Oncolytic Adenovirus Ad11 Enhances Chemotherapy Effect Of Cisplatin On Osteosarcoma Cells By Inhibiting Autophagy

Background Osteosarcoma is the most common primary malignant bone tumor in orthopedic surgery.It originates from mesenchymal cells and is frequently found in children and adolescents.The incidence rate is about 3.4/million^[1].Osteosarcoma is characterized by high invasiveness and early metastasis,and its prognosis is poor.Its 5-year survival rate is about 60%-70%^[2].According to the study,about 20%of patients have metastasized at the time of osteosarcoma diagnosis.90%of metastases occur in the lungs,and patients with distant metastases have a worse prognosis,with a 5-year survival rate of no more than 20%^[3].Prior to the 1970s,the main treatment for osteosarcoma was amputation,but the 5-year survival rate of patients after amputation was not extended.The poor prognosis of osteosarcoma and the lack of effective treatments have caused a huge blow to children and adolescents.After the 1970s,with the development of chemotherapy technology,chemotherapy drugs including cisplatin,doxorubicin,high-dose methotrexate and ifosfamide began to treat osteosarcoma,which greatly improved the curative effect^[4].However,in recent decades,the treatment of osteosarcoma has entered a bottleneck period,and the 5-year survival rate has remained at 60%-70%.Although neoadjuvant chemotherapy can kill tumor micrometastases and limit the role of surgical boundaries,the prognosis of patients with resistance during chemotherapy is still poor.Therefore,it is particularly important to seek ways to improve the chemotherapy resistance of osteosarcoma,thereby improving the prognosis of patients with osteosarcoma and prolonging survival.Oncolytic virus is a type of virus that can specifically replicate in tumor cells,cause cell lysis,and its progeny virus can continue to infect tumor cells,but cannot replicate in normal cells^[5].Oncolytic viruses can be divided into adenovirus,herpes simplex virus,Newcastle disease virus,etc.The most studied at this stage is oncolytic adenovirus^[6],and some studies have entered clinical trials.The most commonly used oncolytic adenovirus is Ad5,which recognizes and infects cells via the Coxsackie receptor（CAR）expressed on the cell surface.However,the infection efficiency is affected by the level of CAR expression on the cell surface.In order to avoid this limitation,chimeric adenovirus has emerged,replacing the cilia of other serotypes with the cilia of the adenovirus type 5,such as the use of CD46-dependent molecular recognition.Group B adenoviruses（types 3,11 and 35）of tumor cells construct chimeric adenoviruses Ad5/F35,Ad5/F11p and Ad5/F3,etc.,which greatly enhance the infection efficiency of adenovirus^[7].At present,chemotherapy is still the basis of osteosarcoma treatment.The first-line chemotherapy drugs for osteosarcoma chemotherapy mainly include cisplatin,doxorubicin,high-dose methotrexate and ifosfamide.And when used,it is often used in combination with three or four chemotherapy drugs^[8].The study of chemoresistance of osteosarcoma has become a hot research topic in the field of osteosarcoma treatment.For example,Chen^[10]and others have discussed the various drug resistance mechanisms of osteosarcoma,and the autophagy-related drug resistance mechanism has a larger research prospects^[11].A number of studies have shown that^[12,13],the anticancer ability of cisplatin is related to the autophagy activity of cancer cells,and the level of autophagy is regulated by various factors.Different regulatory mechanisms have an effect on the drug resistance of cancer cells.Objection To investigate the mechanism of oncolytic adenovirus Ad11 enhancing the sensitivity of osteosarcoma cells to cisplatin and its relationship with autophagy.Methods Immunohistochemical technique was used to detect the expression of CD46 in the oncolytic adenovirus Ad11 in the pathological sections of 20 patients with osteosarcoma.The human osteosarcoma cell lines MG63,U2OS,HOS,SAOS and K7M2 were cultured in vitro,and the half effect concentration（EC50）of the oncolytic adenovirus Ad11 and the 25%inhibitory concentration（IC25）as well as 50%inhibition concentration（IC50）of cisplatin were detected by cytotoxicity assay（MTS method）.The cell viability of the following groups of MG63 and U2OS cells was detected by MTS method,such as blank control group,Ad11 group,cisplatin IC25 group,cisplatin IC50 group,Ad11^first+IC25^second combination group,Ad11^first+IC50^second combination group,IC25^first+Ad11^second joint group,IC50^first+Ad11^second joint group.The expression of autophagy-related antibodies in osteosarcoma cells in the control group and each experimental group was detected by immunofluorescence technique.Western-blot technique was used to detect the expression of autophagy-related proteins Beclin-1 and LC3A/B in the control group and each experimental group.The expression level of green fluorescent protein in oncolytic adenovirus Ad11 was observed by immunofluorescence microscope.The results were analyzed by SPSS21.0 statistical software.The results were expressed as mean±standard deviation（?x±s）,and multiple sample means were used for pairwise comparison and one-way ANOVA.Results CD46 was highly expressed in pathological sections of 20 patients with osteosarcoma,and the positive rate was 75%.The MTS assay was used to detect the killing effect of oncolytic adenovirus Ad11 on five osteosarcoma cell lines.The EC50of the half effect titer was EC50（MG63）=5.26 PFU/cell,EC50（U2OS）=1.92PFU/cell,EC50（HOS）.)=1.90 PFU/cell,EC50（SAOS）=3.46 PFU/cell,EC50（K7M2）=66.11 PFU/cell.The 25%inhibitory concentration IC25 and half-inhibitory concentration IC50 of cisplatin on MG63 and U2OS cells were IC25（MG63）=20μmol/L,IC50（MG63）=30μmol/L,IC25（U2OS）=10μmol/L,IC50（MG63）=25μmol/L.The oncolytic adenovirus Ad11 had a killing effect on osteosarcoma cells MG63 and U2OS.The blank control group and the Ad11 group had a large number of osteosarcoma cells died,MG63 cells:p=0.0015<0.05,U2OS cells:p=0.0015<0.05.The EC50 of oncolytic adenovirus Ad11 was combined with IC25 and IC50 cisplatin to add Ad11 first,then cisplatin was added and cisplatin was added first.After adding Ad11,it had synergistic killing effect on osteosarcoma cells MG63 and U2OS.MG63 cells:cisplatin IC25 group and Ad11first+IC25second combination group p=0.0013<0.05,cisplatin IC50 group and Ad11first+IC25second combination group p=0.0011<0.05,cisplatin IC25 group and IC25 first+Ad11second combination group p=0.0017<0.05,p=0.0009<0.05 for cisplatin IC50 group and IC50 first+Ad11second combination group,p=0.0015<0.05 for U2OS cells:cisplatin IC25 group and Ad11first+IC25second combination group,cisplatin IC50 group and Ad11first p=0.0012<0.05 in the IC25second combination group,p=0.0014<0.05 in the cisplatin IC25 group and the IC25 first+Ad11second combination group,p=0.0008<0.05 in the cisplatin IC50 group and the IC50 first+Ad11second combination group.Immunofluorescence experiments and Western-blot results showed that when Ad11 was combined with cisplatin,the autophagy induced by cisplatin was inhibited when the order of addition was different.Confocal microscopy showed that the addition of cisplatin was followed by the addition of virus treatment,and the replication of Ad11 in MG63 and U2OS cells was inhibited,that is,the reduction of green fluorescent protein was realized.Conclusion 1.High expression of CD46 in pathological sections of patients with osteosarcoma demonstrates the potential of clinical application of oncolytic adenovirus Ad11.2.In the osteosarcoma cell lines MG63 and U2OS,oncolytic adenovirus Ad11combined with cisplatin has a synergistic killing effect.3.This synergistic killing effect is related to the oncolytic adenovirus Ad11 inhibiting autophagy progression in osteosarcoma cells.4.When the oncolytic adenovirus Ad11 is used in combination with cisplatin,it is first treated with a virus,and then treated with cisplatin has a better effect.