Original ArticlesSynergistic antitumor activity of withaferin A combined with oxaliplatin triggers reactive oxygen species-mediated inactivation of the PI3K/AKT pathway in human pancreatic cancer cells
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.
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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)
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These authors contributed equally to this work.