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Induction of HRR genes and inhibition of DNMT1 is associated with anthracycline anti-tumor antibiotic-tolerant breast carcinoma cells

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Abstract

The aim of the study was to understand the role of homologous recombination repair (HRR) pathway genes in development of chemotolerance in breast cancer (BC). For this purpose, chemotolerant BC cells were developed in MCF-7 and MDA MB 231 cell lines after treatment with two anthracycline anti-tumor antibiotics doxorubicin and nogalamycin at different concentrations for 48 h with differential cell viability. The drugs were more effective in MCF-7 (IC50: 0.214–0.242 µM) than in MDA MB 231 (IC50: 0.346–0.37 µM) as shown by cell viability assay. The drugs could reduce the protein expression of PCNA in the cell lines. Increased mRNA/protein expression of the HRR (BRCA1, BRCA2, FANCC, FANCD2, and BRIT1) genes was seen in the cell lines in the presence of the drugs at different concentrations (lower IC50, IC50, and higher IC50) irrespective of the cell viability (68–41%). Quantitative methylation assay showed an increased percentage of hypomethylation of the promoters of these genes after drug treatment in the cell lines. Similarly, chemotolerant neoadjuvant chemotherapy (NACT) treated primary BC samples showed significantly higher frequency of hypomethylation of the genes than the pretherapeutic BC samples. The drugs in different concentrations could reduce m-RNA and protein expression of DNMT1 (DNA methyltransferase 1) in the cell lines. Similar phenomenon was also evident in the NACT samples than in the pretherapeutic BC samples. Thus, our data indicate that reduced DNMT1 expression along with promoter hypomethylation and increased expression of the HRR genes might have importance in chemotolerance in BC.

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Abbreviations

BC:

Breast carcinoma

ER:

Estrogen receptor

PR:

Progesterone receptor

HER2:

Human epidermal growth factor receptor 2

NACT:

Neoadjuvant chemotherapy

HRR:

Homologous recombination repair

UICC:

International Union Against Cancer

TNM:

Tumor size, lymph node, metastasis

MSRA:

Methylation-sensitive restriction analysis

qRT-PCR:

Real-time PCR quantification

DNMT1:

DNA methyltransferase 1

PCNA:

Proliferating cell nuclear antigen

HRP:

Horse-radish-peroxidase

FITC:

Fluorescein isothiocyanate

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Acknowledgements

We thank the director of Chittaranjan National Cancer Institute, Kolkata, India. We are also thankful to the Upjohn Company, USA for gifting nogalamycin. Financial support for this work was provided by UGC-NET Fellowship Grant F.2-3/2000 (SA-I) (Sr. No. 2061030813, Ref. No.: 20-06/2010(i)EU-IV dated 22.10.2010) to H. Dasgupta.

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Authors and Affiliations

  1. Department of Oncogene Regulation, Chittaranjan National Cancer Institute, 37, S.P. Mukherjee Road, Kolkata, West Bengal, 700026, India

    Hemantika Dasgupta, Md. Saimul Islam & Chinmay Kumar Panda

  2. Department of Surgical Oncology, Chittaranjan National Cancer Institute, Kolkata, India

    Neyaz Alam

  3. Department of Pathology, Nil Ratan Sircar Medical College and Hospital, Kolkata, West Bengal, India

    Anup Roy

  4. Saroj Gupta Cancer Center and Research Institute, MG Road, Thakurpukur, Kolkata, India

    Susanta Roychoudhury

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  1. Hemantika Dasgupta
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Correspondence to Chinmay Kumar Panda.

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Informed consent from the patients and approval from the Research Ethics Committee of the institute were obtained for sample collection.

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11010_2018_3442_MOESM1_ESM.tif

Supplementary Figure S1: (a, b) Post real-time representative agarose gel images of m-RNA expression patterns of HRR genes. (a) Untreated and nogalamycin-treated MCF-7 cDNA, 1. untreated/control, 2. 0.1µM NG treated, 3. 0.242µM (IC50) NG treated, 4. 0.4µM NG treated: (b) Untreated and doxorubicin-treated MDA MB 231 cDNA, 1. untreated/control, 2. 0.2µM DX treated, 3. 0.346µM (IC50) DX treated, 4. 0.6µM DX treated. NG = Nogalamycin, DX = Doxorubicin. (TIF 883 KB)

11010_2018_3442_MOESM2_ESM.tif

Supplementary Figure S2: (a, b) Representative promoter methylation patterns of HRR genes after treatment with doxorubicin and nogalamycin in MCF-7 cells. (a) K1: (b) K2 (c) BRCA2: (d) FANCC (e) FANCD2. Lane-wise description of DNA: 1. Undigested, control, 2. HpaII digested, control, 3. HhaI digested, control, 4. Undigested, 0.1µM DX treated, 5. HpaII digested, 0.1µM DX treated, 6. HhaI digested, 0.1µM DX treated, 7. Undigested, 0.214µM (IC50) DX treated, 8. HpaII digested, 0.214µM (IC50) DX treated, 9. HhaI digested, 0.214µM (IC50) DX treated, 10. Undigested, 0.4µM DX treated, 11. HpaII digested, 0.4µM DX treated, 12. HhaI digested, 0.4µM DX treated, 13. Undigested, control, 14. HpaII digested, control, 15. HhaI digested, control, 16. Undigested, 0.1µM NG treated, 17. HpaII digested, 0.1µM NG treated, 18. HhaI digested, 0.1µM NG treated, 19. Undigested, 0.242µM (IC50) NG treated, 20. HpaII digested, 0.242µM (IC50) NG treated, 21. HhaI digested, 0.242µM (IC50) NG treated, 22. Undigested, 0.4µM NG treated, 23. HpaII digested, 0.4µM NG treated, 24. HhaI digested, 0.4µM NG treated. DX=Doxorubicin, NG=Nogalamycin. (TIF 1970 KB)

11010_2018_3442_MOESM3_ESM.tif

Supplementary Figure S3: Post real-time representative agarose gel images of m-RNA expression patterns of DNMT1 cDNA in BC cell lines after doxorubicin/nogalamycin treatment. Lane-wise description of the samples: 1. untreated/control, 2. lower IC50 treated, 3. IC50 treated, 4. higher IC50 treated. (TIF 809 KB)

11010_2018_3442_MOESM4_ESM.tif

Supplementary Table S1: (a) Primers for m-RNA expression analysis of HRR genes (b) Clinicopathological features of pretherapeutic and NACT-treated BC patients. (c) Primers for promoter methylation analysis of HRR genes. (TIF 2391 KB)

11010_2018_3442_MOESM5_ESM.tif

Supplementary Table S2: Concordance of Qualitative methylation status of HRR genes previously determined in pretherapeutic and NACT-treated samples (Dasgupta et al. 2017) with the quantitative methylation status (dCT). + = Methylation positive, − = Methylation negative, ND = Not determined. (TIF 1893 KB)

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Dasgupta, H., Islam, M.S., Alam, N. et al. Induction of HRR genes and inhibition of DNMT1 is associated with anthracycline anti-tumor antibiotic-tolerant breast carcinoma cells. Mol Cell Biochem 453, 163–178 (2019). https://doi.org/10.1007/s11010-018-3442-5

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