Alcohol induces cell proliferation via hypermethylation of ADHFE1 in colorectal cancer cells

Abstract

Background: The hypermethylation of Alcohol dehydrogenase iron containing 1 (ADHFE1) was recently reported to be associated with colorectal cancer (CRC) differentiation. However, the effect of alcohol on ADHFE1 hypermethylation in CRC is still unclear.

Methods: The methylation status and expression levels of ADHFE1 were investigated in primary tumor tissues and adjacent normal tissues of 73 patients with CRC, one normal colon cell line, and 4 CRC cell lines (HT-29, SW480, DLD-1, and LoVo) by quantitative methylation-specific polymerase chain reaction (QMSP) and real-time reverse transcription polymerase chain reaction (real time PCR), respectively. The effect of alcohol on the methylation status of ADHFE1 was analyzed in HT-29, SW480, DLD-1, and CCD18Co cells using QMSP, real-time PCR, immunoblot, and cell proliferation assay.

Results: ADHFE1 was hypermethylated in 69 of 73 CRC tissues (95%) compared to adjacent normal tissues (p<0.05). The mRNA expression of ADHFE1 was significantly reduced in CRC compared to adjacent normal tissues (p<0.05) and its expression was decreased in the alcohol consumption group (p<0.05). ADHFE1 was hypermethylated and its expression was decreased in 4 CRC cell lines compared with normal colon cell line. Alcohol induced hypermethylation of ADHFE1, decreased its expression, and stimulated cell proliferation of HT-29, SW480, and DLD-1cells.

Conclusion: These results demonstrate that the promoter hypermethylation of ADHFE1 is frequently present in CRC and alcohol induces methylation-mediated down expression of ADHFE1 and proliferation of CRC cells.

Figure 1

The methylation status and mRNA expression levels of ADHFE1 in CRC tissues and adjacent normal tissues. The methylation status of ADHFE1 in 73 CRC tissues and adjacent normal tissues is assessed using QMSP. A, C. ADHFE1 is hypermethylated in CRC (A) and also significantly hypermethylated in drinking groups and non-drinking groups (C). B. The expression levels of ADHFE1 in 73 CRC tissues compared with adjacent normal tissues was determined in 73 CRC tissues by real-time PCR. The mRNA expression of ADHFE1 is significantly decreased in CRC compared to adjacent normal tissues. D. The down regulation of ADHFE1 is presented in drinking groups and non-drinking groups. *,†p-Values of < 0.05 were considered statistically significant. AdjN: Adjacent normal tissue; T: Colorectal cancer tissues; PMR,: Percentage of methylated reference.

Figure 2

Changes of ADHFE1 methylation and expression by treatment with 5-aza-dC in CRC cells and normal colon cells. After treatment with 5-aza-dC in cells, the methylation status and expression levels of ADHFE1 are observed using QMSP, real-time PCR and immuno-blotting analysis. A. ADHFE1 is hypermethylated in 4 CRC cells compared to normal colon cells and significantly demethylated in 4 CRC cells by 5-aza-dC. B. The mRNA expression of ADHFE1 is reduced in 4 CRC cells but increased in all cells treated with 5-aza-dC. C. The protein expression of ADHFE1 is not affected by 5-aza-dC in CCD18Co. D. Protein expression of ADHFE1 is restored by 5-aza-dC in 4 CRC cells. Expression of ACTB and GAPDH is used as a loading control. *p-Values of < 0.05 were considered statistically significant. +: Treated with 5-aza-dC; −: Non-treated with 5-aza-dC; PMR: Percentage of methylated reference.

Figure 3

Changes in methylation and expression of ADHFE1 by ethanol treatment. The methylation and expression changes of ADHFE1 are determined in HT-29, SW480, and DLD-1 cells by treatments with ethanol using QMSP, real-time PCR and immunoblot analysis. A. The methylation status of ADHFE1 is significantly increased by ethanol treatment in 2 CRC cells. B. ADHFE1 expression is diminished by ethanol in 3 CRC cells. C. After treatment with ethanol in CRC cells, the protein expression of ADHFE1 is decreased in a concentration-dependent manner. Expression of GAPDH is used as a loading control. *p-Values of < 0.05 were considered statistically significant; PMR: Percentage of methylated reference.

Figure 4

The effect of ADHFE1 down regulation on cell viability and proliferation. The cell viability and proliferation of HT-29, SW480, and DLD-1 cells after ethanol treatment, transfection of ADHFE1 siRNA and combined treatment are determined by MTT, cell counting, and counter staining assay. A. Cell viability of 3 CRC cells is significantly increased by treatment with ethanol, siRNA, and combination of both. B. Cell proliferation of 3 CRC cells is significantly increased by ethanol, siRNA, and co-treatment. C. The captured images of cells using Hoechst 33342 show that the number of 3 CRC cells is increased by ethanol, siRNA, and co-treatment. D. The mRNA expression of ADHFE1 is significantly decreased in 3 CRC cells by treatment with ethanol, siRNA, and combination of both. E. ADHFE1 protein expression is decreased in 3 CRC cells by treatment with ethanol, siRNA, and combination of both. GAPDH was used as a loading control. *p-Values of < 0.05 were considered statistically significant. +: Treated with agent; −: Non-treated with agent.