Cancer Letters

Cancer Letters

Volume 370, Issue 2, 28 January 2016, Pages 185-197
Cancer Letters

Original Articles
In vitro anticancer properties and biological evaluation of novel natural alkaloid jerantinine B

https://doi.org/10.1016/j.canlet.2015.10.013Get rights and content

Highlights

Abstract

Natural products play a pivotal role in medicine especially in the cancer arena. Many drugs that are currently used in cancer chemotherapy originated from or were inspired by nature. Jerantinine B (JB) is one of seven novel Aspidosperma indole alkaloids isolated from the leaf extract of Tabernaemontana corymbosa. Preliminary antiproliferative assays revealed that JB and JB acetate significantly inhibited growth and colony formation, accompanied by time- and dose-dependent apoptosis induction in human cancer cell lines. JB significantly arrested cells at the G2/M cell cycle phase, potently inhibiting tubulin polymerisation. Polo-like kinase 1 (PLK1; an early trigger for the G2/M transition) was also dose-dependently inhibited by JB (IC50 1.5 µM). Furthermore, JB provoked significant increases in reactive oxygen species (ROS). Annexin V+ cell populations, dose-dependent accumulation of cleaved-PARP and caspase 3/7 activation, and reduced Bcl-2 and Mcl-1 expression confirm apoptosis induction. Preclinical in silico biopharmaceutical assessment of JB calculated rapid absorption and bioavailability >70%. Doses of 8–16 mg/kg JB were predicted to maintain unbound plasma concentrations >GI50 values in mice during efficacy studies. These findings advocate continued development of JB as a potential chemotherapeutic agent.

Introduction

Natural products (NPs) have historically been used in the treatment of many diseases and continue to provide pharmacological agents used in pharmaceutical, biological and medical fields [1]. NPs are known for their structural diversity and have played inspirational roles in drug discovery [2]; ~50% of pharmaceuticals are derived from natural sources [3]. Thus it is important to pursue NP drug discovery to identify novel molecules from fragile rainforest habitats which may possess anticancer activity. Success in this field has led to the use of microtubule targeting agents (MTAs) in cancer chemotherapy. Vincristine and vinblastine, isolated from Catharanthus roseus, and taxol, isolated from the Pacific Yew, Taxus brevifolia, remain integral to the treatment of haematological and intractable solid cancers [4]. MTAs can act as stabilising agents (e.g. paclitaxel), promoting microtubule assembly, or destabilising agents (e.g. vincristine) to promote microtubule disassembly, altering microtubule dynamicity by binding to tubulin dimers [5]. Efficacy in the treatment of cancer arises from their ability to bind to tubulin and specifically inhibit mitosis [6], [7]. The cell cycle is governed by a rigorous system of checkpoints that consists of cyclin-dependent kinase (CDK)-cyclin complexes that allow progression from one cell cycle phase to the next [8], [9], [10]. Taxanes and vinca alkaloids arrest cell cycle at G2/M, affecting levels of cyclin B1 [8], [11], [12]. In 2008, seven novel Aspidosperma indole alkaloids were isolated, jerantinines A–G, from a leaf extract of the Malayan plant Tabernaemontana corymbosa [13]. Jerantinine A (JA) evoked potent inhibitory activity against human-derived cancer cells causing profound G2/M block and clear inhibition of tubulin polymerisation [8]. Similarly, jerantinine E (JE) was shown to disrupt microtubules in PtK2 kidney cells [14]. Herein, we report in vitro biological evaluation of JB, a structural analogue of JA. In vitro activities of JB and its acetate derivative have been examined in human-derived colorectal (HCT-116), breast (MCF-7), lung (A549), pancreatic (MIA PaCa-2), vincristine resistant (VR) HCT-116 carcinoma cell lines and MRC5 fibroblasts by MTT assays. Cell counts, clonogenic assays, cell cycle analyses, confocal microscopy, annexin-V/PI apoptosis, caspase 3/7 activity assays and Western blots have been undertaken. Tubulin polymerisation, PLK1 activity and generation of reactive oxygen species (ROS) were also assessed in efforts to elucidate mechanisms of action of JB. In addition, the physicochemical and pharmacokinetic properties of JB were assessed in silico.

Section snippets

Isolation and characterisation of JB and JBA

JB (Fig. 1) was isolated from T. corymbosa leaf extract. JBA (Fig. 1) was prepared by dissolving 20 mg (0.050 mM) in 1 mL of pyridine and 1 mL acetic anhydride. The mixture was stirred for 20 min. Water (10 mL) was added, and pH adjusted to 9 using 10% Na2CO3 solution [13].

Agent stocks

JB and JBA were provided as solids and reconstituted with DMSO to yield concentrations of 10 mM. Stocks were stored as 10 µL aliquots at −80 °C protected from light.

Cell culture

Cells were passaged twice weekly upon reaching 70–80%

MTT assay

Both JB and JBA revealed potent growth inhibitory activity against five cell lines (Fig. 2; Table 1). GI50 values for JB were between 0.7 and 0.9 µM for HCT-116, A549 and MCF-7. However, JBA exhibited more potent growth inhibitory activity against HCT-116, A549 and MCF-7 than JB with GI50 values ranging between 0.36 and 0.6 µM. MIA PaCa-2 cells showed the greatest sensitivity to JB and JBA with GI50 values ~0.25 µM. 2-Way ANOVA between control and treatment groups revealed significant (p

Discussion

JB, a novel aspidosperma indole alkaloid, and its acetate derivative revealed profound growth inhibitory and cytotoxic activity against human-derived HCT-116, VR HCT-116, MCF-7, A549 and MIA PaCa-2 cancer cell lines. Generally, JBA exhibited greater potency against HCT-116, A549 and MCF-7 than JB in the MTT assay. JBA's enhanced potency may be attributed to its stability when compared to JB, which may undergo hydrolysis and degradation thereby impacting activity [8]. Additionally, the presence

Conflict of interest

None.

Acknowledgements

The authors would like to thank Dr. Cornelia de Moor, Dr. Hilary Collins and International Centre for Kinase Profiling in University of Dundee. The VR HCT-116 cell line was generated by V.J. Raja. T.S. Kam and K.H. Lim thank MOHE, Malaysia (HIR-005), for financial support. M.E. Qazzaz thanks the Higher Committee for Education Development (D11000279) in Iraq (HCED). Data represented in Fig. 11, Fig. 12 were generated by GastroPlus™ software provided by Simulations Plus, Inc, Lancaster,

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