Investigating the Impact of Curcumin on the Methylation of DNMT1, CDH1, SMG1, and WT1 Genes in the MIAPaCa2 Cell Line Using High-Resolution Melting Analysis Compared to Methylation-Specific PCR.

BACKGROUND

Curcumin, a natural compound extracted from turmeric, has shown potential in modulating epigenetic mechanisms, including DNA methylation, which is critical in gene regulation and cancer progression. This study investigates the impact of curcumin on the methylation of DNMT1, CDH1, SMG1, and WT1 genes in the MIAPaCa2 pancreatic cancer cell line.

METHODS

MIAPaCa-2 pancreatic cancer cells were cultured and treated with varying concentrations of curcumin (2.5, 10, 20, 40, and 80 μM) for 24, 48, and 72 hours. DNA was extracted and subjected to bisulfite conversion to analyze methylation. The methylation status of DNMT1, CDH1, SMG1, and WT1 promoters was assessed using methylation-specific PCR (MSP) and methylation-sensitive high-resolution melting (MS-HRM) analysis. MSP involved amplifying methylated and unmethylated alleles, while MS-HRM provided quantitative methylation analysis. Standard curves and controls were used to ensure accuracy and validate the results.

RESULTS

MSP analysis revealed that DNMT1, initially hemi-methylated in control cells, exhibited decreased methylation levels across all concentrations of curcumin (2.5 to 80 μM), whereas CDH1 remained consistently unmethylated before and after treatment. MS-HRM employed a standard curve method to quantify methylation, showing that DNMT1 methylation decreased from approximately 50% in control cells to about 20% after exposure to 80 μM curcumin. Meanwhile, CDH1 maintained its unmethylated state throughout. The methylation status of SMG1 was inconclusive in this study, while WT1 initially showed 70% unmethylation, reducing after curcumin treatment.

CONCLUSION

These findings underscore the differential effects of curcumin on DNA methylation patterns in pancreatic cancer-related genes, highlighting its potential as a modulator of epigenetic mechanisms in cancer therapy.