Study On The Mechanism Of β-elemene Inhibiting Osteosarcoma And Enhancing Doxorubicin Sensitivity

Background:Osteosarcoma is one of the most common primary malignant bone tumor in orthopaedics.It usually occurs in children and adolescents and is often transferred through blood.The clinical prognosis is poor,the 5-year survival rate is 60%,but the10-year survival rate is less than 30%,and the disability rate and mortality rate are high,causing great burden to the country and society.At present,the main clinical treatment for osteosarcoma is surgery combined with chemotherapy,that is preoperative chemotherapy+surgery+postoperative chemotherapy.Due to the limitations of chemotherapy drugs,there was not new progress in the treatment of osteosarcoma in the past 40 years.It is urgent to develop new treatment methods or chemotherapy drugs to improve the prognosis of osteosarcoma.Doxorubicin is the main chemotherapy agent in clinical chemotherapy for osteosarcoma.However,one of the main factors limiting the efficacy of chemotherapy for osteosarcoma is that osteosarcoma cells are prone to develop drug resistance to doxorubicin.Therefore,increasing the sensitivity of osteosarcoma cells to doxorubicin would be an effective strategy to improve the therapeutic effect of osteosarcoma.β-elemene is a monomer with anticancer activity extracted from Rhizoma curcumae,which has been developed and approved for clinical use in China.A large number of studies have shown thatβ-elemene has a variety of effects,such as inhibiting tumor development,antioxidant reaction,protecting liver and kidney function from damage,enhancing sensitivity to radiotherapy and chemotherapy,and regulating immune function.Its main feature is that it has anti-cancer effect on many systemic tumors such as glioma,thyroid cancer,esophageal cancer,gastric cancer,lung cancer,leukemia,ovarian cancer and bladder cancer.However,there are few reports aboutβ-elemene on osteosarcoma.The specific anticancer effect and possible mechanism ofβ-elemene on osteosarcoma are not completely clear,and further research is needed.Peroxiredoxin 1（Prx-1）,the most common member of the Peroxiredoxin family,is an antioxidant protein that regulates cell differentiation,proliferation,apoptosis and signal transduction.The mechanism is to regulate the level of H₂O₂ in cells by catalyzing the reduction of H₂O₂ and lipid hydroperoxides,thus regulating cell metabolism and signal transduction.Compared with normal cells,tumor cells have a lot of redox reactions.A large number of studies have shown that Prx-1 is overexpressed in a variety of tumor cells,and is closely related to biological behaviors such as proliferation and apoptosis,as well as sensitivity to radiotherapy and chemotherapy.In addition,studies have confirmed that the expression level of Prx-1 is significantly increased in the pathological tissues of patients with osteosarcoma.However,whetherβ-elemene can regulate the expression of Prx-1 in osteosarcoma cells,and then affect the biological behaviors of osteosarcoma cells such as proliferation,apoptosis,invasion and migration,as well as the sensitivity to chemotherapy drugs,has not been studied.Methods:Osteosarcoma cell lines MG63 and Saos-2 were cultured in vitro,CCK-8assay,flow cytometry,Western blot and Transwell assay were used to detect the effects ofβ-elemene on the proliferation,apoptosis,migration and invasion of osteosarcoma cells,and the expression levels of apoptosis related proteins Bcl-2,Bax and cleaved Caspase3 were detected.The effects ofβ-elemene on the growth of osteosarcoma in xenograft osteosarcoma in mice were observed in vivo.In addition,osteosarcoma cell lines MG63 and Saos-2 were cultured in vitro and doxorubicin resistant osteosarcoma cell lines MG63/Dox and Saos-2/Dox were constructed.CCK-8 assay was used to detect the toxicity ofβ-elemene and doxorubicin on MG63/Dox and Saos-2/Dox cells,and the median inhibitory concentration（IC50）and combination drug index（CI）were calculated.The proliferation and apoptosis of MG63/Dox and Saos-2/Dox cells and the expression levels of apoptosis related proeins Bcl-2,Bax and Cleaved caspase3were detected by Edu staining,flow cytometry,Western blot and Elisa.The effects ofβ-elemene and doxorubicin on the expression levels of ROS and GSH and ERK/P38signaling pathway in MG63/Dox and Saos-2/Dox cells were detected.Finally,Western blot was used to detect the expression levels of Prx-1 in MG63/Dox and Saos-2/Dox cells byβ-elemene and doxorubicin.The MG63/Dox and Saos-2/Dox cells overexpressing Prx-1 were constructed,and the effects ofβ-elemene and doxorubicin on the viability of MG63/Dox and Saos-2/Dox cells and the expression level of Prx-1detected by CCK-8 assay and Western blot.To explore the possible role of Prx-1 in this process;The xenograft drug-resistant osteosarcoma mouse model was constructed to observe the effects ofβ-elemene and doxorubicin on the tumor growth of Saos-2/Dox cells in mice,and the expression level of Prx-1 in tumor tissues and tumor angiogenesis were detected by Western blot and immunohistochemical staining（IHC）.To explore the effect ofβ-elemene on Saos-2/Dox cells and its possible mechanism in mice.Results:β-elemene can significantly reduce the proliferation of osteosarcoma cell lines MG63 and Saos-2,induce the apoptosis of osteosarcoma cells,and inhibit the migration and invasion of osteosarcoma cells.The tumor growth of osteosarcoma was inhibited in nude mice.In addition,the combination ofβ-elemene and doxorubicin significantly inhibited the proliferation and induced apoptosis of MG63/Dox and Saos-2/Dox cells,and it was found that the combination ofβ-elemene and doxorubicin could enhance oxidative stress injury and inhibit ERK/P38 signaling pathway of MG63/Dox and Saos-2/Dox cells.Further studies showed thatβ-elemene combined with doxorubicin could significantly inhibit the expression of Prx-1 in MG63/Dox and Saos-2/Dox cells,and the viability of MG63/Dox and Saos-2/Dox cells was significantly increased after overexpression of Prx-1.In nude mice,the combination can significantly inhibit the growth of Saos-2/Dox cells,and inhibit the expression level of Prx-1 and angiogenesis in tumor tissues.Conclusion:β-elemene can inhibit the proliferation of osteosarcoma cells,induce apoptosis,inhibit the migration and invasion of osteosarcoma cells,and inhibit the growth of osteosarcoma.β-elemene and doxorubicin have synergistic antitumor effects.The combination ofβ-elemene and doxorubicin can significantly inhibit the proliferation and induce the apoptosis of osteosarcoma cells,which may be related to the oxidative stress injury induced by drug-resistant osteosarcoma cells and the inhibition of ERK/P38 signaling pathway.In addition,β-elemene can enhance the sensitivity of drug-resistant osteosarcoma cells to doxorubicin,and reverse the doxorubicin resistance of osteosarcoma cells.The mechanism is related to the inhibition of Prx-1 expression level and angiogenesis.