Abstract
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.
Similar content being viewed by others
References
Singh AD, Borden EC. Metastatic uveal melanoma. Ophthalmol Clin North Am. 2005;18:143–50.
Singh AD, Damato B, Howard P, Harbour JW. Uveal melanoma: genetic aspects. Ophthalmol Clin North Am. 2005;18:85–97.
Ven Murthy MR, Ranjekar PK, Ramassamy C, Deshpande M. Scientific basis for the use of Indian ayurvedic medicinal plants in the treatment of neurodegenerative disorders: Ashwagandha. Cent Nerv Syst Agents Med Chem. 2010;10:238–46.
Mishra LC, Singh BB, Dagenais S. Scientific basis for the therapeutic use of Withania somnifera (ashwagandha): a review. Altern Med Rev. 2000;5:334–46.
Choi MJ, Park EJ, Min KJ, Park JW, Kwon TK. Endoplasmic reticulum stress mediates withaferin A-induced apoptosis in human renal carcinoma cells. Toxicol In Vitro. 2011;25:692–8.
Lee J, Hahm ER, Singh SV. Withaferin A inhibits activation of signal transducer and activator of transcription 3 in human breast cancer cells. Carcinogenesis. 2010;31:1991–8.
Koduru S, Kumar R, Srinivasan S, Evers MB, Damodaran C. Notch-1 inhibition by withaferin-A: a therapeutic target against colon carcinogenesis. Mol Cancer Ther. 2010;9:202–10.
Samadi AK, Mukerji R, Shah A, Timmermann BN, Cohen MS. A novel ret inhibitor with potent efficacy against medullary thyroid cancer in vivo. Surgery. 2010;148:1228–36. discussion 1236.
Samadi AK, Tong X, Mukerji R, Zhang H, Timmermann BN, Cohen MS. Withaferin A, a cytotoxic steroid from Vassobia breviflora, induces apoptosis in human head and neck squamous cell carcinoma. J Nat Prod. 2010;73:1476–81.
Samadi A, Loo P, Mukerji R, O’Donnell G, Tong X, Timmermann BN, Cohen MS. A novel hsp90 modulator with selective activity against thyroid cancers in vitro. Surgery. 2009;146:1196–207.
Cruz 3rd F, Rubin BP, Wilson D, Town A, Schroeder A, Haley A, Bainbridge T, Heinrich MC, Corless CL. Absence of BRAF and NRAS mutations in uveal melanoma. Cancer Res. 2003;63:5761–6.
Kilic E, Bruggenwirth HT, Verbiest MM, Zwarthoff EC, Mooy NM, Luyten GP, de Klein A. The RAS-BRAF kinase pathway is not involved in uveal melanoma. Melanoma Res. 2004;14:203–5.
van den Bosch T, Kilic E, Paridaens D, de Klein A. Genetics of uveal melanoma and cutaneous melanoma: two of a kind? Dermatol Res Pract. 2010;2010:1–13.
Calipel A, Mouriaux F, Glotin AL, Malecaze F, Faussat AM, Mascarelli F. Extracellular signal-regulated kinase-dependent proliferation is mediated through the protein kinase A/B-Raf pathway in human uveal melanoma cells. J Biol Chem. 2006;281:9238–50.
Dhomen N, Marais R. New insight into BRAF mutations in cancer. Curr Opin Genet Dev. 2007;17:31–9.
Lefevre G, Babchia N, Calipel A, Mouriaux F, Faussat AM, Mrzyk S, Mascarelli F. Activation of the FGF2/FGFR1 autocrine loop for cell proliferation and survival in uveal melanoma cells. Invest Ophthalmol Vis Sci. 2009;50:1047–57.
Palmieri G, Capone M, Ascierto ML, Gentilcore G, Stroncek DF, Casula M, Sini MC, Palla M, Mozzillo N, Ascierto PA. Main roads to melanoma. J Transl Med. 2009;7:86.
Onken MD, Worley LA, Long MD, Duan S, Council ML, Bowcock AM, Harbour JW. Oncogenic mutations in GNAQ occur early in uveal melanoma. Invest Ophthalmol Vis Sci. 2008;49:5230–4.
Van Raamsdonk CD, Bezrookove V, Green G, Bauer J, Gaugler L, O’Brien JM, Simpson EM, Barsh GS, Bastian BC. Frequent somatic mutations of GNAQ in uveal melanoma and blue naevi. Nature. 2009;457:599–602.
Babchia N, Calipel A, Mouriaux F, Faussat AM, Mascarelli F. The PI3k/Akt and mTOR/P70S6K signaling pathways in human uveal melanoma cells: interaction with B-raf/ERK. Invest Ophthalmol Vis Sci. 2010;51:421–9.
Mallikarjuna K, Pushparaj V, Biswas J, Krishnakumar S. Expression of epidermal growth factor receptor, ezrin, hepatocyte growth factor, and c-Met in uveal melanoma: an immunohistochemical study. Curr Eye Res. 2007;32:281–90.
Hendrix MJ, Seftor EA, Seftor RE, Kirschmann DA, Gardner LM, Boldt HC, Meyer M, Pe’er J, Folberg R. Regulation of uveal melanoma interconverted phenotype by hepatocyte growth factor/scatter factor (HGF/SF). Am J Pathol. 1998;152:855–63.
Economou MA, All-Ericsson C, Bykov V, Girnita L, Bartolazzi A, Larsson O, Seregard S. Receptors for the liver synthesized growth factors IGF-1 and HGF/SF in uveal melanoma: intercorrelation and prognostic implications. Invest Ophthalmol Vis Sci. 2005;46:4372–5.
Economou MA, All-Ericsson C, Bykov V, Girnita L, Bartolazzi A, Larsson O, Seregard S. Receptors for the liver synthesized growth factors IGF-1 and HGF/SF in uveal melanoma: intercorrelation and prognostic implications. Acta Ophthalmol. 2008;86:20–5.
Peruzzi B, Bottaro DP. Targeting the c-Met signaling pathway in cancer. Clin Cancer Res. 2006;12:3657–60.
Ye M, Hu D, Tu L, Zhou X, Lu F, Wen B, Wu W, Lin Y, Zhou Z, Qu J. Involvement of PI3k/Akt signaling pathway in hepatocyte growth factor-induced migration of uveal melanoma cells. Invest Ophthalmol Vis Sci. 2008;49:497–504.
Abdel-Rahman MH, Yang Y, Zhou XP, Craig EL, Davidorf FH, Eng C. High frequency of submicroscopic hemizygous deletion is a major mechanism of loss of expression of PTEN in uveal melanoma. J Clin Oncol. 2006;24:288–95.
Saraiva VS, Caissie AL, Segal L, Edelstein C, Burnier Jr MN. Immunohistochemical expression of phospho-Akt in uveal melanoma. Melanoma Res. 2005;15:245–50.
Mitsiades N, Chew SA, He B, Riechardt AI, Karadedou T, Kotoula V, Poulaki V. Genotype-dependent sensitivity of uveal melanoma cell lines to inhibition of B-Raf, MEK, and Akt kinases: rationale for personalized therapy. Invest Ophthalmol Vis Sci. 2011;52:7248–55.
Casagrande F, Bacqueville D, Pillaire MJ, Malecaze F, Manenti S, Breton-Douillon M, Darbon JM. G1 phase arrest by the phosphatidylinositol 3-kinase inhibitor LY 294002 is correlated to up-regulation of p27Kip1 and inhibition of G1 CDKs in choroidal melanoma cells. FEBS Lett. 1998;422:385–90.
Harbour JW, Onken MD, Roberson ED, Duan S, Cao L, Worley LA, Council ML, Matatall KA, Helms C, Bowcock AM. Frequent mutation of BAP1 in metastasizing uveal melanomas. Science. 2010;330:1410–3.
Abdel-Rahman MH, Boru G, Massengill J, Salem MM, Davidorf FH. MET oncogene inhibition as a potential target of therapy for uveal melanomas. Invest Ophthalmol Vis Sci. 2010;51:3333–9.
Acknowledgment
This work was supported in part by the Kansas Lion’s Sight Foundation.
Conflicts of interest
None
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Samadi, A.K., Cohen, S.M., Mukerji, R. et al. Natural withanolide withaferin A induces apoptosis in uveal melanoma cells by suppression of Akt and c-MET activation. Tumor Biol. 33, 1179–1189 (2012). https://doi.org/10.1007/s13277-012-0363-x
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s13277-012-0363-x