Evaluation of digestion methods for analysis of trace metals in mammalian tissues and NIST 1577c.

Digestion techniques for ICP analysis have been poorly studied for biological samples. This report describes an optimized method for analysis of trace metals that can be used across a variety of sample types. Digestion methods were tested and optimized with the analysis of trace metals in cancerous as compared to normal tissue as the end goal. Anthropological, forensic, oncological and environmental research groups can employ this method reasonably cheaply and safely whilst still being able to compare between laboratories. We examined combined HNO3 and H2O2 digestion at 170 °C for human, porcine and bovine samples whether they are frozen, fresh or lyophilized powder. Little discrepancy is found between microwave digestion and PFA Teflon pressure vessels. The elements of interest (Cu, Zn, Fe and Ni) yielded consistently higher and more accurate values on standard reference material than samples heated to 75 °C or samples that utilized HNO3 alone. Use of H2SO4 does not improve homogeneity of the sample and lowers precision during ICP analysis. High temperature digestions (>165 °C) using a combination of HNO3 and H2O2 as outlined are proposed as a standard technique for all mammalian tissues, specifically, human tissues and yield greater than 300% higher values than samples digested at 75 °C regardless of the acid or acid combinations used. The proposed standardized technique is designed to accurately quantify potential discrepancies in metal loads between cancerous and healthy tissues and applies to numerous tissue studies requiring quick, effective and safe digestions.

Design, synthesis and evaluation of marinopyrrole derivatives as selective inhibitors of Mcl-1 binding to pro-apoptotic Bim and dual Mcl-1/Bcl-xL inhibitors.

Inhibition of anti-apoptotic Mcl-1 is a promising anticancer strategy to overcome the survival and chemoresistance of a broad spectrum of human cancers. We previously reported on the identification of a natural product marinopyrrole A (1) that induces apoptosis in Mcl-1-dependent cells through Mcl-1 degradation. Here, we report the design and synthesis of novel marinopyrrole-based analogs and their evaluation as selective inhibitors of Mcl-1 as well as dual Mcl-1/Bcl-xL inhibitors. The most selective Mcl-1 antagonists were 34, 36 and 37 with 16-, 13- and 9-fold more selectivity for disrupting Mcl-1/Bim over Bcl-xL/Bim binding, respectively. Among the most potent dual inhibitors is 42 which inhibited Mcl-1/Bim and Bcl-xL/Bim binding 15-fold (IC50 = 600 nM) and 33-fold (500 nM) more potently than (±)-marinopyrrole A (1), respectively. Fluorescence quenching, NMR analysis and molecular docking indicated binding of marinopyrroles to the BH3 binding site of Mcl-1. Several marinopyrroles potently decreased Mcl-1 cellular levels and induced caspase 3 activation in human breast cancer cells. Our studies provide novel "lead" marinopyrroles for further optimization as selective Mcl-1 inhibitors and dual Mcl-1 and Bcl-xL inhibitors.

Transforming growth factor ß signaling overcomes dasatinib resistance in lung cancer.

Lung cancer is the second most common cancer and the leading cause of cancer-related deaths. Despite recent advances in the development of targeted therapies, patients with advanced disease remain incurable, mostly because metastatic non-small cell lung carcinomas (NSCLC) eventually become resistant to tyrosine kinase inhibitors (TKIs). Kinase inhibitors have the potential for target promiscuity because the kinase super family is the largest family of druggable genes that binds to a common substrate (ATP). As a result, TKIs often developed for a specific purpose have been found to act on other targets. Drug affinity chromatography has been used to show that dasatinib interacts with the TGFß type I receptor (TßR-I), a serine-threonine kinase. To determine the potential biological relevance of this association, we studied the combined effects of dasatinib and TGFß on lung cancer cell lines. We found that dasatinib treatment alone had very little effect; however, when NSCLC cell lines were treated with a combination of TGFß and dasatinib, apoptosis was induced. Combined TGFß-1 + dasatinib treatment had no effect on the activity of Smad2 or other non-canonical TGFß intracellular mediators. Interestingly, combined TGFß and dasatinib treatment resulted in a transient increase in p-Smad3 (seen after 3 hours). In addition, when NSCLC cells were treated with this combination, the pro-apoptotic protein BIM was up-regulated. Knockdown of the expression of Smad3 using Smad3 siRNA also resulted in a decrease in BIM protein, suggesting that TGFß-1 + dasatinib-induced apoptosis is mediated by Smad3 regulation of BIM. Dasatinib is only effective in killing EGFR mutant cells, which is shown in only 10% of NSCLCs. Therefore, the observation that wild-type EGFR lung cancers can be manipulated to render them sensitive to killing by dasatinib could have important implications for devising innovative and potentially more efficacious treatment strategies for this disease.

Proteasome inhibitor patents (2010 - present).

INTRODUCTION: Over the past 3 years, numerous patents and patent applications have been submitted and published involving compounds designed to inhibit the proteasome. Proteasome inhibition has been of great interest in cancer research since disruption of proteolysis leads to a significant buildup of cytotoxic proteins and activation of apoptotic pathways, particularly in rapidly proliferating cells. The current standards in proteasome inhibition are the only FDA-approved inhibitors, bortezomib and carfilzomib. Although these drugs are quite effective in treating multiple myeloma and other blood tumors, there are shortcomings, including toxicities and resistance. Most of the current patents attempt to improve on existing compounds, by increasing bioavailability and selectivity, while attempting to reduce toxicity. A general categorization of similar compounds was employed to evaluate and compare drug design strategies. AREAS COVERED: This review focuses on novel compounds and subsequent analogs developed for proteasome inhibition, used in preventing and treating human cancers. A comprehensive description and categorization of patents related to each type of compound and its derivatives, as well as their uses and efficacies as anticancer agents is included. A review of combination therapy patents has also been included. EXPERT OPINION: Although there are many diverse chemical scaffolds being published, there are few patented proteasome inhibitors whose method of inhibition is genuinely novel. Most patents utilize a destructive chemical warhead to attack the catalytic threonine residue of the proteasome active sites. Few patents try to depart from this, emphasizing the need for developing new mechanisms of action and specific targeting.

Cellular and computational studies of proteasome inhibition and apoptosis induction in human cancer cells by amino acid Schiff base-copper complexes.

Proliferation and apoptosis pathways are tightly regulated in a cell by the ubiquitin-proteasome system (UPS) and alterations in the UPS may result in cellular transformation or other pathological conditions. Indeed, the proteasome is often found to be overactive in cancer cells. It has also been found that cancer cells are more sensitive to proteasome inhibition than normal cells, and therefore proteasome inhibitors are pursued as antitumor drugs. The use of the proteasome inhibitor Bortezomib for treatment of multiple myeloma and mantle cell lymphoma has proved this principle. Recent studies have suggested that copper complexes can inhibit proteasome activity and induce apoptosis in some human cancer cells. However, the involved molecular mechanism is unknown. In this study, we investigated the biological activities of four amino acid Schiff base-copper(II) complexes by using human breast (MDA-MB-231 and MCF-7) and prostate (PC-3) cancer cells. The complexes C1 and C3, but not their counterparts C2 and C4, inhibit the chymotrypsin-like activity of purified 20S proteasome and human cancer cellular 26S proteasome, cause accumulation of proteasome target proteins Bax and IκB-α, and induce growth inhibition and apoptosis in concentration- and time-dependent manners. Docking analysis shows that C1, but not C2 has hydrophobic, pi-pi, pi-cation and hydrogen bond interactions with the proteasomal chymotrypsin-like pocket and could stably fit into the S3 region, leading to specific inhibition. Our study has identified the mechanism of action of these copper complexes on inhibiting tumor cell proteasome and suggested their great potential as novel anticancer agents.

Discovery and synthesis of hydronaphthoquinones as novel proteasome inhibitors.

Screening efforts led to the identification of PI-8182 (1), an inhibitor of the chymotrypsin-like (CT-L) activity of the proteasome. Compound 1 contains a hydronaphthoquinone pharmacophore with a thioglycolic acid side chain at position 2 and thiophene sulfonamide at position 4. An efficient synthetic route to the hydronaphthoquinone sulfonamide scaffold was developed, and compound 1 was synthesized in-house to confirm the structure and activity (IC(50) = 3.0 ± 1.6 µM [n = 25]). Novel hydronaphthoquinone derivatives of 1 were designed, synthesized, and evaluated as proteasome inhibitors. The structure-activity relationship (SAR) guided synthesis of more than 170 derivatives revealed that the thioglycolic acid side chain is required and the carboxylic acid group of this side chain is critical to the CT-L inhibitory activity of compound 1. Furthermore, replacement of the carboxylic acid with carboxylic acid isosteres such as tetrazole or triazole greatly improves potency. Compounds with a thio-tetrazole or thio-triazole side chain in position 2, where the thiophene was replaced by hydrophobic aryl moieties, were the most active compounds with up to 20-fold greater CT-L inhibition than compound 1 (compounds 15e, 15f, 15h, 15j, IC(50) values around 200 nM, and compound 29, IC(50) = 150 nM). The synthetic iterations described here not only led to improving potency in vitro but also resulted in the identification of compounds that are more active such as 39 (IC(50) = 0.44 to 1.01 µM) than 1 (IC(50) = 3.54 to 7.22 µM) at inhibiting the proteasome CT-L activity in intact breast cancer cells. Treatment with 39 also resulted in the accumulation of ubiquitinated cellular proteins and inhibition of tumor cell proliferation of breast cancer cells. The hit 1 and its analogue 39 inhibited proteasome CT-L activity irreversibly.

Shp2 protein tyrosine phosphatase inhibitor activity of estramustine phosphate and its triterpenoid analogs.

Shp2 protein tyrosine phosphate (PTP) is a novel target for anticancer drug discovery. We identified estramustine phosphate as a Shp2 PTP inhibitor from the National Cancer Institute Approved Oncology Drug set. A focused structure-activity relationship study indicated that the 17-phosphate group is required for the Shp2 PTP inhibitor activity of estramustine phosphate. A search for estramustine phosphate analogs led to identification of two triterpenoids, enoxolone, and celastrol, having Shp2 PTP inhibitor activity. With the previously reported PTP1B inhibitor trodusquemine, our study reveals steroids and triterpenoids with negatively charged phosphate, carboxylate, or sulfonate groups as novel pharmacophores of selective PTP inhibitors.

Transforming JAK1 mutations exhibit differential signalling, FERM domain requirements and growth responses to interferon-γ.

Recent work has highlighted roles for JAK (Janus kinase) family members in haemopoietic diseases. Although sequencing efforts have uncovered transforming JAK1 mutations in acute leukaemia, they have also identified non-transforming JAK1 mutations. Thus with limited knowledge of the mechanisms of JAK1 activation by mutation, sequencing may not readily identify transforming mutations. Therefore we sought to further understand the repertoire of transforming mutations of JAK1. We identified seven randomly generated transforming JAK1 mutations, including V658L and a deletion of amino acids 629-630 in the pseudokinase domain, as well as L910P, F938S, P960S, K1026E and Y1035C within the kinase domain. These mutations led to differential signalling activation, but exhibited similar transforming abilities, in BaF3 cells. Interestingly, these properties did not always correlate with JAK1 activation-loop phosphorylation. We also identified a JAK1 mutant that did not require a functional FERM (4.1/ezrin/radixin/moesin) domain for transformation. Although we isolated a mutation of JAK1 at residue Val658, which is found mutated in acute leukaemia patients, most of the mutations we identified are within the kinase domain and have yet to be identified in patients. Interestingly, compared with cells expressing JAK1-V658F, cells expressing these mutants had higher STAT1 (signal transducer and activator of transcription 1) phosphorylation and were more sensitive to interferon-γ-mediated growth inhibition. The differential STAT1 activation and interferon-sensitivity of JAK1 mutants may contribute to the determination of which specific JAK1 mutations ultimately contribute to disease and thus are identified in patients. Our characterization of these novel mutations contributes to a better understanding of mutational activation of JAK1.

The ubiquitin-proteasome system and assays to determine responses to inhibitors.

IMPORTANCE OF THE FIELD: Proteasome inhibition is an important therapeutic modality. Additionally, given the toxicities of direct proteasome inhibition, interest is increasing in modulating the ubiquitin ligases in the ubiquitin-proteasome system (UPS). AREAS COVERED IN THIS REVIEW: A detailed examination of the ubiquitin-proteasome pathway and an examination of methods of inhibiting this pathway from a variety of targets including the proteasome, the ubiquitin ligases and molecular biology techniques. Special attention is given to the assays used to measure modulation of the ubiquitin-proteasome pathway. WHAT THE READER WILL GAIN: A thorough examination of the UPS and its role in cells and disease and an overview of several assays for analyzing the effect of inhibitors on the UPS. Significant detail is given to assays of the ligase system and molecular approaches. These assays have their own advantages and disadvantages and will allow investigators to make informed choices on investigating the UPS. TAKE HOME MESSAGE: Interrupting the UPS can have profound consequences for cellular health and disease progression. The ubiquitin-proteasome pathway contains multiple activities that cannot be definitively assayed by a single technique. Assaying the UPS requires investigators to use multiple corroborating techniques and avoid confounding issues within each technique.

Computational validation of the importance of absolute stereochemistry in virtual screening.

Consideration of stereochemistry early in the identification and optimization of lead compounds can improve the efficiency and efficacy of the drug discovery process and reduce the time spent on subsequent drug development. These improvements can result by focusing on specific enantiomers that have the desired potential therapeutic effect (eutomers), while removing from consideration enantiomers that may have no, or even undesirable, effects (distomers). A virtual screening campaign that correctly takes stereochemical information into account can, in theory, be utilized to provide information about the relative binding affinities of enantiomers. Thus, the proper enumeration of the relevant stereoisomers in general, and enantiomeric pairs in particular, of chiral compounds is crucial if one is to use virtual screening as an effective drug discovery tool. As is obvious, in cases where no stereochemical information is provided for chiral compounds in a 2D chemical database, then each possible stereoisomer should be generated for construction of the subsequent 3D database to be used for virtual screening. However, acute problems can arise in 3D database construction when relative stereochemistry is encoded in a 2D database for a chiral compound containing multiple stereogenic atoms but absolute stereochemistry is not implied. In this case, we report that generation of enantiomeric pairs is imperative in database development if one is to obtain accurate docking results. A study is described on the impact of the neglect of enantiomeric pairs on virtual screening using the human homolog of murine double minute 2 (MDM2) protein, the product of a proto-oncogene, as the target. Docking in MDM2 with GLIDE 4.0 was performed using the NCI Diversity Set 3D database and, for comparison, a set of enantiomers we created corresponding to mirror image structures of the single enantiomers of chiral compounds present in the NCI Diversity Set. Our results demonstrate that potential lead candidates may be overlooked when databases contain 3D structures representing only a single enantiomer of racemic chiral compounds.

In silico chemical library screening and experimental validation of a novel 9-aminoacridine based lead-inhibitor of human S-adenosylmethionine decarboxylase.

In silico chemical library screening (virtual screening) was used to identify a novel lead compound capable of inhibiting S-adenosylmethionine decarboxylase (AdoMetDC). AdoMetDC is intimately involved in the biosynthesis of polyamines, which are essential for tumor progression and are elevated in numerous types of tumors. Therefore, inhibition of this enzyme provides an attractive target for the discovery of novel anticancer drugs. We performed virtual screening using a computer model derived from the X-ray crystal structure of human AdoMetDC and the National Cancer Institute's Diversity Set (1990 compounds). Our docking study suggested several compounds that could serve as drug candidates since their docking modes and scores revealed potential inhibitory activity toward AdoMetDC. Experimental testing of the top-scoring compounds indicated that one of these compounds (NSC 354961) possesses an IC50 in the low micromolar range. A search of the entire NCI compound collection for compounds similar to NSC 354961 yielded two additional compounds that exhibited activity in the experimental assay but with significantly diminished potency relative to NSC 354961. In this report, we disclose the activity of NSC 354961 against AdoMetDC and its probable binding mode based on computational modeling. We also discuss the importance of virtual screening in the context of enzymes that are not readily amenable to high-throughput assays, thereby demonstrating the efficacy of virtual screening, combined with selective experimental testing, in identifying new potential drug candidates.

Copper-binding compounds as proteasome inhibitors and apoptosis inducers in human cancer.

The trace element copper is vital to the healthy functioning of organisms. Copper is used in a multitude of cellular activities including respiration, angiogenesis, and immune responses. Recently, copper has become a focus in medical research ranging from Alzheimer's disease to cancer. Copper modulation has been suggested to be a potential modality for therapy in these diseases. Several copper-binding compounds have been found to spontaneously complex with copper and form active proteasome inhibitors and apoptosis inducers. This review examines compounds in the quinoline and dithiocarbamate families and from the National Cancer Institute (NCI) Diversity Set that bind with copper and act as anticancer agents. In each case, it is shown that these compounds can bind with copper, inhibit the proteasome activity, and induce apoptosis in cancer cells. These activities are absent when copper is not present. Compounds alone, clioquinol and pyrrolidinedithiocarbamate as examples, are shown to have no effects in normal breast cells. Current research suggests that a possible therapeutic modality for cancer may be developed using the difference of high copper load in tumors versus low copper load in normal cells. This strategy would convert tumor cellular copper into a potent, specific proteasome inhibitor and apoptosis inducer. Thus, this approach could pave the way for the development of nontoxic anticancer therapy.

Methylation of green tea polyphenols affects their binding to and inhibitory poses of the proteasome beta5 subunit.

Previously, we showed that ester carbon-containing tea polyphenols, including (-)-epigallocatechin gallate [(-)-EGCG] and (-)-epicatechin-3-gallate [(-)-ECG], potently inhibit proteasomal chymotrypsin-like activity. In addition, our in silico docking study suggested that a particular pose of (-)-EGCG could lead to potential covalent modification of the N-terminal threonine (Thr 1) of the proteasome beta5 subunit in the chymotrypsin-like active site. It has been suggested that some major biotransformation reactions, such as methylation, could result in reduced biological activity of (-)-EGCG in vivo. We hypothesize that methylation reduces binding of (-)-EGCG to the beta5 subunit of the proteasome and, therefore, decreases its proteasomal chymotrypsin-like-inhibitory potency. Here, we report that, while methylation has no effect on nucleophilic susceptibility of (-)-EGCG and (-)-ECG, it may disrupt the ability of these polyphenols to interact with Thr 1 of the proteasome beta5 subunit. In silico docking shows that methylation results in the tea polyphenols' ester carbon being moved away or blocked entirely from Thr 1. Additionally, methylation impairs the ability of (-)-EGCG and (-)-ECG to dock in a consistent low energy pose. These observations, no change in nucleophilic susceptibility, moving or blocking the ester carbon from Thr 1, and lack of a consistent docking pose, suggest that methylation disrupts the ability of (-)-EGCG and (-)-ECG to bind to the proteasome beta5 subunit, which may then diminish their proteasomal chymotrypsin-inhibitory and, therefore, other biological activities.

Clioquinol and pyrrolidine dithiocarbamate complex with copper to form proteasome inhibitors and apoptosis inducers in human breast cancer cells.

INTRODUCTION: A physiological feature of many tumor tissues and cells is the tendency to accumulate high concentrations of copper. While the precise role of copper in tumors is cryptic, copper, but not other trace metals, is required for angiogenesis. We have recently reported that organic copper-containing compounds, including 8-hydroxyquinoline-copper(II) and 5,7-dichloro-8-hydroxyquinoline-copper(II), comprise a novel class of proteasome inhibitors and tumor cell apoptosis inducers. In the current study, we investigate whether clioquinol (CQ), an analog of 8-hydroxyquinoline and an Alzheimer's disease drug, and pyrrolidine dithiocarbamate (PDTC), a known copper-binding compound and antioxidant, can interact with copper to form cancer-specific proteasome inhibitors and apoptosis inducers in human breast cancer cells. Tetrathiomolybdate (TM), a strong copper chelator currently being tested in clinical trials, is used as a comparison. METHODS: Breast cell lines, normal, immortalized MCF-10A, premalignant MCF10AT1K.cl2, and malignant MCF10DCIS.com and MDA-MB-231, were treated with CQ or PDTC with or without prior interaction with copper, followed by measurement of proteasome inhibition and cell death. Inhibition of the proteasome was determined by levels of the proteasomal chymotrypsin-like activity and ubiquitinated proteins in protein extracts of the treated cells. Apoptotic cell death was measured by morphological changes, Hoechst staining, and poly(ADP-ribose) polymerase cleavage. RESULTS: When in complex with copper, both CQ and PDTC, but not TM, can inhibit the proteasome chymotrypsin-like activity, block proliferation, and induce apoptotic cell death preferentially in breast cancer cells, less in premalignant breast cells, but are non-toxic to normal/non-transformed breast cells at the concentrations tested. In contrast, CQ, PDTC, TM or copper alone had no effects on any of the cells. Breast premalignant or cancer cells that contain copper at concentrations similar to those found in patients, when treated with just CQ or PDTC alone, but not TM, undergo proteasome inhibition and apoptosis. CONCLUSION: The feature of breast cancer cells and tissues to accumulate copper can be used as a targeting method for anticancer therapy through treatment with novel compounds such as CQ and PDTC that become active proteasome inhibitors and breast cancer cell killers in the presence of copper.

Anti-angiogenic and anti-tumor properties of proteasome inhibitors.

Tumor growth and metastasis depend on the formation of blood vessels, angiogenesis, to supply the developing mass with nutrients, oxygen, and waste removal. The proteasome, a massive multisubunit catabolic body, exerts a regulatory influence on angiogenesis. Inhibition of the proteasome activity has been found to inhibit angiogenesis and induce apoptosis in human cancer cells with limited toxicity to normal cells. Therefore, the dual action of angiogenesis inhibition and cell death induction makes proteasome inhibition an attractive modality for chemotherapy. A variety of proteasome inhibitors have been studied including: antibiotics such as lactacystin, the green tea polyphenols, and the boronic acid Velcade (MLN-341). Most recently, certain classes of copper compounds have been found to act as potent proteasome inhibitors. The potential of particular organic compounds, such as 8-hydroxyquinoline, to spontaneously bind with tumor cellular copper and form proteasome inhibitors provides a new modality of anti-proteasome and anti-angiogenesis chemotherapy. This review examines angiogenesis, the proteasome, representative proteasome inhibitors, and the emerging role of copper. The formation of new blood vessels, or angiogenesis, is an important and necessary function in both embryonic development and wound repair. Therefore, the ability to regenerate or form new vessels for blood flow is essential. The control of angiogenic pathways is tightly regulated in normal differentiated adult cells, which generally do not stimulate blood vessel growth unless injury occurs. However, cancerous tissues stimulate angiogenesis that in turn leads to increased tumor formation and possible metastases. Many of the factors involved in angiogenesis are regulated by the proteasome, which recently has become a focus in anti-cancer therapies due to its involvement in cell cycle and apoptosis control. Here we discuss angiogenesis and its relation to the proteasome. Additionally, current modalities of anti-angiogenic treatment, mainly proteasome inhibitory strategies, are reviewed. Furthermore, proteasome inhibitors, both natural and synthetic, and their anti-angiogenic effects as well as future approaches to anti-angiogenic chemotherapies are also discussed.

Inhibition of prostate cancer cellular proteasome activity by a pyrrolidine dithiocarbamate-copper complex is associated with suppression of proliferation and induction of apoptosis.

Recent research suggests that copper could be used as a novel selective target for cancer therapies. Copper is a co-factor essential for tumor angiogenesis processes and high levels of copper have been found in many types of human cancers, including prostate, breast and brain. We have reported that organic copper-containing compounds, such as 8-hydroxyquinoline-copper(II), are a novel class of proteasome inhibitors and tumor cell apoptosis inducers (Daniel et al., Biochem Pharmacol. 2004;67:1139-51). Most recently, we have found that when complexed with copper, the known antioxidant pyrrolidine dithiocarbamate (PDTC) forms a potent proteasome inhibitor in human breast cancer, but not normal cells (Daniel, Chen, et al., submitted). In the current study, we investigate whether the PDTC-copper complex can play similar roles in inhibiting the proteasomal activity and consequently inducing apoptosis in human prostate cancer cells. We used tetrathiomolybdate (TM), a strong copper chelator currently being tested in clinical trials, as a control. We report here that after binding to copper, PDTC, but not TM, can inhibit the proteasomal chymotrypsin-like activity, suppress proliferation, induce apoptotic cell death, and inhibit uptake of radiopharmaceutical 2-[18F]Fluoro-2-deoxy-D-glucose in cultured human prostate cancer cells. In contrast, PDTC, TM or copper alone or a TM-copper mixture had no such effects. Our study suggests that high copper levels in human prostate cancer in vivo can be targeted by a ligand such as PDTC, resulting in formation of an active proteasome inhibitor and apoptosis inducer specifically in prostate tumor, but not normal cells.

Dietary flavonoids as proteasome inhibitors and apoptosis inducers in human leukemia cells.

It has been shown that proteasome activity is required for cancer cell survival and consumption of fruits and vegetables is associated with decreased cancer risk. Previously, we reported that grape extract could inhibit proteasome activity and induce apoptosis in tumor cells. In this study, we examined the flavonoids apigenin, quercetin, kaempferol and myricetin for their proteasome-inhibitory and apoptosis-inducing abilities in human tumor cells. We report that apigenin and quercetin are much more potent than kaempferol and myricetin at: (i) inhibiting chymotrypsin-like activity of purified 20S proteasome and of 26S proteasome in intact leukemia Jurkat T cells; (ii) accumulating putative ubiquitinated forms of two proteasome target proteins, Bax and Inhibitor of nuclear factor kappabeta-alpha in Jurkat T cells and (iii) inducing activation of caspase-3 and cleavage of poly(ADP-ribose) polymerase in Jurkat T cells. The proteasome-inhibitory abilities of these compounds correlated with their apoptosis-inducing potencies. Results from computational modeling of the potential interactions of these flavonoids to the chymotrypsin site (beta5 subunit) of the proteasome were consistent with the obtained proteasome-inhibitory activities. We found that the C(4) carbon may be a site of nucleophilic attack by the OH group of N-terminal threonine of proteasomal beta5 subunit and that the C(3) hydroxyl may alter the ability of these flavonoids to inhibit the proteasome. Finally, apigenin neither effectively inhibited the proteasome activity nor induced apoptosis in non-transformed human natural killer cells. Our results suggested that the proteasome may be a target of these dietary flavonoids in human tumor cells and that inhibition of the proteasome by flavonoids may be one of the mechanisms responsible for their cancer-preventive effects.

Synthetic peracetate tea polyphenols as potent proteasome inhibitors and apoptosis inducers in human cancer cells.

It has been suggested that proteasome activity is essential for tumor cell proliferation and drug resistance development. We have previously shown that natural and synthetic ester bond-containing tea polyphenols are selective inhibitors of the chymotrypsin-like activity of the proteasome. The most abundant catechin in green tea is (-)-epigallocatechin-3-gallate [(-)-EGCG], which has been found by many laboratories to exhibit the most potent anticancer activity. We have reported that (-)-EGCG is also the most effective proteasome inhibitor among all the natural green tea catechins tested. Unfortunately, (-)-EGCG is very unstable in neutral and alkaline conditions. In an attempt to increase the stability and thus the efficacy, we synthesized several (-)-EGCG analogs with acetyl protected -OH groups as prodrugs. Here we report, for the first time, that these acetylated synthetic tea analogs are much more potent than natural (-)-EGCG in inhibiting the proteasome in cultured tumor cells. Consistently, these protected analogs showed much higher potency than (-)-EGCG to inhibit proliferation and transforming activity and to induce apoptosis in human leukemic, prostate, breast, and simian virus 40-transformed cells. Additionally, these protected analogs had greatly reduced effects on human normal and non-transformed cells. Therefore, these peracetate protected tea polyphenols are more efficacious than (-)-EGCG and possess great potential to be developed into novel anticancer drugs. Identification of the cytosolic metabolite(s) of peracetate-protected polyphenols in cultured tumor cells and examination of their in vivo tumor growth-inhibitory activity are currently underway in our laboratory.

Structure-activity relationships OF N-methylthiolated beta-lactam antibiotics with C3 substitutions and their selective induction of apoptosis in human cancer cells.

The development of novel anti-cancer drugs that induce apoptosis has long been a focus of drug discovery. Beta-lactam antibiotics have been used for over 60 years to fight bacterial infectious diseases with little or no side effects observed. Recently a new class of N-methylthiolated beta-lactams has been discovered that have potent activity against methicillin resistant Staphylococcus aureas. Most recently, we determined the potential effects of these N-thiolated beta-lactams on tumorigenic cell growth and found that they are apoptosis-inducers in human cancer cell lines. In the current study, we further determined the effects of the substitution of the O-methyl moiety on C3 and stereochemistry of the beta-lactams on the anti-proliferative and apoptosis-inducing abilities. We have found that lactam 18, in which C3 is substituted with an acrylate ester group, is a very effective proliferation inhibitor against human premalignant and malignant breast, leukemic, and simian virus 40-transformed fibroblast cells. Generally speaking, increasing the size of the moiety on C3 decreases its anti-proliferation potency, possibly indicating steric hindrance with the cellular target or decreased permeability through the cell membrane. We also found that the stereochemistry of the beta-lactams plays an important role in their potency. The 3S,4R isomers are more effective than their enantiomers (3R,4S), suggesting that 3S,4R configuration is more favorable for target interaction.

Green tea and tea polyphenols in cancer prevention.

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 in the recent decade. 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 affect several signal transduction pathways, including growth factor-mediated, the mitogen-activated protein kinase (MAPK)-dependent, and ubiquitin/proteasome degradation pathways. Epidemiological studies have suggested that the consumption of green tea lowers the risk of cancer. Various animal studies have revealed that treatment by green tea inhibits tumor incidence and multiplicity in different organ sites such as skin, lung, liver, stomach, mammary gland and colon. Phase I and II clinical trials were carried out recently to explore the anticancer effects of green tea in patients with cancer. At this time, more mechanistic research, animal studies, and clinical trials are necessary to further evaluate the role of green tea in cancer prevention.