Nr-CWS Influences The Development Of Cervical Carcinoma By Promoting The Antitumor Effect Of Dendritic Cells And Macrophages

Objective: Cervical cancer is a serious disease affecting the physical and mental health of women in developing countries.Its pathogenesis is the continuous HPV infection and the immune function decline of patients,which determines the important role of immunotherapy in the treatment of cervical cancer.Currently,the common clinical immunotherapy for cervical cancer is anti-PD-1/ PD-L1 monotherapy or combined antivascular therapy.This type of immunotherapy has limited efficacy and serious adverse reactions.Therefore,it is very important to find a safe and effective immunotherapy for patients with cervical cancer.Nocardia rubra cell-wall skeleton(Nr-CWS)is an immune booster,consisting of nocardomycolic acid,arabinogalactan,and mucopeptide.Clinically,Nr-CWS is mainly used for the treatment of high-risk HPV infection and cervical intraepithelial neoplasia.Studies have shown that Nr-CWS can significantly promote the activity of cytokine-induced killer cells(CIKs)and natural killer cells(NKs),and increase the response of CD8+T cells and the activation of CD4+T cells.But there is no evidence that Nr-CWS affects the activity or antitumor role of dendritic cells(DCs)and macrophages(Mφs).DCs are the center that initiate antigen-specific immunity and tolerance.They continuously recognize and collect antigens,present them to T cells through major histocompatibility complex(MHC)I and II,and regulate the activity of other immune cells through intercellular contact and cytokine release,thus activating antitumor immune responses.As the largest immune cell in the microenvironment of the cervical cancer tumor,Mφs not only recognize and phagocytize the tumor cell early on,but also recruit and regulate other effector cells by secreting cytokines.This study explores the influence of Nr-CWS on the activity and function of DCs and Mφs,and further studies the influence of Nr-CWS through the antitumor effect of Mφs on the malignant biological behavior and development of cervical cancer cells.To provide theoretical basis and experimental basis for Nr-CWS to become a new immunotherapy scheme for patients with cervical cancer.Methods: Part 1: Human peripheral blood mononuclear cells(PBMCs)were extracted from the peripheral blood of patients with cervical cancer and cytokines were added to induce the culture of DCs and Mφs.The CCK8 experiment was used to find the optimal concentration and optimal action time of Nr-CWS on PBMC activity.The purity of DCs and Mφs after induction was verified by flow cytometry.DCs and Mφs were stimulated by low-concentration(20 μg/ml)and high-concentration(30 μg/ml)Nr-CWS.The changes of cell viability and apoptosis rate of DCs and Mφs were detected by CCK8 assay and flow cytometry assay.Real Time PCR(RT-PCR)was used to detect the changes of PD-L1 m RNA expression in DCs and Mφs.Two concentrations of Nr-CWS were used to stimulate PBMC,and the changes in the number of CD80+DCs and CD68+Mφs stained cells were compared with those without Nr-CWS stimulation by flow cytometry.ELISA assay was used to detect the effects of low and high concentrations of Nr-CWS on the levels of cytokines IL-6 and IL-12 secreted by DCs,and the levels of cytokines TNF-α and IL-1β secreted by Mφs,compared with the non-Nr-CWS stimulated group.At the same time,the tumor bearing mouse model was established,and the changes of IL-6,IL-12,TNF-α and IL-1β in venous blood of mice were detected by ELISA.The expressions of CD80 and F4/80 in each group were detected by Western Blot.Part 2: The co-culture system of Mφs and cervical cancer cells was established by transwell chamber,and the co-culture system was stimulated by low concentration and high concentration of Nr-CWS.The effects of Nr-CWS on the proliferation,migration and invasion of co-cultured cervical cancer cells were detected by CCK8 assay,scratch assay and transwell assay.The m RNA and protein levels of E6,E7 and p53 genes in co-cultured cervical cancer cells were detected by RT-PCR and Western Blot.The effect of Nr-CWS on cell cycle and apoptosis rate of co-cultured cervical cancer was determined by flow cytometry.The expressions of apoptosis-related proteins Bcl-2,BAX,caspase-3,caspase-8,caspase-9 and Wnt/β-catenin-EMT pathway proteins in co-cultured cervical cancer cells were detected by Western Blot assay.The TNF-α receptor TNFR1 was knocked down in co-cultured cervical cancer cells to detect the change of the influence of Nr-CWS on apoptosis of co-cultured cervical cancer cell lines.The mouse model of cervical cancer bearing tumor was established,and the tumor growth of mice in each group was observed.The expressions of E6,E7,PD-L1,p53 proteins and the cervical cancer proliferationrelated proteins Ki67,P16,and PCNA were detected by immunohistochemistry.Results: Part 1: CCK8 experiment showed that Nr-CWS increased PBMC activity in a dose-dependent and time-dependent manner.The optimal concentration of Nr-CWS on PBMC was 20 μg/ml and the optimal action time was 72 h,P<0.0001.Flow cytometry experiments identified that CD80+DCs and CD68+Mφs reached 97.3% and 96.9%,respectively after cytokine induction.DCs and Mφs were stimulated with lowconcentration(20 μg/ml)and high-concentration(30 μg/ml)Nr-CWS.Compared with no Nr-CWS stimulation,low and high concentration of Nr-CWS increased the cell viability of DCs and Mφs at 72 h,according to CCK8 assay,P<0.0001.Flow cytometry showed that,compared with no Nr-CWS stimulation,low and high concentration of Nr-CWS could reduce the apoptosis rate of DCs and Mφs cells,P<0.05.DCs marker CD80+ and Mφs marker CD68+ expression increased after stimulation of PBMC with low and high concentrations of Nr-CWS compared with no Nr-CWS stimulation,P<0.05.ELISA experiments found that low and high concentrations of Nr-CWS could promote the secretion of cytokines IL-6 and IL-12 by DCs and the cytokines IL-1β and TNF-α by Mφs,P<0.001.RT-PCR experiments showed that both low and high concentrations of Nr-CWS could reduce the expression of PD-L1 m RNA in DCs and Mφs,P<0.01.Serum cytokine secretion levels of mice in each group were detected by ELISA.Compared with the mice treated with no Nr-CWS,low concentration and high concentration of Nr-CWS could increase the serum secretion levels of IL-6,IL-12,TNF-α and IL-1β,P<0.05.Western Blot analysis showed that the expression levels of F4/80 and CD80 in tumor tissue of mice treated with low concentration and high concentration of Nr-CWS were higher than those of the control group.P<0.05.Part 2: In this study,He La/Siha cells were divided into four groups and cultured for 72 h.Cervical cancer cells in He La/Siha-blank group were cultured with ordinary medium.He La/Siha-Mφ group cervical cancer cells were co-cultured with Mφs.He La/Siha-Mφ-Nr-CWs-low group cervical cancer cells were co-cultured with Mφs stimulated by low concentration Nr-CWS.He La/Siha-Mφ-Nr-CWs-high group cervical cancer cells were cocultured with Mφs stimulated by high concentration of Nr-CWS.The results of CCK8 showed that at 72 h,the cell viability of the He La/Siha-Mφ-Nr-CWS-low group and the He La/Siha-Mφ-Nr-CWS-was decreased compared with the He La/Siha-Mφ group,P<0.001.Scratch experiments and transwell experiments showed that compared with He La/Siha-Mφ group,the migration and invasion ability of cells in the He La/Siha-Mφ-NrCWS-low group and He La/Siha-Mφ-Nr-CWS-high group was reduced,P<0.05.The results of RT-PCR and Western Blot showed that compared with the He La/Siha-Mφ group,the expression of E6,E7,PD-L1 m RNA and protein in the He La/Siha-Mφ-Nr-CWS-low group and the He La/Siha-Mφ-Nr-CWS-high group was decreased and the expression of p53 m RNA and protein was increased,P<0.05.The results of flow cytometry showed that Nr-CWS blocked co-cultured cervical cancer cells in the G2/M phase,P<0.05.The results of flow cytometry showed that Nr-CWS blocked co-cultured cervical cancer cells in the G2/M phase,P<0.05.After knocking down TNFR1,compared with the si NC group,the apoptosis rate of cells in the si TNFR1 group was decreased,the expression of Bcl-2 protein was increased,and the expression of BAX,caspase-3,caspase-8,caspase-9 and TNFR1 protein was decreased,P <0.05。 The results of Western Blot experiments showed that compared with the He La/Siha-Mφ group,the expression levels of β-catenin and Ncadherin proteins in the He La/Siha-Mφ-Nr-CWS-low group and the He La/Siha-Mφ-NrCWS-high group were decreased,and the expression levels of E-cadherin proteins were increased,P<0.05.In vivo experiments proved that Nr-CWS could significantly reduce the tumor and volume of subcutaneous tumors in tumor-bearing mice,P<0.01.Immunohistochemical experiments showed that Nr-CWS inhibited the expression of the proliferation related proteins Ki67,P16 and PCNA proteins,and E6,E7 oncoproteins proteins in mouse tumors,P<0.01.Conclusion: 1.Nr-CWS can increase the cell viability and inhibit apoptosis of DCs and Mφs.2.Nr-CWS can promote the secretion of IL-6 and IL-12 in DCs and TNF-α and IL-1β in Mφs,and reduce the expression of PD-L1 m RNA in DCs and Mφs.3.Nr-CWS can inhibit the malignant biological behavior of cervical cancer cells by promoting the antitumor effect of Mφs.4.Nr-CWS can inhibit the tumor growth of cervical cancer bearing mice.