Anti-Osteosarcoma Capacity And Immunomodulatory Effects Of Isorhapontigenin And Resveratrol

Objective To investigate anti-osteosarcoma growth capacity and immunomodulatory effects of isorhapontigenin(ISO)and resveratrol(RES),and to clarify the corresponding cellular and molecular mechanisms to provide a new experimental basis for the treatment of osteosarcoma.Methods K7M2 cells were treated with different concentrations of ISO and RES in vitro.K7M2 cell proliferation inhibition was detected by MTT assay.The effects of ISO and RES on apoptosis and cell cycle of K7M2 cells were measured by flow cytometry.The effects of ISO and RES on the apoptosis and cell cycle-related gene expression of K7M2cells were investigated by real-time quantitative polymerase chain reaction(RT-qPCR).The expressions of the proteins involved in the apoptosis and cell cycle of K7M2 cells treated by ISO and RES were examined by western blot.Flow cytometry was used to check the expression of MHC molecules on K7M2 cells treated in vitro.To evaluate the potency of ISO and RES on anti-osteosarcoma in vivo,the volumes of tumors in the mice transplated with K7M2 cells were monitored daily,and different concentrations of ISO and RES were peritumoral injected according to the body weight of the mice.Mice were sacrificed on the16th day to obtain the liver,kidney and isografts.The liver and kidney tissues were stained to observe the side effects of the drug.The expression of MHC molecules on tumor cells and the percentage of CD8~+T cells,NK cells,NKT cells,MDSCs,and Treg cells infiltrated in transplanted tumor tissues were detected by flow cytometry.Results The proliferation of osteosarcoma K7M2 cells was inhibited at different levels after treated with ISO and RES at 5μM,10μM,20μM,40μM and 80μM for 24 h,48 h and 72 h,and the inhibitory effect of ISO is time-and dose-dependent.RES inhibited the proliferation of osteosarcoma K7M2 cells in a time-dependent manner within 48 h,but the dose-dependent effect was not obvious at 72 h.The apoptosis of K7M2 cells was increased significantly,and the viable cells was decreased significantly after treated with ISO(20μM,40μM,60μM)and RES(20μM,40μM)for 48 h.The G0/G1 phase cells in the group ISO increased significantly,blocking the cell cycle in the G0/G1 phase after treated with ISO(40μM,60μM)for 48 h,the S phase cells in the group RES increased significantly,blocking the cell cycle in the S phase after treated with RES(20μM,40μM)for 48 h.The mRNA expression of Mcl-1,Xiap,Kras and Bcl-2 were decreased and CyclinE1,Bax and Caspase-3 were increased significantly in K7M2 cells after treated with ISO(40μM,60μM)and RES(20μM,40μM)for 48 h.The mRNA expression of CyclinD1 in the group ISO was decreased significantly,and there was no significant change in the group RES.The protein expression of Mcl-1,Xiap,Kras and Bcl-2 were decreased and CyclinE1,Bax and Caspase-3 were increased significantly in K7M2 cells after treated with ISO(40μM,60μM)and RES(20μM,40μM)for 48 h.The protein expression of CyclinD1 in the group ISO was decreased significantly,and there was no significant change in the group RES.The expression of MHC I and MHC II molecules on K7M2 cells was increased significantly after treatment with ISO(40μM,60μM)and RES(20μM,40μM)for 48 h.After 16 days of peritumoral injection of 75mg/kg,150mg/kg ISO and 50 mg/kg,100 mg/kg RES,the volume of transplanted tumors in each treatment group were inhibited significantly compared with the control group,and the drug side effects were not obvious.The expression of MHC I and MHC class II molecules on tumor cells was increased significantly.At the same time,the percentage of CD8~+T cells,NK cells and NKT cells increased significantly,while the percentage of MDSCs and Treg cells decreased significantly in transplanted tumor tissues.Conclusion ISO and RES inhibit the growth of K7M2 cells by regulating the cell cycle and inducing cell apoptosis.Both ISO and RES are able to increase K7M2 cells immunogenicity in vivo and in vitro,and improve the immune microenvironment of tumor tissues in mice transplanted with osteosarcoma cell K7M2,enhancing the anti-tumor ability of the mice,by which the tumor growth is inhibited.Therefore,ISO,like RES,may be one candidate for anti-osteosarcoma.