Cucurbitacin E regulates activation of hepatic stellate cells via AMPK-mTOR pathway

Hepatic fibrosis is a wound-healing response for injury. Activated hepatic stellate cells （HSCs） are the preferred targets of anti-hepatic fibrotic therapies. Cucurbitacin E （CUE） is, one well-known natural compound de- rived from Traditional Chinese Medicine, used in Asian countries for the prevention and treatment of hepatic dis- ease. Therefore, the present study elucidated the mechanism of CuE on inducing apoptosis and attenuating hepatic fibrosis towards activated HSCs. The murine hepatic stellate cells （t-HSC/C1-6） cell line were incubated in 96-well plates and treated with TNF-α and CuE at various concentrations and indicated times. Cell viability was assessed with MTT assay. Another, t-HSC/C1-6 were incubated in 6-well plates and also treated with TNF-α, CuE, AICAR or metformin for the indicated time and concentration. Cell protein and mRNA were prepared using kit and relevant signaling were detected by Western blot and RT-PCR. CuE inhibited cell proliferation of activated HSC/T-6 cells in concentration- and time-dependent manner. CuE triggered the activation of caspase-3, cleaved the PARP, ration of bcl-2/bax, and cytochrom C protein in a time- and concentration-dependent manner. CuE decreased p-Erk/MAPK without effects on p-p38 and p-JNK. CuE inhibited the protein and mRNA expressions of oL-SMA, TIMP-1 and col- lagen I in activated HSC-T6. CuE broadly blocked p-PI3K, p-Akt, p-roTOR and p-p70S6K expressions. CuE sig- nificantly increased phosphorylated AMPK expression as well as AICAR and metoformin. And metformin showed significantly higher activation of AMPK than AICAR. Ability of CuE on activation of AMPK was between AICAR and metformin. It＇s also found that CuE significantly decreased p-roTOR as well as AICAR and metformin. CuE could modulate HSC survival and activation as a potential anti-fibrotic agent for liver fibrosis treatment. The find- ings demonstrate that CuE induced HSC apoptosis via Erk/MAPK and PI3IC/Akt-AMPK-mTOR signaling.

Metformin targets liver tumor-initiating cells through the PI3K/Akt/mTOR survival pathway

Liver tumor-initiating cells(T-ICs) are thought to be inherently resistant to the cytotoxic effects of chemotherapy, and can self-renewal and maintain tumor-initiating potential. Therefore, effective anticancer research strategies should target the unique properties of T-ICs. In this study, we found that metformin, a first-line drug of choice for the treatment of type 2 diabetes, inhibited liver T-ICs both in vivo and in vitro. Metformin inhibited the formation of hepatospheres and epithelial-specific antigen-positive(ESA,CD133?) cell colonies by hepatocellular carcinoma(HCC)cell lines. Metformin also downregulated the expression of several T-IC-related genes which are involved in the signaling pathways, governing the self-renewal, proliferation and differentiation of T-ICs. Furthermore, the targeting of liver T-ICs by metformin was PI-3-kinase-Akt-mTOR(PI3K/Akt/mTOR)-pathway dependent. The PI3K/Akt/mTOR inhibitor LY294002 and rapamycin abolished the inhibitory effect of metformin on CD133?cells, and the PI3K/Akt/mTOR stimulator EGF promoted the inhibitory effect of metformin on CD133?cells. Metformin also dramatically decreased the tumor volume and number of CD133 expressing tumor cells in a xenograft mouse model. Metformin exerted a synergistic effect with cisplatin to target both T-ICs and non-T-ICs, and resulted in the smallest tumor volume and lowest number of CD133 expressing tumor cells. This study indicates that the antidiabetic drug metformin could potentially be used in combination therapy with chemotherapeutic agents to improve the treatment of liver cancer.