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Taimur Safder, Erica Person, Mark Cohen, Ridhwi Mukerji, Abbas Samadi, Patrick Grogan; In Vitro and In Vivo Cytotoxic Effects of Withaferin A on Retinal Epithelial Pigmented Cells. Invest. Ophthalmol. Vis. Sci. 2012;53(14):5364.
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© ARVO (1962-2015); The Authors (2016-present)
Withaferin A (WA) is a naturaceudical product derived from Withania somnifera that has demonstrated anti-cancer activity against several cancer types including leukemia, colon, pancreas, thyroid, head and neck, breast, glioblastoma, and cutaneous and uveal melanoma. Control Retinal Pigment Epithelial (RPE) cells were exposed to WA and revealed an IC50 = 1.8µM in a comparable range to the IC50s of many other cancer lines [Leukemia SUPB15 = 0.13 µM, Thyroid DRO81-1 = 0.65 µM, KAT4B = 0.9 µM, Breast MCF-7 = 0.58 µM, MDA-MB231 = 0.54 µM, Melanoma SKMEL28 = 0.62 µM, UM 92.1= 2.50 µM, OMM 2.3 = 1.0 µM, and Fibroblast MRC-5 = 3.95 µM as systemic control]. Further in vivo and vitro experiments were conducted to observe any cytotoxic effects of Withaferin A on RPE Cells in vitro and in vivo.
In addition to MTS assay, cell proliferation and viability assay were performed through trypan blue exclusion assay to confirm RPE suppression. To determine if apoptosis was the primary mechanism, annexin V/PI staining and Western blot for caspase 3 and PARP were completed. An in vivo experiment compared retinal histology of mice treated with 8mg/kg/day to control. Experimental protocol was in compliance with the ARVO statement for the use of animals in ophthalmic and vision research and institutional animal care and use committee requirements.
Results of trypan blue exclusion confirmed our preliminary findings of dose dependent toxicity of RPE cells to Withaferin A (p =0.006 at 72hrs). Annexin V/PI and Western blot confirmed the mechanism of apoptosis when RPE cells were directly exposed to WA. However, histological evidence from the in vivo experiment suggests no damage was done to the RPE or retina at therapeutic IP dosing of WA.
WA is a promising naturiceudical agent for the treatment of multiple cancer types. While it has a cytotoxic effect on RPE cells in vitro, the same principle does not apply in vivo when applied to a murine model. The retinal-blood barrier could prevent the drug from reaching the retina and RPE and explain the difference between the in vivo and in vitro results. As WA transitions to human trials, close attention to the affects in the retina are indicated.
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