroxyl groups and showed that this peracetate-protected (-)-EGCG [Pro-EGCG (1); formerly named compound 1] could be converted into (-)-EGCG under cell-free conditions. In the current study, we provide evidence that when cultured human breast cancer MDA-MB-231 cells were treated with Pro-EGCG (1), (-)-EGCG was not only converted but also accumulated, accompanied by enhanced levels of proteasome inhibition, growth suppression, and apoptosis induction, compared with cells treated with natural (-)-EGCG. To investigate the potential use of Pro-EGCG (1) as a novel prodrug that converts to a cellular proteasome inhibitor and anticancer agent in vivo, MDA-MB-231 tumors were induced in nude mice, followed by treatment with Pro-EGCG (1) or (-)-EGCG for 31 days. Results of this in vivo study showed a significant inhibition of breast tumor growth by Pro-EGCG (1), compared with (-)-EGCG, associated with increased proteasome inhibition and apoptosis induction in tumor tissues. In conclusion, we have shown that Pro-EGCG (1) increases the bioavailability, stability, and proteasome-inhibitory and anticancer activities of (-)-EGCG in human breast cancer cells and tumors, suggesting its potential use for cancer prevention and treatment.     

Green tea polyphenol (-)-epigallocatechin-3-gallate inhibits cyclooxygenase-2 expression in colon carcinogenesis

Tea, one of the most widely consumed beverages worldwide, has been shown to have anti-cancer activity in various cancers including colon cancer. It has been demonstrated that overexpression of the inducible isoform of cyclooxygenase (COX-2) occurs during colon tumorigenesis and inhibition of COX-2 by non-steroidal anti-inflammatory drugs (NSAIDs) is chemopreventive. To determine whether the anti-cancer effect associated with green tea impacted COX-2 expression levels, human colorectal cancer cell lines HT-29 and HCA-7, were treated with (-)-epigallocatechin-3-gallate (EGCG), the most abundant and effective polyphenol of green tea. EGCG significantly inhibited constitutive COX-2 mRNA and protein overexpression. The inhibitory effects of EGCG on signaling pathways controlling COX-2 expression were examined. We observed that EGCG down regulated the ERK1/2 and Akt pathways in colon cancer cells. The effect of EGCG on COX-2 expression resulted in decreased COX-2 promoter activity via inhibition of nuclear factor kappaB (NF-kappaB) activation. EGCG also promoted rapid mRNA decay mediated through the COX-2 3'untranslated region (3'UTR). In conclusion, these data suggest that inhibition of COX-2 is a mechanism for the anti-proliferative effect of green tea and emphasizes the role that dietary factors have as anti-cancer agents.