Elsevier

Biochemical Pharmacology

Volume 85, Issue 4, 15 February 2013, Pages 583-594
Biochemical Pharmacology

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

https://doi.org/10.1016/j.bcp.2012.12.001Get rights and content

Abstract

Metformin, an extensively used and well-tolerated drug for treating individuals with type 2 diabetes, has recently gained significant attention as an anticancer drug. On the other hand, paclitaxel (Taxol) is a new antineoplastic drug that has shown promise in the treatment of non-small cell lung cancer (NSCLC). High expression levels of excision repair cross-complementary 1 (ERCC1) in cancers have been positively associated with the DNA repair capacity and a poor prognosis in NSCLC patients treated with platinum-containing chemotherapy. In this current study, paclitaxel was found to increase phosphorylation of mitogen-activated protein kinase (MAPK) kinase 3/6 (MKK3/6)-p38 MAPK as well as protein and mRNA levels of ERCC1 in H1650 and H1703 cells. Moreover, paclitaxel-induced ERCC1 protein and mRNA levels significantly decreased via the downregulation of p38 activity by either a p38 MAPK inhibitor SB202190 or p38 knockdown with specific small interfering RNA (siRNA). Specific inhibition of ERCC1 with siRNA was found to enhance the paclitaxel-induced cytotoxic effect and growth inhibition. Furthermore, metformin was able to not only decrease the paclitaxel-induced p38 MAPK-mediated ERCC1 expression, but also augment the cytotoxic effect induced by paclitaxel. Finally, expression of constitutive activate MKK6 or HA-p38 MAPK vectors in lung cancer cells was able to abrogate ERCC1 downregulation by metformin and paclitaxel as well as cell viability and DNA repair capacity. Overall, our results suggest that inhibition of the p38 MAPK signaling by metformin coupled with paclitaxel therapy in human NSCLC cells may be a clinically useful combination, which however will require further validation.

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).

References (55)

  • D.M. Miller et al.

    Mithramycin selectively inhibits transcription of G-C containing DNA

    Am J Med Sci

    (1987)
  • J.P. MacKeigan et al.

    MEK inhibition enhances paclitaxel-induced tumor apoptosis

    J Biol Chem

    (2000)
  • S.V. Bava et al.

    Akt is upstream and MAPKs are downstream of NF-kappaB in paclitaxel-induced survival signaling events, which are down-regulated by curcumin contributing to their synergism

    Int J Biochem Cell Biol

    (2011)
  • S.Y. Oh et al.

    ERK activation by thymosin-beta-4 (TB4) overexpression induces paclitaxel-resistance

    Exp Cell Res

    (2006)
  • X.H. Zhang et al.

    Synergistic antitumor efficacy of sequentially combined paclitaxel with sorafenib in vitro and in vivo NSCLC models harboring KRAS or BRAF mutations

    Cancer Lett

    (2012)
  • W.H. Gotlieb et al.

    In vitro metformin anti-neoplastic activity in epithelial ovarian cancer

    Gynecol Oncol

    (2008)
  • C.H. Bae et al.

    AMPK induces MUC5B expression via p38 MAPK in NCI-H292 airway epithelial cells

    Biochem Biophys Res Commun

    (2011)
  • C.H. Hou et al.

    AMP-activated protein kinase is involved in COX-2 expression in response to ultrasound in cultured osteoblasts

    Cell Signal

    (2008)
  • M.Y. Chang et al.

    AICAR induces cyclooxygenase-2 expression through AMP-activated protein kinase-transforming growth factor-beta-activated kinase 1-p38 mitogen-activated protein kinase signaling pathway

    Biochem Pharmacol

    (2010)
  • A. Jemal et al.

    Cancer statistics

    CA Cancer J Clin

    (2009)
  • B.R. Goldspiel

    Clinical overview of the taxanes

    Pharmacotherapy

    (1997)
  • F.A. Holmes et al.

    Phase II trial of Taxol, an active drug in the treatment of metastatic breast cancer

    J Natl Cancer Inst

    (1991)
  • K. Torres et al.

    Mechanisms of Taxol-induced cell death are concentration dependent

    Cancer Res

    (1998)
  • S.S. Bacus et al.

    Taxol-induced apoptosis depends on MAP kinase pathways (ERK and p38) and is independent of p53

    Oncogene

    (2001)
  • P.Y. Chan-Hui et al.

    Human mitogen-activated protein kinase kinase kinase mediates the stress-induced activation of mitogen-activated protein kinase cascades

    Biochem J

    (1998)
  • S. Correia et al.

    Metformin protects the brain against the oxidative imbalance promoted by type 2 diabetes

    Med Chem

    (2008)
  • S. Correia et al.

    Mechanisms of action of metformin in type 2 diabetes and associated complications: an overview

    Mini Rev Med Chem

    (2008)
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