Phytochemical study of Dryobalanops from Malaysian Dipterocarpaceae, and structure - activity relationship studies / Agustono Wibowo

distributed as major species in emergent canopy of Lambir Forest and Sarawak lowland dipterocarps forest. The genus is very unique, as there are only seven species available in the whole world, which confined to the tropical forests of West Malesia. The chemical constituents of Dipterocarpaceae are reported to possess various biological activities such as cytotoxicity, antiviral, antibacterial and anti-inflammatory activities. The aims of this study are to isolate secondary metabolites, to determine their antibacterial, DPPH scavenging and cytotoxic activities, to study structure-activity relationship, and to propose biogenesis pathway and chemotaxonomic significance in Dryobalanops. The dried powder of the stem bark of D. aromatica, D. lanceolata, D. rappa and D. becarii were macerated with acetone and evaporated under reduced pressure. The crude acetone extract was subjected to vacuum liquid chromatography to give several fractions

Dillenia suffruticosa dichloromethane root extract induced apoptosis towards MDA-MB-231 triple negative breast cancer cells

Ethnopharmacological Relevance: Dillenia suffruticosa is traditionally used for treatment of cancerous growth including breast cancer in Malaysia. Aim of The Study: Dillenia suffruticosa is a well-known medicinal plant in Malaysia for the treatment of cancer. Nevertheless, no study has been reported the cytotoxicity of this plant towards MDA-MB-231 triple-negative breast cancer cells. The present study was designed to investigate the mode of cell death and signalling pathways of MDA-MB-231 cells treated with dichloromethane Dillenia suffruticosa root extract (DCM-DS). Methods: Extraction of Dillenia suffruticosa root was performed by the use of sequential solvent procedure. The cytotoxicity of DCM-DS was determined by using MTT assay. The mode of cell death was evaluated by using an inverted light microscope and flow cytometry analysis using Annexin-V/PI. Cell cycle analysis and measurement of reactive oxygen species level were performed by using flow cytometry. The cells were treated with DCM-DS and antioxidants α-tocopherol or ascorbic acid to evaluate the involvement of ROS in the cytotoxicity of DCM-DS. Effect of DCM-DS on the expression of antioxidant, apoptotic, growth, survival genes and proteins were analysed by using GeXP-based multiplex system and Western blot, respectively. The cytotoxicity of compounds isolated from DCM-DS was evaluated towards MDA-MB-231 cells using MTT assay. Results: DCM-DS induced apoptosis, G2/M phase cell cycle arrest and oxidative stress in MDA-MB-231 cells. The induction of apoptosis in MDA-MB-231 cells by DCM-DS is possibly due to the activation of pro-apoptotic JNK1 and down-regulation of anti-apoptotic ERK1, which in turn down-regulates anti-apoptotic BCL-2 to increase the BAX/BCL-2 ratio to initiate the mitochondrial apoptotic pathway. The cell cycle arrest in DCM-DS-treated MDA-MB-231 cells is possibly via p53-independent but p21-dependent pathway. A total of 3 triterpene compounds were isolated from DCM-DS. Betulinic acid appears to be the most major and most cytotoxic compound in DCM-DS. Conclusion: The data suggest the potential application of DCM-DS in the treatment of triple-negative breast cancer

Ampelopsin E reduces the invasiveness of the triple negative breast cancer cell line, MDA-MB-231

Breast cancer is the most common and the second leading cause of cancer-related deaths in women. It has two distinctive hallmarks: rapid abnormal growth and the ability to invade and metastasize. During metastasis, cancer cells are thought to form actin-rich protrusions, called invadopodia, which degrade the extracellular matrix. Current breast cancer treatments, particularly chemotherapy, comes with adverse effects like immunosuppression, resistance development and secondary tumour formation. Hence, naturally-occurring molecules claimed to be less toxic are being studied as new drug candidates. Ampelopsin E, a natural oligostilbene extracted from Dryobalanops species, has exhibited various pharmacological properties, including anticancer and anti-inflammatory activities. However, there is yet no scientific evidence of the effects of ampelopsin E towards metastasis. Scratch assay, transwell migration and invasion assays, invadopodia and gelatin degradation assays, and ELISA were used to determine the effects of ampelopsin E towards the invasiveness of MDA-MB-231 cells. Strikingly in this study, ampelopsin E was able to halt migration, transmigration and invasion in MDA-MB-231 cells by reducing formation of invadopodia and its degradation capability through significant reduction (p < 0.05) in expression levels of PDGF, MMP2, MMP9 and MMP14. In conclusion, ampelopsin E reduced the invasiveness of MDA-MB-231 cells and was proven to be a potential alternative in treating TNBC

Induction of Apoptosis in MCF-7 Cells via Oxidative Stress Generation, Mitochondria-Dependent and Caspase-Independent Pathway by Ethyl Acetate Extract of Dillenia suffruticosa and Its Chemical Profile.

Dillenia suffruticosa, which is locally known as Simpoh air, has been traditionally used to treat cancerous growth. The ethyl acetate extract of D. suffruticosa (EADs) has been shown to induce apoptosis in MCF-7 breast cancer cells in our previous study. The present study aimed to elucidate the molecular mechanisms involved in EADs-induced apoptosis and to identify the major compounds in the extract. EADs was found to promote oxidative stress in MCF-7 cells that led to cell death because the pre-treatment with antioxidants α-tocopherol and ascorbic acid significantly reduced the cytotoxicity of the extract (P<0.05). DCFH-DA assay revealed that treatment with EADs attenuated the generation of intracellular ROS. Apoptosis induced by EADs was not inhibited by the use of caspase-inhibitor Z-VAD-FMK, suggesting that the cell death is caspase-independent. The use of JC-1 dye reflected that EADs caused disruption in the mitochondrial membrane potential. The related molecular pathways involved in EADs-induced apoptosis were determined by GeXP multiplex system and Western blot analysis. EADs is postulated to induce cell cycle arrest that is p53- and p21-dependent based on the upregulated expression of p53 and p21 (P<0.05). The expression of Bax was upregulated with downregulation of Bcl-2 following treatment with EADs. The elevated Bax/Bcl-2 ratio and the depolarization of mitochondrial membrane potential suggest that EADs-induced apoptosis is mitochondria-dependent. The expression of oxidative stress-related AKT, p-AKT, ERK, and p-ERK was downregulated with upregulation of JNK and p-JNK. The data indicate that induction of oxidative-stress related apoptosis by EADs was mediated by inhibition of AKT and ERK, and activation of JNK. The isolation of compounds in EADs was carried out using column chromatography and elucidated using the nuclear resonance magnetic analysis producing a total of six compounds including 3-epimaslinic acid, kaempferol, kaempferide, protocatechuic acid, gallic acid and β-sitosterol-3-O-β-D-glucopyranoside. The cytotoxicity of the isolated compounds was determined using MTT assay. Gallic acid was found to be most cytotoxic against MCF-7 cell line compared to others, with IC50 of 36 ± 1.7 μg/mL (P<0.05). In summary, EADs generated oxidative stress, induced cell cycle arrest and apoptosis in MCF-7 cells by regulating numerous genes and proteins that are involved in the apoptotic signal transduction pathway. Therefore, EADs has the potential to be developed as an anti-cancer agent against breast cancer

Induction of apoptosis and G2/M arrest by ampelopsin E from Dryobalanops towards triple negative breast cancer cells, MDA-MB-231

Background: Several compounds isolated from Dryobalanops have been reported to exhibit cytotoxic effects to several cancer cell lines. This study investigated the cytotoxic effects, cell cycle arrest and mode of cell death in ampelopsin E-treated triple negative cells, MDA-MB-231. Methods: Cytotoxicity of ampelopsin E, ampelopsin F, flexuosol A, laevifonol, Malaysianol A, Malaysianol D and nepalensinol E isolated from Dryobalanops towards human colon cancer HT-29, breast cancer MDA-MB-231 and MCF-7, alveolar carcinoma HeLa and mouse embryonic fibroblast NIH/3 T3 cells were determined by MTT assay. The cells were treated with the compounds (0.94–30 μM) for 72 h. The mode of cell death was evaluated by using an inverted light microscope and annexin V/PI analysis. Cell cycle analysis was performed by using a flow cytometer. Results: Data showed that ampelopsin E was most cytotoxic toward MDA-MB-231 with the IC50 (50 % inhibition of cell viability compared to control) of 14.5 ± 0.71 μM at 72 h. Cell shrinkage, membrane blebbing and formation apoptotic bodies characteristic of apoptosis were observed following treatment with ampelopsin E. The annexin V/PI flow cytometric analysis further confirmed that ampelopsin E induced apoptosis in MDA-MB-231 cells. Cell cycle analysis revealed that ampelopsin E induced G2/M phase cell cycle arrest in the cells. Conclusion: Ampelopsin E induced apoptosis and cell cycle arrest in MDA-MB-231 cells. Therefore, ampelopsin E has the potential to be developed into an anticancer agent for treatment of triple negative breast cancer

Chemical structure of the compounds isolated from EADs.

<p>Structures of the isolated compounds were elucidated using 1^H and 13^C NMR spectroscopy. The isolated compounds were identified as kaempferide (<b>1</b>), kaempferol (<b>2</b>), protocatechuic acid (<b>3</b>), gallic acid (<b>4</b>), 3-epimaslinic acid (<b>5</b>) and β-sitosterol-3-O-β-D-glucopyranoside (<b>6</b>).</p

Involvement of oxidative stress in EADs-induced apoptosis in MCF-7 cells.

<p>(A) and (B) represent the percentage of viability of MCF-7 cells pre-treated with vitamin C, α-tocopherol or EADs alone, at 24 and 48 hours, respectively. (C) Level of ROS in MCF-7 cells as determined using DCFH-DA assay. Data showed that pre-treatment of MCF-7 cells with α-tocopherol and ascorbic acid significantly reduced the cytotoxicity of EADs (P<0.05). On the other hand, EADs attenuated the intracellular ROS in MCF-7 cells in a concentration-dependent manner (P<0.05). The data are presented as mean±standard deviation of three replicates from at least three independent tests. An asterisk * indicates statistically significantly different from the untreated control (P<0.05).</p

Mitochondria membrane potential of MCF-7 cells treated with EADs as determined by JC-1 fluorescent dye using flow cytometry analysis.

<p>The increment of green fluorescence indicates the loss of ΔΨm in the mitochondria of EADs-treated MCF-7 cells. The data are presented as dot plots of JC-1 red fluorescence (Y-axis) against JC-1 green fluorescence (X-axis) of at least three independent tests.</p

Expression level of the apoptotic-related genes in MCF-7 cells treated with EADs as determined by GeXP analysis.

<p>EADs upregulated the expression of <i>Bax</i> and <i>p21</i> and downregulated the expression of <i>Bcl-2</i> and <i>caspase-9</i>. The expression of genes was normalized against beta actin and compared to the control. The data are represented as relative expression of genes in bars±SD of at least three replicates from three independent tests. An asterisk * indicates statistically significantly different from the untreated control (P<0.05).</p

Extraction and isolation of ethyl acetate extract of <i>Dillenia suffruticosa</i>.

<p>Sequential solvent extraction using solvents with increasing polarity (hexane< dichloromethaneD. <i>suffruticosa</i>. The extract obtained was subjected to isolation using column chromatography and thin layer chromatography by using different solvent systems.</p