Effect Of Oral Cancer Microenvironment On Neutrophil Oxidative Mitochondrial DNA

Objective:(1)To compare the expression of oxidative mt DNA in the neutrophils,which extracted from human peripheral blood and co-cultured normal human keratocytes(HOK)and human squamous cell carcinoma(SCC-9cell line)cells,and explore the mechanism of its elevation.(2)To discuss the possibility of detecting oxidative mtDNA levels in peripheral blood as a clinically diagnose method to early oral cancer in nude mice injected with SCC-9 cells in oral tissue.Methods:(1)Human neutrophils were co-cultured with HOK cells and SCC-9 cells by transwell chambers,and then cytokines IL-8(IL-8)and IL-8inhibitor curcumin were added.The expression of neutrophil oxidation mt DNA in different microenvironments were detected by Immunofluorescence of8-OHdG,H3 cit,and DNA complexes,and by 8-OHdG antibody Elisa assay through extraction of oxidized mtDNA on neutronphil extracellular traps(NET).(2)The SCC-9 nude mouse xenograft tumor models was constructed by injecting SCC-9 cells in mouse tongue tissue.Injecting human gingival fibroblasts(HGF)and culture medium(DMEM)medium were used as a normaland blank control group,respectively.Peripheral blood and tongue tissue of each group was collected at 48 h or/and 72 h.The neutrophil infiltration in tissues was observed by HE staining,and detected by neutrophils specific antibodies Ly6 G immunofluorescence staining.In addition,the expression of oxidized mtDNA in tissues was detected by 8-OHdG,H3 cit and DNA complexes immunofluorescence staining.Furtherly,the neutrophils ox-mtDNA in peripheral blood of each group was extracted;and detected by 8-OHdG antibody Elisa method.Results:(1)Immunofluorescence analysis showed that co-culture of neutrophils with SCC-9 increased the expression of ox-mtDNA,compared with co-cultured neutrophil with HOK cells.After further stimulation with IL-8 in the same co-culture system,the expression of oxidized mtDNA was obviously increased than the groups without IL-8 stimulation.On the other hand,Elisa results of neutrophils co-cultured with the SCC-9 cells,which pretreated with the IL-8 inhibitor curcumin,showed that the amount of oxidized mt DNA released by neutrophils was decreased,compared with curcumin no-treated group(P < 0.05)while there was no significant changes between the HOK cells which were pretreated with curcumin or not(P>0.05).(2)In nude mice injected with SCC-9 group,after 72 h,HE staining of the tongue injection area showed tumor-like changes,disorder of striated muscle arrangement,and increased infiltration of neutrophils were observed in or around the microenvironment of SCC-9 cells injection region.However,in the HGF injection group,some striated muscle disorder and neutrophil infiltration were observed in the tissues,whereas in the DMEM injection group,the striated muscle was arranged orderly,and no neutrophil infiltration was observed.(3)Compared to the nude mice with HGF injection group and DMEMinjection group,the neutrophils in SCC-9 injection group which detected by neutrophil-specific antibody Ly6 G immunofluorescent staining was increased.(4)The immunofluorescence detection of 8-OHdG,H3 cit and DNA complexes showed that the expression of oxidative mtDNA in neutrophils in SCC-9 injection group was significantly higher compared with controls.Furtherly,for 72 hours after surgical removal of early SCC-9 transplanted tumors,and HGF or DMEM injection regions in nude mice tongue,the oxidative mtDNA in the tongue tissues was significantly decreased in SCC-9group,while there was no significant change in HGF or DMEM group.(5)8-OHd G antibody Elisa results showed that the expression level of oxidized mtDNA in SCC-9 injection group was significantly higher than the two control groups,respectively(P<0.01;P<0.001),while the DMEM injection group had less oxidized mtDNA levels than the HGF injection group(P< 0.05).Oxidative mtDNA levels for 72 h after surgical removal in SCC9 group was significantly lower than that before surgical removal(P<0.001).Conclusions: IL-8 released by SCC-9 induces neutrophils to release oxidized mtDNA.The presence of early OSCC and its microenvironment increase the expression of neutrophil oxidized mtDNA in cancer tissue and peripheral blood.It suggests that oxidative mtDNA detection in peripheral blood may be used to clinical diagnosis of early OSCC.