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Epigenetic mechanisms involved in differential MDR1 mRNA expression between gastric and colon cancer cell lines and rationales for clinical chemotherapy.

Lee TB, Park JH, Min YD, Kim KJ, Choi CH - BMC Gastroenterol (2008)

Bottom Line: Methylation status was explored by quantification PCR-based methylation and bisulfite DNA sequencing analyses.These 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Research Center for Resistant Cells, Chosun University, Gwangju 501-759, Korea. tblee@chosun.ac.kr

ABSTRACT

Background: The 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.

Methods: The 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.

Results: The 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.

Conclusion: These 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.

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The quantification PCR-based methylation analysis of gastric and colon cancer cell lines. (A) CpG sites and Hpa II/Msp I sites in the human MDR1 promoter region. Top: The CpG sites are represented by the short vertical bars. The positions of exons 1 and 2 are indicated as closed boxes. The position corresponding to these fragments are indicated. Middle: Hpa II/Msp I recognition sites are represented by short vertical bars. Bottom, PCR primers used in methylation analysis. (B) Representative methylation status of the MDR1 promoter region by quantification PCR-based methylation analysis in SNU-5 (gastric) and HT-29 (colon). 1: MN, Never-methylated Hpa II/Msp I site at the triosephosphate isomerase gene promoter region (negative control) (240 bp); 2: MC, the positive control primer pair (240 bp); 3: MS1, Hpa II/Msp I site 1 (121 bp); 4: MS2, Hpa II/Msp I site 2 (206 bp).
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Figure 5: The quantification PCR-based methylation analysis of gastric and colon cancer cell lines. (A) CpG sites and Hpa II/Msp I sites in the human MDR1 promoter region. Top: The CpG sites are represented by the short vertical bars. The positions of exons 1 and 2 are indicated as closed boxes. The position corresponding to these fragments are indicated. Middle: Hpa II/Msp I recognition sites are represented by short vertical bars. Bottom, PCR primers used in methylation analysis. (B) Representative methylation status of the MDR1 promoter region by quantification PCR-based methylation analysis in SNU-5 (gastric) and HT-29 (colon). 1: MN, Never-methylated Hpa II/Msp I site at the triosephosphate isomerase gene promoter region (negative control) (240 bp); 2: MC, the positive control primer pair (240 bp); 3: MS1, Hpa II/Msp I site 1 (121 bp); 4: MS2, Hpa II/Msp I site 2 (206 bp).

Mentions: The methylation status was determined by quantification PCR-based methylation analysis for a CpG-rich domain to be approximately 1 Kb containing exon 1 and intron 1 among the MDR1 promoter. To determine the degree of methylation of the MDR1 gene promoter region, two primers (MS1 and MS2) containing the Msp I/Hpa II sites were designed from exon 1 and intron 1, respectively. The primer pair MC was used as a positive control to determine the quality of the source genomic DNA. In contrast, the MN that crosses the Msp I/Hpa II site at the triosephosphate isomerase gene promoter region and is never methylated was used as the negative control. Figure 5B shows typical quantification PCR-based methylation analysis images of the SNU-5 (gastric) and HT-29 (colon) cells. The quantification PCR-based methylation analysis revealed that any PCR products for the MS1 and MS2 were not produced from Pst1-digested genomic DNA treated with Msp I (methylation-insensitive enzyme). On the other hand, PCR products for both MS1 and MS2 in the SNU-5 cells but a PCR product for the MS1 alone in the HT-29 cells were obtained after Hpa II (methylation-sensitive enzyme) treatment, indicating methylation of CpG at the MS1 and MS2 sites in the SNU-5 cells and only at the MS2 site in the HT-29 cells. As summarized in Table 1, methylation was detected at the MS1 and MS2 sites of the 9 gastric cancer cell lines with the exception of SNU-484 but 2 (SNU-C5 and HCT-116) of the 9 colon cancer cell lines. On the other hand, the HT-29 cells were methylated only at the MS2 site. The SNU-C5, HT-29 (colon) and SNU-16 (gastric) cells not expressing MDR1 mRNA were methylated. Bisulfite DNA sequencing analysis was performed to confirm the methylation. As show in Table 1, methylation degree (%) of 10 CpG sites on the expended MS1 site is completely matched with results obtained by quantification PCR-based methylation analysis.


Epigenetic mechanisms involved in differential MDR1 mRNA expression between gastric and colon cancer cell lines and rationales for clinical chemotherapy.

Lee TB, Park JH, Min YD, Kim KJ, Choi CH - BMC Gastroenterol (2008)

The quantification PCR-based methylation analysis of gastric and colon cancer cell lines. (A) CpG sites and Hpa II/Msp I sites in the human MDR1 promoter region. Top: The CpG sites are represented by the short vertical bars. The positions of exons 1 and 2 are indicated as closed boxes. The position corresponding to these fragments are indicated. Middle: Hpa II/Msp I recognition sites are represented by short vertical bars. Bottom, PCR primers used in methylation analysis. (B) Representative methylation status of the MDR1 promoter region by quantification PCR-based methylation analysis in SNU-5 (gastric) and HT-29 (colon). 1: MN, Never-methylated Hpa II/Msp I site at the triosephosphate isomerase gene promoter region (negative control) (240 bp); 2: MC, the positive control primer pair (240 bp); 3: MS1, Hpa II/Msp I site 1 (121 bp); 4: MS2, Hpa II/Msp I site 2 (206 bp).
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Figure 5: The quantification PCR-based methylation analysis of gastric and colon cancer cell lines. (A) CpG sites and Hpa II/Msp I sites in the human MDR1 promoter region. Top: The CpG sites are represented by the short vertical bars. The positions of exons 1 and 2 are indicated as closed boxes. The position corresponding to these fragments are indicated. Middle: Hpa II/Msp I recognition sites are represented by short vertical bars. Bottom, PCR primers used in methylation analysis. (B) Representative methylation status of the MDR1 promoter region by quantification PCR-based methylation analysis in SNU-5 (gastric) and HT-29 (colon). 1: MN, Never-methylated Hpa II/Msp I site at the triosephosphate isomerase gene promoter region (negative control) (240 bp); 2: MC, the positive control primer pair (240 bp); 3: MS1, Hpa II/Msp I site 1 (121 bp); 4: MS2, Hpa II/Msp I site 2 (206 bp).
Mentions: The methylation status was determined by quantification PCR-based methylation analysis for a CpG-rich domain to be approximately 1 Kb containing exon 1 and intron 1 among the MDR1 promoter. To determine the degree of methylation of the MDR1 gene promoter region, two primers (MS1 and MS2) containing the Msp I/Hpa II sites were designed from exon 1 and intron 1, respectively. The primer pair MC was used as a positive control to determine the quality of the source genomic DNA. In contrast, the MN that crosses the Msp I/Hpa II site at the triosephosphate isomerase gene promoter region and is never methylated was used as the negative control. Figure 5B shows typical quantification PCR-based methylation analysis images of the SNU-5 (gastric) and HT-29 (colon) cells. The quantification PCR-based methylation analysis revealed that any PCR products for the MS1 and MS2 were not produced from Pst1-digested genomic DNA treated with Msp I (methylation-insensitive enzyme). On the other hand, PCR products for both MS1 and MS2 in the SNU-5 cells but a PCR product for the MS1 alone in the HT-29 cells were obtained after Hpa II (methylation-sensitive enzyme) treatment, indicating methylation of CpG at the MS1 and MS2 sites in the SNU-5 cells and only at the MS2 site in the HT-29 cells. As summarized in Table 1, methylation was detected at the MS1 and MS2 sites of the 9 gastric cancer cell lines with the exception of SNU-484 but 2 (SNU-C5 and HCT-116) of the 9 colon cancer cell lines. On the other hand, the HT-29 cells were methylated only at the MS2 site. The SNU-C5, HT-29 (colon) and SNU-16 (gastric) cells not expressing MDR1 mRNA were methylated. Bisulfite DNA sequencing analysis was performed to confirm the methylation. As show in Table 1, methylation degree (%) of 10 CpG sites on the expended MS1 site is completely matched with results obtained by quantification PCR-based methylation analysis.

Bottom Line: Methylation status was explored by quantification PCR-based methylation and bisulfite DNA sequencing analyses.These 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Research Center for Resistant Cells, Chosun University, Gwangju 501-759, Korea. tblee@chosun.ac.kr

ABSTRACT

Background: The 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.

Methods: The 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.

Results: The 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.

Conclusion: These 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.

Show MeSH
Related in: MedlinePlus