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Lata Singh, Seema Kashyap, Neelam Pushker, Seema Sen, Sameer Bakhshi, Anjana Sharma, Neeru Saini, Jasbir Kaur; Role of Apoptotic Regulatory Proteins in Retinoblastoma. Invest. Ophthalmol. Vis. Sci. 2013;54(15):4987.
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Deregulation of apoptosis has been demonstrated in cancer development, proliferation and treatment resistance. Bcl-2 protein family plays a key role in the initiation of the apoptotic pathways. Bax and PUMA are pro-apoptotic proteins whereas Bcl-2 is an anti-apoptotic protein which together influence apoptosis. However, we aim to study the expression of these apoptotic regulatory proteins Bax, PUMA and Bcl-2 in retinoblastoma (Rb) and correlate them with histopathological parameters.
Prospective analysis of immunohistochemical expression of Bax, PUMA and Bcl-2 proteins in 45 paraffin embedded sections of primary enucleated Rb cases were performed. Their results were validated in fresh tumor tissue by western blotting. Cytoplasmic staining was graded as weak/negative (1+), moderate (2+) and strong (3+). Protein expressions were correlated with tumor differentiation, necrosis, calcification and histopathological high risk factors (HRFs) like massive choroidal invasion, optic nerve invasion, sclera, anterior chamber, iris & ciliary body invasion.
A total of 45 cases were analyzed with a male preponderance (25/45) and their age ranged from 6mths to 8 years. Higher tumor staging (T2aNoMo-T4bNoMo) was found in 25 cases. Poorly differentiated retinoblastoma constituted 71.1% and necrosis and calcification was found in 28/45 (62.5%) and 11/45 (25%) respectively. One or more than one HRFs were found in 55.5%. There were 16 cases with massive choroidal invasion, 27 optic nerve invasion, 7 scleral invasion and 6 iris & ciliary body invasion. Immunohistochemistry showed strong expression of Bax (66.6%) and PUMA (62.2%), whereas no Bcl-2 expression was found in 32/45 (71.1%) cases. The western blotting results correlated with the immunohistochemical expression of Bax (21kDa), PUMA (24kDa) and Bcl-2 (25kDa) proteins. Overexpression of Bax significantly correlated with HRFs (P=0.0274) and poor differentiation (P=0.0163). Similarly, expression of PUMA also correlated with extensive necrosis (p=0.0038) and HRFs (p=0.0007). However, no significant correlation was found with Bcl-2 expression.
Immunohistochemistry and western blotting showed negative correlation between pro-apoptotic and anti-apoptotic proteins in patients with histopathological HRFs. Our findings suggest that these apoptotic regulatory proteins may serve as poor prognostic markers and can be used as a therapeutic target for the treatment of Retinoblastoma.
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