Emodin enhances cisplatin-induced cytotoxicity via down-regulation of ERCC1 and inactivation of ERK1/2

Abstract

Emodin, a tyrosine kinase inhibitor, is a natural anthraquinone derivative found in the roots and rhizomes of numerous plants; it exhibits an anticancer effect on many malignancies. The most important chemotherapeutic agent for patients with advanced non-small cell lung cancer (NSCLC) is a platinum-containing compound such as cisplatin or carboplatin. The molecular mechanism underlying decreased NSCLC cell viability after treatment with emodin and cisplatin is unclear. Therefore, the aim of this study was to assess the cytotoxic effect of combined emodin and cisplatin on NSCLC cell lines and to clarify underlying molecular mechanisms. Exposure of human NSCLC cells to emodin decreased cisplatin-elicited ERK1/2 activation and ERCC1 protein induction by increasing instability of ERCC1 protein. Cisplatin alone did not affect expression of ERCC1 mRNA. However, emodin alone or combined with cisplatin significantly decreased expression of ERCC1 mRNA levels. Enhancement of ERK1/2 activation by transfection with constitutively active MKK1/2 (MKK1/2-CA) vector increased ERCC1 protein levels and protein stability, as well as increasing viability of NSCLC cells treated with emodin and cisplatin. In contrast, blocking ERK1/2 activation by U0126 (an MKK1/2 inhibitor) decreased cisplatin-elicited ERCC1 expression and enhanced cisplatin-induced cytotoxicity. Depletion of endogenous ERCC1 expression by si-ERCC1 RNA transfection significantly enhanced cisplatin's cytotoxic effect. In conclusion, ERCC1 protein protects NSCLC cells from synergistic cytotoxicity induced by emodin and platinum agents. Further investigation of combined emodin and cisplatin may lead to novel therapy in the future for NSCLC through down-regulating expression of ERCC1.

Introduction

Lung cancer is one of the most commonly occurring malignant tumors in the world, and non-small cell lung cancer (NSCLC) is the most common type of lung cancer, accounting for approximately 85% of all cases. Most patients with advanced lung cancer develop and die of distant metastasis. To improve overall survival, the current first-line therapeutic option for patients with advanced NSCLC is chemotherapy with a platinum-containing compound such as cisplatin or carboplatin combined with a second- or third-generation cytotoxic agent such as mitomycin C, paclitaxel, gemcitabine, vinorelbine, irinotecan, or docetaxel [1]. When combined with a third-generation chemotherapeutic agent, platinum-based compounds produce a higher response rate and improve overall survival compared with treatment with a third-generation chemotherapeutic agent alone [2], [3].

Cisplatin, a DNA-damaging agent, can covalently bind to DNA with preferential binding to the N-7 position of guanine and adenine; this binding interferes with DNA replication by inducing production of DNA adducts, accumulation of which leads to cell death [4]. Moreover, cisplatin causes intra-strand and inter-strand cross-links in DNA [5]. In mammalian cells, DNA intra-strand cross-links are repaired by the nucleotide excision repair pathway [6], [7]. Removal of adducts from genomic DNA is mediated by the enzyme called excision repair cross-complementation 1 (ERCC1), the structure-specific DNA repair endonuclease responsible for incision at the 5′ site of damaged DNA [8], [9].

Increased expression of ERCC1 in several cancers has been associated with more efficient removal of DNA adducts induced by platinum, leading to clinical resistance to platinum-based agents [10], [11], [12], [13]. For instance, transfecting ERCC1 into an ERCC1-deficient Chinese hamster ovary cell line increased the cells’ ability to repair DNA adducts, leading to platinum resistance [14]. On the other hand, patients with NSCLC who had a lower level of ERCC1 were shown to have longer overall survival after treatment with platinum-based chemotherapy than patients who had a higher level of ERCC1 [15], [16].

Emodin (1,3,8-trihydroxy-6-methylanthraquinone) is an active component in the root and rhizome of Rheum palmatum L. (Polygonaceae)[17], [18]. Emodin has shown potential as a treatment for several proliferative diseases such as liver cirrhosis, diabetic vascular complications, and a variety of tumors [19], [20]. Emodin has antimicrobial, antiviral, anti-inflammatory, anti-ulcerogenic, immunosuppressive and chemopreventive activities [21], [22]. Emodin was first discovered as a strong inhibitor of a protein tyrosine kinase (p56lck) [23]. Emodin was also shown to block phosphorylation of HER2/neu and ERK1/2 in PC3 prostate cancer cells [24]. In A549 lung carcinoma cells, emodin antagonized the cytoprotective signaling pathways ERK and AKT and triggered generation of reactive oxygen species and mitochondrial dysfunction through modulation of Bcl-2/Bax expression, with resultant release of cytochrome-c from mitochondria, caspase activation, and cell apoptosis [25], [26]. However, the effect of emodin on expression of ERCC1 participating in the nucleotide excision repair pathway in NSCLC cells remains unknown.

The aim of the current study was to determine whether emodin could be used as an adjunct to enhance the growth-inhibiting effect of cisplatin on NSCLC cells. Our results show that emodin has an additive effect on cisplatin-induced cell death and also has a suppressive effect on ERCC1 expression in human NSCLC cells through regulation of the ERK1/2 signaling pathway. The ERK1/2 signaling pathway is the upstream signal involved in the regulation of expression of ERCC1 and maintenance of its protein stability. This is the first report to document that the synergistic cytotoxicity induced by cisplatin and emodin is exerted through suppression of expression of the ERCC1 repair protein in NSCLC cells. The combination of emodin and cisplatin may be the basis for a novel future therapy to overcome drug resistance to cisplatin in patients with NSCLC.