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Final Report – 2008-09 Research & Creative Works Grants Principal Investigator: Nicholas E. Burgis, Chemistry/Biochemistry Dept. Project Title: Developing a Less Toxic Chemotherapeutic Cocktail to Treat Multiple Myeloma. This report serves to document the scholarly research activities performed by the principal investigator and collaborators during the term of the Faculty Research Grant Award (summer of 2009). All research was performed on campus by me and two very talented EWU undergraduates, Richard Sipes and Xue (Susie) Xia. My proposal included two objectives: (1) Determine if a drug cocktail which includes an
alkylating agent, proteasome inhibitor and lysosome inhibitor results in decreased survival and
growth for noncancerous human cells. (2) Determine if the alkylating agent-induced reduction in
protein breakdown observed in multiple myeloma cells is modulated by exposure to proteasome
inhibitors and/or lysosome inhibitors. Both objectives and additional experiments were
completed within the timeframe of the proposal and generated exciting results.
Objective 1: Results obtained in the previous funding period (summer 2008) suggested that the toxic effect of alkylating agent on multiple myeloma cells may result from a modulation of protein breakdown in these cells. Experiments performed during the current funding period aimed to determine the toxic effects of a clinically relevant alkylating agent, melphalan, in conjunction with clinically relevant drugs which are known to modulate protein breakdown, MG132 (proteasome inhibitor) and chloroquine (lysosome inhibitor). These experiments were performed with at least four replicates with both multiple myeloma cells and noncancerous B-cells (multiple myeloma is a cancer of B cells in blood) allowing for a side by side comparison. Surprisingly we observed that in general the noncancerous cells were more sensitive to varying combinations of these drugs. For treatment of these cells with melphalan or MG132 alone both cell lines showed an increase in cytotoxicity, however, the differences between the two cell lines was not statistically significant. Conversely, exposure of both melphalan and MG132 resulted in an increased level of cytotoxicity for both cells but the noncancerous cell line was approximately two-fold more sensitive than the multiple myeloma cell line. This difference was statistically significant. This difference was further exasperated by a three drug combination of melphalan, MG132 and chloroquine. For the three drug treatment a statistically significant three-fold difference in cytotoxicity was observed. Again the cell killing was greater for the noncancerous cell line. Objective 2: Because our cytotoxicity tests in Objective 1 were not anticipated we decided to do a more thorough investigation of the effects of these drugs on protein breakdown than we initially intended and we chose to again do a side by side comparison of both multiple myeloma cells and noncancerous B-cells with at least three replicate experiments. The results we obtained in Objective 2 nicely complemented results we obtained in Objective 1. Overall we observed that protein breakdown was less severely modulated by various drug cocktails for the multiple myeloma cells than noncancerous cells. Melphalan had no effect on protein breakdown for the multiple myeloma cell line but the noncancerous cell line had a reduction in protein breakdown that was greater than two-fold reduced compared to the untreated cells. Treatment with MG132 or chloroquine alone resulted in a reduction of protein breakdown for both cell lines with the noncancerous cell line showing the greatest reduction in both cases. Chloroquine treatment demonstrated the greatest disparity in the level of protein breakdown suggesting that lysosome-mediated protein breakdown is the more dysregulated mechanism for protein breakdown. For the three possible two-drug combinations and the three-drug combination the multiple myeloma cells exhibited no statistical difference in protein breakdown that if treated with MG132 or chloroquine alone. This is in stark contrast to the results with the noncancerous cell line. For the two-drug treatments with melphalan/MG132 and MG132/chloroquine and the three-drug treatment protein breakdown was severely diminished for the noncancerous cell line. No difference was observed between cell lines for the melphalan/chloroquine two-drug treatment; however, significant error was present in the measurement of the noncancerous cell line for this two-drug treatment. As anticipated, the greatest effect was seen for the MG132/chloroquine treatment, where both mechanisms for protein breakdown in the cell are inhibited. Compared to untreated cells, a roughly 20-fold reduction in protein breakdown was observed, and, compared to the multiple myeloma cell line treated in the same manner a 10-fold reduction in protein breakdown was observed. Additional analytical techniques were performed which demonstrated that rate of protein synthesis and breakdown were much greater for the multiple myeloma cell line than the noncancerous cell line. For our experiments to measure protein breakdown in a cell we allow the cells to incorporate a radioactive amino acid into the newly synthesized proteins for one-hour and then stop protein synthesis which allows us to determine the rate of protein breakdown by measuring the levels of radioactivity in the intact proteins over a one-hour time course. From this protocol we have determined that the rate of protein synthesis for the multiple myeloma cells is almost 2.5-fold greater than the rate of protein synthesis for the noncancerous cell line. In addition the multiple myeloma cells show a rate of protein breakdown that is five-fold higher than the rate for the noncancerous cell line. It should be noted that the growth rates for these cells is roughly equivalent with the noncancerous B-cells growing at a somewhat higher rate. Therefore the observed differences cannot be attributed to differences in cell growth. In summary, the multiple myeloma cells have a much greater rate of protein turnover than noncancerous B-cells. In general our results were quite surprising. Melphalan and a MG132-like agent, bortezomib, are currently administered to multiple myeloma patients and result in successful outcomes, albeit drug toxicity is common. It is currently not know what the mechanism of action is for this drug cocktail. Clearly, from my data it does not seem likely that the beneficial effects of this drug cocktail are due to a modulation of protein breakdown. What is clear is that multiple aspects of protein metabolism are dysregulated in multiple myeloma cells. Overall, the data obtained in Objective 2 shows that the protein breakdown machinery in multiple myeloma cells is much more resistant to modulation by clinically relevant drugs. The outcome of this dysregulation results in higher levels of protein breakdown, despite drug treatment, and a decreased sensitivity to the three-drug cocktail compared to noncancerous cells. In addition we have demonstrated that protein metabolism in general is elevated for the multiple myeloma cells and that this cannot be attributed to a difference in cell growth. In conclusion, my data nicely demonstrates that protein breakdown is dysregulated in multiple myeloma cells compared to normal B-cells. Results obtained during this funding period resulted in a nice body of work. Work on this project will continue throughout the school year to shore up some loose ends in preparation for submission of a manuscript to an internationally recognized peer-reviewed journal. One of the students (Richard Sipes) that performed this work with me over the summer plans on continuing with this project during the school year and he expects this work to be the basis for his senior thesis. I am grateful to the Research and Scholarship Committee and Teaching & Learning Center for providing this excellent opportunity for unfettered faculty research! The experiments I performed included lengthy protocols which would be impossible for me or a student to perform during the school year. I simply would not have been able to “start something big” without this grant. It would be remiss to not mention that this grant provided an opportunity for undergraduates to really sink their teeth into a cancer research project and work collaboratively with a faculty mentor to learn valuable skills which will undoubtedly help them achieve their goals post-graduation. As stated this work will continue throughout the year with undergraduate researchers and it is expected to be incorporated into student presentations both on campus at the Student Research and Creative Works Symposium and at a regional meeting of the American Chemical Society.


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