Crude Methanol Extract of Rosin Gum Exhibits Specific Cytotoxicity against Human Breast Cancer Cells via Apoptosis Induction

Abstract

Background: Rosin (Colophony) is a natural resin derived from species of the pine family Pinaceae. It has wide industrial applications including printing inks, photocopying paper, adhesives and varnishes, soap and soda. Rosin and its derivatives are employed as ingredients in various pharmaceutical products such as ointments and plasters. Rosin-based products contain allergens that may exert some occupational health problems such as asthma and contact dermatitis. Objective: Our knowledge on the pharmaceutical and medicinal properties of rosin is limited. The current study aims at investigating the cytotoxic potential of rosin-derived crude methanolic extract (RDCME) and elucidation of its mode-of-action against breast cancer cells (MCF-7 and MDAMB231). Methods: Crude methanol extract was prepared from rosin. Its phenolic contents were analyzed by reversed-phase High-Performance Liquid Chromatography (RP-HPLC). Antioxidant activity was evaluated by DPPH radical-scavenging assay. Antiproliferation activity against MCF-7 and MDA-MB231 cancerous cells was investigated by MTT assay; its potency compared with doxorubicin as positive control and specificity was assessed compared to two non-cancerous cell lines (BJ-1 and MCF-12F). Selected apoptosis protein markers were assayed by western blotting. Cell cycle analysis was performed by Annexin V-FITC/PI FACS assay. Results: RD-CME exhibited significant and selective cytotoxicity against the two tested breast cancer cells (MCF-7 and MDA-MB231) compared to normal cells as revealed by MTT assay. ELISA and western blotting indicated that the observed antiproliferative activity of RD-CME is mediated via the engagement of an intrinsic apoptosis signaling pathway, as judged by enhanced expression of key pro-apoptotic protein markers (p53, Bax and Casp 3) relative to vehicle solvent-treated MCF-7 control cells. Conclusion: To our knowledge, this the first report to investigate the medicinal anticancer and antioxidant potential of crude methanolic extract derived from colophony rosin. We provided evidence that RD-CME exhibits a strong antioxidant and anticancer effects. The observed cytotoxic activity against MCF-7 is proposed to take place via G2/M cell cycle arrest and apoptosis. Colophony resin has a great potential to join the arsenal of plant-derived natural anticancer drugs. Further thorough investigation of the potential cytotoxicity of RD-CME against various cancerous cell lines is required to assess the spectrum and potency of its novel activity.

Biochemical and Molecular Investigation of In Vitro Antioxidant and Anticancer Activity Spectrum of Crude Extracts of Willow Leaves Salix safsaf

“…Over the past two decades, our research group has published several reports that extended understanding of the antioxidant and anticancer properties of crude extracts of plants as well as their inherent active constituents and their modes-of-actions [ 14 , 15 , 17 , 19 , 25 , 26 , 27 , 28 , 29 , 30 ]. In this context, we were the first to report that aqueous and ethanolic crude extracts derived from young, emerging leaves of willow ( S. safsaf ) exhibit potent anti-leukemic properties [ 14 , 15 ].…”

“…Caspases are effector proteins that are vital for initiation and sustaining apoptosis. Upregulation of caspase 3 protein and mRNA transcripts ( Figure 8 A,B) is indicative of execution of the main intrinsic pathway of apoptosis; which is characterized by the collapse of the mitochondrial membrane with Bax-induced cytochrome c release, and activation of caspase 9 leading to the subsequent engagement of caspase 3 [ 17 , 19 ]. Up-regulation of Casp3, p53 and Bax, along with reduction of Bcl-2, expression in our study reveals that F3 induces the intrinsic pathway of apoptosis, where upregulation of p53 will stimulate expression of Bax, which, in turn, will induce cytochrome c release, followed by caspase-9 and -3 activation.…”

“…Early changes in cell surfaces, associated with apoptosis and cell cycle arrest of cancer cells, treated with willow organic-soluble fractions were investigated by use of flow cytometry with the FITC-Annexin V/Propidium Iodide (PI) Apoptosis Detection Kit I (BD Biosciences Company, NJ, USA) by use of fluorescence-activated cell sorter analysis (FACS) Canto II flow cytometry system as previously described by us [ 19 ]. MCF-7 cells were treated with IC 50 concentrations of F2 (128.1 µg/mL) and F3 (111.72 µg/mL) for 72 h. F2 and F3 were chosen as they exhibited the most potent anti-proliferation potential out of all the seven prepared S. salix fractions and extracts.…”

“…Western blot analyses were conducted as previously described [ 19 ]. Collected cells were frozen and then homogenized in extraction buffer containing 150 mM NaCl, 50 mM Tris-HCl pH 7.5, 10 mM MgCl 2 , 1 mM PMSF, 0.1% NP-40 and 1× complete protease inhibitor (Roche), by use of a stick “pellet pestle blue” (Sigma).…”

“…This second supernatant was transferred to a new tube and the protein content was quantified by the Bradford protein assay method (Bio-Rad ® ). Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunodetection of selected pro-apoptotic (p53, Bax, Caspase-3) and anti-apoptotic (Bcl-2) protein markers were essentially conducted as detailed in our recently published protocol [ 19 ].…”