Selective cytotoxic and anti-metastatic activity in DU-145 prostate cancer cells induced by Annona muricata L. bark extract and phytochemical, annonacin

Abstract

Background Annona muricata L. was identified as a popular medicinal plant in treatment regimens among cancer patients in Jamaica by a previously conducted structured questionnaire. Ethnomedically used plant parts, were examined in this study against human prostate cancer cells for the first time and mechanisms of action elucidated for the most potent of them, along with the active phytochemical, annonacin. Methods Nine extracts of varying polarity from the leaves and bark of A. muricata were assessed initially for cytotoxicity using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay on PC-3 prostate cancer cells and the ethyl acetate bark (EAB) extract was identified as the most potent. EAB extract was then standardized for annonacin content using High-performance Liquid Chromatography - Mass Spectrometry (HPLC-MS) and shown to be effective against a second prostate cancer cell line (DU-145) also. The mode of cell death in DU-145 cells were assessed via several apoptotic assays including induction of increased reactive oxygen species (ROS) production, reduction of mitochondrial membrane potential, activation of caspases and annexin V externalization combined with morphological observations using confocal microscopy. In addition, the potential to prevent metastasis was examined via inhibition of cell migration, vascular endothelial growth factor (VEGF) and angiogenesis using the chorioallantoic membrane assay (CAM). Results Annonacin and EAB extract displayed selective and potent cytotoxicity against the DU-145 prostate carcinoma cells with IC50 values of 0.1 ± 0.07 μM and 55.501 ± 0.55 μg/mL respectively, without impacting RWPE-1 normal prostate cells, in stark contrast to chemotherapeutic docetaxel which lacked such selectivity. Docetaxel’s impact on the cancerous DU-145 was improved by 50% when used in combination with EAB extract. Insignificant levels of intracellular ROS content, depolarization of mitochondrial membrane, Caspase 3/7 activation, annexin V content, along with stained morphological evaluations, pointed to a non-apoptotic mode of cell death. The extract at 50 μg/mL deterred cell migration in the wound-healing assay, while inhibition of angiogenesis was displayed in the CAM and VEGF inhibition assays for both EAB (100 μg /mL) and annonacin (0.5 μM). Conclusions Taken together, the standardized EAB extract and annonacin appear to induce selective and potent cell death via a necrotic pathway in DU-145 cells, while also preventing cell migration and angiogenesis, which warrant further examinations for mechanistic insights and validity in-vivo.

Antitumoral effect of Striga hermonthica (Delile) Benth. methanolic extract is mediated by alterations on procaspase-3 and cyclin B expression in prostate cancer cell lines

“…Cytotoxicity activities of plants extracts were described in previous investigations on prostate cancer and/or urinary tract related-diseases (Turan et al, 2015;Foster et al, 2020). Some plant species showed anti-proliferative activities against various prostate cancer cell lines (DU-145, PC-3, and LNCaP, Pca and hTert-PrEC) (Shabbeer et al, 2009;Petiwala et al, 2013;Foster et al, 2020), respectively, Trigonella foenum-graecum (IC 50 = 100 µg/mL on MDA-MB-231), Rosmarinus officinalis (IC 50 = 41.1 µg/mL on PC-3) and Annona muricata L (IC 50 = 0.1 µg/mL on DU145 and 55 µg/mL on DU145) using MTT Curcumim from Curcuma longa L. species exhibited an IC 50 between 0.68 and 0.78 µg/mL on prostate cancer cell lines (Chen et al, 2018). assay.…”

“…Tumor cells exhibited a resistance to apoptosis associated with the overexpression of procaspases. Thereafter, the reactivation of apoptotic cascades was a strategic approach to new anti-cancer drug discovery (Lamas et al, 1993;Saleli et al, 2019;Foster et al, 2020;Dell'Oste et al, 2021). The conversion of procaspase-3 to caspase-3 is a crucial node of apoptosis and is often considered as "the end executioner caspase of no return" for a cell.…”

Natural biomolecules and derivatives as anticancer immunomodulatory agents

“…Graviola leaf extract gavage of male rats increased apoptosis in the testes and glandular epithelium, decreased seminal index and reduced prostate size ( 212 ). Graviola synergized with doxorubicin to induce ROS production, apoptosis and potentially inhibit metastasis by downregulating VEGF ( 213 ). Furthermore, synergistic effects of graviola and low-dose radiation resulted in reduced TGF-β and CD44 expression, increased apoptosis, and overall reduced solid tumor burden in mouse xenografts ( 214 ).…”

Current Situation and Perspectives of Fruit Annonaceae in Mexico: Biological and Agronomic Importance and Bioactive Properties

“…Cytotoxic activity has been studied in fruit, leaves, root, and stem bark extracts or isolated acetogenins of some Annona species ( Table 4 ). Annonacin at a very low dose (extracted from A. muricata leaves) decreased the induced-tumors on mice [ 147 , 148 ]; moreover, acetogenins have a potent cytotoxicity against many cancer cell lines (DU-145, A-549, MCF-7, HT-29, A-498, PC-3, HL60, HCT, Caco-2, and HepG-2) [ 88 , 96 , 119 , 137 , 143 , 149 , 150 , 151 , 152 ]. The mechanism of action of acetogenins on cancer cell lines or in vivo cancer models is attributed to inhibition mitochondrial complex I (ubiquinone-linked NADH oxidase) to present in cell membranes; the ATP decrease cause apoptosis [ 123 ].…”