The ubiquitin-proteasome pathway is involved in intracellular protein turnover and plays an essential function in many cellular processes, thus contributing to cellular homeostasis. The observation that proteasome inhibitors are able to induce apoptosis preferentially in tumor cells opened the way to their use as potential drugs. One of these drugs, bortezomib, was introduced in cancer therapy and its use was approved for the treatment of multiple myeloma and mantle cell lymphoma. The investigation of the mechanisms through which bortezomib causes cell death of cancer cells showed that this drug affects many cellular pathways and many of its effects cannot be related to proteasome inhibition. These observations, as well as the occurrence of bortezomib resistance observed in some myeloma patients treated with this drug, prompted the study of new proteasome inhibitors. These studies have lead to a new generation of proteasome inhibitors, some of them exhibiting the property of selective proteasome inhibition and efficacy in cancer therapy without inducing the side effects elicited by bortezomib. The ensemble of these drugs offers the scenario of the possible development of a family of proteasome inhibitors, contributing a drug arsenal for the therapy of cancer and other diseases.
Cancer is characterized by sequential and progressive genetic and epigenetic alterations in key proto-oncogenes and tumor suppressor genes, which ultimately lead to tumor development. Advances in the technology of analysis of molecular mechanisms have increased the efficiency of clinical management of cancer patients. Recent years have witnessed a progressive development in technologies that enable the detection of specific molecular abnormalities associated with various types of solid tumors in body fluids, a process that is globally known as "liquid biopsy". Liquid biopsy is largely based on the circulating free DNA (cfDNA) present in the plasma of healthy individuals and derived either from cell apoptosis or from the active secretion of microvesicles mediated by white blood cells (WBCs). The plasma of cancer patients contains DNA, which is referred to as circulating tumor DNA (ctDNA) and is released by the tumor cells in the form of DNA fragments of various sizes bearing the various types of genetic abnormalities specific to the tumors from which were derived. Sequencing studies conducted with several thousands of cancer patients have revealed that ctDNA accounts for only a fraction of the total DNA, and the size of this fraction varies in relation to tumor burden, tumor site, tumor subtypes, and several other biological properties of the tumor cells. Therefore, the levels of ctDNA are extremely low in several earlystage tumors, requiring highly sensitive methods for the detection of genetic alterations
Despite recent advances in therapy, Multiple myeloma (MM) remains incurable because of the high resistance to apoptosis and both intrinsic and acquired drug resistance. Therefore, new therapeutic strategies are needed to improve patient outcome. We recently demonstrated that blockade of the MEK/ERK signaling module, using the small-molecule inhibitors PD184352 or PD0325901 (PD), strikingly enhances arsenic trioxide (ATO)-induced cytotoxicity in MM cells through a multiple modulation of apoptotic regulatory proteins, including p53 family proteins, tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) receptors, several Bcl-2 family proteins and caspases, that depend on the functionality of the p53 pathway (Blood prebublished on line june 26, 2008). Furthermore, we also demonstrated that PD plus ATO treatment induces early tumor (volume approximately 200 mm3) regression, prolongs survival and is well tolerated in vivo in a human plasmacytoma xenograft model. The aim of this study was to investigate whether the combined treatment with PD and ATO is effective in animals with more advanced tumors; thus we used a murine model in which MM RPMI 8226 cells were injected subcutaneously into NOD-SCID mice and when the tumors reached approximately 1000mm3, mice were randomized (n=6/group) to receive vehicle or PD0325901 at 10 mg/kg administered by oral gavage or ATO (3.75 mg/kg) injected intraperitoneally or PD/ATO on a 5-days-a-week schedule for 3 consecutive weeks. Treatment of RPMI 8226 MM-advanced tumor-bearing mice with PD0325901 (10mg/kg) significantly reduced MM-tumor growth as compared to control (P <.01 Tukey-Kramer test), ATO (3.75 mg/kg) had minimal effect on the growth of tumors, which increased as in control mice. Importantly, when PD (10mg/kg) was combined with ATO (3.75mg/Kg), there was a significant reduction in tumor size and growth rate relative to untreated or PD treated mice (P<.001 for PD/ATO versus control, and P<.01 for PD/ATO versus PD Tukey-Kramer test). The combination of PD and ATO (3.75mg/Kg) significantly prolonged survival compared with treatment with either drug alone and was well tolerated in vivo because no differences in body weight and general appearance was noted in mice during the treatment. We next investigated the in vivo effects of the drug combination on proliferation and apoptosis; whole tumor-cell tissues and tumor lysates from mice treated for five days (n=2/group) were subjected to immunohistochemical staining and immunoblotting to assess in vivo phosphorylation of ERK, the proliferative antigen, Ki-67, and cleaved caspase-3. Tumor tissues from PD0325901 (10mg/kg) treatments resulted in profound p-ERK inhibition compared with tumor tissues from vehicle control or ATO-treated animals. In agreement with these data, a significant decrement in the number of Ki-67 positive plasma cells was noted in tumor sections from PD-treated mice relative to tumors from mice receiving either vehicle control or ATO (3.75mg/Kg) treatment alone thereby confirming the tumors growth retardation observed in PD-treated mice. Either PD (10mg/kg) or ATO (3.75mg/Kg) alone did not increase caspase activation compared with tumors from control cohorts. However, the combination PD/ATO dramatically activated caspase-3 in advanced tumors. Notably, consistent with our previous in vitro study demonstrating the involvement of the Bim pathway in MM PD/ATO-induced apoptosis, immunoblotting of MM tumors form PD plus ATO-treated mice showed an elevated ratio of proapoptotic Bim to antiapoptotic Mcl-1 compared with treatment with either drug alone. Collectively, our previous and present findings suggest that combining PD with ATO induces both cytostatic and cytotoxic responses in vivo, resulting in regression of early or advanced tumors, prolongs survival in vivo, and is well tolerated in vivo. In conclusion, our preclinical in vivo studies provide the framework for testing PD0325901 and ATO combination therapy in clinical trials aimed to improve patient outcome in MM.
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