Antineoplastic properties of zafirlukast against hepatocellular carcinoma via activation of mitochondrial mediated apoptosis

https://doi.org/10.1016/j.yrtph.2019.104489Get rights and content

Highlights

  • Anti-HCC potential of zafirlukast.

  • Zafirlukast triggers iNOS/eNOS mediated mitochondrial apoptosis pathway.

  • Restoration of perturbated metabolites to normal using 1H-NMR based metabolomics.

Abstract

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwideand haslimited treatment options. In view of this, zafirlukast (ZAF) was administered orally to DEN-induced HCC rats to evaluate its antineoplastic properties. ELISA, qRT-PCR and Western blot were used to determine the molecular mechanism associated with ZAF therapy for HCC. We found that HCC developed as a result of lower expression of caspases 3 and 9, but their levels returned to normal when the expression of eNOS, BAX, BAD, and Cyt C was decreased and when the expression of iNOS, Bcl-xl, and Bcl-2 was increased. Again, ZAF (80 mg/kg dose) treatment normalized the expression of caspase-mediated apoptotic factors, i.e. BAX and Bcl-2 proteins, as established through Western blot analysis. Later, 1H NMR-based serum metabolomics study revealed that levels of perturbed metabolites in DEN-induced rat serum returned to normal after ZAF administration. Altogether, the antineoplastic potential of ZAF was found to be comparable, and to some degree better, than the marketed chemotherapeutic 5-flurouracil, which may be beneficial for anti-HCC treatment from a future drug design perspective.

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).

References (48)

  • A.K. Singh

    Isolated mangiferin and naringenin exert antidiabetic effect via PPARγ/GLUT4 dual agonistic action with strong metabolic regulation

    Chem. Boil. Interact.

    (2018)
  • H.N. Xu

    Is higher lactate an indicator of tumor metastatic risk? A pilot MRS study using hyperpolarized 13C-pyruvate

    Acad. Radiol.

    (2014)
  • M.C. Yu

    Alkaline phosphatase: does it have a role in predicting hepatocellular carcinoma recurrence?

    J. Gastrointest. Surg.

    (2011)
  • E. Anadol

    The changes of inducible nitric oxide synthase, endothelial nitric oxide synthase and cyclooxygenase-2 mRNA expressions in intrauterine tissues of pregnant rats

    Ankara Univ. Vet. Fak. Derg.

    (2012)
  • A. Bhattacharyya

    Oxidative stress: an essential factor in the pathogenesis of gastrointestinal mucosal diseases

    Physiol. Rev.

    (2014)
  • M. Frederich

    Metabolomics as a challenging approach for medicinal chemistry and personalized medicine

    J. Med. Chem.

    (2016)
  • J. Ferlay

    Estimates of worldwide burden of cancer in 2008: globocan 2008

    Int. J. Cancer

    (2010)
  • A. Fages

    Metabolomic profiles of hepatocellular carcinoma in a European prospective cohort

    BMC Med.

    (2015)
  • H. Gao

    Application of 1H NMR‐based metabonomics in the study of metabolic profiling of human hepatocellular carcinoma and liver cirrhosis

    Cancer Sci.

    (2009)
  • A.I. Jafari

    Evaluation of Bcl-2 family gene expression and Caspase-3 activity in hippocampus STZ-induced diabetic rats

    Exp. Diabetes Res.

    (2008)
  • A. Karaman

    Hepatic damage in biliary-obstructed rats is ameliorated by leflunomide treatment

    Pediatr. Surg. Int.

    (2006)
  • A.K. Keshari

    5H-benzo [h] thiazolo [2, 3-b] quinazolines ameliorate NDEA-induced hepatocellular carcinogenesis in rats through IL-6 downregulation along with oxidative and metabolic stress reduction

    Drug Des. Dev. Ther.

    (2017)
  • A. Kumar

    Silibinin inhibits the hepatocellular carcinoma in NDEA-induced rodent carcinogenesis model: an evaluation through biochemical and bio-structural parameters

    J. Cancer Sci. Ther.

    (2015)
  • P. Kumar

    6, 7-dimethoxy-1, 2, 3, 4-tetrahydro-isoquinoline-3-carboxylic acid attenuates heptatocellular carcinoma in rats with NMR-based metabolic perturbations

    Future Sci. OA

    (2017)
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