Identification of mTOR as a primary resistance factor of the IAP antagonist AT406 in hepatocellular carcinoma cells.

Dysregulation of inhibitor of apoptosis (IAP) proteins (IAPs) in hepatocellular carcinoma (HCC) is often associated with poor prognosis. Here we showed that AT406, an IAP antagonist, was cytotoxic and pro-apoptotic to both established (HepG2, SMMC-7721 lines) and primary HCC cells. Activation of mTOR could be a key resistance factor of AT406 in HCC cells. mTOR inhibition (by OSI-027), kinase-dead mutation or knockdown remarkably enhanced AT406-induced lethality in HCC cells. Reversely, forced-activation of mTOR by adding SC79 or exogenous expressing a constitutively active S6K1 (T389E) attenuated AT406-induced cytotoxicity against HCC cells. We showed that AT406 induced degradation of IAPs (cIAP-1 and XIAP), but didn't affect another anti-apoptosis protein Mcl-1. Co-treatment of OSI-027 caused simultaneous Mcl-1 downregulation to overcome AT406's resistance. Significantly, shRNA knockdown of Mcl-1 remarkably facilitated AT406-induced apoptosis in HCC cells. In vivo, AT406 oral administration suppressed HepG2 tumor growth in nude mice. Its activity was potentiated with co-administration of OSI-027. We conclude that mTOR could be a key resistance factor of AT406 in HCC cells.

[Mechanism of combined use of cyclopamine and hydroxycamptothecin in inducing the apoptosis of human oral squamous cell carcinoma cell line].

OBJECTIVE: To study the mechanism underlying the effect of combined use of cyclonpamine and hydroxycamptothecin in inducing the apoptosis of human oral squamous cell carcinoma cell line (OSCC) HSQ-89. METHODS: CCK8 assay was used to investigate the inhibitory effect of cyclopamine on HSQ-89 cells. Flow cytometry (FCM) was employed to examine the cell apoptosis following combined treatment with cyclonpamine and hydroxycamptothecin. Reverse transcription polymerase chain reaction (RT-PCR) was applied to detect the mRNA expressions of Bcl-2, Bcl-xl, and Bid in HSQ-89 cells after the treatments. RESULTS: Combined treatment with cyclonpamine and hydroxycamptothecin significantly inhibited the cell proliferation compared with hydroxycamptothecin treatment alone, also resulting in a significantly higher apoptosis rate of the cells (P<0.05). The mRNA level of Bcl-2 was significantly decreased after the treatments, especially after the combined treatment. Cyclopamine produced no significant effect on the mRNA levels of Bcl-xl and Bid in the cells. CONCLUSION: The combined use of cyclopamine and hydroxycamptothecin significantly down-regulates the expression on of bcl-2 to induce the apoptosis of human OSCC cell line HSQ-89.

[Epigallocatechin-3-gallate induces G1 phase cell cycle arrest in KB cells].

OBJECTIVE: To explore the effects of epigallocatechin-3-gallate (EGCG) on the proliferation of human oral epithelial cancer cell line KB cells and the molecular mechanisms. METHOD: KB cells were treated with various concentrations of EGCG for 24 or 48 h. MTT assay was used to test the cell viability. The changes of cell cycle in KB cells treated with EGCG for 48 h were analyzed using flow cytometry. The expressions of cyclin A, cyclin D1 and cyclin E were detected by RT-PCR and Western blotting. RESULT: The viability of KB cells treated with various concentrations of EGCG (25, 50, 100, 200, 400, and 800 micromol/L) for 48 h were decreased to (85.4-/+2.4)%, (80.4-/+2.8)%, (51.5-/+4.5)%, (30.2-/+1.9)%, (25.3-/+1.5)%, (20.0-/+1.1)%, respectively, showing significant difference from that of the control group [(100.0-/+2.2)%, P<0.05). EGCG decreased the viabilities of KB cells in a dose-dependent manner. Flow cytometry demonstrated that treatment with EGCG significantly increased the cell percentage in sub-G1 phase, which was (73.5-/+4.4)% after a 48-h EGCG treatment, significantly different from that in the control group [(47.3-/+3.5)%, P<0.05). EGCG-induced G1 phase arrest was correlated to the down-regulation of cyclin A and cyclin E. CONCLUSION: EGCG inhibits the proliferation of KB cells by inducing G1 phase arrest, which involves the downregulation of cyclin E.

[Epigallocatechin-3-gallate induces apoptosis in human hepatocellular carcinoma cells].

OBJECTIVE: To investigate the effects of epigallocatechin-3-gallate (EGCG) on human hepatocellular carcinoma (HCC) cells and mechanism thereof. METHOD: Human HCC cells of the lines HepG2 and SMMC-7721 were cultured and treated with of EGCG of the concentrations of 6.25, 12.5, 25, 50, 100, 200, and 400 microg/ml respectively for 24 h and 48 h. The cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. Trypan blue staining was used to count the cells. Flow cytometry was conducted to detect the cell apoptosis. The protein levels of Bcl-2, an anti-apoptosis factor, and cyclooxygenase-2 (COX-2), an up-regulator of Bcl-2. The activities of caspase-9 and caspase-3 hat promote the apoptosis of HCC cells, were measured using colorimetric method. RT-PCR was used to detect the mRNA expression of COX-2 and Bcl-2 family. RESULTS: The viabilities of the HepG2 and SMMC-7721 cells treated with EGCG of the concentrations of 50 - 400 microg/ml for 48 h reduced to 93.8% +/- 2.8%, 62.3% +/- 5.4%, 33.9% +/- 2.5%, and 17.6% +/- 3.2% respectively, all significantly lower than that of the control group [(100.0% +/- 2.8%), all P < 0.05]; and the viabilities of the SMMC-772 cells treated with EGCG of the concentrations of 50 - 400 microg/ml for 48 h reduced to 49.6% +/- 3.5%, 30.3% +/- 3.8%, 17.7% +/- 2.2%, and 13.0% +/- 2.5% respectively, all significantly lower than that of the control group [(100.0% +/- 0.8%), all P < 0.05]. After treatment with 100 microg/ml EGCG for 24 h, 48 h, 72 h, and 96 h, the live HepG2 cell numbers were (8.0 +/- 1.5), (22.0 +/- 3.1), (37.0 +/- 5.4), and (61.0 +/- 8.7) 10(4) respectively, all significantly lower than those of the control cells [(15.0 +/- 2.5), (45.0 +/- 5.3), (86.0 +/- 11.0), and (210.0 +/- 23.0) 10(4) respectively, all P < 0.05]; and the live SMMC-7721 cell numbers were (7.0 +/- 2.2), (13.0 +/- 2.5), (20.0 +/- 3.7), and (31.0 +/- 4.0) 10(4) respectively, all significantly lower than those of the control cells [(15.0 +/- 2.5), (45.0 +/- 5.3), (86.0 +/- 11.0), and (210.0 +/- 23.0) 10(4) respectively, all P < 0.05]. The apoptotic rates of HepG2 cells treated with EGCG of the concentrations of 50, 100, and 200 microg/ml for 12 h were 8.7% +/- 0.4%, 18.1% +/- 1.1%, and 22.1% +/- 1.8% respectively, all significantly higher than that of the control group (3.3% +/- 0.3%, P < 0.05); and the apoptotic rates of SMMC-7721 cells were 5.9% +/- 0.3%, 7.8% +/- 0.6%, and 12.2% +/- 0.8% respectively, all significantly higher than that of the control group (3.7% +/- 0.4%, P < 0.05). After treatment with EGCG of the concentrations of 100 and 200 microg/ml for 12 h, the caspase-9 activities of the HepG2 cells increased to (1.8 +/- 0.4) and (2.5 +/- 0.4) respectively, both significantly higher than that of the control group (1.0 +/- 0.1, both P < 0.05); and the caspase-3 activities of the HepG2 cells increased to (2.0 +/- 0.4) and (2.8 +/- 0.5) respectively, both significantly higher than that of the control group (1.0 +/- 0.2, P < 0.05) ; and the caspase-9 activities of the SMMC-7721 cells increased to (1.7 +/- 0.4) and (2.5 +/- 0.4), both significantly higher than that of the control group (1.0 +/- 0.1, both P < 0.05), and the caspase-3 activities of the SMMC-7721 cells increased to (1.9 +/- 0.4) and (2.6 +/- 0.3) respectively, both significantly higher than that of the control group [ (1.0 +/- 0.2), both P < 0.05]. When the concentration of EGCG was over 200microg/ml, it down-regulated the expression of COX-2 and Bcl-2 in both cell lines, however, EGCG resulted in no significant changes of Bcl-xl, Bax, Bad, and Bid. CONCLUSION: EGCG induces apoptosis in HCC cells through down-regulation of COX-2 and Bcl-2 and consequently activating caspase-9 and caspase-3.

Establishment of multiplexed, microsphere-based flow cytometric assay for multiple human tumor markers.

AIM: The multiplexed, microsphere-based flow cytometric assay (MFCA) for multiple human tumor markers was established for the early screening and detection of suspected cancer patients. METHODS: Covalent coupling of capture antibodies directed against their respective tumor markers to fluorescent microspheres was performed by following the protocols recommended by a commercial corporation with some modifications. The coupling efficiency and cross-reactivity were identified by the Luminex 100 system and associated software. The standard curve was constructed by using serial dilution of recombinant tumor marker standards and was validated by comparison with ELISA for quantifying the tumor markers in serum samples. RESULTS: The identifications revealed that the coupling procedures were successful without non-specific cross-reactivity and the standard curve was highly efficient. However, it was necessary to ensure the quality control of the coupling process since slight variations in the coupling procedures could profoundly affect the density of capture reagents coupled to the microspheres and consequently adversely affect the assay precision. In addition to its multi-analyte capability, the MFCA system had definite advantages, such as higher reproducibility, greater dynamic range of measurement, and considerably less preparation time and labor over the conventional "gold standard", which was the ELISA. CONCLUSION: The successful establishment of the MFCA system for the simultaneous detection of multiple tumor markers will provide the foundation for the further study of clinical applications.

Upregulation of PTEN involved in rosiglitazone-induced apoptosis in human hepatocellular carcinoma cells.

AIM: To investigate the effects of rosiglitazone, a peroxisome proliferator-activated receptor gamma (PPARgamma) agonist, on the expression of the phosphatase and tensin homologue deleted on chromosome 10 gene (PTEN) and cell growth in hepatocellular carcinoma cells, as well as the underlying mechanisms of these effects. METHODS: RT-PCR and Western blotting analyses were performed to detect transcription and the expression of PTEN in Hep3B cells treated with rosiglitazone. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay was used to evaluate cell growth. Flow cytometry, DNA fragmentation analysis, caspase enzymatic assay, and Hoechst 33258 staining were used to determine cell apoptosis. Furthermore, small interfering RNA was used to suppress PTEN expression. RESULTS: Rosiglitazone increased the expression of PTEN in a dose- and time-dependent manner through the PPARgamma-dependent signal transduction pathway. PTEN upregulation was concomitant with a decreased level of Akt phosphorylation, subsequently resulting in cell growth inhibition and apoptosis in Hep3B cells. PTEN knockdown dramatically blocked these effects of rosiglitazone. Moreover, the exposure of cells to rosiglitazone activated caspases-9 and -3 during apoptotic proceeding. CONCLUSION: Thus, upregulation of PTEN is involved in the inhibition of cell growth and the induction of cell apoptosis by rosiglitazone, suggesting that rosiglitazone may be useful in liver cancer therapy via apoptosis.

Green tea polyphenol epigallocatechin-3-gallate inhibits oxidative damage and preventive effects on carbon tetrachloride-induced hepatic fibrosis.

The aim of the study was to examine the effects of epigallocatechin-3-gallate (EGCG) on hepatic fibrogenesis and on cultured hepatic stellate cells (HSCs). The rat model of carbon tetrachloride (CCl(4))-induced hepatic fibrosis was used to assess the effect of daily intraperitoneal injections of EGCG on the indexes of fibrosis. Histological and hepatic hydroxyproline examination revealed that EGCG significantly arrested progression of hepatic fibrosis. EGCG caused significant amelioration of liver injury (reduced activities of serum alanine aminotransferase and aspartate aminotransferase). The development of CCl(4)-induced hepatic fibrosis altered the redox state with a decreased hepatic glutathione and increased the formation of lipid peroxidative products, which were partially normalized by treatment with EGCG, respectively. Moreover, EGCG markedly attenuated HSC activation as well as matrix metalloproteinase (MMP)-2 activity. In cultured stellate cell, the expression of MMP-2 mRNA and protein were substantially reduced by EGCG treatment. Concanavalin A-induced activation of secreted MMP-2 was inhibited by EGCG through the influence of membrane type 1-MMP activity. These results demonstrate that administration of EGCG may be useful in the treatment and prevention of hepatic fibrosis.

Rab23 is a potential biological target for treating hepatocellular carcinoma.

AIM: To elucidate the role of Rab23 in hepatocellular carcinoma (HCC) by assessing the expression of Rab23 in HCC tissue and in HCC cell lines. METHODS: Primary tumors (n = 100) were stained with Rab23 antibodies using immunohistochemistry and in situ hybridization in tissue microarrays. Relationships between gene expression and pathology parameters were analysed. The biological significance of Rab23 in Hep-3B cells was examined by knocking down Rab23 gene expression. We designed a pair of double-stranded RNAs against human rab23 and transfected siRNA into Hep-3B cells. Rab23 expression in these cells was examined using RT-PCR and Western blots. We investigated cell growth by MTT assays and fluorescence-activated cell sorting. RESULTS: High cytoplasmic and nuclear expression of Rab23 was found in 38 of 71 (53.5%) and in 49 of 68 HCC patients (72%) respectively, which correlated with tumor size. HCC cell lines expressed Rab23. In Hep3B cells, siRNA for Rab23 decreased Rab23 mRNA by 4.5-fold and protein expression by 2-fold. Survival rates at 24 and 48 h for Hep-3B cells transfected with siRNA were lower and about 30% Hep-3B cells were apoptotic. Knocking down rab23 suppressed Hep3B cell growth, suggesting that rab23 could play an important role in Hep3B cell growth. CONCLUSION: Rab23 is overexpressed and/or activated in HCC. Rab23 may be both a HCC predictor and a target for treating HCC.

[15-deoxy-Delta(12,14)-prostaglandin J(2) induces anoikis of hepatocellular carcinoma cells: an in vitro experiment].

OBJECTIVE: To investigate the effect of 15-deoxy-Delta(12,14)-prostaglandin J(2) (15-d-PGJ(2)) on the anoikis of hepatocellular carcinoma (HC) cells and mechanisms thereof. METHODS: Fibronectin or polyhydroxyethyl methacrylate (poly-HEMA) were coated onto tissue culture plates, cell growth status and morphological changes were detected by optical microscope. DNA fragmentation analysis and Flow cytometry were used to measure cell apoptotic activity. Western blotting analysis was performed to detect the levels of focal adhesion kinase (FAK) and phosphorylated FAK(p-FAK). Furthermore, small interfering RNA (siRNA) was used to suppress FAK expression. RESULTS: The adhesion rate of the BEL-7402 cells treated with 15-d-PGJ(2) began to decrease 12 h after the treatment, time- and dose-dependently compared with the HC cell control group (all P < 0.05); when the concentration of 15-d-PGJ2 was 20 micromol/L, the adherent cells ratio at 24 h and 48 h later were (66.0 +/- 3.6)% and (35.0 +/- 5.0)% respectively. Anoikis of BEL-7402 cells was observed by flow cytometry and DNA fragmentation analysis. Western blotting showed that the p-FAK level of the BEL-7402 cells treated with 15-d-PGJ2 for 24 h decreased dose-dependently, however, the total FAK protein did not change. CONCLUSION: 15-d-PGJ(2) induces anoikis and decreases the phosphorylated FAK expression of the hepatocellular carcinoma cells.

Green tea polyphenol epigallocatechin-3-gallate suppresses rat hepatic stellate cell invasion by inhibition of MMP-2 expression and its activation.

AIM: Epigallocatechin-3-gallate (EGCG) is the major component of green tea polyphenols, whose wide range of biological properties includes anti-fibrogenic activity. Matrix metalloproteinases (MMP) that participate in extracellular matrix degradation are involved in the development of hepatic fibrosis. The present study investigates whether EGCG inhibits activation of the major gelatinase matrix metalloproteinase-2 (MMP-2) in rat hepatic stellate cells (HSC). METHODS: The expression of MMP-2, tissue inhibitors of metalloproteinases-2 (TIMP-2), and membrane-type 1-MMP (MT1-MMP) was assessed by RT-PCR and Western blot analyses. MMP-2 activity was evaluated by zymography and MT1-MMP activity was assessed by an enzymatic assay. HSC migration was measured by a wound healing assay and cell invasion was performed using Transwell cell culture chambers. RESULTS: The expression of MMP-2 mRNA and protein in HSC was substantially reduced by EGCG treatment. EGCG treatment also reduced concanavalin A (ConA)-induced activation of secreted MMP-2 and reduced MT1-MMP activity in a dose-dependent manner. In addition, EGCG inhibited either HSC migration or invasion. CONCLUSION: The abilities of EGCG to suppress MMP-2 activation and HSC invasiveness suggest that EGCG may be useful in the treatment and prevention of hepatic fibrosis.