Experimental Study Of MEK Inhibitor Combined With Oxaliplatin In Inhibiting Cervical Cancer Based On Immunoregulatory Pathway

Objective Immune escape is a key factor for tumor occurrance and development.Tumor cells escape immune survellance through negativelly signaling pathways.This topic will study the oxaliplatin induced CRT ectropion of cervical cancer U-14 cells,resulting in the death of tumor cells immunogenicity;Exploring MEK inhibitor PD-0325901 may reduce the expression of PD-L1 in tumor microenvironment by inhibiting MAPK signaling pathway and enhance the antitumor immune activity of chemotherapeutic drugs.Thus,this study sheds new lights on the the immune therapy of cervical cancer.Methods(1)Detecting oxaliplatin or MEK inhibitor PD-0325901 monotherapy and combination therapy to inhibit the proliferation activity of cervical cancer U-14 cells by MTT.(2)Detecting of immunogenic death of U-14 cells by flow cytometry.(3)Detecting to analysis immunogenic activity of tumor cells by flow cytometry.(4)Studying on cervical cancer bearing mice combined of tumor immunotherapy: Construct the U-14tumor-bearing 40 Babl/C mice model about cervical cancer,the diameter of tumor grew to5 mm,were randomly divide into 4 groups,each group has 10 mice,then the treatment starts,group Ⅰ: control group,PBS injection;group Ⅱ: injection of MEK inhibitor;groupⅢ: injection of oxaliplatin;group Ⅳ: MEK inhibitor+oxaliplatin joint suitable for injection;above all by intraperitoneal injection of drugs for treatment of growth curve,calculate the tumor volume and render tumor growth curve,weight and calculate the inhibition rate of tumor speciments at the end of treatment.1)After the treatment,the mice were sacrificed and the tumor tissues were fixed,dehydrated,paraffin embedded and sectioned,respectively.The expression of CD34 in tumor tissues was detected by immunohistochemistry.2)Using Western blot to detect the expression of Bcl-2,Bax and CDK6 related apoptotic proteins in each group of tumor tissues.3)The expression of CRT and PD-L1 in tumor cells and the infiltration of immune cells in tumor tissues by immunofluorescence.Results(1)In vitro experiments showed that oxaliplatin and MEK inhibitors PD-0325901 could inhibit the growth of U-14 cells.When combined with them,the effect of cell inhibition is more obvious with the increase of drug concentration,When the concentration of oxaliplatin was 5 μmol and the concentration of PD-0325901 was 0.01nmol/L,the inhibition efficiency reached 55.90%,indicating that PD-0325901 combined with oxaliplatin has synergistic inhibition effect.(2)In vivo results showed that: 1)the tumor inhibition rates in each group were: group I:(-);group II:(37%);group III:(27.7%);group IV:(64.1%);all subcutaneous transplanted tumors in mice were round,oval or irregular nodular,with poor mobility.2)immunohistochemistry showed that all the positive cells were brown.CD34 was mainly expressed in the surrounding vessels of cancer cells.The average optical density of CD34 in combination group was significantly weaker than that in the other three groups,the difference was statistically significant.3)Western blot detection showed that the protein expression level of Bcl-2 and CDK6 in combination group was lower than that in the other three groups,and the expression level of Bax protein was higher than that of the other three groups,the difference was statistically significant.4)immunofluorescence showed that the combined treatment group CRT was higher than that of the other three groups,the expression of PD-L1 was significantly lower than the other three groups,and the combination group was significantly more than in the CD8 infiltration in the other three groups,and the expression of macrophages was significantly lower than that of the other three groups,the difference was statistically significant.Conclusion The application of oxaliplatin induced CRT membrane eversion,promote macrophage recognition and phagocytosis of tumor cells and cause tumor cell immunogenicity by death;MEK inhibitor via MAPK pathway targeting inhibition of expression of tumor micro environment of PD-L1,and the release of tumor microenvironment immune tolerance state,so as to promote the cytotoxic T cells the proliferation and cytotoxic activity of chemotherapeutic drugs,enhance antitumor immunity,so as to achieve the purpose of treatment of cervical cancer.