Studies of the Anti-Tumoral Mechanisms of Proteasome Inhibitor by Proteomic Techniques

The proteasome is an abundant multi-enzyme complex that provides the main roles for selective degradation of intracellular unfolding proteins in eukaryotic cells. Previous studies have demonstrated that Bortezomib, a strong proteasome inhibitor, inhibited tumor cells proliferation and induced apoptosis through multiple pathways. In addition, Bortezomib is the first proteasome inhibitor approved by FDA to enter clinical trials in therapy of relapsed/refractory multiple myeloma patients. However, the detail mechanism of Bortezomib on the inhibition of cancer cells proliferation and induction of cancer cells apoptosis are still not well understood. Up until now, there is no study to investigate the effects of Bortezomib on the anti-tumor effects by proteomic techniques. First year of this proposal, we will employ a quantitative proteomic approach to examine the alteration in protein levels associated with Bortezomib-treated human Burkitt’s lymphoma CA46 and Daudi cells, which overexpress c-myc oncogene. Our preliminary data found that Bortezomib significantly inhibited both cells proliferation and induced both cell apoptosis in a dose-dependent manner. Quantitative 2-DE was used to determine the changing protein levels with MALDI-TOF MS identification of proteins. All of those proteins have been reported to be associated with multiple cellular functions, including unfolded protein response (UPS), RNA processing, protein biosynthesis and targeting, oxidative stress, apoptosis, and signal transduction. Of the changing proteins analysed, the sumoylated heterogeneous nuclear ribonucleoprotein K (sumo-hnRNP K) proteins was significantly decrease and phospho-14-3-3β/α protein showed a dramatic increase in level in Bortezomib-treated cells. In view of the facts that hnRNP K has been implicated in c-Myc mRNA translation and the sumoylated site located in RNA-binding domain of hnRNP K, we propose that the sumoylation status might affect it binding to target RNA and affect c-Myc mRNA translation activity. In addition, the decrease of sumo-hnRNP K might be associated with the down-regulation of c-myc mRNA translation and afterwards decreasing cell proliferation in Bortezomib-treated cells. Therefore, the sumoylation status of hnRNP K might be important in the regulation of tumor cell proliferation, and we will investigate the molecular mechanisms of non-sumoylated hnRNP K on the regulation of Burkitt’s lymphoma proliferation in 2nd year of plan. It is well known that JNK was activated during ER stress induced by proteosome inhibitor. Recent studies showed that phosphorylation of 14-3-3 proteins by JNK results in dissociation of proapoptotic Bax from 14-3-3 proteins, leading to enter apoptotic cascade. Indeed, our preliminary data indicated that 14-3-3 protein was phosphorylated after treatment of Bortezomib, suggesting phospho-14-3-3 protein might play a critical step in the regulation of Burkitt’s lymphoma apoptosis in Bortezomib-treated cells. We propose that the JNK-mediated phosphorylation of 14-3-3 regulates the proapoptotic proteins in concert and makes cells go into apoptotic cascade in Bortezomib-treated Burkitt’s lymphoma. Based on our recent findings and hypothesis, we would like to fully understand the molecular mechanisms of Bortezomib on the inhibition of Burkitt’s lymphoma, and hope to achieve the following Specific Goals: 1. To examine the alteration in protein levels associated with Bortezomib-treated human Burkitt’s lymphoma by a quantitative proteomic techniques. 2. To investigate the role of hnRNP K sumoylation on regulation of the c-myc mRNA translation and the proliferation in Bortezomib-treated Burrkitt’s lymphoma. 3. To investigate the role of phosphor-14-3-3 β/α protein on the induction of apoptosis in Bortezomib-treated Burrkitt’s lymphoma. This study of alterations in protein expression will allow us to identify the molecular mechanisms involved in the anti-tumoral activity of proteosome inhibitor. The information could be useful for design of new anti-tumoral drugs and treatment of cancer patient in the future.