Cancer Letters

Cancer Letters

Volume 357, Issue 1, 1 February 2015, Pages 219-230
Cancer Letters

Original Articles
Synergistic antitumor activity of withaferin A combined with oxaliplatin triggers reactive oxygen species-mediated inactivation of the PI3K/AKT pathway in human pancreatic cancer cells

https://doi.org/10.1016/j.canlet.2014.11.026Get rights and content

Highlights

  • The combination of oxaliplatin and withaferin A has a synergistic antitumor activity in PC cells in vitro and in vivo.

  • This synergistic effect occurred primarily through increased generation of intracellular ROS.

  • The generation of ROS was involved in the induction of mitochondrial dysfunction and inhibition of PI3K/AKT signaling.

Abstract

Application of oxaliplatin for the treatment of pancreatic cancer (PC) is restricted owing to its toxic side effects and drug resistance. We investigated how withaferin A (WA), a bioactive component isolated from the medicinal plant Withania somnifera, acts synergistically with oxaliplatin on human PC in vitro and in vivo. We found that WA enhanced oxaliplatin-induced growth suppression and apoptosis in PC cells dramatically through a mechanism involving mitochondrial dysfunction and inactivation of the PI3K/AKT pathway. Combination treatment resulted in significant accumulation of intracellular reactive oxygen species (ROS). Pretreatment of cells with the ROS scavenger N-acetylcysteine completely blocked the apoptosis induced by combination treatment, and recovered expression of AKT inactivation, which revealed the important role of ROS in apoptosis and AKT regulation. In vivo, combination therapy showed the strongest anti-tumor effects compared with single agents, without obvious additional toxicity. These results support the notion that combination treatment with oxaliplatin and WA could facilitate development of an effective strategy for PC treatment.

Introduction

Pancreatic cancer (PC) carries a very poor prognosis. Despite advances in early diagnosis and treatment, 5-year survival is <5% and median survival is 6 months [1]. Resection can be curative, but 80% of patients have locally advanced or metastatic cancer at the time of diagnosis and only 10–15% of patients are eligible for surgery [2]. Thus, chemotherapy remains the mainstay for management of PC.

The nucleoside analog gemcitabine is considered first-line palliative treatment. However, it improves only the symptoms of PC and has failed to show clinically significant survival benefit [3]. The platinum-based compound oxaliplatin has been used frequently to treat PC that is refractory to gemcitabine therapy [4], [5], [6]. Paradoxically, a high dose of oxaliplatin eliminates PC effectively but causes severe side effects, whereas a low dose of oxaliplatin elicits a poor response and drug resistance (“chemoresistance”).

Recently, combination therapies have been developed to reduce side effects and overcome chemoresistance. Combination of oxaliplatin with conventional chemotherapy drugs such as gemcitabine and fluorouracil has improved the survival of PC patients [7], [8]. Moreover, oxaliplatin has been combined with the poly (adenosine diphosphate-ribose) polymerase-1 inhibitor BSI-401 to achieve enhanced PC cell death while attenuating oxaliplatin-induced acute neurotoxicity [9]. Therefore, evaluation of chemotherapy agents that could improve the efficacy of oxaliplatin without additional toxicity to normal tissue would lead to novel therapeutic applications against PC.

Withaferin A (WA) is a purified steroidal lactone isolated from the plant Withania somnifera. WA has been used for centuries in Indian Ayurvedic medicine, and is available as a dietary supplement in the USA owing to its anti-inflammatory and antibacterial effects. The antitumor activity of WA has been identified in various types of cancer cells [10]. The mechanisms of action of WA have yet to be elucidated fully, but the following phenomena are involved in the growth-inhibitory and pro-apoptotic effects of WA: inactivation of nuclear factor-kappa B (NF-κB) and AKT pathways; induction of expression of Par-4, FOXO3a, and Bim; down-regulation of expression of estrogen receptor alpha and Notch-1; generation of reactive oxygen species (ROS) [11], [12], [13], [14]. Additionally, recent studies have shown that WA could be efficacious as an adjunct agent for enhancing the antitumor activity of chemotherapy drugs [15], [16], [17]. However, the combined effect of WA and oxaliplatin in PC has not been studied. Therefore, we evaluated the combined effect of oxaliplatin and WA on human PC cells in vitro and in vivo and investigated the molecular mechanisms involved.

Section snippets

Cell culture and reagents

Human pancreatic cancer cell lines Panc-1, MIAPaCa-2, and SW1990 were purchased from American Type Culture Collection (Manassas, VA, USA). The Immortalized human pancreatic ductal epithelial cell HPDE was obtained from Beijing North Carolina Chuanglian Biotechnology Research Institute (Beijing, China). All cells were maintained in Dulbecco's modified Eagle's medium (DMEM) or Roswell Park Memorial Institute 1640 (RPMI-1640) medium supplemented with 10% fetal bovine serum (FBS), 2 mmol/L of

Combination of oxaliplatin and WA inhibited proliferation of PC cells in a synergistic manner

The effect of oxaliplatin or WA on PC cells was evaluated by the MTS assay. Oxaliplatin and WA decreased the survival of three human PC cell lines (Panc-1, MIAPaCa-2, SW1990) in a dose- and time-dependent manner (Supplementary Fig. S1). Next, we evaluated the effect of the combination of oxaliplatin and WA on these cell lines. Interestingly, combination treatment significantly increased growth-inhibitory effects on these cell lines compared with the agent alone (Fig. 1B). Conversely, the

Discussion

Combination with agents that sensitize cancer cells to conventional chemotherapy drugs has gained much attention recently in efforts to reduce side effects and chemoresistance. Naturally occurring products from medicinal plants or diets are, in general, safe and have low toxicity, making them ideal candidates of “chemotherapy sensitizers” for PC therapy [34], [35].

Here, we investigated if treatment of human PC cells with WA (a bioactive component isolated from Withania somnifera) could act with

Authors' contributions

  • Conception and design: X. Li, F. Zhu, J. Jiang, M. Wang, R. Qin

  • Development of methodology: R. Qin, X. Li, M. Wang,

  • Acquisition of data (provided animals, provided facilities, etc.): X. Li, F. Zhu, R. Tian, F. Peng, X. Guo, C. Shi, M. Xu, R. Qin

  • Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): X. Li, J. Jiang, R. Qin

  • Drafting of the manuscript: X. Li, M. Wang, R. Qin

  • Administrative, technical, or material support (i.e., reporting or organizing

Conflict of interest

The authors do not have any possible conflicts of interest.

Acknowledgements

This study was funded by The National Natural Science Foundation of China (No. 81272659) to R. Qin, (No. 81101621) to M. Wang, (No. 81160311) to J. Jiang, (No. 81372353) to X. Wang, (No. 81172064) to M. Shen and (No. 81301860) to C. Shi; National “Eleventh Five-Year” Scientific and Technological Support Projects (No. 2006BAI02A13-402) to Renyi Qin; Projects of Science Foundation of Hubei Province (No. 2012FFB02401) to Feng Zhu; International S&T Cooperation Projects of China (No. 2014DFA31420)

References (50)

  • H. Oettle et al.

    Adjuvant chemotherapy with gemcitabine vs observation in patients undergoing curative-intent resection of pancreatic cancer: a randomized controlled trial

    JAMA

    (2007)
  • H.L. Kindler et al.

    Gemcitabine plus bevacizumab compared with gemcitabine plus placebo in patients with advanced pancreatic cancer: phase III trial of the Cancer and Leukemia Group B (CALGB 80303)

    J. Clin. Oncol

    (2010)
  • N. Androulakis et al.

    Oxaliplatin for pretreated patients with advanced or metastatic pancreatic cancer: a multicenter phase II study

    Cancer Invest

    (2005)
  • M.W. Saif et al.

    Role of platinum agents in the management of advanced pancreatic cancer

    Expert Opin. Pharmacother

    (2007)
  • A. Novarino et al.

    Oxaliplatin, 5-fluorouracil, and leucovorin as second-line treatment for advanced pancreatic cancer

    Am. J. Clin. Oncol

    (2009)
  • K.H. Lee et al.

    Gemcitabine and oxaliplatin combination as first-line treatment for advanced pancreatic cancer: a multicenter phase II study

    Cancer Chemother. Pharmacol

    (2009)
  • D. Melisi et al.

    Oral poly(ADP-ribose) polymerase-1 inhibitor BSI-401 has antitumor activity and synergizes with oxaliplatin against pancreatic cancer, preventing acute neurotoxicity

    Clin. Cancer Res

    (2009)
  • S. Srinivasan et al.

    Par-4-dependent apoptosis by the dietary compound withaferin A in prostate cancer cells

    Cancer Res

    (2007)
  • S.D. Stan et al.

    Withaferin A causes FOXO3a- and Bim-dependent apoptosis and inhibits growth of human breast cancer cells in vivo

    Cancer Res

    (2008)
  • S. Koduru et al.

    Notch-1 inhibition by Withaferin-A: a therapeutic target against colon carcinogenesis

    Mol. Cancer Ther

    (2010)
  • M.Y. Fong et al.

    Withaferin A synergizes the therapeutic effect of doxorubicin through ROS-mediated autophagy in ovarian cancer

    PLoS ONE

    (2012)
  • S.M. Cohen et al.

    A novel combination of withaferin A and sorafenib shows synergistic efficacy against both papillary and anaplastic thyroid cancers

    Am. J. Surg

    (2012)
  • E. Crescenzi et al.

    Low doses of cisplatin or gemcitabine plus Photofrin/photodynamic therapy: disjointed cell cycle phase-related activity accounts for synergistic outcome in metastatic non-small cell lung cancer cells (H1299)

    Mol. Cancer Ther

    (2006)
  • G. Kroemer et al.

    Mitochondrial membrane permeabilization in cell death

    Physiol. Rev

    (2007)
  • S.W. Tait et al.

    Mitochondria and cell death: outer membrane permeabilization and beyond

    Nat. Rev. Mol. Cell Biol

    (2010)
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