Withaferin A Causes FOXO3a- and Bim-Dependent Apoptosis and Inhibits Growth of Human Breast Cancer Cells In vivo

Department of Pharmacology and Chemical Biology, and University of Pittsburgh Cancer Institute, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
Cancer Research (Impact Factor: 9.33). 10/2008; 68(18):7661-9. DOI: 10.1158/0008-5472.CAN-08-1510
Source: PubMed


Withaferin A (WA) is derived from the medicinal plant Withania somnifera, which has been safely used for centuries in Indian Ayurvedic medicine for treatment of different ailments. We now show, for the first time, that WA exhibits significant activity against human breast cancer cells in culture and in vivo. The WA treatment decreased viability of MCF-7 (estrogen-responsive) and MDA-MB-231 (estrogen-independent) human breast cancer cells in a concentration-dependent manner. The WA-mediated suppression of breast cancer cell viability correlated with apoptosis induction characterized by DNA condensation, cytoplasmic histone-associated DNA fragmentation, and cleavage of poly-(ADP-ribose)-polymerase. On the other hand, a spontaneously immortalized normal mammary epithelial cell line (MCF-10A) was relatively more resistant to WA-induced apoptosis compared with breast cancer cells. The WA-mediated apoptosis was accompanied by induction of Bim-s and Bim-L in MCF-7 cells and induction of Bim-s and Bim-EL isoforms in MDA-MB-231 cells. The cytoplasmic histone-associated DNA fragmentation resulting from WA exposure was significantly attenuated by knockdown of protein levels of Bim and its transcriptional regulator FOXO3a in both cell lines. Moreover, FOXO3a knockdown conferred marked protection against WA-mediated induction of Bim-s expression. The growth of MDA-MB-231 cells implanted in female nude mice was significantly retarded by 5 weekly i.p. injections of 4 mg WA/kg body weight. The tumors from WA-treated mice exhibited reduced cell proliferation and increased apoptosis compared with tumors from control mice. These results point toward an important role of FOXO3a and Bim in regulation of WA-mediated apoptosis in human breast cancer cells.

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    • "It is available in US over-the-counter as a dietary supplement and is known to treat various disorders due to its anti-inflammatory [11], [12], anti-bacterial [13], and cardio protective properties [14]. In recent years, WFA has been suggested as a potential anti-cancer compound shown to prevent tumor growth, angiogenesis, and metastasis [15], [16] in various types of cancer [17]–[26]. Mechanisms by which WFA attains its anticancer activity include inactivation of Akt and NF-κB [27] to achieve apoptosis, decrease in pro-survival protein Bcl-2 [28], G2/M cell cycle arrest [29], [30], generation of reactive oxygen species (ROS) [31], [32], induction of Par-4 [17], activation of caspase 3 and 9 activities, DNA damage [10], inhibition of HSP90 [20], regulation of FOXO3a and Bim [15] inhibition of Notch-1 [33] and down regulation of expression of HPV E6 and E7 oncoproteins [19]. "
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    ABSTRACT: Currently, the treatment for ovarian cancer entails cytoreductive surgery followed by chemotherapy, mainly, carboplatin combined with paclitaxel. Although this regimen is initially effective in a high percentage of cases, unfortunately within few months of initial treatment, tumor relapse occurs because of platinum-resistance. This is attributed to chemo-resistance of cancer stem cells (CSCs). Herein we show for the first time that withaferin A (WFA), a bioactive compound isolated from the plant Withania somnifera, when used alone or in combination with cisplatin (CIS) targets putative CSCs. Treatment of nude mice bearing orthotopic ovarian tumors generated by injecting human ovarian epithelial cancer cell line (A2780) with WFA and cisplatin (WFA) alone or in combination resulted in a 70 to 80% reduction in tumor growth and complete inhibition of metastasis to other organs compared to untreated controls. Histochemical and Western blot analysis of the tumors revealed that inclusion of WFA (2 mg/kg) resulted in a highly significant elimination of cells expressing CSC markers - CD44, CD24, CD34, CD117 and Oct4 and downregulation of Notch1, Hes1 and Hey1 genes. In contrast treatment of mice with CIS alone (6 mg/kg) had opposite effect on those cells. Increase in cells expressing CSC markers and Notch1 signaling pathway in tumors exposed to CIS may explain recurrence of cancer in patients treated with carboplatin and paclitaxel. Since, WFA alone or in combination with CIS eliminates putative CSCs, we conclude that WFA in combination with CIS may present more efficacious therapy for ovarian cancer.
    PLoS ONE 09/2014; 9(9):e107596. DOI:10.1371/journal.pone.0107596 · 3.23 Impact Factor
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    • "Therefore, inhibition of Notch signaling may have important therapeutic applications for treating and preventing osteosarcoma metastasis. Recently, withaferin A has been shown to possess potent antineoplastic activity in vitro and in vivo against a variety of malignancies, including prostate, breast and colon cancer (Srinivasan et al., 2007; Stan et al., 2008; Koduru et al., 2010 "
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    ABSTRACT: Human osteosarcoma cells were treated with withaferin A and the anti-proliferative activity was evaluated by the 3-(4, 5 dimethyl-thiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assays. Real-time RT-PCR and western blot were used to investigate whether the down-regulation of Notch-1 contributes to withaferin A-induced inhibition of proliferation in osteosarcoma cells. The results showed that withaferin A caused marked inhibition of osteosarcoma cell growth and G2/M phase cell cycle arrest. This was associated with concomitant attenuation of Notch-1 and down-regulation of its downstream genes, such as matrix metalloproteinases. These results suggest that antitumor activity of withaferin A is mediated through a novel mechanism involving inactivation of the Notch-1 signalling pathway. Our data provide the first evidence that the down regulation of Notch-1 by withaferin A may be an effective approach for the treatment of osteosarcoma.
    Bangladesh Journal of Pharmacology 07/2014; 9(3). DOI:10.3329/bjp.v9i3.19568 · 1.05 Impact Factor
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    • "In vivo research on Ashwagandha as an antitumor agent confirms its usefulness in slowing tumor growth and increasing survival time. Recently, the ability of withaferin-A in the inhibition of human breast cancer cells growth in vivo through G2 and mitotic arrest has been reported (Silvia et al., 2008). Presently, withanolides have been commercially obtained by solvent extraction of roots of the plants collected from the natural sources. "
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    ABSTRACT: Transformation of Withania somnifera was carried out by using three Agrobacterium rhizogenes strains (ATCC 15834, R1000 and K599) for hairy root induction. Induction of hairy root was carried out in leaf, petiole and internodal explants. Hairy root induction was successful only in ATCC 15834 and R1000. The highest frequency of hairy root was obtained in petiole explants (64%) infected with R1000 and it resulted in five distinct morpho-types (callus (fragile), callus (hard), callus + hairy roots, hairy roots and callusing roots). The frequency of R1000 transformation was increased up to 93.2% by the addition of acetosyringone during various steps of infection. Molecular identification through PCR analysis of rolC confirmed the presence of Ri T-DNA. The half strength Murashige and Skoog (MS) liquid medium was found to be the best medium that supports the high root biomass accumulation than the other tested medium types (MS full strength, Gamborg B5 medium (B5) full strength and B5 half strength). High performance liquid chromatography (HPLC) analysis of hairy roots revealed the accumulation of withaferin-A (72.3 mg/g dw). This study reports the influence of Agrobacterium strains, explant types and acetosyringone in the hairy root induction of W. somnifera.
    AFRICAN JOURNAL OF BIOTECHNOLOGY 12/2012; 11(98):16412-16420. · 0.57 Impact Factor
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