Molecular and cellular effects of bortezomib on Epstein-Barr virus positive nasopharyngeal carcinoma

Abstract

Nasopharyngeal carcinoma (NPC) is a common cancer in Southeast Asia. While external radiotherapy is the mainstay of treatment, adjuvant chemotherapy is required in advanced disease. Current chemotherapy heavily relies on cisplatin and docetaxel. The disease relapse rate is relatively high with poor survival chance for recurrent or metastatic disease. Development of novel therapeutic strategies against the disease is clearly needed.

Bortezomib and suberoylanilide hydroxamic acid are respectively classified as proteasome inhibitor and histone deacetylase inhibitor. Bortezomib and SAHA induce apoptosis in various cancers including renal cell carcinoma, hepatoma and mantle cell lymphoma. However, the effect of bortezomib and SAHA on NPC cells was not mentioned. We sought to study the molecular and cellular effects of the bortezomib and SAHA on NPC cells hoping to look for drug alternatives in NPC treatment. Since SAHA reactivates EBV in NPC cells, the combined effect of bortezomib and SAHA on EBV lytic cycle was also evaluated.

NPC proliferation was assessed by MTT assay. 5 EBV-positive NPC cell lines authenticated by Short Tandem Repeats (STR) profiling were used as most NPC in Chinese contains EBV. Isobologram and combination index analysis confirmed that the anti-proliferative effect on NPC mediated by the drug combination was synergistic.

30 nM bortezomib and 5μM SAHA were chosen for further studies on apoptosis because the synergism of the drugs was maximal at these concentrations. NA and C666-1 were chosen for further studies because C666-1 was the only NPC cell line that consistently harboured native NPC and the combination index was lowest in NA among the rest of the NPC cell lines. Bortezomib led to apoptosis in NPC cells. The effect was more pronounced after the addition of SAHA as evidenced by greater TUNEL positive population and earlier cleavage of poly ADP ribose polymerase (PARP).

In previous cancer studies, ROS induction was commonly suggested pathways of bortezomib and SAHA’s antiproliferative effects. Staining with dichlorofluorescein diacetate (DCFH-DA) revealed enhanced reactive oxygen species (ROS) level in cells treated with both drugs. At the same time, addition of N-acetyl cysteine, a ROS scavenger, markedly reduced their effect on cytotoxicity.

SAHA is known for its effect on EBV lytic cycle induction. Yet, the addition of bortezomib diminished SAHA-induced viral load, lytic protein expression and EBV infectivity. The expression of Latent Membrane Protein 1 (LMP1) was much lower in NPC treated with both drugs than in NPC treated with SAHA alone, which would reduce NF-κB activation. This, together with reduced EBNA1 expression upon treatment with both drugs, would theoretically reduce oncogenic activity.

In conclusion, bortezomib and SAHA induced ROS-driven apoptosis of NPC in a synergistic manner and bortezomib inhibited SAHA-induced EBV lytic cycle. It suggests that bortezomib and SAHA are potential drug candidates for the treatment of nasopharyngeal carcinoma.