Global DNA hypermethylation-associated cancer chemotherapy resistance and its reversion with the demethylating agent hydralazine

Abstract

Background: The development of resistance to cytotoxic chemotherapy continues to be a major obstacle for successful anticancer therapy. It has been shown that cells exposed to toxic concentrations of commonly used cancer chemotherapy agents develop DNA hypermethylation. Hence, demethylating agents could play a role in overcoming drug resistance.

Methods: MCF-7 cells were rendered adriamycin-resistant by weekly treatment with adriamycin. Wild-type and the resulting MCF-7/Adr cells were analyzed for global DNA methylation. DNA methyltransferase activity and DNA methyltransferase (dnmt) gene expression were also determined. MCF-7/Adr cells were then subjected to antisense targeting of dnmt1, -3a, and -b genes and to treatment with the DNA methylation inhibitor hydralazine to investigate whether DNA demethylation restores sensitivity to adriamycin.

Results: MCF-7/Adr cells exhibited the multi-drug resistant phenotype as demonstrated by adriamycin resistance, mdr1 gene over-expression, decreased intracellular accumulation of adriamycin, and cross-resistance to paclitaxel. The mdr phenotype was accompanied by global DNA hypermethylation, over-expression of dnmt genes, and increased DNA methyltransferase activity as compared with wild-type MCF-7 cells. DNA demethylation through antisense targeting of dnmts or hydralazine restored adriamycin sensitivity of MCF-7/Adr cells to a greater extent than verapamil, a known inhibitor of mdr protein, suggesting that DNA demethylation interferes with the epigenetic reprogramming that participates in the drug-resistant phenotype.

Conclusion: We provide evidence that DNA hypermethylation is at least partly responsible for development of the multidrug-resistant phenotype in the MCF-7/Adr model and that hydralazine, a known DNA demethylating agent, can revert the resistant phenotype.

Figure 1

MCF-7/Adr cells show the mdr phenotype. a: Wild-type MCF-7 cells exposed to IC90 of adriamycin show expected growth inhibition, whereas MCF-7/Adr cells are resistant to adriamycin as evaluated by the MTT assay; b: RT-PCR analysis of the mdr1 gene demonstrates that MCF-7/Adr cells over-express the mdr1 transcript; c: Flow cytometric analysis of adriamycin-treated cells for 1 h show that wild-type MCF-7 cells retain the majority of adriamycin inside the cell (M1); however, MCF-7/Adr cells have essentially no drug accumulation, indicative of the presence of a functional mdr1 protein, and d: MCF-7/adr cells demonstrate cross-resistance to paclitaxel but not to 5-fluorouracil and cisplatin.

Figure 2

Global hypermethylation in MCF-7/Adr cells. a) 5-mC content was evaluated by capillary electrophoresis. Percentage of 5-mC was 3.6% in MCF-7, while this increased to 5.6% in MCF-7/Adr cells; b) cytosine-extension assay based on use of methylation-sensitive restriction endonuclease that leaves a 5' guanine overhang after DNA cleavage with subsequent single-nucleotide extension with radiolabeled [(3)H]dCTP shows a 40% decrease in incorporation of radiolabeled [(3)H]dCTP by the DNA of HpaII-digested MCF-7/Adr cells, indicative of hypermethylation, and c), a polyclonal antibody against 5-methylcytosine was incubated with the digested DNA spotted in a nitrocellulose membrane. Spot intensity is 30% higher as evaluated by densitometric analysis in the DNA of MCF-7/Adr cells, also indicative of DNA hypermethylation.

Figure 3

Expression of DNA methyltransferase genes and DNA methyltransferase activity. a) MCF-7 and MCF-7/Adr cells were growth-arrested and dnmts analyzed by RT-PCR. Wild- type cells had very low or no expression of dnmt1, -3a, and -b as compared with MCF-7/Adr cells; b) corresponding DNA methyltransferase activity was increased by 30% in resistant cells.

Figure 4

Effects of DNA methyltransferase antisense treatment on drug-induced hypermethylation and chemotherapy resistance. a) MCF-7/Adr cells had no expression of dnmts transcripts after 72 h of antisense treatments; b) transfected cells with the antisense oligonucleotides had a 5-mC content between 2 and 4%, as compared with 5.6% in untreated MCF-7Adr cells, and c) MTT assays demonstrate that cells transfected with mismatch (MM) oligonucleotides showed no reduced cell viability and only a small reduction when challenged with adriamycin. The sole transfection with oligonucleotides against dnmts led to a reduction in viability between 30 and 40%, and when treated with adriamycin strong cytotoxicity was observed being higher for dnmt1 followed by -3a and -b.

Figure 5

Effects of hydralazine on DNA methylation and DNA methyltransferase activity. a) MCF-7/Adr cells were treated with increasing concentrations of hydralazine for 4 days and then DNA methyltransferase activity assayed. Hydralazine at 10 μM decreased enzymatic activity by 30%; b) 5-mC content was also reduced by hydralazine from 5.6–2.7%; c) cytosine-extension assay also showed higher incorporation into hydralazine treated cells as reflected by the bar marked HpaII, as did (d), dot-blot evaluation of hydralazine-treated MCF7/Adr cells.

Figure 6

Drug resistance and DNA hypermethylation are reversed with hydralazine. MCF-7/Adr cell viability was evaluated with the MTT assay after treatment with adriamycin and paclitaxel. Cells pretreated with hydralazine had their sensitivity to these drugs restored.

Figure 7

The mdr phenotype of MCF-7/Adr cells and its reversion with hydralazine and verapamil. a) Untreated MCF-7/Adr cells had no intracellular retention of adriamycin as indicated by MI, whereas verapamil and hydralazine clearly led to drug retention, and b) hydralazine decreased mdr1 gene expression as evaluated by RT-PCR. Figure 7c demonstrates that pre-treatment with verapamil led to only a 50% decrease in viability when cells were exposed to adriamycin as compared with an 85% reduction achieved with hydralazine pre-treatment.