Abstract
Background
Multi-drug resistance to chemotherapeutic agents is a major cause of treatment failure in breast cancer. In this study, we investigated the effects of emodin on reversing the multi-drug resistance, examined the ERCC1 protein expression in breast cancer cell line, and explored the relationship between reversal of multi-drug resistance and ERCC1 protein expression.
Methods
MTT assay was conducted to test the cytotoxicity of adriamycin and cisplatin to MCF-7/Adr cells with and without emodin pretreatment, and Western blot was performed to examine the ERCC1 protein expression.
Results
MCF-7/Adr cells had 21-fold and 11-fold baseline resistances to adriamycin and cisplatin, respectively. When emodin was added to the cell culture at the concentration of 10 μg/ml, the drug resistance was reduced from 21 folds to 2.86 folds for adriamycin, and from 11 folds to 1.79 folds for cisplatin. MCF-7/Adr cells treated with two concentrations (10μg/mL and 20μg/mL) of emodin, after 2, 4, 6, 10 days, the trend of ERCC1 expression was gradually decreased and the reduction was more obvious comparatively at the concentration of 20μg/mL.
Conclusions
Emodin could reverse the multi-drug resistance in MCF-7/Adr cells and down-regulate ERCC1 protein expression.
Background
Excision repair cross complementation group 1 (ERCC1) protein encoded by ERCC1 gene is a key player in nucleotide excision repair (NER), and our previous work and others have demonstrated that this protein is also expressed in breast cancer [29].
Patients with lower DNA repair capacity are more chemosensitive than those who carry a proficient DNA repair system. In early, it was shown that elevated DNA repair capacity is associated with drug resistance in lung cancer cell lines [30], and it was suggested that modulation of DNA repair mechanisms, such as the incorporation of specific DNA repair inhibitors in therapeutic regimens. Although low expression of ERCC1 is related to carcinogenesis, high expression could enhance the NER, leading to rapid repair of the damaged tumor DNA after chemotherapy, a plausible mechanism of multi-drug resistance in many cancers [31]. Thus, we processed a serial studies about ERCC1 in breast cancer and look for novel anti-cancer strategies to avoid drug resistance and improve treatment outcomes.
EMD is a chemical compound of the anthraquinone family mainly derived from the root of Rheum palmatum, a widely used herb in traditional Chinese medicine, with a variety of anti-bacteria, anti-tumor and anti-constipation properties. Studies at the cellular level have shown that EMD affects cell proliferation by inhibiting DNA synthesis, prolonging the cell cycle duration, and suppressing mitosis. In our study, when the two breast cancer cells were exposed to 0~120 μg/ml of EMD, the IC50 was 100 ± 0.35 μg/ml for MCF-7 and 79 ± 0.28 μg/ml for MCF-7/Adr. At 20μg/ml EMD, cell viability was over 85% for both MCF-7 and MCF-7/Adr. Therefore, we selected 10μg/ml EMD at which no cell growth inhibition was observed as the starting dose for the reversal test. Our results showed that in MCF-7/Adr cells, EMD could reduce the ADM-resistance by 2.86 folds, and the DDP resistance was reduced by 1.79 folds. These results confirm that EMD is a highly effective drug-resistance reversal agent with low toxicity.
High expression of NER repairing ability enhancement is considered as one of the mechanisms of tumor drug resistance [32, 33]. In this study, two concentrations of emodin, 20μg/mL and 10μg/mL, were used to deal with low toxicity concentration MCF-7/Adr cell line, in 2, 4, 6 and10 days and the expression of ERCC1 protein was examined by Western blotting. The results showed higher levels of ERCC1 protein expression in MCF-7/Adr cell lines than in MCF-7 ones, but after emodin treatment, the ERCC1 protein levels decreased further and is significantly time-dependent and possibly dose-dependent as greater inhibitory effect was observed in the concentration of 20μg/ml. Given that only two concentration were studied, it may still have a certain concentration range to explore further the true dose-effect relationship.
Many new anti-cancer agents is being developed in recent years, but many studies have now focused on the reversal of chemoresistance in tumor in order to maximize the treatment capacity of existing chemotherapeutic agents. Some medicinal herbs may hold great potential in this field. This in vitro study found that EMD can significantly reverse the multi-drug resistance and reduce ERCC1 expression in breast cancer cells. Further studies are warranted to explore how ERCC1 plays role in reversing drug resistance and more pre-clinical evidence is necessary before the use emodin will be studied in clinical trials.
Conclusions
In this in vitro study, we noticed that ERCC1 expression might be associated with drug resistance and that emodin might play a possible role of reversing drug resistance. This is a preliminary finding which needs further investigation to explore the use of emodin in breast cancer treatment. However, we must emphasize that with recent advances of molecular biology, breast cancer was categorized into many different molecular subtypes which might possess different characteristics from carcinogenesis to metastasis. Therefore, the study of a single biomarker might be further complicated. Nevertheless, we believe further studies on ERCC1 expression in breast cancer and emodin in facilitating the reversal of drug resistance are warranted.
References
Fu JM, Zhou J, **e JJ, Li H: Effect of neoadjuvant chemotherapy on ERCC1 gene expression in breast cancer. J South Med Univ. 2008, 28: 603-605.
Juhasz A, Frankel P, Cheng C, Rivera H, Vishwanath R, Chiu A, Margolin K, Yen Y, Newman EM, Synold T, Wilczynski S, Lenz HJ, Gandara D, Albain KS, Longmate J, Doroshow JH: Quantification of chemotherapeutic target gene mRNA expression in human breast cancer biopsies: comparison of real-time reverse transcription-PCR vs. relative quantification reverse transcription-PCR utilizing DNA sequencer analysis of PCR products. J Clin Lab Anal. 2003, 17: 184-194. 10.1002/jcla.10091.
Ferry KV, Hamilton TC, Johnson SW: Increased nucleotide excision repair in cisplatin-resistant ovarian cancer cells: role of ERCC1-XPF. Biochem Pharmacol. 2000, 60: 1305-1313. 10.1016/S0006-2952(00)00441-X.
Selvakumaran M, Pisarcik DA, Bao R, Yeung AT, Hamilton TC: Enhanced cisplatin cytotoxicity by disturbing the nucleotide excision repair pathway in ovarian cancer cell lines. Cancer Res. 2003, 63: 1311-1316.
Vandenput I, Capoen A, Coenegrachts L, Verbist G, Moerman P, Vergote I, Amant F: Expression of ERCC1, p53, and Class III β-Tubulin Do Not Reveal Chemoresistance in Endometrial Cancer: Results From an Immunohistochemical Study. Int J Gynecol Cancer. 2011, 21: 1071-1077. 10.1097/IGC.0b013e318218f28b.
Krivak TC, Darcy KM, Tian C, Bookman M, Gallion H, Ambrosone CB, Deloia JA: Single nucleotide polypmorphisms in ERCC1 are associated with disease progression, and survival in patients with advanced stage ovarian and primary peritoneal carcinoma; a Gynecologic Oncology Group study. Gynecol Oncol. 2011, 122: 121-126. 10.1016/j.ygyno.2011.03.027.
Roth JA, Carlson JJ: Prognostic Role of ERCC1 in Advanced Non-Small-Cell Lung Cancer: A Systematic Review and Meta-Analysis. Clin Lung Cancer.
Tepeli E, Caner V, Büyükpınarbaşılı N, Cetin GO, Düzcan F, Elmas L, Bağcı G: Expression of ERCC1 and its clinicopathological correlations in non-small cell lung cancer. Mol Biol Rep.
Tsai MS, Weng SH, Kuo YH, Chiu YF, Lin YW: Synergistic effect of curcumin and cisplatin via down-regulation of thymidine phosphorylase and excision repair cross-complementary 1 (ERCC1). Mol Pharmacol. 2011, 80: 136-146. 10.1124/mol.111.071316.
Sun JM, Ahn MJ, Park MJ, Lee HY, Ahn JS, Lee S, Kang G, Han J, Son YI, Baek CH, Ahn YC, Park K: Expression of excision repair cross-complementation group 1 as predictive marker for nasopharyngeal cancer treated with concurrent chemoradiotherapy. Int J Radiat Oncol Biol Phys. 2011, 80: 655-660. 10.1016/j.ijrobp.2010.02.061.
Kaira K, Serizawa M, Koh Y, Miura S, Kaira R, Abe M, Nakagawa K, Ohde Y, Okumura T, Naito T, Murakami H, Takahashi T, Kondo H, Nakajima T, Endo M, Yamamoto N: Expression of excision repair cross-complementation group 1, breast cancer susceptibility 1, and β III-tubulin in thymic epithelial tumors. J Thorac Oncol. 2011, 6: 606-613. 10.1097/JTO.0b013e31820b9b35.
Knez L, Sodja E, Kern I, Košnik M, Cufer T: Predictive value of multidrug resistance proteins, topoisomerases II and ERCC1 in small cell lung cancer: a systematic review. Lung Cancer. 2011, 72: 271-279. 10.1016/j.lungcan.2011.02.014.
Kim D, Jung W, Koo JS: The expression of ERCC1, RRM1, and BRCA1 in breast cancer according to the immunohistochemical phenotypes. J Korean Med Sci. 2011, 26: 352-359.
Huang Z, Chen G, Shi P: Emodin-induced apoptosis in human breast cancer BCap-37 cells through the mitochondrial signaling pathway. Arch Pharm Res. 2008, 31: 742-748. 10.1007/s12272-001-1221-6.
**g X, Ueki N, Cheng J, Imanishi H, Hada T: Induction of apoptosis in hepatocellular carcinoma cell lines by emodin. Jpn J Cancer Res. 2002, 93: 874-882. 10.1111/j.1349-7006.2002.tb01332.x.
Zhang L, Lau YK, **a W, Hortobagyi GN, Hung MC: Tyrosine kinase inhibitor emodin suppresses growth of HER-2/neu-overexpressing breast cancer cells in athymic mice and sensitizes these cells to the inhibitory effect of paclitaxel. Clin Cancer Res. 1999, 5: 343-353.
Jiang XF, Zhen YS: Reversal of multidrug resistance by emodin in cancer cells. Acta Pharmaceutica Sinica. 1999, 34: 164-167.
Fu ZY, Han JX, Huang HY: Effects of emodin on gene expression profile in small cell lung cancer NCI-H446 cell. Chin Med J (Engl). 2007, 120: 1710-1715.
Falo C, Moreno A, Varela M, Lloveras B, Figueras A, Escobedo A: HER-2/neu status and response to CMF: retrospective study in a series of operable breast cancer treated with primary CMF chemotherapy. J Cancer Res Clin Oncol. 2007, 133: 423-429. 10.1007/s00432-006-0176-7.
Gan HZ, Zhang GZ, Zhao JS, Zhang FC, Bu LS, Yang SJ, Piao SL, Du ZW, Gao S, Zheng DM: Reversal of MDR1 gene-dependent multidrug resistance using short hairpin RNA expression vectors. Chin Med J (Engl). 2005, 118: 893-902.
Rosell R, Felip E, Paz-Ares L: How could pharmacogenomics help improve patient survival?. Lung Cancer. 2007, 57 (Suppl 2): S35-S41.
Geyer FC, Weigelt B, Natrajan R, Lambros MB, de Biase D, Vatcheva R, Savage K, Mackay A, Ashworth A, Reis-Filho JS: Molecular analysis reveals a genetic basis for the phenotypic diversity of metaplastic breast carcinomas. J Pathol. 2010, 220: 562-573. 10.1002/path.2675.
Li WT, Zhou GY, Wang CL, Guo CH, Song XR, Chi WL: Modulation of breast cancer resistance protein mediated atypical multidrug resistance using RNA interference delivered by adenovirus. Chin Med J (Engl). 2005, 118: 1123-1126.
Burness ML, Grushko TA, Olopade OI: Epidermal growth factor receptor in triple-negative and basal-like breast cancer: promising clinical target or only a marker?. Cancer J. 2010, 16: 23-32. 10.1097/PPO.0b013e3181d24fc1.
Rosell R, Moran T, Fernanda Salazar M, Mendez P, De Aguirre I, Ramirez JL, Isla D, Cobo M, Camps C, Lopez-Vivanco G, Alberola V, Taron M: The place of targeted therapies in the management of non-small cell bronchial carcinoma. Molecular markers as predictors of tumor response and survival in lung cancer. Rev Mal Respir. 2006, 23: 16S131-16S136.
Yao GY, Zhou JL, Zhao ZS, Ruan J: Biological characteristics of breast carcinomas with neuroendocrine cell differentiation. Chin Med J (Engl). 2004, 117: 1536-1540.
Zhou W, Liu G, Park S, Wang Z, Wain JC, Lynch TJ, Su L, Christiani DC: Gene-smoking interaction associations for the ERCC1 polymorphisms in the risk of lung cancer. Cancer Epidemiol Biomarkers Prev. 2005, 14: 491-496. 10.1158/1055-9965.EPI-04-0612.
Olaussen KA, Dunant A, Fouret P, Brambilla E, André F, Haddad V, Taranchon E, Filipits M, Pirker R, Popper HH, Stahel R, Sabatier L, Pignon JP, Tursz T, Le Chevalier T, Soria JC, IALT Bio Investigators: DNA repair by ERCC1 in non-small-cell lung cancer and cisplatin-based adjuvant chemotherapy. N Engl J Med. 2006, 355: 983-991. 10.1056/NEJMoa060570.
Wachters FM, Wong LS, Timens W, Kam**a HH, Groen HJ: ERCC1, hRad51, and BRCA1 protein expression in relation to tumour response and survival of stage III/IV NSCLC patients treated with chemotherapy. Lung Cancer. 2005, 50: 211-219. 10.1016/j.lungcan.2005.06.013.
Zeng-Rong N, Paterson J, Alpert L, Tsao MS, Viallet J, Alaoui-Jamali MA: Elevated DNA repair capacity is associated with intrinsic resistance of lung cancer to chemotherapy. Cancer Res. 1995, 55: 4760-4764.
Vilmar A, Sørensen JB: Excision repair cross-complementation group 1 (ERCC1) in platinum-based treatment of non-small cell lung cancer with special emphasis on carboplatin: a review of current literature. Lung Cancer. 2009, 64: 131-139. 10.1016/j.lungcan.2008.08.006.
Joyce EF, Tanneti SN, McKim KS: Drosophila hold'em is required for a subset of meiotic crossovers and interacts with the dna repair endonuclease complex subunits MEI-9 and ERCC1. Genetics. 2009, 181: 335-340.
Bhargava R, Beriwal S, Dabbs DJ, Ozbek U, Soran A, Johnson RR, Brufsky AM, Lembersky BC, Ahrendt GM: Immunohistochemical surrogate markers of breast cancer molecular classes predicts response to neoadjuvant chemotherapy: a single institutional experience with 359 cases. Cancer. 2010, 116: 1431-1439. 10.1002/cncr.24876. Erratum in: Cancer 2011, 117:2238
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This article has been published as part of Journal of Translational Medicine Volume 10 Supplement 1, 2012: Selected articles from the Organisation for Oncology and Translational Research (OOTR) 7th Annual Conference. The full contents of the supplement are available online at http://www.translational-medicine.com/supplements/10/S1.
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Authors' contributions
JF, JZ, JS, JX and LH carried out the pre-clinical research. JF, AY, WL and LC participated in writing the manuscript. WL performed statistical analysis. JF and LC provided expert opinion for the study. JF was the initiator of the study.
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Fu, Jm., Zhou, J., Shi, J. et al. Emodin affects ERCC1 expression in breast cancer cells. J Transl Med 10 (Suppl 1), S7 (2012). https://doi.org/10.1186/1479-5876-10-S1-S7
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DOI: https://doi.org/10.1186/1479-5876-10-S1-S7