The Role And Mechanism Of Sufentanil And Opioid Growth Factor Receptor On The Growth Of Human Non-small Cell Lung Cancer

Objective: Lung cancer is still the leading cause of cancer related death.Non-small cell lung cancer(NSCLC)accounts for more than 85% of the total lung cancer population.As the early clinical symptoms of NSCLC were not obvious,60%-70% of NSCLC patients have advanced disease at diagnosis,and followed with a poor prognosis.Therapeutic strategies for NSCLC,such as surgery,chemotherapy,radiotherapy,targeted therapy and immunotherapy show promising outcome.However,because of the genetic heterogeneity of NSCLC,the 5-year overall survival(OS)rate still remains poor.Opioid growth factor(OGF)-opioid growth factor receptor(OGFR)axis has recently received extensive attention due to its physiological anti-tumor properties in diverse human cancers.OGF,chemically termed [Met5]-enkephalin,is an important endogenous opioid peptide widely expressed in almost all human tissues.OGFR,an integral membrane protein associated with the nucleus,is expressed on or near the outer nuclear membrane.It contains specific nuclear localization signals(NLS).When OGF specifically binds to OGFR,the complex can be transported into the nucleus to regulate DNA synthesis,and plays an important role in tissue organization,cellular renewal,tumorigenesis and development,angiogenesis,wound healing and immunoregulation.OGF-OGFR axis has been widely identified in many human tumor cell lines and tumor tissues,including NSCLC.Studies suggested that the functional OGFR expression was significantly down-regulated in tumor cells or tissues.Moreover,the OGFR expression can regulate the malignant biological behaviors of tumors.Overexpression of OGFR can inhibit the proliferation of tumor cells,while OGFR silencing can promote the proliferation of tumor cells.There are limited studies focused on the effect of OGFR on NSCLC tumor progression and clinical prognosis.A large scale of studies have shown that opioids and opioid receptors might affect the occurrence,development and prognosis of NSCLC.Opioid receptors include ? opioid receptor(MOR),? opioid receptor(KOR),? opioid receptor(DOR)and zeta opioid receptor(OGFR).Studies have demonstrated that OGFR and MOR modulated the tumor malignant porgression,and interacted with opioids to affect the tumor outcome.MOR is highly expressed in NSCLC tumor tissues.The activation and overexpression of MOR affect the proliferation,migration and epithelial mesenchymal transition(EMT)of lung cancer cells.Previous study suggested that morphine can selectively combine to MOR to promote the growth of tumor cells.Moreover,morphine was found to interact with OGFR to inhibit the growth of NSCLC cells.At present,there is little relevant research on the effect of interaction of OGFR and other opioids on the growth of NSCLC tumor cells.There is still no research on the possible relationship between OGFR and MOR.Therefore,in this study OGFR was considered as a potential therapeutic target for NSCLC.We studied the regulation and the underlying molecular mechanism of OGFR expression on the malignant physiological behaviors of NSCLC tumor cells.We also detected the expression of OGFR and MOR in NSCLC tumor tissues and analyzed the correlation between OGFR or MOR expression and the clinicopathological factors and OS to illustrate the clinical siginifcance of both opioid receptors in modulation and prediction of the prognosis of NSCLC.We also studied the influence of sufentanil on the growth of NSCLC tumor cells with different expression levels of OGFR.Methods: 1.In this study,human lung fibroblast-like cell line(HLF),human NSCLC cell lines A549,H460,H322,H358 and H1299 were selected.The expressions of OGFR in different cell lines were detected using Western Blot analysis.Lentivirus were used to construct OGFR low-expression cell lines in H1299 and OGFR overexpression cell lines in A549.The transfection efficiency was evaluated using Western Blot analysis.MTT assay and plate clonony formation assay were used to detect transfected cell proliferation ability.The expressions of PI3K/Akt signaling pathway related proteins were detected using Western Blot analysis.AV/PI staining and FACS analysis were used to evaluate the apoptosis status of transfected cells.Immunofluorescence assay was used to detect the release of cytochrome c(Cyt-c)from mitochondria.The expression of apoptosis related proteins were also detected using Western Blot analysis.Wound scratch assay and Transwell assay were used to evaluate the migration and invasion ability of OGFR silencing tumor cells.EMT-related proteins were also detected by Western Blot analysis.Sphere formation assay and detection of cell stemness related proteins using Western Blot were employed to evaluate the tumor cell stemness.2.NSCLC tumor biopsies were obtained from 4 cases undergoing lobectomy at the First Affiliated Hospital of Dalian Medical University between November and December.Proteins were extracted and the OGFR expressions were detected using Western Blot analysis.Then a tissue microarray of 98 human lung adenocarcinoma cases was employed and immunohistochemical(IHC)method was performed to detect the expression level of OGFR and MOR.Chi square test was used to compare the expression of OGFR or MOR with different clinical pathological factors,such as age,pathological differentiation and clinical stages.Kaplan-Meier method was used to analyze the survival curve of NSCLC.Log rank test was used to compare the difference of OS of NSCLC patients with different levels of OGFR or MOR expression.The correlation between expressions of OGFR and MOR was evaluated as well.3.Lentivirus were used to construct OGFR overexpression cell lines and si RNA was used to construct OGFR low-expression cell lines in A549 cells.The transfection efficiency was evaluated using q RT-PCR.A549 cells were treated with different concentrations of sufentanil(0n M,0.5n M,5n M,50 n M,500 n M).6,12,24 and48 h later the effects of sufentanil on the proliferation of A549 cells were observed using CCK-8 method.Results:1.This study demonstrated that OGFR was down-regulated in NSCLC cell lines compared with HLF cell line(P<0.05),and in NSCLC cell lines the OGFR expressions were at various level.H1299 and H322 cells exhibited a relative higher expression,while H358,H460 and A549 cells showed a lower expression of OGFR.OGFR was knockdown using its specific sh RNAs in H1299 cells and overexpressed with OGFR overexpression vectors in A549 cells.MTT assay and plate colony formation assay were done and found that OGFR silencing enhanced cell proliferation in H1299 cells,while OGFR overexpression inhibited cell proliferation in A549 cells compared to control groups(P<0.05).PI3K/Akt signaling pathway related proteins p110?,p110? and p-Akt were found decreased in OGFR over-expressed A549 cells,and increased in OGFR silencing H1299 cells.AV/PI staining and FACS analysis were used to evaluate the apoptotic status of transfected H1299 cells,and the results suggested that OGFR knockdown significantly inhibited the apoptosis of H1299 cells.Immunofluorescence assay showed decreased release of Cyt-c in the OGFR silencing cells(P<0.05).Consistently the expression of pro-apoptotic protein cleaved caspase-7,cleaved caspase-9 and cleaved poly ADP-ribose polymerase(PARP)were significantly decreased using Western Blot analysis(P<0.05).Wound scratch assay and Transwell assay were done in transfected H1299 cells,and demonstrated that OGFR knockdown significantly enhanced the migration and invasion abilities of NSCLC cells(P<0.05).The expressions of EMT-related markers E-cadherin was down-regulated(P<0.05),while N-cadherin and Vimentin were both up-regulated(P<0.05).The tumor sphere formation assay showed that OGFR silencing significantly increased the sphere formation number and size.OGFR knockdown also increased the expression of the stemness markers CD133,CD44 and OCT4 evaluated by Western Blot analysis(P<0.05).2.We detected OGFR expression in tumor specimens collected from 4 human NSCLC cases undergoing thoracoscopic surgery using Western Blot analysis.The results indicated that OGFR expression was down-regulated in tumor tissues relative to adjacent normal lung tissues(P<0.05).A NSCLC tissue microarray with a cohort of 98 cases was also employed to evaluate OGFR or MOR expression using IHC assay.82 cases owned both tumor tissues and corresponding adjacent normal tissues.The results indicated that OGFR was down-expressed in tumor tissues relative to normal lung tissues in 72 cases(P<0.05).The relationship between OGFR expression in tumor tissues and corresponding clinicopathologic characteristics of NSCLC cases were analyzed.The data illustrated that OGFR expression was negative relevant to the pathological differentiation(P=0.046),T stage(P=0.049)and clinical stage(P=0.05),but not age(P>0.05).In addition,the Kaplan-Meier method was used to analyze the life curve and indicated that the patients with lower expression of OGFR owned lower OS rate(P<0.05).75 cases of NSCLC tissue microarray demonstrated higher expression level of MOR in tumor tissues compared to normal tissues(P<0.05).The MOR expression was positively relevant to the pathological differentiation(P=0.032),T stage(P=0.043),N stage(P<0.0001)and clinical stage(P<0.0001),but not age(P>0.05).The patients with higher expression of MOR owned lower OS rate(P=0.00011).There was no significant difference of MOR expression in tumor tissues with different OGFR expression(P=0.105).3.Different concentration of sufentanil(0n M,0.5n M,5n M,50 n M and 500 n M)were used to treat A549 cells,and the proliferation abilities of A549 cells were evalulated 6h,12 h,24h and48 h after medication.The results suggested that sufentanil at the concentration of 5n M or above showed inhibition role on the prolliferation of A549 cells,which was enhanced as the medication duration and concentration increased.In OGFR overexpression A549 cell line,sufentanil at all the concentration from 0.5n M to 500 n M inhibited cell proliferative abilities.Overexpression of OGFR enhanced the inhibition role of sufentanil.In OGFR silencing A549 cell line,the proliferation abilities of transfected cells increased 12 h after the treatment of sufentanil,and gradually decreased as the concentration of sufentanil and treatment time increasing.Sufentanil exhibited the inhibition role on cell proliferation at the concentration of 500 n M 6h,24 h,48h later,50 n M and 5n M under 24 h and 48 h treatment time,and 0.5n M with treatment time of 48 h.Conclusion:1.The expressions of OGFR are low in NSCLC cell lines relative to HLF cell,and varied in different NSCLC cell lines.OGFR can inhibit the proliferation,migration,invasion and cell stemness of NSCLC cell lines,and promote apoptosis.The PI3K/Akt pathway might be involved in the regulation of OGFR on the growth of NSCLC cells.2.The expressions of OGFR are low in NSCLC tumor tissues compared to adjacent normal tissues.The expression of OGFR is negatively correlated with malignant biological characteristics and OS rate.The low expression of OGFR indicates poor prognosis of NSCLC.MOR is highly expressed in NSCLC tumor tissues.The expressions of MOR are positively correlated with malignant biological characteristics and OS rate of NSCLC.The high expression of MOR indicates poor prognosis of NSCLC.There was no correlation between the expression of OGFR and MOR in NSCLC tumor tissues.3.Sufentanil exhibits a suppression role on the proliferation of A549 cells,and the influence is enhanced as the medication duration and concentration increased.OGFR facilitates the supression of sufentanil on the proliferation of A549 cells.