Longjuan Zhang

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).

Sonic hedgehog signaling pathway induces cell migration and invasion through focal adhesion kinase/AKT signaling-mediated activation of matrix metalloproteinase (MMP)-2 and MMP-9 in liver cancer

The aberrant activation of sonic hedgehog (SHH) pathway contributes to initiation and progression of various malignancies. However, the roles and underlying mechanisms of SHH signaling pathway in invasion and metastasis of liver cancer have not been well understood. In this study, we found that SHH signaling was activated and correlated with invasion and metastasis in hepatocellular carcinoma (HCC). Enhanced SHH signaling by recombinant human SHH N-terminal peptide (rSHH-N) promoted hepatoma cell adhesion, migration and invasion, whereas blockade of SHH signaling with SHH neutralizing antibody or cyclopamine suppressed hepatoma cell adhesion, migration and invasion. Furthermore, matrix metalloproteinase (MMP)-2 and MMP-9 expressions and activities were upregulated and downregulated by rSHH-N and SHH signaling inhibitor, respectively. The rSHH-N-mediated hepatoma cell migration and invasion was blocked by MMP-specific inhibitors or neutralizing antibodies to MMP-2 and MMP-9. In addition, phosphorylations of AKT and focal adhesion kinase (FAK) were increased and decreased by rSHH-N and SHH signaling inhibitor, respectively. Further investigations showed that activation of AKT and FAK were required for rSHH-N-mediated upregulation of MMP-2 and MMP-9, cell migration and invasion. Finally, we found that SHH protein expression was positively correlated with phosphorylatd FAK Tyr397, phosphorylatd AKT Ser473, MMP-2 and MMP-9 protein expressions in HCC samples. Taken together, our findings suggest that SHH pathway induces cell migration and invasion through FAK/AKT signaling-mediated MMP-2 and MMP-9 production and activation in liver cancer.

miR-338-3p suppresses invasion of liver cancer cell by targeting smoothened†

MicroRNAs are involved in human carcinogenesis and cancer progression. Our previous study has shown that loss of miR‐338‐3p expression is associated with clinical aggressiveness of hepatocellular carcinoma (HCC). However, the exact roles and mechanisms of miR‐338‐3p remain unknown in HCC. To determine whether and how miR‐338‐3p influences liver cancer cell invasion, we studied miR‐338‐3p in the liver cancer cell lines, and we found that miR‐338‐3p is down‐regulated in treated cells. Forced expression of miR‐338‐3p in SK‐HEP‐1 cells suppressed cell migration and invasion, whereas inhibition of miR‐338‐3p in SMMC‐7721 cells induced cell migration and invasion. Furthermore, smoothened (SMO) was identified as a direct target of miR‐338‐3p. Forced expression of miR‐338‐3p down‐regulated SMO and matrix metalloproteinase (MMP)‐9 expression, but inhibition of miR‐338‐3p up‐regulated SMO and MMP9 expression. However, small interfering RNA targeted SMO reversed the effects induced by blockade of miR‐338‐3p. SMO and MMP9 were overexpressed and associated with invasion and metastasis in HCC tissues. These data indicate that miR‐338‐3p suppresses cell invasion by targeting the smoothened gene in liver cancer in vitro and miR‐338‐3p might be a novel potential strategy for liver cancer treatment. Copyright

FAK is involved in invasion and metastasis of hepatocellular carcinoma

Studies have shown that focal adhesion kinase (FAK) is overexpressed in several human tumors and plays an important role in tumor progression. However, the role and underlying mechanisms of FAK in hepatocellular carcinoma (HCC) progression remains to be elucidated. In this study, we examined FAK and phosphorylated FAK Tyr397 expression in a large series of HCCs. We found that both FAK and phosphorylated FAK Tyr397 were overexpressed in HCC samples and HCC cell lines. Increased FAK and phosphorylated FAK Tyr397 expressions were correlated with tumor stage, vascular invasion and intrahepatic metastasis in HCC. Furthermore, HCC cell adhesion, migration and invasion were substantially impaired by siRNA-mediated knockdown of FAK expression, whereas cell growth, apoptosis and cell cycle distribution were not affected. In addition, depletion of FAK induced a significant reduction in expressions and activities of both MMP-2 and MMP-9. Taken together, FAK contributes to invasion and metastasis of HCC partly through regulating expressions and activations of both MMP-2 and MMP-9, suggesting FAK could be a promising therapeutic target for 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.

miR‐145 suppresses cell invasion in hepatocellular carcinoma cells: miR‐145 targets ADAM17

miR‐145 is a candidate tumor suppressor miRNA. However, it is unknown whether miR‐145 is involved in the invasion of hepatocellular carcinoma (HCC). Therefore, we aimed to explore the effect and mechanism of miR‐145 in the control of HCC cell invasion.

In vitro and in vivo induction of bone formation based on adeno-associated virus-mediated BMP-7 gene therapy using human adipose-derived mesenchymal stem cells

AbstractAim:To determine whether adeno-associated virus (AAV)-2-mediated, bone morphogenetic protein (BMP)-7-expressing human adipose-derived mesenchymal stem cells (ADMS) cells would induce bone formation in vitro and in vivo.Methods:ADMS cells were harvested from patients undergoing selective suction-assisted lipectomy and transduced with AAV carrying the human BMP-7 gene. Non-transduced cells and cells transduced with AAV serotype 2 carrying the enhanced green fluorescence protein gene served as controls. ADMS cells were qualitatively assessed for the production of BMP-7 and osteocalcin, and subjected to alkaline phosphatase (ALP) and Chinalizarin staining. A total of 2.5×106 cells mixed with type I collagen were implanted into the hind limb of severe combined immune-deficient (SCID) mice and subjected to a histological analysis 3 weeks post implantation.Results:Transfection of the ADMS cells achieved an efficiency of 99% at d 7. Transduction with AAV2-BMP-7 induced the expression of BMP-7 until d 56, which was markedly increased by d 7. The cells were positively stained for ALP. Osteocalcin production and matrix mineralization further confirmed that these cells differentiated into osteoblasts and induced bone formation in vitro. A histological examination demonstrated that implantation of BMP-7-expressing ADMS cells could induce new bone formation in vivo.Conclusion:The present in vitro and in vivo study demonstrated that human ADMS cells would be a promising source of autologous mesenchymal stem cells for BMP gene therapy and tissue engineering.