Effect Of Dihydromyricetin Isolated From Ampelosis Grossedentata On The Proliferation, Apoptosis And Invasion Metastasis Of Breast Cancer Cells

Tumor is the most life-threaten disease in human being. Chemotherapy, as one of thetreatment methods is essential for the management of advanced tumor and prevention of itsrecurrence. Patients suffered from hematological malignancies have good probability of longterm survival, thanks to the clinical application of chemotherapy. However, chemotherapy hasnot been able to achieve satisfactory result to those solid tumors, which take account for90%of malignant tumors. Chemotherapeutic drugs have poor selectivity on tumor cells and normalcells, and have a certain side effects on human body. Therefore, it remains an importantproject in tumor research to look for efficient and low toxic drugs with specific activityagainst tumor cells. Natural products have attracted even more attention for the properties ofrich sources, diverse, lower adverse effects and great potential to being adjuvant drugs ofchemotherapy drug synergism and attenuation.Dihydromyricetin (DMY) have many physiological activities, and its source is broad.The stem and leaves of Ampelosis grossedentata possess DMY with high content. Veryrecently, the anti-tumor action of DMY is drawing increasing attention. In order to make thebest use of DMY, it is very necessary to study on the anti-tumor effect and its actionmechanisms of DMY. For the moment, the studies of DMY anti-tumor effect is focused onanti-proliferation action in several cancer cell lines in vitro. But the presentation of anti-tumormechanisms of DMY is varied, and in vivo experiment datas is lacking. Those aren't suitableto clinical application. In view of the problems mentioned above, a series of experimentstudies were conducted, with active component DMY isolated from Ampelosis grossedentata.MTT assay results showed that the sensitivity sequence of three cancer cells lines fromhigh to low was MDA-MB-231, MCF-7/A and MCF-7, and DMY inhibited the proliferationof MDA-MB-231cells in a dose-dependent manner, and the IC50value was about73.6μg/mLafter48h treatment. While the IC50value of DMY on human normal embryo kidney cell(HEK-293) and liver cell (L-02) were475.3μg/mL and324.8μg/mL, respectively. In addition,when the concentration of DMY was40or80μg/mL, it induced G2/M phase arrest,enhanced cell late apoptosis rate and decreased secrete gelatin enzyme activity. These resultsshowed that DMY possess doughty cell specificity, ane it's anti-tumor action mechanismsmay be cell phase arrest, induction of cell apoptosis and inhibition of invasion migration.Cell morphological observasion, DAPI staining, and Annexin V-FITC/PI stainingfollowed flow cytometry detection powerfully testify that DMY can induce the apoptosis of MDA-MB-231cells in a concentration-dependent way. Fluo-3-AM staining and JC-1stainingcombined with Flow cytometry detection and Western blot assay furtherly shown that theapoptpsis of MDA-MB-231, which was induced by DMY, were kept campany with a rise inintracellular calcium concentration, descent in mitochondrial membrane potential, andup-regulation of Caspase-3/-9proteins expression. Those results DMY could inducted theapoptosis of MDA-MB-231cell by the mitochondrial passway.Erasion trace test and cell invasion assay by Transwell furtherly indicated DMY caninhibit invasion metastasis of MDA-MB-231cells without cytotoxicity against the cancercells. Real-time PCR results showed that after treated with DMY for48h, during metastasisrelated genes of MDA-MB-231cell, the change of MMP-9/-2mRNA expression levels weresignificantly higher than that of TIMP-2,VEGF and TGF-β1mRNA expression levels, butthere had no obvious concentration gradient relationship. Western blot assay results showedDMY (20,40and80μg/mL) suppressesed MMP-2/-9protein expression levels in adose-dependent manner. Invasion inhibition mechanisms of DMY on MDA-MB-231cellsmay be directly related to the down-regulation of the expression levels of MMP-2/-9proteins.Also, DMY inhibited proliferation of mouse breast cancer4T1cells in a dose-dependentmanner, and the IC50value was102.1μg/mL. In addition, DMY could inhibit cell migrationand invasion metastasis of4T1cells without cytotoxicity against the cancer cells. Also,invasion inhibition mechanisms of DMY on4T1cells may be related to the down-regulationof the expression levels of MMP-2/-9proteins. Anti-proliferation action mechanisms ofDMY may be S phase arrest, induction of cell apoptosis and inhibition of invasion migration.Median effect principle indicated that there were significant synergistic effect in growthinhibition of DMY combined with ADM on MDA-MB-231or4T1cells when the fractionaffected (fa) was less than or equal to0.75and0.6respectively. At the same fa, the dose ofDMY and ADM was obviously reduced compared with their independent application.Finally, we innoculated4T1cells into Balb/C mice to establish mouse breast cancerpulmonary metastasis model. Anti-proliferation and anti-metastasis effects of DMY in vivowere valued. Results showed: Compared with the control group, the tumor weight, lungmetastatic loci and blood geometric clonogenic cells of100mg/(kg d)DMY group wasobviously lower than that of50mg/(kg d)DMY group. In addition, there was not significanttoxic effect.2mg/(kg d)ADM significantly inhibited4T1cells growth and metastasis, butserum biochemical analysis, spleen index calculation and HE tissue staining showed that2 mg/(kg d)ADM drug delivery associated with marked heart, liver toxicity and immunesuppression. The inhibition effects of100mg/(kg d)DMY and2mg/(kg d)ADM jointadministration on tumor cells growth and metastasis was stronger than that of two separateadministration group. In addition, tumor cell density in the tumor tissue of jointadministration group was obviously lower than that of two separate administration group, andit associated with more necrotic foci and fewer vascular. Serum LDH,CK,ALT and ASTlevels of the joint administration group were markedly lower than that of2mg/(kg d)ADMgroup. And the infiltration of leukocytes were obviously observed in tumor tissue of the jointadministration, but it's not found in the tumor tissue of2mg/(kg d)ADM group. Thoseresults suggested that: DMY could inhibit mouse breast cancer cells growth and metastasis invivo, and it exerted low toxicity.2mg/(kg d)ADM could markedly inhibit the tumor cellsgrowth and metastasis, however it exhibited cardiotoxicity,hepatotoxicity and immunesuppression to a certain degree. DMY combined with ADM group exhibited synergy andattenuation.