DNA Methyltransferase Expression and Proliferation Status of Metastatic Breast Cancer Cell Line After Prolonged and Repeated Rapamycin and Melatonin Application

Amaç: Bu çalışmanın amacı; Rapamisin, Melatoninin ve bunların birlikte kullanımlarının, östrojen reseptörü (ER)-negatif meme kanseri hücre hattının (4T1 hücre hattı) deoksiribonükleikasit (DNA) metilasyonu ve hücre proliferasyonu üzerine olan etkilerini incelemekti.Gereç ve Yöntemler: İlaç kombinasyonlarına (kontrol, Rapamisin, Melatonin, Rapamisin + Melatonin) ve bu ilaçların farklı zaman dilimlerinde (24, 48 ve 72 saat) uygulanmalarına bağlı olarak; 4T1 hücre hattı ile dört farklı grup oluşturuldu. İlaçlar, bu zaman dilimleri için sırasıyla; 1, 2 ve 3 defa uygulanmıştır. Tüm örnekler ile hücre sayımı, immun-boyama (Ki67, DNA metiltranferaz-1 (DNMT-1), DNA metiltransferaz-3a (DNMT-3a) ve p53 antikorları ile) ve Real-time PCR (polimeraz zincir reaksiyonu) (DNMT-1 ve DNMT-3a primerleri ile) yapıldı.Bulgular: Rapamisin ve Rapamisin + Melatonin uygulanmış olan gruplarda canlı/ölü hücre oranı düşüş göstermiştir. İlaç uygulanan gruplarda 24. saate kıyasla 48. saatte, Ki67 immun-boyaması ile proliferasyonda düşüş gözlenmiştir. Ayrıca DNMT-1 ekspresyonları; 72. saatte, 24. saate kıyasla tüm gruplarda, özellikle rapamisin uygulanan grupta, düşüş göstermiştir. DNMT-3a ekspresyonu ise 72. saatte, 24. saate kıyasla tüm gruplarda, özellikle rapamisin uygulanan grupta, artış göstermiştir. İlaç verilen gruplarda (en çok rapamisin uygulanmış olan grupta), ilaç uygulaması arttıkça ve süre geçtikçe artan p53 ekspresyonu gözlendi. DNMT-1 gen ekspresyonunun Real-time RT-PCR sonuçlarına göre, DNMT-1 gen ekspresyonu Melatonin uygulanan grupta kontrol grubuna kıyasla düşme eğilimi gösterirken, rapamisin ve Rapamisin + Melatonin uygulanan gruplarda kontrol grubuna göre artmıştır. DNMT-3a gen ekspresyonunun Real-time RT-PCR sonuçlarına göre ise, DNMT-3a gen ekspresyonu, tüm ilaç uygulanan gruplarda kontrol grubuna kıyasla artmıştır. Sonuç: Rapamisinin, metastatik meme kanseri hücrelerinde hem hücre canlılığı açısından hem de Ki67, DNMT-1, DNMT-3a ve p53'ün ekspresyonel değişimleri açısından Melatoninden daha etkili olduğu bulunmuştur.

Uzun Süreli ve Tekrarlı Rapamisin ve Melatonin Uygulaması Sonrası Metastatik Meme Kanseri Hücre Hattında DNA Metiltransferaz Ekspresyon ve Proliferasyon Durumu

Objective: The aim of this study was to investigate the effects of Rapamycin and Melatonin and their combination on deoxyribonucleic acid (DNA) methylation and cell proliferation in a estrogen receptor (ER)-negative breast cancer cell line (4T1 cell line).Materials and Methods: Four groups were designed with 4T1 cell line depending on drug combination (control, Rapamycin, Melatonin, Rapamycin + Melatonin) and their administration on different time periods (24, 48 and 72 hours). The drugs were administrated for 1, 2 and 3 times, respectively for these time periods. All samples were counted; immunostained (Ki67, DNA methyltransferase-1 (DNMT-1), DNA methyltransferase-3a (DNMT-3a) and p53) and real-time polymerase chain reaction (PCR) (DNMT-1 and DNMT-3a) was performed.Results: The live/dead cell ratios were decreased in the Rapamycin and Rapamycin + Melatonin applied groups. Ki67 immunostaining showed that there was a decreased proliferation in the drug applied groups at 48th hours compared to the 24th hours. Also DNMT-1 expressions were decreased at 72th hour compared to that at 24thhour in all groups, especially in the Rapamycin administrated group. Adversely, DNMT-3a expression was increased at 72th hour compared to that at 24th hour in the groups, especially in the Rapamycin administrated group. Furthermore, an increased expression of p53 was seen in the drug given groups (highest in the Rapamycin applied group) when the time prolonged. Real-time RT-PCR analysis of DNMT-1 gene expression showed a decreased expression level in the Melatonin given group compared to the control group and an increased expression level was seen in the Rapamycin and Rapamycin + Melatonin administrated groups compared to the control group.Conclusion: As a result, it was found that Rapamycin is more effective in metastatic breast cancer cells than Melatonin, both in the manner of cell viability and expressional changes of Ki67, DNMT-1, DNMT-3a and p53

PDF

___

Widodo I, Dwianingsih EK, Triningsih E, Utoro T, Soeripto. Clinicopathological features of indonesian breast cancers with different molecular subtypes. Asian Pac J Cancer Prev 2014; 15: 6109-13.
Haffty BG, Yang Q, Reiss M, Kearney T, Higgins SA, Weidhaas J, et al. Locoregional relapse and distant metastasis in conservatively managed triple negative early-stage breast cancer. J Clin Oncol 2006; 24: 5652-7.
DeBerardinis RJ, Lum JJ, Hatzivassiliou G, Thompson CB. The biology of cancer: metabolic reprogramming fuels cell growth and proliferation. Cell Metab 2008; 7: 11-20.
van Diest PJ, van der Wall E, Baak JP. Prognostic value of proliferation in invasive breast cancer: a review. Journal Clin Pathol 2004; 57: 675-81.
Rodríguez-Paredes M, Esteller M. Cancer epigenetics reaches mainstream oncology. Nat Med 2011; 17: 330-9.
Denis H, Ndlovu MN, Fuks F. Regulation of mammalian DNA methyltransferases: a route to new mechanisms. EMBO Rep 2010; 12: 647-56.
Seto B. Rapamycin and mTOR: a serendipitous discovery and implications for breast cancer. Clin Transl Med 2012; 1: 29.
Baldwin WS, Travlos GS, Risinger JI, Barrett JC. Melatonin does not inhibit estradiol-stimulated proliferation in MCF-7 and BG-1 cells. Carcinogenesis 1998; 19: 1895-900.
Cos S, González A, Martínez-Campa C, Mediavilla MD, Alonso- González C, et al. Estrogen-signaling pathway: a link between breast cancer and melatonin oncostatic actions. Cancer Detect Prev 2006; 30: 118-28.
Cos S, Fernández R, Güézmes A, Sánchez-Barceló EJ. Influence of melatonin on invasive and metastatic properties of MCF-7 human breast cancer cells. Cancer Res 1998; 58: 4383-90.
Cos S, Blask DE, Lemus-Wilson A, Hill AB. Effects of melatonin on the cell cycle kinetics and estrogen-rescue of MCF-7 human breast cancer cells in culture. J Pineal Res 1991; 10: 36-42.
Jardim-Perassi BV, Arbab AS, Ferreira LC, Borin TF, Varma NR, Iskander AS, et al. Effect of melatonin on tumor growth and angiogenesis in xenograft model of breast cancer. PLoS One 2014; 9: 85311.
Lv D, Zhang Y, Kim HJ, Zhang L, Ma X. CCL5 as a potential immunotherapeutic target in triple-negative breast cancer. Cel Mol Immunol 2013; 10: 303-10.
Basu S, Chen W, Tchou J, Mavi A, Cermik T, Czerniecki B, et al. omparison of triple-negative and estrogen receptor-positive/ progesterone receptor-positive/HER2-negative breast carcinoma using quantitative fluorine-18 fluorodeoxyglucose/positron emission tomography imaging parameters: a potentially useful method for disease characterization. Cancer 2008; 112: 995-1000.
Russo IH, Russo J. Role of hormones in mammary cancer initiation and progression. J Mammary Gland Biol Neoplasia 1998; 3: 49-61.
Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell 2011; 5: 646-74.
De Carvalho DD, Sharma S, You JS, Su SF, Taberlay PC, Kelly TK, et al. DNA methylation screening identifies driver epigenetic events of cancer cell survival. Cancer Cell 2012; 21: 655-67.
Bading JR, Shields AF. Imaging of cell proliferation: status and prospects. J Nucl Med 2008; 49: 64-80.
Kulis M, Esteller M. DNA methylation and cancer. Adv Genet 2010; 70: 27-56.
Kim H, Park J, Jung Y, Song SH, Han SW, Oh DY, et al. DNA methyltransferase 3-like affects promoter methylation of thymine DNA glycosylase independently of DNMT1 and DNMT3B in cancer cells. Int J Oncol 2010; 36: 1563-72.
Portela A, Esteller M. Epigenetic modifications and human disease. Nat Biotechnol 2010; 28: 1057-68.
Easton JB, Houghton PJ. mTOR and cancer therapy. Oncogene 2006; 25: 6436-46.
Bjornsti MA, Houghton PJ. The TOR pathway: a target for cancer therapy. Nat Rev Cancer 2004; 4: 335-48.
Cos S, Alvarez-García V, González A, Alonso-González C, Martínez-Campa C. Melatonin modulation of crosstalk among malignant epithelial, endothelial and adipose cells in breast cancer (Review). Oncol Lett 2014; 8: 487-92.
Mao L, Yuan L, Slakey LM, Jones FE, Burow ME, Hill SM. Inhibition of breast cancer cell invasion by melatonin is mediated through regulation of the p38 mitogen-activated protein kinase signaling pathway. Breast Cancer Res 2010; 12: 107.
Sánchez-Barceló EJ, Cos S, Fernández R, Mediavilla MD. Melatonin and mammary cancer: a short review. Endocr Relat Cancer 2003; 10: 153-9.
Liu T, Yacoub R, Taliaferro-Smith LD, Sun SY, Graham TR, Dolan R, et al. Combinatorial effects of lapatinib and rapamycin in triple- negative breast cancer cells. Mol Cancer Ther 2011; 10: 1460-9.
Zeng Q, Yang Z, Gao YJ, Yuan H, Cui K, Shi Y, et al. Treating triple- negative breast cancer by a combination of rapamycin and cyclophosphamide: an in vivo bioluminescence imaging study. Eur J Cancer 2010; 46: 1132-43.
Georgia S, Kanji M, Bhushan A. DNMT1 represses p53 to maintain progenitor cell survival during pancreatic organogenesis. Genes Dev 2013; 15: 372-7.
Wang YA, Kamarova Y, Shen KC, Jiang Z, Hahn MJ, Wang Y, et al. DNA methyltransferase-3a interacts with p53 and represses p53- mediated gene expression. Cancer Biol Ther 2005; 4: 1138-43.