Study Of Pharmacodynamics And Functional Mechanism Of Sargentgloryvine Stem Polyterpene Macrolide Acid Sodium In Hepatoma

Hepatoma is one of the most commonly diagnosed cancers in our humankind, of which 90% are hepatocellular carcinoma. Although many advancement have been achieved in the therapeutic strategies against HCC, which make the survival rate and survival quality of the patient being improved obviously, the total curative effect of HCC is still out of satisfaction. Genesis of HCC is a complicated process in which multifactor and multiple genes are involved. The precise mechanism underlying this process is still elusive. The clinical biological behavior of hepatoma is relatively vicious and the prognosis is not good. Nonage endo-liver metastasis and recurrence after surgical operation mostly observed in HCC patients directly influence the selection of therapeutic methods and survival quality and live time. Recently, following the development of nanometer molecular medicine, molecular biological techniques, explaining of the mechanism of tumorigenesis and discovery of new tumor therapy targets, development of neotype anti-cancer drugs and combing usage of multiple-therapeutic strategies have been exploited. Among numerous anti-cancer drugs, plant-derived natural products which account for a great proportion play important roles in cancer therapy.Objective Accompanying with the appearance of more and more side effects causing by radio- or chemo-therapeutic drugs, researchers have pay their attentions on development of neotype, high performance and low-poison anti-cancer drugs. Currently, the function of macrolides drugs in tumor therapy is gradually drawing the attentions of the researchers. Sargentgloryvine stem polyterpene macrolide acid is a metamer extracted from Sargentgloryvine stem of Sargentodoxa cunneata. Salification of sargentgloryvine stem polyterpene macrolide acid is named as Sargentgloryvine stem polyterpene macrolide acid sodium (SPMA) with molecular formula of C38H46O8Na. Our preliminary experimental results showed that SPMA could inhibit proliferation and promote apoptosis of multiple malignant tumors. In this study, we explored the pharmacodynamics and toxicological effects of SPMA with the aims of identifying the anti-cancer mechanism of SPMA, providing data for further pre-clinical study and establishing foundation for acquirement of high performance/low-poison anti-cancer drug.Methods (1)HepG-2 and HHCC cells were treated with different concentrations of SPMA. By detecting the proliferation curve, cell cycle, apoptosis of SPMA treated cells and tumor growth inhibition and side toxic effect in xenograft mice, the pharmacodynamics effects of SPMA was identified. (2)By analyzing the difference genes using microarray technique and analyzing the pathway in which SPMA is involved, the pathway through which the SPMA inhibits the proliferation of hepatoma were identified. (3)Through gene transfection and gene silencing targeting molecules involved in the above pathway, the targets of SPMA were identified. (4)By detecting the blood pressure and heart rate of the SD rats treated with master single test administration and detecting blood routine, biochemistry indexes and HE staining of main organs of SD rats treated with successive administration for one month, the influence of SPMA on normal metabolism of rat was identified.Results(1)SPMA could significantly inhibit growth and proliferation of hepatoma cells with a time and dose dependent style, while the control normal PBMCs was not inhibited. In addition, cells treated with SPMA were blocked in S stage. SPMA treatment also induced apoptosis of HepG-2 and HHCC in vitro. In vivo experiments results showed that the volumes of xenograft tumor in SPMA treatment group were obviously diminished compared with control groups. HE staining results showed that there was no obvious pathological change in main organs of SPMA treatment rat. (2)Microarray analysis results showed that there were 4349 difference genes between SPMA treated HepG-2 cell and control cell. Through analyzing the related pathway in which obvious difference genes are involved, we obtained two pathways that may be related with SPMA treatment in hepatoma cells. One is endogenous mitochondria pathway: bcl-2-mitochondria-cyt-caspase 9- apoptosis, the other is apoptosis pathway mediated by skp2.(3)SPMA could down-regulate the expressions of bcl-xl, mcl-1 and up-regulate the expression of bak and induce the aggregation of cytochrome C. (4)By silencing the expression of Skp2 using siRNA technique, we observed obvious proliferation inhibition in SPMA treated group. However, cell apoptosis was not observed in SPMA treated cells. (5)After being successive administrated for one month (once per 3 days), the main biochemical indicators of SPMA treated rats were in their normal reference scopes and there was no obvious pathological change in the HE staining results of their main organs.Conclusion(1)The pharmacodynamics results showed that SPMA can inhibit proliferation and promote apoptosis of hepatoma cells and does not produce obvious side effect on the body within its effective therapeutical dose. (2)SPMA can active the endogeneous mitochondria apoptotic pathway through down-regulating the expressions of bcl-xl and mcl-1 and up-regulating the expression bak. And then hepatoma cell apoptosis is induced by releasing of cytochrome C and activation of caspase 9. (3)SPMA can block the hepatoma cells in S stage through targeting skp2, which promotes the apoptosis of cells. (4)SPMA treatment with effective therapeutical dose could not produce obvious side effect on haematogenesis system, liver function and kidney function of the rat. In summary, SPMA have potiential to be a kind of neotype metamer anti-ancer drug with characteristics of high performance, low-poison and safety.