Combination of HDAC and proteasome inhibitors up-regulates P16INK4A and P21WAF1 and induces apoptosis of EBV-Transformed lymphoblastoid cell lines

Abstract

Epstein-Barr virus (EBV) transforms B cells through its unique set of latent genes into continually proliferating lymphoblastoid cell lines (LCLs). Amongst the latent genes, EBV nuclear antigen (EBNA)-3A and -3C exert antiapoptotic effects on the LCLs through down-regulation of p16INK4A and p21WAF1. Histone deacetylase (HDAC) inhibitors induce apoptosis of various types of cancer cells through up-regulation of p16INK4A and p21WAF1 and proteasome inhibitors can enhance the anti-tumour effect of HDAC inhibitors through the generation of reactive oxygen species (ROS). Here, we hypothesise that combination of HDAC and proteasome inhibitors can counteract the anti-apoptotic effects of EBNA-3A and -3C in LCLs. We found that combination of HDAC (SAHA or romidepsin) and proteasome inhibitors (bortezomib or carfilzomib) could synergistically inhibit cell proliferation and mediate apoptosis, as evidenced by the annexin V-positive and sub-G1 populations as well as proteolytic cleavage of PARP and caspase-3, in three LCLs. The drug combinations did not affect the expression level of EBNA-3A and -3C whereas they directly induced the expression of p16INK4A and p21WAF1. Both up-regulation of p16INK4A and p21WAF1 and induction of apoptosis were dependent on the generation of ROS. We conclude that combination of HDAC and proteasome inhibitors may counteract the anti-apoptotic effects of EBNA-3A and -3C by the up-regulation of p16INK4A and p21WAF1, thereby inducing apoptosis of EBVtransformed LCLs.