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

Background: Centratherum anthelminticum (L.) Kuntze (scientific synonyms: Vernonia anthelmintica; black cumin) is one of the ingredients of an Ayurvedic preparation, called Kayakalpâ, 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. Methods: A 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. Results: The 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. Conclusions: Together, our findings suggest CACF as a potential therapeutic agent against human melanoma malignancy

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

Vernodalin induces cell cycle arrest at G0/G1 stage.

<p>MCF-7 and MDA-MB-231 cells were treated with indicated dosages of verdonalin for 24 hours. Cells were ethanol-permeabilized and stained with propidium iodide before subjected to flow cytometry analysis. Representative figures of cell cyle distribution (G0/G1, S, and G2/M) showing accumulation of vernodalin-treated cells in G0–G1 stage. Data were mean ± SD of two independent experiments.</p

Photo of <i>Centratherum anthelminticum (L.)</i> seeds.

<p>Photo of <i>Centratherum anthelminticum (L.)</i> seeds.</p

Vernodalin reduces expression of pro-survival molecules.

<p>MCF-7 and MDA-MB-231 cells were treated with control DMSO, standard drug doxorubicin (12.5 µg/ml) or various concentrations of vernodalin (3.125, 6.25, 12.5 µg/ml). Western blot showing the expression levels of the pro-survival molecules Bcl-2 and Bcl-xL in untreated and treated breast cancer cells. β-actin served as a loading control. Decreased Bcl-2 and Bcl-xL protein levels were observed upon doxorubicin or vernodalin treatment. Data were representative of at least two similar experiments.</p

Morphological assessment of CACF-treated MCF-7 cells.

<p>(A) Representative figures of MCF-7 cells were treated with CACF for 12 hours. Cells were stained with apoptosis marker annexin V (green) and nucleus marker Hoechst 33258 (blue). Histogram shows mean fluorescence intensities of annexin V in MCF-7 cells treated with various concentration of CACF. Data were mean ± SD, *<i>P</i><0.05. (B) Representative figures of cytoskeletal F-actin formation in control or CACF-treated MCF-7 cells. Cells were fixed, stained with DY544-phalloidin (red) and Hoechst 33258 (blue) after treated with 6.25 µg/ml CACF or solvent DMSO for 12 hours. Histogram shows mean fluorescence intensities of phalloidin in MCF-7 cells treated with various concentration of CACF. Data were mean ± SD, *<i>P</i><0.05. (C). Representative figures of MCF-7 cells treated with DMSO (control), 6.25 or 12.5 µg/ml of CACF for 24 hours. Cells were also treated with a standard drug doxorubixin (DOX) as positive control of apoptosis induction. Cells were stained with Hoechst 33258 dye (blue). All images were visualized and captured using Cellomic HCS array scan reader (objective 20 ×).</p

CACF inhibits MCF-7 cells proliferation in a time- and dose-dependent manner.

<p>(A) MCF-7 cells were treated with control DMSO, various concentrations (0.195, 0.39, 0.78, 1.56, 3.125, 6.25, 12.5, 25, 50 µg/ml) of CACF or anti-cancer drug doxorubicin for 24 hours. Cell viability was determined by MTT assays. (B) Real-time cell proliferation was measured using xCELLigence Real-Time Cellular Analysis (RTCA) system. MCF-7 cells were treated with DMSO (control), indicated concentration of CACF or doxorubicin (DOX) and normalized cell index for 3 consecutive treatment days was shown. Data were mean ± SD. Arrow showing time-point of CACF administration.</p

Isolation of active compound from CACF.

<p>A. Flow chart of bioassay guided isolation of <i>Centratherum anthelminticum</i>. B. HPLC chromatogram of the fraction of CACF-A of the chloroform extract of <i>C. anthelminticum</i>.</p

Vernodalin induces apoptosis in human breast cancer cells.

<p>(A) Flow cytometry analysis of MCF-7 and MDA-MB-231 cells treated with 3.125, 6.25 and 12.5 µg/ml verdonalin for 24 hours. Representative figures showing population of viable (annexin V- PI-), early apoptotic (annexin V+ PI-), late apoptotic (annexin V+ PI+) and necrotic (annexin V- PI+) cells. (B) Bar chart showing increased proportion of early and late apoptotic cells after vernodalin administration. Data were mean ± SD of two independent experiments. (*<i>P</i><0.05).</p