Sirtuin 6 promotes transforming growth factor-ß1/H2O2/HOCl-mediated enhancement of hepatocellular carcinoma cell tumorigenicity by suppressing cellular senescence.

Sirtuin 6 (SIRT6) can function as a tumor suppressor by suppressing aerobic glycolysis and apoptosis resistance. However, the negative effect of SIRT6 on cellular senescence implies that it may also have the potential to promote tumor development. Here we report that the upregulation of SIRT6 expression was required for transforming growth factor (TGF)-ß1 and H2O2/HOCl reactive oxygen species (ROS) to promote the tumorigenicity of hepatocellular carcinoma (HCC) cells. Transforming growth factor-ß1/H2O2/HOCl could upregulate SIRT6 expression in HCC cells by inducing the sustained activation of ERK and Smad pathways. Sirtuin 6 in turn abrogated the inducing effect of TGF-ß1/H2O2/HOCl on cellular senescence of HCC cells, and was required for the ERK pathway to efficiently suppress the expression of p16 and p21. Sirtuin 6 altered the effect of Smad and p38 MAPK pathways on cellular senescence, and contributed to the inhibitory effect of the ERK pathway on cellular senescence. However, SIRT6 was inefficient in antagonizing the promoting effect of TGF-ß1/H2O2 HOCl on aerobic glycolysis and anoikis resistance. Intriguingly, if SIRT6 expression was inhibited, the promoting effect of TGF-ß1/H2O2/HOCl on aerobic glycolysis and anoikis resistance was not sufficient to enhance the tumorigenicity of HCC cells. Suppressing the upregulation of SIRT6 enabled TGF-ß1/H2O2/HOCl to induce cellular senescence, thereby abrogating the enhancement of HCC cell tumorigenicity by TGF-ß1/H2O2/HOCl. These results suggest that SIRT6 is required for TGF-ß1/H2O2/HOCl to enhance the tumorigenicity of HCC cells, and that targeting the ERK pathway to suppress the upregulation of SIRT6 might be a potential approach in comprehensive strategies for the therapy of HCC.

ß3 integrin promotes TGF-ß1/H2O2/HOCl-mediated induction of metastatic phenotype of hepatocellular carcinoma cells by enhancing TGF-ß1 signaling.

In addition to being an important mediator of migration and invasion of tumor cells, ß3 integrin can also enhance TGF-ß1 signaling. However, it is not known whether ß3 might influence the induction of metastatic phenotype of tumor cells, especially non-metastatic tumor cells which express low level of ß3. Here we report that H2O2 and HOCl, the reactive oxygen species produced by neutrophils, could cooperate with TGF-ß1 to induce metastatic phenotype of non-metastatic hepatocellular carcinoma (HCC) cells. TGF-ß1/H2O2/HOCl, but not TGF-ß1 or H2O2/HOCl, induced ß3 expression by triggering the enhanced activation of p38 MAPK. Intriguingly, ß3 in turn promoted TGF-ß1/H2O2/HOCl-mediated induction of metastatic phenotype of HCC cells by enhancing TGF-ß1 signaling. ß3 promoted TGF-ß1/H2O2/HOCl-induced expression of itself via positive feed-back effect on p38 MAPK activation, and also promoted TGF-ß1/H2O2/HOCl-induced expression of α3 and SNAI2 by enhancing the activation of ERK pathway, thus resulting in higher invasive capacity of HCC cells. By enhancing MAPK activation, ß3 enabled TGF-ß1 to augment the promoting effect of H2O2/HOCl on anoikis-resistance of HCC cells. TGF-ß1/H2O2/HOCl-induced metastatic phenotype was sufficient for HCC cells to extravasate from circulation and form metastatic foci in an experimental metastasis model in nude mice. Inhibiting the function of ß3 could suppress or abrogate the promoting effects of TGF-ß1/H2O2/HOCl on invasive capacity, anoikis-resistance, and extravasation of HCC cells. These results suggest that ß3 could function as a modulator to promote TGF-ß1/H2O2/HOCl-mediated induction of metastatic phenotype of non-metastatic tumor cells, and that targeting ß3 might be a potential approach in preventing the induction of metastatic phenotype of non-metastatic tumor cells.

[Membrane cholesterol mediates the endocannabinoids-anandamide affection on HepG2 cells].

OBJECTIVE: To study the effect of anandamide (AEA) on necrosis in HepG2 cells and to explore the role of AEA in progression of liver cancer. METHODS: Localization of the fatty acid hydrolytic enzyme (FAAH), cannabinoid receptors 1(CB1) and cannabinoid receptors 2 (CB2) proteins was detected in L02 and HepG2 cells using immunofluorescence. L02 and HepG2 cells were treated with different concentrations of AEA and methyl-beta-cyclodextrin, and the rates of cells necrosis were examined by PI stain. Meanwhile, the expression levels of FAAH, CB1 and CB2 receptor proteins, as well as P38 mitogen-activated protein kinase (p-P38 MAPK) and c-Jun-NH2-terminal kinase (p-JNK) proteins, were analyzed by Western blot. RESULTS: The FAAH, CB1 and CB2 receptor proteins were observed both in cytoplasm and on membrane in L02 and HepG2 cells. The expression level of FAAH protein was higher in HepG2 than in L02 cells. The expression level of CB1 receptor protein was very low in both L02 and HepG2 cells. The expression level of CB2 receptor protein was high in both L02 and HepG2 cells. AEA treatment induced necrosis in HepG2 cells but not in L02 cells. Methyl-beta-cyclodextrin treatment prevented necrosis in HepG2 cells (t = 3.702; 5.274; 3.503, P less than 0.05). The expression patterns of FAAH, CB1 and CB2 receptor protein in L02 and HepG2 cells were confirmed by western blot, which were consistent with the immunofluorescence results. AEA treatment increased the levels of p-P38MAPK and p-JNK proteins in a dose-dependent manner in HepG2 cells (F = 11.908; 26.054, P less than 0.05) and the increase can be partially by prevented by MCD (t = 2.801; t = 12.829, P less than 0.05). CONCLUSION: AEA treatment induces necrosis in HepG2 cells via CB1 and CB2 receptors and lipid rafts.