Steroidal Lactone Research Articles

Antiproliferation potential of withaferin A on human osteosarcoma cells via the inhibition of G2/M checkpoint proteins.

Withaferin A (WA) is a well-known steroidal lactone of the medicinally important plant, Withania somnifera. This secondary metabolite has been noted for its anticancer effects against a number of human cancer cell lines. However, there are a limited number of studies investigating the growth inhibitory potential of WA against human osteosarcoma cells and the underlying molecular mechanisms. Thus, in the present study, the antiproliferative activities of WA, along with the underlying mechanisms of action, were investigated using flow cytometry for cell cycle distribution and western blot analysis for the assessment of various checkpoint proteins. In addition, the antiproliferative activity was evaluated using a sulforhodamine B assay, where MG-63 and U2OS human osteosarcoma cell lines were treated with different concentrations of WA. Furthermore, the mRNA expression levels of the checkpoint proteins in the WA-treated MG-63 and U2OS cells were examined. The results obtained corresponded with the western blot analysis results. Furthermore, WA was shown to significantly inhibit the proliferation of the two types of treated cell lines (MG-63 and U2OS). Flow cytometric analysis revealed that WA induced cell cycle arrest at the G2/M phase, which was associated with the inhibition of cyclin B1, cyclin A, Cdk2 and p-Cdc2 (Tyr15) expression and an increase in the levels of p-Chk1 (Ser345) and p-Chk2 (Thr68). In conclusion, the present study found that the antiproliferative potential of WA was associated with the induction of cell cycle arrest at the G2/M phase, which was a result of the attenuation of the expression levels of G2/M checkpoint proteins.

Comprehensive Report on Phytochemistry and Pharmacological Prominence of Withania somnifera

Medicinal plants have been one of the prominent sources of remedies since the inception of human civilization. In rural area herbal medicines are considered to be best healthcare products due to its easily availability. A large proportion of the world population, especially in the developing countries relies mainly on the traditional system of medicine. Withania somnifera is known as Ashwagandha belonging to the Solanaceae family. W. somnifera is used as adaptogen, antiarthritic, antispasmodic, anti-inflammatory, nervine tonic, nerve soothing, sedative, hypotensive, antioxidant, immunomodulator, free radical scavenger, anti-stress and anti-cancer agent. From chemistry point of view, the drug contains group of biologically active constituents known as withanolides. The major active constituents of W. somnifera root are steroidal alkaloids and steroidal lactones in a class of constituents called withanolides. The present review is therefore, an effort to give a detailed overview of the published writings on phytochemistry and pharmacological activities of W. somnifera; this information will be beneficial in developing new formulations, which are more effective and have more therapeutic values.

Improved Agrobacterium rhizogenes-mediated hairy root culture system of Withania somnifera (L.) Dunal using sonication and heat treatment.

An improved Agrobacterium rhizogenes-mediated genetic transformation of Withania somnifera (L.) Dunal was developed using the bacterial strain R1000 with leaf segment explants of in vitro raised plantlets. Out of the three strains used (R1000, MTCC 2364 and MTCC 532), the strain R1000 proved to be more efficient than others. Among the different conditions tested, the highest (93.3 %) transformation rate was observed after 3 weeks when the explants were subjected to sonication (15 s) and heat treatment (41 °C for 5 min). Transgenic status of the hairy roots was confirmed by PCR using rol B-specific primers. HPLC analysis showed the ability of hairy roots to synthesize withaferin A and withanolide A, both steroidal lactones of medicinal value. This protocol offers new avenue in A. rhizogenes-mediated hairy root induction and is useful for large-scale production of these bioactive compounds from W. somnifera.

Virus-induced gene silencing of Withania somnifera squalene synthase negatively regulates sterol and defence-related genes resulting in reduced withanolides and biotic stress tolerance.

Withania somnifera (L.) Dunal is an important Indian medicinal plant that produces withanolides, which are triterpenoid steroidal lactones having diverse biological activities. To enable fast and efficient functional characterization of genes in this slow-growing and difficult-to-transform plant, a virus-induced gene silencing (VIGS) was established by silencing phytoene desaturase (PDS) and squalene synthase (SQS). VIGS of the gene encoding SQS, which provides precursors for triterpenoids, resulted in significant reduction of squalene and withanolides, demonstrating its application in studying withanolides biosynthesis in W. somnifera leaves. A comprehensive analysis of gene expression and sterol pathway intermediates in WsSQS-vigs plants revealed transcriptional modulation with positive feedback regulation of mevalonate pathway genes, and negative feed-forward regulation of downstream sterol pathway genes including DWF1 (delta-24-sterol reductase) and CYP710A1 (C-22-sterol desaturase), resulting in significant reduction of sitosterol, campesterol and stigmasterol. However, there was little effect of SQS silencing on cholesterol, indicating the contribution of sitosterol, campesterol and stigmasterol, but not of cholesterol, towards withanolides formation. Branch-point oxidosqualene synthases in WsSQS-vigs plants exhibited differential regulation with reduced CAS (cycloartenol synthase) and cycloartenol, and induced BAS (β-amyrin synthase) and β-amyrin. Moreover, SQS silencing also led to the down-regulation of brassinosteroid-6-oxidase-2 (BR6OX2), pathogenesis-related (PR) and nonexpressor of PR (NPR) genes, resulting in reduced tolerance to bacterial and fungal infection as well as to insect feeding. Taken together, SQS silencing negatively regulated sterol and defence-related genes leading to reduced phytosterols, withanolides and biotic stress tolerance, thus implicating the application of VIGS for functional analysis of genes related to withanolides formation in W. somnifera leaves.

Formation of hydroxylated steroid lactones from dehydroepiandrosterone by Spicaria fumoso-rosea F-881

Formation of hydroxylated steroid lactones from dehydroepiandrosterone by Spicaria fumoso-rosea F-881

Withanolide E sensitizes renal carcinoma cells to TRAIL-induced apoptosis by increasing cFLIP degradation.

Withanolide E, a steroidal lactone from Physalis peruviana, was found to be highly active for sensitizing renal carcinoma cells and a number of other human cancer cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis. Withanolide E, the most potent and least toxic of five TRAIL-sensitizing withanolides identified, enhanced death receptor-mediated apoptotic signaling by a rapid decline in the levels of cFLIP proteins. Other mechanisms by which TRAIL sensitizers have been reported to work: generation of reactive oxygen species (ROS), changes in pro-and antiapoptotic protein expression, death receptor upregulation, activation of intrinsic (mitochondrial) apoptotic pathways, ER stress, and proteasomal inhibition proved to be irrelevant to withanolide E activity. Loss of cFLIP proteins was not due to changes in expression, but rather destabilization and/or aggregation, suggesting impairment of chaperone proteins leading to degradation. Indeed, withanolide E treatment altered the stability of a number of HSP90 client proteins, but with greater apparent specificity than the well-known HSP90 inhibitor geldanamycin. As cFLIP has been reported to be an HSP90 client, this provides a potentially novel mechanism for sensitizing cells to TRAIL. Sensitization of human renal carcinoma cells to TRAIL-induced apoptosis by withanolide E and its lack of toxicity were confirmed in animal studies. Owing to its novel activity, withanolide E is a promising reagent for the analysis of mechanisms of TRAIL resistance, for understanding HSP90 function, and for further therapeutic development. In marked contrast to bortezomib, among the best currently available TRAIL sensitizers, withanolide E's more specific mechanism of action suggests minimal toxic side effects.

Formation of hydroxylated steroid lactones from Dehydroepiandrosterone by Spicaria fumoso-rosea F-881

The transformation of dehydroepiandrosterone by Spicaria fumoso-rosea VKM F-881 produced 7alpha- and 7beta-hydroxy-dehydroepiandrosterone, 3beta,7alpha-dihydroxy-17a-oxa-D-homo-androst-5-en-17-one, and 3beta,7beta-dihydroxy- 17a-oxa-D-homo-androst-5-en-17-one. The yield of the main product-3beta,7beta-dihydroxy-17a-oxa-D-homo-androst-5-en-17-one-was 49.5-72 mol % at substrate loadings of 5-20 g/L. Lactone formation proceeded through 7alpha- and 7beta-hydroxy derivatives of dehydroepiandrosterone. The structure of the products was determined by mass spectrometry, 1H-NMR spectroscopy, and 13C-NMR spectroscopy. The proposed microbiological method for producing steroid lactones opens prospects for the syn- thesis of novel steroid compounds.

Influence of indole-3-butyric acid and triazole compounds on the growth and antioxidant constituents in Ashwagandha (Withania somnifera L.) Dunal

Withania somnifera (L.) Dunal belongs to the family Solanaceae and commonly known as Ashwagandha, Indian ginseng and winter cherry is major ayurvedic medicinal plant cultivated specifically for its root portion, which has medicinal and nutritional values due the presence of clinically important active compounds such as steroidal lactones, alkaloids, flavonoids, and tannins etc. Ashwagandha root is the medicinal part of the plant and is used in the ayurvedic formulations and pharmaceutical industries. The present study was undertaken to investigate the influence of indole-3-butyric acid (IBA) and triazole compounds viz., triadimefon (TDM) and propiconazole (PCZ) on the growth and non-enzymatic antioxidant content of Ashwagandha. Plants were treated with IBA 2.5 mg/L, TDM 20 mg/L and PCZ 20 mg/L separately by soil drenching on 50, 90, 130 days after sowing (DAS). Plants were analyzed on 60, 100 and 140 DAS and it’s parameters such as shoot length, internodal length, number of leaves, total leaf area, fresh and dry weight of shoot, root length, root diameter, number of roots, fresh and dry weight of root, ascorbic acid (AA), α-tocopherol (α-toc) and total phenol (TP) contents were determined. It was observed that shoot length, internodal length, the number of leaves, total leaf area, fresh and dry weight of shoot decreased, but the root length, root diameter, number of roots fresh and dry weight of roots were increased higher level. Likewise, the non-enzymatic antioxidants constituents such as AA, α-toc and TP contents were also increased by TDM and PCZ treatments. But IBA treatment increased all the parameters studied. Among the treatments, triazole showed beneficial growth by increasing the biomass of root system antioxidant constituents when compared to IBA treatment. This finding suggested that the triazole compounds showed a great significant for the cultivation of medicinally important root crops, which is helpful to meet the needs of root production by enhancing antioxidant constituents in Ashwagandha.

Withania somnifera: a pre-clinical study on neuroregenerative therapy for stroke.

Stroke management exerts insurmountable societal and economic burden on the patient as well as their caregivers. In the year 2010 alone, the direct and indirect costs of stroke care amounted to 36.5 billion dollars (Go et al., 2014). Despite concentrated efforts to develop a safe, effective drug for stroke, we have not discovered one since the introduction of recombinant tissue plasminogen activator (rtPA)—the standalone FDA-approved therapy for stroke. While rtPA is highly effective, it needs to be given within 3–4.5 hours of the onset of stroke symptoms (Zivin, 2009). This is often complicated by the delay in the commencement of treatment due to preliminary inclusion parameters that are required to be ascertained before rtPA administration.

Withaferin A Inhibits Nuclear Factor-κB-Dependent Pro-Inflammatory and Stress Response Pathways in the Astrocytes.

Several lines of evidence suggest that astrocytes play a key role in modulating the immune responses of the central nervous system (CNS) to infections, injuries, or pathologies. Yet, their contribution to these processes remains mostly elusive. Astroglia are endowed with a wide range of toll-like receptors (TLR) by which they can sense infectious agents as well as endogenous danger signals released by damaged cells. Here we demonstrate that the activation of astrocytic TLR4 by bacterial lipopolysaccharide (LPS) challenge can promote nuclear factor κB (NF-κB)-dependent induction of pro-inflammatory and stress response mediators, particularly Tumor Necrosis Factor α (TNFα), cyclooxygenase 2 (COX-2), and inducible nitric oxide synthase (iNOS). Since the steroid lactone Withaferin A was described to inhibit NF-κB activity in different cell types, we next determined the impact of this natural compound towards the identified astrocytic signalling pathway. Innate immune activation was induced by stimulation of the LPS/TLR4 axis in spinal cord astrocytes. We provide evidence that both pre-treating and post-treating the cells with Withaferin A attenuate astrocytic NF-κB activity as well as the consequent production of TNFα, COX-2, and iNOS induced by stimulation of the LPS/TLR4 pathway. This study suggests that Withaferin A may be an eligible candidate for the treatment of neuroinflammatory and stress conditions characterized by an important astrocytic input.

Anchorage-dependent multicellular aggregate formation induces CD44 high cancer stem cell-like ATL cells in an NF-κB- and vimentin-dependent manner

Adult T-cell leukemia/lymphoma (ATL) is an intractable T-cell malignancy accompanied by massive invasion of lymphoma cells into various tissues. We demonstrate here that ATL cells cultured on a layer of epithelial-like feeder cells form anchorage-dependent multicellular aggregates (Ad-MCAs) and that a fraction of MCA-forming ATL cells acquire CD44 high cancer stem cell-like phenotypes. ATL cells forming Ad-MCAs displayed extracellular microvesicles with enhanced expression of CD44v9 at cell synapses, augmented expression of multidrug resistance protein 1, and increased NF-κB activity. Blockade of the NF-κB pathway dramatically reduced Ad-MCA formation by ATL cells and the emergence of CD44 high ATL cells, but left a considerable number of ATL cells adhering to the feeder layer. Disruption of vimentin cytoskeleton by treatment with withaferin A, a natural steroidal lactone, suppressed not only the adhesion of ATL cells to the feeder layer but also subsequent Ad-MCA formation by ATL cells, suggesting the involvement of vimentin in anchoring ATL cells to the feeder layer. Ad-MCA formation by ATL cells on a layer of epithelial-like feeder cells may mimic critical events that occur in metastatic colonization.

DD-03 * THE NATURALLY OCCURRING STEROID, WITHAFERIN A, IN SYNERGISTIC CONCERT WITH HER2/EGFR INHIBITORS ABROGATES PROLIFERATION OF HUMAN GLIOBLASTOMA CELL CULTURES AT NANOMOLAR CONCENTRATIONS

INTRODUCTION: Withaferin A, a steroidal lactone that is isolated from the winter cherry (Withania sominifera), confers abrogatory effects on proliferating cancer cells. This is achieved, partly though Withaferin A's ability to retard angiogenesis along with VEGF and calcium signaling. At the level of transcription, Withaferin A inhibits both Nfkb and Sp1 Transcription factor activity. In glioblastomas, Withaferin A promotes cytotoxicity by impinging on Akt/mTOR-associated transduction pathways. In this report, we probe the relationship of Withaferin A with Her2- and EGFR-directed signaling (through the use of receptor-targeted inhibitors such as Afatinib and Lapatinib) in human glioblastomas. METHODS: Human parietal-cortical glioblastoma cells (pcGBM2, GBM39, LN229) were derived from primary tumors. Genomic material (both RNA and DNA) was isolated via standardized methods so as to study the mutational and expression status of relevant gene targets (PTEN, p53, MGMT, EGFRvIII). Proliferative potential of these cells was assessed by the alamar blue bioassay. RESULTS: The human glioblastomas studied displayed both wild-type and mutant p53. Both MGMT and PTEN expressions were usually suppressed. Expression of EGFRvIII was highly variegated with both wild type and vIII receptors being present. GBM39 expressed EGFRvIII levels 20-fold higher than pcGBM2. Growth of GBM39 and pcGBM2 were inhibited by Withaferin A with an IC50 of 0.3 + /-0.05 µM. The EGFR inhibitors, Afatinib and Lapatinib, abrogated glioblastoma growth at IC50s of 4.5/-1.0 uM and 18.0 + /-2.0 uM respectively. Inclusion of 0.15 µM, 0.3 µM and 0.6 µM of Withaferin A on top of Afatinib or Lapatinib resulted in dose-dependent, 2 to 4-fold sensitization of glioblastomas to the Her2/EGFR inhibitors. Addition of the Akt inhibitor, BKM120 to Afatinib and Withaferin A did not add further to the sensitization. CONCLUSIONS: The results suggest that Withaferin A exerts an inhibitory effect on glioblastoma proliferation and that it provides a synergistic effect on inhibition when co-administered with Afatinib or Lapatinib.

Covalent Modification of the NF-κB Essential Modulator (NEMO) by a Chemical Compound Can Regulate Its Ubiquitin Binding Properties in Vitro

Post-translational modification by ubiquitin plays important roles in multiple physiological and pathological processes. Ubiquitin-binding proteins play a critical role in recognizing and relaying polyubiquitin-based signaling. NEMO (NF-κB Essential Modulator) is a central player in canonical NF-κB signaling whose major function is to bind to Lys-63- and/or M1- (or linear) linked polyubiquitin chains generated in response to cell stimulation. Here we show that Withaferin A (WA), a steroidal lactone, causes a change in NEMO's interaction with specific types of polyubiquitin chains in vitro. WA induces full-length recombinant NEMO to bind to long Lys-48-linked polyubiquitin chains but not tetra-ubiquitin species. Significantly, the UBAN (ubiquitin binding in ABIN and NEMO) domain, essential for the ability of NEMO to bind M1/Lys-63-linked polyubiquitin, is dispensable for the WA-induced gain-of-function activity. Mass spectrometric analysis demonstrated that WA covalently modifies NEMO on a cysteine residue within the C-terminal zinc finger (ZF) domain. Point mutations to the ZF can reverse the WA-induced Lys-48-polyubiquitin binding phenotype. Our study demonstrates the feasibility of directly altering the ubiquitin interaction properties of an ubiquitin-binding protein by a chemical compound, thereby shedding light on a novel drug class to potentially alter polyubiquitin-based cellular processes.

ET-71 * THE ANTITUMOR EFFECTS OF WITHAFERIN A IN GLIOBLASTOMA STEM CELLS

INTRODUCTION: Glioblastoma Multiforme (GBM) is the most common and malignant primary brain tumors. Recent studies indicate that GBM stem cells (GSCs), are chemotherapy and radiotherapy resistant and are responsible for tumor cell invasion into normal brain, tumor growth and tumor reoccurrence following treatment. Withaferin A (WA), a steroidal lactone isolated from Withania somnifera, has demonstrated anti-tumor potential including inhibiting tumor growth, metastasis and angiogenesis. However, effects of WA in GSCs have not been previously explored. METHODS: GSCs were generated from patient's tumors and analyzed for stem cell markers CD133, CD44, GFAP, Nestin, Musashi, SOX2 and BMi1 by immunocytochemistry. Effect of WA on GSC viability was determined by MTS and/or LDH assay. Morphological effects of WA were evaluated by transmission electron microscopy. Effect of WA on cell signaling pathways was evaluated with flow cytometry, western blot analysis and fluorescent microscopy. ROS was detected using MitoSOX Red. RESULTS: WA induced both dose and time dependent increase in cell death in GSC lines (IC50 average 1.27+ 0.12uM for 6 GSC lines). In contrast, primary neurons were not affected by these concentrations and had a much higher IC50 (5.78uM). Treatment of GSCs with WA significantly increased the diameter of endoplasmic reticulum (ER) and western blot analysis confirmed ER stress demonstrating increased grp78, IRE1, and CHOP protein levels. WA treatment increased mitochondrial ROS production and induced apoptosis as indicated by increased levels of cleaved PARP, caspase 4/3/7/12, Bax, and bak. Pre-treatment with the antioxidant N-acetyl-L-cysteine (NAC) or caspase inhibitor (z-vad-fmk) attenuated WA-induced apoptosis. Lastly WA increased both velcade and NVP-BEZ235 induced cell death. CONCLUSION: Our data indicates that WA mediates anti-tumor effects via ROS generation, ER stress activation, and apoptosis induction in GSCs and potentially sensitizes these cells to additional chemotherapy. These findings warrant further investigation of WA as an adjuvant therapy for GBM.

Withaferin A induces apoptosis through the generation of thiol oxidation in human head and neck cancer cells.

Withaferin A is a steroidal lactone purified from the Indian medicinal plant, Withania somnifera. Withaferin A has been shown to inhibit the proliferation, metastasis, invasion and angiogenesis of cancer cells. In the present study, we investigated whether withaferin A induces apoptosis in the human head and neck cancer cells, AMC-HN4. Withaferin A markedly increased the sub-G1 cell population and the cleavage of poly(ADP-ribose) polymerase (PARP), which are markers of apoptosis. Pan-caspase inhibitor, z-VAD-fmk (z-VAD), markedly inhibited the withaferin A-induced apoptosis. However, the withaferin A-induced increase in the expression of COX-2 was not affected by treatment with z-VAD. Furthermore, withaferin A upregulated cyclooxygenase-2 (COX-2) expression. The COX-2 inhibitor, NS-398, reduced the withaferin A-induced production of prostaglandin E2. However, treatment with NS-398 did not affect the sub-G1 population and the cleavage of PARP. In addition, the withaferin A-induced apoptosis was independent of reactive oxygen species production. Thiol donors [N-acetylcysteine (NAC) and dithiothreitol (DTT)] reversed withaferin A-induced apoptosis. Therefore, our data suggest that withaferin A induces apoptosis through the mechanism of thiol oxidation in head and neck carcinoma cells.

Withaferin A disrupts ubiquitin-based NEMO reorganization induced by canonical NF-κB signaling

The NF-κB family of transcription factors regulates numerous cellular processes, including cell proliferation and survival responses. The constitutive activation of NF-κB has also emerged as an important oncogenic driver in many malignancies, such as activated B-cell like diffuse large B cell lymphoma, among others. In this study, we investigated the impact and mechanisms of action of Withaferin A, a naturally produced steroidal lactone, against both signal-inducible as well as constitutive NF-κB activities. We found that Withaferin A is a robust inhibitor of canonical and constitutive NF-κB activities, leading to apoptosis of certain lymphoma lines. In the canonical pathway induced by TNF, Withaferin A did not disrupt RIP1 polyubiquitination or NEMO–IKKβ interaction and was a poor direct IKKβ inhibitor, but prevented the formation of TNF-induced NEMO foci which colocalized with TNF ligand. While GFP-NEMO efficiently formed TNF-induced foci, a GFP-NEMOY308S mutant that is defective in binding to polyubiquitin chains did not form foci. Our study reveals that Withaferin A is a novel type of IKK inhibitor which acts by disrupting NEMO reorganization into ubiquitin-based signaling structures in vivo.

Abstract 228: Withaferin A downregulates tubulins and covalently binds β-tubulin at cysteine-303 in human breast cancer cells

Abstract We have shown previously that withaferin A (WA), a C5,C6-epoxy steroidal lactone derived from an Ayurvedic medicine plant (Withania somnifera), not only inhibits growth of human breast cancer cells in vitro and in vivo but also prevents mammary cancer development in a transgenic mouse model without any toxicity. However, the mechanisms underlying cancer preventive effect of WA are not fully understood. Herein, we report that tubulin is a novel target of WA in human breast cancer cells. WA treatment resulted in G2 and mitotic arrest in MCF-7 (estrogen-receptor positive), SK-BR-3 (estrogen-receptor negative), and SUM159 (triple-negative) cells in association with a marked decrease in protein level of β-tubulin. These effects were not observed with the naturally-occurring C6,C7-epoxy analogs of WA (withanone and withanolide A). In addition, a non-tumorigenic normal human mammary epithelial cell line (MCF-10A) was markedly more resistant to mitotic arrest and tubulin downregulation by WA treatment compared with breast cancer cells. Downregulation of tubulin protein in WA-treated cells was due to transcriptional repression as well as proteasomal degradation. Vehicle-treated control breast cancer cells exhibited a normal bi-polar spindle with chromosomes aligned along the metaphase plate. In contrast, WA treatment led to a severe disruption of the normal spindle morphology in breast cancer cells. Unlike breast cancer cells, WA-treated MCF-10A cells exhibited aggregation of tubulin around nucleus. NMR analyses revealed that the A-ring enone in WA, but not in withanone or withanolide A, was highly reactive with cysteamine and rapidly succumbed to irreversible nucleophilic addition. Mass spectrometry demonstrated direct covalent binding of WA at cysteine-303 of β-tubulin in MCF-7 cells. Molecular docking studies indicated that the WA-binding pocket was located on the surface of the β-tubulin and characterized by a hydrophobic floor, a hydrophobic wall, and a charge-balanced hydrophilic entrance. These results provide novel insights into the mechanism of anticancer effect of WA. This work was supported by the grants RO1 CA142604 and P30 CA047904 awarded by the National Cancer Institute. Citation Format: Shivendra V. Singh, Marie Lue Antony, Joomin Lee, Eun-Ryeong Hahm, Su-Hyeong Kim, Guillermo Romero, Adam I. Marcus, Zhen Yang, Vandana Kumari, Xinhua Ji, Courtney L. Vowell, Peter Wipf, Guy T. Uechi, Nathan A. Yates. Withaferin A downregulates tubulins and covalently binds β-tubulin at cysteine-303 in human breast cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 228. doi:10.1158/1538-7445.AM2014-228

Mammary cancer chemoprevention by withaferin A is accompanied by in vivo suppression of self-renewal of cancer stem cells.

Current dogma favors elimination of therapy-resistant cancer stem cells for chemoprevention of breast cancer. We showed recently that mammary cancer development in a transgenic mouse model (mouse mammary tumor virus-neu; MMTV-neu) was inhibited significantly upon treatment with withaferin A (WA), a steroidal lactone derived from a medicinal plant. Herein, we demonstrate that the mammary cancer prevention by WA is accompanied by in vivo suppression of breast cancer stem cells (bCSC). In vitro mammosphere formation was dose-dependently inhibited by WA treatment in MCF-7 and SUM159 human breast cancer cells. Other markers of bCSC, including aldehyde dehydrogenase 1 (ALDH1) activity and CD44(high)/CD24(low)/epithelial-specific antigen-positive (ESA+) fraction, were also decreased significantly in the presence of plasma achievable doses of WA. However, WA exposure resulted in cell line-specific changes in Oct4, SOX-2, and Nanog mRNA expression. WA administration to MMTV-neu mice (0.1 mg/mouse, 3 times/week for 28 weeks) resulted in inhibition of mammosphere number and ALDH1 activity in vivo. Mechanistic studies revealed that although urokinase-type plasminogen activator receptor overexpression conferred partial protection against bCSC inhibition by WA, Notch4 was largely dispensable for this response. WA treatment also resulted in sustained (MCF-7) or transient (SUM159) downregulation of Bmi-1 (B-cell-specific Moloney murine leukemia virus insertion region-1) protein. Ectopic expression of Bmi-1 conferred partial but significant protection against ALDH1 activity inhibition by WA. Interestingly, WA treatment caused induction of Kruppel-like factor 4 (KLF4) and its knockdown augmented bCSC inhibition by WA. In conclusion, this study shows in vivo effectiveness of WA against bCSC.

Cloning and Functional Characterization of Three Branch Point Oxidosqualene Cyclases from Withania somnifera (L.) Dunal

Oxidosqualene cyclases (OSCs) positioned at a key metabolic subdividing junction execute indispensable enzymatic cyclization of 2,3-oxidosqualene for varied triterpenoid biosynthesis. Such branch points present favorable gene targets for redirecting metabolic flux toward specific secondary metabolites. However, detailed information regarding the candidate OSCs covering different branches and their regulation is necessary for the desired genetic manipulation. The aim of the present study, therefore, was to characterize members of OSC superfamily from Withania somnifera (Ws), a medicinal plant of immense repute known to synthesize a large array of biologically active steroidal lactone triterpenoids called withanolides. Three full-length OSC cDNAs, β-amyrin synthase (WsOSC/BS), lupeol synthase (WsOSC/LS), and cycloartenol synthase (WsOSC/CS), having open reading frames of 2289, 2268, and 2277 bp, were isolated. Heterologous expression in Schizosaccharomyces pombe, LC-MS analyses, and kinetic studies confirmed their monofunctionality. The three WsOSCs were found to be spatially regulated at transcriptional level with WsOSC/CS being maximally expressed in leaf tissue. Promoter analysis of three WsOSCs genes resulted in identification of distinct cis-regulatory elements. Further, transcript profiling under methyl jasmonate, gibberellic acid, and yeast extract elicitations displayed differential transcriptional regulation of each of the OSCs. Changes were also observed in mRNA levels under elicitations and further substantiated with protein expression levels by Western blotting. Negative regulation by yeast extract resulted in significant increase in withanolide content. Empirical evidence suggests that repression of competitive branch OSCs like WsOSC/BS and WsOSC/LS possibly leads to diversion of substrate pool toward WsOSC/CS for increased withanolide production.

Withanolides array of Withania ashwagandha sp. novo populations from India

Withania ashwagandha (Solanaceae) is an annual herb of immense medicinal value largely due to the presence of array of steroidal lactones called withanolides. The present study was conducted to determine chemical and morphological variability among five W. ashwagandha (WA) populations collected from different regions of India. Variation in the contents of three promising bioactive withanolide, namely withanolide A (WS-1), withanone (WS-2) and withaferin A (WS-3) and morphological characters including plant height, number of shoots, root yield, and leaf biomass per plant were investigated. A considerable degree of variation in these bioactive withanolides and morphological characters was detected among the populations. Plant height and plant biomass were the highest in plants collected from Manasa population followed by Hyderabad. Leaves were found to be the principle organ for WS-3 accumulation while roots mainly accumulate WS-1, suggesting a spatial variation of withanolides. Plants of Manasa population alone showed the presence of WS-2. Withanolide accumulation was also the highest in Manasa population, with 1.312% WS-3 in the leaves and 0.083% WS-1 in roots, suggesting that plants from Manasa (WA02) are elite with regard to the parameters investigated. Further, ontogenetic variation of bioactive withanolides was studied in WA02 at five developmental stages. Withanolide accumulation correlated positively with developmental stages and highest content of these withanolides was found at maturity in both roots and leaves indicating that plants be harvested at maturity stage for maximum economic benefit. These results would offer a suitable alternative for unwise random harvesting that leads to rapid reduction of the existing natural populations of W. ashwagandha and for use as potential pharmacological agents.