withaferin-a and Melanoma

Although selective BRAF inhibitors and novel immunotherapies have improved short-term treatment responses in metastatic melanoma patients, acquired resistance to these therapeutics still represent a major challenge in clinical practice. In this study, we evaluated the efficacy of Withaferin A (WFA), derived from the medicinal plant Withania Somnifera, as a novel therapeutic agent for the treatment of melanoma. WFA showed selective toxicity to melanoma cells compared to non-malignant cells. WFA induced apoptosis, significantly reduced cell proliferation and inhibited migration of melanoma cells. We identified that repression of the tumour suppressor TRIM16 diminished WFA cytotoxicity, suggesting that TRIM16 was in part responsible for the cytotoxic effects of WFA in melanoma cells. Together our data indicates that WFA has potent cytopathic effects on melanoma cells through TRIM16, suggesting a potential therapeutic application of WFA in the disease.

Topics: Antineoplastic Agents; Apoptosis; Cell Movement; Cell Proliferation; Gene Expression Regulation, Neoplastic; Humans; Melanoma; Tripartite Motif Proteins; Tumor Cells, Cultured; Ubiquitin-Protein Ligases; Withanolides

Paracrine interactions between keratinocytes and melanocytes via cytokines play an essential role in regulating pigmentation in epidermal hyperpigmentary disorders. There is an urgent need for a human epidermal model in which melanogenic paracrine interactions between UVB-exposed keratinocytes and melanocytes can be precisely evaluated because human epidermal equivalents consisting of multilayered keratinocytes and melanocytes have significant limitations in this respect.. To resolve this challenge, we established a co-culture system with cell inserts using human keratinocytes and human melanocytes that serves as an appropriate new model for UVB-induced hyperpigmentation. Using that new model, we examined the blocking effects of two natural chemicals, astaxanthin and withaferin A, on paracrine cytokine interactions between UVB-exposed keratinocytes and melanocytes and characterized their mechanisms of action.. RT-PCR analysis showed that co-culture of human keratinocytes that had been exposed to UVB significantly stimulated human melanocytes to increase their expression of genes encoding microphthalmia-associated transcription factor, tyrosinase and tyrosinase-related protein 1. The catalytic activity of tyrosinase was also increased. ELISA assays revealed that UVB significantly increased the secretion of interleukin-1α, interleukin-6/8, granulocyte macrophage stimulatory factor and endothelin-1 but not α-melanocyte stimulating hormone. The addition of an endothelin-1 neutralizing antibody significantly abrogated the increase of tyrosinase activity. Post-irradiation treatment with astaxanthin or withaferin A significantly abolished the up-regulation of tyrosinase activity induced by UVB. Treatment with astaxanthin or withaferin A significantly reduced the increased levels of interleukin-1α, interleukin-6/8, granulocyte macrophage stimulatory factor and endothelin-1. Withaferin A but not astaxanthin also significantly abrogated the endothelin-1-stimulated activity of tyrosinase in melanocytes. Western blot analysis of intracellular signaling factors revealed that withaferin A but not astaxanthin significantly abolished the endothelin-1-stimulated phosphorylation of Raf-1, MEK, ERK, MITF and CREB in human melanocytes.. These results demonstrate that this co-culture system is an appropriate model to characterize melanogenic paracrine interactions and that astaxanthin and withaferin A serve as potent inhibitors of those interactions. Their effects are caused not only by down-regulating the increased secretion of an intrinsic melanogenic cytokine, endothelin-1, by UVB-exposed human keratinocytes, but also by interrupting the endothelin-1-triggered downstream intracellular signaling between protein kinase C and Raf-1 in human melanocytes (only for withaferin A).

Topics: Antibodies; Calcium; Cell Line, Tumor; Coculture Techniques; Cytokines; Dithiothreitol; Endothelin-1; Gene Expression Regulation; Humans; Intracellular Space; Keratinocytes; Melanocytes; Melanoma; Monophenol Monooxygenase; Paracrine Communication; Phosphorylation; Signal Transduction; Ultraviolet Rays; Withanolides; Xanthophylls

Uveal melanoma (UM) is the most common primary intraocular malignancy in adults. While effective therapy exists for the primary tumor, there is a lack of effective treatment for metastatic disease currently. Natural withanolide withaferin A (WA) has shown efficacy in cancers demonstrating upregulation of pro-survival pathways. The purpose of the present study is to investigate the effect of WA as a potential therapeutic agent for UM in vitro as well as in vivo. UM cells were treated with WA and several cell-based assays, such as MTS, trypan blue exclusion assay, clonogenic, wound healing, cell cycle shift, annexin V/propidium iodide, and Western blot, were performed. In vivo experiments utilized the 92.1 cells in a xenograft murine model. WA inhibits cell proliferation of uveal melanoma cells with an IC50 of 0.90, 1.66, and 2.42 μM for OMM2.3, 92.1, and MEL290 cells, respectively. Flow cytometry analysis demonstrates G2/M cell cycle arrest and apoptosis at 1 μM WA in treated cells. WA induced apoptosis partly through the suppression of c-Met, Akt, and Raf-1 signaling activation. In vivo studies using WA reduced tumor growth in 100% of animals (p = 0.015). Our observation indicates that WA is a potent drug that inhibits cell proliferation, shifts cell cycle arrest, and induces apoptosis in multiple UM cell lines in vitro. WA-mediated apoptosis in UM cells is partly mediated though the suppression of c-Met and Akt activation. WA significantly decreases UM tumor growth in vivo and justifies further evaluation of this drug for the treatment of metastatic uveal melanoma.

Topics: Animals; Apoptosis; Blotting, Western; Caspase 3; Cell Cycle; Cell Line, Tumor; Cell Movement; Cell Proliferation; Dose-Response Relationship, Drug; Enzyme Activation; Female; Humans; Inhibitory Concentration 50; Melanoma; Mice; Mice, SCID; Molecular Structure; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-met; Time Factors; Tumor Burden; Uveal Neoplasms; Withanolides; Xenograft Model Antitumor Assays

A high resistance and heterogeneous response to conventional anti-cancer chemotherapies characterize malignant cutaneous melanoma, the most aggressive and deadly form of skin cancer. Withaferin A (WFA), a withanolide derived from the medicinal plant Withania somnifera, has been reported for its anti-tumorigenic activity against various cancer cells. For the first time, we examined the death-inducing potential of WFA against a panel of four different human melanoma cells and investigated the cellular mechanisms involved. WFA induces apoptotic cell death with various IC50 ranging from 1.8 to 6.1 μM. The susceptibility of cells toward WFA-induced apoptosis correlated with low Bcl-2/Bax and Bcl-2/Bim ratios. In all cell lines, the apoptotic process triggered by WFA involves the mitochondrial pathway and was associated with Bcl-2 down regulation, Bax mitochondrial translocation, cytochrome c release into the cytosol, transmembrane potential (ΔΨm) dissipation, caspase 9 and caspase 3 activation and DNA fragmentation. WFA cytotoxicity requires early reactive oxygen species (ROS) production and glutathione depletion, the inhibition of ROS increase by the antioxidant N-acetylcysteine resulting in complete suppression of mitochondrial and nuclear events. Altogether, these results support the therapeutic potential of WFA against human melanoma.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Caspase 9; Cell Line, Tumor; DNA Fragmentation; Down-Regulation; Humans; Melanoma; Mice; Mitochondria; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Withanolides

The effect of withaferin A, a plant withanolide, alone or in combination with acute and fractionated radiotherapy and/or hyperthermia, was tested on two mouse tumors, B16F1 melanoma and fibrosarcoma, grown in C57BL and Swiss albino mice, respectively. Tumors were exposed locally to 30 or 50 Gy gamma radiation as acute dose, or 5 fractions of 10 Gy. Withaferin A, 40 mg/kg, was injected intraperitoneally, 1h before acute irradiation, or 30 mg/kg before every 10 Gy fraction. Local hyperthermia, 43 degrees C for 30 min, followed acute RT or first fraction of 10 Gy. Withaferin A, radiation and hyperthermia, individually and in bimodality treatments, produced no complete response (CR) in melanoma. Some CR were seen in fibrosarcoma, which increased after bimodality treatments. Trimodality treatment synergistically increased CR to 37% in melanoma and to 64% in fibrosarcoma. Fractionated radiotherapy (10 Gy x 5) was more effective (25% CR) than acute dose of 50 Gy (0% CR) on melanoma, while there was no difference between the response of fibrosarcoma to the two regimens. Withaferin A with fractionated radiotherapy synergistically increased the CR of both tumors; hyperthermia further enhanced this effect. Utility of withaferin A in increasing the clinical response of radioresistant tumors to fractionated radiotherapy has to be explored.

Topics: Animals; Combined Modality Therapy; Dose Fractionation, Radiation; Ergosterol; Female; Fibrosarcoma; Hyperthermia, Induced; Melanoma; Mice; Mice, Inbred C57BL; Radiation-Sensitizing Agents; Withanolides