Resistance to chemotherapy and molecularly targeted therapies is a major problem facing current cancer research. The mechanisms of resistance to 'classical' cytotoxic chemotherapeutics and to therapies that are designed to be selective for specific molecular targets share many features, such as alterations in the drug target, activation of prosurvival pathways and ineffective induction of cell death. With the increasing arsenal of anticancer agents, improving preclinical models and the advent of powerful high-throughput screening techniques, there are now unprecedented opportunities to understand and overcome drug resistance through the clinical assessment of rational therapeutic drug combinations and the use of predictive biomarkers to enable patient stratification.

Cancer drug resistance: an evolving paradigm

The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay is based on the conversion of MTT into formazan crystals by living cells, which determines mitochondrial activity. Since for most cell populations the total mitochondrial activity is related to the number of viable cells, this assay is broadly used to measure the in vitro cytotoxic effects of drugs on cell lines or primary patient cells. In this chapter the protocol of the assay is described including important considerations relevant for each step of the assay as well as its limitations and possible applications.

Cell Sensitivity Assays: The MTT Assay

The ubiquitin proteasome pathway was discovered in the 1980s to be a central component of the cellular protein-degradation machinery with essential functions in homeostasis, which include preventing the accumulation of misfolded or deleterious proteins. Cancer cells produce proteins that promote both cell survival and proliferation, and/or inhibit mechanisms of cell death. This notion set the stage for preclinical testing of proteasome inhibitors as a means to shift this fine equilibrium towards cell death. Since the late 1990s, clinical trials have been conducted for a variety of malignancies, leading to regulatory approvals of proteasome inhibitors to treat multiple myeloma and mantle-cell lymphoma. First-generation and second-generation proteasome inhibitors can elicit deep initial responses in patients with myeloma, for whom these drugs have dramatically improved outcomes, but relapses are frequent and acquired resistance to treatment eventually emerges. In addition, promising preclinical data obtained with proteasome inhibitors in models of solid tumours have not been confirmed in the clinic, indicating the importance of primary resistance. Investigation of the mechanisms of resistance is, therefore, essential to further maximize the utility of this class of drugs in the era of personalized medicine. Herein, we discuss the advances and challenges resulting from the introduction of proteasome inhibitors into the clinic.

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Proteasome inhibitors in cancer therapy

Targeting the ubiquitin-proteasome pathway has emerged as a rational approach in the treatment of human cancer. Based on positive preclinical and clinical studies, bortezomib was subsequently approved for the clinical use as a front-line treatment for newly diagnosed multiple myeloma patients and for the treatment of relapsed/refractory multiple myeloma and mantle cell lymphoma, for which this drug has become the staple of treatment. The approval of bortezomib by the US Food and Drug Administration (FDA) represented a significant milestone as the first proteasome inhibitor to be implemented in the treatment of malignant disease. Bortezomib has shown a positive clinical benefit either alone or as a part of combination therapy to induce chemo-/radio-sensitization or overcome drug resistance. One of the major mechanisms of bortezomib associated with its anticancer activity is through upregulation of NOXA, which is a proapoptotic protein, and NOXA may interact with the anti-apoptotic proteins of Bcl-2 subfamily Bcl-XL and Bcl-2, and result in apoptotic cell death in malignant cells. Another important mechanism of bortezomib is through suppression of the NF-κB signaling pathway resulting in the down-regulation of its anti-apoptotic target genes. Although the majority of success achieved with bortezomib has been in hematological malignancies, its effect toward solid tumors has been less than encouraging. Additionally, the widespread clinical use of bortezomib continues to be hampered by the appearance of dose-limiting toxicities, drug-resistance and interference by some natural compounds. These findings could help guide physicians in refining the clinical use of bortezomib, and encourage basic scientists to generate next generation proteasome inhibitors that broaden the spectrum of efficacy and produce a more durable clinical response in cancer patients. Other desirable applications for the use of proteasome inhibitors include the development of inhibitors against specific E3 ligases, which act at an early step in the ubiquitin-proteasome pathway, and the discovery of less toxic and novel proteasome inhibitors from natural products and traditional medicines, which may provide more viable drug candidates for cancer chemoprevention and the treatment of cancer patients in the future.

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Bortezomib as the First Proteasome Inhibitor Anticancer Drug: Current Status and Future Perspectives

Transcription Factor Nrf1 Mediates the Proteasome Recovery Pathway after Proteasome Inhibition in Mammalian Cells

Molecular basis of bortezomib resistance: proteasome subunit beta5 (PSMB5) gene mutation and overexpression of PSMB5 protein

and Gerrit Jansen Heijden, Bauke Ylstra, Godefridus J. Peters, Gertjan L. Kaspers, Ben A. C. Dijkmans, Rik J. Scheper Berkers, George L. Scheffer, Kabir Debipersad, Katharina Vojtekova, Clara Lemos, Joost W. van der Ruud Oerlemans, Niels E. Franke, Yehuda G. Assaraf, Jacqueline Cloos, Ina van Zantwijk, Celia R. ) gene mutation and overexpression of PSMB5 protein PSMB5 Molecular basis of bortezomib resistance: proteasome subunit 25 (

Kabir Debipersad

Papers

Molecular basis of bortezomib resistance: proteasome subunit beta5 (PSMB5) gene mutation and overexpression of PSMB5 protein

) gene mutation and overexpression of PSMB5 protein PSMB5 Molecular basis of bortezomib resistance: proteasome subunit ²5 (