Epigenetic and Cellular Reprogramming of Doxorubicin-Resistant MCF-7 Cells Treated with Curcumin

Abstract

The MCF-7R breast cancer cell line, developed by treating the parental MCF-7 cells with increasing doses of doxorubicin, serves as a model for studying acquired multidrug resistance (MDR). MDR is a major challenge in cancer therapy, often driven by overexpression of the efflux pump P-glycoprotein (P-gp) and epigenetic modifications. While many P-gp inhibitors show promise in vitro, their nonspecific effects on the efflux pump limit in vivo application. Curcumin, a natural compound with pleiotropic action, is a nontoxic P-gp inhibitor capable of modulating multiple pathways. To explore curcumin's molecular effects on MCF-7R cells, we analyzed the expression of genes involved in DNA methylation and transcription regulation, including ABCB1/MDR1. Reduced representation bisulfite sequencing further unveiled key epigenetic changes induced by curcumin. Our findings indicate that curcumin treatment not only modulates critical cellular processes, such as ribosome biogenesis and cytoskeletal dynamics, but also reverses the resistant phenotype, toward that of sensitive cells. This study highlights curcumin's potential as an adjuvant therapy to overcome chemoresistance, offering new avenues for pharmacological strategies targeting epigenetic regulation to re-sensitize resistant cancer cells.

Keywords: DNA methylation; P-glycoprotein; breast cancer; curcumin; cytoskeletal dynamics; multidrug resistance; ribosome biogenesis; translation.

Figure 1

Western blotting analysis of P-gp levels. Cells were treated with curcumin (30 µM) for 24 h. “CTR” refers to the MCF-7 doxorubicin-resistant cells and “CUR” refers to the MCF-7 doxorubicin-resistant cells treated with curcumin for 24 h. The results are expressed as the mean ± standard error of two different experiments. Differences when treatment is compared to control: * p < 0.05 (one-way ANOVA followed by Tukey’s test).

Figure 2

Evaluation of P-gp mRNA expression levels by qRT-PCR. For each condition, N = 3 technical replicates were used. Data are expressed as mean ± standard error of two experiments. Cells were treated with curcumin (30 µM) at the indicated time. ** (p < 0.01) represent significant differences among the times (one-way ANOVA followed by Tukey’s test).

Figure 3

Reduced representation bisulfite sequencing (RRBS) results. Statistical analysis of global data of a representative MCF-7R untreated cell line (R1, (a)) and an MCF-7R sample treated with curcumin (RC2, (b)).

Figure 4

Annotations of all DMRs (aggregated DMCs and tiles). (A) CpG island annotation, (B) ENCODE candidate Cis-Regulatory Elements annotation, (C) gene annotation, (D) gene class annotation.

Figure 5

GO enrichment analysis performed on 33 differentially methylated genes belonging to Chromatin or Chromosome GO terms.

Figure 6

Distribution of differentially methylated CpGs per chromosome.

Figure 7

UCSC genome browser view of the 45S ribosomal DNA. This gene represents a copy of the 45S ribosomal RNA on chromosome 21 (chr21:8,204,556–8,220,997). The 45S rDNA repeat unit encodes a 45S rRNA precursor, transcribed by RNA polymerase I, which is processed to form the 18S, 5.8S and 28S rRNAs. Under the chromosome scale, the following are shown: RRBS data (methylation difference R-S: significant DMCs, 200 bp tiles and 100 bp tiles; methylation difference RC-R: significant DMCs, 200 bp tiles and 100 bp tiles), HUGO gene annotation, CGI, Fantom5 CAGE peaks (mapped TSS: total counts in several cell types and MCF-7 data—forward in red and reverse in blue), ReMap Atlas of Regulatory Regions filtered by MCF-7, and DNAseI hypersensitivity in MCF-7.

Figure 8

UCSC genome browser view of the 5S ribosomal DNA locus. In this region, there are 17 copies of the 5S ribosomal RNA (chr1:228,607,600–228,650,600). The 5S rDNA is transcribed by RNA polymerase III. On the right, the upstream TSS for RHOU is visible. Under the chromosome scale, the following are shown: RRBS data (methylation difference R-S: significant DMCs, 200 bp tiles and 100 bp tiles; methylation difference RC-R: significant DMCs, 200 bp tiles and 100 bp tiles), HUGO gene annotation, CGI, ENCODE cCRE, Fantom5 CAGE peaks (mapped TSS: total counts in several cell types and MCF-7 data—forward in red and reverse in blue), ReMap Atlas of Regulatory Regions complete and filtered by MCF-7, and DNAseI hypersensitivity in MCF-7.

Figure 9

UCSC genome browser view of the differentially methylated CGI on chromosome 1 containing 23 tRNA genes (chr1: 161,440,000–161,472,000). Transfer RNA precursors are transcribed by RNA polymerase III. Under the chromosome scale, the following are shown: RRBS data (methylation difference R-S: significant DMCs, 200 bp tiles and 100 bp tiles; methylation difference RC-R: significant DMCs, 200 bp tiles and 100 bp tiles), HUGO gene annotation, CGI, CTCF binding sites in MCF-7, and cCRE (CTCF-only ENCODE Classification is blue).

Figure 10

Results of RT-qPCR experiments performed on ABCB1 regulators and chromatin regulators. ***: p value < 0.0005, *: p value < 0.05.