Azithromycin’s Cytotoxic Effects and CASP8 Expression Modulation in HT-29 Colorectal Cancer Cells In Vitro

Document Type : Original Article

Authors

Department of Biology, Ra.C., Islamic Azad University, Rasht, Iran.

Abstract

Colorectal cancer has been one major cause of cancer deaths all around the world, thus demanding some new therapeutic approaches to face the problems posed by resistance and side effects in the present conventional treatments. In this study, azithromycin was explored to determine whether it could be repurposed—by interfering with cytotoxicity in HT-29 CRC cells—and to examine CASP8 gene expression, an important signal-transducing actor in extrinsic apoptotic pathways. The cells were cultured in DMEM, supplemented with FBS and antibiotics, and treated with azithromycin concentrations ranging from 10 to 1000 μg/ml. Post-treatment at 24 hours, cytotoxicity was studied by MTT assays, while CASP8 was examined through qRT-PCR employing GAPDH as the reference gene. Statistical analyses included one-way ANOVA followed by Dunnett post-hoc test, ROC curve plotting for viability, and unpaired t-test for gene expression. Dose-dependent cytotoxicity from azithromycin was observed with cell viability declining from 100% in controls to approximately 2-3% in concentrations ≥100 μg/ml (P < 0.0001), which was so much so that it plateaued with increasing concentration, with a perfect discriminatory power level (ROC AUC = 1.000). Transcript levels of CASP8 revealed no significant difference between groups (treated versus controls) with a P-value of 0.5106. These results indicate an azithromycin potential for antiproliferative effects on CRC, potentially through CASP8-independent mechanisms such as the inhibition of autophagy; however, mRNA data alone are insufficient to confirm pathway involvement, justifying further in vivo, mechanistic, and protein-level investigations for its repositioning as an adjunctive treatment.

Keywords

Main Subjects

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Journal of Epigenetics
Volume 6, Issue 2
November 2025
Pages 53-62

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History

  • Receive Date: 10 October 2025
  • Revise Date: 06 November 2025
  • Accept Date: 12 November 2025

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