Screening of Human Gut Bacterial Culture Collection Identifies Species That Biotransform Quercetin into Metabolites with Anticancer Properties.

We previously demonstrated that flavonoid metabolites inhibit cancer cell proliferation through both CDK-dependent and -independent mechanisms. The existing evidence suggests that gut microbiota is capable of flavonoid biotransformation to generate bioactive metabolites including 2,4,6-trihydroxybenzoic acid (2,4,6-THBA), 3,4-dihydroxybenzoic acid (3,4-DHBA), 3,4,5-trihyroxybenzoic acid (3,4,5-THBA) and 3,4-dihydroxyphenylacetic acid (DOPAC). In this study, we screened 94 human gut bacterial species for their ability to biotransform flavonoid quercetin into different metabolites. We demonstrated that five of these species were able to degrade quercetin including Bacillus glycinifermentans, Flavonifractor plautii, Bacteroides eggerthii, Olsenella scatoligenes and Eubacterium eligens. Additional studies showed that B. glycinifermentans could generate 2,4,6-THBA and 3,4-DHBA from quercetin while F. plautii generates DOPAC. In addition to the differences in the metabolites produced, we also observed that the kinetics of quercetin degradation was different between B. glycinifermentans and F. plautii, suggesting that the pathways of degradation are likely different between these strains. Similar to the antiproliferative effects of 2,4,6-THBA and 3,4-DHBA demonstrated previously, DOPAC also inhibited colony formation ex vivo in the HCT-116 colon cancer cell line. Consistent with this, the bacterial culture supernatant of F. plautii also inhibited colony formation in this cell line. Thus, as F. plautii and B. glycinifermentans generate metabolites possessing antiproliferative activity, we suggest that these strains have the potential to be developed into probiotics to improve human gut health.

Mechanisms of Colorectal Cancer Prevention by Aspirin-A Literature Review and Perspective on the Role of COX-Dependent and -Independent Pathways.

Aspirin, synthesized and marketed in 1897 by Bayer, is one of the most widely used drugs in the world. It has a well-recognized role in decreasing inflammation, pain and fever, and in the prevention of thrombotic cardiovascular diseases. Its anti-inflammatory and cardio-protective actions have been well studied and occur through inhibition of cyclooxygenases (COX). Interestingly, a vast amount of epidemiological, preclinical and clinical studies have revealed aspirin as a promising chemopreventive agent, particularly against colorectal cancers (CRC); however, the primary mechanism by which it decreases the occurrences of CRC has still not been established. Numerous mechanisms have been proposed for aspirin's chemopreventive properties among which the inhibition of COX enzymes has been widely discussed. Despite the wide attention COX-inhibition has received as the most probable mechanism of cancer prevention by aspirin, it is clear that aspirin targets many other proteins and pathways, suggesting that these extra-COX targets may also be equally important in preventing CRC. In this review, we discuss the COX-dependent and -independent pathways described in literature for aspirin's anti-cancer effects and highlight the strengths and limitations of the proposed mechanisms. Additionally, we emphasize the potential role of the metabolites of aspirin and salicylic acid (generated in the gut through microbial biotransformation) in contributing to aspirin's chemopreventive actions. We suggest that the preferential chemopreventive effect of aspirin against CRC may be related to direct exposure of aspirin/salicylic acid or its metabolites to the colorectal tissues. Future investigations should shed light on the role of aspirin, its metabolites and the role of the gut microbiota in cancer prevention against CRC.

Do Aspirin and Flavonoids Prevent Cancer through a Common Mechanism Involving Hydroxybenzoic Acids?-The Metabolite Hypothesis.

Despite decades of research to elucidate the cancer preventive mechanisms of aspirin and flavonoids, a consensus has not been reached on their specific modes of action. This inability to accurately pinpoint the mechanism involved is due to the failure to differentiate the primary targets from its associated downstream responses. This review is written in the context of the recent findings on the potential pathways involved in the prevention of colorectal cancers (CRC) by aspirin and flavonoids. Recent reports have demonstrated that the aspirin metabolites 2,3-dihydroxybenzoic acid (2,3-DHBA), 2,5-dihydroxybenzoic acid (2,5-DHBA) and the flavonoid metabolites 2,4,6-trihydroxybenzoic acid (2,4,6-THBA), 3,4-dihydroxybenzoic acid (3,4-DHBA) and 3,4,5-trihydroxybenzoic acid (3,4,5-THBA) were effective in inhibiting cancer cell growth in vitro. Limited in vivo studies also provide evidence that some of these hydroxybenzoic acids (HBAs) inhibit tumor growth in animal models. This raises the possibility that a common pathway involving HBAs may be responsible for the observed cancer preventive actions of aspirin and flavonoids. Since substantial amounts of aspirin and flavonoids are left unabsorbed in the intestinal lumen upon oral consumption, they may be subjected to degradation by the host and bacterial enzymes, generating simpler phenolic acids contributing to the prevention of CRC. Interestingly, these HBAs are also abundantly present in fruits and vegetables. Therefore, we suggest that the HBAs produced through microbial degradation of aspirin and flavonoids or those consumed through the diet may be common mediators of CRC prevention.

Formulation and Nanotechnology-Based Approaches for Solubility and Bioavailability Enhancement of Zerumbone.

About 40-70% of drug molecules in the clinical development pipeline suffer from one of either low aqueous solubility, poor absorption, or extremely low bioavailability. Approximately 75% of the world population relies on traditional therapies and therefore there has been a growing interest in the utilization of natural compounds. Zerumbone is one such natural compound, classified as a sesquiterpenoid that is extracted from the essential volatile oils of rhizomes from Zingiber zerumbet. It possesses strong antitumor, antioxidant, antimicrobial, and anti-inflammatory activity. However, despite promising preclinical studies demonstrating the therapeutic utility of zerumbone, its clinical development has been limited due to its low aqueous solubility, poor absorption, or associated low bioavailability. Multiple reviews demonstrating the pharmacological effects of zerumbone for various diseases have been published. However, to our knowledge, no review demonstrates the various formulation strategies developed to overcome the biopharmaceutical challenges of zerumbone. The purpose of this review is to provide a comprehensive perspective on zerumbone as a molecule for formulation development. A section related to pharmacokinetics, toxicity, and patents of zerumbone is included. This review provides the importance of developing novel formulations of zerumbone to overcome its biopharmaceutical challenges thereby advance its potential in the treatment of various diseases.

Aspirin and salicylic acid decrease c-Myc expression in cancer cells: a potential role in chemoprevention.

Epidemiological studies have demonstrated a significant correlation between regular aspirin use and reduced colon cancer incidence and mortality; however, the pathways by which it exerts its anti-cancer effects are still not fully explored. We hypothesized that aspirin's anti-cancer effect may occur through downregulation of c-Myc gene expression. Here, we demonstrate that aspirin and its primary metabolite, salicylic acid, decrease the c-Myc protein levels in human HCT-116 colon and in few other cancer cell lines. In total cell lysates, both drugs decreased the levels of c-Myc in a concentration-dependent fashion. Greater inhibition was observed in the nucleus than the cytoplasm, and immunofluorescence studies confirmed these observations. Pretreatment of cells with lactacystin, a proteasome inhibitor, partially prevented the downregulatory effect of both aspirin and salicylic acid, suggesting that 26S proteasomal pathway is involved. Both drugs failed to decrease exogenously expressed DDK-tagged c-Myc protein levels; however, under the same conditions, the endogenous c-Myc protein levels were downregulated. Northern blot analysis showed that both drugs caused a decrease in c-Myc mRNA levels in a concentration-dependent fashion. High-performance liquid chromatography (HPLC) analysis showed that aspirin taken up by cells was rapidly metabolized to salicylic acid, suggesting that aspirin's inhibitory effect on c-Myc may occur through formation of salicylic acid. Our result suggests that salicylic acid regulates c-Myc level at both transcriptional and post-transcription levels. Inhibition of c-Myc may represent an important pathway by which aspirin exerts its anti-cancer effect and decrease the occurrence of cancer in epithelial tissues.

Aspirin acetylates wild type and mutant p53 in colon cancer cells: identification of aspirin acetylated sites on recombinant p53.

Aspirin's ability to inhibit cell proliferation and induce apoptosis in cancer cell lines is considered to be an important mechanism for its anti-cancer effects. We previously demonstrated that aspirin acetylated the tumor suppressor protein p53 at lysine 382 in MDA-MB-231 human breast cancer cells. Here, we extended these observations to human colon cancer cells, HCT 116 harboring wild type p53, and HT-29 containing mutant p53. We demonstrate that aspirin induced acetylation of p53 in both cell lines in a concentration-dependent manner. Aspirin-acetylated p53 was localized to the nucleus. In both cell lines, aspirin induced p21(CIP1). Aspirin also acetylated recombinant p53 (rp53) in vitro suggesting that it occurs through a non-enzymatic chemical reaction. Mass spectrometry analysis and immunoblotting identified 10 acetylated lysines on rp53, and molecular modeling showed that all lysines targeted by aspirin are surface exposed. Five of these lysines are localized to the DNA-binding domain, four to the nuclear localization signal domain, and one to the C-terminal regulatory domain. Our results suggest that aspirin's anti-cancer effect may involve acetylation and activation of wild type and mutant p53 and induction of target gene expression. This is the first report attempting to characterize p53 acetylation sites targeted by aspirin.

Efficacy of acetylsalicylic acid (aspirin) in skin B16-F0 melanoma tumor-bearing C57BL/6 mice.

Several epidemiological studies show that aspirin can act as a chemopreventive agent and decrease the incidences of various cancers including melanoma. In this work, we investigated the in vitro and in vivo efficacy of acetylsalicylic acid (ASA) as an antimelanoma agent in B16-F0 cells and skin B16-F0 melanoma tumor mouse model. Our findings indicate that the IC50 (48 h) for ASA in B16-F0 melanoma cells was 100 µM and that ASA caused a dose- and time-dependent GSH depletion and increase in reactive oxygen species (ROS) formation in B16-F0 melanoma cells. Male C57BL/6 mice were inoculated s.c. with 1 × 10(6) B16-F0 melanoma cells. ASA (80, 100, and 150 mg/kg) was initiated on day 1 or day 7, or day 9 after cell inoculation and continued daily for 13, 7, and 5 days, respectively. Animals were weighed daily and sacrificed on day 13. The tumors were excised and weighed. The animals receiving 13 days of ASA therapy at 80, 100, and 150 mg/kg demonstrated tumor growth inhibition by 1 ± 12%, 19 ± 22%, and 50 ± 29%, respectively. Animals receiving 7 days of therapy at 80, 100, and 150 mg/kg demonstrated tumor growth inhibition by 12 ± 14%, 27 ± 14%, and 40 ± 14%, respectively. No significant tumor growth inhibition was observed with 5 days of therapy. ASA at 100 and 150 mg/kg caused significant tumor growth inhibition in C57BL/6 mice when administered for 13 and 7 days, respectively. The results obtained in this study are consistent with the recent epidemiologically based report that aspirin is associated with lower melanoma risk in humans.

Positive Synergistic Effects of Quercetin and Rice Bran on Human Gut Microbiota Reduces Enterobacteriaceae Family Abundance and Elevates Propionate in a Bioreactor Model.

Dietary fiber and flavonoids have substantial influence on the human gut microbiota composition that significantly impact health. Recent studies with dietary supplements such as quercetin and rice bran have shown beneficial impacts on the host alongside a positive influence of the gut microbiota. The specific bacterial species impacted by quercetin or rice bran in the diet is not well understood. In this study, we used a minibioreactor array system as a model to determine the effect of quercetin and rice bran individually, as well as in combination, on gut microbiota without the confounding host factors. We found that rice bran exerts higher shift in gut microbiome composition when compared to quercetin. At the species level, Acidaminococcus intestini was the only significantly enriched taxa when quercetin was supplemented, while 15 species were enriched in rice bran supplementation and 13 were enriched when quercetin and rice bran were supplemented in combination. When comparing the short chain fatty acid production, quercetin supplementation increased isobutyrate production while propionate dominated the quercetin and rice bran combined group. Higher levels of propionate were highly correlated to the lower abundance of the potentially pathogenic Enterobacteriaceae family. These findings suggest that the combination of quercetin and rice bran serve to enrich beneficial bacteria and reduce potential opportunistic pathogens. In vivo studies are necessary to determine how this synergy of quercetin and rice bran on microbiota impact host health.

Aspirin metabolites 2,3­DHBA and 2,5­DHBA inhibit cancer cell growth: Implications in colorectal cancer prevention.

Although compelling evidence exists on the ability of aspirin to treat colorectal cancer (CRC), and numerous theories and targets have been proposed, a consensus has not been reached regarding its mechanism of action. In this regard, a relatively unexplored area is the role played by aspirin metabolites 2,3­dihydroxybenzoic acid (2,3­DHBA) and 2,5­dihydroxybenzoic acid (2,5­DHBA) in its chemopreventive actions. In a previous study, we demonstrated that 2,3­DHBA and 2,5­DHBA inhibited CDK1 enzyme activity in vitro. The aim of the present study was to understand the effect of these metabolites on the enzyme activity of all CDKs involved in cell cycle regulation (CDKs 1, 2, 4 and 6) as well as their effect on clonal formation in three different cancer cell lines. Additionally, in silico studies were performed to determine the potential sites of interactions of 2,3­DHBA and 2,5­DHBA with CDKs. We demonstrated that 2,3­DHBA and 2,5­DHBA inhibits CDK­1 enzyme activity beginning at 500 µM, while CDK2 and CDK4 activity was inhibited only at higher concentrations (>750 µM). 2,3­DHBA inhibited CDK6 enzyme activity from 250 µM, while 2,5­DHBA inhibited its activity >750 µM. Colony formation assays showed that 2,5­DHBA was highly effective in inhibiting clonal formation in HCT­116 and HT­29 CRC cell lines (250­500 µM), and in the MDA­MB­231 breast cancer cell line (~100 µM). In contrast 2,3­DHBA was effective only in MDA­MB­231 cells (~500 µM). Both aspirin and salicylic acid failed to inhibit all four CDKs and colony formation. Based on the present results, it is suggested that 2,3­DHBA and 2,5­DHBA may contribute to the chemopreventive properties of aspirin, possibly through the inhibition of CDKs. The present data and the proposed mechanisms should open new areas for future investigations.

A functional role for sodium-dependent glucose transport across the blood-brain barrier during oxygen glucose deprivation.

In the current study, we determined the functional significance of sodium-dependent/-independent glucose transporters at the neurovasculature during oxygen glucose deprivation (OGD). Confluent brain endothelial cells cocultured with astrocytes were exposed to varying degrees of in vitro stroke conditions. Glucose transporter (GLUT) 1 and sodium glucose cotransporter (SGLT) activity were investigated by luminal membrane uptake and transport studies using [(3)H]D-glucose and also by [(14)C]alpha-methyl D-glucopyranoside (AMG), a specific, nonmetabolized substrate of SGLT. In vivo middle cerebral artery occlusion experiments were tested to determine whether blood-brain barrier (BBB) SGLT activity was induced during ischemia. Increases in luminal D-glucose and AMG uptake and transport were observed with in vitro stroke conditions. Specific inhibitor experiments suggest a combined role for both SGLT and GLUT1 at the BBB during OGD. A time-dependent increase in the uptake of AMG was also seen in mice exposed to permanent focal ischemia, and this increase was sensitive to the SGLT inhibitor, phlorizin. Infarct and edema ratio during ischemia were significantly decreased by the inhibition of this transporter. These results show that both GLUT1 and SGLT play a role at the BBB in the blood-to-brain transport of glucose during ischemic conditions, and inhibition of SGLT during stroke has the potential to improve stroke outcome. Pharmacological modulation of this novel BBB transporter could prove to be a brain vascular target in stroke.

Aspirin inhibits camptothecin-induced p21CIP1 levels and potentiates apoptosis in human breast cancer cells.

The ability of aspirin to trigger apoptosis in cancer cells is well known and is consistent with the clinical and epidemiological evidence on its chemopreventive effects in curtailing epithelial cancers, including breast cancer. We hypothesized that the anticancer effects of aspirin may involve acetylation of the tumor suppressor protein p53, a known regulator of apoptosis. In the present study, we determined if aspirin at the physiologically achievable concentration of 100 microM acetylates p53 and modulates the expression of p21CIP1, a protein involved in cell cycle arrest, and Bax, a pro-apoptotic protein. Using MDA-MB-231 human breast cancer cells, we demonstrate that aspirin at 100 microM concentration markedly acetylated the p53 protein, which was primarily localized to the nucleus. Aspirin induced p21CIP1 protein levels in a transient fashion in contrast to the sustained induction of Bax. The induction of p21CIP1 protein levels began at 3 h and was maximal at 6-8 h; however, it decreased to control levels by 30 h. In contrast, the anticancer drug, camptothecin (CPT) induced a steady accumulation of p21CIP1 protein. Remarkably, when cells were co-treated with aspirin and CPT, p21CIP1 levels were drastically downregulated, and this phenomenon was observed in many cancer cell lines. Incubation of recombinant p21 with cytoplasmic extracts from aspirin-treated cells caused its degradation suggesting the involvement of proteases in the disappearance of p21CIP1. Consistent with this data, aspirin decreased the survival of CPT-treated cells and greatly increased the extent of apoptosis. Our observation that aspirin has the ability to inhibit p21CIP1 after its initial induction has important implications in chemotherapy, and suggests its potential use to increase the efficacy of anticancer agents.

The Flavonoid Metabolite 2,4,6-Trihydroxybenzoic Acid Is a CDK Inhibitor and an Anti-Proliferative Agent: A Potential Role in Cancer Prevention.

Flavonoids have emerged as promising compounds capable of preventing colorectal cancer (CRC) due to their anti-oxidant and anti-inflammatory properties. It is hypothesized that the metabolites of flavonoids are primarily responsible for the observed anti-cancer effects owing to the unstable nature of the parent compounds and their degradation by colonic microflora. In this study, we investigated the ability of one metabolite, 2,4,6-trihydroxybenzoic acid (2,4,6-THBA) to inhibit Cyclin Dependent Kinase (CDK) activity and cancer cell proliferation. Using in vitro kinase assays, we demonstrated that 2,4,6-THBA dose-dependently inhibited CDKs 1, 2 and 4 and in silico studies identified key amino acids involved in these interactions. Interestingly, no significant CDK inhibition was observed with the structurally related compounds 3,4,5-trihydroxybenzoic acid (3,4,5-THBA) and phloroglucinol, suggesting that orientation of the functional groups and specific amino acid interactions may play a role in inhibition. We showed that cellular uptake of 2,4,6-THBA required the expression of functional SLC5A8, a monocarboxylic acid transporter. Consistent with this, in cells expressing functional SLC5A8, 2,4,6-THBA induced CDK inhibitory proteins p21Cip1 and p27Kip1 and inhibited cell proliferation. These findings, for the first time, suggest that the flavonoid metabolite 2,4,6-THBA may mediate its effects through a CDK- and SLC5A8-dependent pathway contributing to the prevention of CRC.

Salicylic acid metabolites and derivatives inhibit CDK activity: Novel insights into aspirin's chemopreventive effects against colorectal cancer.

Aspirin's potential as a drug continues to be evaluated for the prevention of colorectal cancer (CRC). Although multiple targets for aspirin and its metabolite, salicylic acid, have been identified, no unifying mechanism has been proposed to clearly explain its chemopreventive effects. Our goal here was to investigate the ability of salicylic acid metabolites, known to be generated through cytochrome P450 (CYP450) enzymes, and its derivatives as cyclin dependent kinase (CDK) inhibitors to gain new insights into aspirin's chemopreventive actions. Using in vitro kinase assays, for the first time, we demonstrate that salicylic acid metabolites, 2,3-dihydroxybenzoic acid (2,3-DHBA) and 2,5-dihydroxybenzoic acid (2,5-DHBA), as well as derivatives 2,4-dihydroxybenzoic acid (2,4-DHBA), 2,6-dihydroxybenzoic acid (2,6-DHBA), inhibited CDK1 enzyme activity. 2,3-DHBA and 2,6-DHBA did not inhibit CDK2 and 4; however, both inhibited CDK-6 activity. Interestingly, another derivative, 2,4,6-trihydroxybenzoic acid (2,4,6-THBA) was highly effective in inhibiting CDK1, 2, 4 and 6 activity. Molecular docking studies showed that these compounds potentially interact with CDK1. Immunoblotting experiments showed that aspirin acetylated CDK1, and pre-incubation with salicylic acid and its derivatives prevented aspirin-mediated CDK1 acetylation, which supported the data obtained from molecular docking studies. We suggest that intracellularly generated salicylic acid metabolites through CYP450 enzymes within the colonic epithelial cells, or the salicylic acid metabolites generated by gut microflora may significantly contribute to the preferential chemopreventive effect of aspirin against CRC through inhibition of CDKs. This novel hypothesis and mechanism of action in aspirin's chemopreventive effects opens a new area for future research. In addition, structural modification to salicylic acid derivatives may prove useful in the development of novel CDK inhibitors in cancer prevention and treatment.

In Vitro and In Vivo Antimetastatic Effect of Glutathione Disulfide Liposomes.

Cancer metastasis is the major cause of cancer mortality. Despite extensive research efforts, effective treatment for cancer metastasis is still lacking. Cancer metastasis involves 4 essential steps: cell detachment, migration, invasion, and adhesion. Detachment is the first and required step for metastasis. Glutathione disulfide (GSSG) is derived from the oxidation of glutathione (GSH), which is present in biological systems in millimolar concentration. Although GSSG is commercially available, the impact of GSSG on cell functions/dysfunctions has not been fully explored due to the fact that GSSG is not cell membrane permeable and a lack of method to specifically increase GSSG in cells. We have developed GSSG liposomes that effectively deliver GSSG to cells. Unexpectedly, cells treated with GSSG liposomes were resistant to detachment by trypsinization. This observation led to the investigation of the antimetastatic effect of GSSG liposomes. Our data demonstrate that GSSG liposomes at 1 mg/mL completely blocked cell detachment and migration, and significantly inhibited cancer cell invasion. Aqueous GSSG showed no such effect, confirming that the effects on cell detachment, migration, and invasion were caused by the intracellular delivery of GSSG. An in vivo experiment with a murine melanoma experimental metastasis model showed that GSSG liposomes prevented melanoma lung metastasis. The unique antimetastatic mechanism through the effects on detachment and migration, and effective in vitro and in vivo metastasis inhibition, warrants further investigation of the GSSG liposomes as a potential treatment for cancer metastasis.

In Vitro and In Vivo Tumor Growth Inhibition by Glutathione Disulfide Liposomes.

Glutathione disulfide (GSSG) is an endogenous peptide and the oxidized form of glutathione. The impacts of GSSG on cell function/dysfunction remain largely unexplored due to a lack of method to specifically increase intracellular GSSG. We recently developed GSSG liposomes that can specifically increase intracellular GSSG. The increase affected 3 of the 4 essential steps (cell detachment, migration, invasion, and adhesion) of cancer metastasis in vitro and, accordingly, produced a significant inhibition of cancer metastasis in vivo. In this investigation, the effect of GSSG liposomes on cancer growth was investigated with B16-F10 and NCI-H226 cells in vitro and with B16-F10 cells in C57BL/6 mice in vivo. Experiments were conducted to elucidate the effect on cell death through promotion of apoptosis and the effect on the cell cycle. The in vivo results with C57BL/6 mice implanted subcutaneously with B16-F10 cells showed that GSSG liposomes retarded tumor proliferation more effectively than that of dacarbazine, a chemotherapeutic drug for the treatment of melanoma. The GSSG liposomes by intravenous injection (GLS IV) and GSSG liposomes by intratumoral injection (GLS IT) showed a tumor proliferation retardation of 85% ± 5.7% and 90% ± 3.9%, respectively, compared with the phosphate-buffered saline (PBS) control group. The median survival rates for mice treated with PBS, blank liposomes, aqueous GSSG, dacarbazine, GLS IV, and GLS IT were 7, 7, 7.5, 7.75, 11.5, and 16.5 days, respectively. The effective antimetastatic and antigrowth activities warrant further investigation of the GSSG liposomes as a potentially effective therapeutic treatment for cancer.

Aspirin inhibits glucose­6­phosphate dehydrogenase activity in HCT 116 cells through acetylation: Identification of aspirin-acetylated sites.

Glucose-6-phosphate dehydrogenase (G6PD) catalyzes the first reaction in the pentose phosphate pathway, and generates ribose sugars, which are required for nucleic acid synthesis, and nicotinamide adenine dinucleotide phosphate (NADPH), which is important for neutralization of oxidative stress. The expression of G6PD is elevated in several types of tumor, including colon, breast and lung cancer, and has been implicated in cancer cell growth. Our previous study demonstrated that exposure of HCT 116 human colorectal cancer cells to aspirin caused acetylation of G6PD, and this was associated with a decrease in its enzyme activity. In the present study, this observation was expanded to HT­29 colorectal cancer cells, in order to compare aspirin­mediated acetylation of G6PD and its activity between HCT 116 and HT­29 cells. In addition, the present study aimed to determine the acetylation targets of aspirin on recombinant G6PD to provide an insight into the mechanisms of inhibition. The results demonstrated that the extent of G6PD acetylation was significantly higher in HCT 116 cells compared with in HT­29 cells; accordingly, a greater reduction in G6PD enzyme activity was observed in the HCT 116 cells. Mass spectrometry analysis of aspirin­acetylated G6PD (isoform a) revealed that aspirin acetylated a total of 14 lysine residues, which were dispersed throughout the length of the G6PD protein. One of the important amino acid targets of aspirin included lysine 235 (K235, in isoform a) and this corresponds to K205 in isoform b, which has previously been identified as being important for catalysis. Acetylation of G6PD at several sites, including K235 (K205 in isoform b), may mediate inhibition of G6PD activity, which may contribute to the ability of aspirin to exert anticancer effects through decreased synthesis of ribose sugars and NADPH.

Molecular complexation of curcumin with pH sensitive cationic copolymer enhances the aqueous solubility, stability and bioavailability of curcumin.

Curcumin is a natural dietary compound with demonstrated potential in preventing/treating several chronic diseases in animal models. However, this success is yet to be translated to humans mainly because of its poor oral bioavailability caused by extremely low water solubility. This manuscript demonstrates that water insoluble curcumin (~1µg/ml) forms highly aqueous soluble complexes (>2mg/ml) with a safe pH sensitive polymer, poly(butyl-methacrylate-co-(2-dimethylaminoethyl) methacrylate-co-methyl-methacrylate) when precipitated together in water. The complexation process was optimized to enhance curcumin loading by varying several formulation factors. Acetone as a solvent and polyvinyl alcohol as a stabilizer with 1:2 ratio of drug to polymer yielded complexes with relatively high loading (~280µg/ml) and enhanced solubility (>2mg/ml). The complexes were amorphous in solid and were soluble only in buffers with pHs less than 5.0. Hydrogen bond formation and hydrophobic interactions between curcumin and the polymer were recorded by infrared spectroscopy and nuclear magnetic resonance spectroscopy, respectively. Molecular complexes of curcumin were more stable at various pHs compared to unformulated curcumin. In mice, these complexes increased peak plasma concentration of curcumin by 6 times and oral bioavailability by ~20 times. This is a simple, economic and safer strategy of enhancing the oral bioavailability of curcumin.

Cyclin A2 and CDK2 as Novel Targets of Aspirin and Salicylic Acid: A Potential Role in Cancer Prevention.

UNLABELLED: Data emerging from the past 10 years have consolidated the rationale for investigating the use of aspirin as a chemopreventive agent; however, the mechanisms leading to its anticancer effects are still being elucidated. We hypothesized that aspirin's chemopreventive actions may involve cell-cycle regulation through modulation of the levels or activity of cyclin A2/cyclin-dependent kinase-2 (CDK2). In this study, HT-29 and other diverse panel of cancer cells were used to demonstrate that both aspirin and its primary metabolite, salicylic acid, decreased cyclin A2 (CCNA2) and CDK2 protein and mRNA levels. The downregulatory effect of either drugs on cyclin A2 levels was prevented by pretreatment with lactacystin, an inhibitor of proteasomes, suggesting the involvement of 26S proteasomes. In-vitro kinase assays showed that lysates from cells treated with salicylic acid had lower levels of CDK2 activity. Importantly, three independent experiments revealed that salicylic acid directly binds to CDK2. First, inclusion of salicylic acid in naïve cell lysates, or in recombinant CDK2 preparations, increased the ability of the anti-CDK2 antibody to immunoprecipitate CDK2, suggesting that salicylic acid may directly bind and alter its conformation. Second, in 8-anilino-1-naphthalene-sulfonate (ANS)-CDK2 fluorescence assays, preincubation of CDK2 with salicylic acid dose-dependently quenched the fluorescence due to ANS. Third, computational analysis using molecular docking studies identified Asp145 and Lys33 as the potential sites of salicylic acid interactions with CDK2. These results demonstrate that aspirin and salicylic acid downregulate cyclin A2/CDK2 proteins in multiple cancer cell lines, suggesting a novel target and mechanism of action in chemoprevention. IMPLICATIONS: Biochemical and structural studies indicate that the antiproliferative actions of aspirin are mediated through cyclin A2/CDK2.

Aspirin acetylates multiple cellular proteins in HCT-116 colon cancer cells: Identification of novel targets.

Epidemiological and clinical observations provide consistent evidence that regular intake of aspirin may effectively inhibit the occurrence of epithelial tumors; however, the molecular mechanisms are not completely understood. In the present study, we determined the ability of aspirin to acetylate and post-translationally modify cellular proteins in HCT-116 human colon cancer cells to understand the potential mechanisms by which it may exerts anti-cancer effects. Using anti-acetyl lysine antibodies, here we demonstrate that aspirin causes the acetylation of multiple proteins whose molecular weight ranged from 20 to 200 kDa. The identity of these proteins was determined, using immuno-affinity purification, mass spectrometry and immuno-blotting. A total of 33 cellular proteins were potential targets of aspirin-mediated acetylation, while 16 were identified as common to both the control and aspirin-treated samples. These include enzymes of glycolytic pathway, cytoskeleton proteins, histones, ribosomal and mitochondrial proteins. The glycolytic enzymes which were identified include aldolase, glyceraldehyde-3-phosphate dehydrogenase, enolase, pyruvate kinase M2, and lactate dehydrogenase A and B chains. Immunoblotting experiment showed that aspirin also acetylated glucose-6-phosphate dehydrogenase and transketolase, both enzymes of pentose phosphate pathway involved in ribonucleotide biosynthesis. In vitro assays of these enzymes revealed that aspirin did not affect pyruvate kinase and lactate dehydrogenase activity; however, it decreased glucose 6 phosphate dehydrogenase activity. Similar results were also observed in HT-29 human colon cancer cells. Selective inhibition of glucose-6-phosphate dehydrogenase may represent an important mechanism by which aspirin may exert its anti-cancer effects through inhibition of ribonucleotide synthesis.

Cys-141 glutathionylation of human p53: Studies using specific polyclonal antibodies in cancer samples and cell lines.

Previously, we reported that human p53 is functionally inactivated by S-glutathionylation at Cys-141 during oxidative and DNA-damaging treatments. Here, we describe the presence of thiolated p53 and the dynamic nature of this modification in human tissues using unique and specific polyclonal antibodies raised against a 12-residue p53 peptide bearing a mixed disulfide at Cys-141. The affinity- purified antibodies (glut-p53) were sequence-specific in that they recognized the antigenic peptide but not the unthiolated peptide or a scrambled glutathionylated peptide in ELISAs. On immunoblots, the purified antibodies did not react with native p53 or recombinant p53 (rp53), but readily detected the glutathionylated or cysteinylated or ethanethiol-treated rp53 only under nonreducing conditions. Untreated HCT116 cells showed low levels of glut-p53, which increased markedly after H(2)O(2), diamide, cisplatin, and doxorubicin treatments. Glut-p53 levels decreased sharply after cells were passed into oxidant-free medium, suggesting efficient dethiolation. The mutant p53 present in HT29 and T47D human cancer cells was also recognized. In vitro, the glut-p53 was rapidly degraded by rabbit reticulocyte lysates. Human prostate and prostate cancer tissues showed an abundant presence of glut-p53 in luminal epithelium, a site well known to generate ROS. Melanoma and colon cancer samples were also positive for glut-p53. The availability of the thiolation-specific antibodies should enhance our knowledge of p53 regulation in redox-perturbed states found in various diseases including cancer.