Advertisement

Molecular and Cellular Biochemistry

, Volume 453, Issue 1–2, pp 163–178 | Cite as

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

  • Hemantika Dasgupta
  • Md. Saimul Islam
  • Neyaz Alam
  • Anup Roy
  • Susanta Roychoudhury
  • Chinmay Kumar PandaEmail author
Article

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.

Keywords

HRR pathway MCF-7/MDA MB 231 Doxorubicin/nogalamycin Promoter methylation DNMT1 

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

Notes

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.

Compliance with ethical standards

Conflict of interest

There are no conflicts of interest.

Informed consent

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

Supplementary material

11010_2018_3442_MOESM1_ESM.tif (884 kb)
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 (1.9 mb)
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 (809 kb)
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 (2.3 mb)
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 (1.8 mb)
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)

References

  1. 1.
    Ochayon L, Tunin R, Yoselis A, Kadmon I (2014) Symptoms of hormonal therapy and social support: is there a connection? Comparison of symptom severity, symptom interference and social support among breast cancer patients receiving and not receiving adjuvant hormonal treatment. Eur J Oncol Nurs 14:192–196Google Scholar
  2. 2.
    Sinha S, Chunder N, Mukherjee N, Alam N, Roy A, Roychoudhury S, Panda CK (2008) Frequent deletion and methylation in SH3GL2 and CDKN2A loci are associated with early- and late-onset breast carcinoma. Ann Surg Oncol 15:1070–1080CrossRefGoogle Scholar
  3. 3.
    Gampenrieder SP, Rinnerthaler G, Greil R (2013) Neoadjuvant chemotherapy and targeted therapy in breast cancer: past, present, and future. J Oncol 2013:732047Google Scholar
  4. 4.
    Asakawa H, Koizumi H, Koike A, Takahashi M, Wu W, Iwase H, Fukuda M, Ohta T (2010) Prediction of breast cancer sensitivity to neoadjuvant chemotherapy based on status of DNA damage repair proteins. Breast Cancer Res 12:R17CrossRefGoogle Scholar
  5. 5.
    Kriege M, Seynaeve C, Meijers-Heijboer H, Collee JM, Menke-Pluymers MB, Bartels CC, Tilanus-Linthorst MM, Blom J, Huijskens E, Jager A, van den Ouweland A, van Geel B, Hooning MJ, Brekelmans CT, Klijn JG (2009) Sensitivity to first-line chemotherapy for metastatic breast cancer in BRCA1 and BRCA2 mutation carriers. J Clin Oncol 27:3764–3771CrossRefGoogle Scholar
  6. 6.
    Kennedy RD, D’Andrea AD (2006) DNA repair pathways in clinical practice: lessons from pediatric cancer susceptibility syndromes. J Clin Oncol 24:3799–3808CrossRefGoogle Scholar
  7. 7.
    Mulligan JM, Hill LA, Deharo S, Irwin G, Boyle D, Keating KE, Raji OY, McDyer FA, O’Brien E, Bylesjo M, Quinn JE, Lindor NM, Mullan PB, James CR, Walker SM, Kerr P, James J, Davison TS, Proutski V, Salto-Tellez M, Johnston PG, Couch FJ, Paul Harkin D (2014) Identification and validation of an anthracycline/cyclophosphamide-based chemotherapy response assay in breast cancer. J Natl Cancer Inst 106:djt335CrossRefGoogle Scholar
  8. 8.
    Rassool FV, Tomkinson AE (2010) Targeting abnormal DNA double strand break repair in cancer. Cell Mol Life Sci 67:3699–3710CrossRefGoogle Scholar
  9. 9.
    Bhattacharya N, Mukherjee N, Singh RK, Sinha S, Alam N, Roy A, Roychoudhury S, Panda CK (2013) Frequent alterations of MCPH1 and ATM are associated with primary breast carcinoma: clinical and prognostic implications. Ann Surg Oncol 3:S424–S432CrossRefGoogle Scholar
  10. 10.
    Rai R, Dai H, Multani AS, Li K, Chin K, Gray J, Lahad JP, Liang J, Mills GB, Meric-Bernstam F, Lin SY (2006) BRIT1 regulates early DNA damage response, chromosomal integrity, and cancer. Cancer Cell 10:145–157CrossRefGoogle Scholar
  11. 11.
    Chunder N, Mandal S, Roy A, Roychoudhury S, Panda CK (2004) Differential association of BRCA1 and BRCA2 genes with some breast cancer-associated genes in early and late onset breast tumors. Ann Surg Oncol 11:1045–1055CrossRefGoogle Scholar
  12. 12.
    Dasgupta H, Mukherjee N, Islam S, Bhattacharya R, Alam N, Roy A, Roychoudhury S, Biswas J, Panda CK (2017) Frequent alterations of HRR pathway in primary and chemotolerant breast carcinomas: clinical importance. Future Oncol 13:159–174CrossRefGoogle Scholar
  13. 13.
    Sinha S, Singh RK, Alam N, Roy A, Roychoudhury S, Panda CK (2008) Alterations in candidate genes PHF2, FANCC, PTCH1 and XPA at chromosomal 9q22.3 region: pathological significance in early- and late-onset breast carcinoma. Mol Cancer 7:84CrossRefGoogle Scholar
  14. 14.
    van der Groep P, Hoelzel M, Buerger H, Joenje H, de Winter JP, van Diest PJ (2008) Loss of expression of FANCD2 protein in sporadic and hereditary breast cancer. Breast Cancer Res Treat 107:41–47CrossRefGoogle Scholar
  15. 15.
    Sharma SV, Lee DY, Li B, Quinlan MP, Takahashi F, Maheswaran S, McDermott U, Azizian N, Zou L, Fischbach MA, Wong KK, Brandstetter K, Wittner B, Ramaswamy S, Classon M, Settleman J (2010) A chromatin-mediated drug-tolerant state in cancer cell subpopulations. Cell 141:69–80CrossRefGoogle Scholar
  16. 16.
    Glasspool RM, Teodoridis JM, Brown R (2006) Epigenetics as a mechanism driving polygenic clinical drug resistance. Br J Cancer 94:1087–1092CrossRefGoogle Scholar
  17. 17.
    Trudeau M, Charbonneau F, Gelmon K, Laing K, Latreille J, Mackey J, McLeod D, Pritchard K, Provencher L, Verma S (2005) Selection of adjuvant chemotherapy for treatment of node-positive breast cancer. Lancet Oncol 6:886–898CrossRefGoogle Scholar
  18. 18.
    Siitonen V, Selvaraj B, Niiranen L, Lindqvist Y, Schneider G, Metsä-Ketelä M (2016) Divergent non-heme iron enzymes in the nogalamycin biosynthetic pathway. Proc Natl Acad Sci 113:5251–5256CrossRefGoogle Scholar
  19. 19.
    Andres JL, Fan S, Turkel GJ, Wang JA, Twu NF, Yuan RQ, Lamszus K, Goldberg ID, Rosen EM (1998) Regulation of BRCA1 and BRCA2 expression in human breast cancer cells by DNA-damaging agents. Oncogene 16:2229–2241CrossRefGoogle Scholar
  20. 20.
    Chen JS, Konopleva M, Andreeff M, Multani AS, Pathak S, Mehta K (2004) Drug-resistant breast carcinoma (MCF-7) cells are paradoxically sensitive to apoptosis. J Cell Physio 200:223–234CrossRefGoogle Scholar
  21. 21.
    Sur S, Pal D, Banerjee K, Mandal S, Das A, Roy A, Panda CK (2015) Amarogentin regulates self renewal pathways to restrict liver carcinogenesis in experimental mouse model. Mol Carcinog 55:1138–1149CrossRefGoogle Scholar
  22. 22.
    Khafif A, Schantz SP, Chou TC, Edelstein D, Sacks PC (1998) Quantitation of chemopreventive synergism between (-)-epigallocatechin-3-gallate and curcumin in normal, premalignant and malignant human oral epithelial cells. Carcinogenesis 19:419–424CrossRefGoogle Scholar
  23. 23.
    Veroni C, Marnetto F, Granieri L, Bertolotto A, Ballerini C, Repice AM, Schirru L, Coghe G, Cocco E, Anastasiadou E, Puopolo M, Aloisi F (2015) Immune and Epstein-Barr virus gene expression in cerebrospinal fluid and peripheral blood mononuclear cells from patients with relapsing-remitting multiple sclerosis. J Neuroinflammation 14:12:132Google Scholar
  24. 24.
    Di Napoli A, Al-Jadiri MF, Talerico C, Duranti E, Pilozzi E, Trivedi P, Anastasiadou E, Alsaadawi AR, Al-Darraji AF, Al-Hadad SA, Testi AM, Uccini S, Ruco L (2013) Epstein-Barr virus (EBV) positive classical Hodgkin lymphoma of Iraqi children: an immunophenotypic and molecular characterization of Hodgkin/Reed-Sternberg cells. Pediatr Blood Cancer 60:2068–2072CrossRefGoogle Scholar
  25. 25.
    Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25:402–408CrossRefGoogle Scholar
  26. 26.
    Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by the comparative CT method. Nat Protoc 3:1101–1108CrossRefGoogle Scholar
  27. 27.
    Dasgupta S, Mukherjee N, Roy S, Roy A, Sengupta A, Roychowdhury S, Panda CK (2002) Mapping of the candidate tumor suppressor genes’ loci on human chromosome 3 in head and neck squamous cell carcinoma of an Indian patient population. Oral Oncol 38:6–15CrossRefGoogle Scholar
  28. 28.
    Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring HarborGoogle Scholar
  29. 29.
    Loginov VI, Maliukova AV, SereginIu A, Khodyrev DS, Kazubskaia TP, Ermilova VD, Gar’kavtseva RF, Kiselev LL, Zabarovskii ER, Braga EA (2004) Methylation of the promoter region of the RASSF1A gene, a candidate tumor suppressor, in primary epithelial tumors. MolBiol (Mosk) 38:654–667Google Scholar
  30. 30.
    Ivanova T, Petrenko A, Gritsko T, Vinokourova S, Eshilev E, Kobzeva V, Kisseljov F, Kisseljova N (2002) Methylation and silencing of the retinoic acid receptor-beta 2 gene in cervical cancer. BMC Cancer 2:4CrossRefGoogle Scholar
  31. 31.
    Thomassin H, Kress C, Grange T (2004) MethylQuant: a sensitive method for quantifying methylation of specific cytosines within the genome. Nucleic Acids Res 32:e168CrossRefGoogle Scholar
  32. 32.
    Perrone F, Suardi S, Pastore E, Casieri P, Orsenigo M, Caramuta S, Dagrada G, Losa M, Licitra L, Bossi P, Staurengo S, Oggionni M, Locati L, Cantu G, Squadrelli M, Carbone A, Pierotti MA, Pilotti S (2006) Molecular and cytogenetic subgroups of oropharyngeal squamous cell carcinoma. Clin Cancer Res 12:6643–6651CrossRefGoogle Scholar
  33. 33.
    Klein CJ, Botuyan MV, Wu Y, Ward CJ, Nicholson GA, Hammans S, Hojo K, Yamanishi H, Karpf AR, Wallace DC, Simon M, Lander C, Boardman LA, Cunningham JM, Smith GE, Litchy WJ, Boes B, Atkinson EJ, Middha S, Dyck B, Parisi PJ, Mer JE, Smith G, Dyck DI PJ (2011) Mutations in DNMT1 cause hereditary sensory neuropathy with dementia and hearing loss. Nat Genet 43:595–600CrossRefGoogle Scholar
  34. 34.
    Perou CM (2010) Molecular stratification of triple-negative breast cancers. Oncologist 16:61–70CrossRefGoogle Scholar
  35. 35.
    Alfaro Y, Delgado G, Carabez A, Anguiano B, Aceves C (2013) Iodine and doxorubicin, a good combination for mammary cancer treatment: antineoplastic adjuvancy, chemoresistance inhibition, and cardioprotection. Mol Cancer 12:45CrossRefGoogle Scholar
  36. 36.
    Vissac-Sabatier C, Bignon YJ, Bernard-Gallon DJ (2003) Effects of the phytoestrogens genistein and daidzein on BRCA2 tumor suppressor gene expression in breast cell lines. Nutr Cancer 45:247–255CrossRefGoogle Scholar
  37. 37.
    Chai KM, Wang CY, Liaw HJ, Fang KM, Yang CS, Tzeng SF (2014) Downregulation of BRCA1-BRCA2-containing complex subunit 3 sensitizes glioma cells to temozolomide. Oncotarget 5:10901–10915Google Scholar
  38. 38.
    Liedtke S, Biebernick S, Radke TF, Stapelkamp D, Coenen C, Zaehres H, Fritz G, Kogler G (2015) DNA damage response in neonatal and adult stromal cells compared with induced pluripotent stem cells. Stem Cells Transl Med 4:576–589CrossRefGoogle Scholar
  39. 39.
    Ray R, Chakraborty BK, Ray K, Mukherji S, Chowdhury JR, Panda CK (1996) Effect of anthracycline antitumor antibiotics (adriamycin and nogalamycin) and cycloheximide on the biosynthesis and processing of major UsnRNAs. Mol Cell Biochem 162l:75–82Google Scholar
  40. 40.
    Bosviel R, Durif J, Déchelotte P, Bignon YJ, Bernard-Gallon D (2012) Epigenetic modulation of BRCA1 and BRCA2 gene expression by equol in breast cancer cell lines. Br J Nutr 108:1187–1193CrossRefGoogle Scholar
  41. 41.
    Takabatake M, Blyth BJ, Daino K, Imaoka T, Nishimura M, Fukushi M, Shimada Y (2016) DNA methylation patterns in rat mammary carcinomas induced by pre-and post-pubertal irradiation. PLoS ONE 11:e0164194CrossRefGoogle Scholar
  42. 42.
    Ignatov T, Poehlmann A, Ignatov A, Schinlauer A, Costa SD, Roessner A, Kalinski T, Bischoff J (2013) BRCA1 promoter methylation is a marker of better response to anthracycline-based therapy in sporadic TNBC. Breast Cancer Res Treat 141:205–212CrossRefGoogle Scholar
  43. 43.
    Yokochi T, Robertson KD (2004) Doxorubicin inhibits DNMT1, resulting in conditional apoptosis. Mol Pharmacol 66:1415–1420CrossRefGoogle Scholar
  44. 44.
    Tesei A, Brigliadori G, Carloni S, Fabbri F, Ulivi P, Arienti C, Sparatore A, Del Soldato P, Pasini A, Amadori D, Silvestrini R, Zoli W (2012) Organosulfur derivatives of the HDAC inhibitor valproic acid sensitize human lung cancer cell lines to apoptosis and to cisplatin cytotoxicity. J Cell Physiol 227:3389–3396CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Hemantika Dasgupta
    • 1
  • Md. Saimul Islam
    • 1
  • Neyaz Alam
    • 2
  • Anup Roy
    • 3
  • Susanta Roychoudhury
    • 4
  • Chinmay Kumar Panda
    • 1
    Email author
  1. 1.Department of Oncogene RegulationChittaranjan National Cancer InstituteKolkataIndia
  2. 2.Department of Surgical OncologyChittaranjan National Cancer InstituteKolkataIndia
  3. 3.Department of PathologyNil Ratan Sircar Medical College and HospitalKolkataIndia
  4. 4.Saroj Gupta Cancer Center and Research InstituteKolkataIndia

Personalised recommendations