Peter J. Elliott

Enhancement of radiosensitivity by proteasome inhibition: Implications for a role of NF-κB

PURPOSE NF-kappaB is activated by tumor necrosis factor, certain chemotherapeutic agents, and ionizing radiation, leading to inhibition of apoptosis. NF-kappaB activation is regulated by phosphorylation of IkappaB inhibitor molecules that are subsequently targeted for degradation by the ubiquitin-proteasome pathway. PS-341 is a specific and selective inhibitor of the proteasome that inhibits NF-kappaB activation and enhances cytotoxic effects of chemotherapy in vitro and in vivo. The objective of this study was to determine if proteasome inhibition leads to enhanced radiation sensitivity. METHODS AND MATERIALS Inhibition of NF-kappaB activation in colorectal cancer cells was performed by treatment of LOVO cells with PS-341 or infection with an adenovirus encoding IkappaB super-repressor, a selective NF-kappaB inhibitor. Cells were irradiated at 0, 2, 4, 6, 8, and 10 Gy with or without inhibition of NF-kappaB. NF-kappaB activation was determined by electrophoretic mobility gel shift assay, and apoptosis was evaluated using the TUNEL assay. Growth and clonogenic survival data were obtained to assess effects of treatment on radiosensitization. In vitro results were tested in vivo using a LOVO xenograft model. RESULTS NF-kappaB activation was induced by radiation and inhibited by pretreatment with either PS-341 or IkappaBalpha super-repressor in all cell lines. Inhibition of radiation-induced NF-kappaB activation resulted in increased apoptosis and decreased cell growth and clonogenic survival. A 7-41% increase in radiosensitivity was observed for cells treated with PS-341 or IkappaBalpha. An 84% reduction in initial tumor volume was obtained in LOVO xenografts receiving radiation and PS-341. CONCLUSIONS Inhibition of NF-kappaB activation increases radiation-induced apoptosis and enhances radiosensitivity in colorectal cancer cells in vitro and in vivo. Results are encouraging for the use of PS-341 as a radiosensitizing agent in the treatment of colorectal cancer.

Proteasome Inhibition: a New Strategy in Cancer Treatment

The ubiquitin proteasome pathway is a highly conservedintracellular pathway for the degradation of proteins. Many of theshort-lived regulatory proteins which govern cell division, growth,activation, signaling and transcription are substrates that aretemporally degraded by the proteasome. In recent years, new andselective inhibitors of the proteasome have been employed in cellculture systems to examine the anti-tumor potential of theseagents. This review covers the chemistry of selected proteasomeinhibitors, possible mechanisms of action in cell culture and thein vivo examination of proteasome inhibitors in murine andhuman xenograft tumor models in mice. One inhibitor, PS-341, hasrecently entered Phase I clinical trials in cancer patients withadvanced disease to further test the potential of this approach.

26S proteasome inhibition induces apoptosis and limits growth of human pancreatic cancer

The 26S proteasome degrades proteins that regulate transcription factor activation, cell cycle progression, and apoptosis. In cancer, this may allow for uncontrolled cell division, promoting tumor growth, and spread. We examined whether selective inhibition of the 26S proteasome with PS‐341, a dipeptide boronic acid analogue, would block proliferation and induce apoptosis in human pancreatic cancer. Proteasome inhibition significantly blocked mitogen (FCS) induced proliferation of BxPC3 human pancreatic cancer cells in vitro, while arresting cell cycle progression and inducing apoptosis by 24 h. Accumulation of p21Cip1‐Waf‐1, a cyclin dependent kinase (CDK) inhibitor normally degraded by the 26S proteasome, occurred by 3 h and correlated with cell cycle arrest. When BxPC3 pancreatic cancer xenografts were established in athymic nu/nu mice, weekly administration of 1 mg/kg PS‐341 significantly inhibited tumor growth. Both cellular apoptosis and p21Cip1‐Waf‐1 protein levels were increased in PS‐341 treated xenografts. Inhibition of tumor xenograft growth was greatest (89%) when PS‐341 was combined with the tumoricidal agent CPT‐11. Combined CPT‐11/PS‐341 therapy, but not single agent therapy, yielded highly apoptotic tumors, significantly inhibited tumor cell proliferation, and blocked NF‐κB activation indicating this systemic therapy was effective at the cancer cell level. 26S proteasome inhibition may represent a new therapeutic approach against this highly resistant and lethal malignancy. J. Cell. Biochem. 82: 110–122, 2001.

Proteasome inhibition: a new anti-inflammatory strategy

The ubiquitin-proteasome pathway has a central role in the selective degradation of intracellular proteins. Among the key proteins modulated by the proteasome are those involved in the control of inflammatory processes, cell cycle regulation, and gene expression. Consequently proteasome inhibition is a potential treatment option for cancer and inflammatory conditions. Thus far, proof of principle has been obtained from studies in numerous animal models for a variety of human diseases including cancer, reperfusion injury, and inflammatory conditions such as rheumatoid arthritis, asthma, multiple sclerosis, and psoriasis. Two proteasome inhibitors, each representing a unique chemical class, are currently under clinical evaluation. Velcade (PS-341) is currently being evaluated in multiple phase II clinical trials for several solid tumor indications and has just entered a phase III trial for multiple myeloma. PS-519, representing another class of inhibitors, focuses on the inflammatory events following ischemia and reperfusion injury. Since proteasome inhibitors exhibit anti-inflammatory and antiproliferative effects, diseases characterized by both of these processes simultaneously, as is the case in rheumatoid arthritis or psoriasis, might also represent clinical opportunities for such drugs.

Proteasome Inhibitor PS519 Reduces Infarction and Attenuates Leukocyte Infiltration in a Rat Model of Focal Cerebral Ischemia

BACKGROUND AND PURPOSE Reperfusion brain injury after cerebral ischemia is associated with a developing inflammatory response at the site of infarction. Proteasome inhibitors block nuclear factor-kappaB activation and provide anti-inflammatory effects in several animal models of peripheral inflammation. We tested the novel proteasome inhibitor PS519 in a rat model of transient focal ischemia to establish its pharmacodynamics as a neuroprotection treatment and related effects on leukocyte infiltration. METHODS Rats were subjected to 2 hours of focal cerebral ischemia by means of the filament method of middle cerebral artery occlusion (MCAo). After either 22 or 70 hours of reperfusion, infarct size was measured and neurological function, electroencephalographic (EEG) activity, and/or neutrophil and macrophage infiltration was quantified. PS519 was administered in a single intravenous bolus at 2 hours after MCAo. In addition, the therapeutic window for PS519 was estimated by delaying treatment for 4 or 6 hours after MCAo. RESULTS Dose-response analysis of infarct volume at 24 hours revealed that PS519 neuroprotection approached 60%, and clinical evaluations showed significant improvements in neurological function and EEG activity. Neutrophil infiltration at 24 hours was also significantly decreased in cortical and striatal infarcted tissue of PS519-treated rats. Delaying the PS519 treatment up to 4 hours continued to result in significant neuroprotection. In the 72-hour injury model, infarction was reduced 40% by PS519, and significant improvements in neurological function and EEG recovery were again measured. Considerable reductions in both neutrophil and macrophage infiltration were evident. CONCLUSIONS PS519 mitigates infarction and improves neurological recovery in brain-injured rats, an effect in part caused by a reduction in the leukocyte inflammatory response.

The Proteasome A New Target for Novel Drug Therapies

The proteasome is an enzyme present in all cells, from yeast to human, and has a central role in the proteolytic degradation of the vast majority of intracellular proteins. Among the key proteins modulated by the proteasome are those involved in controlling inflammatory processes, cell cycle regulation, and gene expression. As such, agents that inhibit the proteasome have been shown to be active in numerous animal models of inflammation and cancer Two proteasome inhibitors are under clinical evaluation. PS-519 is being studied for the treatment of reperfusion injury that occurs following cerebral ischemia and myocardial infarction. The other, PS-341, has recently entered multiple phase 2 clinical trials for the treatment of multiple myeloma, chronic lymphocytic leukemia, and a variety of solid tumors. The proteasome may have an important role in the evolution of HIV-related disorders including AIDS and inflammatory disorders. Therapeutic strategies using proteasome inhibitors for the treatment of these conditions have now entered preclinical development.

Proteasome inhibition reduces superantigen-mediated T cell activation and the severity of psoriasis in a SCID-hu model.

There is increasing evidence that bacterial superantigens contribute to inflammation and T cell responses in psoriasis. Psoriatic inflammation entails a complex series of inductive and effector processes that require the regulated expression of various proinflammatory genes, many of which require NF-kappa B for maximal trans-activation. PS-519 is a potent and selective proteasome inhibitor based upon the naturally occurring compound lactacystin, which inhibits NF-kappa B activation by blocking the degradation of its inhibitory protein I kappa B. We report that proteasome inhibition by PS-519 reduces superantigen-mediated T cell-activation in vitro and in vivo. Proliferation was inhibited along with the expression of very early (CD69), early (CD25), and late T cell (HLA-DR) activation molecules. Moreover, expression of E-selectin ligands relevant to dermal T cell homing was reduced, as was E-selectin binding in vitro. Finally, PS-519 proved to be therapeutically effective in a SCID-hu xenogeneic psoriasis transplantation model. We conclude that inhibition of the proteasome, e.g., by PS-519, is a promising means to treat T cell-mediated disorders such as psoriasis.

Proteasome inhibition: A novel mechanism to combat asthma

BACKGROUND Nuclear factor-kappaB (NF-kappaB) is a critical transcription factor required for the regulation of many genes involved in inflammatory responses to noxious stimuli. On activation, NF-kappaB induces the transcription of numerous proinflammatory cytokines, enzymes, and cellular adhesion molecules. Blockade of the proteasome with selective inhibitors attenuates the effects of NF-kappaB, leading to suppression of the inflammatory response. OBJECTIVE We sought to determine whether proteasome inhibitors would be active in a model of asthma. METHODS The mouse delayed-type hypersensitivity model was used to screen a panel of compounds for in vivo activity. The proteasome inhibitor, PS-519, was shown to be the most active in this model and was selected for further development. Allergen-induced pulmonary eosinophilia in Brown Norway rats was used subsequently to determine anti-inflammatory activity in an animal model. RESULTS Direct administration of PS-519 into the lungs significantly reduced leukocyte numbers, particularly the selective increase in eosinophils. Because steroids are the mainstay anti-inflammatory therapy in asthma, and data is available to suggest their possible interaction to suppress the activation of NF-kappaB, rats were also treated by inhalation with combinations of a steroid and the proteasome inhibitor. In both the delayed-type hypersensitivity and the animal eosinophil model, low doses of proteasome inhibitors were shown to be effective when given with low doses of steroids. CONCLUSION Taken together, the present data suggest that proteasome inhibition may represent a novel strategy for the treatment of inflammatory lung diseases such as asthma.

INHIBITION OF NF-KAPPA B ACTIVATION IN VITRO AND IN VIVO : ROLE OF 26S PROTEASOME

It is becoming increasingly apparent that NF-kappa B plays a critical role in regulating the inflammatory response. Data obtained from studies in our laboratories demonstrate that the proteasome plays an important role in the inflammatory cascade by regulating the activation of NF-kappa B. Indeed, the availability of selective and orally active proteasome inhibitors should prove useful in delineating the roles of the proteasome and NF-kappa B in other pathophysiological conditions such as cancer and heart disease.

New agents in cancer clinical trials

With advances in modern technologies and growing understanding of cellular biology, a variety of new mechanisms and molecular antagonists are currently being pursued in the ®ght against cancer. The result of these advances has spawned dozens of new exciting small molecule agents which are slowly progressing through the arduous process of clinical trials. We attempt in this review to highlight some of the new and exciting molecules representing previously unexplored targets. While it is by no means certain that novelty in a molecular target assures that it is a good one, our reading of the current literature would suggest that the seven molecules discussed in this review represent potentially new therapies which may add to the much needed armamentarium of cancer drugs (Figure 1).