Apigenin Suppresses Melanoma Growth By Regulating Immune System To Elicit Synergistic Therapeutic Effects

Malignant melanoma(MM)is the most aggressive and fatal malignant tumor of skin cancers.Early melanoma can be removed by surgery and the 5-year survival rate can reach 95%.But advanced or unresectable melanoma remains a great challenge for melanoma treatment.Prior to 2011,chemotherapy and targeted therapy were the main treatments for advanced melanoma,and can be only applied in 40%-50%of melanoma patients due to the limit of specific target and some serious side effects.In recent years,with the development of research on immunotherapy,the treatment of advanced melanoma has entered into a new stage.Immunotherapy has become an effective treatment option which is based on two reasons for advanced melanoma:first,immunotherapy can be a therapeutic option independent of the mutational status and its therapeutic effect mainly depends on the immune status of patients.Moreover,melanoma arises from malignant transformation of normal melanocytes in the epidermis,meaning that tumor cells are surrounded by abundant immune cells.Therefore,it is considered that immunotherapy may be the most promising treatment for advanced melanoma.Current immunotherapy are mainly including tumor vaccines,cytokines,oncolytic viruses,adaptive cell therapy and immune checkpoint inhibitors,among which immune checkpoint inhibitors,such as ipilimumab,nivolumab and pembrolizumab,have achieved a high-profile therapeutic effects in advanced melanoma and become a milestone in human treatment of cancer.The current researches show that blocking the PD-L1/PD-1 pathway can significantly enhanc the host anti-tumor immune response and is the best target for the treatment of'advanced malignant melanoma.Since PD-L1 is simultaneously expressed in tumor cells and APCs,targeting PD-L1 can exert a dual effect to enhance anti-tumor immune response in the initial and effector stages of T cell activation.At the same time,clinical studies also suggest that anti-PD-Ll mAbs compare with anti-PD-1 mAbs,showed a more sustained response rate and lower drug toxicity,so PD-L1 is considered to be a more effective immunotherapeutic target for the treatment of advanced melanoma.However,the current immune checkpoint inhibitors used in clinical treatment still lack high specificity,and the side effects and expensive treatment costs of the drugs also limit their application.Therefore,it is urgent to find and develop natural and low-toxic drugs to solve these problems.Natural plants and their extracts have been widely studied as very important therapeutic drugs in the treatment of tumors,exhibiting low toxicity,accessibility and low price.Among them,the natural and edible small molecular such as flavonoids,which are thought to have remarkable biological activity,low toxicity and non-mutagenic properties,have received more and more attention in anti-tumor treatment.Apigenin(APG),which is abundant in vegetables,fruits and beverages,has been widely studied as a classic flavonoid in cancer therapy.Studies have shown that it can exert anti-tumor effect in various tumor types,including melanoma..In recent years,a number of studies have shown that apigenin can exert an anti-tumor effect by regulating the host immune response.Above all highlight that apigenin can be used as a promising agent for melanoma therapy.In the present study,we first mentioned that apigenin can inhibit the growth of melanoma by regulating the T cell induced immune response.The apigenin manifested a dual effect to restore and enhance T cell immunity by inhibiting the expression of PD-L1 both in melanoma cells and dendritic cells.Therefore,this study illustrates a new direction of anti-tumor effect of apigenin,which may have potential clinical significance for the clinical treatment of malignant melanoma.Experiment 1:Expression of Programmed Cell Death Ligand-1 in Melanoma CellsObjective:This part investigated the expression level of PD-L1 in human melanoma cell lines and the influence of various cytokines and growth factors on the expression of PD-L1 in the tumor microenvironment.Method:Seven classical human melanoma cell lines were selected and the expression level of PD-L1 in melanoma cell line was detected by Western blot.The PD-L1 high expression cell lines were selected for subsequent experiments.A375 cell line was treated with the cytokines and growth factors which can regulate immune function in tumor microenvironment and the expression level of cytokines and growth factors induced PD-LI was detected by Western blot.The cytokine or growth factor that regulates PD-L1 was selected and the induction of PD-L1 was further verified by Western bolt.Results:The results of Western bolt showed that the expression level of PD-L 1 from high to low in the melanoma cell lines was RPMI-7951,A2058,A375,Me Wo,SK-MEL-3,G361,SK-MEL-28 and RPMI-7951,A2058 and A375 cell lines with higher expression level of PD-L 1 was selected for subsequent experiments.cytokine and growth factor induction experiments showed that IFN-y compared with the blank could significantly up-regulate PD-Ll expression in A375 cell line and the difference was statistically significant,and further Western blot results showed that IFN-? also up-regulated PD-L1 expression in other melanoma cell lines.Conclusions:PD-L1 is common expressed in melanoma cells;IFN-?-induced up-regulation of PD-L1 plays an important role in tumor immune escape;PD-L1 plays an important role in tumor immunotherapy.Experiment 2:Apigenin Suppresses PD-L1 Expression in Melanoma to Elicit the Function of ImmunomodulatoryObjective:This part investigated the effects and mechanism of flavonoids on PD-L1 expression in melanoma and further to determine its influence on T cell immune response.Method:The influence of flavonoids on melanoma cell proliferation and cell cycle was detected using cell proliferation and flow cytometry analyses.The influence of flavonoids on melanoma cell apoptosis was detected by immunofluorescence and apoptosis kits.The IFN-? induced PD-L1 expression were examined in apigenin and curcumin-treated melanoma cells using western blot or immunofluorescence and cell surface PD-L1 expression was detected by flow cytometry.Western blot was used to detect the levels of STAT1 and p-STAT1.The effects of flavonoid treatment on melanoma cells sensitivity towards T cells were detected using T cell killing assay,cytotoxicity assay and IL-2 ELISA kit.Melanoma xenograft model was used to evaluate the influence of flavonoids on tumorigenesis in vivo.Results:Flavonoids showed growth-suppressive and pro-apoptosis effects on melanoma cells.The IFN-y-induced PD-L1 upregulation was significantly inhibited by flavonoids,especially apigenin,with correlated reductions in STAT1 phosphorylation.Apigenin and curcumin-treated A375 cells enhance the ability of T cell-mediated killing and promote T cell proliferation.Flavonoids also exhibits inhibition of tumor growth and promotes T cells infiltration into tumor tissue in vivo.Conclusions:Apigenin restricted melanoma growth through its suppression of PD-L1 expression which restored and enhanced the ability of T cell-mediated killing and lymphocytes infiltration into tumor tissues to exert the effects of immunoregulatory.Experiment 3:Apigenin Suppresses PD-L1 Expression in host Dendritic Cells to Elicit the Anti-tumor Function of ImmunomodulatoryObjective:This part investigated the effect of flavonoids on PD-L1 expression in dendritic cells and its regulation of host immunity.Method:Dendritic cells of flavonoids-treated mice spleen were isolated and PD-L1 expression was detected by Western blot.Dendritic cells from human peripheral blood mononuclear cells were isolated and treated with flavonoids using Western blot to detect the PD-L1 expression.DC and CIK were isolated from human peripheral blood mononuclear cells and using cytokine to induce maturity,then flavonoids-treated DC were co-cultured with CIK to detect ability of T cell-mediated killing by colony formation and cytotoxicity assay.Results:Western bolt results showed that flavonoids,especially apigenin,significantly inhibited PD-L1 expression in mouse and human peripheral blood dendritic cells.Clonal formation and cytotoxicity assay indicated that DC could enhance the ability of CIK-mediated killing.Flavonoid-treated DC cells co-cultured with CIK can enhance the ability of DC-CIK against melanoma cells.Conclusions:Apigenin restricted melanoma growth though PD-L1 expression of host DC to exerted the effect via regulating host immune response.