Metformin-mediated downregulation of p38 mitogen-activated protein kinase-dependent excision repair cross-complementing 1 decreases DNA repair capacity and sensitizes human lung cancer cells to paclitaxel
Graphical abstract
Introduction
Lung cancer is the most lethal form of cancers in both men and women, with non-small cell lung cancer (NSCLC) comprising the majority (∼85%) [1]. Paclitaxel (Taxol) is a chemotherapeutic drug used in the treatment of human NSCLC as well as breast and ovarian cancers [2], [3], [4]. The biological activity of paclitaxel, based on its ability to bind to the β-subunit of tubulin, results in the formation of stabilized microtubule bundles that interfere with mitosis [5]. Paclitaxel has also been reported to induce activation of the mitogen-activated protein kinase (MAPK) signaling pathway, including p38 MAPK [6], [7], [8], [9], JNK [10], and ERK [6], [8], [9]. However, the effects of MAPK activation are highly controversial; they exert both protective and apoptotic functions depending on the cell type and stimuli [6], [11].
On the other hand, metformin (N,N-dimethylbiguanide) is an oral antidiabetic drug that is regularly used for the treatment of type 2 diabetes [12], [13]. In addition to its antidiabetic properties, metformin has been recently considered to be used for anticancer therapy [14]. Specifically, metformin was found to improve the chemotherapy response in diabetic patients with breast cancer [15]. Additionally, metformin has also been shown to inhibit in vitro cell growth of breast, colon, and prostate cancers, as well as gliomas [16], [17], [18], [19]. Moreover, metformin was reported to have antitumor effects in mouse xenografts [20], [21]. However, the cytotoxic efficacy of the combination of metformin and paclitaxel has not been assessed in human NSCLC cells.
In mammalian cells, to prevent DNA damage-induced cell death, the breaks must be repaired by different components of the DNA repair machinery [22], [23], [24]. Protein excision repair cross complementing 1 (ERCC1), a DNA endonuclease required for repairing DNA damages, forms a heterodimer with XPF which acts as a 5′ endonuclease and executes the 5′ excision in the DNA strand [25]. The relationship between low ERCC1 expression and better response to platinum-based chemotherapy has already been confirmed in NSCLC [26], [27]. A possible implication of ERCC1 in decreasing sensitivity to platinum agents has been reported because of its ability to repair platinum-induced DNA breaks [28]. In addition, the p38 MAPK signaling-mediated ERCC1 expression was shown to play a protective role in human lung cancer cells that were exposed to a topoisomerase II inhibitor, etoposide [29]. The p38 MAPK activation in response to paclitaxel has also been observed in both human breast cancer cell line MCF-7 [30] and HeLa cells [31]. However, whether p38 MAPK signaling is also involved in the paclitaxel-induced cytotoxic effect and ERCC1 expression in NSCLC is yet to be defined.
In this study, we used 2 human NSCLC cell lines challenged with physiological concentrations of paclitaxel, and we showed that paclitaxel induced ERCC1 mRNA and protein expression accompanied with p38 MAPK activation. Moreover, metformin was found to reduce viability of paclitaxel-treated H1650 and H1703 cells via ERCC1 downregulation as well as decreased DNA repair capacity mediated by the p38 MAPK inactivation. Overall, our results suggest that paclitaxel-mediated p38 MAPK activation plays a role in ERCC1 expression and thereby increases both DNA repair ability and viability of NSCLC cells.
Section snippets
Cell lines and reagents
Human lung carcinoma H1650 and H1703 cell lines obtained from the American Type Culture Collection (Manassas, VA) were cultured at 37 °C in a humidified atmosphere containing 5% CO2 in RPMI-1640 complete medium, supplemented with sodium bicarbonate (2.2%, w/v), l-glutamine (0.03%, w/v), penicillin (100 units/mL), streptomycin (100 μg/mL), and fetal calf serum (10%).
Metformin, paclitaxel, mithramycin A, and cycloheximide were purchased from Sigma Chemical (Sigma–Aldrich, St. Louis, MO), whereas
Paclitaxel increased protein and mRNA levels of phospho-MKK3/6-p38 MAPK and ERCC1 in NSCLC cell lines
We first examined the effect of paclitaxel on the ercc1 mRNA expression in 2 NSCLC cell lines, H1650 and H1703. Paclitaxel (0.1–1 μM) was added to H1650 or H1703 cells for 4–24 h, and total RNA was isolated and subjected to real-time PCR and RT-PCR analyses. As shown in Fig. 1A and B, paclitaxel increased endogenous ercc1 mRNA levels in human lung cancer cells. Moreover, paclitaxel stimulated ERCC1 protein expression, which was accompanied by increased protein levels of phosphorylated MKK3/6-p38
Discussion
In this current report, we provide evidence for a novel mechanism, wherein paclitaxel activates the p38 MAPK pathway and subsequently mediates ERCC1 upregulation, which ultimately plays a protective role in paclitaxel-induced cytotoxicity. The results of our studies show that either a pharmacological p38 MAPK inhibitor or specific p38-siRNA enhanced the paclitaxel-induced cytotoxic effect in human lung cancer cells. On the other hand, previous study indicates that paclitaxel combined with the
Acknowledgements
We thank Dr. Jia-Ling Yang for providing us with expression plasmids for transfection. This work was supported by the National Science Council of Taiwan, Grants no. NSC NSC 99-2320-B-415-001-MY3 (to Y.-W. Lin).
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These authors contributed equally.