Effect Of GRP78-GPX4 Pathway On Enhancing Ferroptosis Tolerance And Promoting Its Migration In Cutaneous Melanoma Cells

Objective: Cutaneous melanoma is one of the most aggressive tumors on the skin,with a poor prognosis and high mortality.At present,the pathogenesis of melanoma is still being explored.Ferroptosis is a new form of cell death,which is involved in the malignant progression of various tumors.To explore the regulation of ferroptosis on melanoma and the mechanism of melanoma ferroptosis has become one of the focuses of melanoma research.GRP78(glucose regulated protein 78)is a member of the HSP70(heat shock protein 70)family.It participates in endoplasmic reticulum stress and is associated with higher mortality of melanoma.In this study,we explored the role and regulatory mechanism of GRP78 on ferroptosis in melanoma,providing a theoretical basis for precise treatment of melanoma.Methods: 1.The expression of GRP78 was detected by immunohistochemistry in the samples of nevus,melanoma in situ and lymph node metastasis melanoma in our hospital.2.For different melanoma cell lines,their ferroptosis-induced survival was measured using CCK8,GRP78 protein level was detected by Western blot,then analyze its correlation with ferroptosis tolerance.3.Melanoma cells with interference/overexpression of GRP78 protein were constructed by lentivirus.4.CCK8 was used to detect the ferroptosis sensitivity of melanoma cells with GRP78 interfered/overexpressed.5.The level of glutathione peroxidase 4(GPX4)in melanoma cells during ferroptosis was detected by WB;MDA assay kit was used to detect the level of intracellular lipid peroxide accumulation;The intracellular GSH and GSSG levels were detected with GSSG/GSH assay kit.6.Scratch test and transwell migration test were used to detect the migration ability of melanoma cells after interference/overexpression of GRP78.Results: 1.Immunohistochemical results showed that GRP78 was expressed in both in situ melanoma and lymph node metastasis,and the level of GRP78 expression was elevated in melanoma compared with benign nevi.2.CCK-8 results suggest that the sensitivity of different melanoma cell lines to ferroptosis was different.Further,the content of GRP78 protein in melanoma cell lines A375 and UACC257,which were relatively tolerant to ferroptosis,was higher than that in 451 lu and A2058,which were relatively sensitive to ferroptosis.3.By using CCK-8 to detect the sensitivity of melanoma cells with interference/overexpression of GRP78 to ferroptosis,we found that more cells survived when ferroptosis occurred in melanoma cell lines with overexpression of GRP78,and GRP78 interfered would cause ferroptosis sensitivity.4.The intracellular MDA and GSH levels induced by ferroptosis were detected by MDA assay kit and GSSG/GSH assay kit,the results showed that compared with the control group,melanoma cells overexpressing GRP78 had a lower intracellular GSH decline and lower lipid peroxide accumulation when stimulated by ferroptosis.Correspondingly,Melanoma cells that interfere with GRP78 show increased lipid peroxide accumulation and glutathione consumption.5.WB experiment found that the level of GPX4 decreased when melanoma cells stimulated by ferroptosis,overexpression of GRP78 could inhibit GPX4 level decline,and interference of GRP78 could promote the decline of GPX4.6.The results of scratch test and transwell migration test showed that interference with GRP78 inhibited A375 cell migration.Conclusion: 1.The expression of GRP78 is significantly increased in cutaneous melanoma and is closely related to its metastasis.2.Melanoma with high expression of GRP78 showed more tolerance to ferroptosis.3.The GRP78-GPX4 pathway ultimately promotes ferroptosis tolerance in melanoma by reversing the decline in intracellular GSH level and reducing lipid peroxide accumulation in cells.4.Inhibition of GRP78 expression in melanoma leads to migration ability decreased in melanoma.