Studies On The Effects Of Double-Edged Sword Of Dimethyl Fumarate In Melanoma Therapy

Objective:To investigate the effects and mechanisms of Dimethyl Fumarate(DMF) on tumor inhibition and cytoprotection in human melanoma cells, and develop strategy enhancing the cytotoxicity of DMF to melanoma cells.Methods: MTT assay and anchorage-dependent colony formation assay were used to determine the proliferation status of A375 melanoma cells treated with a different dose of DMF. Cell cycle distribution, apoptosis, and intracellular GSH level were detected by Flow cytometer analysis after cell staining with Propidium Iodide(PI) or Annexin-V/PI, and labeling with CMFDA respectively. The expression of proteins and their transcripts related to this study was examined by western blotting and quantitative RT-PCR respectively. Immunofluorescence staining was performed to observe the features of autophagy. Transfection of Nrf2 reporter plasmid together with dual luciferase bioluminescence assay was used to detect Nrf2 activity, and transfection of specific Si RNAs was employed to silence p62 and Nrf2 gene expression.Results: DMF inhibited A375 cell proliferation in a dose-dependent fashion. The ability of colony formation was also impaired by single DMF treatment in a dose range from 10 μmol/L to 100 μmol/L, and an almost complete inhibition was achieved at 50 μmol/L. Cell cycle analysis showed that a lower dose of DMF(50 μmol/L) mainly led to reduced cell fraction in S phase and a higher dose(100 μmol/L) caused noteble G2/M growth arrest. DMF also induced moderate apoptosis demonstrated by increased percent of the total dead cell(about 40% cell death at the dose of 100 μmol/L), which was companied by PARP-1 cleavage and caspase-3 degradations. All these data indicated that DMF has significant anti-tumor effects. GSH depletion by DMF was confirmed by the reduction of fluorescence in CMFDA labeled cells by FACS analysis. Pretreatment with thiol-containing antioxidant, N-Acetylcysteine, restored the GSH level in DMF-treated cells and reversed the G2/M arrested cells back to normal cell cycle as seen in untreated control cells. However, simultaneously analyzing the GSH depletion and cell death in single cell level by FACS revealed that only half the GSH deplete cells underwent apoptosis. To characterize the cell-protection effects of DMF, we focused on autophagy and Nrf2 activity. DMF promoted autophagy of A375 cells, which was evidenced by increased LC3-II protein conversion, higher rate of cells with LC3 punctate that co-localized with a lysosome marker LAMP-1. Furthermore, DMF also enhanced the Nrf2 activity in A375 cells, demonstrated by increased Nrf2 protein expression, reporter gene activity and transcripts of downstream genes of Nrf2(HO-1 and NQO1). A concurrent upregulation of p62 and Nrf2 by DMF indicated the existence of a positive feedback loop between p62 and Nrf2. In addition, combined treatment with DMF(50 μmol/L) and Nrf2 Si RNA, but not p62 Si RNA, enhanced the cell death rate of A375 from 40% in the group of control Si RNA plus DMF to 60%, suggesting that mitigating the cell protection mediated by Nrf2 signaling can improve the efficacy of DMF to kill melanoma cells.Conclusion:The current study demonstrated that DMF has significant antitumor effect to melanoma cells, but in other hand, also triggers the cell-protection response mediated by Nrf2 signaling pathway and enhanced autophagy. Thus, DMF alone may work as a double-edged sword regarding to its potential as a new anti-cancer reagent. Targeting the cell protection mediated by Nrf2 signaling can enhance the efficacy of DMF to kill tumor cells, providing a concept of principle for developing strategies to take full advantages of DMF for cancer treatment.