withanolides and Neoplasm-Metastasis

Cancer, being the second leading cause of mortality, exists as a formidable health challenge. In spite of our enormous efforts, the emerging complexities in the molecular nature of disease progression limit the real success in finding an effective cancer cure. It is now conceivable that cancer is, in fact, a progressive illness, and the morbidity and mortality from cancer can be reduced by interfering with various oncogenic signaling pathways. A wide variety of structurally diverse classes of bioactive phytochemicals have been shown to exert anticancer effects in a large number of preclinical studies. Multiple lines of evidence suggest that withaferin-A can prevent the development of cancers of various histotypes. Accumulating data from different rodent models and cell culture experiments have revealed that withaferin-A suppresses experimentally induced carcinogenesis, largely by virtue of its potent anti-oxidative, anti-inflammatory, anti-proliferative and apoptosis-inducing properties. Moreover, withaferin-A sensitizes resistant cancer cells to existing chemotherapeutic agents. The purpose of this review is to highlight the mechanistic aspects underlying anticancer effects of withaferin-A.

Topics: Angiogenesis Inhibitors; Animals; Anti-Inflammatory Agents; Anticarcinogenic Agents; Antineoplastic Agents, Phytogenic; Apoptosis; Cell Movement; Cell Proliferation; Humans; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasms; Withania; Withanolides

Lung cancer is the leading cause of cancer-related deaths worldwide and in the United States. Despite recent advancements in treatment approaches, metastasis remains a major therapeutic challenge in lung cancer and explains the extremely poor prognosis. Epithelial to mesenchymal transition (EMT), a complex process of cellular reprogramming has become an attractive drug target because it plays a crucial role in the metastasis of non-small cell lung cancer (NSCLC). In the present study, we examined the effects of withaferin A (WFA), a plant-derived steroidal lactone on EMT in human NSCLC cell lines. First, we demonstrated that WFA displayed time- and concentration-dependent cytotoxicity on A549 and H1299 NSCLC cells. Then, cells were exposed to ≤ 0.5 µM WFA for ≤ 4 h to minimize cytotoxicity and determined its effects on EMT, cell adhesion, motility, migration, and invasion. EMT induction was performed by culturing cells in serum-free media containing TGFβ1 (5 ng/mL) and TNFα (25 ng/mL) for 48 h. We observed that pretreatment of cells with WFA inhibited cell adhesion, migration, and invasion of A549 and H1299 cells. Using western blot, immunofluorescence, and qRT-PCR analysis, we demonstrated that WFA suppressed TGFβ1 and TNFα-induced EMT in both cell lines. Mechanistically, WFA suppressed the phosphorylation and nuclear translocation of Smad2/3 and NF-κB in A549 and H1299 cells. Together, our study provides additional evidence demonstrating the inhibitory effects of WFA on EMT induction in NSCLC cells and further demonstrates the therapeutic potential of WFA against the metastasis in NSCLC.

Topics: Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Cell Adhesion; Cell Line, Tumor; Cell Movement; Epithelial-Mesenchymal Transition; Humans; Neoplasm Invasiveness; Neoplasm Metastasis; Withanolides

The eukaryotic translation initiation factor 4E (eIF4E) is considered as a key survival protein involved in cell cycle progression, transformation and apoptosis resistance. Herein, we demonstrate that medicinal plant derivative 3-AWA (from Withaferin A) suppressed the proliferation and metastasis of CaP cells through abrogation of eIF4E activation and expression via c-FLIP dependent mechanism. This translational attenuation prevents the de novo synthesis of major players of metastatic cascades viz. c-FLIP, c-Myc and cyclin D1. Moreover, the suppression of c-FLIP due to inhibition of translation initiation complex by 3-AWA enhanced FAS trafficking, BID and caspase 8 cleavage. Further ectopically restored c-Myc and GFP-HRas mediated activation of eIF4E was reduced by 3-AWA in transformed NIH3T3 cells. Detailed underlying mechanisms revealed that 3-AWA inhibited Ras-Mnk and PI3-AKT-mTOR, two major pathways through which eIF4E converges upon eIF4F hub. In addition to in vitro studies, we confirmed that 3-AWA efficiently suppressed tumor growth and metastasis in different mouse models. Given that 3-AWA inhibits c-FLIP through abrogation of translation initiation by co-targeting mTOR and Mnk-eIF4E, it (3-AWA) can be exploited as a lead pharmacophore for promising anti-cancer therapeutic development.

Topics: Adaptor Proteins, Signal Transducing; Adenosine Triphosphatases; Animals; Carcinoma, Ehrlich Tumor; Carrier Proteins; CASP8 and FADD-Like Apoptosis Regulating Protein; Caspase 8; Cation Transport Proteins; Cell Cycle Proteins; Cell Line, Tumor; Cell Movement; Copper-Transporting ATPases; Disease Models, Animal; Eukaryotic Initiation Factor-4E; Eukaryotic Initiation Factors; fas Receptor; Humans; Male; Mice; Neoplasm Metastasis; Phosphatidylinositol 3-Kinases; Phosphoproteins; Phosphorylation; Protein Biosynthesis; Protein Transport; Proto-Oncogene Proteins c-akt; ras Proteins; Signal Transduction; TOR Serine-Threonine Kinases; Withanolides

Ashwagandha is an important herb used in the Indian system of traditional home medicine, Ayurveda. Alcoholic extract (i-Extract) from its leaves and its component, withanone, were previously shown to possess anticancer activity. In the present study, we developed a combination of withanone and withaferin A, major withanolides in the i-Extract, that retained the selective cancer cell killing activity and found that it also has significant antimigratory, -invasive, and -angiogenic activities, in both in vitro and in vivo assays. Using bioinformatics and biochemical approaches, we demonstrate that these phytochemicals caused downregulation of migration-promoting proteins hnRNP-K, VEGF, and metalloproteases and hence are candidate natural drugs for metastatic cancer therapy.

Topics: Animals; Antineoplastic Agents, Phytogenic; Cell Line, Tumor; Cell Movement; Cell Proliferation; Disease Models, Animal; Heterogeneous-Nuclear Ribonucleoprotein K; Human Umbilical Vein Endothelial Cells; Humans; Mice; Models, Molecular; Molecular Conformation; Neoplasm Metastasis; Neoplasms; Neovascularization, Pathologic; Plant Extracts; Reproducibility of Results; Triterpenes; Tumor Burden; Withanolides; Xenograft Model Antitumor Assays

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.

Topics: Animals; Biomarkers, Tumor; Carcinoma, Ovarian Epithelial; Cell Division; Cell Line, Tumor; Cisplatin; Female; Humans; Mice; Mice, Nude; Neoplasm Metastasis; Neoplasms, Glandular and Epithelial; Neoplastic Stem Cells; Ovarian Neoplasms; Withanolides

The stem cell marker nestin is an intermediate filament protein that plays an important role in cell integrity, migration, and differentiation. Nestin expression occurs in approximately one third of pancreatic ductal adenocarcinoma (PDAC), and its expression strongly correlates with tumor staging and metastasis. Little is known about the mechanisms by which nestin influences PDAC progression. Here, nestin overexpression in PDAC cells increased cell motility and drove phenotypic changes associated with the epithelial-mesenchymal transition (EMT) in vitro; conversely, knockdown of endogenous nestin expression reduced the migration rate and reverted cells to a more epithelial phenotype. Mouse xenograft studies showed that knockdown of nestin significantly reduced tumor incidence and volume. Nestin protein expression was associated with Smad4 status in PDAC cells; hence, nestin expression might be regulated by the TGF-β1/Smad4 pathway in PDAC. We examined nestin expression after TGF-β1 treatment in human pancreatic cancer PANC-1 and PANC-1 shSmad4 cells. The TGF-β1/Smad4 pathway induced nestin protein expression in PDAC cells in a Smad4-dependent manner. Moreover, increased nestin expression caused a positive feedback regulator of the TGF-β1 signaling system. In addition, hypoxia was shown to induce nestin expression in PDAC cells, and the hypoxia-induced expression of nestin is mediated by the TGF-β1/Smad4 pathway. Finally, the antimicrotubule inhibitors, cytochalasin D and withaferin A, exhibited anti-nestin activity; these inhibitors might be potential antimetastatic drugs. Our findings uncovered a novel role of nestin in regulating TGF-β1-induced EMT. Anti-nestin therapeutics may serve as a potential treatment for PDAC metastasis.

Topics: Adenocarcinoma; Animals; Biomarkers, Tumor; Carcinogenesis; Carcinoma, Pancreatic Ductal; Cell Movement; Cytochalasin D; Disease Progression; Epithelial-Mesenchymal Transition; Gene Knockdown Techniques; Humans; Male; Mice; Mice, SCID; Microtubules; Neoplasm Metastasis; Nestin; Pancreas; Pancreatic Neoplasms; Signal Transduction; Smad4 Protein; Stem Cells; Transforming Growth Factor beta1; Up-Regulation; Withanolides; Xenograft Model Antitumor Assays

The medicinal plant Withania somnifera has been used for over centuries in Indian Ayurvedic Medicine to treat a wide spectrum of disorders. Withaferin A (WA), a bioactive compound that is isolated from this plant, has anti-inflammatory, immuno-modulatory, anti-angiogenic, and anti-cancer properties. Here we investigated malignant pleural mesothelioma (MPM) suppressive effects of WA and the molecular mechanisms involved. WA inhibited growth of the murine as well as patient-derived MPM cells in part by decreasing the chymotryptic activity of the proteasome that resulted in increased levels of ubiquitinated proteins and pro-apoptotic proteasome target proteins (p21, Bax, IκBα). WA suppression of MPM growth also involved elevated apoptosis as evidenced by activation of pro-apoptotic p38 stress activated protein kinase (SAPK) and caspase-3, elevated levels of pro-apoptotic Bax protein and cleavage of poly-(ADP-ribose)-polymerase (PARP). Our studies including gene-array based analyses further revealed that WA suppressed a number of cell growth and metastasis-promoting genes including c-myc. WA treatments also stimulated expression of the cell cycle and apoptosis regulatory protein (CARP)-1/CCAR1, a novel transducer of cell growth signaling. Knock-down of CARP-1, on the other hand, interfered with MPM growth inhibitory effects of WA. Intra-peritoneal administration of 5 mg/kg WA daily inhibited growth of murine MPM cell-derived tumors in vivo in part by inhibiting proteasome activity and stimulating apoptosis. Together our in vitro and in vivo studies suggest that WA suppresses MPM growth by targeting multiple pathways that include blockage of proteasome activity and stimulation of apoptosis, and thus holds promise as an anti-MPM agent.

Topics: Animals; Anticarcinogenic Agents; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Caspase 7; Cell Line, Tumor; Cell Proliferation; Enzyme Activation; Female; Gene Expression Regulation, Neoplastic; Humans; Mesothelioma; Mice; Mice, Inbred BALB C; Neoplasm Metastasis; p38 Mitogen-Activated Protein Kinases; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Withanolides; Xenograft Model Antitumor Assays

Interleukin-6 (IL-6), involved in cancer-related inflammation, acts as an autocrine and paracrine growth factor, which promotes angiogenesis, metastasis, and subversion of immunity, and changes the response to hormones and to chemotherapeutics. We explored transcription mechanisms involved in differential IL-6 gene expression in breast cancer cells with different metastatic properties. In weakly metastatic MCF7 cells, histone H3 K9 methylation, HP1 binding, and weak recruitment of AP-1 Fra-1/c-Jun, NF-kappaB p65 transcription factors, and coactivators is indicative of low chromatin accessibility and gene transcription at the IL-6 gene promoter. In highly metastatic MDA-MB231 cells, strong DNase, MNase, and restriction enzyme accessibility, as well potent constitutive transcription of the IL-6 gene promoter, coincide with increased H3 S10 K14 phosphoacetylation and promoter enrichment of AP-1 Fra-1/c-Jun and NF-kappaB p65 transcription factors and MSK1, CBP/p300, Brg1, and Ezh2 cofactors. Complementation, silencing, and kinase inhibitor experiments further demonstrate involvement of AP-1 Fra-1/c-Jun and NF-kappaB p65/RelB members, but not of the alpha estrogen receptor in promoting chromatin accessibility and transcription across the IL-6 gene promoter in metastatic breast cancer cells. Finally, the natural withanolide Withaferin A was found to repress IL-6 gene transcription in metastatic breast cancer cells upon dual inhibition of NF-kappaB and AP-1 Fra-1 transcription factors and silencing of IL-6 promoter chromatin accessibility.

Topics: Breast Neoplasms; Cell Line, Tumor; Chromatin; Ergosterol; Gene Expression Regulation, Neoplastic; Histones; Humans; Interleukin-6; Neoplasm Metastasis; Promoter Regions, Genetic; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-jun; RNA, Small Interfering; Transcription Factor AP-1; Transcription Factor RelA; Withanolides