A novel nickel complex works as a proteasomal deubiquitinase inhibitor for cancer therapy.

Based on the central role of the ubiquitin-proteasome system (UPS) in the degradation of cellular proteins, proteasome inhibition has been considered an attractive approach for anticancer therapy. Deubiquitinases (DUBs) remove ubiquitin conjugates from diverse substrates; therefore, they are essential regulators of the UPS. DUB inhibitors, especially the inhibitors of proteasomal DUBs are becoming a research hotspot in targeted cancer therapy. Previous studies have shown that metal complexes, such as copper and zinc complexes, can induce cancer cell apoptosis through inhibiting UPS function. Moreover, we have found that copper pyrithione inhibits both 19S proteasome-associated DUBs and 20S proteasome activity with a mechanism distinct from that of the classical 20S proteasome inhibitor bortezomib. In the present study, we reveal that (i) nickel pyrithione complex (NiPT) potently inhibits the UPS via targeting the 19S proteasome-associated DUBs (UCHL5 and USP14), without effecting on the 20S proteasome; (ii) NiPT selectively induces proteasome inhibition and apoptosis in cultured tumor cells and cancer cells from acute myeloid leukemia human patients; and (iii) NiPT inhibits proteasome function and tumor growth in nude mice. This study, for the first time, uncovers a nickel complex as an effective inhibitor of the 19S proteasomal DUBs and suggests a potentially new strategy for cancer treatment.

Green tea polyphenols as proteasome inhibitors: implication in chemoprevention.

Next to water, tea is the most popular beverage in the world. The most abundant and active compound in green tea is (-)-epigallocatechin-3-gallate (EGCG), which is extensively studied for its cancer-preventive and anti-cancer activities as well as its cellular targets. One potential molecular target of EGCG is the proteasome. While molecular docking and structure-activity relationship (SAR) analysis suggests that the ester carbon of EGCG is important for mediating its proteasome-inhibitory activity, EGCG is very unstable under physiological conditions. Therefore, a series of analogs were synthesized aiming to improve stability and bioavailability of EGCG. Among them, peracetate-protected or the prodrug of EGCG was found to have increased bioavailability, stability, and proteasome-inhibitory activities against various human cancer cells and tumors compared to EGCG, suggesting its potential use for cancer prevention and treatment. Epidemiological studies have indicated that green tea consumption is associated with the reduced risk of cancers, especially associated with the reduced risk of late stage of cancers. This risk reduction may be attributed not only to proteasome inhibition, but also to numerous other intracellular molecules targeted by EGCG that are involved in cell cycle regulation, apoptosis, angiogenesis, and metastasis.

Bortezomib as the first proteasome inhibitor anticancer drug: current status and future perspectives.

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-X(L) 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.

Turning tumor-promoting copper into an anti-cancer weapon via high-throughput chemistry.

Copper is an essential element for multiple biological processes. Its concentration is elevated to a very high level in cancer tissues for promoting cancer development through processes such as angiogenesis. Organic chelators of copper can passively reduce cellular copper and serve the role as inhibitors of angiogenesis. However, they can also actively attack cellular targets such as proteasome, which plays a critical role in cancer development and survival. The discovery of such molecules initially relied on a step by step synthesis followed by biological assays. Today high-throughput chemistry and high-throughput screening have significantly expedited the copper-binding molecules discovery to turn "cancer-promoting" copper into anti-cancer agents.

The challenge of developing green tea polyphenols as therapeutic agents.

The health benefits of green tea and its main constituent (-)-epigallocatechin gallate [(-)-EGCG] have been widely supported by results from epidemiological, cell culture, animal and clinical studies. On the other hand, there are a number of issues, such as stability, bioavailability and metabolic transformations under physiological conditions, facing the development of green tea polyphenols into therapeutic agents. We previously reported that the synthetic peracetate of (-)-EGCG has improved stability and better bioavailability than (-)-EGCG itself and can act as pro-drug under both in vitro and in vivo conditions. Analogs of catechins have been synthesized and their structure activity relationship provides an understanding to the mechanism of proteasome inhibition. Metabolic methylation of catechins leading to methylated (-)-EGCG may alter the biological activities of these compounds.

Green tea polyphenols as a natural tumour cell proteasome inhibitor.

The cancer-preventive effects of green tea and its main constituent (-)-epigallocatechin gallate [(-)-EGCG] are widely supported by results from epidemiological, cell culture, animal and clinical studies although the molecular target has not been well defined. We previously reported that ester bond-containing tea polyphenols, e. g. (-)-EGCG, and their synthetic analogs potently and specifically inhibited the proteasomal activity. Subsequently, we further demonstrated that methylation on green tea polyphenols under physiological conditions decreased their proteasome-inhibitory activity, contributing to decreased cancer-preventive effects of tea consumption. Since (-)-EGCG is unstable under physiological conditions, we also developed the peracetate-protected or prodrug form of (-)-EGCG, Pro-EGCG (1), and shown that Pro-EGCG (1) increases the bioavailability, stability, and proteasome-inhibitory and anticancer activities of (-)-EGCG in human breast cancer cells and xenografts, suggesting its potential use for cancer prevention and treatment.

Natural compounds with proteasome inhibitory activity for cancer prevention and treatment.

The proteasome is a multicatalytic protease complex that degrades most endogenous proteins including misfolded or damaged proteins to ensure normal cellular function. The ubiquitin-proteasome degradation pathway plays an essential role in multiple cellular processes, including cell cycle progression, proliferation, apoptosis and angiogenesis. It has been shown that human cancer cells are more sensitive to proteasome inhibition than normal cells, indicating that a proteasome inhibitor could be used as a novel anticancer drug. Indeed, this idea has been supported by the encouraging results of the clinical trials using the proteasome inhibitor Bortezomib (Velcade, PS-341), a drug approved by the US Food and Drug Administration (FDA). Several natural compounds, including the microbial metabolite lactacystin, green tea polyphenols, and traditional medicinal triterpenes, have been shown to be potent proteasome inhibitors. These findings suggest the potential use of natural proteasome inhibitors as not only chemopreventive and chemotherapeutic agents, but also tumor sensitizers to conventional radiotherapy and chemotherapy. In this review, we will summarize the structures and biological activities of the proteasome and several natural compounds with proteasome inhibitory activity, and will discuss the potential use of these compounds for the prevention and treatment of human cancers.

Tea polyphenols, their biological effects and potential molecular targets.

Tea is the most popular beverage in the world, second only to water. Tea contains an infusion of the leaves from the Camellia sinensis plant rich in polyphenolic compounds known as catechins, the most abundant of which is (-)-EGCG. Although tea has been consumed for centuries, it has only recently been studied extensively as a health-promoting beverage that may act to prevent a number of chronic diseases and cancers. The results of several investigations indicate that green tea consumption may be of modest benefit in reducing the plasma concentration of cholesterol and preventing atherosclerosis. Additionally, the cancer-preventive effects of green tea are widely supported by results from epidemiological, cell culture, animal and clinical studies. In vitro cell culture studies show that tea polyphenols potently induce apoptotic cell death and cell cycle arrest in tumor cells but not in their normal cell counterparts. Green tea polyphenols were shown to affect several biological pathways, including growth factor-mediated pathway, the mitogen-activated protein (MAP) kinase-dependent pathway, and ubiquitin/proteasome degradation pathways. Various animal studies have revealed that treatment with green tea inhibits tumor incidence and multiplicity in different organ sites such as skin, lung, liver, stomach, mammary gland and colon. Recently, phase I and II clinical trials have been conducted to explore the anticancer effects of green tea in humans. A major challenge of cancer prevention is to integrate new molecular findings into clinical practice. Therefore, identification of more molecular targets and biomarkers for tea polyphenols is essential for improving the design of green tea trials and will greatly assist in a better understanding of the mechanisms underlying its anti-cancer activity.

Polyphenols: biological activities, molecular targets, and the effect of methylation.

Polyphenolic compounds are widely distributed in the plant kingdom and the anticancer benefits obtained from their consumption have been studied extensively. However, polyphenols are subject to various biotransformation reactions within the human body including methylation. Likewise, naturally occurring polyphenols may contain O-methylations in place of the hydroxyls of the parent compounds. While some studies suggest that methylations can increase the bioavailability of polyphenols, other studies indicate a decrease in the anticancer benefits of methylated polyphenols. This review will focus on the cellular activities of polyphenols, their potential molecular targets and their biological effects after enzymatic methylation. Furthermore, an assessment of the positive and negative aspects of polyphenol methylation on the anticancer activity will be discussed. Finally, the future of polyphenols in both cancer prevention and cancer intervention will be addressed.

Gold complexes as prospective metal-based anticancer drugs.

Medical and therapeutic value of gold has been recognized thousands of years ago, but its rational use in medicine has not begun until the early 1920s. Cisplatin is one of the first metal-containing compounds with anti-cancer activity discovered in the 1960s. Despite the fact that cisplatin treatment is efficient for several types of solid tumors, its effectiveness is limited by toxic side effects and tumor resistance that often leads to the occurrence of secondary malignancies. Since gold(III) is isoelectronic with platinum(II) and tetracoordinate gold(III) complexes have the same square-planar geometries as cisplatin, the anticancer activity of gold(III) compounds has been investigated. Previous studies suggested that, in contrast to cisplatin, gold complexes target proteins but not DNA. Recently, we have investigated gold(III) dithiocarbamates for their anticancer activity and showed that their primary target is the proteasome. Treatment of human breast tumor-bearing nude mice with a gold(III) dithiocarbamate complex resulted in significant inhibition of tumor growth, associated with proteasome inhibition and massive apoptosis induction in vivo. Better understanding of physiological processing of gold compounds will provide a rational basis for their further development into novel anticancer drugs.

Tannic acid potently inhibits tumor cell proteasome activity, increases p27 and Bax expression, and induces G1 arrest and apoptosis.

Animal studies have demonstrated that a dietary polyphenol known as tannic acid (TA) exhibits anticarcinogenic activity in chemically induced cancers, although the involved molecular target remains unknown. In addition, proteasome inhibitors have been shown to suppress human tumor growth in nude mice. Most recently, we have reported that ester-bond-containing tea polyphenols are potent proteasome inhibitors in vitro and in vivo. We have hypothesized that TA, which contains multiple similar gallate moieties linked by ester bonds, should inhibit the proteasome activity. Here, we report that indeed TA potently and specifically inhibits the chymotrypsin-like activity of purified 20S proteasome (IC(50) = 0.06 microg/ml), 26S proteasome of Jurkat T-cell extracts, and 26S proteasome of living Jurkat cells. Inhibition of the proteasome by TA in Jurkat cells results in accumulation of two natural proteasome substrates, the cyclin-dependent kinase inhibitor p27(Kip1) and the proapoptotic protein Bax, followed by growth arrest in G1 and induction of apoptotic cell death. Our present study suggests that TA targets and inhibits the proteasome in tumor cells, which may contribute to the previously observed anticarcinogenic activity of TA.

Sequential two-step cleavage of the retinoblastoma protein by caspase-3/-7 during etoposide-induced apoptosis.

During cellular apoptosis, retinoblastoma protein (RB) is subjected to cleavage near the carboxyl terminus by a caspase-3-like protease. In addition, an heretofore unidentified protease cleaves RB internally, generating fragments of 68 and 48 kDa. Internal cleavage abrogates the ability of RB to associate with E2F. To investigate the mechanism of RB internal cleavage, we developed and employed an in vitro cleavage assay. Incubation of in vitro translated (35)S-RB with apoptotic cell extracts led to RB cleavage at the C-terminus, followed by internal cleavage. The caspase peptide inhibitors z-VAD-FMK or z-DEVD-FMK blocked both cleavage events. Rapid C-terminal and internal cleavage were also observed when recombinant caspase-3 was added to (35)S-RB. Moreover, when caspase-3 was added to nonapoptotic cell extract, efficient internal cleavage of cellular RB was observed. Caspase-mediated internal cleavage occurred following RB residue aspartate(349) in the sequence DSID(349). This sequence is consistent with a DXXD recognition motif for caspase-3-like enzymes. Interestingly, we also observed RB internal cleavage in caspase-3-deficient MCF-7 cells, indicating that other caspases are capable of cleaving RB internally. Indeed, caspase-7, a member of the caspase-3 subfamily, was found to cleave (35)S-RB at both the carboxyl terminus, and following aspartate(349). By contrast, caspases that are not members of the caspase-3 subfamily failed to cleave RB. Taken together, our findings demonstrate that during apoptosis, a caspase-3-like protease is responsible for degradation and functional inactivation of RB by cleaving the protein internally following aspartate(349).

Green tea polyphenol epigallocatechin inhibits DNA replication and consequently induces leukemia cell apoptosis.

Consistent with the putative role of green tea in cancer prevention, tea polyphenols have previously been shown to inhibit tumor cell proliferation by inducing G1 or G2/M cell cycle arrests, also documented is their ability to induce apoptosis (programmed cell death). However, it is unclear whether or not the cell cycle effects of polyphenols are related to their cell death-inducing ability. Here we report that the tea polyphenol (-)-epigallocatechin (EGC) inhibits DNA replication in three leukemia cancer cell lines, Jurkat T, HL-60 and K562. Among all the tested tea polyphenols, EGC was found to be the most potent in accumulation of S phase cells and inhibition of the S-G2 progression. In addition, EGC-mediated inhibition of S phase progression results in induction of apoptosis, as determined by sub-G1 cell population, breakage of endonuclear DNA, cleavage of poly(ADP-ribose) polymerase and loss of cell viability. When used in cells containing low S and high G1 and G2/M populations, EGC did not induce apoptosis. Furthermore, EGC did not inhibit M-G1 transition. Our finding that EGC inhibits S phase progression that results in leukemia cell death provides a novel and plausible molecular mechanism for how green tea may inhibit the growth of rapidly proliferating neoplastic cells.

Ester bond-containing tea polyphenols potently inhibit proteasome activity in vitro and in vivo.

It has been discovered that proteasome inhibitors are able to induce tumor growth arrest or cell death and that tea consumption is correlated with cancer prevention. Here, we show that ester bond-containing tea polyphenols, such as (-)-epigallocatechin-3-gallate (EGCG), potently and specifically inhibit the chymotrypsin-like activity of the proteasome in vitro (IC(50) = 86-194 nm) and in vivo (1-10 microm) at the concentrations found in the serum of green tea drinkers. Atomic orbital energy analyses and high performance liquid chromatography suggest that the carbon of the polyphenol ester bond is essential for targeting, thereby inhibiting the proteasome in cancer cells. This inhibition of the proteasome by EGCG in several tumor and transformed cell lines results in the accumulation of two natural proteasome substrates, p27(Kip1) and IkappaB-alpha, an inhibitor of transcription factor NF-kappaB, followed by growth arrest in the G(1) phase of the cell cycle. Furthermore, compared with their simian virus-transformed counterpart, the parental normal human fibroblasts were much more resistant to EGCG-induced p27(Kip1) protein accumulation and G(1) arrest. Our study suggests that the proteasome is a cancer-related molecular target of tea polyphenols and that inhibition of the proteasome activity by ester bond-containing polyphenols may contribute to the cancer-preventative effect of tea.

CEP1612, a dipeptidyl proteasome inhibitor, induces p21WAF1 and p27KIP1 expression and apoptosis and inhibits the growth of the human lung adenocarcinoma A-549 in nude mice.

The ubiquitin proteasome system is responsible for the proteolysis of important cell cycle and apoptosis-regulatory proteins. In this paper we report that the dipeptidyl proteasome inhibitor, phthalimide-(CH2)8CH-(cyclopentyl) CO-Arg(NO2)-Leu-H (CEP1612), induces apoptosis and inhibits tumor growth of the human lung cancer cell line A-549 in an in vivo model. In cultured A-549 cells, CEP1612 treatment results in accumulation of two proteasome natural substrates, the cyclin-dependent kinase inhibitors p21WAF1 and p27KIP1, indicating its ability to inhibit proteasome activity in intact cells. Furthermore, CEP1612 induces apoptosis as evident by caspase-3 activation and poly(ADP-ribose) polymerase cleavage. Treatment of A-549 tumor-bearing nude mice with CEP1612 (10 mg/kg/day, i.p. for 31 days) resulted in massive induction of apoptosis and significant (68%; P < 0.05) tumor growth inhibition, as shown by terminal deoxynucleotidyltransferase-mediated UTP end labeling. Furthermore, immunostaining of tumor specimens demonstrated in vivo accumulation of p21WAF1 and p27KIP1 after CEP1612 treatment. The results suggest that CEP1612 is a promising candidate for further development as an anticancer drug and demonstrate the feasibility of using proteasome inhibitors as novel antitumor agents.

Regulation of tumor cell apoptotic sensitivity during the cell cycle (Review).

Understanding how current chemotherapeutic modalities induce apoptosis is critical to designing better anti-cancer agents. This review is concerned with how pharmacological agents induce tumor cell apoptosis in a cell cycle-dependent manner. Recent experiments demonstrate that expression of several apoptotic regulatory proteins (such as Bcl-2, Bax, p53, and Survivin) are differentially regulated according to the phases of the cell cycle. This cell cycle-dependent regulation in turn contributes to increased drug sensitivity in different phases of the cell cycle. It is therefore likely that the cell cycle-dependent expression of cell death proteins plays a role in regulating chemosensitivity and apoptotic commitment of human tumor cells.

N-terminal cleavage of bax by calpain generates a potent proapoptotic 18-kDa fragment that promotes bcl-2-independent cytochrome C release and apoptotic cell death.

Upon apoptosis induction, the proapoptotic protein Bax is translocated from the cytosol to mitochondria, where it promotes release of cytochrome c, a caspase-activating protein. However, the molecular mechanisms by which Bax triggers cytochrome c release are unknown. Here we report that before the initiation of apoptotic execution by etoposide or staurosporin, an active calpain activity cleaves Bax at its N-terminus, generating a potent proapoptotic 18-kDa fragment (Bax/p18). Both the calpain-mediated Bax cleavage activity and the Bax/p18 fragment were found in the mitochondrial membrane-enriched fraction. Cleavage of Bax was followed by release of mitochondrial cytochrome c, activation of caspase-3, cleavage of poly(ADP-ribose) polymerase, and fragmentation of DNA. Unlike the full-length Bax, Bax/p18 did not interact with the antiapoptotic Bcl-2 protein in the mitochondrial fraction of drug-treated cells. Pretreatment with a specific calpain inhibitor calpeptin inhibited etoposide-induced calpain activation, Bax cleavage, cytochrome c release, and caspase-3 activation. In contrast, transfection of a cloned Bax/p18 cDNA into multiple human cancer cell lines targeted Bax/p18 to mitochondria, which was accompanied by release of cytochrome c and induction of caspase-3-mediated apoptosis that was not blocked by overexpression of Bcl-2 protein. Therefore, Bax/p18 has a cytochrome c-releasing activity that promotes cell death independent of Bcl-2. Finally, Bcl-2 overexpression inhibited etoposide-induced calpain activation, Bax cleavage, cytochrome c release, and apoptosis. Our results suggest that the mitochondrial calpain plays an essential role in apoptotic commitment by cleaving Bax and generating the Bax/p18 fragment, which in turn mediates cytochrome c release and initiates the apoptotic execution.

G(1) phase-dependent expression of bcl-2 mRNA and protein correlates with chemoresistance of human cancer cells.

Recent experiments suggest an interconnection between cell proliferation and programmed cell death (apoptosis), although the detailed molecular mechanisms remain unclear. We have hypothesized that expression of some apoptosis regulators is cell cycle-dependent, which in turn influences tumor cell chemosensitivity in a cell cycle-dependent fashion. To test these hypotheses, we synchronized human leukemia Jurkat T, Neo (using aphidicolin), breast cancer MCF-7, normal fibroblast, and simian virus 40-transformed cells (by aphidicolin or serum starvation), and measured levels of several Bcl-2 family proteins. The highest expression of Bcl-2 protein was found in the G(1) phase of all the five cell lines tested. In contrast, levels of Bax protein remained relatively unchanged in four of the cell lines, and levels of Bcl-X(L), Bcl-X(S), and Bak proteins showed little or no cell cycle-dependent changes in Jurkat T cells. Similar to the changes in Bcl-2 protein levels, its mRNA expression was also G(1) phase-specific, whereas the level of a Bcl-2 cleavage activity remained constitutive. When treated with an anticancer drug (etoposide or cisplatin) or the kinase inhibitor staurosporin, the cells containing a high G(1) population and a high Bcl-2 protein level were much more resistant to the induced apoptosis than the cells containing a high S phase population and a low Bcl-2 protein level. Constitutive overexpression of Bcl-2 protein in Jurkat T cells completely blocked the S phase-associated sensitivity to these apoptosis stimuli. The cell cycle-dependent Bcl-2 protein expression seems to contribute to the regulation of chemosensitivity and apoptotic commitment of human tumor cells.

Interleukin-2 expression in human carcinoma cell lines and its role in cell cycle progression.

Human carcinomas were shown to express mRNA and protein for IL-2R alpha, beta and gamma chains. Recently, human carcinomas were also shown to constitutively express protein and mRNA for IL-2 in vivo and in vitro. Here we report that the expression levels of cytoplasmic IL-2 as well as IL-2Rbeta- and gamma-chain in human carcinoma cells change during the cell cycle progression. Carcinoma cells synchronized in the G2/M phase of the cell cycle expressed significantly more intracytoplasmic IL-2 as well as IL-2Rbeta and gamma proteins than tumor cells in the G0/G1 phase. The level of mRNA for IL-2 was 5-10-fold higher in the M phase than in the G0/G1-phase, as shown by quantitative competitive RT-PCR. Expression of the cyclin-dependent kinase (CDK) inhibitor p27kip1 in these carcinoma cells was found to be high in the G0/G1 phase, nearly absent in the S phase, and it increased again in the G2/M phase of the cell cycle. In synchronized cells, the decrease in p27 expression coincided with high levels of expression of IL-2. Using the IL-2 specific antisense oligonucleotide to block synthesis of endogenous IL-2 in tumor cells, we observed increased levels of p27 as well as p21. The antisense oligonucleotides specific for p27 or p21 blocked expression of these proteins but not of IL-2. Thus, endogenous IL-2 is important in regulating expression of p27 as well as p21 and, therefore, in controlling cell cycle progression of tumor cells, while its own expression remains independent of the CDK inhibitors.

Bax degradation by the ubiquitin/proteasome-dependent pathway: involvement in tumor survival and progression.

Previously we reported that proteasome inhibitors were able to overcome Bcl-2-mediated protection from apoptosis. Here we show that inhibition of the proteasome activity in Bcl-2-overexpressing cells accumulates the proapoptotic Bax protein to mitochondria/cytoplasm, where it interacts to Bcl-2 protein. This event was followed by release of mitochondrial cytochrome c into the cytosol and activation of caspase-mediated apoptosis. In contrast, proteasome inhibition did not induce any apparent changes in Bcl-2 protein levels. In addition, treatment with a proteasome inhibitor increased levels of ubiquitinated forms of Bax protein, without any effects on Bax mRNA expression. We also established a cell-free Bax degradation assay in which an in vitro-translated, (35)S-labeled Bax protein can be degraded by a tumor cell protein extract, inhibitable by addition of a proteasome inhibitor or depletion of the proteasome or ATP. The Bax degradation activity can be reconstituted in the proteasome-depleted supernatant by addition of a purified 20S proteasome or proteasome-enriched fraction. Finally, by using tissue samples of human prostate adenocarcinoma, we demonstrated that increased levels of Bax degradation correlated well with decreased levels of Bax protein and increased Gleason scores of prostate cancer. Our studies strongly suggest that ubiquitin/proteasome-mediated Bax degradation is a novel survival mechanism in human cancer cells and that selective targeting of this pathway should provide a unique approach for treatment of human cancers, especially those overexpressing Bcl-2.