Targeting retinoblastoma binding protein 6 (RBBP6) as an anti-ovarian cancer therapeutic strategy
Date
2015-05-07
Authors
Ubanako, Philemon Njende
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Abstract
Ovarian cancer is the most lethal gynaecological cancer. About 90% of ovarian cancers are
epithelial (ovarian carcinomas), thought to arise from the ovarian surface
epithelium. Diagnosed usually at clinically advanced stages, many patients show poor
response to chemotherapy, with resistance and recurrent disease being prevalent. siRNA
technology is currently being explored in clinical trials as a form of targeted therapeutic
strategy in the disease. RBBP6 is a 250kD protein that enhances MDM2-mediated
ubiquitination of p53 and also plays a role in cell cycle regulation and cell differentiation. It
is upregulated in numerous cancers such as lung, oesophageal, colorectal and cervical cancer.
RBBP6 suppresses p53 binding to DNA thereby inhibiting p53-dependent gene transcription.
RBBP6 was knocked down using 30 nM siRNA in RMG-1 cells for 48 hours, after which the
cells were treated with 50 nM paclitaxel and 0.5μM camptothecin for 24 hours. xCELLigence
real time cell analysis was used to evaluate cell proliferation. qPCR and western blot were
used to evaluate both gene expression and protein expressions respectively, of Bax, Bcl-2,
MDM2, p53 and p21. Flow cytometry was used to determine the mode of cell death elicited
apoptosis and also analyse changes in cell cycle progression.
qPCR and Western blot analyses showed that RBBP6 expression reduced by approximately
57%. There was a significant upregulation of p53 and a significant downregulation of Bcl-2
in siRBBP6 transfected cells (p<0.05). Knockdown of RBBP6 resulted in a 37±5.8% cell
death. There was a significant increase in cell death in paclitaxel and siRBBP6 co-treated
cells (81.6±0.79%) as compared to cells treated with paclitaxel only (76.±1.14%).
siRNA-mediated knock down of RBBP6 induces cell death in RMG-1 ovarian carcinoma
cells. In addition, paclitaxel-induced cell death in RMG-1 cells is potentiated by RBBP6
siRNA transfection. A combination of chemotherapy with paclitaxel or camptothecin and
RBBP6 siRNA could be a possible therapeutic strategy in combatting ovarian carcinomas.
Description
A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of requirements for the degree of Master of Science. Johannesburg 2015.
Keywords
Ovarian cancer, Paclitaxel, MDM2, siRNA, Apoptosis, RBBP6, Camptothecin, p53