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.

Site-directed non-covalent polymer-drug complexes for inflammatory bowel disease (IBD): Formulation development, characterization and pharmacological evaluation.

Inflammatory Bowel Diseases (IBD) is a debilitating condition that affects ~70,000 new people every year and has been described as "an expensive disease with no known cure". In addition, IBD increases the risk of developing colon cancer. The current therapeutics for IBD focus on the established disease where the immune dysfunction and bowel damage have already occurred but do not prevent or delay the progression. The current work describes a polymer-based anti-inflammatory technology (Ora-Curcumin-S) specifically targeted to the luminal side of the colon for preventing and/or treating IBD. Ora-Curcumin-S was prepared by molecular complexation of curcumin with a hydrophilic polymer Eudragit® S100 using co-precipitation method. Curcumin interacted with the polymer non-covalently and existed in an amorphous state as demonstrated by various physicochemical techniques. Ora-Curcumin-S is a polymer-drug complex, which is different than solid dispersions in that the interactions are retained even after dissolving in aqueous buffers. Ora-Curcumin-S was >1000 times water soluble than curcumin and importantly, the enhanced solubility was pH-dependent, which was observed only at pHs above 6.8. In addition, around 90% of Ora-Curcumin-S was stable in phosphate buffer, pH 7.4 and simulated intestinal fluid after 24 h, in contrast to 10-20% unformulated curcumin. Ora-Curcumin-S inhibited Monophosphoryl Lipid-A and E. coli induced inflammatory responses in dendritic cells and cells over expressing Toll-Like Receptor-4 (TLR-4) suggesting that Ora-Curcumin-S is a novel polymer-based TLR-4 antagonist. Preliminary pharmacokinetics in mice showed targeted delivery of soluble curcumin to the colon lumen without exposing to the systemic circulation. Furthermore, Ora-Curcumin-S significantly prevented colitis and associated injury in a mouse model of ulcerative colitis estimated using multiple preclinical parameters: colonoscopy pictures, body weight, colon length, colon edema, spleen weight, pro-inflammatory signaling and independent pathological scoring. Overall, the outcome of this innovative proof-of-concept study provides an exciting and locally-targeted pathway for a dietary therapeutic option for IBD patients to help limit colonic inflammation and thus susceptibility to colitis-associated colorectal cancer.

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.

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 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.