| Background and Object:Cervical malignancy is the first and fourth most common malignancy in women.There were 569,847 new cases and 311,365 deaths caused by cervical malignancy every year,85%of which occurred in developing countries,ranking first among female malignancies in terms of mortality.Currently,the main treatment methods are surgery,radiotherapy,chemotherapy and targeting therapy.However,most patients still face recurrence and metastasis within 2 years after initial treatment,with poor prognosis and median survival of only 7 months.Despite increasingly prominent anti-tumor effects appear such as targeted immunotherapy,adoptive cell immunotherapy and vaccines,including programmed cell death protein-1（PD-1）and its monoclonal antibodies,programmed cell death ligand protein-1（PD-L1）,the overall survival has not been significantly improved.Therefore,it is urgent to explore more effective treatment methods to improve the quality of life and prognosis of patients with cervical cancer.Methionine enkephalin（MENK）,also known as Opioid Growth Factor（OGF）,has precursor of proenkephalin and derminogen.Opioid Growth Factor（OGF）and Opioid Growth Factor Receptor（OGFr）,binding to three classical opioid receptors calledμ,κ,δ.Some studies suggest that MENK has significant application in inflammation,chronic pain,autoimmune diseases,chronic hepatitis,diabetes and other diseases.A growing number of research suggests that MENK exert function as a kind of bidirectional immune regulator on immune cells,on the one hand,enhancing immune efficiency and release of effector molecules,on the other hand,it also has strong inhibition and killing effect on the tumor,because of MENK-OGFr are expressed in many tumor tissues,OGFr,as receptor of MENK,play a role on regulating nuclear transcription factors,inhibiting tumor cell proliferation,promoting cell apoptosis,and so on in the tumor research.In previous studies of our team,OGFr had been found expressed in cervical malignant cells,but the relevant studies on the effect of MENK on the biological behavior of cervical cancer have not been found.This study mainly aimed at the effect of MENK on cervical cancer biological behavior and apoptosis,and discussed the change in the tumor microenvironment（TME）,revealing the machnism of MENK on cervical cancer regulation,TME alteration,finally,trying to explore its targeting molecular application for the treatment of cervical cancer.Methods:1.In vitro,different concentrations of MENK（0,1,2.5,5,7.5,10,12.5,15,17.5,20 mg/m L）were used to treat cervical cancer Hela and Siha cells,for 24,48,72 h,respectively.10 mg/m L MENK for 48h were selected as the appropriate dose and time by MTS experiment.Next,based on this MENK administration,different concentrations of naltrexone(10-2,10-3,10-4,10-5,10-6,10-7,10-8,10-9,10-10,10-11,10-12 M)were added for 48 h to detect the blocking effect of MENK.10-4 M naltrexone was selected as the dose to block MENK,and the subsequent functional experiments of cervical cancer cells were carried out.Comparison of the MENK treatment group and the negtive control（nc）group was observed,and the clone formation test was used to detect the changes of the clone formation of Hela and Siha cells.Cell scratch assay and Transwell invasion assay were used to detect the changes of migration and invasion ability of Hela and Siha cells.Cell cycle changes were detected by flow cytometry.Flow cytometry and Hoechst 33258 were used to detect the apoptosis of cervical cancer cells.The morphological changes of Hela and Siha cells were observed by light microscopy.After that,Cells in MENK group and nc group were collected to extract mRNA and protein,caspase 3,caspase 8,caspase 9,and vegfa were detected by qRT-PCR.Western blot was used to detect the protein expression levels of caspase/cleaved caspase 3,caspase/cleaved caspase 8,caspase/cleaved caspase 9,and vegfa.2.In vivo,subcutaneous xenograft tumor model of human cervical cancer was established in nude mice.After Siha cells were transplanted into the right posterior back of nude mice and tumor formation was observed,the nude mice were randomly divided into two groups:MENK administration group（8 mg/time,every other day）and blank control group（equal volume of isotonic saline,every other day）.The vital factors of nude mice were observed daily,and the changes of body weight and subcutaneous tumor volume were dynamically monitored.About 5 weeks after treatment,the nude mice were killed by cervical vertebra dislocated.Subcutaneous tumors and major organs（heart,liver,spleen,lung,kidney）were removed and weighed.The paraffin section of tumor was prepared and the following tests were performed:H&E was used to detect histopathology.The expression of OGFr protein in tissues was detected by immunohistochemistry.3.In vivo,nude mouse model transplanted with cervical cancer was established to furtherly detect the changes in the immune microenvironment.Siha cells were transplanted into the right and posterior back of nude mice.We observed tumor formation and then measured tumor size every other day.The mice were sacrificed by cervical dislocation,and peripheral blood was collected to detect the levels of CD11b and GR-1,as well as Arg-1,i NOS,IL-10,TGF-βand vegfa.The expression of PD-1 and PD-L1 protein was detected by immunohistochemistry.4.In vitro experiment,another group of MENK combined with naltrexone was set up for each experiment to furtherly clarify effects of the combination of MENK on the proliferation,apoptosis and biological behavior of cervical cancer cells.The mRNA and protein expression of OGFR and apoptosis-related indexes were detected by qRT-PCR and western blot.Results:1.In vitro,the results showed that compared with the control group,MENK significantly inhibited the proliferation of Hela and Siha cells in a dose-dependent manner.MENK could arrest cervical cancer cells in G0/G1 phase of cell cycle.Hela and Siha cells were induced apoptosis by MENK.The ability of clone formation,migration and invasion of Hela and Siha cells was inhibited.MENK up-regulated mRNA and protein expression of OGFr,fas,and bax,protein expression of cleaved caspase 3,cleaved caspase 8,cleaved caspase 9 while decreased mRNA and protein expression of vegfa.2.MENK inhibited the tumor growth in subcutaneous xenograft and induced apoptosis of xenograft tumor cells in nude mice.There were no significant differences in the weight of main organs（heart,liver,spleen,lung,kidney）between the two groups.Immunohistochemical results showed that MENK could up-regulate the expression levels of OGFR,PD-1 and PD-L1.3.In vivo,flow cytometry results indicated that MENK could decrease the levels of CD11b,Gr-1（marker of bone marrow derived suppressor cells）,as well as the immunosuppressive molecules Arg-1,IL-10,TGF-βand vegfa in peripheral blood.Immunohistochemical results indicated that the expression of OGFr,PD-1 and PD-L1 was significantly increased in tumor tissue.4.After treatment by combining MENK and naltrexone,the inhibition of proliferation,invasion and metastasis of Hela and Siha cells,the effect of cell cycle and the induction of apoptosis by MENK were significantly weakened.The results of qRT-PCR and western blot indicated that MENK combining with naltrexone could decrease the expression of OGFr,fas/caspase 8/caspase 3 and bax/caspase 9/caspase 3 pathways,reversing the effect of MENK administration group on Hela and Siha cells.Conclusions:1.Both in vivo and in vitro experiments confirmed that MENK significantly inhibited the proliferation of cervical cancer with appropriate dose.2.MENK could block the cell cycle in G0/G1,induce cell apoptosis,inhibit cell migration and invasion activity,and improve immunosuppressive state to remodel the components in the tumor microenvironment,and enhance the anti-tumor immune activity of the body,which played a role on inhibiting cervical cancer.3.MENK significantly upregulated the level of OGFr in cervical cancer cells.MENK,combined with OGFr,triggered anti-tumor mechanism and regulated exogenous fas/caspase 8/caspase 3 and endogenous bax/caspase 9/caspase 3signaling pathways to induce apoptosis of cervical cancer Hela and Siha cells. |
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