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Laura Asnaghi, Qian Yang, Arushi Tripathy, Allison Hanaford, Harpreet Kaur, Charles Eberhart; Hypoxia and Notch inhibition both enhance sensitivity of Retinoblastoma cells to Melphalan. Invest. Ophthalmol. Vis. Sci. 2016;57(12):3662.
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© ARVO (1962-2015); The Authors (2016-present)
To determine the effects of hypoxia and Notch pathway inhibition in modulating the response of retinoblastoma to Melphalan, an alkylating agent commonly used to treat this tumor.
Notch pathway inhibition was achieved in WERI Rb1 and Y79 retinoblastoma lines using the γ-secretase inhibitor MRK003. Cell viability was measured by Cell Counting kit-8 (CCK-8) or trypan blue exclusion cell dye. Induction of apoptosis was determined by immunofluorescence, using cleaved-caspase-3 assay. CompuSyn software was used to define the interaction between MRK003 and Melphalan. For the hypoxia experiments, cells were incubated for 1-7 days in 1% pO2 using an oxygen controller glove box. Cell viability was determined by CCK-8 assay in cells exposed to normoxia or hypoxia in the presence of Melphalan at 0.5, 1, 2, 4 µM. DMSO-treated cells were used as a control for MRK003 and Melphalan treatment.
We have recently found that Notch pathway is active and promotes growth and clonogenicity in retinoblastoma cells. Combination treatment with the Notch pathway inhibitor MRK003 (5 µM) and Melphalan (0.5-1 µM) produced a 70% greater reduction in growth and 40% increase in the percentage of cells positive for the apoptotic marker cleaved-caspase-3 as compared to single agents. The effect on viable cell number was at least additive, with a combination index < 1 when using these drugs at concentrations equal to their IC50. Since low oxygen tension (hypoxia) is a common phenomenon in retinoblastoma, we also investigated the effects that hypoxia might have on tumor growth and response to treatment. 1% pO2 conditions potently inhibited growth in both lines, with more prominent effect in Y79 cells. In addition, Melphalan resulted in an even stronger reduction in cell survival when cells were treated in hypoxia as compared to normoxia.
Inhibition of Notch and exposure to hypoxia significantly increased the sensitivity of retinoblastoma cells to Melphalan. Thus the combination with Notch antagonists or hypoxia modulators may represent a strategy to more effectively treat retinoblastoma or reduce doses in order to limit side effects.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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