p53 enhances elesclomol-Cu-induced cuproptosis in hepatocellular carcinoma via FDXR-mediated FDX1 upregulation

Abstract

Background: Cuproptosis, a novel cell death pathway mediated by ferredoxin 1 (FDX1) and protein lipoylation, has emerged as a valuable target in cancer therapy. Although the findings of previous research have indicated a potential correlation between p53 and cuproptosis, the precise role and underlying mechanisms of p53 in cuproptosis, particularly within the context of hepatocellular carcinoma (HCC), remain unclear.

Methods: To evaluate cuproptosis, three HCC cell lines (HepG2, PLC/PRF/5, and Hep3B2.1-7) with distinct p53 statuses were treated with elesclomol-Cu. p53 overexpression/knockdown, siRNA-mediated ferredoxin reductase (FDXR)/FDX1 knockdown, and the p53 activators CP-31398 and nutlin-3 were employed to elucidate the associated molecular mechanisms. Cell viability, protein expression [FDX1, dihydrolipoyl transacetylase (DLAT), FDXR], and DLAT oligomerization were assessed via Cell Counting Kit-8 (CCK-8), western blotting, and immunofluorescence analyses. A PLC/PRF/5 xenograft mouse model was used to assess combined the therapeutic efficacy of elesclomol-Cu and CP-31398.

Results: Elesclomol-Cu triggered cuproptosis in HCC cells, as evidenced by a dose-dependent suppression of proliferation, FDX1 upregulation, DLAT oligomerization, and rescue by the copper chelator tetrathiomolybdate (TTM). p53 activation enhanced FDXR expression, promoting FDX1 upregulation and subsequent DLAT oligomerization, thereby sensitizing HCC cells to elesclomol-Cu, whereas FDXR knockdown reversed these effects, demonstrating its role in p53-mediated potentiation of cuproptosis sensitivity. In mutant p53-R249S cells, CP-31398 functioned synergistically with elesclomol-Cu to suppress proliferation. In vivo, elesclomol-Cu and CP-31398 combination therapy significantly reduced tumor growth and Ki67 expression whilst upregulating FDXR levels.

Conclusions: These findings revealed that p53 enhances elesclomol-Cu-induced cuproptosis in HCC via FDXR-mediated FDX1 upregulation. This study provides mechanistic insights into p53's role in cuproptosis and may serve as a basis for targeting copper metabolism in therapeutic strategies for HCC.

Keywords: cuproptosis; dihydrolipoyl transacetylase; elesclomol-Cu; ferredoxin 1; ferredoxin reductase; hepatocellular carcinoma; p53.

Figure 1

Elesclomol-Cu-induced cell death is distinct compared to various recognized forms of cell death. (A) Viability of HepG2 cells after pulse treated with various concentrations of elesclomol (ES) and 10 µM CuCl₂ for 2 h and then cultured for an additional 48 h (B) Viability of HepG2 cells pretreated with ferrostatin-1 (10 μM), Z-VAD-FMK (30 μM), necrostatin-1 (20 μM), NAC (1 mM), TTM (20 μM) overnight and then pulse treated with ES (40 nM)-CuCl₂ (10 μM). Control cells were treated with ES (40 nM) + CuCl₂ (10 μM) alone. (C, D) Viability of PLC/PRF/5 (C) and Hep3B2.1-7 (D) cells pretreated with or without TTM, followed by pulse treatment with ES-Cu ((PLC/PRF/5: 40 nM ES + 10 μM CuCl₂; Hep3B2.1-7: 30 nM ES + 10 μM CuCl₂) for 2 h and then cultured for an additional 48 h. *** p < 0.001; **** p < 0.0001.

Figure 2

ES-Cu treatment upregulates FDX1 expression and promotes DLAT oligomerization. (A-F) Western blot analysis of FDX1 in HepG2, Hep3B2.1–7 and PLC/PRF/5 cells 24 h after 2h-pulse-treatment with ES-Cu. (G-L) Protein oligomerization was analyzed by western blot (G-I) and immunofluorescence imaging (J-L, DLAT-green, MitoTracker-red, DAPI-blue) in HepG2, Hep3B2.1–7 and PLC/PRF/5 cells 24 h after 2h-pulse-treatment with ES-Cu. (M, N) Protein oligomerization analyzed by immunofluorescence imaging (N) in FDX1 knockdown PLC/PRF/5 cells. (O) Viability of FDX1-knockdown PLC/PRF/5 cells after pulse treated with ES-Cu. * p < 0.05; ** p < 0.01; **** p < 0.0001.

Figure 3

p53 upregulates FDX1 expression and enhances cuproptosis in HCC cells. (A-F) Western blot analysis of FDX1, FDXR and DLAT in p53-overexpressing HepG2 and Hep3B2.1–7 cells 24 h after 2h-pulse-treatment with ES-Cu. (G, H) Western blot analysis of FDX1 in p53-knockdown HepG2 cells with ES-Cu treatment. (I) Viability of p53-knockdown HepG2 cells 24 h following 2h-pulse-treatment with ES-Cu. (J, K) Viability of p53-overexpressing HepG2 (J) and Hep3B2.1–7 cells (K) after pulse treatment with ES-Cu. (L, M) Protein oligomerization was analyzed by immunofluorescence imaging in p53-overexpressing HepG2 (L) and Hep3B2.1–7 cells (M). (N, O) Western blot analysis of FDX1 treated with ES-Cu, CP-31398 or a combination of both compounds in PLC/PRF/5 cells. (P) Viability of PLC/PRF/5 cells following exposure to ES-Cu, CP-31398 or a combination of both compounds. (Q) Protein oligomerization was analyzed by immunofluorescence imaging. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Figure 4

p53 targets FDXR to enhance cuproptosis in HCC cells. (A, B) Western blot analysis of FDXR in p53-overexpressing HepG2 cells 24 h after 2h-pulse-treatment with ES-Cu. (C-F) Western blot analysis of FDX1 in FDXR-knockdown HepG2 cells with or without ES-Cu treatment. (G, H) Western blot analysis of FDX1 in FDXR-knockdown HepG2 cells with nutlin-3 (10 μM) treatment. (I) Viability of FDXR-knockdown HepG2 cells 24 h following 2h-pulse-treatment with ES-Cu. (J) Protein oligomerization was analyzed by immunofluorescence imaging in FDXR-knockdown HepG2 cells. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Figure 5

Combination of p53 activator CP-31398 and elesclomol-Cu significantly suppresses tumor growth. (A) Gross images of tumors at the end (14 days after treatment). (B) The weight of the resected tumors was measured in the control, ES-Cu, and ES-Cu+CP-31398 groups. (C) Graph of tumor volume changes in the control, ES-Cu, and ES-Cu+CP-31398 groups. (D) Tumor volume fold changes in mice treated with the indicated regimens. Each bar represents an individual mouse (n=5 per group). Calculation formula: fold change = ((V1-V2)/V2) * 100% (V1: tumor volume at endpoint; V2: tumor volume measured on the initial day of treatment). (E) Tumor samples were stained immunohistochemically for Ki67 and FDXR. ** p < 0.01; **** p < 0.0001.