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Induction of apoptosis through oxidative stress-related pathways in MCF-7, human breast cancer cells, by ethyl acetate extract of Dillenia suffruticosa.

ABSTRACT: BACKGROUND: Breast cancer is one of the most dreading types of cancer among women. Herbal medicine has becoming a potential source of treatment for breast cancer. Herbal plant Dillenia suffruticosa (Griff) Martelli under the family Dilleniaceae has been traditionally used to treat cancerous growth. In this study, the anticancer effect of ethyl acetate extract of D. suffruticosa (EADs) was examined on human breast adenocarcinoma cell line MCF-7 and the molecular pathway involved was elucidated. METHODS: EADs was obtained from the root of D. suffruticosa by using sequential solvent extraction. Cytotoxicity was determined by using MTT assay, mode of cell death by cell cycle analysis and apoptosis induction by Annexin-FITC/PI assay. Morphology changes in cells were observed under inverted light microscope. Involvement of selected genes in the oxidative stress-mediated signaling pathway was explored using multiplex gene expression analysis. RESULTS: The treatment of EADs caused cytotoxicity to MCF-7 cells in a dose- and time-dependent manner at 24, 48 and 72 hours with IC50 of 76?±?2.3, 58?±?0.7 and 39?±?3.6 ?g/mL, respectively. The IC50 of tamoxifen-treated MCF-7 cells was 8?±?0.5 ?g/mL. Induction of apoptosis by EADs was dose- and time- dependent. EADs induced non-phase specific cell cycle arrest at different concentration and time point. The multiplex mRNA expression study indicated that EADs-induced apoptosis was accompanied by upregulation of the expression of SOD1, SOD2, NF-?B, p53, p38 MAPK, and catalase, but downregulation of Akt1. CONCLUSION: It is suggested that EADs induced apoptosis in MCF-7 cells by modulating numerous genes which are involved in oxidative stress pathway. Therefore, EADs has the potential to act as an effective intervention against breast cancer cells.

SUBMITTER: Tor YS

PROVIDER: S-EPMC3927215 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Induction of apoptosis through oxidative stress-related pathways in MCF-7, human breast cancer cells, by ethyl acetate extract of Dillenia suffruticosa.

Tor Yin Sim YS Yazan Latifah Saiful LS Foo Jhi Biau JB Armania Nurdin N Cheah Yoke Kqueen YK Abdullah Rasedee R Imam Mustapha Umar MU Ismail Norsharina N Ismail Maznah M

BMC complementary and alternative medicine 20140214

<h4>Background</h4>Breast cancer is one of the most dreading types of cancer among women. Herbal medicine has becoming a potential source of treatment for breast cancer. Herbal plant Dillenia suffruticosa (Griff) Martelli under the family Dilleniaceae has been traditionally used to treat cancerous growth. In this study, the anticancer effect of ethyl acetate extract of D. suffruticosa (EADs) was examined on human breast adenocarcinoma cell line MCF-7 and the molecular pathway involved was elucid ...[more]

PMID: 24524627

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Project description:BackgroundDillenia suffruticosa root dichloromethane extract (DCM-DS) has been reported to exhibit strong cytotoxicity towards breast cancer cells. The present study was designed to investigate the cell cycle profile, mode of cell death and signalling pathways of DCM-DS-treated human caspase-3 deficient MCF-7 breast cancer cells.MethodsDillenia suffruticosa root was extracted by sequential solvent extraction. The anti-proliferative activity of DCM-DS was determined by using MTT assay. The mode of cell death was evaluated by using inverted light microscope and Annexin-V/PI-flow cytometry analysis. Cell cycle analysis and measurement of intracellular reactive oxygen species (ROS) were performed by using flow cytometry. MCF-7 cells were co-treated with antioxidants α-tocopherol and ascorbic acid to evaluate whether the cell death was mainly due to oxidative stress. GeXP-based multiplex system was employed to investigate the expression of apoptotic, growth and survival genes in MCF-7 cells. Western blot analysis was performed to confirm the expression of the genes.ResultsDCM-DS was cytotoxic to the MCF-7 cells in a time-and dose-dependent manner. The IC50 values of DCM-DS at 24, 48 and 72 hours were 20.3 ± 2.8, 17.8 ± 1.5 and 15.5 ± 0.5 μg/mL, respectively. Cell cycle analysis revealed that DCM-DS induced G0/G1 and G2/M phase cell cycle arrest in MCF-7 cells at low concentration (12.5 and 25 μg/mL) and high concentration (50 μg/mL), respectively. Although Annexin-V/PI-flow cytometry analysis has confirmed that DCM-DS induced apoptosis in MCF-7 cells, the distinct characteristics of apoptosis such as membrane blebbing, chromatin condensation, nuclear fragmentation and formation of apoptotic bodies were not observed under microscope. DCM-DS induced formation of ROS in MCF-7 cells. Nevertheless, co-treatment with antioxidants did not attenuate the cell death at low concentration of DCM-DS. The pro-apoptotic gene JNK was up-regulated whereby anti-apoptotic genes AKT1 and ERK1/2 were down-regulated in a dose-dependent manner. Western blot analysis has confirmed that DCM-DS significantly up-regulated the expression of pro-apoptotic JNK1, pJNK and down-regulated anti-apoptotic AKT1, ERK1 in MCF-7 cells.ConclusionDCM-DS induced cell cycle arrest and apoptosis in MCF-7 cells via multiple signalling pathways. It shows the potential of DCM-DS to be developed to target the cancer cells with mutant caspase-3.

Project description:Potentilla fulgens root traditionally used as a folk remedy in Meghalaya, India. However, systematic evaluation of its anticancer efficacy was limited. We investigated the anticancer potentials of the various extracts prepared by partitioning of the methanol extract of the root with the aim to discover major contributing factors from the most effective fractions. Methanol extract of P. fulgens roots (PRE) was prepared by maceration which was subsequently fractionated into hexane, ethyl-acetate (EA) and n-butanol soluble fractions. Various assays (clonogenic assay, Flow cytometry analysis, western blot, semiquantitative RT-PCR and the level of endogenous glutathione) were used to evaluate different parameters, such as Cell survivability, PARP-1 proteolysis, expression pattern of anti-apoptotic and γ-glutamyl-cysteine synthetase heavy subunit (GCSC) genes in both MCF-7 and U87 cancer cell lines. Since the EA-fraction showed most efficient growth inhibitory effect, it was further purified and a total of nine compounds and some monomeric and dimeric flavan-3-ols were identified and characterized. Three compounds viz., epicatechin (EC), gallic acid (GA) and ursolic acid (UA) were taken on the basis of their higher yield and 10 μg/ml of each was mixed together. The concentration used in this study for PRE, EA- and Hex-fraction was 100 μg/ml, which was higher than the IC50 value. Apoptotic cell death in the PRE, EA-fraction and EC+GA+UA treated cancer cell cultures was significantly greater than in normal cells due to suppression of anti-apoptotic protein Bcl2 following treatment. Depletion of glutathione by downregulating GCSC was also observed. Induction of apoptosis and lowering the level of glutathione are considered to be positive activity for an anticancer agent. Therefore, modulation of GSH concentration in tumor cells by PRE and its EA-fraction opened up the possibility of a new therapeutic approach because these plant products are not harmful to normal cells and may regulate the tumor cellular response to different anticancer treatments. Thus, it would be interesting to examine efficacy of these plant products or EA-fraction in human cancer treatment.

Project description:Objective: To investigate the effect of ethyl acetate extract from Celastrus orbiculatus (COE) on gastric cancer cell apoptosis and reveal its underlying molecular mechanism. In addition, it was aimed to stablish a theoretical basis for the clinical application of Celastrus orbiculatus in the gastric cancer treatment. Material and Methods: Western blot and RT-qPCR were used to detect mRNA and protein expression of PHB in gastric cancer and adjacent tissues. MTT method was used to detect the COE effect on the proliferation of AGS cells and to determine the 50% inhibitory concentration COE on these cells. COE effect on AGS apoptosis was evaluated by flow cytometry. Changes in apoptosis-related proteins expression in AGS cells were detected by western blot and changes in mitochondrial membrane potential were detected by JC-1 fluorescence staining. PHB expression was knocked down in AGS cells by lentiviral-mediated RNA interference. The COE antitumor effect was assessed in vivo using a subcutaneous transplantation tumor model in nude mice and in vivo fluorescence tracing technique in small animals. Results: The clinical samples analysis results showed that the PHB expression in gastric cancer samples was significantly higher than in corresponding adjacent tissues. MTT results showed that the AGS cell proliferation was significantly inhibited. RT-qPCR and western blot results showed that COE can significantly inhibit the PHB mRNA and protein expression, respectively. Flow cytometry analysis showed that COE was able to significantly promote AGS cell apoptosis. Western blot results also indicated that apoptosis-related protein expression changed significantly; BCL-2 expression significantly reduced while the Caspase-3 and Bax expression significantly increased after COE treatment. JC-1 fluorescence staining results showed that COE changed the mitochondrial membrane potential and activated the mitochondrial apoptosis pathway. Furthermore, in vivo experiments results demonstrated that the growth of subcutaneous transplanted tumor was significantly inhibited by the PHB knockdown and by the COE intragastric administration. Conclusion: COE can significantly promote apoptosis of human gastric cancer cells, which can be achieved by inhibiting PHB expression, thus altering the structure and function of mitochondria and activating the mitochondria apoptosis pathway. The antitumor effect of COE has also been proved in vivo.

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Induction of apoptosis through oxidative stress-related pathways in MCF-7, human breast cancer cells, by ethyl acetate extract of Dillenia suffruticosa.

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Induction of apoptosis through oxidative stress-related pathways in MCF-7, human breast cancer cells, by ethyl acetate extract of Dillenia suffruticosa.

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