withanolides and Mouth-Neoplasms

Abnormal expression of claudin-1 (CLDN1) has important roles in carcinogenesis and metastasis in various cancers. The role of CLDN1 in human oral squamous cell carcinoma (OSCC) remains unknown. Here, we report the functional role of CLDN1 in metastasis of human OSCC, as a potential target regulated by withaferin A. From gene expression profiling with microarray technology, we found that the majority of notable differentially expressed genes were classified into migration/invasion category. Withaferin A impaired the motility of human OSCC cells in vitro and suppressed metastatic nodule formation in an in vivo metastasis model, both associated with reduced CLDN1. CLDN1 overexpression enhanced metastatic nodule formation in vivo, resulting in severe metastatic lesions in lung tissue. Moreover, CLDN1 expression was positively correlated to lymphatic metastasis in OSCC patients. The impaired motility of human OSCC cells upon withaferin A treatment was restored by CLDN1 overexpression. Furthermore, upregulation of let-7a induced by withaferin A was inversely correlated to CLDN1 expression. Overall, these give us an insight into the function of CLDN1 for prognosis and treatment of human OSCC, substantiating further investigation into the use of withaferin A as good anti-metastatic drug candidate.

Topics: Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Movement; Claudin-1; Gene Expression Regulation, Neoplastic; Head and Neck Neoplasms; Humans; Mouth Neoplasms; Squamous Cell Carcinoma of Head and Neck; Withanolides

The aim of this study was to determine the apoptotic activity of methanol extract of Ashwagandha (MEAG) and in human head and neck squamous cell carcinoma (HNSCC) cells and to investigate the underlying mechanisms.. We investigated the effects of MEAG on programmed cell death in HNSCC cells using a Live/Dead assay, detection of nuclear morphologic changes, Mitotracker, siRNA knockdown, and RT-PCR.. Treatment with MEAG showed dose-dependent growth-inhibitory activity that attribute to caspase-dependent apoptosis. Loss of mitochondrial membrane potential, release of cytochrome c, and activation of caspase 9 suggested that MEAG leads to activation of mitochondria-mediated apoptosis. MEAG selectively upregulated the expression of Bim protein at the transcriptional level and induced the translocation of Bim into the mitochondria. Knockdown of Bim by siRNA partially blocked MEAG-mediated apoptosis. MEAG also caused an increase in truncated Bid (t-Bid), cleaved caspase-8, and death receptor 5 (DR5). Interestingly, withaferin A (WA), a bioactive component of MEAG, clearly induced apoptosis accompanied by upregulation of Bim, t-Bid, caspase-8, and DR5 similar to the effects of MEAG.. These suggest that MEAG and WA may be potential natural materials for the treatment of HNSCC.

Topics: Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Bcl-2-Like Protein 11; Carcinoma, Squamous Cell; Caspase 8; Caspase 9; Cell Line, Tumor; Cytochromes c; Enzyme Activation; Head and Neck Neoplasms; Humans; Membrane Potential, Mitochondrial; Mitochondrial Membranes; Mouth Neoplasms; Plant Extracts; Receptors, TNF-Related Apoptosis-Inducing Ligand; Squamous Cell Carcinoma of Head and Neck; Up-Regulation; Withanolides

Most chemotherapeutic drugs for killing cancer cells are highly cytotoxic in normal cells, which limits their clinical applications. Therefore, a continuing challenge is identifying a drug that is hypersensitive to cancer cells but has minimal deleterious effects on healthy cells. The aims of this study were to evaluate the potential of 4β-hydroxywithanolide (4βHWE) for selectively killing cancer cells and to elucidate its related mechanisms.. Changes in survival, oxidative stress, DNA damage, and apoptosis signaling were compared between 4βHWE-treated oral cancer (Ca9-22) and normal fibroblast (HGF-1) cells. At 24 h and 48 h, the numbers of Ca9-22 cells were substantially decreased, but the numbers of HGF-1 cells were only slightly decreased. Additionally, the IC50 values for 4βHWE in the Ca9-22 cells were 3.6 and 1.9 µg/ml at 24 and 48 h, respectively. Time-dependent abnormal increases in ROS and dose-responsive mitochondrial depolarization can be exploited by using 4βHWE in chemotherapies for selectively killing cancer cells. Dose-dependent DNA damage measured by comet-nuclear extract assay and flow cytometry-based γ-H2AX/propidium iodide (PI) analysis showed relatively severer damage in the Ca9-22 cells. At both low and high concentrations, 4βHWE preferably perturbed the cell cycle in Ca9-22 cells by increasing the subG1 population and arrest of G1 or G2/M. Selective induction of apoptosis in Ca9-22 cells was further confirmed by Annexin V/PI assay, by preferential expression of phosphorylated ataxia-telangiectasia- and Rad3-related protein (p-ATR), and by cleavage of caspase 9, caspase 3, and poly ADP-ribose polymerase (PARP).. Together, the findings of this study, particularly the improved understanding of the selective killing mechanisms of 4βHWE, can be used to improve efficiency in killing oral cancer cells during chemoprevention and therapy.

Topics: Apoptosis; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Survival; Comet Assay; DNA Damage; Drug Screening Assays, Antitumor; Flow Cytometry; Histones; Humans; Membrane Potential, Mitochondrial; Models, Biological; Mouth Neoplasms; Physalis; Propidium; Reactive Oxygen Species; Signal Transduction; Withanolides

Oral cancer, the fifth most frequent cancer worldwide, is a major health problem and accounts for highest morbidity and mortality in human populations. This form of cancer accounts for 40-50% of all cancers in developing countries including India. Despite recent advancement in the treatment, imaging and diagnosis of oral carcinoma, a 5-year survival and mortality rate for this cancer is still at 50%. Our aim was to study the protective effect of Withaferin-A on molecular pathogenesis of oral cancer by evaluating the immunoexpression of p53 and bcl-2 proteins. Oral squamous cell carcinoma was developed in the left buccal pouch of golden Syrian hamsters by painting with 0.5% 7,12-dimethylbenz(a)anthracene (DMBA), three times a week for 14 weeks. We observed 100% tumor formation with high tumor volume and burden in the DMBA alone painted hamsters as compared to control hamsters. We also observed markedly altered expression of p53 and bcl-2 proteins in tumor tissues of oral cancer bearing hamsters. Oral administration of Withaferin-A to DMBA-painted hamsters not only completely prevented oral squamous cell carcinoma formation but also significantly prevented the alterations of p53 and bcl-2 expressions. Our results thus suggest that Withaferin-A has significant protective role against DMBA induced molecular alterations in the buccal mucosa of golden Syrian hamsters.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Carcinogens; Carcinoma, Squamous Cell; Cricetinae; Disease Models, Animal; Ergosterol; Humans; Male; Mesocricetus; Molecular Structure; Mouth Neoplasms; Phytotherapy; Plant Extracts; Proto-Oncogene Proteins c-bcl-2; Tumor Suppressor Protein p53; Withanolides

With an aim to investigate the protective effect of Withaferin-A on 7,12-dimethylbenz[a]anthracene (DMBA) induced oral carcinogenesis in Syrian golden hamsters, tumour incidence, tumour volume and tumour burden and status of detoxication agents, lipid peroxidation and antioxidants in DMBA administered (3 times/week for 14 weeks) hamsters were assessed. Hundred percent tumour formation in DMBA alone administered animal was observed. Oral administration of Withaferin-A (20 mg/kg body weight) to DMBA administered animals for 14 weeks completely prevented the tumour incidence, tumour volume and tumour burden. Also, Withaferin-A showed significant anti-lipid peroxidative and antioxidant properties and maintained the status of phase-I and phase-II detoxication agents during DMBA-induced oral carcinogenesis. The results thus indicate that the protective effect of Withaferin-A is probably due to its anti-lipid peroxidative and antioxidant functions as well as modulating effect on carcinogen detoxication during DMBA-induced oral carcinogenesis.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Antioxidants; Cricetinae; Drug Screening Assays, Antitumor; Ergosterol; Erythrocytes; Liver; Metabolic Detoxication, Phase I; Metabolic Detoxication, Phase II; Mouth Neoplasms; Phytotherapy; Protective Agents; Thiobarbituric Acid Reactive Substances; Withanolides