The interaction of berberine with chemodrugs for treating EBV-associated nasopharyngeal carcinomas (NPC)

Abstract

Nasopharyngeal carcinoma (NPC) is a type of head and neck cancer closely associated with Epstein-Barr virus (EBV) infection and has high incident rate in Southern China including Hong Kong. In Chinese community, cancer patients are prone to consume medical herbs in a hope to enhance survival rate and relieve side effects of chemotherapy. A natural alkaloid, berberine, is commonly found in various traditional medical herbs or remedies used for treating cancers. It was also previously reported to suppress invasiveness and in vivo tumor growth in mice model, and therefore suggesting its potential use as an adjuvant therapeutics with chemotherapy. However, no preclinical studies have been perform to assess if berberine affects the efficacy of western conventional chemodrugs in the treatment of NPC. In this study, we aimed to investigate the interaction of berberine with chemodrugs including cisplatin and suberanilohydroxamic acid (SAHA).

In the in vitro experiments, the cytotoxicity of three individual treatments: berberine, cisplatin, berberine and two combined treatments: berberine+SAHA and berberine+cisplatin was assessed in three NPC cell lines by MTT assay. Combined treatments were shown to have additional cytotoxicity compared to treatment of individual drug. Since previous studies have shown that chemodrugs could induce lytic EBV cycle in cancer cells for additional cytotoxicity, we then investigated if berberine would promote chemodrugs-induced lytic reactivation. In this study, we confirmed the previous research about the potent ability of SAHA to induce lytic infection in vitro, while berberine was found to suppress SAHA-induced lytic reactivation in NPC cells. It suggests that lytic reactivation was not involved in the additional growth inhibition of the combined treatments. Interestingly, we found that berberine interfered with the cell-cycle arrest induced by cisplatin or SAHA, but how this modulates the killing effects of both chemodrugs remains elusive. We further elucidated that berberine did not affect the DNA damage response strongly evoked by cisplatin. Nevertheless, we observed that berberine could induce differentiation of NPC cells as indicated by the high expression of involucrin (marker for epithelial differentiation) and downregulated expressions of inhibitor of differentiation (Id-1), p63, p-src and p-STAT3. Similar effects were shown in the treatments of berberine+cisplatin and berberine+SAHA, suggesting berberine might impose alternate strategy to stop the tumor growth through differentiation induction. In the in vivo experiments, mice co-treated with berberine and cisplatin suffered from great weight loss. Unintentional interaction of berberine and cisplatin might happen which caused loss of appetite or malfunctions of organs. The tumor growth was suppressed under all treatments and SAHA-treated tumors showed cystic phenotype. It is also the first report showing the lytic activity of in vivo tumor cells at single cell level using RNAscope. There were only less than 1% of cells could express lytic transcripts across all treatments, indicating lytic reactivation did not play any major role in the in vivo tumor suppression process. At last, our results demonstrated that berberine and SAHA co-treated tumor exhibited differentiated phenotype with more than 35% of tumor cells express high level of involucrin.