Study On The Mechanism Of Calcium Channel Blocker Azelnidipine In Inhibiting The Growth Of Melanoma Cells

Background and Objective:The incidence of melanoma is increasing rapidly around the world.It is highly aggressive and is the malignant tumor with the highest lethality among skin tumors.The treatment methods for advanced melanoma mainly include chemotherapy,targeted therapy and immunotherapy.Dacarbazine is the only chemotherapy drug approved by the FDA for the treatment of advanced melanoma,but the objective response rate is less than 15%;targeted therapy represented by the BRAF inhibitor vemurafenib significantly prolongs the overall survival and progression-free survival of patients,but are highly susceptible to drug resistance;the immunotherapy represented by PD1 monoclonal antibody also has problems such as low response rate,large side effects,and high price.Therefore,there is a need to find new melanoma treatment strategies.In this study,azelnidipine was found to have significant anti-melanoma effects.Azelnidipine is a dihydropyridine antihypertensive agent that selectively inhibits L-type calcium channels.This study aims to explore the mechanism of azelnidipine in the treatment of melanoma and provide a new therapeutic strategy for clinical treatment of melanoma.Method:(1)Cell viability was detected by CCK-8,cell clone formation ability was detected by plate cloning assay,cell migration ability was detected by wound healing test,cell invasion ability was detected by Transwell test,changes in autophagy flux were detected by RFP-GFP-LC3 indicator technology,and cell apoptosis was detected by flow cytometry.(2)Construct a BALB/c nude mouse xenograft tumor model to study the in vivo effects of azelnidipine on melanoma.(3)Changes in the m RNA levels of target molecules were detected by q PCR,and protein levels of target molecules were detected by immunoblotting.(4)Fluo-4 AM fluorescent probe to detect intracellular calcium ion concentration.(5)Detect changes in cellular gene expression by transcriptome sequencing.Result:(1)Azelnidipine inhibits the proliferation,migration,invasion and clone formation of melanoma cells in a dose-dependent manner.(2)The level of LC3-II in melanoma was significantly increased after azelnidipine treatment.Autophagosomes and autolysosomes were observed after transfection with RFP-GFP-LC3 plasmid.Azelnidipine treatment significantly increased the number of apoptotic melanoma cells and induced PARP cleavage.(3)Azelnidipine treatment can reduce the intracellular calcium concentration of melanoma cells,and KCl treatment weakens the inhibitory effect of azelnidipine on melanoma cells.(4)Through transcriptome sequencing,it was found that azelnidipine affects a variety of cancer pathways,among which the HER3/PI3K/Akt pathway has the most obvious changes.It was proved by q PCR and western blotting that azelnidipine can significantly inhibit the HER3/PI3K/Akt pathway.(5)The combined use of azelnidipine and MK2206 has a greater inhibitory effect on melanoma than any single drug,and the two have a synergistic effect.(6)Azelnidipine can inhibit the expression of HER3 and MK2206 can induce the expression of HER3,and azelnidipine can inhibit the expression of HER3 induced by MK2206.Conclusion:(1)Azelnidipine induces autophagy and apoptosis by inhibiting the HER3/PI3K/Akt pathway,thereby inhibiting the growth of melanoma cells(2)Azelnidipine enhanced the inhibitory effect of MK2206 on melanoma cells by inhibiting MK2206-induced HER3 expression.