Q. Ping Dou

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.

Novel Metals and Metal Complexes as Platforms for Cancer Therapy

Metals are essential cellular components selected by nature to function in several indispensable biochemical processes for living organisms. Metals are endowed with unique characteristics that include redox activity, variable coordination modes, and reactivity towards organic substrates. Due to their reactivity, metals are tightly regulated under normal conditions and aberrant metal ion concentrations are associated with various pathological disorders, including cancer. For these reasons, coordination complexes, either as drugs or prodrugs, become very attractive probes as potential anticancer agents. The use of metals and their salts for medicinal purposes, from iatrochemistry to modern day, has been present throughout human history. The discovery of cisplatin, cis-[Pt(II) (NH(3))(2)Cl(2)], was a defining moment which triggered the interest in platinum(II)- and other metal-containing complexes as potential novel anticancer drugs. Other interests in this field address concerns for uptake, toxicity, and resistance to metallodrugs. This review article highlights selected metals that have gained considerable interest in both the development and the treatment of cancer. For example, copper is enriched in various human cancer tissues and is a co-factor essential for tumor angiogenesis processes. However the use of copper-binding ligands to target tumor copper could provide a novel strategy for cancer selective treatment. The use of nonessential metals as probes to target molecular pathways as anticancer agents is also emphasized. Finally, based on the interface between molecular biology and bioinorganic chemistry the design of coordination complexes for cancer treatment is reviewed and design strategies and mechanisms of action are discussed.

Clioquinol, a therapeutic agent for alzheimer's disease, has proteasome-inhibitory, androgen receptor-suppressing, apoptosis-inducing, and antitumor activities in human prostate cancer cells and xenografts

Tumor growth and metastasis depend on angiogenesis that requires the cofactor copper. Consistently, high levels of copper have been found in many types of human cancers, including prostate, breast, colon, and lung. Recent studies suggest that copper could be used as a novel selective target for cancer therapies. Clioquinol is capable of forming stable complexes with copper and currently used in clinics for treatment of Alzheimers disease. Most recently, it has been reported that clioquinol possesses antitumor effects. However, the underlying molecular mechanism is unclear. We report here that after binding to copper, clioquinol can inhibit the proteasomal chymotrypsin-like activity, repress androgen receptor (AR) protein expression, and induce apoptotic cell death in human prostate cancer LNCaP and C4-2B cells. In addition, clioquinol alone exhibits similar effects in prostate cancer cell lines with elevated copper at concentrations similar to those found in patients. Addition of dihydrotestosterone did not affect clioquinol-mediated proteasome inhibition in both prostate cancer cell lines. However, dihydrotestosterone partially inhibited clioquinol-induced AR suppression and apoptosis only in androgen-dependent LNCaP cells. Animal studies show that clioquinol treatment significantly inhibits the growth of human prostate tumor C4-2B xenografts (by 66%), associated with in vivo proteasome inhibition, AR protein repression, angiogenesis suppression, and apoptosis induction. Our study provides strong evidence that clioquinol is able to target tumor proteasome in vivo in a copper-dependent manner, resulting in formation of an active AR inhibitor and apoptosis inducer that is responsible for its observed antiprostate tumor effect.

Androgen receptor- and E2F-1-targeted thymoquinone therapy for hormone-refractory prostate cancer

Relapse of prostate cancer after androgen ablation therapy is hormone-refractory, with continued tumor growth being dependent on the androgen receptor (AR). E2F-1, a regulator of cell proliferation and viability, reportedly plays a role in the development of hormone-refractory prostate cancer. Thymoquinone is a component of Nigella sativa, an herb used for thousands of years for culinary and medicinal purposes in Asian and Middle Eastern countries and has been reported to have an antineoplastic effect both in vitro and in vivo. We observed that thymoquinone inhibited DNA synthesis, proliferation, and viability of cancerous (LNCaP, C4-B, DU145, and PC-3) but not noncancerous (BPH-1) prostate epithelial cells by down-regulating AR and E2F-1. In LNCaP cells, this was associated with a dramatic increase in p21(Cip1), p27(Kip1), and Bax. Thymoquinone blunted progression of synchronized LNCaP cells from G1 to S phase, with a concomitant decrease in AR and E2F-1 as well as the E2F-1-regulated proteins necessary for cell cycle progression. In a xenograft prostate tumor model, thymoquinone inhibited growth of C4-2B-derived tumors in nude mice. This in vivo suppression of tumor growth, as with C4-2B cell growth in culture, was associated with a dramatic decrease in AR, E2F-1, and cyclin A as determined by Western blot of tissue extracts. Tissue immunohistochemical staining confirmed a marked reduction in E2F-1 and showed induction of apoptosis on terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling assay. These findings show that thymoquinone suppresses the expression of AR and E2F-1 necessary for proliferation and viability of androgen-sensitive as well as androgen-independent prostate cancer cells both in vitro and in vivo and, moreover, produced no noticeable side effects in mice. We conclude that thymoquinone, a naturally occurring herbal product, may prove to be effective in treating hormone-sensitive as well as hormone-refractory prostate cancer. Furthermore, because of its selective effect on cancer cells, we believe that thymoquinone can also be used safely to help prevent the development of prostate cancer.

Targeting the ubiquitin–proteasome system for cancer therapy

Introduction: The ubiquitin–proteasome system (UPS) degrades 80 – 90% of intracellular proteins. Cancer cells take advantage of the UPS for their increased growth and decreased apoptotic cell death. Thus, the components that make up the UPS represent a diverse group of potential anti-cancer targets. The success of the first-in-class proteasome inhibitor bortezomib not only proved that the proteasome is a feasible and valuable anti-cancer target, but also inspired researchers to extensively explore other potential targets of this pathway. Areas covered: This review provides a broad overview of the UPS and its role in supporting cancer development and progression, especially in aspects of p53 inactivation, p27 turnover and NF-κB activation. Also, efforts toward the development of small molecule inhibitors (SMIs) targeting different steps in this pathway for cancer treatment are reviewed and discussed. Expert opinion: Whereas some of the targets in the UPS, such as the 20S proteasome, Nedd8 activating enzyme and HDM2, have been well-established and validated, there remains a large pool of candidates waiting to be investigated. Development of SMIs targeting the UPS has been largely facilitated by state-of-the-art technologies such as high-throughput screening and computer-assisted drug design, both of which require a better understanding of the targets of interest.

Shikonin Exerts Antitumor Activity via Proteasome Inhibition and Cell Death Induction in vitro and in vivo

Dysregulation of the ubiquitin‐proteasome pathway plays an essential role in tumor growth and development. Shikonin, a natural naphthoquinone isolated from the traditional Chinese medicine Zi Cao (gromwell), has been reported to possess tumor cell‐killing activity, and results from a clinical study using a shikonin‐containing mixture demonstrated its safety and efficacy for the treatment of late‐stage lung cancer. In this study, we reported that shikonin is an inhibitor of tumor proteasome activity in vitro and in vivo. Our computational modeling predicts that the carbonyl carbons C1 and C4 of shikonin potentially interact with the catalytic site of β5 chymotryptic subunit of the proteasome. Indeed, shikonin potently inhibits the chymotrypsin‐like activity of purified 20S proteasome (IC50 12.5 μmol/L) and tumor cellular 26S proteasome (IC50 between 2–16 μmol/L). Inhibition of the proteasome by shikonin in murine hepatoma H22, leukemia P388 and human prostate cancer PC‐3 cultures resulted in accumulation of ubiquitinated proteins and several proteasome target proapoptotic proteins (IκB‐α, Bax and p27), followed by induction of cell death. Shikonin treatment resulted in tumor growth inhibition in both H22 allografts and PC‐3 xenografts, associated with suppression of the proteasomal activity and induction of cell death in vivo. Finally, shikonin treatment significantly prolonged the survival period of mice bearing P388 leukemia. Our results indicate that the tumor proteasome is one of the cellular targets of shikonin and inhibition of the proteasome activity by shikonin contributes to its antitumor property.

Ni(II), Cu(II), and Zn(II) Diethyldithiocarbamate Complexes Show Various Activities Against the Proteasome in Breast Cancer Cells

A series of three complexes with diethyldithiocarbamate ligand and three different metals (Ni, Cu, Zn) was prepared, confirmed by X-ray crystallography, and tested in human breast cancer MDA-MB-231 cells. Zinc and copper complexes, but not nickel complex, were found to be more active against cellular 26S proteasome than against purified 20S proteasome core particle. One of the possible explanations is inhibition of JAMM domain in the 19S proteasome lid.

Regulation of FOXO3a/β-catenin/GSK-3β signaling by 3,3'-diindolylmethane contributes to inhibition of cell proliferation and induction of apoptosis in prostate cancer cells

Previous studies from our laboratory have shown anti-proliferative and pro-apoptotic effects of 3,3′-diindolylmethane (DIM) through regulation of Akt and androgen receptor (AR) in prostate cancer cells. However, the mechanism by which DIM regulates Akt and AR signaling pathways has not been fully investigated. It has been known that FOXO3a and glycogen synthase kinase-3β (GSK-3β), two targets of activated Akt, interact with β-catenin, regulating cell proliferation and apoptotic cell death. More importantly, FOXO3a, GSK-3β, and β-catenin are all AR coregulators and regulate the activity of AR, mediating the development and progression of prostate cancers. Here, we investigated the molecular effects of B-DIM, a formulated DIM with higher bioavailability, on Akt/FOXO3a/GSK-3β/β-catenin/AR signaling in hormone-sensitive LNCaP and hormone-insensitive C4-2B prostate cancer cells. We found that B-DIM significantly inhibited the phosphorylation of Akt and FOXO3a and increased the phosphorylation of β-catenin, leading to the inhibition of cell growth and induction of apoptosis. We also found that B-DIM significantly inhibited β-catenin nuclear translocation. By electrophoretic mobility shift and chromatin immunoprecipitation assays, we found that B-DIM inhibited FOXO3a binding to the promoter of AR and promoted FOXO3a binding to the p27KIP1 promoter, resulting in the alteration of AR and p27KIP1 expression, the inhibition of cell proliferation, and the induction of apoptosis in both androgen-sensitive and -insensitive prostate cancer cells. These results suggest that B-DIM-induced cell growth inhibition and apoptosis induction are partly mediated through the regulation of Akt/FOXO3a/GSK-3β/β-catenin/AR signaling. Therefore, B-DIM could be a promising non-toxic agent for possible treatment of hormone-sensitive but most importantly hormone-refractory prostate cancers.

EGCG, green tea polyphenols and their synthetic analogs and prodrugs for human cancer prevention and treatment.

Cancer-preventive effects of tea polyphenols, especially epigallocatechin-3-gallate (EGCG), have been demonstrated by epidemiological, preclinical, and clinical studies. Green tea polyphenols such as EGCG have the potential to affect multiple biological pathways, including gene expression, growth factor-mediated pathways, the mitogen-activated protein kinase-dependent pathway, and the ubiquitin/proteasome degradation pathway. Therefore, identification of the molecular targets of EGCG should greatly facilitate a better understanding of the mechanisms underlying its anticancer and cancer-preventive activities. Performing structure-activity relationship (SAR) studies could also greatly enhance the discovery of novel tea polyphenol analogs as potential anticancer and cancer-preventive agents. In this chapter, we review the relevant literature as it relates to the effects of natural and synthetic green tea polyphenols and EGCG analogs on human cancer cells and their potential molecular targets as well as their antitumor effects. We also discuss the implications of green tea polyphenols in cancer prevention.