The Experimental Study Of The Anti-Breast Cancer Efficacy And Corresponding Mechanisms Of Iridoid Esters Compound Jatamanvaltrate P From Valeriana Jatamansi Jones

Breast cancer is the most common malignancy among women in the world,with the highest incidence rate among all female tumors and second among all tumors.Breast cancer treatment is composed of surgery,radiotherapy,chemotherapy,endocrine therapy,targeted therapy and other treatment programs.In China,serious drug resistance and great side effects of traditional therapy in some types of high-risk breast cancer have contributed to the increasing death rate,this promoted people to look for a new drug to treat breast cancer.Valeriana jatamansi Jones.is a herbs used in traditional medicine.Chinese medicine theory which is believed to taste Ku,Xing Wen and is related to heart,spleen,stomach.It is used to relieve pain,promote digestion,and has sedative and anti-diarrhea effect.Many clinical formulas against neurological diseases contain Valeriana jatamansi Jones.Furthermore,some studies have shown that Valerian plants have anti-cancer effects,such as inducing apoptosis in gastric cancer and leukemia.However,little is known about the specific anti-cancer components of Valeriana jatamansi Jones.,and how one of its active compounds affects the function of tumor cell cycle and autophagy.Our in vitro screening experiments showed that Jatamanvaltrate P has anti-cancer potential and strongly inhibits the proliferation of breast cancer cell.Therefore,this study focused on the inhibitory effects of Jatamanvaltrate P on the growth of breast cancer cells in vitro and in vivo,and explored the underlying molecular mechanisms.Objective:The aim of this study was to investigate the inhibitory effect of the compound Jatamanvaltrate P from Valeriana jatamansi Jones.on the growth of human breast cancer cells,and to elucidate its possible mechanisms through the induction of apoptosis and autophagy and cell cycle arrest.Methods:(1)MTT assay was used to detect the effect of iridoid ester on the proliferation of tumor cells,and the effect of compound Jatamanvaltrate P on the proliferation of breast cancer cells as well as mammary epithelial cells was determined.(2)The number and morphology of breast cancer cells treated with Jatamanvaltrate P were observed.(3)The effect of Jatamanvaltrate P on cell proliferation in breast cancer cells were determined by plate colony formation assays.(4)Jatamanvaltrate P induced apoptotic bodies of breast cancer cells were determined by DAPI staining assay.(5)The effect of Jatamanvaltrate P on apoptosis and cell cycle of breast cancer cells was determined by flow cytometry.(6)The effect of Jatamanvaltrate P on autophagy of breast cancer cells was observed by fluorescence microscopy.(7)The expression of related proteins of apoptosis,autophagy and cell cycle were detected by Western Blotting.(8)We added the relevant inhibitors(Z-VAD-FMK,3-MA),and then used flow cytometry to detect apoptosis and Western Blotting to detect the changes of related proteins.(9)The effect of Jatamanvaltrate P on breast cancer was investigated by using nude mice transplanted tumor model.The apoptosis of tumor tissue was detected by TUNEL staining.The expression of autophagy marker protein LC3 was detected by Western Blotting.Results:(1)Jatamanvaltrate P from Valeriana jatamansi Jones significantly inhibited 12 human tumor cells,and the results showed that Jatamanvaltrate P has little toxicity against normal human breast epithelial cells MCF-10A while showed significant potency against the proliferation of breast cancer cell lines MDA-MB-231,MDA-MB-468,MDA-MB-453 and MCF-7(with IC50 of about 1/10 of that of MCF-10A cells).(2)Microscopy analysis showed that Jatamanvaltrate P was able to significantly inhibit the growth of breast cancer cells and induce morphological changes.(3)Plate colony formation assay also showd that Jatamanvaltrate P could inhibit colony formation of breast cancer cells.These results suggest that Jatamanvaltrate P can inhibit the proliferation of breast cancer cells.(4)DAPI staining showed that Jatamanvaltrate P induced apoptotic bodies in breast cancer cells.(5)Flow cytometry analysis revealed that the apoptosis rate of breast cancer cells increased in a dosedependent manner after the treatment of Jatamanvaltrate P.(6)Western Blotting analysis showed the active cleavage of PARP,Caspase-3 or Caspase-7,Caspase-8 and Caspase-9 in breast cancer cells after treated with Jatamanvaltrate P.(7)The proportion of apoptotic cells was significantly decreased after adding Caspase inhibitor Z-VAD-FMK.The addition of Caspase inhibitor Z-VAD-FMK significantly inhibited the cleavage of PARP,Caspase-3 or Caspase-7 and partially rescued breast cancer cell viability,which indicates that Jatamanvaltrate P-induced apoptosis is Caspase-dependent.(8)AO staining showed that the degree of autophagy increased in a dose-dependent,with the transformation of the intracellular autophagic marker protein LC3 from I to II,which partially can be reversed by the addition of the autophagic inhibitor 3-MA.These results suggest that Jatamanvaltrate P can inhibit the activity of breast cancer cells by inducing autophagy.(9)Cell cycle analysis revealed that Jatamanvaltrate P treatment arrested MDA-MB-231,MDA-MB-468 and MDA-MB-453 cells at G2/M phase,and upregulated the expression of G2/M-associated protein p-cdc-2 while downregulated the expression of cdc-2 and Cyclin B1.MCF-7 cells were arrested at G0/G1 phase,with downregulated the expression of Cyclin D1.The results suggest that anti-breast cancer effect of Jatamanvaltrate P is associated with cell cycle arrest.(10)Nude mice transplanted tumor model was established.The results showed that Jatamanvaltrate P could significantly reduce the tumor volume of nude mice while exhibiting no significant toxic effect.TUNEL staining revealed that Jatamanvaltrate P could induce apoptosis in tumor tissue.Western Blotting assay showed that Jatamanvaltrate P could transform LC3 from I to II.Conclusion:The experiments showed that Jatamanvaltrate P inhibited breast cancer proliferation through the induction of apoptosis,autophagy and cell cycle arrest both in vitro and in vivo.These results deepen our understanding of the anti-breast cancer efficacy of Jatamanvaltrate P and lay the theoretical foundation for the development of novel anti-breast cancer drugs.