Research On The Inhibitory Effect Of Oncolytic Adenovirus CD55-SMAD4 Combined With Gemcitabine On NSCLC

Lung cancer has become a major killer that threatens human life and health.In 2020,there were 1.8 million people worldwide died of lung cancer,ranking first in the number of cancer deaths.Among all primary lung cancer cases,80% are non-small cell lung cancer(NSCLC).The prognosis of patients with traditional NSCLC treatment is not optimistic due to many conditions,drug resistance and high treatment costs.In recent years,immunotherapy represented by oncolytic virus(OV)is emerging,some of which have entered clinical trials for the treatment of patients with unresectable advanced NSCLC.The TGF-β signaling pathway controls many life behaviors including cell proliferation,differentiation,migration,early embryonic development and adult homeostasis.SMAD4,also known as DPC4,is a key mediator of the TGF-β signaling pathway,which is responsible for transmitting signals from outside the nucleus to the nucleus.The role of SMAD4 in different types of cancers and different stages of cancer is not the same,so far there is no research on the specific role of SMAD4 in NSCLC.In this paper,we first analyzed the expression of SMAD4 in NSCLC population through TCGA and GEPIA databases.The results showed that SMAD4 was significantly down-regulated in NSCLC tissues,and NSCLC lung cancer patients with high SMAD4 expression tended to have longer disease-free survival.Afterwards,we compared the expression of SMAD4 in several NSCLC cell lines and normal lung epithelial cells BEAS-2B,and constructed NSCLC stable cell lines that knockdown/overexpress SMAD4.By comparing the proliferation,migration,and colony formation abilities of NSCLC stably transformed cell lines,it was found that the proliferation,migration,invasion,and colony formation abilities of H1299 were significantly enhanced after SMAD4 knockdown.In mechanism,SMAD4 deletion promoted the further growth of H1299 by regulating epithelial-mesenchymal transition(EMT),JNK and AKT signaling pathways,and was accompanied by increased expression of proto-oncogenes such as MMP2,MMP9,and STAT3.After overexpressing SMAD4,the growth of H460 was inhibited to some extent.Thus,the results suggest that SMAD4 functions as a tumor suppressor in NSCLC.Further,we used adenovirus type 5 as the oncolytic virus vector to construct an oncolytic adenovirus CD55-SMAD4 expressing SMAD4 regulated by the carcinoembryonic Antigen(CEA)promoter.The results of in vitro experiments showed that compared with the control virus CD55-EGFP,CD55-SMAD4 had a stronger inhibitory effect on A549 and H460,induced massive apoptosis of NSCLC cells,and had no obvious inhibitory effect on normal lung cells BEAS-2B.As detected by q RT-PCR,it was found that this was related to the activation of TGF-β/SMADs,EMT inhibition and inhibition of other cancer-related pathways after virus infection.In order to further overcome the limitations of the tumor microenvironment and break the single-drug resistance of tumors,we combined the first-line chemotherapy drug gemcitabine(Gem)for NSCLC.The combination of CD55-SMAD4 and gemcitabine has a stronger inhibitory effect on NSCLC cell proliferation and invasion in vitro than single drug.In addition,through flow cytometry detection,it was found that more NSCLC cells in the combined treatment group underwent apoptosis;cell cycle results showed that a large number of tumor cells in the combined treatment group were inhibited in the G1/S phase.In summary,our results show that the SMAD4 is generally downregulated in NSCLC cells and tissues.There is a correlation between SMAD4 level and clinical stage and survival time of NSCLC patients.The overexpression of SMAD4 inhibits the proliferation and metastasis of NSCLC cells and may be used as a potential NSCLC inhibitor.It is further found that the recombinant oncolytic adenovirus CD55-SMAD4 can inhibit the growth of NSCLC cells,and the combination of CD55-SMAD4 and gemcitabine exerts stronger anti-tumor effect than that of gemcitabine alone.The results of this study may provide a new idea for the clinical treatment of NSCLC and have good clinical research value.