Mechanistic insights into proteasome inhibitor MG132 induced apoptosis in melanoma A375 cells

Abstract

Despite advancements in melanoma therapy, the prognosis remains unfavorable for many patients. The proteasome inhibitor MG132 has shown therapeutic potential through pathway regulation, yet its precise mechanisms in melanoma require systematic elucidation. Using A375 melanoma cells, we conducted multi-modal investigations combining cytotoxicity assessment (CCK8), migration analysis (wound healing), apoptosis quantification (flow cytometry), and proteomic profiling (western blot) to dissect MG132's molecular mechanisms. Our findings revealed MG132's potent anti-tumor activity with an IC₅₀ of 1.258 ± 0.06 µM, significantly suppressing cellular migration at therapeutic concentrations. Apoptosis assays demonstrated concentration-dependent effects, with 2 µM treatment inducing early apoptosis in 46.5% and total apoptotic response in 85.5% of cells within 24 h. Mechanistic studies uncovered MG132's dual regulatory capacity: (1) Through MDM2 inhibition, it activated p53/p21/caspase-3 axis while suppressing CDK2/Bcl2, triggering cell cycle arrest and DNA damage cascades; (2) MAPK pathway activation emerged as a critical apoptosis driver. Notably, western blot analysis established dose-responsive modulation of these molecular targets, confirming pathway specificity. Our results position MG132 as a multi-target agent capable of simultaneously disrupting proliferative signaling and activating apoptotic machinery. The observed MAPK-mediated apoptosis mechanism provides novel insights for melanoma therapeutics, suggesting that combinatorial targeting of proteasomal and MAPK pathways may enhance treatment efficacy.

Keywords: Antitumor; Apoptosis effects; MG132; Melanoma.

Fig. 1

Unexpected discovery that MG132 inhibits the growth of cells. (A) MG132 chemical structure. (B) Influence of different concentrations of MG132 on A375, A549, MCF-7 and Hela cells (48 h). (C) Influence of different processing times of MG132 on A375. The results represent the average of data from three independent experiments and were expressed as mean ± standard error of the mean (^nsP>0.05, ****P < 0.0001).

Fig. 2

Wound healing assay to investigate the ability of MG132 to inhibit A375 cell migration. (A) MG132 (0.125 µM, 0.25 µM and 0.5 µM) inhibited the migration and invasion of A375 cells, the area in the yellow box indicates the empty area. (B) Empty areas were analyzed using image J. The results represent the average of data from three independent experiments and were expressed as mean ± standard error of the mean. The control group was cultured with DMSO treatment. (^nsP>0.05, **P<0.01)

Fig. 3

Apoptosis of A375 cells after treatment with MG132 (0.5 µM, 1 µM and 2 µM) was analyzed by flow cytometry assay. The control group was cultured with DMSO treatment.

Fig. 4

Analysis of A375 cell cycle changes after treatment with MG132 (0.5 µM, 1 µM and 2 µM) by flow cytometry assay. (A) The graphs show the representative cell cycle distribution of propidium-iodide stained cells assessed by flow cytometry. (B) Quantification of cells at various stages of the cell cycle. The control group was cultured with DMSO treatment.

Fig. 5

Western-blot experiment to investigate the anti-melanoma mechanism of MG132. (A) MG132 (0.5 µM, 1 µM and 2 µM) affects the expression of apoptosis-related proteins, cell cycle-related proteins and MAPKs in A375 cells. (B–D) Quantitative analysis of Apoptosis-related proteins, Cell cycle-related proteins and MAPK signaling pathway.