The Effects Of Triptonide On Hepatocellular Carcimoma Cell Growth And Oroxin A Inhibits Breast Cancer Cell Growth By Inducing Senescence And Endoplasmic Reticulum

Aim: In this study, we are prepared to investigate the effect of Triptonide(TN) on cell proliferation of human hepatocellular carcinoma(HCC) cell QGY-7703 and the effect of Oroxin A(OA) on cell proliferation and cell senescence on human breast cancer cell MDA-MB-231,and to learn its molecular mechanisms.Methods: AlamarBlue assay was used to determine the effects of both TN on the proliferation of human hepatocellular carcinoma cell QGY-7703 and OA on the proliferation of human breast cancer cell MDA-MB-231.Wright-Giemsa staining assay was used to observe the cell morphological of QGY-7703 treated with TN and MDA-MB-231 treated with OA. The effect of TN and the effect of OA on cell cycle were examined by PI staining and FACS analysis, the expression of cell cycle-related genes and proteins were detected by RT-PCR and Western blot. The cellular senescence induced by Oroxin A in human breast cancer cell MDA-MB-231 was determined by SA β-Gal staining and SAHF staining. The effect of OA on the expression of cell senescence-related genes and proteins were assessed by RT-PCR and Western blot. ER stress in MDA-MB-231 cells treated with OA was measured using a specific ER-Tracker Red fluorescent probe. The level of intracellular ROS was assessed by flow cytometry.Results:1.AlamarBlue results showed that TN could obviously inhibit QGY-7703 cell proliferation with IC50 19.2nmol/l at 72 h. The PI staining and the flow cytometry assay showed tha TN could induced QGY-7703 cell cycle arrest at G0/G1 phase.2.Alamarblue results showed that OA could inhibit MDA-MB-231 cell proliferation with IC50 16μmol/l at 72 h. The β-Gal assay indicated that treatment of MDA-MB-231 cells with OA for 72 h resulted in an obvious increase inβ-Gal-positive cells. In addition, the SAHF staining indicated that OA-treated cells showed classic SAHF. Accumulated date and flow cytometry have shown that an increase in intracellular ROS levels can induce intracellular ER stress and senescence. PI staining and FACS analysis showed that OA increased the cell population of the G2/M phase of the MDA-MB-231 cells, whereas Annexin V staining showed that OA did not significantly induce apoptosis at lower doses. In addition, we explored the effect of OA on the expression of cell cycle-related gene p21 by RT-PCR. The signal pathway assay showed that OA significantly increase p38 phosphorylation. The RT-PCR showed that OA markly increased the expression of ER stress-related genes ATF4 and GRP78. Western blot result showed that OA could increase ATF4 and GRP78 protein levels.Furthermore, OA has slight inhibition effect on human peripheral blood cell and other cancer cells(human chronic myelogenous leukemia cell lines and human glioma cell cells), and it indicated that OA had very low toxicity.Conclusion:1.TN could effectively inhibit cell proliferation on QGY-7703 with very low toxicity.2.OA could specifically inhibit cell proliferation on MDA-MB-231 with low toxicity.3.OA may inhibit breast cancer cell growth by inducing cellular senescence through activation of ER stress by an increase in ROS levels, overexpression in ATF4 and GRP78 genes, and activation of the p38 signaling pathway. Thus, OA appears to be a potential new antibreast cancer drug candidate.