The Study Of The Effect Of GSP On Proliferation,Migration,Apoptosis And Cell Cycle Of Osteosarcoma Cells And Their Mechanisms

BackgroundOsteosarcoma(OS)is a malignant bone tumor commonly seen in children and adolescents with high metastasis potential.Although surgery combined with adjuvant chemotherapy can significantly increase the 5-year survival rate of the patient to 70%,the efficacy is still urgent to be improved,so it is necessary to explore new potential drugs for the treatment of osteosarcoma.At present,the strong toxic side effects of conventional chemotherapeutic drugs may lead to poor prognosis of patients.Therefore,it is clinically significant to find new,safe and effective anti-osteosarcoma drugs.Grape seed procyanidins(GSP)is a class of polyphenols extracted from grape seeds.At present,the studies have shown that GSP has low toxicity,and can regulate immunity,reduce body damage,anti-oxidation and anti-inflammation,etc.It can also inhibit the growth of various tumors such as lung cancer,gastric cancer and breast cancer,and play ananti-tumor effect.However,its effect on osteosarcoma and its mechanism remain unclear.PurposeWe intends to study the effect of GSP on proliferation,migration,apoptosis of osteosarcoma cells,xenograft osteosarcoma growth of nude mice,and its molecular mechanism to explore whether it has anti-osteosarcoma effect.Methods1 The effect of GSP on proliferation,migration,apoptosis and cell cycle of osteosarcoma cellsThe morphological changes of osteosarcoma cell lines 143 B and MG63 were observed using an ordinary inverted microscope.The effect of GSP on the proliferation and colony forming ability of osteosarcoma cells was detected by MTT assay and colony formation assay;The effect of GSP on the migration of osteosarcoma cells was tested by the wound healing assay and transwell assay.The effect of GSP on apoptosis and cell cycle of osteosarcoma cells was analyzed by flow cytometry.2 The mechanism of GSP affecting the proliferation and migration,and apoptosis of osteosarcoma cells(1)The mechanism of GSP affecting the proliferation and migration of osteosarcoma cellsAfter treating the cell lines 143 B and MG63 by GSP,Western Blotanalysis was used to test the signal molecules of Wnt/?-catenin pathways(GSK3?,p-GSK3?,and ?-catenin)and its downstream target molecules(c-myc,MMP-7,and cyclin D1).The recombinant adenovirus Ad?-catenin was used to infect 143 B and MG63 cells to overexpress ?-catenin;MTT assay and Transwell assay were used to detect the effect of ?-catenin overexpression on GSP inhibition on proliferation and migration of osteosarcoma cells;Western Blot was used to examine the molecules of Wnt/?-catenin signaling pathway.(2)The effect of GSP on MET in osteosarcoma cellsWestern Blot and RT-PCR were used to detect the expression of E-cadherin,Vimentin,Snail and Twist separately.(3)The mechanism of GSP affecting the apoptosis of osteosarcoma cellsWestern Blot assay was used to detect the changes of apoptosis-related molecules Cleaved caspase-8 and Cleaved PARP in osteosarcoma cells after GSP treatment.3 The effect of GSP on xenograft osteosarcoma in nude miceHuman osteosarcoma 143 B cells with good condition in vitro were inoculated into the right tibia to establish osteosarcoma xenograft model in nude mice.All model rats were randomly divided into a control group and a GSP group with 3 mice in each group.Nude mice were intragastrically administered with physiological saline or GSP everyday.The growth of thenude mice in each group was observed,and body weight and tumor volume were measured every 5 days.After 30 days,the nude mice were sacrificed;the tumors were removed,embedded in paraffin and sectioned.The morphology of the tumor cells was observed after HE staining.The expression of ?-catenin and Caspase-3 were detected by immunohistochemical staining.Results1 The effect of GSP on proliferation,migration,apoptosis and cell cycle of osteosarcoma cellsGSP inhibited the proliferation of osteosarcoma cells in a time-and dose-dependent manner.At the same time,GSP decreased the colony-forming ability of osteosarcoma cells,inhibited their migration,promoted their apoptosis,and arrested the cells in G0/G1 phase.2 The mechanism of GSP affecting the proliferation and migration,and apoptosis of osteosarcoma cells(1)The mechanism of GSP affecting the proliferation and migration of osteosarcoma cellsThe level of GSK3? protein in osteosarcoma cells in GSP groups,was up-regulated and the levels of p-GSK3?,?-catenin,cyclin D1,c-myc,and MMP-7 proteins were significantly decreased,suggesting that GSP can inhibite the Wnt/?-catenin signaling pathway in osteosarcoma cells.?-catenin overexpression by infecting osteosarcoma cells usingAd?-catenin significantly reversed the inhibition of GSP on proliferation and migration of osteosarcoma cells,and also attenuated the down-regulation of GSP on ?-catenin and downstream target molecules.It suggested that the inhibition of Wnt/?-catenin signaling pathway is involved in the inhibitory effect of GSP on the proliferation and migration in osteosarcoma cells.(2)The effect of GSP on MET in osteosarcoma cellsGSP up-regulated E-cadherin and down-regulated Vimentin levels in osteosarcoma cells,and also down-regulated Snail and Twist mRNA levels,suggesting that GSP can promote MET in osteosarcoma cells.(3)The mechanism of GSP affecting the apoptosis of osteosarcoma cellsThe apoptosis-related molecules Cleaved caspase-8 and Cleaved PARP were significantly increased in osteosarcoma cells after GSP treatment,suggesting that GSP can activate the caspase apoptotic pathway.3 The effect of GSP on xenograft osteosarcoma in nude miceThere was no significant difference in body weight between GSP group and control group,but the growth rate of osteosarcoma in GSP group was slower and the tumor size was smaller,suggesting that GSP inhibited the growth of xenograft osteosarcoma in nude mice.Compared with the control group,the cells arrangement in the GSP group was more orderly and compactly,the size was uniform,and the heterotypicity was lower,itsuggested that GSP can reduce the malignancy of osteosarcoma.IHC staining results showed that ?-catenin in the GSP group was decreased while Caspase-3 was increased,suggesting that the mechanism of GSP inhibiting osteosarcoma growth include the inhibition of Wnt/?-catenin signaling pathway and activation of caspase apoptotic pathway.All results in vivo were consistent with the results in vitro.Conclusions1 GSP inhibited osteosarcoma cell proliferation,migration and Wnt/?-catenin signaling pathway activity,promoted apoptosis and MET,and induced G0/G1 phase arrest.2 The inhibition of Wnt/?-catenin signaling pathway is one of the mechanisms which GSP inhibit proliferation and migration in osteosarcoma cells.The mechanisms of GSP inhibiting cell migration and promoting apoptosis are respectively related to MET promotion and activation of Caspase signaling pathway.3 GSP inhibits the growth of osteosarcoma xenografts in nude mice.The mechanism included the inhibition of Wnt/?-catenin signaling pathway and the activation of caspase apoptotic pathway,All results in vivo were consistent with the results in vitro.In summary,GSP has an anti-osteosarcoma effect.This study suggests that low toxicity of GSP may be a potential anti-osteosarcoma drug.