| ObjectiveMelanoma is the common and highly lethal type of skin cancer for which conventional treatments have failed to achieve ideal and durable clinical outcomes.Suicide gene therapy,a promising approach for comprehensive oncology treatment,also suffers from low transfection efficiency or insufficient targeting.In our study,we found that dioscin enhanced the bystander effect of suicide gene therapy.Therefore,we combined in vivo and in vitro experiments with cell biology technique,molecular biology technique,immunological technique and high-throughput sequencing of RNA transcriptome to elucidate the potential regulatory process of dioscin on the efficacy of suicide gene therapy from the perspective of immune mechanism,providing new targets and ideas for the combination of Traditional Chinese Medicine with modern medicine.Methods1.In vivo drug efficacy studies(1)Forty male C57BL/6 mice aged 5-6 weeks were randomly divided into the blank group,GCV group,Dio group and GCV+Dio group,with 10 mice in each.The melanoma graft models were constructed by subcutaneous inoculation of B16 and B16-Tk cells under the axillae of both forelimbs,respectively,and the corresponding drug intervention was started on the first day after inoculation and samples were collected on the 18th day after inoculation.(2)The body weight of the mice was recorded daily during the drug intervention,and the tumor length and diameter were measured to estimate the volume and then assess the tumor suppression effect.(3)On the day of extraction,the eyeball was taken for blood,and the levels of IFN-y,TNF-α cytokines and granzyme B within the serum of C57BL/6 mice were detected via enzyme-linked immuno sorbent assay(ELISA);spleens were removed,single cell suspensions were prepared,and the percentage of splenic CD8+T lymphocytes and CD8+CD69+T lymphocytes,a marker of early activation,were measured by flow cytometry to predict the immune activation of secondary lymphoid organs in mice.The tumors were removed and visualized by HE staining,and Cx43 expression and CD8+T lymphocyte infiltration were assessed by immunohistochemistry,and Cx43 protein expression in melanoma bodies was detected by Western blot.2.In vitro drug efficacy studies(1)Toxicity assay and concentration screening of dioscin were performed by cell counting kit 8(CCK8);the apoptosis rate of GCV-intervened B16-Tk cells was determined by CCK8 and Annexin V/PI staining to screen the subsequent intervention concentration.(2)The mouse bone marrow cells were extracted and induced to differentiate into DCs under the condition of GM-CSF and IL-4.Then the DCs were treated with different concentrations of LPS,and the morphological changes of DCs during in vitro culture were observed by light microscope,and the CD11C and MHC-Ⅱ double positive cells ratio was detected by flow cytometry to predict cell purity.Cell maturity was predicted by DCs surface costimulatory molecules CD80,CD86 and CD40,and antigen presentation was detected by cell surface MHC-I/OVA antigenic peptides,so that the purity and maturity conditions of DCs could meet the subsequent experiments.(3)In vitro tumor microenvironment was simulated using B16-OVA-Tk cells,DCs cells,OT-I initial CD8+T lymphocytes co-culture system,and cell killing of B16-OVA by OT-I CD8+T lymphocytes detected by Hoechst/PI co-staining assay.3.Verification of mechanism(1)In vitro experiments were performed to detect Cx43 protein and gene in B16 cells after dioscin intervention by Western blot and real-time fluorescence PCR quantification.The expression of Cx43 protein on the membrane of B16 cells after dioscin intervention was observed by immunofluorescence assay.(2)By confocal microscopy and flow cytometry,we visualized and quantified the changes in intercellular communication function of intercellular gap junctions in B16 cells after dioscin intervention by indirect fluorescence transmission,dye transfer and fluorescent bleaching recovery experiments.(3)By transient transfection constructs of overexpressed wild-type Cx43 and mutant Cx43,directly co-cultured with DCs after GCV action,the effect of GJIC function on the immunocidal effect of activation of OT-I initial CD8+T lymphocytes by antigen delivery to DCs was examined by flow cytometry and immunofluorescence assays.(4)After blocking GJIC with the blocker Gap26,the effect of dioscin-mediated GJIC on the immunocidal effect of DCs antigen delivery activation of OT-I initial CD8+T lymphocytes was examined by flow cytometry,immunofluorescence assay and enzyme-linked immunosorbent assay.(5)The core regulatory signaling pathway for the change in Cx43 protein content in B16 cells after dioscin intervention was predicted by cellular level RNA high-throughput sequencing method and preliminary validation of the target proteins on the pathway was performed by Western blot.Results1.In vivo drug efficacy studies(1)In C57BL/6 mouse subcutaneous B16 and B16-Tk melanoma transplantation models,dioscin combined with GCV exerted a significant synergistic immune killing effect compared to the blank group,GCV and Dio alone group,as shown by a significant decrease in tumor volume and mass on the B16 side(P<0.01).(2)In HE staining,the tumor was richly vascularized in the blank and Dio groups,while significant nuclear consolidation and massive necrosis of tumor cells were seen in the Dio combined with GCV group.Immunohistochemical results suggested that Cx43 protein expression in tumor cells was upregulated in the Dio alone and combined groups;there was no obvious CD8+ T lymphocyte infiltration in the blank group,while there was an increase in tumor infiltrating CD8+ T lymphocytes in the Dio combined with GCV group(P<0.05).Western blot results showed that Cx43 protein expression was significantly increased in dioscin group and combined group(P<0.01).(3)Dioscin promotes immune activation of peripheral lymphoid organs in a transplantation tumor model:flow cytometric detection of the proportion of CD8+ T lymphocytes and the surface marker CD69 in mouse spleen suggested that dioscin combined with GCV significantly upregulated CD8-T lymphocytes in mouse spleen(P<0.01),and the early activation marker CD69 was significantly upregulated(P<0.01).(4)Dioscin can affect the levels of immune-related cytokines in the serum of mouse subcutaneous suppressor tumor model:the results of ELISA for immune-related factor levels suggested that the levels of IFN-y,TNF-α cytokines and granzyme B were significantly upregulated in the dioscin combined with GCV group compared with the blank group and single drug group(P<0.01).2.In vitro drug efficacy studies(1)The cell activity assay by CCK8 on concentration gradient dioscin action on B16 cells after 24h,48h and 72h,the cell viability after 1 μM and 2 μM action for 48h was 98.16%±0.64 and 83.99%± 5.44 respectively at low toxic concentration and action time of diosgenin could be used for the follow-up study.By the cell activity and apoptosis assay after GCV action on B16-OVA-Tk,50 μg/ml of GCV with apoptosis rate at 19.78%± 2.35 and cell inhibition rate at 21.65%±0.65%was selected as the concentration of suicide gene precursor drug for subsequent in vitro experiments.(2)Optical microscopy showed that DCs changed from an adnate state to a suspended colony state.Over time,the dendritic protrusions of DCs became obvious and gradually increased.The purity of bone marrow-derived DCs extracted from mouse bone marrow and cultured in vitro was high(CD11C+ MHC-Ⅱ+ DC>85%);the results of flow cytometric detection of co-stimulatory molecules CD80,CD86,CD40 on the cell surface of DCs and the degree of MHC-Ⅰ/OVA antigenic peptide presentation on the cell surface of DCs after incubation with OVA antigenic peptide suggested that 500ng/ml LPS was the appropriate concentration for induction of DC maturation in subsequent experiments.The purity of OT-Ⅰ CD8+ T lymphocytes(CD8α+T>90%)obtained by immunomagnetic bead sorting of spleen monocytes was high and met the experimental requirements.(3)The results of Hoechst/PI apoptosis assay in the in vitro co-culture system showed that 1μM and 2 μM Dio synergistic GCV significantly upregulated the immunocidal effect of OT-I CD8+ T lymphocytes(P<0.05).3.Verification of mechanism(1)The results of Cx43 gene and protein expression in B16 cells suggested that dioscin significantly increased Cx43 gene expression and protein expression,and Cx43 protein was up-regulated by dioscin in a concentration-dependent and time-dependent manner(P<0.05).the expression of Cx43 was significantly different at 1 μM and 2 μM of dioscin,and the difference was most significant at 48 h of dioscin action(P<0.05).Compared with the blank group,the fluorescence intensity of Cx43 on the cell membrane was gradually enhanced with the upregulation of dioscin concentration.(2)The results of dye transfer,indirect fluorescence transmission and fluorescence bleaching recovery experiments suggested that significant fluorescent dye transmission between cells could be visually observed in the dioscin intervention group and that 2μM upregulated fluorescence transmission more significantly than 1 μM dioscin(P<0.05).(3)Transient transfection constructs overexpressing wild-type Cx43 and mutant Cx43,and flow results assaying eBio25-D1.16 showed that B16-OVA-Tk Cx43G21R antigen presentation function was significantly diminished in B16-OVA-Tk Cx43 with impaired GJIC function compared to wild-type Cx43 with upregulated GJIC function(P<0.01).The fluorescence intensity results of CD69,showed that the immune activating effect of mutant Cx43 was significantly diminished compared to wild-type Cx43(P<0.01).In addition,PI apoptotic cell staining experiments showed that the immunocidal effect of mutant Cx43 activation was significantly weaker than that of wild-type Cx43(P<0.05).(4)By blocking GJIC function,the flow cytometry results suggested that the intervention of Gap26 reversed the upregulation of DC antigen presentation by dioscin to some extent(P<0.05),further confirming the facilitation of DC antigen cross-presentation by Dio-mediated GJIC.ELISA results suggested that the intervention of Gap26 reversed the upregulation of DC-released cytokines by Dio to some extent(P<0.05),further confirming the facilitation of DC antigen cross-presentation by Dio-mediated GJIC.Further confirming the facilitation of Dio-mediated GJIC on DC antigen cross-presentation.(5)The results of CD69 showed that Dioscin-coordinated GCV promoted OT-I CD8+ T lymphocyte activation and that blockade of GJIC function partially reversed this activation effect.1 μM and 2 μM of Dio-coordinated GCV significantly upregulated the immunocidal effect of OT-I CD8-T cells(P<0.05).inhibition of GJIC function partially reversed the immunocidal effect of OT-I CD8+T cells.partially reversed the immunocidal effect of OT-I CD8+T cells.(6)B16 cells with 1μM dioscin intervention in B16 cells for 48h were subjected to RNA high-throughput sequencing differential expression threshold set at |logFC|>1 and p value<0.05,and 344 differential genes were obtained,including 173 down-regulated genes and 171 up-regulated genes,and 171 signaling by KEGG analysis such as PI3K/Akt,JAK/STAT pathways.The WB results of 10 target proteins on PI3K/Akt and JAK/STAT pathways confirmed that dioscin may affect the content of Cx43 protein in B16 cells through these two pathways.Conclusion1.The in vivo efficacy results of the drug combination booster confirmed that dioscin synergistically potentiated the immune killing effect of HSV-Tk/GCV suicide gene therapy on B16 cells and upregulated the expression of Cx43 in tumor cells in vivo,as well as the number of tumor-infiltrating CD8+T and the proportion and activation of splenic CD8+T cells,and significantly upregulated serum IFN-γ,Gzms-B,and TNF-α levels in mice.It indicates that the distal killing effect of dioscin on B16 cells may be related to gap junctions and immune mechanisms.2.The in vitro efficacy results of the drug combination potentiation confirmed that in an in vitro simulated tumor microenvironment co-culture system of tumor cells,DCs and OT-I CD8+T cells,dioscin in concert with GCV suicide gene therapy promoted the immunocidal effect of OT-I initial CD8+T lymphocytes on B 16-OVA cells.3.Drug combination potentiation mechanism found in exploration suggested a potential regulatory role of GJIC function on the activation of OT-I CD8+T lymphocyte immune response by antigen cross-presentation in DCs.Dioscin could activate the antitumor immunocidal potency of OT-I initial CD8+ T cells by mediating GJIC upregulation of DCs antigen cross-presentation.Dioscin was found to have the effect of upregulating Cx43 protein expression and improving GJIC function,and the specific mechanism may be related to the negative regulation of PI3K/Akt and JAK/STAT signaling pathways. |
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