Study On The Effect And Mechanism Of Tirapazamine In Hypoxic Osteosarcoma

Osteosarcoma(OS)is one of the most common primary malignant bone neoplasms.It is more likely to happen in adolescents,and prone to lung metastasis early.The current treatment for osteosarcoma is surgery combined with perioperative chemotherapy.With the development of artificial joint reconstruction technology,as well as the amelioration of chemotherapy strategy,both the limb salvage rate and survival rate of osteosarcoma patients have been significantly improved.However,for osteosarcoma patients with local recurrence and distant metastasis,the prognosis is still poor,and the 5-year survival rate is only about 20%.The chemotherapy resistance,and the residual tumor after the radical surgery,may be related to the recurrence,metastasis,and poor prognosis of osteosarcoma.Hypoxia is one of the characteristics of the tumor microenvironment.As the rapid growth rate of the tumor exceeds the extension of oxygen supply vessels,hypoxia would occur in most solid tumors.The hypoxic microenvironment has been proven to be one of the factors of increased malignancy,enhanced metastasis ability,and drug resistance,which leads to tumor progression,metastasis,and poor prognosis.Hypoxia-inducible factor-1α(HIF-1α)is an important hypoxic marker and is often highly expressed in hypoxic cells.HIF-1αhas been proven to be helpful to improve the survivability of tumor cells in a hypoxic environment by promoting proliferation and invasion.Tirapazamine(TPZ)is one of the hypoxia-activated drugs.In hypoxic cells,tirapazamine can produce free radicals that bind with DNA,resulting in DNA damage and cell death.However,the cytotoxicity of tirapazamine to cells in a normoxic environment is limited.Tirapazamine can also enhance the effect of chemotherapy drugs such as doxorubicin and cisplatin.For the effect of tirapazamine on osteosarcoma,the first study was reported in 2021 and there is little relevant literature available.At present,there is a great research prospect for the effect and mechanism of tirapazamine on osteosarcoma.In addition,as orthopedic neoplasms are often adjacent to crucial blood vessels and nerves,and the balance between retaining enough soft tissue and obtaining adequate surgical margins needs to be considered,an effective method is needed to inactivate the potential residual tumor cells to reduce the risk of recurrence in the radical surgery.According to the characteristics and clinical needs of malignant bone neoplasms such as osteosarcoma,it may be worth exploring the new ways of tirapazamine and other hypoxia-activated prodrugs(such as a local application).In this study,we explored the effect of hypoxia on osteosarcoma both in vitro and in vivo,illustrated the effect and mechanism of tirapazamine on both normoxic and hypoxic osteosarcoma cells,and demonstrated the therapeutic effect of tirapazamine for the local application on solid osteosarcoma.Part I:Proliferation and invasion of hypoxic osteosarcoma cells in vivoObjectives and Significance:To our knowledge,there is no reported animal experiment identifying the differences between osteosarcoma cells that survive in hypoxia and those cultured in normoxia in animal models.In this part,we aimed to compare the expression of proliferation,invasion,and other related proteins between solid osteosarcoma formed by hypoxic and normoxic cells.Methods:HOS osteosarcoma cells that could grow stably in a hypoxic environment were cultured.Twelve nude mice were equally divided into the normoxia and the hypoxia group.The nude mice in the normoxia group were subcutaneously implanted with HOS osteosarcoma cells cultured in the normoxic environment.The nude mice in the hypoxia group were subcutaneously implanted with HOS osteosarcoma cells pretreated by hypoxia.The growth curve of subcutaneous osteosarcoma in the two groups was drawn.After the nude mice were sacrificed,the tumors were weighed.The Western blot was adopted to compare the expression of Ki-67,MMP2,and HIF-1αin tumor tissues of the two groups.Results:The solid osteosarcoma of the hypoxia group grew faster and had higher quality.A higher expression of Ki-67 and MMP2 was presented in the solid osteosarcoma of the hypoxia group.No difference was found in HIF-1αexpression between the two groups.Conclusions:The solid osteosarcoma formed by cells that survived in a hypoxic environment had a stronger proliferative ability and invasive potential in animal models.The expression of HIF-1αin solid osteosarcoma might not fully reflect the proliferation and invasion ability in solid tumors.PartⅡ:The effects and mechanism of hypoxia and tirapazamine on osteosarcoma cellsObjectives and Significance:In this part,we aimed to explore the effects of hypoxia,tirapazamine,and the interaction between them on osteosarcoma.This is a relatively comprehensive study on the effects and mechanism of tirapazamine on osteosarcoma nowadays.This study also demonstrated the changes in the expression of HIF-1αand Akt phosphorylation levels induced by tirapazamine in osteosarcoma cells.As far as we know,there have been no previous reports of the change in Akt pathways caused by tirapazamine.Methods:The HOS and K7M2-WT osteosarcoma cell lines were selected.The MTT assay,Ed U assay,Transwell assay,cell apoptosis assay,cell cycle assay,and Western blot were adopted to evaluate the effects of hypoxia and tirapazamine on proliferation,migration,invasion,apoptosis,cell cycle,cell pathway,and the HIF-1αexpression of osteosarcoma cells.Results:The osteosarcoma cells that survived in a hypoxic environment had greater proliferation,migration,and invasion ability,as well as higher HIF-1αexpression and Akt phosphorylation levels.Hypoxia made a greater proportion of osteosarcoma cells to be in the S phase.In the hypoxic environment,tirapazamine could inhibit proliferation,migration,and invasion,promote apoptosis,reduce the proportion of S phase cells,and downregulate HIF-1αexpression and Akt phosphorylation levels in osteosarcoma cells.In the hypoxic environment with tirapazamine,upregulating Akt phosphorylation levels could improve the cell viability and invasive ability of osteosarcoma.The interaction between hypoxia and tirapazamine was presented.In a normoxic environment,tirapazamine had relatively low toxicity to osteosarcoma cells.Tirapazamine at the concentration used in this experiment promoted the HIF-1αexpression of osteosarcoma cells in the normoxic environment.Conclusions:The osteosarcoma cells that survived in a hypoxic environment tended to be more malignant.Tirapazamine reversed the effect of hypoxia on osteosarcoma cells to some extent.The Akt pathways participated in the effects of hypoxia and tirapazamine on osteosarcoma cells.PartⅢ:Local application of tirapazamine in solid hypoxic osteosarcomaObjectives and Significance:In this part,we aimed to explore the effects of intratumoral injection of tirapazamine on osteosarcoma.As far as we know,there has been no study on the local application of tirapazamine in solid osteosarcoma.The local application of tirapazamine seems to be closer to the clinical requirements of radical surgery for osteosarcoma,and more feasible in clinical practice.Methods:The HOS osteosarcoma cells that could grow stably in the hypoxic environment were implanted subcutaneously in twelve nude mice.The nude mice were equally divided into the TPZ and the DMSO group.The tirapazamine solution(for the TPZ group)or DMSO solution(for the DMSO group,with the same DMSO concentration as the TPZ group)was intratumorally injected when the subcutaneous tumors were about 90～110 mm~3.The nude mice were sacrificed on the fifth day after the first intratumoral injection.The volume and quality of osteosarcoma in nude mice were compared.The differences in HE staining sections between the two groups were observed.Western bolt was adopted to evaluate the expression of Cleaved-caspase 3,Ki-67,and HIF-1αin tumor tissues of the two groups.Results:The volume and weight of osteosarcoma in the TPZ group were lower than that in the DMSO group.Compared with the DMSO group,the TPZ group had larger necrotic areas in HE staining sections.The upregulated expression of Cleaved-caspase 3 and downregulated expression of Ki-67 and HIF-1αwas exhibited in the osteosarcoma tissue of the TPZ group.Conclusions:Tirapazamine could inhibit proliferation,promote apoptosis and necrosis,and downregulate HIF-1αexpression when applied locally to solid hypoxic osteosarcoma.