Jen-Kun Lin

Rotenone induces apoptosis in MCF-7 human breast cancer cell-mediated ROS through JNK and p38 signaling.

Rotenone is an inhibitor of the mitochondrial electron transport chain complex I, resulting in the generation of reactive oxygen species (ROS). Rotenone has been shown to display anticancer activity through the induction of apoptosis in various cancer cells. However, the underlying mechanism is still not fully understood. Here, rotenone showed a strong growth inhibitory effect against human breast cancer MCF‐7 cells. DNA flow cytometric analysis, chromatin condensation, and poly (ADP‐ribose) polymerase (PARP) cleavage indicated rotenone actively induced apoptosis in MCF‐7 cells. The antiapoptotic protein, Bcl‐2, was decreased, whereas the apoptotic protein, Bax, was increased in a time‐dependent manner in rotenone‐induced apoptosis. Moreover, the treatment of rotenone in MCF‐7 cells caused the activation of c‐jun N‐terminal kinase (JNK) and p38 mitogen‐activated protein kinases (MAPKs), and the inactivation of extracellular signal‐regulated protein kinase 1/2 (ERK1/2). The pharmacological inhibition of JNK and p38 MAPK revealed significant protection against rotenone‐induced apoptosis. Taken together, these results indicate rotenone may induce apoptosis through ROS and JNK/p38 MAPKs activation in MCF‐7 cells.

Suppression of nitric oxide synthase and the down‐regulation of the activation of NFκB in macrophages by resveratrol

Resveratrol, naringenin and naringin are naturally occurring flavonoids in grapes and grapefruits. The anti‐inflammatory effects of these flavonoids have been well documented, but the mechanism is poorly characterized. High concentration of NO are produced by inducible NO synthase (iNOS) in inflammation, and the prevention of the expression of iNOS may be an important anti‐inflammatory mechanism. In this study, the effects of these flavonoids on the induction of NO synthase (NOS) in RAW 264.7 cells activated with bacterial lipopolysaccharide (LPS, 50 ng ml−1) were investigated. Resveratrol was found strongly to inhibit NO generation in activated macrophages, as measured by the amount of nitrite released into the culture medium, and resveratrol strongly reduced the amount of cytosolic iNOS protein and steady state mRNA levels. However, the inhibitory abilities of naringenin were lower, and the inhibitory abilities of naringin were almost negligible. In electrophoretic mobility shift assays, the activation of NFκB induced by LPS for 1 h was inhibited by resveratrol (30 μM). Furthermore, in immunoblotting analysis, cells treated with LPS plus resveratrol showed an inhibition of phosphorylation as well as degradation of IκBα, and a reduced nuclear content of NFκB subunits. The flavonoids may be of value for inhibiting the enhanced expression of iNOS in inflammation through down‐regulation of NFκB binding activity.

Involvement of reactive oxygen species and caspase 3 activation in arsenite-induced apoptosis

Recent studies indicate that arsenic may generate reactive oxygen species to exert its toxicity. However, the mechanism is still unclear. In this study, we demonstrate that arsenite is able to induce apoptosis in a concentration‐ and time‐dependent manner; however, arsenate is unable to do so. An increase of intracellular peroxide levels was accompanied with arsenite‐induced apoptosis, as demonstrated by flow cytometry using DCFH‐DA. N‐Acetyl‐L‐cysteine (a thiol‐containing antioxidant), diphenylene iodonium (an inhibitor of NADPH oxidase), 4,5‐dihydro‐1,3‐benzene disulfonic acid (a selective scavenger of O  2− ) and catalase significantly inhibit arsenite‐induced apoptosis and intracellular fluorescence intensity. In contrast, allopurinol (an inhibitor of xanthine oxidase), indomethacin (an inhibitor of cyclooxygenase), superoxide dismutase, or PDTC had no effect on arsenite‐induced cell death. Activation of CPP32 activity, PARP (a DNA repair enzyme) degradation, and release of cytochrome c from mitochondria to the cytosol are involved in arsenite‐induced apoptosis, and Bcl‐2 antagonize arsenite‐induced apoptosis by a mechanism that interferes in the activity of CPP32. These results lead to a working hypothesis that arsenite‐induced apoptosis is triggered by the generation of hydrogen peroxide through activation of flavoprotein‐dependent superoxide‐producing enzymes (such as NADPH oxidase), and hydrogen peroxide might play a role as a mediator to induce apoptosis through release of cytochrome c to cytosol, activation of CPP32 protease, and PARP degradation. J. Cell. Physiol. 177:324–333, 1998.

Induction of apoptosis by apigenin and related flavonoids through cytochrome c release and activation of caspase-9 and caspase-3 in leukaemia HL-60 cells

The aim of this study was to investigate the mechanism of flavonoid-induced apoptosis in HL-60 leukaemic cells. Thus, the effect of structurally related flavonoids on cell viability, DNA fragmentation and caspase activity was assessed. Loss of membrane potential and reactive oxygen species generation were also monitored by flow cytometry. The structurally related flavonoids, such as apigenin, quercetin, myricetin, and kaempferol were able to induce apoptosis in human leukaemia HL-60 cells. Treatment with flavonoids (60 microM) caused a rapid induction of caspase-3 activity and stimulated proteolytic cleavage of poly-(ADP-ribose) polymerase (PARP). Furthermore, these flavonoids induced loss of mitochondrial transmembrane potential, elevation of reactive oxygen species (ROS) production, release of mitochondrial cytochrome c into the cytosol, and subsequent induction of procaspase-9 processing. The potency of these flavonoids on these features of apoptosis were in the order of: apigenin > quercetin > myricetin > kaempferol in HL-60 cells treated with 60 microM flavonoids. These results suggest that flavonoid-induced apoptosis is stimulated by the release of cytochrome c to the cytosol, by procaspase-9 processing, and through a caspase-3-dependent mechanism. The induction of apoptosis by flavonoids may be attributed to their cancer chemopreventive activity. Furthermore, the potency of flavonoids for inducing apoptosis may be dependent on the numbers of hydroxyl groups in the 2-phenyl group and on the absence of the 3-hydroxyl group. This provides new information on the structure-activity relationship of flavonoids.

Curcumin, an antioxidant and anti-tumor promoter, induces apoptosis in human leukemia cells

Curcumin, widely used as a spice and coloring agent in food, possesses potent antioxidant, anti-inflammatory and anti-tumor promoting activities. In the present study, curcumin was found to induce apoptotic cell death in promyelocytic leukemia HL-60 cells at concentrations as low as 3.5 micrograms/ml. The apoptosis-inducing activity of curcumin appeared in a dose- and time-dependent manner. Flow cytometric analysis showed that the hypodiploid DNA peak of propidium iodide-stained nuclei appeared at 4 h after 7 micrograms/ml curcumin treatment. The apoptosis-inducing activity of curcumin was not affected by cycloheximide, actinomycin D, EGTA, W7 (calmodulin inhibitor), sodium orthovanadate, or genistein. By contrast, an endonuclease inhibitor ZnSO4 and proteinase inhibitor N-tosyl-L-lysine chloro-methyl ketone (TLCK) could markedly abrogate apoptosis induced by curcumin, whereas 12-O-tetradecanoylphorbol-13-acetate (TPA) had a partial effect. The antioxidants, N-acetyl-L-cysteine (NAC), L-ascorbic acid, alpha-tocopherol, catalase and superoxide dismutase, all effectively prevented curcumin-induced apoptosis. This result suggested that curcumin-induced cell death was mediated by reactive oxygen species. Immunoblot analysis showed that the level of the antiapoptotic protein Bcl-2 was decreased to 30% after 6 h treatment with curcumin, and was subsequently reduced to 20% by a further 6 h treatment. Furthermore, overexpression of bcl-2 in HL-60 cells resulted in a delay of curcumin-treated cells entering into apoptosis, suggesting that bcl-2 plays a crucial role in the early stage of curcumin-triggered apoptotic cell death.

Comparative studies on the suppression of nitric oxide synthase by curcumin and its hydrogenated metabolites through down-regulation of IκB kinase and NFκB activation in macrophages

Nitric oxide (NO) plays an important role in inflammation and in the multiple stages of carcinogenesis. In this study, we investigated the inhibitory effects of curcumin and its metabolites, tetrahydrocurcumin, hexahydrocurcumin, and octahydrocurcumin, on the induction of NO synthase (NOS) in RAW 264.7 cells activated with lipopolysaccharide (LPS). Western blotting and northern blotting analyses demonstrated that curcumin strongly reduced 130-kDa protein and 4.5-kb mRNA levels of iNOS in LPS-activated macrophages compared with its metabolites, tetrahydrocurcumin, hexahydrocurcumin, and octahydrocurcumin. Moreover, electrophoretic mobility shift assay (EMSA) experiments indicated that curcumin blocked the LPS-induced binding of nuclear factor-kappaB (NFkappaB), a transcription factor necessary for iNOS induction to its (32)P-labeled double-stranded oligonucleotide probe. The inhibition of NFkappaB activation occurred through the prevention of inhibitor kappaB (IkappaB) degradation. Transient transfection experiments also showed that curcumin inhibited NFkappaB-dependent transcriptional activity. Curcumin blocked the disappearance of inhibitory kappaBalpha (IkappaBalpha) and p65 from the cytosolic fraction, and inhibited the phosphorylation of IkappaBalpha. Furthermore, we showed that curcumin could inhibit the IkappaB kinase 1 (IKK1) and IkappaB kinase 2 (IKK2) activities induced by LPS, but tetrahydrocurcumin, hexahydrocurcumin, and octahydrocurcumin were less active. These results suggest that curcumin may exert its anti-inflammatory and anti-carcinogenic properties by suppressing the activation of NFkappaB through inhibition of IKK activity.

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.

Curcumin induces apoptosis in immortalized NIH 3T3 and malignant cancer cell lines

Curcumin, which is a widely used dietary pigment and spice, has been demonstrated to be an effective inhibitor of tumor promotion in mouse skin carcinogenesis. We report that curcumin induces cell shrinkage, chromatin condensation, and DNA fragmentation, characteristics of apoptosis, in immortalized mouse embryo fibroblast NIH 3T3 erb B2 oncogene-transformed NIH 3T3, mouse sarcoma S180, human colon cancer cell HT-29, human kidney cancer cell 293, and human hepatocellular carcinoma Hep G2 cells, but not in primary culture of mouse embryonic fibroblast C3H 10T1/2, rat embryonic fibroblast, and human foreskin fibroblast cells in a concentration- and time-dependent manner. Many cellular and biochemical effects of curcumin in mouse fibroblast cells have been reported, such as inhibition of protein kinase C (PKC) activity induced by phorbol 12-myristate 13-acetate treatment, inhibition of tyrosine protein kinase activity, and inhibition of arachidonic acid (AA) metabolism. Treatment of NIH 3T3 cells with the PKC inhibitor staurosporine, the tyrosine kinase inhibitor herbimycin A, and the AA metabolism inhibitor quinacrine induces apoptotic cell death. These results suggest that, in some immortalized and transformed cells, blocking the cellular signal transduction might trigger the induction of apoptosis.

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.