Liang-qi Cao

Involvement of PI3K/PTEN/AKT/mTOR pathway in invasion and metastasis in hepatocellular carcinoma: Association with MMP‐9

Aim:  To investigate the status of Phosphatidylinositol 3‐kinase (PI3K)/PTEN/AKT/mammalian target of rapamycin (mTOR) pathway and its correlation with clinicopathological features and matrix metalloproteinase‐2, ‐9 (MMP‐2, 9) in human hepatocellular carcinoma (HCC).

Bead‐based microarray analysis of microRNA expression in hepatocellular carcinoma: miR‐338 is downregulated

Aim:  Recent studies have underlined causative links between microRNA (miRNA) deregulation and cancer development. However, the relevance of abnormally expressed miRNA to tumor biology has not been well understood in hepatocellular carcinoma (HCC).

Clinical significance of miR-221 and its inverse correlation with p27Kip1 in hepatocellular carcinoma

The aim of the present study is to explore possible role of miR-221 in the pathogenesis of HCC. Matched HCC and adjacent non-cancerous samples were assayed for the expression of miR-221 and three G1/S transition inhibitors: p27Kip1, p21WAF1/Cip1and TGF-β1 by in situ hybridization and immunohistochemistry respectively. p27Kip1 is one of miR-221’s proven targets. Real time qRT-PCR was used to investigate miR-221 and p27Kip1 transcripts in different clinical stages. Western blotting was used to analyze the expression levels of p27Kip1 protein in different clinical stages. In result, miR-221 and TGF-β1 are frequently up-regulated in HCC, while p27Kip1 and p21WAF1/Cip1 proteins are frequently down-regulated. Moreover, miR-221 and p27Kip1’s expression correlated with metastasis and miR-221’s expression also correlated with tumor size. Both of p21WAF1/Cip1and TGF-β1’s expression correlated with tumor differentiations. miR-221’s upregulation and p27Kip1’s downregulation were significantly associated with tumor stages and metastasis. In conclusion, miR-221 is important in tumorigenesis of HCC, possibly by specifically down-regulating p27Kip1, a cell-cycle inhibitor. These results indicate miR-221 as a new therapeutic target in HCC.

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

AbstractAim:To investigate the effects of rosiglitazone, a peroxisome proliferator-activated receptor gamma (PPARγ) 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 PPARγ-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 suppresses rat hepatic stellate cell invasion by inhibition of MMP-2 expression and its activation

AbstractAim: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 con-canavalin 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.

Expression of sonic hedgehog signaling components in hepatocellular carcinoma and cyclopamine-induced apoptosis through Bcl-2 downregulation in vitro.

BACKGROUND AND AIMS Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. Aberrant activation of sonic hedgehog (Shh) signaling pathway plays important roles in tumorigenesis and progression of several tumors. Cyclopamine, an important inhibitor of Shh signaling pathway, can induce cell apoptosis. However, the mechanisms underlying cyclopamine-induced apoptosis are not well understood. The aim of this study is to determine the expression of the Shh signaling pathway components in HCC and to investigate the mechanisms underlying cyclopamine-induced apoptosis in HCC cells. METHODS Shh signaling components (Shh, Ptch, Smo and Gli-1) expression levels were evaluated by immunohistochemistry on tissue microarrays containing 98 HCCs with paired adjacent noncancerous liver tissues. The relationships between sonic hedgehog signal pathway and clinicopathological factors were analyzed in HCC. Cell viability was analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Apoptosis was detected by flow cytometry. mRNA and protein levels were analyzed by RT-PCR and Western blot, respectively. RESULTS Shh, Ptch, Smo and Gli-1 were overexpressed in HCC tissues compared with paired adjacent noncancerous liver tissue. Activated Shh signaling pathway was associated with tumor size, capsular invasion and vascular invasion in HCC. Cyclopamine remarkably decreased cell viability, induced apoptosis and downregulated Bcl-2 expression in HCC cells. CONCLUSIONS Shh signaling pathway plays an important role in HCC tumorigenesis and progression, indicating that Shh signaling pathway is a potential therapeutic target for HCC. Cyclopamine induces apoptosis through downregulating Bcl-2 in HCC.

Rosiglitazone sensitizes hepatocellular carcinoma cell lines to 5-fluorouracil antitumor activity through activation of the PPARγ signaling pathway

AbstractAim:Resistance to 5-fluorouracil (5-FU) is a major cause of chemotherapy failure in advanced hepatocellular carcinoma (HCC).Rosiglitazone, a peroxisome proliferator-activated receptor γ (PPARγ) agonist, has a crucial role in growth inhibition and induction of apoptosis in several carcinoma cell lines. In this study, we examine rosiglitazone-induced sensitization of HCC cell lines (BEL-7402 and Huh-7 cells) to 5-FU.Methods:The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was used to evaluate cell viability. Western blotting analysis was performed to detect the protein expression (PPARγ, PTEN, and COX-2) in BEL-7402 cells. Immunohistochemistry staining was used to examine the expression of PTEN in 100 advanced HCC tissues and paracancerous tissues. In addition, small interfering RNA was used to suppress PPARγ, PTEN, and COX-2 expression.Results:Rosiglitazone facilitates the anti-tumor effect of 5-FU in HCC cell lines, which is mediated by the PPARγ signaling pathway. Activation of PPARγ by rosiglitazone increases PTEN expression and decreases COX-2 expression. Since distribution of PTEN in HCC tissues is significantly decreased compared with the paracancerous tissue, over-expression of PTEN by rosiglitazone enhances 5-FU-inhibited cell growth of HCC. Moreover, down-regulation of COX-2 is implicated in the synergistic effect of 5-FU.Conclusion:Rosiglitazone sensitizes hepatocellular carcinoma cell lines to 5-FU antitumor activity through the activation of PPARγ. The results suggest potential novel therapies for the treatment of advanced liver cancer.

Expression patterns and polymorphisms of PTCH in Chinese hepatocellular carcinoma patients

Aberrant activation of Hedgehog signaling pathway leads to pathological consequences in a variety of human tumors. PTCH (PTCH1), the receptor of Hedgehog pathway, is reported to function as a gatekeeper in tumor formation. Here we report, by semi-quantitative RT-PCR, PTCH expression was found in 38 hepatocellular carcinoma (HCC) patients (66%). Evidences from real time quantitative RT-PCR further indicate that compared to their matched nontumorous liver tissue, PTCH exhibit a higher expression in well and moderate differentiated tumor, but a lower expression in poorly differentiated tumor. Immunohistochemical staining showed PTCH protein was detected in the cytoplasm of 56.3% HCC samples (9/16). For the first time, we investigate the polymorphisms of PTCH in HCC. First we sequenced the recognized mutation hot spots regions of PTCH of 38 HCC samples. Two previously reported single nucleotide polymorphisms (SNPs) and a novel SNP A1056G were identified. Then we examined these three SNPs in 171 HCC samples and 162 normal liver samples. However, statistic analysis showed none of these SNPs was statistically significant for association with HCC. In conclusion, our data suggest that PTCH is involved in early stage tumor development and the Hh pathway in Chinese HCC is activated by ligand expression but not by mutation.

Hematoporphyrin derivative-mediated photodynamic therapy inhibits tumor growth in human cholangiocarcinoma in vitro and in vivo

Aim:  To investigate the effects of hematoporphyrin derivative‐mediated photodynamic therapy (HPD‐PDT) on cell growth in human cholangiocarcinoma in vitro and in vivo, as well as the underlying mechanisms of these effects.