Yu Chih Liang

Cancer chemoprevention by tea polyphenols through mitotic signal transduction blockade

Tea is a popular beverage. The consumption of green tea is associated with a lower risk of several types of cancer, including stomach, esophagus, and lung. The cancer chemopreventive effect of tea has been attributed to its major phytopolyphenols. The tea polyphenols comprise about one-third of the weight of the dried leaf, and they show profound biochemical and pharmacological activities including antioxidant activities, modulation of carcinogen metabolism, inhibition of cell proliferation, induction of cell apoptosis, and cell cycle arrest. They intervene in the biochemical and molecular processes of multistep carcinogenesis, comprising tumor initiation, promotion, and progression. Several studies demonstrate that most tea polyphenols exert their scavenging effects against reactive oxygen species (ROS); excessive production of ROS has been implicated for the development of cardiovascular diseases, neurodegenerative disorders, and cancer. Recently, we have found that the major tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) suppresses extracellular signals and cell proliferation through epidermal growth factor receptor binding in human A431 epidermoid carcinoma cells; EGCG also blocks the induction of nitric oxide synthase by down-regulating lipopolysaccharide-induced activity of the transcription factor NFKB in macrophages. Furthermore, EGCG blocks the cell cycle at the G1 phase in MCF-7 cells. We have demonstrated that EGCG inhibits the activities of cyclin-dependent kinases 2 and 4; meanwhile, EGCG induces the expression of the Cdk inhibitors p21 and p27. These results suggest that tumor promotion can be enhanced by ROS and oxidative mitotic signal transduction, and this enhancement can be suppressed by EGCG or other tea polyphenols.

Suppression of extracellular signals and cell proliferation through EGF receptor binding by (−)-epigallocatechin gallate in human A431 epidermoid carcinoma cells

Tea polyphenols are known to inhibit a wide variety of enzymatic activities associated with cell proliferation and tumor progression. The molecular mechanisms of antiproliferation are remained to be elucidated. In this study, we investigated the effects of the major tea polyphenol (−)‐epigallocatechin gallate (EGCG) on the proliferation of human epidermoid carcinoma cell line, A431. Using a [3H]thymidine incorporation assay, EGCG could significantly inhibit the DNA synthesis of A431 cells. In vitro assay, EGCG strongly inhibited the protein tyrosine kinase (PTK) activities of EGF‐R, PDGF‐R, and FGF‐R, and exhibited an IC50 value of 0.5–1 μg/ml. But EGCG scarcely inhibited the protein kinase activities of pp60v‐src, PKC, and PKA (IC50 > 10 μg/ml). In an in vivo assay, EGCG could reduce the autophosphorylation level of EGF‐R by EGF. Phosphoamino acid analysis of the EGF‐R revealed that EGCG inhibited the EGF‐stimulated increase in phosphotyrosine level in A431 cells. In addition, we showed that EGCG blocked EGF binding to its receptor. The results of further studies suggested that the inhibition of proliferation and suppression of the EGF signaling by EGCG might mainly mediate dose‐dependent blocking of ligand binding to its receptor, and subsequently through inhibition of EGF‐R kinase activity. J. Cell. Biochem. 67:55–65, 1997.

Inhibition of cyclin‐dependent kinases 2 and 4 activities as well as induction of cdk inhibitors p21 and p27 during growth arrest of human breast carcinoma cells by (−)‐epigallocatechin‐3‐gallate

(−)‐Epigallocatechin‐3‐gallate (EGCG) potently inhibits cell proliferation and suppresses tumor growth both in vitro and vivo, but little is known regarding the cell cycle regulatory proteins mediating these effects. This study investigated the effects of EGCG and other catechins on the cell cycle progression. DNA flow cytometric analysis indicated that 30 μM of EGCG blocked cell cycle progression at G1 phase in asynchronous MCF‐7 cells. In addition, cells exposed to 30 μM of EGCG remained in the G1 phase after release from aphidicolin block. Over a 24‐h exposure to EGCG, the Rb protein changed from hyper‐ to hypophosphorylated form and G1 arrest developed. The protein expression of cyclin D1, and E reduced slightly under the same conditions. Immunocomplex kinase experiments showed that EGCG inhibited the activities of cyclin‐dependent kinase 2 (Cdk2) and 4 (Cdk4) in a dose‐dependent manner in the cell‐free system. As the cells were exposed to EGCG (30 μM) over 24 h a gradual loss of both Cdk2 and Cdk4 kinase activities occurred. EGCG also induced the expression of the Cdk inhibitor p21 protein and this effect correlated with the increase in p53 levels. The level of p21 mRNA also increased under the same conditions. In addition, EGCG also increased the expression of the Cdk inhibitor p27 protein within 6 h after EGCG treatment. These results suggest that EGCG either exerts its growth‐inhibitory effects through modulation of the activities of several key G1 regulatory proteins such as Cdk2 and Cdk4 or mediates the induction of Cdk inhibitor p21 and p27. J. Cell. Biochem. 75:1–12, 1999.

Suppression of TNFα-mediated NFκb activity by myricetin and other flavonoids through downregulating the activity of IKK in ECV304 cells

Flavonoids are a group of naturally‐occurring phenolic compounds in the plant kingdom, and many flavonoids are found with vascular protective properties. Nevertheless how the protective response is exerted by flavonoids is not well characterized. In view of the nuclear factor‐κB (NFκB) may play a central role in the initiation of atherosclerosis, prevention of the activation of NFκB represents an important role in protecting vascular injury. In this study, the effects of flavonoids on NFκB/inhibitor‐κB (IκB) system in ECV304 cells activated with tumor necrosis factor‐α (TNFα) were examined. We investigated the inhibitory action of six flavonoids on IκB kinase (IKK) activity, an enzyme recently found to phosphorylate critical serine residues of IκB for degradation. Of six flavonoids tested, myricetin was found to strongly inhibit IKK kinase activity, and prevent the degradation of IκBα and IκBβ in activated endothelial cells. Furthermore, myricetin was also found to inhibit NFκB activity correlated with suppression of monocyte adhesion to ECV304 cells. Therefore we conclude that flavonoids may be of therapeutic value for vascular disease through down regulation of NFκB/IκB system. J. Cell. Biochem. 74:606–615, 1999.

Osthole, a potential antidiabetic agent, alleviates hyperglycemia in db/db mice

Osthole is an agent isolated from Cnidium monnieri (L.) Cusson and Angelica pubescens and has been used to treat several diseases, including metabolic syndromes. To investigate the hypoglycemic effects of osthole in diabetic db/db mice and the underlying mechanisms of these effects by in vitro assay, diabetic db/db mice and cell experiments were utilized to understand its possible effects. Osthole significantly activated both PPARalpha and PPARgamma in a dose-dependent manner based on the results of the transition transfection assay. The activation of PPARalpha and PPARgamma by osthole also resulted in an increase in the expression of PPAR target genes such as PPAR itself, adipose fatty acid-binding protein 2, acyl-CoA synthetases, and carnitine palmitoyltransferase-1A. In vitro results suggested that osthole might be a dual PPARalpha/gamma activator, but its chemical structure differed from that of the thiazolidinedione class of antidiabetic drugs. In addition, osthole markedly activated the AMP-activated protein kinase and its downstream acetyl CoA carboxylase molecules by increasing their phosphorylation levels. Finally, obese diabetic db/db mice were treated with osthole by different administered routes, and osthole was found to markedly reduce blood glucose level. Interestingly, osthole did not reduce the blood insulin or lipid levels, two phenomena that did occur in animals treated with insulin sensitizers like PPAR agonists. These results suggest that osthole can alleviate hyperglycemia and could be potentially developed into a novel drug for treatment of diabetes mellitus.

Suppression of inducible nitric oxide synthase and cyclooxygenase-2 in downregulating nuclear factor-kappa B pathway by Garcinol

Garcinol is a polyisoprenylated benzophenone derivative of Garcinia indica fruit rind and other species. Recent studies have demonstrated that garcinol exhibited antioxidative effects in vitro. In this study, we found that garcinol inhibited the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase‐2 (COX‐2) in lipopolysaccharide (LPS)‐activated macrophages. Western blot analyzes and gel‐shift assays revealed that garcinol strongly blocks the activation of eukaryotic transcription factor nuclear factor‐kappa B (NF‐κB)‐induced by LPS. Moreover, transient transfection experiments showed that garcinol inhibited the NF‐κB‐dependent transcriptional activity. Based on these data, we demonstrated that inhibition of LPS‐induced NF‐κB activation occurred through suppressing the phosphorylation of IκBα and p38 mitogen‐activated kinase (MAPK). Garcinol also lowers the LPS‐induced increase of intracellular reactive oxygen species (ROS), which contributes to the activation of NF‐κB. The NF‐κB signaling pathway leads to inflammatory reaction and our results suggest that garcinol suppresses the expression of iNOS in this pathway.

Death signaling pathway induced by pyrrolidine dithiocarbamate-Cu2+ complex in the cultured rat cortical astrocytes

The chelating and antioxidant effects of pyrrolidine dithiocarbamate (PDTC) have been investigated extensively for preventing cell death induced by different insults. However, the toxic effects of PDTC have been studied only recently and fewer studies on the toxic effects on astrocytes have been reported. In our study, we demonstrated that both PDTC and Cu2+ alone were rated as only weakly toxic in inducing cell death in cortical astrocytes with IC50 of 300 μM and 180 μM, respectively. However, PDTC and Cu2+ in the complex form markedly potentiated with each other by about 1,000‐fold with IC50 of 0.3 μM PDTC plus 10 μM Cu2+. Other metals at concentrations of 3–10 μM (VO45+, Cr6+, Mn2+, Fe2+, Co2+, Ni2+, Zn2+, Pb2+, Bi2+, Ba2+, UO2+, Cs+, SeO42−, La3+) had no such potentiating effects on PDTC. Changes in morphology (nuclear condensation), apoptotic body formation, and hypodiploidity of DNA suggested that the PDTC‐Cu2+ complex induced cell death through an apoptotic process. Further studies showed that the PDTC‐Cu2+ complex decreased mitochondrial membrane potential, increased hydrogen peroxide production, and depleted GSH contents. After the increased oxidative stress, PDTC‐Cu2+ complex differentially activated JNKs, ERK, p38 and caspase 3, which caused PARP degradation in a time‐dependent manner. All these effects were consistent with the increased cellular Cu contents. The nonpermeable copper‐specific chelator bathocuproine disulfonate (BCPS), but not the permeable Cu2+ chelator neocuproine, abolished all the observed effects. Antioxidants (N‐acetylcysteine [NAC], vitamin C), catalase, and Cu2+‐binding proteins (albumin, hemoglobin, and higher serum) reduced the cytotoxic effects of PDTC‐Cu2+ complex. We concluded that the death signaling pathway of PDTC‐Cu2+ complex was mediated by oxidative stress and subsequent JNK activation. These findings imply that PDTC, a widely used pesticide and medicine that is capable of penetrating the blood‐brain barrier, may cause neurotoxicity through astrocyte dysfunction. GLIA 31:249–261, 2000.

Inhibition of 1,2,4-benzenetriol-generated active oxygen species and induction of phase II enzymes by green tea polyphenols

Autooxidation of polyphenolic metabolites of benzene, such as hydroquinone (HQ), catechol (CT), 1,2,4-benzenetriol (BT) and pyrogallol (PG), produced several kinds of active oxygen species (AOS). BT and PG induced DNA breaks in the absence of metal ions, especially when producing AOS such as H2O2, O2-, HO. or 1 delta gO2. HQ and CT did not result in double-strand DNA breaks, except when ferrous ion was added, indicating the participation of the Fenton reaction. Polyphenolic fractions isolated from green tea (GTP) exerted inhibitory effects on the autooxidation of BT and suppressive effects on H2O2 or HO. generated from phenolic metabolites of benzene in the presence of S9 or an in vivo system. Additionally, although the activities of antioxidant and phase II enzymes were elevated by both GTP and phenolic metabolites of benzene, GTP counteracted the lowering GSH caused by phenolic metabolites of benzene in rat liver. The above results suggest that GTP and phenolic metabolites of benzene are antagonistic in their response to AOS, especially hydroxyl radical.

In vitro and in vivo studies of the anticancer action of terbinafine in human cancer cell lines: G0/G1 p53‐associated cell cycle arrest

Terbinafine (TB) (Lamisil®), a promising oral antifungal agent used worldwide, has been used in the treatment of superficial mycosis. In our study, we demonstrated that TB dose‐dependently decreased cell number in various cultured human malignant cells. Flow cytometry analysis revealed that TB interrupts the cell cycle at the G0/G1 transition. The TB‐induced cell cycle arrest in colon cancer cell line (COLO 205) occurred when the cyclin‐dependent kinase (cdk) system was inhibited just as the levels of p53, p21/Cip1 and p27/Kip1 proteins were augmented. In the TB‐treated COLO 205, the binding between p53 protein and p53 consensus binding site in p21/Cip1 promoter DNA probe was increased. Pretreatment of COLO 205 with p53‐specific antisense oligodeoxynucleotide decreased the TB‐induced elevations of p53 and p21/Cip1 proteins, which in turn led to arrest in the cell cycle at the G0/G1 phase. Moreover, in the p53 null cells, HL60, TB treatment did not induce cell cycle arrest. Taken together, these results suggest an involvement of the p53‐associated signaling pathway in the TB‐induced antiproliferation in COLO 205. We further examined whether administration of TB could affect the growth of tumors derived from human colon cancer cells in an in vivo setting. COLO 205 cells implanted subcutaneously in nude mice formed solid tumor; subsequent intraperitoneal injections of TB (50 mg/kg) led to obvious decline in tumor size, up to 50–60%. In these tumors, increases in the p21/Cip1, p27/Kip1 and p53 proteins and the occurrence of apoptosis were observed. Combined treatment with TB and nocodazole (ND), a clinically used anticancer agent, potentiated the apoptotic effect in COLO 205. These findings demonstrate for the first time that TB can inhibit the proliferation of tumor cells in vitro and in vivo.

Molecular mechanisms of G0/G1 cell-cycle arrest and apoptosis induced by terfenadine in human cancer cells.

Terfenadine (TF), a highly potent histamine H1 receptor antagonist, has been shown to exert no significant central nervous system side effects in clinically effective doses. In this study, we demonstrated that TF induced significant growth inhibition of human cancer cells, including Hep G2, HT 29, and COLO 205 cells, through induction of G0/G1 phase cell‐cycle arrest. The minimal dose of TF induced significant G0/G1 arrest in these cells was 1–3 μM. The protein levels of p53, p21/Cip1, and p27/Kip1 were significantly elevated, whereas the kinase activities of cyclin‐dependent kinase 2 (CDK2) and CDK4 were inhibited simultaneously in the TF‐treated cells. On the other hand, significant apoptosis, but not G0/G1 arrest, was induced in the HL 60 (p53‐null) or Hep 3B (with deleted p53) cells when treated with TF (3–5 μM). To clarify the roles of p21/Cip1 and p27/Kip1 protein expression, which was involved in G0/G1 arrest and apoptosis induced by TF in human cancer cells, antisense oligodeoxynucleotides (ODNs) specific to p21/Cip1 and p27/Kip1 were used, and the expression of the p21/Cip1 and p27/Kip1 were monitored by immunoblotting analysis. Our data demonstrated that the percentage of the apoptotic cells detected by annexin V/PI analysis in the TF‐treated group was clearly attenuated by pretreatment with p27/Kip1–specific ODNs. These results indicated that p27/Kip1 (but not p21/Cip1) protein indeed played a critical role in the TF‐induced apoptosis. We also demonstrated that the TF‐induced G0/G1 cell‐cycle arrest effect was not reversed by TF removal, and this growth inhibition lasted for at least 7 d. Importantly, the occurrence of apoptosis and cell growth arrest was not observed in the TF‐treated normal human fibroblast, even at a dose as high as 25 μM. Our study showed the molecular mechanisms for TF‐induced cell growth inhibition and the occurrence of apoptosis in human cancer cells.