Verapamil synergistically enhances cytotoxicity of bortezomib in mantle cell lymphoma via induction of reactive oxygen species production.

Mantle cell lymphoma (MCL) is a subtype of non-Hodgkin lymphoma. Most MCL patients show a poor response to conventional therapy, and therapeutic interventions, such as monoclonal antibody-based therapy and stem cell transplantation, have shown only minor effects on overall survival rates (Jares & Campo, 2008). Despite approval by the US Food and Drug Administration (FDA) for the treatment of patients with relapsed or refractory MCL, bortezomib has shown limited effects (Kane et al, 2007), and various clinical studies demonstrated that the combination of bortezomib with other agents could enhance its efficacy (Yang et al, 2008). Therefore, we investigated whether a calcium blocker, verapamil, could increase the cell cytotoxicity of bortezomib. We first compared cell proliferation after drug treatment using four different MCL cell lines and primary cells. Drugs were serially diluted from the maximal drug doses of verapamil (240 μM) and fixed amounts of bortezomib (10 nM) were added during culture. SP-53 and Jeko-1 cell lines, which are bortezomib susceptible MCL cells, and Mino and REC-1 cell lines, which are bortezomib-resistant MCL cells, were tested. Compared to treating cells with verapamil only, combining these drug treatments significantly increased cell cytotoxicity (Figure S1). Regardless of susceptibility to bortezomib, cell cytotoxicity was increased upon the combination of bortezomib and verapamil regimens in different cell lines. We also tested the verapamil and bortezomib combinatory regimen on MCL primary cells. A higher concentration of bortezomib (100 nM) was used for 4 h in primary cells because prolonged cell culture of primary cells resulted in higher cell death. The bortezomib and verapamil combination also increased cytotoxicity in apheresis patient cells (Figure S1). The synergistic cytotoxic effects of the combined regimens were analysed by combination index (CI) plots. Treatment of MCL cells with the combined regimens, resulted in CI values < 1.0, suggesting synergy between verapamil and bortezomib (Figure 1). These findings indicate that verapamil could be used to increase the cell cytotoxicity of bortezomib. Next, the effects of verapamil on the expression of caspases 3 and 9 (members of the caspase family that play central roles in cell apoptosis) were examined. Immunohistochemistry analyses showed that the majority of MCL cells expressed activated caspase-3 and -9 after drug treatments (Figure S2A). The effects of verapamil on the expression of cleaved poly(ADP-ribose) polymerase (PARP) were then investigated. Bortezomib, both alone and in combination with verapamil, increased the levels of PARP in our cells (Figure S2B). A gradual increase of caspase-3 and -7 activity was noted upon addition of bortezomib and verapamil (Figure S2C). Figure 1 Verapamil has synergic cytotoxic effects with bortezomib in MCL cells In an effort to find biological mechanisms, we investigated the activity of nuclear factor (NF)-κB after bortezomib and verapamil treatment. The transcription factor NF-κB is constitutively activated in MCL and is important for selective growth and survival of stem-like cells in MCL (Jung et al, 2012). The combination of bortezomib and verapamil significantly decreased the NF-κB DNA-binding activities of p65 and p50 compared with bortezomib or verapamil single treatment alone (P<0.05) (Figure S3A). The effects of combination therapies on NF-κB DNA binding activities were greater in MCL cell lines. We then performed reverse transcription polymerase chain reaction (RT-PCR) analysis of interleukin (IL) 6, IL8, and cellular inhibitor of apoptosis protein 2 (c-IAP2), which are well-known NF-κB-linked transcriptional targets (Wang et al, 1998). In addition, NF-κB complexes regulate the gene expression of IL6 and IL8, which are representative inflammatory cytokines (Libermann & Baltimore, 1990). Figure S3B demonstrates RT-PCR analysis for these NF-κB-regulated genes in MCL treated with bortezomib or verapamil monotherapy or combinatory therapies. ACTB gene expression was monitored as a loading control. The NF-κB-downstream target genes were readily detectable in MCL. Importantly, significant down-regulation of target genes was detected in the presence of verapamil and bortezomib. Chronic or unresolved endoplasmic reticulum (ER) stress can lead to apoptosis. Factors that contribute to ER stress development include increase in protein synthesis or protein misfolding rates that exceed the capacity of protein chaperones, oxidative stress and changes to redox balance in the ER lumen. ER stress can be initiated by upstream signalling proteins, which begin a cascade of corrective actions (Obeng et al, 2006). The activity of these pathways constitutes an ER-specific unfolded protein response (UPR). To delineate mechanisms of increased apoptosis after combinatory drug treatments, we analysed the accumulation of reactive oxygen species (ROS). ROS were generated in response to verapamil treatment, but their levels decreased upon treatment with the antioxidant, N-acetylcysteine (Figure 2). To investigate whether the combination of bortezomib and verapamil induced UPR signals, UPR components were analysed in MCL lysates. Because increased levels of chaperone proteins indicate UPR activation, we measured several genes involved in this process by real-time PCR. The ER-resident chaperone μ-heavy chain binding protei n (HSPA5, BiP) was increased upon bortezomib treatment but not after verapamil treatment (Figure S4). The levels of transmembrane factor ATF6, which induces transcription of several genes including chaperones, XBP1, and the levels of CEB/P homologous protein (DDIT3, CHOP) were increased in response to ER stress. Other genes involved in ER stress, such as HSP90B1 (GRP94), HSPA1A (HSP70), ATF6, and DDIT3 were all increased upon verapamil and bortezomib treatment. Figure 2 Verapamil enhances the bortezomib-induced UPR response in MCL cells Together, these analyses showed that bortezomib and verapamil induced UPR and the ER perturbation. It is also worth noting that both bortezomib and verapamil can reduce blood pressure, suggesting that patient doses may need to be re-evaluated. Verapamil treatment also induced ROS production, which can activate calcium release from the ER and cause protein folding. A previous report showed that ROS production and protein misfolding together activated calcium-dependent kinases, such as Jun N-terminal kinase (JNK), leading to cell apoptosis (Malhotra et al, 2007). Our results provide new potential mechanisms for the increased cytotoxicity of bortezomib in combination with verapamil; this new drug combination could decrease adverse effects from bortezomib by reducing its required dosage.