Daniel Boring

Hydrogen sulfide-releasing NSAIDs inhibit the growth of human cancer cells: a general property and evidence of a tissue type-independent effect.

Hydrogen sulfide-releasing non-steroidal anti-inflammatory drugs (HS-NSAIDs) are an emerging novel class of compounds with significant anti-inflammatory properties. They consist of a traditional NSAID to which an H(2)S-releasing moiety is covalently attached. We examined the effects of four different HS-NSAIDs on the growth properties of eleven different human cancer cell lines of six different tissue origins. Human colon, breast, pancreatic, prostate, lung, and leukemia cancer cell lines were treated with HS-aspirin, -sulindac, -iburofen, -naproxen, and their traditional counterparts. HS-NSAIDs inhibited the growth of all cancer cell lines studied, with potencies of 28- to >3000-fold greater than that of their traditional counterparts. HS-aspirin (HS-ASA) was consistently the most potent. HS-NSAIDs inhibited cell proliferation, induced apoptosis, and caused G(0)/G(1) cell cycle block. Metabolism of HS-ASA by colon cells showed that the acetyl group of ASA was hydrolyzed rapidly, followed by hydrolysis of the ester bond linking the salicylate anion to the H(2)S releasing moiety, producing salicylic acid and ADT-OH from which H(2)S is released. In reconstitution studies, ASA and ADT-OH were individually less active than the intact HS-ASA towards cell growth inhibition. Additionally, the combination of these two components representing a fairly close approximation to the intact HS-ASA, was 95-fold less active than the intact HS-ASA for growth inhibition. Taken together, these results demonstrate that HS-NSAIDs have potential anti-growth activity against a wide variety of human cancer cells.

Hydrogen sulfide-releasing aspirin suppresses NF-κB signaling in estrogen receptor negative breast cancer cells in vitro and in vivo

Hormone-dependent estrogen receptor positive (ER+) breast cancers generally respond well to anti-estrogen therapy. Unfortunately, hormone-independent estrogen receptor negative (ER-) breast cancers are aggressive, respond poorly to current treatments and have a poor prognosis. New approaches and targets are needed for the prevention and treatment of ER- breast cancer. The NF-κB signaling pathway is strongly implicated in ER- tumor genesis, constituting a possible target for treatment. Hydrogen sulfide-releasing aspirin (HS-ASA), a novel and safer derivative of aspirin, has shown promise as an anti-cancer agent. We examined the growth inhibitory effect of HS-ASA via alterations in cell proliferation, cell cycle phase transitions, and apoptosis, using MDA-MB-231 cells as a model of triple negative breast cancer. Tumor xenografts in mice, representing human ER- breast cancer, were evaluated for reduction in tumor size, followed by immunohistochemical analysis for proliferation, apoptosis and expression of NF-κB. HS-ASA suppressed the growth of MDA-MB-231 cells by induction of G(0)/G(1) arrest and apoptosis, down-regulation of NF-κB, reduction of thioredoxin reductase activity, and increased levels reactive oxygen species. Tumor xenografts in mice, were significantly reduced in volume and mass by HS-ASA treatment. The decrease in tumor mass was associated with inhibition of cell proliferation, induction of apoptosis and decrease in NF-κB levels in vivo. HS-ASA has anti-cancer potential against ER- breast cancer and merits further study.

NO-releasing NSAIDs suppress NF-κB signaling in vitro and in vivo through S-nitrosylation

NO-NSAIDs are promising anticancer drugs, comprising an NSAID, an NO-releasing moiety, and a spacer linking them. Although the effect of NO-NSAIDs on a wide variety of signaling and other cellular mechanisms has been deciphered, a key question remains unanswered, that being the role of NO to the overall biological effect of these agents. It has been shown that NO can directly modify sulfhydryl residues of proteins through S-nitrosylation and induce apoptosis. We studied 3 NO-NSAIDs having a different NSAID, spacer, and NO-releasing moiety. In vitro: aspirin, NO-ASA, naproxen, and NO-naproxen inhibited HT-29 human colon cancer cell growth, the IC(50)s being >5000, 192±6, 2800±210 and 95±5μM at 24h, respectively. NO-Aspirin and NO-naproxen reduced NF-κB protein levels, and activated caspase-3 enzyme in a dose- and time-dependent manner. Based on the biotin switch assay, NO-ASA and NO-naproxen S-nitrosylated NF-κB p65 in a time-dependent manner. Pretreatment of the cells with carboxy-PTIO, abrogated the S-nitrosylation of NF-κB p65. In vivo: rats treated with NO-ASA, NONO-ASA, and NO-naproxen showed S-nitrosylation of NF-κB p65 in the stomach tissue, increases in plasma TNF-α, and reductions in mucosal PGE(2) levels. These data provide a mechanistic role for NO and a rational for the chemopreventive effects of NO-NSAIDs.

Lack of Efficacy of the Statins Atorvastatin and Lovastatin in Rodent Mammary Carcinogenesis

The statins are highly effective in lowering cholesterol by inhibiting 3-hydroxy-3-methylglutaryl CoA reductase. Recently, there has been conflicting epidemiologic data indicating that statins decrease the incidence of certain types of cancer, including breast cancer. Atorvastatin and lovastatin, statins with different lipophicilities, were administered in diet either as single agents or in combination with suboptimal doses of tamoxifen or the retinoid X receptor agonist bexarotene were evaluated for prevention of estrogen receptor–positive mammary cancers induced in the rat with methylnitrosourea. Atorvastatin (125 or 500 mg/kg diet) alone did not significantly alter cancer incidence or multiplicity. Suboptimal doses of tamoxifen (0.4 mg/kg diet) or bexarotene (80 mg/kg diet) reduced cancer multiplicity from 3.8 (control) to 2.9 and 0.9, respectively. Combining atorvastatin (500 mg/kg diet) with either of these effective agents minimally altered their efficacy. Although this dose of atorvastatin did not decrease serum triglyceride levels in control rats, it significantly decreased triglyceride levels that had been increased in bexarotene-treated rats. Experiments done with a second statin, lovastatin (100 and 400 mg/kg diet), yielded similar results: (a) limited activity when administered alone, (b) no obvious synergy with bexarotene, and (c) an ability to decrease bexarotene-induced increases in serum triglycerides. Thus, the statins had minimal activity in this model of mammary cancer in which approximately half of the cancers are mutated in the Ha Ras oncogene. Similarly, atorvastatin failed to alter the development of estrogen receptor–negative mammary carcinomas in a new animal model using bitransgenic mice (MMTV-Neu+/−/p53KO+/−), whereas bexarotene (250 mg/kg diet) was effective.

Hydrogen sulfide-releasing aspirin modulates xenobiotic metabolizing enzymes in vitro and in vivo.

The balance between phase-I carcinogen-activating and phase-II detoxifying xenobiotic metabolizing enzymes is critical to determining an individuals risk for cancer. We evaluated the effect of Hydrogen sulfide-releasing aspirin (HS-ASA) on xenobiotic metabolizing enzymes in HT-29 human colon and Hepa 1c1c7 mouse liver adenocarcinoma cells and in Wistar rats. HS-ASA inhibited the growth of HT-29 and Hepa 1c1c7 cells, with an IC(50) of 3.2 ± 0.3 μM and 4.2 ± 0.4 μM, respectively. The IC(50) for ASA in both cell lines was greater than 5000 μM at 24h. In these cell lines, HS-ASA caused a dose-dependent increase in activity and expression of the phase-II enzymes glutathione S-transferase (GST) and NAD(P)H:quinoneoxireductase (NQO1). It also caused an increase in UDP-glucuronosyltransferase (UGT) expression. The levels of CYP 1A1 a phase-I enzyme was increased by HS-ASA in both cell lines. Pretreatment of cells with NaF, an esterase inhibitor, abrogated the HS-ASA-mediated increases in NQO1 enzyme activity. HS-ASA increased the protein levels of the transcription factor Nrf2, which is a regulator of the phase-II enzymes. In vivo, HS-ASA at 100mg/kg/day had no effect on rats weights; it induced a 3.4-fold and 1.4-fold increase in hepatic GST and NQO1 enzyme activities, respectively. GST and NQO1 protein levels were also increased. In contrast to that in cultured cells, CYP 1A1 protein levels were not altered in vivo. Therefore, HS-ASA induces phase-II enzymes, at least in part, through the action of H(2)S and by modulating Nrf2; these effects may be part of its mechanism of action against carcinogenesis.

Preventive Effects of NSAIDs, NO-NSAIDs, and NSAIDs Plus Difluoromethylornithine in a Chemically Induced Urinary Bladder Cancer Model

Urinary bladder cancer prevention studies were performed with the nonsteroidal anti-inflammatory drugs (NSAID) naproxen (a standard NSAID with a good cardiovascular profile), sulindac, and their nitric oxide (NO) derivatives. In addition, the effects of the ornithine decarboxylase inhibitor, difluoromethylornithine (DFMO), alone or combined with a suboptimal dose of naproxen or sulindac was examined. Agents were evaluated at their human equivalent doses (HED), as well as at lower doses. In the hydroxybutyl(butyl)nitrosamine (OH-BBN) model of urinary bladder cancer, naproxen (400 or 75 ppm) and sulindac (400 ppm) reduced the incidence of large bladder cancers by 82%, 68%, and 44%, respectively, when the agents were initially given 3 months after the final dose of the carcinogen; microscopic cancers already existed. NO-naproxen was highly effective, whereas NO-sulindac was inactive. To further compare naproxen and NO-naproxen, we examined their effects on gene expression in rat livers following a 7-day exposure. Limited, but similar, gene expression changes in the liver were induced by both agents, implying that the primary effects of both are mediated by the parent NSAID. When agents were initiated 2 weeks after the last administration of OH-BBN, DFMO at 1,000 ppm had limited activity, a low dose of naproxen (75 ppm) and sulindac (150 ppm) were highly and marginally effective. Combining DFMO with suboptimal doses of naproxen had minimal effects, whereas the combination of DMFO and sulindac was more active than either agent alone. Thus, naproxen and NO-naproxen were highly effective, whereas sulindac was moderately effective in the OH-BBN model at their HEDs. Cancer Prev Res; 7(2); 246–54. ©2013 AACR.

Abstract 4608: Hydrogen sulfide-releasing aspirin inhibits the growth of leukemic Jurkat cells and modulates β-catenin expression

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Introduction: Acute leukemia is the primary cause of cancer-related mortality in children. s -catenin, a regulator of cell-cell adhesion and transcription, is expressed in T-acute lymphoblastic leukemia cells, tumor lines of hematopoietic origin and primary leukemia cells but not in normal peripheral blood T cells. s -catenin may promote leukemia cell proliferation, adhesion, and survival. Hydrogen sulfide-releasing aspirin (HS-ASA) is a novel compound with significant potential for the control of cancer. It consists of a traditional ASA molecule covalently bound to an H2S-releasing moiety (5-(4-hydroxyphenyl)-3H-1,2-dithiole-3-thione). Here we examined the effect of HS-ASA on the growth of Jurkat T-acute lymphoblastic leukemia cells and on the fate of s -catenin. Methods: HS-ASA was synthesized and purified at our lab with 1H-NMR verification. Cell line: Jurkat T-leukemia. Quantification of cell growth: MTT assay. Cell kinetics: cell cycle phase distribution by flow cytometry; apoptosis by subdiploid (sub-G0/G1) peak in DNA content histograms; and proliferation by PCNA. s-Catenin and caspase-3 expression was assayed by immunoblotting in cells treated with HS-ASA at 24 hr. Caspase-3 enzyme activity, and H2S levels was also measured. Results: 1) The IC50 for HS-ASA was 1.5 ± 0.2 µM, whereas that of ASA was >5000 µM strongly suggesting that HS-ASA is at least 3000-fold more potent than traditional ASA. 2) At 0.5xIC50, 1xIC50, and 2xIC50 the following observations were made: Proliferation was inhibited by 88 ± 2%, 63 ± 2%, 42 ± 3%; apoptosis was increased by 15 ± 2%, 39 ± 3%, 54 ± 4%; and cells in the G0/G1 phase increased dose-dependently whereas those in the S and G2/M phases decreased, suggesting a G0/G1 block. 3) HS-ASA dose-dependently reduced the levels of s-catenin, whereas ASA up to 5 mM had no effect on s-catenin expression. HS-ASA also increased caspase-3 protein levels and increased its activity dose-dependently, at 0 (no treatment), 0.5xIC50, 1xIC50, and 2xIC50 the values were 3.9 ± 0.3, 12.3 ± 0.5, 23.3 ± 0.7, and 42.6 ± 1.2 pmol/min/mg protein. H2S levels also increased as a function of HS-ASA concentration. Conclusion: HS-ASA strongly inhibits the growth of Jurkat T cells and causes inhibition of s-catenin expression. These studies suggest one mechanism by which HS-ASA inhibits the growth of Jurkat T cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4608. doi:10.1158/1538-7445.AM2011-4608

Population pharmacokinetic model for cancer chemoprevention with sulindac in healthy subjects.

Sulindac is a prescription‐based non‐steroidal anti‐inflammatory drug (NSAID) that continues to be actively investigated as a candidate cancer chemoprevention agent. To further current understanding of sulindac bioavailability, metabolism, and disposition, we developed a population pharmacokinetic model for the parent compound and its active metabolites, sulindac sulfide, and exisulind. This analysis was based on data from 24 healthy subjects who participated in a bioequivalence study comparing two formulations of sulindac. The complex disposition of sulindac and its metabolites was described by a seven‐compartment model featuring enterohepatic recirculation and is the first reported population pharmacokinetic model for sulindac. The derived model was used to explore effects of clinical variables on sulindac pharmacokinetics and revealed that body weight, creatinine clearance, and gender were significantly correlated with pharmacokinetic parameters. Moreover, the model quantifies the relative bioavailability of the sulindac formulations and illustrates the utility of population pharmacokinetics in bioequivalence assessment. This novel population pharmacokinetic model provides new insights regarding the factors that may affect the pharmacokinetics of sulindac and the exisulind and sulindac sulfide metabolites in generally healthy subjects, which have implications for future chemoprevention trial design for this widely available agent.

Prevention of Chemically Induced Urinary Bladder Cancers by Naproxen: Protocols to Reduce Gastric Toxicity in Humans Do Not Alter Preventive Efficacy

The COX inhibitors (NSAID/Coxibs) are a major focus for the chemoprevention of cancer. The COX-2–specific inhibitors have progressed to clinical trials and have shown preventive efficacy in colon and skin cancers. However, they have significant adverse cardiovascular effects. Certain NSAIDs (e.g., naproxen) have a good cardiac profile, but can cause gastric toxicity. The present study examined protocols to reduce this toxicity of naproxen. Female Fischer-344 rats were treated weekly with the urinary bladder–specific carcinogen hydroxybutyl(butyl)nitrosamine (OH-BBN) for 8 weeks. Rats were dosed daily with NPX (40 mg/kg body weight/day, gavage) or with the proton pump inhibitor omeprazole (4.0 mg/kg body weight/day) either singly or in combination beginning 2 weeks after the final OH-BBN. OH-BBN–treated rats, 96% developed urinary bladder cancers. While omeprazole alone was ineffective (97% cancers), naproxen alone or combined with omeprazole-prevented cancers, yielding 27 and 35% cancers, respectively. In a separate study, OH-BBN–treated rats were administered naproxen: (A) daily, (B) 1 week daily naproxen/1week vehicle, (C) 3 weeks daily naproxen/3 week vehicle, or (D) daily vehicle beginning 2 weeks after last OH-BBN treatment. In the intermittent dosing study, protocol A, B, C, and D resulted in palpable cancers in 27%, 22%, 19%, and 96% of rats (P < 0.01). Short-term naproxen treatment increased apoptosis, but did not alter proliferation in the urinary bladder cancers. Two different protocols that should decrease the gastric toxicity of NSAIDs in humans did not alter chemopreventive efficacy. This should encourage the use of NSAIDs (e.g., naproxen) in clinical prevention trials. Cancer Prev Res; 8(4); 296–302. ©2015 AACR.

Synthesis and biological activity of acetyl-protected hydroxybenzyl diethyl phosphates (EHBP) as potential chemotherapeutic agents.

Several acetyl-protected hydroxybenzyl diethyl phosphates (EHBPs) that are capable of forming quinone methide intermediates were synthesized and their cell growth inhibitory properties were evaluated in four different human cancer cell lines. Compounds 1, 1a, and 1b, corresponding to (4-acetyloxybenzyl diethylphosphate), (3-methyl-4-acetyloxybenzyl diethylphosphate), and (3-chloro-4-acetyloxybenzyl diethylphosphate), were significantly more potent than compounds 2 and 3, (2-acetyloxybenzyl diethylphosphate) and (3-acetyloxybenzyl diethylphosphate), respectively. Using HT-29 human colon cancer cells, compounds 1 and 3 increased apoptosis, inhibited proliferation, and caused a G(2)/M block in the cell cycle. Our data suggest that these compounds merit further investigation as potential anti-cancer agents.