Molecular pathway for (-)-epigallocatechin-3-gallate-induced cell cycle arrest and apoptosis of human prostate carcinoma cells

Epigallocatechin-3-gallate (EGCG), the major polyphenolic constituent present in green tea, is a promising chemopreventive agent. We recently showed that green tea polyphenols exert remarkable preventive effects against prostate cancer in a mouse model and many of these effects are mediated by the ability of polyphenols to induce apoptosis in cancer cells [Proc. Natl. Acad. Sci. USA 98 (2001) 10350]. Earlier, we showed that EGCG causes a G0/G1 phase cell cycle arrest and apoptosis of both androgen-sensitive LNCaP and androgen-insensitive DU145 human prostate carcinoma cells, irrespective of p53 status [Toxicol. Appl. Pharmacol. 164 (2000) 82]. Here, we provide molecular understanding of this effect. We tested a hypothesis that EGCG-mediated cell cycle dysregulation and apoptosis is mediated via modulation of cyclin kinase inhibitor (cki)-cyclin-cyclin-dependent kinase (cdk) machinery. As shown by immunoblot analysis, EGCG treatment of LNCaP and DU145 cells resulted in significant dose- and time-dependent (i) upregulation of the protein expression of WAF1/p21, KIP1/p27, INK4a/p16, and INK4c/p18, (ii) down-modulation of the protein expression of cyclin D1, cyclin E, cdk2, cdk4, and cdk6, but not of cyclin D2, (iii) increase in the binding of cyclin D1 toward WAF1/p21 and KIP1/p27, and (iv) decrease in the binding of cyclin E toward cdk2. Taken together, our results suggest that EGCG causes an induction of G1 phase ckis, which inhibits the cyclin-cdk complexes operative in the G0/G1 phase of the cell cycle, thereby causing an arrest, which may be an irreversible process ultimately leading to apoptotic cell death. This is the first systematic study showing the involvement of each component of cdk inhibitor-cyclin-cdk machinery during cell cycle arrest and apoptosis of human prostate carcinoma cells by EGCG.     

Green tea polyphenol epigallocatechin-3-gallate differentially modulates nuclear factor kappaB in cancer cells versus normal cells

Green tea has shown remarkable anti-inflammatory and cancer chemopreventive effects in many animal tumor bioassays, cell culture systems, and epidemiological studies. Many of these biological effects of green tea are mediated by epigallocatechin 3-gallate (EGCG), the major polyphenol present therein. We have earlier shown that EGCG treatment results in apoptosis of several cancer cells, but not of normal cells (J. Natl. Cancer Inst. 89, 1881-1886 (1997)). The mechanism of this differential response of EGCG is not known. In this study, we investigated the involvement of NF-kappaB during these differential responses of EGCG. EGCG treatment resulted in a dose-dependent (i) inhibition of cell growth, (ii) G0/G1-phase arrest of the cell cycle, and (iii) induction of apoptosis in human epidermoid carcinoma (A431) cells, but not in normal human epidermal keratinocytes (NHEK). Electromobility shift assay revealed that EGCG (10-80 microM) treatment results in lowering of NF-kappaB levels in both the cytoplasm and nucleus in a dose-dependent manner in both A431 cells and NHEK, albeit at different concentrations. EGCG treatment was found to result in a dose-based differential inhibition of TNF-alpha- and LPS-mediated activation of NF-kappaB in these cells. The inhibition of NF-kappaB constitutive expression and activation in NHEK was observed only at high concentrations. The immunoblot analysis also demonstrated a similar pattern of inhibition of the constitutive expression as well as activation of NF-kappaB/p65 nuclear protein. This inhibition of TNF-alpha-caused NF-kappaB activation was mediated via the phosphorylative degradation of its inhibitory protein IkappaBalpha. Taken together, EGCG was found to impart differential dose-based NF-kappaB inhibitory response in cancer cells vs normal cells; i.e., EGCG-mediated inhibition of NF-kappaB constitutive expression and activation was found to occur at much higher dose of EGCG in NHEK as compared to A431 cells. This study suggests that EGCG-caused cell cycle deregulation and apoptosis of cancer cells may be mediated through NF-kappaB inhibition.     

Epigallocatechin-3-gallate inhibits epidermal growth factor receptor signaling pathway. Evidence for direct inhibition of ERK1/2 and AKT kinases

Epidermal growth factor receptor (EGFR) activation is absolutely required for cervical cell proliferation. This suggests that EGFR-inhibitory agents may be of therapeutic value. In the present study, we investigated the effects of epigallocatechin-3-gallate (EGCG), a bioactive green tea polyphenol, on EGFR signaling in cervical cells. EGCG inhibits epidermal growth factor-dependent activation of EGFR, and EGFR-dependent activation of the mitogen-activated protein kinases ERK1/2. EGCG also inhibits EGFR-dependent AKT activity. The EGCG-dependent reduction in ERK and AKT activity is associated with reduced phosphorylation of downstream substrates, including p90RSK, FKHR, and BAD. These changes are associated with increased p53, p21(WAF-1), and p27(KIP-1) levels, reduced cyclin E level, and reduced CDK2 kinase activity. Consistent with these findings, flow cytometry and TUNEL (terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling) staining revealed EGCG-dependent G(1) arrest. Moreover, sustained EGCG treatment caused apoptotic cell death. In addition to inhibiting EGFR, cell-free studies demonstrated that EGCG directly inhibits ERK1/2 and AKT, suggesting that EGCG acts simultaneously at multiple levels to inhibit EGF-dependent signaling. Importantly, the EGCG inhibition is selective, as EGCG does not effect the EGFR-dependent activation of JNK. These results suggest that EGCG acts to selectively inhibit multiple EGF-dependent kinases to inhibit cell proliferation.     

Phosphorylation of the cyclin-dependent kinase inhibitor p21Cip1 on serine 130 is essential for viral cyclin-mediated bypass of a p21Cip1-imposed G1 arrest

K cyclin encoded by Kaposi's sarcoma-associated herpesvirus confers resistance to the cyclin-dependent kinase (cdk) inhibitors p16Ink4A, p21Cip1, and p27Kip1 on the associated cdk6. We have previously shown that K cyclin expression enforces S-phase entry on cells overexpressing p27Kip1 by promoting phosphorylation of p27Kip1 on threonine 187, triggering p27Kip1 down-regulation. Since p21Cip1 acts in a manner similar to that of p27Kip1, we have investigated the subversion of a p21Cip1-induced G1 arrest by K cyclin. Here, we show that p21Cip1 is associated with K cyclin both in overexpression models and in primary effusion lymphoma cells and is a substrate of the K cyclin/cdk6 complex, resulting in phosphorylation of p21Cip1 on serine 130. This phosphoform of p21Cip1 appeared unable to associate with cdk2 in vivo. We further demonstrate that phosphorylation on serine 130 is essential for K cyclin-mediated release of a p21Cip1-imposed G1 arrest. Moreover, we show that under physiological conditions of cell cycle arrest due to elevated levels of p21Cip1 resulting from oxidative stress, K cyclin expression enabled S-phase entry and was associated with p21Cip1 phosphorylation and partial restoration of cdk2 kinase activity. Thus, expression of the viral cyclin enables cells to subvert the cell cycle inhibitory function of p21Cip1 by promoting cdk6-dependent phosphorylation of this antiproliferative protein.     

Cyclin D3 is dispensable for human diffuse large B-cell lymphoma survival and growth: evidence for redundancy with cyclin E

Genomic changes disrupting the expression of cyclin D3 are associated with aberrant growth of several human B-lymphoid malignancies. We demonstrate that the human diffuse large B-cell lymphoma (DLBCL), OCI-LY18 (LY18) expresses cyclin D3 but not cyclins D1 and D2. RNA interference was used to deplete cyclin D3 from LY18 cells. Surprisingly, knockdown of cyclin D3 did not inhibit pRb phosphorylation on cdk4/6- and cdk2-specific residues or measurably affect viability and proliferation. These results suggest that cyclin D3 is dispensable in LY18 cell proliferation and survival. Similar results were obtained following depletion of cyclin E. By contrast, combined knockdown of cyclins D3 and E had substantial consequences leading to G1-phase arrest and inhibition of proliferation. Whereas cell cycle distribution was not affected following individual depletion of cdk4, cdk6, or cdk2, the combined knockdown of cdk4 and cdk6 led to accumulation of LY18 cells in G1-phase of the cell cycle and inhibition of proliferation. Likewise treatment of LY18 cells with 2-Bromo-12,13-dihydro-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione, a selective inhibitor of cdk4/6, led to inhibition of proliferation. Taken together, these results uncover a built-in redundancy with cyclins D3 and E for G1-S progression. Moreover these findings highlight the rationale for simultaneous disruption of cdk4/6 as a potential therapeutic cancer strategy.     

Green tea constituent (−)-Epigallocatechin-3-gallate inhibits hep G2 cell proliferation and induces apoptosis through p53-dependent and fas-mediated pathways

(-)-Epigallocatechin-3-gallate (EGCG) is a polyphenolic compound found in green tea. It has been reported to possess a wide range of pharmacological properties, and is one of the most promising chemopreventive agents for cancer. To provide a better understanding of the preventive effect of EGCG on liver cancer, we examined EGCG for its effect on proliferation and cell cycle progression in a human liver cancer cell line, Hep G2. The results showed that EGCG inhibited the proliferation of Hep G2 by inducing apoptosis and blocking cell cycle progression in the G1 phase. ELISA showed that EGCG significantly increased the expression of p53 and p21/WAF1 protein, and this contributed to cell cycle arrest. An enhancement in Fas/APO-1 and its two form ligands, membrane-bound Fas ligand (mFasL) and soluble Fas ligand (sFasL), as well as Bax protein, was responsible for the apoptotic effect induced by EGCG. Taken together, our study suggests that the induction of p53 and the activity of the Fas/FasL apoptotic system play major roles in the antiproliferative activity of EGCG in Hep G2 cells.     

Role for cyclin D1 in UVC-induced and p53-mediated apoptosis.

DNA damaging agents such as ultraviolet (UV) induce cell cycle arrest followed by apoptosis in cells where irreparable damage has occurred. Here we show that during early phase G1 arrest which occurs in UV-irradiated human U343 glioblastoma cells, there are (1) decreases in cyclin D1 and cdk4 levels which parallel a loss of S-phase promoting cyclin D1/cdk4 complexes, and (2) increases in p53 and p21 protein levels. We also show that the late phase UV-induced apoptosis of U343 cells occurs after cell cycle re-entry and parallels the reappearance of cyclin D1 and cdk4 and cyclin D1/cdk4 complexes. These findings suggest that cyclin D1 can abrogate UV-induced G1 arrest and that the p53-mediated apoptosis that occurs in these cells is dependent on cyclin D1 levels. We examined these possibilities using U343 cells that ectopically express cyclin D1 and found that indeed cyclin D1 can overcome the cell cycle arrest caused by UV. Moreover, the appearance of p53 protein and the induction of apoptosis in UV-irradiated cells was found to be dependent on the level of ectopically expressed cyclin D1. These findings, therefore, indicate that expression of cyclin D1 following DNA damage is essential for cell cycle re-entry and p53-mediated apoptosis.     

Fucoxanthin induces cell cycle arrest at G0/G1 phase in human colon carcinoma cells through up-regulation of p21WAF1/Cip1

Fucoxanthin, a natural carotenoid, has been reported to have antitumorigenic activity in mouse colon, skin and duodenum models. The present study was designed to evaluate the molecular mechanisms of fucoxanthin against colon cancer using the human colon adenocarcinoma cell lines. Fucoxanthin reduced the viability of WiDr cells in a dose-dependent manner accompanied by the induction of cell cycle arrest during the G0/G1 phase at 25 microM and apoptosis at 50 microM. Fucoxanthin at 25 microM inhibited the phosphorylation of the retinoblastoma protein (pRb) at Ser780 and Ser807/811 24 h after treatment without changes in the protein levels of the D-types of cyclin and cyclin-dependent kinase (cdk) 4, whose complexes are responsible for the phosphorylation of pRb at these sites. A cdk inhibitory protein, p21WAF1/Cip1 increased 24 h after the treatment with 25 microM of fucoxanthin, but not p27Kip1. In addition, the mRNA of p21WAF1/Cip1 also increased in a dose-dependent manner. According to the experiments using the isogenic human colon adenocarcinoma cell lines, fucoxanthin failed to induce G0/G1 arrest in the p21-deficient HCT116 cells, but not in HCT116 wild-type cells. All of these findings showed that fucoxanthin inhibited proliferation of colon cancer cells. The inhibitory mechanism is due to the cell cycle arrest during the G0/G1 phase mediated through the up-regulation of p21WAF1/Cip1, which may be related to the antitumorigenic activity.     

Inhibition of G1 phase cyclin dependent kinases by transforming growth factor β1

Transforming growth factor β1 (TGFβ1) inhibits epithelial cell proliferation late in the G1 phase of the cell cycle. We examined the effect of TGFβ1 on known late G1 cell cycle regulators in an attempt to determine the molecular mechanism of growth inhibition by this physiological inhibitor. The results demonstrate the TGFβ1 inhibits the late G1 and S phase specific histone H1 kinase activity of p33^cdk2. This inhibitiion is not dur to TGFβ1's effect on p33^cdk2 synthesis, but rather due to its negative effect on the late G1 phosphorylation of p33^cdk2. It is also shown that TGFβ1 inhibits both late G1 cyclin A and cyclin E associated histon H1 kinase activities. The inhibitor has no effects on the synthesis of cyclin E but to inhibit the synthesis of cyclin A protein in a cell cycle dependent manner. If TGFβ1 is added to cells which have progressed futher than 8 hours into G1, then it is without inhibitory effect on cyclin A synthesis. These effect on TGFβ1 on late G1 cell cycle regulators correlate well with its inhibitory effects on cellular growth and suggest that these G1 cyclin dependent kinases might serve as targets for TGFβ1-mediated growth arrest.     

Molecular mechanisms for inhibition of colon cancer cells by combined epigenetic-modulating epigallocatechin gallate and sodium butyrate

Bioactive compounds are considered safe and have been shown to alter genetic and epigenetic profiles of tumor cells. However, many of these changes have been reported at molecular concentrations higher than physiologically achievable levels. We investigated the role of the combinatorial effects of epigallocatechin gallate (EGCG), a predominant polyphenol in green tea, and sodium butyrate (NaB), a dietary microbial fermentation product of fiber, in the regulation of survivin, which is an overexpressed anti-apoptotic protein in colon cancer cells. For the first time, our study showed that the combination treatment induced apoptosis and cell cycle arrest in RKO, HCT-116 and HT-29 colorectal cancer cells. This was found to be regulated by the decrease in HDAC1, DNMT1, survivin and HDAC activity in all three cell lines. A G2/M arrest was observed for RKO and HCT-116 cells, and G1 arrest for HT-29 colorectal cancer cells for combinatorial treatment. Further experimentation of the molecular mechanisms in RKO colorectal cancer (CRC) cells revealed a p53-dependent induction of p21 and an increase in nuclear factor kappa B (NF-κB)-p65. An increase in double strand breaks as determined by gamma-H2A histone family member X (γ-H2AX) protein levels and induction of histone H3 hyperacetylation was also observed with the combination treatment. Further, we observed a decrease in global CpG methylation. Taken together, these findings suggest that at low and physiologically achievable concentrations, combinatorial EGCG and NaB are effective in promoting apoptosis, inducing cell cycle arrest and DNA-damage in CRC cells.     

Vitamin E succinate inhibits human prostate cancer cell growth via modulating cell cycle regulatory machinery

Several epidemiological studies have demonstrated that vitamin E is a chemopreventative agent for prostate cancer. alpha-Tocopheryl succinate (VES), a derivative of vitamin E, effectively modulates prostate cancer cell growth. However, little is known about the mechanisms regarding this action. Here we show that VES causes human prostate cancer cell LNCaP arrest at G1 phase. This effect is accomplished through VES significantly decreasing expression of the cell cycle regulatory proteins cyclin D1, D3, and E, cdk2 and 4, but not cdk6. Furthermore, VES reduces cdk4 kinase activity, Rb phosphorylation, and cyclin E mRNA expression. Recently there is increasing interest in the protective effect of the VES and selenium combination on prostate cancer. Here we show that VES and selenium work through different mechanisms to exert their inhibitory effects on prostate cancer cells. Taken together, our studies suggest that VES-mediated prostate cancer cell G1/S arrest is a consequence of the regulation of multiple molecules of the cell cycle regulatory machinery.     

Differential regulation of the cyclin-dependent kinase inhibitors p21(Cip1) and p27(Kip1) by phosphorylation directed by the cyclin encoded by Murine Herpesvirus 68

Members of the gamma2-herpesvirus family encode cyclin-like proteins that have the ability to deregulate mammalian cell cycle control. Here we report the key features of the viral cyclin encoded by Murine Herpesvirus 68, M cyclin. M cyclin preferentially associated with and activated cdk2; the M cyclin/cdk2 holoenzyme displayed a strong reliance on phosphorylation of the cdk T loop for activity. cdk2 associated with M cyclin exhibited substantial resistance to the cdk inhibitor proteins p21(Cip) and p27(Kip). Furthermore, M cyclin directed cdk2 to phosphorylate p27(Kip1) on threonine 187 (T187) and cellular expression of M cyclin led to down-regulation of p27(Kip1) and the partial subversion of the associated G1 arrest. Mutation of T187 to a non-phosphorylatable alanine rendered the p27(Kip1)-imposed G1 arrest resistant to M cyclin expression. Unlike the related K cyclin, M cyclin was unable to circumvent the G1 arrest associated with p21(Cip1) and was unable to direct its associated catalytic subunit to phosphorylate this cdk inhibitor. These results imply that M cyclin has properties that are distinct from other viral cyclins and that M cyclin expression alone is insufficient for S phase entry.     

Tuning Cell Cycle Regulation with an Iron Key

Iron (Fe) is essential for cellular metabolism e.g., DNA synthesis and its depletion causes G1/S arrest and apoptosis. Considering this, Fe chelators have been shown to be effective anti-proliferative agents. In order to understand the anti-tumor activity of Fe chelators, the mechanisms responsible for G1/S arrest and apoptosis after Fe-depletion have been investigated. These studies reveal a multitude of cell cycle control molecules are regulated by Fe. These include p53, p27Kip1, cyclin D1 and cyclin-dependent kinase 2 (cdk2). Additionally, Fe-depletion up-regulates the mRNA levels of the cdk inhibitor, p21CIP1/WAF1, but paradoxically down-regulates its protein expression. This effect could contribute to the apoptosis observed after Fe-depletion. Iron-depletion also leads to proteasomal degradation of p21CIP1/WAF1 and cyclin D1 via an ubiquitin-independent pathway. This is in contrast to the mechanism in Fe-replete cells, where it occurs by ubiquitin-dependent proteasomal degradation. Up-regulation of p38 mitogen-activated protein kinase (MAPK) after Fe-depletion suggests another facet of cell cycle regulation responsible for inhibition of proliferation and apoptosis induction. Elucidation of the complex effects of Fe-depletion on the expression of cell cycle control molecules remains at its infancy. However, these processes are important to dissect for complete understanding of Fe-deficiency and the development of chelators for cancer treatment.     

A preliminary study: the anti-proliferation effect of salidroside on different human cancer cell lines

Salidroside (p-hydroxyphenethyl-beta-d-glucoside), which is present in all species of the genus Rhodiola, has been reported to have a broad spectrum of pharmacological properties. The present study, for the first time, focused on evaluating the effects of the purified salidroside on the proliferation of various human cancer cell lines derived from different tissues, and further investigating its possible molecular mechanisms. Cell viability assay and [³H] thymidine incorporation were used to evaluate the cytotoxic effects of salidroside on cancer cell lines, and flow cytometry analyzed the change of cell cycle distribution induced by salidroside. Western immunoblotting further studied the expression changes of cyclins (cyclin D1 and cyclin B1), cyclin-dependent kinases (CDK4 and Cdc2), and cyclin-dependent kinase inhibitors (p21^Cip1 and p27^Kip1). The results showed that salidroside inhibited the growth of various human cancer cell lines in concentration- and time-dependent manners, and the sensitivity to salidroside was different in those cancer cell lines. Salidroside could cause G1-phase or G2-phase arrest in different cancer cell lines, meanwhile, salidroside resulted in a decrease of CDK4, cyclin D1, cyclin B1 and Cdc2, and upregulated the levels of p27^Kip1 and p21^Cip1. Taken together, salidroside could inhibit the growth of cancer cells by modulating CDK4-cyclin D1 pathway for G1-phase arrest and/or modulating the Cdc2-cyclin B1 pathway for G2-phase arrest.     

Low levels of cyclin D and nonfunctional Rb protein affect cdk6 association with cyclin-dependent kinase inhibitor p27(Kip1)

p27(Kip1) associates with cyclin/cdk complexes and inhibiting cdk activity, and overexpression of p27(Kip1) induces G1 arrest. We found that p27(Kip1) overexpression inhibits cdk2 kinase activity, but not cdk6 kinase activity in HeLa cells. The amount of p27(Kip1) associated with cdk2 was significantly higher than that associated with cdk6. cdk6 complexes contained detectable amounts of p27(Kip1) in all human cell lines examined, except in HeLa cells where p27(Kip1) preferentially associated with cdk2. It appears that in HeLa cells overexpressed p27(Kip1) fails to inhibit cdk6 kinase activity because of low binding affinity of cdk6 to p27(Kip1). The low binding affinity is due to a low level of the cdk6/cyclin D complexes. Functional inactivation of pRb has an effect on p27(Kip1) association with cdk6/cyclin D complexes.