Antineoplastic properties of zafirlukast against hepatocellular carcinoma via activation of mitochondrial mediated apoptosis
Graphical abstract
Introduction
HCC is the fifth and seventh most common cause of cancer in men and women, respectively, and is the third most common cause of cancer-related death worldwide (Ferlay et al., 2010; Lin et al., 2016). The molecular mechanisms of HCC are still unknown, and thus medical doctors are challenged as the demand for HCC treatment increases each day. Synthetic chemotherapeutic agents have been shown to have good efficacy for HCC treatment, but researchers have achieved very little success due to chemo-resistance (Keshari et al., 2017). Sorafenib is the only drug of choice for HCC treatment, but it has a poor efficacy with possibility of resistance (Park, 2015; Wilhelm et al., 2006). Therefore, it is necessary to explore some newer anticancer drugs to prolong the survival of patients with HCC.
A previous literature review suggested that zafirlukast (ZAF) is used to cure asthma via agonistic action on cysteinyl leukotriene receptor-1 (CysLT1) (Kahnt et al., 2013). CysLT1 triggers colitis-associated colon cancer in a mouse model (Osman et al., 2017), and a CysLT1 antagonist, montelukast, inhibited tumor growth in a xenograft mouse model of colon cancer (Savari et al., 2013). Therefore, we questioned whether ZAF exerts antineoplastic effects since it is structurally similar to montelukast and has a similar mechanism of action, i.e. CysLT1 antagonistic properties. Before performing an in vivo study, we investigated the anti-HCC potential of ZAF using a human liver cancer cell line (Hep-G2 cells) and demonstrated its strong antiproliferative potential against Hep-G2 cells (IC50 ~13.95 μM) (Supplementary Data Sheet, Fig. S1). Inspired by the aforementioned finding, we speculated that ZAF might be an effective agent for the treatment of HCC.
An earlier report documented that oxidative stress and liver injury have important roles in HCC development and that the carcinogenic action of diethylnitrosamine (DEN) has a specific role in inflammation and oxidative stress in liver tissue (Kumar et al., 2015). Therefore, to evaluate the molecular mechanism of the anti-HCC potential of ZAF, we performed an in vivo experiment using a DEN-induced HCC rat model. Various apoptotic (caspases-3 and 9) and inflammatory (IL-2, IL-6, IL-10 and IL-1β) mediators in liver tissues were analyzed through enzyme linked immunosorbent assay (ELISA). Apoptotic markers exhibited promising effects as compared with inflammatory mediators, and thus, we decided to perform a mechanistic study on apoptotic pathways using gene and protein expression analyses at the molecular level. Later, a proton nuclear magnetic resonance (1H-NMR)-based serum metabolomics study was performed to differentiate the metabolic perturbations associated with HCC before and after ZAF treatment.
Section snippets
Drugs and reagents
All chemicals were purchased from Sigma-Aldrich, Bangalore and Loba Chemicals, New Delhi, India. The chemicals used in qRT-PCR and Western blot were procured from Genetix Biotech Asia Pvt. Ltd., New Delhi, and Thermo-Fisher Scientific, Bangalore, India. All the solvents and chemicals were analytical grade with 99% purity; in-house distilled water was used for all experiments.
Experimental design
Male albino Wistar rats (100–120 g) were used for this experiment (Approval No 1648/PO/Re/S/12/CPCSEA/54). Animals were
Evaluation of physiological and biochemical parameters, catabolic by-products in the liver and various enzyme levels in the serum
Various physiological parameters (body and liver weights, tumor incidence) were improved after oral ZAF administration (Table 1). It is obvious from the data that the DEN-treated groups experienced a major loss in body weight and improved condition after ZAF treatment, particularly at the 80 mg/kg dose. Similar trends were found for liver weight and tumor incidence (Table 1).
In the later stage, various oxidative stress parameters were measured in liver tissue to evaluate the antioxidant
Discussion
The liver plays a vital role in various biochemical and physiological actions in the human body, and for the body to be healthy, the liver should always function properly. HCC has been the third most common cause of cancer-related death worldwide in the past five years (Ferlay et al., 2010; Lin et al., 2016), but chemotherapy against HCC is limited due to systemic toxicities and various side effects (Anadol et al., 2012). Sorafenib is the only FDA-approved drug for HCC, but it has a poor
Conclusion
The discovery of a novel drug for HCC treatment has been very difficult since the molecular mechanism of HCC is still unknown. According to the current demands of HCC treatment, we proposed a cellular function for ZAF in HCC treatment at the molecular level. The current study supported a clear link among biochemical, pathophysiological, molecular and metabolic parameters after treatment with 5-FU and ZAF. The molecular insights observed in this study could explain the role of ZAF in HCC
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
Dr. Sudipta Saha would like to thank to UGC, India, for MRP grant [Project no. 42-680/2013(SR)] and DST, India for SERB project (Ref. No. DST/SB/EMEQ-320/2014). The authors also express their sincere gratitude to CBMR, Lucknow for providing the NMR facilities. Mr. Pranesh Kumar acknowledged DST India for the INSPIRE Fellowship (DST/INSPIRE Fellowship/2016/IF160364).
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All the authors contributed equally.