Decreased proliferation of HepG2 liver cancer cells in vitro and exhibited proteomic changes in vivo in subjects with metabolic syndrome and metabolic dysfunction-associated steatotic liver disease who performed four-week dawn-to-dusk dry fasting.

Abstract

Background: Four-week dawn-to-dusk dry fasting (DDDF) was previously shown to have a potent anti-inflammatory effect and induce an anti-tumorigenic proteome in the serum and peripheral blood mononuclear cells in subjects without cancer. The study goal was to determine if serum obtained from these subjects without cancer who underwent 4-week DDDF has an anti-tumorigenic effect.

Methods: HepG2 cells were treated with serum collected from four individuals with metabolic syndrome and metabolic dysfunction-associated steatotic liver disease (MASLD) and four healthy individuals who performed 4-week DDDF. The objective was to assess cell proliferation/viability in HepG2 cells treated with non-fasted and dry-fasted serum and determine proteomic changes in human serum. We comparatively performed 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) cell proliferation assay and untargeted proteomic analysis using nano ultra-high performance liquid chromatography coupled with tandem mass spectrometry.

Results: Serum collected from 3 out of 4 subjects with metabolic syndrome and MASLD at the end of 4-week DDDF (dry-fasted serum/V2) significantly reduced proliferation/viability in HepG2 cells compared with the serum collected before 4-week DDDF (non-fasted serum/V1). A similar reduction effect on cell proliferation was not observed when HepG2 cells were treated with dry-fasted serum collected from healthy subjects. In addition to the in vitro changes observed, the following circulating gene protein products (GP) demonstrated significant increases or decreases in subjects with metabolic syndrome and MASLD after a 4-week DDDF regimen, compared with their GP levels before the 4-week DDDF: CD248 molecule (mean log2 fold = 8.124, P = 0.001), dipeptidyl peptidase 4 (mean log2 fold = 0.937, P = 0.027), lymphatic vessel endothelial hyaluronan receptor 1 (mean log2 fold = 1.054, P = 0.029), LDL receptor related protein 1 (mean log2 fold = 1.401, P = 0.031), and beta-2-microglobulin (mean log2 fold= -0.977, P = 0.033) at the end of 4-week DDDF compared with the GP levels before 4-week DDDF.

Conclusion: This study demonstrated that dry-fasted serum collected from subjects with metabolic syndrome and MASLD decreased HepG2 cell proliferation in vitro and showed that proteomic changes occurred in vivo. These findings suggest that DDDF may be an effective intervention for inducing proteomic responses that could assist in the prevention and adjunct treatment of cancers associated with metabolic syndrome.