Kristin R. Landis-Piwowar

A Novel Anticancer Gold(III) Dithiocarbamate Compound Inhibits the Activity of a Purified 20S Proteasome and 26S Proteasome in Human Breast Cancer Cell Cultures and Xenografts

Although cisplatin has been used for decades to treat human cancer, some toxic side effects and resistance are observed. It has been suggested that gold(III) complexes, containing metal centers isoelectronic and isostructural to cisplatin, are promising anticancer drugs. Gold(III) dithiocarbamate complexes were shown to exhibit in vitro cytotoxicity, comparable with and even greater than cisplatin; however, the involved mechanism of action remained unknown. Because we previously reported that copper(II) dithiocarbamates are potent proteasome inhibitors, we hypothesized that gold(III) dithiocarbamate complexes could suppress tumor growth via direct inhibition of the proteasome activity. Here, for the first time, we report that a synthetic gold(III) dithiocarbamate (compound 2) potently inhibits the activity of a purified rabbit 20S proteasome and 26S proteasome in intact highly metastatic MDA-MB-231 breast cancer cells, resulting in the accumulation of ubiquitinated proteins and the proteasome target protein p27 and induction of apoptosis. The compound 2-mediated proteasome inhibition and apoptosis induction were completely blocked by addition of a reducing agent DTT or N-acetyl-L-cysteine, showing that process of oxidation is required for proteasome inhibition by compound 2. Treatment of MDA-MB-231 breast tumor-bearing nude mice with compound 2 resulted in significant inhibition of tumor growth, associated with proteasome inhibition and massive apoptosis induction in vivo. Our findings reveal the proteasome as a primary target for gold(III) dithiocarbamates and support the idea for their potential use as anticancer therapeutics.

Curcumin inhibits the proteasome activity in human colon cancer cells in vitro and in vivo

Curcumin (diferuloylmethane) is the major active ingredient of turmeric (Curcuma longa) used in South Asian cuisine for centuries. Curcumin has been shown to inhibit the growth of transformed cells and to have a number of potential molecular targets. However, the essential molecular targets of curcumin under physiologic conditions have not been completely defined. Herein, we report that the tumor cellular proteasome is most likely an important target of curcumin. Nucleophilic susceptibility and in silico docking studies show that both carbonyl carbons of the curcumin molecule are highly susceptible to a nucleophilic attack by the hydroxyl group of the NH(2)-terminal threonine of the proteasomal chymotrypsin-like (CT-like) subunit. Consistently, curcumin potently inhibits the CT-like activity of a purified rabbit 20S proteasome (IC(50) = 1.85 micromol/L) and cellular 26S proteasome. Furthermore, inhibition of proteasome activity by curcumin in human colon cancer HCT-116 and SW480 cell lines leads to accumulation of ubiquitinated proteins and several proteasome target proteins, and subsequent induction of apoptosis. Furthermore, treatment of HCT-116 colon tumor-bearing ICR SCID mice with curcumin resulted in decreased tumor growth, associated with proteasome inhibition, proliferation suppression, and apoptosis induction in tumor tissues. Our study shows that proteasome inhibition could be one of the mechanisms for the chemopreventive and/or therapeutic roles of curcumin in human colon cancer. Based on its ability to inhibit the proteasome and induce apoptosis in both HCT-116 and metastatic SW480 colon cancer cell lines, our study suggests that curcumin could potentially be used for treatment of both early-stage and late-stage/refractory colon cancer.

A Novel Prodrug of the Green Tea Polyphenol (−)-Epigallocatechin-3-Gallate as a Potential Anticancer Agent

The most abundant and biologically active green tea catechin, (-)-epigallocatechin-3-gallate or (-)-EGCG, has been shown to act as a proteasome inhibitor and tumor cell death inducer. However, (-)-EGCG is unstable under physiologic conditions and has poor bioavailability. Previously, in an attempt to increase the stability of (-)-EGCG, we introduced peracetate protections to its reactive hydroxyl groups and showed that this peracetate-protected (-)-EGCG [Pro-EGCG (1); formerly named compound 1] could be converted into (-)-EGCG under cell-free conditions. In the current study, we provide evidence that when cultured human breast cancer MDA-MB-231 cells were treated with Pro-EGCG (1), (-)-EGCG was not only converted but also accumulated, accompanied by enhanced levels of proteasome inhibition, growth suppression, and apoptosis induction, compared with cells treated with natural (-)-EGCG. To investigate the potential use of Pro-EGCG (1) as a novel prodrug that converts to a cellular proteasome inhibitor and anticancer agent in vivo, MDA-MB-231 tumors were induced in nude mice, followed by treatment with Pro-EGCG (1) or (-)-EGCG for 31 days. Results of this in vivo study showed a significant inhibition of breast tumor growth by Pro-EGCG (1), compared with (-)-EGCG, associated with increased proteasome inhibition and apoptosis induction in tumor tissues. In conclusion, we have shown that Pro-EGCG (1) increases the bioavailability, stability, and proteasome-inhibitory and anticancer activities of (-)-EGCG in human breast cancer cells and tumors, suggesting its potential use for cancer prevention and treatment.

Inhibition of proteasome activity by the dietary flavonoid apigenin is associated with growth inhibition in cultured breast cancer cells and xenografts

IntroductionProteasome inhibition is an attractive approach to anticancer therapy and may have relevancy in breast cancer treatment. Natural products, such as dietary flavonoids, have been suggested as natural proteasome inhibitors with potential use for cancer prevention and therapeutics. We previously reported that apigenin, a flavonoid widely distributed in many fruits and vegetables, can inhibit proteasome activity and can induce apoptosis in cultured leukemia Jurkat T cells. Whether apigenin has proteasome-inhibitory activity in the highly metastatic human breast MDA-MB-231 cells and xenografts, however, is unknown.MethodsMDA-MB-231 breast cancer cell cultures and xenografts were treated with apigenin, followed by measurement of reduced cellular viability/proliferation, proteasome inhibition, and apoptosis induction. Inhibition of the proteasome was determined by levels of the proteasomal chymotrypsin-like activity, by ubiquitinated proteins, and by accumulation of proteasome target proteins in extracts of the treated cells or tumors. Apoptotic cell death was measured by capase-3/caspase-7 activation, poly(ADP-ribose) polymerase cleavage, and immunohistochemistry for terminal nucleotidyl transferase-mediated nick end labeling positivity.ResultsWe report for the first time that apigenin inhibits the proteasomal chymotrypsin-like activity and induces apoptosis not only in cultured MDA-MB-231 cells but also in MDA-MB-231 xenografts. Furthermore, while apigenin has antibreast tumor activity, no apparent toxicity to the tested animals was observed.ConclusionWe have shown that apigenin is an effective proteasome inhibitor in cultured breast cancer cells and in breast cancer xenografts. Furthermore, apigenin induces apoptotic cell death in human breast cancer cells and exhibits anticancer activities in tumors. The results suggest its potential benefits in breast 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.

The challenge of developing green tea polyphenols as therapeutic agents

Abstract.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.

Relationship between the methylation status of dietary flavonoids and their growth‐inhibitory and apoptosis‐inducing activities in human cancer cells

Flavonoids are polyphenolic compounds widely distributed in the plant kingdom. Compelling research indicates that flavonoids have important roles in cancer chemoprevention and chemotherapy possibly due to biological activities that include action through anti‐inflammation, free radical scavenging, modulation of survival/proliferation pathways, and inhibition of the ubiquitin–proteasome pathway. Plant polyphenols including the green tea polyphenol (‐)‐epigallocatechin gallate or (‐)‐EGCG, and the flavonoids apigenin, luteolin, quercetin, and chrysin have been shown to inhibit proteasome activity and induce apoptosis in human leukemia cells. However, biotransformation reactions to the reactive hydroxyl groups on polyphenols could reduce their biological activities. Although methylated polyphenols have been suggested to be metabolically more stable than unmethylated polyphenols, the practical use of methylated polyphenols as cancer preventative agents warrants further investigation. In the current study, methylated and unmethylated flavonoids were studied for their proteasome‐inhibitory and apoptosis‐inducing abilities in human leukemia HL60 cells. Methylated flavonoids displayed sustained bioavailability and inhibited cellular proliferation by arresting cells in the G1 phase. However, they did not act as proteasome inhibitors in either an in vitro system or an in silico model and only weakly induced apoptosis. In contrast, unmethylated flavonoids exhibited inhibition of the proteasomal activity in intact HL60 cells, accumulating proteasome target proteins and inducing caspase activation and poly(ADP‐ribose) polymerase cleavage. We conclude that methylated flavonoids lack potent cytotoxicity against human leukemia cells and most likely have limited ability as chemopreventive agents. J. Cell. Biochem. 105: 514–523, 2008.

Pristimerin induces apoptosis by targeting the proteasome in prostate cancer cells

Pristimerin is a natural product derived from the Celastraceae and Hippocrateaceae families that were used as folk medicines for antiinflammation in ancient times. Although it has been shown that pristimerin induces apoptosis in breast cancer cells, the involved mechanism of action is unknown. The purpose of the current study is to investigate the primary target of pristimerin in human cancer cells, using prostate cancer cells as a working model. Nucleophilic susceptibility and in silico docking studies show that C6 of pristimerin is highly susceptible towards a nucleophilic attack by the hydroxyl group of N‐terminal threonine of the proteasomal chymotrypsin subunit. Consistently, pristimerin potently inhibits the chymotrypsin‐like activity of a purified rabbit 20S proteasome (IC50 2.2 µmol/L) and human prostate cancer 26S proteasome (IC50 3.0 µmol/L). The accumulation of ubiquitinated proteins and three proteasome target proteins, Bax, p27 and IκB‐α, in androgen receptor (AR)‐negative PC‐3 prostate cancer cells supports the conclusion that proteasome inhibition by pristimerin is physiologically functional. This observed proteasome inhibition subsequently led to the induction of apoptotic cell death in a dose‐ and kinetic‐dependent manner. Furthermore, in AR‐positive, androgen‐dependent LNCaP and AR‐positive, androgen‐independent C4‐2B prostate cancer cells, proteasome inhibition by pristimerin results in suppression of AR protein prior to apoptosis. Our data demonstrate, for the first time, that the proteasome is a primary target of pristimerin in prostate cancer cells and inhibition of the proteasomal chymotrypsin‐like activity by pristimerin is responsible for its cancer cell death‐inducing property. J. Cell. Biochem. 103: 234–244, 2008.

Methylation suppresses the proteasome -inhibitory function of green tea polyphenols

Under physiological conditions, biotransformation reactions, such as methylation, can modify green tea polyphenols (GTPs) and therefore limit their in vivo cancer‐preventive activity. Although a recent study suggested that methylated polyphenols are less cancer‐protective, the molecular basis is unknown. We previously reported that ester bond‐containing GTPs, for example (−)‐epigallocatechin‐3‐gallate [(−)‐EGCG] or (−)‐epicatechin‐3‐gallate [(−)‐ECG], potently and specifically inhibit the proteasomal chymotrypsin‐like activity. In this study, we hypothesize that methylated GTPs have decreased proteasome‐inhibitory abilities. To test this hypothesis, methylated (−)‐EGCG and (−)‐ECG analogs that can be found in vivo were synthesized and studied for their structure‐activity relationships (SARs) using a purified 20S proteasome. The addition of a single methyl group on (−)‐EGCG or (−)‐ECG led to decreased proteasome inhibition and, as the number of methyl groups increased, the inhibitory potencies further decreased. These SARs were supported by our findings from in silico docking analysis published recently. Previously, we synthesized a peracetate‐protected (−)‐EGCG molecule, Pro‐EGCG (1), to enhance its cellular permeability and stability, and current HPLC analysis confirms conversion of Pro‐EGCG (1) to (−)‐EGCG in cultured human leukemic Jurkat T cells. Furthermore, in this study, peracetate‐protected forms of methylated GTPs were added in intact Jurkat T cells to observe the intracellular effects of methylation. Peracetate‐protected, monomethylated (−)‐EGCG induced greater cellular proteasome inhibition and apoptosis than did peracetate‐protected, trimethylated (−)‐EGCG, consistent with the potencies of the parent methylated analogs against a purified 20S proteasome. Therefore, methylation on GTPs, under physiological conditions, could decrease their proteasome‐inhibitory activity, contributing to decreased cancer‐preventive effects of tea consumption. J. Cell. Physiol. 213: 252–260, 2007.

Maspin augments proteasome inhibitor‐induced apoptosis in prostate cancer cells

Proteasome inhibitors are known to induce apoptosis in a variety of cancer cells. On the other hand, maspin, a non‐inhibitory serine protease inhibitor, is shown to sensitize cancer cells to therapeutic agents that induce apoptosis. We examined the consequence of maspin expression in prostate cancer cells targeted for treatment with various proteasome inhibitors. We observed that proteasome inhibitors induced apoptosis more effectively in maspin transfected human prostate cancer DU145 cells than in control cells. Interestingly, increased apoptosis in these cells was associated with a significant induction of maspin expression. MG‐132, a proteasome inhibitor, induced endogenous and ectopic [cytomegalovirus promoter (CMV)‐driven] maspin expression, and maspin siRNA attenuated MG‐132‐induced apoptosis. Proteasome inhibitor‐induced maspin expression was inhibited by actinomycin D (Act D) and cyclohexamide (CHX), and by the inhibitors of p38MAPK, but not ERK1/2 or NF‐κB. Electrophoretic mobility‐shift assay (EMSA) and promoter‐reporter activity analyses suggested that p38MAPK activated transcription factor AP‐1 is responsible for proteasome inhibitor‐induced maspin expression. Taken together, these observations demonstrate that proteasome inhibitors induce maspin expression by activating p38MAPK pathway, and that maspin thus expressed, in turn, augments proteasome inhibitor‐induced apoptosis in prostate cancer cells. Our results suggest that gene therapy involving ectopic maspin expression may dramatically improve the efficacy of proteasome inhibitors for the treatment of prostate cancer. J. Cell. Physiol. 212: 298–306, 2007.