INTERMITTENT FASTING-INDUCED AUTOPHAGY AND APOPTOSIS IN COLORECTAL CARCINOMA: THE ROLE OF BECLIN-1 AND BCL-2
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Abstract
Colorectal cancer (CRC) remains one of the leading causes of cancer-related mortality worldwide, primarily due to
uncontrolled cell proliferation and dysregulation of autophagy and apoptosis. The present study aimed to evaluate the
effect of Embinin under serum withdrawal (intermittent fasting conditions) on autophagy and apoptosis in HT-29 human
colorectal adenocarcinoma cells, with a focus on the expression of Beclin-1 and Bcl-2. Cells were cultured in Dulbecco’s
Modified Eagle Medium (DMEM) and subjected to serum deprivation for 6, 12, 18, and 24 hours, followed by Embinin
and 5-fluorouracil (5-FU) treatments. A series of analyses—MTT assay, flow cytometry-based apoptosis and cell cycle
assessment, quantitative PCR, western blotting, and immunofluorescence—were performed. Results from the MTT
assay revealed that Embinin significantly inhibited HT-29 cell proliferation in a dose- and time-dependent manner,
comparable to 5-FU at 6 h and 24 h of treatment. Annexin V-FITC/PI staining confirmed that Embinin induced both
early and late apoptosis, with higher apoptotic indices at 12.5 µM concentration. Cell cycle analysis indicated an increase
in the sub-G1 phase population, suggesting apoptosis-mediated cell death, accompanied by a G2/M phase arrest. qPCR
analysis demonstrated upregulation of Beclin-1 and downregulation of Bcl-2 expression, suggesting activation of
autophagy and suppression of anti-apoptotic mechanisms. Collectively, the findings indicate that Embinin enhances cell
death through coordinated modulation of autophagy and apoptosis pathways, particularly under nutrient-deprived
(fasting) conditions, highlighting its potential as a novel adjuvant in CRC therapy
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