Anti-tumor Effect And Mechanism Of Venlafaxine In Mice Burdened Melanoma

BackgroundMelanoma is a most aggressive skin cancer,with a highly lethal and drug resistance.Find new treatment methods to improve the survival rates is a hot topic of current research.PD-L1,programmed death protein ligand-1,binds to its receptor PD-1 on activated T cells to suppress anti-tumor immunity,resulting in accelerated tumor growth.In recent years,monoclonal antibodies against PD-1/PD-L1 have shown excellent antitumor efficacy in the treatment of patients with melanoma.However,monoclonal antibodies are expensive and time-consuming,finding a safe alternative method with inexpensive PD-L1 inhibitor is worth exploring.Venlafaxine hydrochloride,a novel anti-depression medication,significantly suppressed the growth of melanoma and the expression of PD-L1.In this study,we detected the effect of venlafaxine hydrochloride on melanoma in vitro and in vivo,and explored the mechanism involved.ObjectiveTo investigate the enhancement therapeutic effect of venlafaxine hydrochloride on melanoma in vitro and in vivo,and explored the mechanism involved.Methods1.CCK-8 and wound healing scratch assaysB16 cells were seeded into 96-well plates or 6-well plates and incubated for 16-18h,and then were treated with the indicated concentrations of venlafaxine hydrochloride.CCK-8and wound healing scratch assay were carried out to detect the cell ability and migration of the cells.2.Western blotting assayB16 cells were seeded into 6-well plates for 16-18h and treated with the different concentrations of venlafaxine hydrochloride for another 24h or 48h.Western blotting was applied to analyze the expression of tumor relative proteins.3.Establish the melanoma bearing tumor model and the treatment with venlafaxine hydrochlorideFemale C57BL/6 mice,with 6-8 weeks,were used as experimental animals.B16 cells（1×10~6）were injected subcutaneously into each mouse.After inoculation for 7 days,the mice were randomly divided into four groups,control group（PBS group）low-dose group（100μg venlafaxine hydrochloride/mouse）,medium-dose group（200μg venlafaxine hydrochloride/mouse）and high-dose group（400μg venlafaxine hydrochloride/mouse）.The mice were received treatment for 7 consecutive days and sacrificed on day 7post-treatment.The effect of venlafaxine hydrochloride on tumor growth in melanoma bearing mice was observed.The effect of venlafaxine hydrochloride on expression of tumor relative proteins in tumor tissues was detect by WB.Immunofluorescence technique was used to detect the effect of venlafaxine hydrochloride on lymphocyte infiltration in tumor tissues.The effect of venlafaxine hydrochloride on the ratio of related immune cells in spleen was detected by flow cytometry.Results1.The results from CCK8 assay showed that venlafaxine hydrochloride had no obvious killing effect on B16 cells after treatment for 24 hours,while when the concentration of venlafaxine hydrochloride above 20μg/ml and last for 48h,it exhibited the killing ability.2.Wound healing scratch assay was carried out to detect migration of the cells.The effect of migrative inhibition was shown when the treatment of venlafaxine hydrochloride at the concentration of 2.5μg/ml,and last for 24h or 48h.3.The effect of venlafaxine hydrochloride on the expression of related proteins in B16 cells was detected by WB experiment.It was found that venlafaxine hydrochloride significantly inhibited the expression of PD-L1,MMP2 and p-STAT3 in B16 cells.4.The results showed in vivo,only the treatment of venlafaxine hydrochloride with the dose of 200μg/mouse significantly inhibited the growth of tumor bearing mice,as compared with PBS group.5.As shown by WB,the expression of PD-L1,MMP2 and p-STAT3 was significantly inhibited in tumor tissue in all the three venlafaxine hydrochloride treatment groups（100μg/mouse,200μg/mouse or 400μg/mouse）,as compared in that of PBS group.6.It was found by immunofluorescence that the infiltration of CD4⁺or CD8⁺T lymphocytes were significantly increased,and the expression of PD-L1 was significantly inhibited in tumor tissues when the dose of venlafaxine hydrochloride was 200μg/mouse or 400μg/mouse.7.The therapeutic concentration of venlafaxine hydrochloride with 200μg/mouse or 400μg/mouse resulted in the increase of the ratio of CD4⁺and CD8⁺T lymphocytes,and NK cells in the spleen of tumor bearing mice.ConclusionsVenlafaxine hydrochloride effectively inhibited the expression of MMP2 and p-STAT3in melanoma B16 cells and tumor tissues of tumor bearing mice,and inhibited the tumor growth,suggesting that venlafaxine hydrochloride has the potential to become an anti-melanoma drug.In animal study,venlafaxine hydrochloride significantly inhibited the expression of PD-L1 in tumor tissues,which could enhance the overall anti-tumor immune response of tumor bearing mice by increasing the infiltration of T lymphocytes in tumor tissues and the ratio of T lymphocytes in spleen.This provides a experimental basis for finding a safe and effective PD-L1 inhibitor to treat melanoma.