High-dose vitamin C promotes mitochondrial biogenesis in HCT116 colorectal cancer cells by regulating the AMPK/PGC-1α signaling pathway

Abstract

Background: Mitochondrial dysfunction is closely associated with cancer development. Colorectal cancer (CRC) cells often exhibit altered energy metabolism, characterized by increased glycolysis and reduced oxidative phosphorylation. Enhancing mitochondrial biogenesis and function may represent a promising therapeutic approach. High-dose vitamin C has demonstrated anti-tumor properties and the ability to reverse the Warburg effect, but its role in regulating mitochondrial biogenesis and function remains unclear.

Methods: We evaluated the altered mitochondrial functional status of HCT116 colorectal cancer cells compared to FHC colorectal epithelial cells, assessed the effects of high-dose vitamin C on mitochondrial biogenesis and function in HCT116 cells, and explored the underlying regulatory mechanisms.

Results: HCT116 cells exhibited mitochondrial dysfunction compared to FHC cells, including decreased expression of electron transport chain complexes III and IV, reduced TFAM levels, and lower mtDNA content. Vitamin C treatment significantly enhanced mitochondrial biogenesis and function, as reflected by increased AMPK phosphorylation, upregulation of PGC-1α, SOD2, NRF2, TFAM, MT-CYB, and MTCO1, elevated mtDNA content, restored membrane potential, enhanced oxidative phosphorylation, and reduced glycolytic activity. Furthermore, vitamin C markedly suppressed HCT116 cell viability and clonogenic capacity, while these effects were substantially diminished by cotreatment with Compound C.

Conclusion: This study demonstrates that high-dose vitamin C ameliorates mitochondrial dysfunction and promotes mitochondrial biogenesis and function in colorectal cancer cells through activation of the AMPK-PGC-1α signaling pathway, thereby suppressing tumor cell proliferation. These findings suggest that vitamin C may serve as a promising therapeutic agent for targeting mitochondrial metabolism in colorectal cancer.

Keywords: AMPK; Colorectal cancer; Mitochondrial biogenesis; PGC-1α; Vitamin C.

Fig. 1

HCT116 cells have reduced mitochondrial function and biogenesis and increased mitochondrial damage compared to FHC cells. A wb bands of MT-CYB, MTCO1 and TFAM in FHC and HCT116, quantitative analysis of protein levels normalized to β-Actin (mean ± SD).; B Protein levels and mRNA levels of TFAM; C Mitochondrial-to-nuclear DNA (mtDNA/nDNA) ratio; D Representative transmission electron microscopy images of mitochondria in FHC and HCT116 with Scale bars of 2 μm, 1 μm (magnification, × 4,000, × 15,000); E Quantification of mitochondrial mean length, number, presented as mean ± SEM based on observations from three cells

Fig. 2

VC up-regulated mitochondrial biogenesis related genes (A) mRNA levels and WB bands of PGC-1α with increasing concentrations of VC; B WB bands of NRF2 and TFAM; C mRNA levels of NRF2 and TFAM; D mtDNA/nDNA ratio; E Representative transmission electron microscopy images of mitochondria from HCT116 treated with or without VC. Scale bars 2 μm, 1 μm (magnification, × 4,000, × 15,000); F Quantitative analysis of mitochondrial parameters, including mean length, count, proportion of damaged mitochondria, and mitochondria-containing autophagosomes, presented as mean ± SEM from three individual cells

Fig. 3

Vitamin C Activates AMPK Signaling to Enhance Mitochondrial Biogenesis. H = Blank Control; VC = 5 mM Vitamin C; CC = 5 μM Compound C; CCV = 5 mM Vitamin C + 5 μM Compound C. A WB bands of Phosphorylated AMPK, AMPK, PGC-1α, NRF2 and SOD2. The p-AMPK/t-AMPK ratios were quantified, and normalized protein levels relative to β-Actin are presented as mean ± SD; B mRNA levels of PGC-1α, NRF2 and TFAM and SOD2; C Relative Value of mtDNA/nDNA; D Mitochondrial oxidative phosphorylation related protein expression, semi-quantified and normalized to GAPDH, are shown as mean ± S

Fig. 4

AMPK-mediated effects of VC on mitochondrial energy metabolism and function in HCT116. H = Blank Control; VC = 5 mM VC; CC = 5 μM Compound C; CCV = 5 mM VC + 5 μM Compound C. A Oxygen consumption rate (OCR) displayed as mean ± SEM from three independent measurements. B Extracellular acidification rate (ECAR). C Cellular ATP content. D JC-1 staining (40 ×) to evaluate mitochondrial membrane potential, with the red/green fluorescence ratio representing ΔΨm. Results are shown as mean ± SEM from three independent experiments. E mPTP opening assay (40 ×), with green fluorescence intensity indicating mPTP activation. F Cell viability was measured by MTS assay. G Colony formation ability was assessed by clonogenic assay. Data are presented as mean ± SD from at least three independent experiments

Fig. 5

The schematic representation of the AMPK-PGC-1α-NRF2-TFAM pathway activation by high-dose vitamin C