Suberoylanilide Hydroxamic Acid and Bortezomib Synergistically Induce Apoptosis of EBV-Positive Burkitt Lymphoma Cells of Wp-Restricted or Type III Latency

Abstract

Background: Endemic Burkitt lymphoma (BL) is strongly associated with Epstein-Barr virus (EBV) with variable latent viral gene expression patterns (type I, Wprestricted and type III latency). We reported that bortezomib, a proteasome inhibitor, could potentiate the anti-tumour effects of suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, on EBV-positive nasopharyngeal carcinoma. Here, we aim to investigate the anti-tumour effects of SAHA, bortezomib and combination of the two drugs on EBV-positive BL cells of different viral latency. Methods: Cytotoxic effect of SAHA, bortezomib or combination of the two drugs (SAHA/bort) on BL cells of type I, Wp-restricted or type III latency was determined by MTT assay. Effects on apoptosis and cell cycle were measured by flow cytometry. Expression of apoptotic markers, EBV latent proteins and tumour suppressor genes were analyzed by western blotting.

Results: BL cells of Wp-restricted or type III latency, which express the EBV nuclear antigen (EBNA)-3 proteins, were more resistant to killing by SAHA than those of type I latency. However, adding bortezomib to SAHA synergistically enhanced the killing of BL cells of these two latency types. Compared with SAHA or bortezomib, SAHA/bort triggered enhanced apoptosis in the BL cells as indicated by the higher percentages of AV/PI-positive and sub-G1 populations as well as stronger proteolytic cleavage of apoptotic markers, PARP, caspase-3 and caspase-9, and induced the expression of two tumour suppressor genes, p16INK4A and p21WAF1, which are known to be down-regulated by the EBNA3 proteins. Furthermore, SAHA/bort suppressed the growth of BL xenografts in nude mice. Conclusions: Combination of SAHA and bortezomib synergistically induces the apoptosis of BL cells of Wprestricted or type III latency and possibly overcomes the resistance to apoptosis conferred by the EBNA3 proteins by up-regulation of p16INK4A and p21WAF1 genes. Clinical application of this drug combination to the treatment of primary EBV-driven lymphoproliferative disease such as post-transplant lymphoproliferative disorder should be further explored.