Abstract
BackgroundThe membrane transporters such as P-glycoprotein (Pgp), the MDR1 gene product, are one of causes of treatment failure in cancer patients. In this study, the epigenetic mechanisms involved in differential MDR1 mRNA expression were compared between 10 gastric and 9 colon cancer cell lines.MethodsThe MDR1 mRNA levels were determined using PCR and real-time PCR assays after reverse transcription. Cytotoxicity was performed using the MTT assay. Methylation status was explored by quantification PCR-based methylation and bisulfite DNA sequencing analyses.ResultsThe MDR1 mRNA levels obtained by 35 cycles of RT-PCR in gastric cancer cells were just comparable to those obtained by 22 cycles of RT-PCR in colon cancer cells. Real-time RT-PCR analysis revealed that MDR1 mRNA was not detected in the 10 gastric cancer cell lines but variable MDR1 mRNA levels in 7 of 9 colon cancer cell lines except the SNU-C5 and HT-29 cells. MTT assay showed that Pgp inhibitors such as cyclosporine A, verapamil and PSC833 sensitized Colo320HSR (colon, highest MDR1 expression) but not SNU-668 (gastric, highest) and SNU-C5 (gastric, no expression) to paclitaxel. Quantification PCR-based methylation analysis revealed that 90% of gastric cancer cells, and 33% of colon cancer cells were methylated, which were completely matched with the results obtained by bisulfite DNA sequencing analysis. 5-aza-2'-deoxcytidine (5AC, a DNA methyltransferase inhibitor) increased the MDR1 mRNA levels in 60% of gastric cells, and in 11% of colon cancer cells. Trichostatin A (TSA, histone deacetylase inhibitor) increased the MDR1 mRNA levels in 70% of gastric cancer cells and 55% of colon cancer cells. The combined treatment of 5AC with TSA increased the MDR1 mRNA levels additively in 20% of gastric cancer cells, but synergistically in 40% of gastric and 11% of colon cancer cells.ConclusionThese results indicate that the MDR1 mRNA levels in gastric cancer cells are significantly lower than those in colon cancer cells, which is at least in part due to different epigenetic regulations such as DNA methylation and/or histone deacetylation. These results can provide a better understanding of the efficacy of combined chemotherapy as well as their oral bioavailability.
Highlights
The membrane transporters such as P-glycoprotein (Pgp), the multidrug resistance 1 (MDR1) gene product, are one of causes of treatment failure in cancer patients
The MDR1 mRNA was not detected after 22 cycles of PCR in the 10 gastric cancer cell lines but could be detected at variable levels after 35 cycles of PCR with the exception of SNU-16, suggesting a significantly low level of MDR1 mRNA expression in the gastric cancer cells tested (Figure 1)
Of the 9 colon cancer cell lines, variable MDR1 mRNA levels could be detected in 7 colon cancer cell lines after 22 cycles of PCR but not in the SNU-C5 and HT-29 cells
Summary
The membrane transporters such as P-glycoprotein (Pgp), the MDR1 gene product, are one of causes of treatment failure in cancer patients. The epigenetic mechanisms involved in differential MDR1 mRNA expression were compared between 10 gastric and 9 colon cancer cell lines. The relationships between the transcriptional expression of MDR1 gene expression and epigenetic mechanisms in gastric and colon cancer cells have not been compared. It is unclear why chemotherapy regimens have been differently used to treat gastric and colorectal cancers and why MDR1 mRNA is expressed differentially in gastric and colorectal cancer cells. This study examined whether or not the degree of methylation at the promoter site of the MDR1 gene is closely associated with MDR1 gene expression in both cancer cells
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