The effect of high dose ascorbic acid on necroptosis pathway activation in HT29 colon cancer cell line

Dayer, Dian; Shokati Eshkiki, Zahra; Abbasi Samie, Kosar

doi:10.22118/jsmj.2026.544167.3978

The effect of high dose ascorbic acid on necroptosis pathway activation in HT29 colon cancer cell line

Document Type : Original Article

Authors

Dian Dayer

zahra shokati Eshkiki

Kosar Abbasi Samie

Cellular and Molecular Research Center,, Medical Basic Sciences Institute, Ahvaz Jundishapur University of Medical Sciences

10.22118/jsmj.2026.544167.3978

Abstract

Objective: The effect of high dosages of ascorbic acid on the expression of major necroptosis pathway genes in HT-29 colon cancer cells was studied. Materials and methods: HT-29 cells were cultured in DMEM-HG medium containing 10% FBS and 1% Pen/Strep and incubated at 37°C with 5% CO2. After reaching 90% density, the cells were trypsinised and cultured in triplicate in 12-well culture plates at 30,000 cells per well. The cells were divided into two groups: a control group that received only basal culture medium and a test group that received 5 mM ascorbic acid for 3 hours. After 24 hours, the cell supernatant and the cell pellet were collected for further analysis. LDH activity was assessed using the kinetic technique, and the proportion of apoptosis and necrosis was calculated using flow cytometry. Gene expression analysis was performed using real-time PCR. Statistical analysis was performed using GraphPad Prism software version 8 and a t-test. Results: Microscopic images and flow cytometry results confirmed the induction of necroptosis by ascorbic acid. In addition, ascorbic acid treatment significantly increased key necroptosis gene expression and LDH activity. Conclusion: High dosages of ascorbic acid activate necroptosis in HT-29 cells.

Keywords

Ascorbic acid

necroptosis

colon cancer

HT29 cell line

Subjects