Previous studies have shown that all-trans retinoic acid (ATRA) suppresses growth of hepatocarcinoma cell in vitro. To understand the underlying mechanisms, we investigated the protein expression profiles by 2-DE in hepatocarcinoma cell line SMMC-7721 treated with ATRA. Our results reveal that six proteins were differently expressed in response to ATRA. Using MS and database searching, they were identified as profilin 1, phosphoglycerate kinase 1, RuvB-like 1, alpha-enolase, pyridoxal kinase and F-actin capping protein. We selected the up-regulated protein, profilin 1 (PFN1), for further studies. The PFN1 expression was increased in response to ATRA in a dose- and time-dependent manner. The PFN1 expression was reduced dramatically in four hepatoma cell lines compared to L02 cell line of non-tumor origin. The PFN1 expression was also examined in 4 cases of primary hepatocarcinoma tissues by Western blot and 30 cases by tissues microarray. It was found that the protein level of PFN1 was lower in hepatocarcinoma tissues compared to that in the adjacent tissues. Similar to ATRA, overexpression of PFN1 led to inhibition of cell proliferation and migration. Furthermore, RNAi-based PFN1 knockdown could rescue the inhibitory effect of ATRA on cell proliferation and migration. In conclusion, ATRA inhibited cell proliferation and migration through up-regulation of PFN1.

No abstract

PTEN is a major tumor suppressor gene that has been shown to inhibit cell invasion. Its mutation has been found in 20-40% of malignant gliomas. Meanwhile, the type III EGFR mutation (EGFRvIII), which was frequently found in gliomas, promoted cell invasion. In the present study, the effects of PTEN on cell invasion were investigated in U87DEGFR glioblastoma cells with EGFRvIII expression but missing PTEN. The cell invasion was downregulated by transfection of phosphatase-active forms of PTEN (wild-type and G129E) but not by PTEN (C124A) with an inactive phosphatase domain; the effects were correlated with decreased tyrosine phosphatase levels of FAK at Tyr 397 , which was increased by EGFRvIII. Overexpression of FAK mutant (Y397F) could partially mimic the effect of PTEN on cell invasion. Although EGFRvIII increased the levels of P-Akt and PTEN eliminated it, PI-3K inhibitors, wortmannin or Ly294002, could not decrease the cell invasion. In conclusion, PTEN could inhibit cell invasion even in the presence of the constitutively active EGFR; this inhibition depended on its protein phosphatase activity, partially by dephosphorylating FAK, but not depended on its lipid phosphatase activity. ' 2005 Wiley-Liss, Inc.

No abstract

Evidence has been emerging to suggest that integrin could induce growth inhibition in some cell types. Some of the molecular mechanisms underlying growth arrest have been elucidated. We reported here that overexpression of integrin L L1 imposed a growth inhibitory e¡ect on the hepatocellular carcinoma cell line SMMC-7721, and this phenomenon was mainly attributed to the cyclin-dependent kinase inhibitor p21 CIP1 . Furthermore, our ¢ndings suggested that transcription activity of the p21 CIP1 gene could be upregulated in the integrin L L1-overexpressing cells, and possibly controlled by the cis-elements in the core region of the p21 CIP1 promoter.

In this study, an enzymatic inactive mutant of GnT-V (DcGnT-V) was constructed and transfected in SMMC 7721 cell line. Integrin b1 in DcGnT-V transfectants (Dc-7721) showed attenuation of the number of b1-6 GlcNAc branching, whereas those in wtGnT-V transfectants (wt-7721) presented a b1-6 GlcNAc-rich pattern. High integrin b1 expression was observed in wt-7721 compared with mock cells (7721 cell transfected with the vector pcDNA3), while transfection of DcGnT-V decreased the integrin b1 expression, despite of no significant changes on integrin b1 mRNA level in these cell lines. Pulse-chase experiment showed that Integrin b1 in Dc-7721 was prone to quick degradation and its halflife was less than 3 h, on the contrary, the alleviating degradation of b1 subunit was observed in wt-7721 where the b1 subunit half-life was about 16 h, meanwhile, the degradation rate of b1 subunit in mock cells was in between, about 10 h. More effective in promoting cell migration toward fibronectin and invasion through Matrigel was observed in wt-7721 while this was almost suppressed in Dc-7721. Our results suggest that the addition of b1-6 GlcNAc branching caused more fully glycosylated mature form on integrin b1 and inhibited b1 protein degradation. Glycosylation caused by GnT-V directs integrin b1 stability and more delivery to plasma membrane, subsequently promotes Fn-based cell migration and invasion.

No abstract

DNA methyltransferases (DNMTs) are essential for maintenance of aberrant methylation in cancer cells and play important roles in the development of cancers. Unregulated activation of PI3K/Akt pathway is a prominent feature of many human cancers including human hepatocellular carcinoma (HCC). In present study, we found that DNMT3B mRNA and protein levels were decreased in a dose- and time-dependent manner in HCC cell lines with LY294002 treatment. However, we detected that LY294002 treatment did not induce increase of the degradation of DNMT3B protein using protein decay assay. Moreover we found that Akt induced alteration of the expression of DNMT3B in cells transfected with myristylated variants of Akt2 or cells transfected with small interfering RNA respectively. Based on DNMT3B promoter dual-luciferase reporter assay, we found PI3K pathway regulates DNMT3B expression at transcriptional level. And DNMT3B mRNA decay analysis suggested that down-regulation of DNMT3B by LY294002 is also post-transcriptional control. Furthermore, we demonstrated that LY294002 down-regulated HuR expression in a time-dependent manner in BEL-7404. In summary, we have, for the first time, demonstrate that PI3K/Akt pathway regulates the expression of DNMT3B at transcriptional and post-transcriptional levels, which is particularly important to understand the effects of PI3K/Akt and DNMT3B on hepatocarcinogenesis.