New halogenated constituents from Mangifera zeylanica Hook.f. and their potential anti-cancer effects in breast and ovarian cancer cells.

Mangifera zeylanica Hook.f. (Anacardiaceae) is a plant endemic to Sri Lanka. Its bark has been used in traditional and Ayurvedic medicine for the treatment of various diseases including some cancers. This study was planned to isolate and identify potentially cytotoxic compounds from the bark of M. zeylanica, which may have contributed to its ethno pharmacological use in the treatment of cancer. The chloroform extract of M. zeylanica bark which is cytotoxic to breast and ovarian cancer cells was fractionated using column chromatography and preparative reversed phase high performance liquid chromatography to isolate four compounds. Structures of the isolated compounds were elucidated by means of (1)H- and (13)C NMR spectroscopy, and mass spectrometric techniques. Cytotoxic potential of the isolated compounds was tested in MDA-MB-231 (triple negative breast cancer), MCF-7 (estrogen receptor positive breast cancer), SKOV-3 (ovarian epithelial cancer) and MCF-10A (normal mammary epithelial) cells by SRB assay. Human cancer drug target real-time PCR array was carried out to analyze regulation of possible cancer drug target genes in compound 2 treated triple negative breast cancer cells. DPPH radical scavenging and caspase 3 and 7 induction in response to isolated compounds were also studied. Two new halogenated compounds, bromomangiferic acid (1), and chloromangiferamide (2) along with two known compounds quercetin (3), and catechin (4), were isolated from the bark of Mangifera zeylanica for the first time. Interestingly, chloromangiferamide showed cytotoxicity only to triple negative breast cancer cells [IC50:73.19±0.87µM (24h), 56.29±0.86µM (48h)] with no cytotoxicity to other two cancer cell lines or to normal mammary epithelial cells. Quercetin and catechin were cytotoxic to all three cancer cell lines while bromomangiferic acid had no effect. Chloromangiferamide significantly regulated expression of genes associated with apoptosis, drug metabolism, cell cycle, receptor tyrosine kinase signaling, protein kinases, histone deacetylases, growth factors and receptors, topoisomerases, PI-3 kinases and phosphatases in triple negative breast cancer cells. Selective cytotoxic activity in triple negative breast cancer cells and regulation of some cancer drug target genes by chloromangiferamide indicate that it can be used to develop a potential chemotherapeutic agent for triple negative breast cancer cells.

Ediriweera MK ，Tennekoon KH ，Adhikari A ，Samarakoon SR ，Thabrew I ，de Silva ED ... -  《-》

Isolation of a new resorcinolic lipid from Mangifera zeylanica Hook.f. bark and its cytotoxic and apoptotic potential.

Mangifera zeylanica is a plant endemic to Sri Lanka and its bark has been used in traditional medicine to treat some cancers. This study was aimed to isolate potentially cytotoxic compound/s from the hexane extract of the bark of M. zeylanica by bio-activity guided fractionation. The structure of the isolated compound (1) was elucidated using 1H, 13C NMR and mass spectrometric techniques. Compound 1 was identified as a new resorcinolic lipid (5-((8Z, 11Z, 14Z)-hexatriaconta-8, 11, 14-trienyl) benzene-1,3-diol). Apoptotic potential of the isolated compound was determined only in MCF-7 (estrogen receptor positive) breast cancer cells to which it was more cytotoxic than to normal mammary epithelial cells. Oxidative stress markers [reactive oxygen species (ROS), glutathione levels (GSH) and glutathione-S-transferase (GSH)] were also determined in MCF-7 cells treated with compound 1. Treatment with compound 1 led to an increase in caspase 7 activity, morphological features of apoptosis and DNA fragmentation in MCF-7 cells. Furthermore, it also led to an increase in ROS and GST levels while depleting GSH levels. Results of this study suggest that isolated new resorcinolic lipid can induce apoptosis in MCF-7 cells, possibly via oxidative stress mechanism.

Ediriweera MK ，Tennekoon KH ，Samarakoon SR ，Adhikari A ，Thabrew I ，Dilip de Silva E ... -  《-》

A study of the potential anticancer activity of Mangifera zeylanica bark: Evaluation of cytotoxic and apoptotic effects of the hexane extract and bioassay-guided fractionation to identify phytochemical constituents.

Ediriweera MK ，Tennekoon KH ，Samarakoon SR ，Thabrew I ，Dilip DE Silva E ... -  《-》

Chloroform Fraction of Methanolic Extract of Seeds of Annona muricata Induce S Phase Arrest and ROS Dependent Caspase Activated Mitochondria-Mediated Apoptosis in Triple-Negative Breast Cancer.

Triple Negative Breast Cancers (TNBCs) have high morbidity and shorter survival rate in the population. These types of cancers have high aggressiveness, lymphatic invasion, and absence of receptors. The treatment options for these types of cancers are also scarce. Several studies have been conducted to investigate the effectiveness of seeds of Annona muricata for its anti-cancer activities in various cancer cell lines, such as lung A549, breast MCF7, colon HT-29, oral KB, and human hepatoma cell lines. But works related to its anti-cancer effect and mechanism of action in TNBCs have not been elucidated. The present study was undertaken to evaluate the in vitro, in vivo, and in silico anti-cancer potential of chloroform fraction of methanolic extract of seeds of Annona muricata (CMAM) against TNBC along with elucidation of its mechanistic pathway. In vitro cytotoxicity- and antiproliferative- studies in three triple-negative breast cancer cell lines were conducted using the MTT and SRB assays, respectively. The mechanism through which CMAM exerts its pharmacological effect was elucidated in vitro employing cell morphological assessment studies using Acridine Orange/Ethidium Bromide (AO/EB), intracellular reactive oxygen species assay, DNA fragmentation assay, agarose gel electrophoresis, terminal deoxynucleotidyl Transferase dUTP Nick End Labeling (TUNEL) assay, cell cycle analysis, annexin binding assay, and caspase-activated mitochondria-mediated apoptotic assays using western blot. In vivo evaluation in 4T1 induced murine mammary tumor model was also conducted. Phytoconstituents in CMAM were analyzed using liquid chromatography mass spectroscopy. In silico binding studies with various annonaceous acetogenins against BCL-2 and cyclin E were performed. Cytotoxicity studies in MDA-MD-231, 4TI, and BT-549 revealed the IC50 value of CMAM to be 2.5±0.14, 4.8±0.3, and 4.5±0.16μg/mL, respectively. Anti-proliferative studies in 4T1, MDA-MB-231, and BT- 549 revealed the GI50 values to be 0.128+0.03, 18.03+0.20, 0.95+0.04μg/mL, respectively. CMAM exhibited its cytotoxicity through the lysis of cell membrane, ROS dependent caspase-activated mitochondria-mediated apoptosis, and arresting the S phase of the cell cycle. In vivo evaluation also supported the tumoricidal property of CMAM, as evidenced by a reduction in tumor volume and serum biomarkers. Histopathologically, there was a marked reduction in cellularity, nuclear chromatin condensation, and a few normal cells in the group treated with CMAM at a dose of 31mg/Kg. Phytoconstituent evaluation has revealed the presence of annonaceous acetogenins in CMAM. Among the various annonaceous acetogenins, muricatacin alone showed lipophilicity and binding affinity towards BCL-2 and cyclin E1. The current study shows the effectiveness of CMAM against TNBC both in vitro and in vivo. This anticancerous effect of CMAM could be by virtue of its ROS dependent caspase-activated mitochondriamediated apoptosis and the S-phase arrest of the cell cycle in the TNBCs. Our results indicate that the presence of annonaceous acetogenins, especially muricatacin, could be contributing to this anticancerous effect of CMAM. Thus, muricatacin could be a potential candidate for the targeted therapy of TNBCs.

Kariyil BJ ，Ayyappan UPT ，Gopalakrishnan A ，George AJ ... -  《-》

Isolation of a New Sesquiterpene Lactone From Vernonia Zeylanica (L) Less and its Anti-Proliferative Effects in Breast Cancer Cell Lines.

Vernonia zeylanica (L) less is an endemic plant to Sri Lanka. The present study was designed to isolate potential cytotoxic compound/s from chloroform and ethyl acetate extracts of V. zeylanica by bio-activity guided isolation and to evaluate its anti-proliferative effects in three breast cancer phenotypes (MCF -7, MDA-MB-231, SKBR-3). Combined chloroform and ethyl acetate extracts were subjected to chromatographic separations to isolate a compound (1) and the structure of the isolated compound was elucidated using 1H, 13C and mass spectroscopic techniques. Cytotoxic effects of the compound were evaluated by the sulforhodamine B (SRB) and the MTT (3- (4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays. Effects of the compound on apoptosis were evaluated by fluorescent microscopy, caspase 3/7 activation, DNA fragmentation and real time PCR. Effects of the compound on the expression of heat shock protein complex were also evaluated by real time PCR and immunofluorescence. Isolated compound was identified as a new sesquiterpene lactone (vernolactone). The compound mediated significant cytotoxic effects in SKBR-3 and MDA-MB-231 breast cancer cells, with little effect in MCF-7 and normal mammary epithelial MCF-10A cells. Morphological changes, DNA fragmentation, increased caspase 3/7 activities and up-regulation of p53, Bax and down regulation of Survivin confirmed the proapoptotic effects of the compound. Significant inhibition of HSP complex related genes were also observed in SKBR-3 and MDA-MB-231 breast cancer cells. Overall results indicate that vernolactone can mediate its cytotoxic effects via apoptosis and modulating the HSP complex.

Mendis AS ，Thabrew I ，Ediriweera MK ，Samarakoon SR ，Tennekoon KH ，Adhikari A ，de Silva ED ... -  《-》