Khalit Mohamad

Induction of Apoptosis in Human Breast Cancer Cells via Caspase Pathway by Vernodalin Isolated from Centratherum anthelminticum (L.) Seeds

Background Centratherum anthelminticum (L.) seeds (CA) is a well known medicinal herb in Indian sub-continent. We recently reported anti-oxidant property of chloroform fraction of Centratherum anthelminticum (L.) seeds (CACF) by inhibiting tumor necrosis factor-α (TNF-α)-induced growth of human breast cancer cells. However, the active compounds in CACF have not been investigated previously. Methodology/Principal Findings In this study, we showed that CACF inhibited growth of MCF-7 human breast cancer cells. CACF induced apoptosis in MCF-7 cells as marked by cell size shrinkage, deformed cytoskeletal structure and DNA fragmentation. To identify the cytotoxic compound, CACF was subjected to bioassay-guided fractionation which yielded 6 fractions. CACF fraction A and B (CACF-A, -B) demonstrated highest activity among all the fractions. Further HPLC isolation, NMR and LC-MS analysis of CACF-A led to identification of vernodalin as the cytotoxic agent in CACF-A, and -B. 12,13-dihydroxyoleic acid, another major compound in CACF-C fraction was isolated for the first time from Centratherum anthelminticum (L.) seeds but showed no cytotoxic effect against MCF-7 cells. Vernodalin inhibited cell growth of human breast cancer cells MCF-7 and MDA-MB-231 by induction of cell cycle arrest and apoptosis. Increased of reactive oxygen species (ROS) production, coupled with downregulation of anti-apoptotic molecules (Bcl-2, Bcl-xL) led to reduction of mitochondrial membrane potential (MMP) and release of cytochrome c in both human breast cancer cells treated with vernodalin. Release of cytochrome c from mitochondria to cytosol triggered activation of caspase cascade, PARP cleavage, DNA damage and eventually cell death. Conclusions/Significance To the best of our knowledge, this is the first comprehensive study on cytotoxic and apoptotic mechanism of vernodalin isolated from the Centratherum anthelminticum (L.) seeds in human breast cancer cells. Overall, our data suggest a potential therapeutic value of vernodalin to be further developed as new anti-cancer drug.

Ceramicines B-D, new antiplasmodial limonoids from Chisocheton ceramicus

Three new limonoids, ceramicines B-D (1-3), have been isolated from the bark of Chisocheton ceramicus. Structures and stereochemistry of 1-3 were fully elucidated and characterized by 2D NMR analysis. Ceramicines exhibited a moderate antiplasmodial activity.

Induction of apoptosis in melanoma A375 cells by a chloroform fraction of Centratherum anthelminticum (L.) seeds involves NF-kappaB, p53 and Bcl-2-controlled mitochondrial signaling pathways

BackgroundCentratherum anthelminticum (L.) Kuntze (scientific synonyms: Vernonia anthelmintica; black cumin) is one of the ingredients of an Ayurvedic preparation, called “Kayakalp”, commonly applied to treat skin disorders in India and Southeast Asia. Despite its well known anti-inflammatory property on skin diseases, the anti-cancer effect of C. anthelminticum seeds on skin cancer is less documented. The present study aims to investigate the anti-cancer effect of Centratherum anthelminticum (L.) seeds chloroform fraction (CACF) on human melanoma cells and to elucidate the molecular mechanism involved.MethodsA chloroform fraction was extracted from C. anthelminticum (CACF). Bioactive compounds of the CACF were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Human melanoma cell line A375 was treated with CACF in vitro. Effects of CACF on growth inhibition, morphology, stress and survival of the cell were examined with MTT, high content screening (HSC) array scan and flow cytometry analyses. Involvement of intrinsic or extrinsic pathways in the CACF-induced A375 cell death mechanism was examined using a caspase luminescence assay. The results were further verified with different caspase inhibitors. In addition, Western blot analysis was performed to elucidate the changes in apoptosis-associated molecules. Finally, the effect of CACF on the NF-κB nuclear translocation ability was assayed.ResultsThe MTT assay showed that CACF dose-dependently inhibited cell growth of A375, while exerted less cytotoxic effect on normal primary epithelial melanocytes. We demonstrated that CACF induced cell growth inhibition through apoptosis, as evidenced by cell shrinkage, increased annexin V staining and formation of membrane blebs. CACF treatment also resulted in higher reactive oxygen species (ROS) production and lower Bcl-2 expression, leading to decrease mitochondrial membrane potential (MMP). Disruption of the MMP facilitated the release of mitochondrial cytochrome c, which activates caspase-9 and downstream caspase-3/7, resulting in DNA fragmentation and up-regulation of p53 in melanoma cells. Moreover, CACF prevented TNF-α-induced NF-κB nuclear translocation, which further committed A375 cells toward apoptosis.ConclusionsTogether, our findings suggest CACF as a potential therapeutic agent against human melanoma malignancy.

An ethnobotanical study of medicinal plants used by tribal and native people of Madhupur forest area, Bangladesh.

ETHNOPHARMACOLOGICAL RELEVANCE Madhupur forest area, Tangail is one of early human settlements in Bangladesh. Having abode in the vicinity of the forest, a strong ethnobotanical practice has prevailed in this area since ancient time. Due to the rapid deforestation during the last few decades, many plants have already disappeared or are facing extinction. Thus we attempted to document the medicinal plant use of Madhupur forest area with a view to preserve the ethnobotanical knowledge and in order to protect the biodiversity of this area. MATERIALS AND METHODS The fieldwork was conducted during a period of 1 year. Data was collected by interview, questionnaire, and group discussion with randomly selected informants including indigenous, tribal people, and Traditional Health Practitioners (THPs) living in the study area. Recorded plants are listed along with their indication, part used, form of preparation and use value (UV). Results were also analysed to determine informant consensus factor (ICF) and fidelity level (FL) of the plants on the basis of their use under various ailment categories. RESULTS The present study has documented 78 medicinal plant species from 45 families used for the treatment of at least 77 different major and minor ailments and conditions. Medicinal plant species were categorised as tree, shrub, tuber, herb, and climber. Leaves were found to be the most frequently used plant part while decoction is the major form of preparation. In most cases preparations are either administered orally or applied topically. CONCLUSION The present study revealed that some of the well-known medicinal plants are used extensively demonstrating an effective ethnobotanical practice in the study area. Plants with high ICF and FL values can be subjected to bioassay guided investigation while plants which scored low UVs require bioactivity screening to justify their use for the reported ailment.

Dammarane triterpenes and pregnane steroids from Aglaia lawii and A. tomentosa

Three known dammaranes, cabraleone, eichlerianic acid and shoreic acid, together with two new ones, aglinins A and B, were isolated from the bark of Aglaia lawii. A. tomentosa also contained dammarane triterpenes: the known, cabraleone, cabraleadiol 3-acetate, 3-epiocotillol, cabralealactone, cabralealactone 3-acetate and the new aglinins C and D. In addition, two new pregnane steroids, aglatomins A and B, were isolated from A. tomentosa along with the known cytotoxic cyclopentatetrahydrobenzofuran, rocaglaol. The structure of the new compounds was determined by spectral means, essentially 2D NMR experiments.

7α-Hydroxy-β-Sitosterol from Chisochetontomentosus Induces Apoptosis via Dysregulation of Cellular Bax/Bcl-2 Ratio and Cell Cycle Arrest by Downregulating ERK1/2 Activation

In continuation of our interest towards the elucidation of apoptotic pathways of cytotoxic phytocompounds, we have embarked upon a study on the anticancer effects of 7α-hydroxy-β-sitosterol (CT1), a rare natural phytosterol oxide isolated from Chisocheton tomentosus. CT1 was found to be cytotoxic on three different human tumor cell lines with minimal effects on normal cell controls, where cell viability levels were maintained ≥80% upon treatment. Our results showed that cell death in MCF-7 breast tumor cells was achieved through the induction of apoptosis via downregulation of the ERK1/2 signaling pathway. CT1 was also found to increase proapoptotic Bax protein levels, while decreasing anti-apoptotic Bcl-2 protein levels, suggesting the involvement of the intrinsic pathway. Reduced levels of initiator procaspase-9 and executioner procaspase-3 were also observed following CT1 exposure, confirming the involvement of cytochrome c-mediated apoptosis via the mitochondrial pathway. These results demonstrated the cytotoxic and apoptotic ability of 7α-hydroxy-β-sitosterol and suggest its potential anti-cancer use particularly on breast adenocarcinoma cells.

Tirucallane triterpenes from Dysoxylum macranthum

Abstract Eleven new tirucallane-type triterpenes, dymacrins A–K, were isolated from the bark of Dysoxylum macranthum together with two known tetracyclic triterpenes and two known pregnane steroids. The structures of the new compounds were determined by spectral means, essentially 2D NMR experiments. Dymacrins B, C, H and J showed moderate cytotoxicity against KB cells (IC 50 5.6, 5.0, 8.3 and 1.0 μg/ml, respectively).

Triterpenes and steroids from the leaves of Aglaia exima (Meliaceae)

A study on the leaves of Aglaia exima led to the isolation of one new and seven known compounds: six triterpenoids and two steroids. Their structures were elucidated and analyzed mainly by using spectroscopic methods; 1D and 2D NMR, mass spectrometry, UV spectrometry and X-ray. All the triterpenoids and steroids were measured in vitro for their cytotoxic activities against eight cancer cell lines; lung (A549), prostate (DU-145), skin (SK-MEL-5), pancreatic (BxPC-3), liver (Hep G2), colon (HT-29), breast (MCF-7) and (MDA-MB-231). The new cycloartane triterpenoid, 24(E)-cycloart-24-ene-26-ol-3-one 1, showed potent cytotoxic activity against colon (HT-29) cancer cell line (IC(50) 11.5μM).

Chisomicines A-C, limonoids from Chisocheton ceramicus.

Three new limonoids, chisomicines A-C (1-3), have been isolated from the bark of Chisocheton ceramicus. Their structures were determined by 2D NMR, CD spectroscopic methods, and X-ray analysis. Chisomicine A (1) exhibited NO production inhibitory activity in J774.1 cells stimulated by LPS dose-dependently at high cell viability.

Bioactivity-guided fractionation of the lipoxygenase and cyclooxygenase inhibiting constituents from Chisocheton polyandrus Merr.

Lipoxygenase (LOX)-inhibiting compounds from the leaves of Chisocheton polyandrus Merr. were isolated in this study using a bioactivity-guided fractionation technique. Two dammarane triterpenoids, dammara-20,24-dien-3-one (1) (IC(50)=0.69±0.07 μM) and 24-hydroxydammara-20,25-dien-3-one (2) (IC(50)=1.11±0.38 μM), were isolated and identified based on the soybean LOX assay. Dammara-20,24-dien-3-one (1) exhibited dual inhibition of both human 5-LOX (IC(50)=24.27±2.92 μM) and cyclooxygenase-2 (COX-2) (IC(50)=3.17±0.90 μM), whereas 24-hydroxydammara-20,25-dien-3-one (2) did not exhibit any significant inhibitory effects. This report is the first to detail the inhibition of LOX and COX by both C. polyandrus and its isolated compounds.