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. 2022 Jul 19:2022:5361241.
doi: 10.1155/2022/5361241. eCollection 2022.

Vitamin C Sensitizes Pancreatic Cancer Cells to Erastin-Induced Ferroptosis by Activating the AMPK/Nrf2/HMOX1 Pathway

Yawen Liu  1 , Pu Huang  2   3 , Zheng Li  1 , Chunhui Xu  1 , Huizhi Wang  1 , Baoqing Jia  3 , Aihua Gong  4 , Min Xu  1
Affiliations

Vitamin C Sensitizes Pancreatic Cancer Cells to Erastin-Induced Ferroptosis by Activating the AMPK/Nrf2/HMOX1 Pathway

Yawen Liu et al. Oxid Med Cell Longev. .

Abstract

Ferroptosis is a type of regulated cell death that displays a promising therapeutic pathway for drug-resistant tumor cells. However, some pancreatic cancer (PC) cells are less sensitive to erastin-induced ferroptosis, and normal pancreatic cells are susceptible to this newly discovered cell death. Therefore, there is an urgent need to find drugs to enhance the sensitivity of these PC cells to erastin while limiting side effects. Here, we found that the oxidized form of vitamin C-dehydroascorbic acid (DHA) can be transported into PC cells expressing high levels of GLUT1, resulting in ferroptosis. Moreover, pharmacological vitamin C combined with erastin can synergistically induce ferroptosis of PC cells involving glutathione (GSH) reduction and ferrous iron accumulation while inhibiting the cytotoxicity of normal cells. Mechanistically, as a direct system Xc- inhibitor, erastin can directly suppress the synthesis of GSH, and the recycling of vitamin C and DHA is performed through GSH consumption, which is denoted as the classical mode. Furthermore, oxidative stress induced by erastin and vitamin C could enhance the expression of HMOX1 via the AMP-activated protein kinase (AMPK)/nuclear factor erythroid 2-related factor 2 (NRF2) pathway to increase the labile iron level, which is named the nonclassical mode. In vivo experiments showed that erastin and vitamin C can significantly slow tumor growth in PC xenografts. In summary, the combination of erastin and vitamin C exerts a synergistic effect of classical and nonclassical modes to induce ferroptosis in PC cells, which may provide a promising therapeutic strategy for PC.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Vitamin C selectively induces ferroptosis in pancreatic cancer cells. (a) Cell viability was assessed by CCK-8 assay after treatment with various concentrations of vitamin C. (b) Cell viability was detected by CCK-8 assay after treatment with vitamin C with or without different cell death inhibitors. (c) GSH levels were measured in BxPC3 cells treated with vitamin C in the presence or absence of DFO. (d–f) Flow cytometry was performed to detect lipid ROS levels, and the quantification of fluorescence intensity is shown.
Figure 2
Figure 2
Vitamin C selectively kills pancreatic cancer cells via GLUT1. (a) TCGA database analysis showed that GLUT1 expression was higher in PC tumor tissues than in normal tissues. (b) The Kaplan–Meier survival curves indicated a significant correlation between high GLUT1 expression and low overall survival of PC patients (P = 0.016). (c, d) The relative expression of GLUT1 mRNA and protein in H6C7 cells, MEFs, and different PC cell lines. (e) Cell viability was detected by CCK-8 assay after treatment with vitamin C and/or STF-31. (f) Cell viability was detected in GLUT1-downregulated PaTu8988 and BxPC3 cells treated with vitamin C or not (∗∗P < 0.01, ∗∗∗P < 0.001).
Figure 3
Figure 3
The synergistic effect after combination treatment with vitamin C and erastin. (a–d) PaTu8988 and BxPC3 cells were treated with a mono- or combination of erastin and vitamin C for 24 h. Cell viability (a), GSH level (b), and lipid ROS levels (c, d) were analyzed (∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001).
Figure 4
Figure 4
Vitamin C protected MEFs and H6C7 cells from erastin-induced ferroptosis. (a) Cell viability was assessed by CCK-8 assay after treatment with various concentrations of erastin. (b, c) H6C7 and MEF cells were treated with erastin, vitamin C, or a combination of both for 24 h. Cell viability (b) and lipid ROS levels (c) were analyzed (∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001).
Figure 5
Figure 5
Erastin and vitamin C increase the intracellular ferrous iron level. (a, b) The intracellular ferrous iron level was detected by an iron assay kit in PaTu8988 and BxPC3 cell lines under treatment with erastin and vitamin C. (c, d) Flow cytometry was performed to measure ferrous iron levels, and the quantification of fluorescence intensity is shown (∗P < 0.05, ∗∗∗P < 0.001). (e, f) Heat map and volcano map analyses showed differentially expressed genes in response to erastin and vitamin C treatment measured by RNA-seq, respectively. Blue: low expression levels. Red: high expression levels.
Figure 6
Figure 6
Erastin and vitamin C trigger ferroptosis by regulating the AMPK/NRF2/HMOX1 pathway. (a, b) The mRNA levels of CP, HMOX1, and FTH1 were analyzed in Patu8988 and BxPC3 cell lines treated with erastin and/or vitamin C. (c, d) Ferroptosis and iron metabolism-related proteins were detected by Western blot analysis. (e, f) Western blotting analysis of the nuclear/cytoplasmic fraction showed NRF2 nuclear localization in Patu8988 and BxPC3 cells after treatment with erastin and/or vitamin C (∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001).
Figure 7
Figure 7
Vitamin C enhances erastin-induced ferroptosis in pancreatic cancer cells in vivo. (a, b) Images and weights of xenograft tumors from various mouse groups after 14 days of treatment. (c, d) The tumor volume and mouse body weight were measured every two days. (e–g) Combination treatment with erastin and vitamin C reduced GSH (e) and increased the MDA (e) and ferrous iron levels (g). (h) A schematic showing that vitamin C sensitizes PC cells to erastin-induced ferroptosis via GSH depletion and ferrous iron overload (∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001).
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