Therefore, we next analyzed the effect on cell viability in Hep3B cells of various treatment combinations: VO-OHpic with the multi-kinase inhibitor sorafenib, with the MEK inhibitor U0126, with the dual PI3K/mTOR inhibitor BEZ235. of p21 and p16 mRNAs were analyzed by quantitative RT-PCR in HCC cells. Hep3B, PLC/PRF/5 and SNU475 cells were treated with the 500?nM of VO-OHpic for 72?hours. Relative expression was calculated as ratio of drug-treated samples versus control (DMSO) and corrected by the quantified expression level of -actin. The results shown are the means SD of three ARMD5 experiments, each performed in triplicate. Cell cycle phase progression is usually regulated by a number of the cyclin-dependent kinases (CDKs) and cyclins which can be negatively regulated by kinase inhibitor proteins, such as p21 and p16, two well known CDK inhibitors involved in the control of cellular senescence. BAZ2-ICR To further elucidate the mechanism of VO-OHpic induced cell cycle arrest in HCC cells, we decided the levels p16 and p21 mRNAs in all cell lines exposed to different concentrations of VO-OHpic (Fig.?4B). The levels of p16 mRNA were only slightly increased in Hep3B and SNU475 cells, whereas p21 BAZ2-ICR mRNA was increased only in Hpe3B cells, but not in BAZ2-ICR PLC/PRF/5 and SNU475 cells, suggesting that it may play a role in VO-OHpic-induced senescence. VO-OHpic synergizes with PI3K/mTOR and Raf/MEK/ERK inhibitors The observation that treatment with VO-OHpic altered AKT and ERK1/2 signaling prompted us to investigate the functional functions of the activation of these signaling pathways. Therefore, we next analyzed the effect on cell viability in Hep3B cells of various treatment combinations: VO-OHpic with the multi-kinase inhibitor sorafenib, with the MEK inhibitor U0126, with the dual PI3K/mTOR inhibitor BEZ235. According to the combination index (CI), the combination of varying concentrations of VO-OHpic with all these inhibitors resulted in a synergistic inhibition of cell viability in Hep3B cells, as evaluated by MTS assay after 72?hours of treatment (Table?1). Table 1. VO-OHpic in combination with sorafenib, U0126, and BEZ235 elicited synergistic inhibition of cell viability in Hep3B cells. The combination index (CI) values are indicated. effectiveness of VO-OHpic on HCC, a mouse xenograft tumor model of Hep3B cells was used. Treatment with VO-OHpic significantly reduced tumor volume BAZ2-ICR when compared with tumors of the untreated group (Fig.?5A). Open in a separate window Physique 5. The effect of VO-OHpic on xenograft models of Hep3B cells. (A) Effect of VO-OHpic on tumor growth. Once tumors were engrafted and palpable, mice (experiments (Fig.?1C). Immunohistochemical analysis showed a lower expression of cell proliferation marker Ki-67 in tumor tissues from animals treated with VO-OHpic, than in the tissues of the untreated animals (Fig.?5D-E), confirming data obtained using an proliferation assay (BrdU assays) (Fig.?2B). Conversation In the present study using human HCC cells expressing different levels of PTEN, we present a new insight into the antitumor effects of the PTEN inhibitor VO-OHpic, as well as the putative mechanisms involved. First, we exhibited the effect of VO-OHpic by analyzing expression of PTEN-regulated phosphoproteins (p-AKT, p-ERK1/2). We then decided that VO-OHpic inhibited the cell viability, cell proliferation and colony-forming ability of HCC cells in relation to PTEN levels. Although some reports have reported that VO-OHpic is usually a specific and potent inhibitor of PTEN,21,25-29 others have raised issues about its specificity.30 In particular, Spinelli (exhibited that complete acute loss of did not give a proliferative advantage as would be expected, but.