Pym-5 Targeting GSK-3? Induces Melanogenesis In Murine Melanoma Cells

Bcakground and PurposeGlycogen Synthase Kinase-3(GSK-3)is a multifunctional serine/threonine protein kinase that can be involved in multiple signaling pathways such as Wnt/?-catenin and NF-?B in tumorigenesis,and GSK-3 expression.The intracellular localization and activity are closely related to the growth,proliferation,apoptosis and differentiation of tumor cells.It is one of the important targets in tumor research.Pym-5 is a novel 1H-pyrazole-3-carboxamide derivative,and its in vitro kinase screening showed strong selectivity for GSK-3p.This study selected the compound pym-5 as an inhibitor of GSK-3 ? to study a variety of tumor cell types,confirming its mode of action and elucidating its exact mechanism of action.Research Contents and Results1.In this experiment,the effects of pym-5 on the differentiation of tumor cells were first investigated using mouse melanoma cells B16 and B16BL6.The proliferation of melanoma cells in vitro was confirmed by proliferation experiments.Inhibitory effects of pym-5 on the cell cycle of mouse melanoma cells were examined by flow cytometry.2.Based on the determination of dose and time,the inhibitory effect of Pym-5 on GSK-3? in mouse melanoma cells was observed by Western Blot.It was observed by immunofluorescence that Pym-5 could affect the downstream of GSK-3?.-catenin entry.3.Through the bioinformatics analysis of GSK-3P and ?-catenin in melanoma,it was confirmed that MITF in melanoma is most closely related to GSK-3? and ?-catenin,and the effect of GSK-3? on melanoma is concentrated.In the aspect of wnt signaling pathway and cell differentiation cycle,it is suggested that pym-5 may affect the expression of downstream MITF through inhibiting GSK-3?,and ultimately affect the differentiation of mouse melanoma cells.4.The above conjecture was confirmed by tests such as rt-qPCR,melanin production assay,and tyrosinase activity assay.Finally,knocking out GSK-3? in cells by the Crispr/cas 9 method confirmed that GSK-3 ? is a target for pym-5 to affect the differentiation of melanoma cells in mice.5.Further studies were conducted on human non-small cell lung cancer cell A549 and human breast cancer cell MDA-MB-231 for autophagy induced by pym-5.In the cell morphology and protein level,it was verified that pym-5 can also promote the GSK-3?-mediated degradation of Mcl-1 by influencing the phosphorylation of ?-catenin,a downstream substrate of GSK-3?,and promote human non-small cell Lung cancer cells A549 and human breast cancer cells MDA-MB-231 autophagy.Conclusion and SignificanceIn conclusion,on the one hand,pym-5 can affect the nuclear import of p-catenin by inhibiting GSK-3?,which in turn influences the transcription and expression of downstream MITF,and ultimately affects the differentiation of mouse melanoma cells;On the other hand,pym-5 promotes GSK-3?-mediated degradation of Mcl-1 by influencing the phosphorylation of ?-catenin,a downstream substrate of GSK-3?,and promotes autophagy in many tumor cells.The subject confirms that the main target of pym-5 to exert anti-tumor effect is GSK-3?,and clarifies the molecular mechanism of pym-5 promoting the differentiation and autophagy,which provides a strong evidence for pym-5 to enter new drug research.