Nengtai Ouyang

Nitric Oxide–Donating Aspirin Prevents Pancreatic Cancer in a Hamster Tumor Model

To evaluate the chemopreventive effect of nitric oxide-donating aspirin (NO-ASA), an ASA bearing a NO-releasing moiety, against pancreatic cancer, we studied six groups of female Syrian golden hamsters: groups 1 to 3 (n = 12 each) were given saline and groups 4 to 6 (n = 17) the carcinogen N-nitrosobis(2-oxopropyl)amine (BOP) s.c. in five weekly injections (the first, 70 mg/kg, and the remaining, 20 mg/kg each). Control and BOP-treated hamsters were fed a NO-ASA 3,000 ppm or conventional ASA 3,000 ppm or control diet for 19 weeks. Groups 1 to 3 had no tumors. Compared with the BOP/vehicle group, NO-ASA reduced the incidence (88.9%, P < 0.003) and multiplicity (94%, P < 0.05) of pancreatic cancer; ASA had no statistically significant effect. NO-ASA arrested the transition from PanIN2 to PanIN3 and carcinoma. The proliferation (proliferating cell nuclear antigen) / apoptosis (terminal deoxyribonucleotide transferase-mediated nick-end labeling) ratio of ductal cells increased with the histologic severity of the ductal lesion; NO-ASA suppressed it significantly during all stages except PanIN1A. p21(WAF1/CIP1), undetectable in normal cells, was progressively induced in neoplastic cells and suppressed by NO-ASA up to PanIN3. Nuclear factor-kappaB activation, absent in normal tissue, increased progressively (17-fold in cancer); NO-ASA suppressed it throughout and significantly in PanIN1B and PanIN2. Cyclooxygenase-2 expression, absent during early stages, was induced 6-fold in carcinoma and suppressed by NO-ASA in PanIN3 and carcinoma. Conventional ASA had no effect on these molecular markers. Thus, NO-ASA profoundly prevented pancreatic cancer and modulated multiple molecular targets in this model system; conventional ASA had no such effects. NO-ASA merits further evaluation as a chemopreventive agent against pancreatic cancer.

Phospho-Sulindac (OXT-328), a Novel Sulindac Derivative, Is Safe and Effective in Colon Cancer Prevention in Mice

BACKGROUND & AIMS Nonsteroidal anti-inflammatory drugs (NSAIDs) are effective cancer chemopreventive agents. However, chronic administration of NSAIDs is associated with significant side effects, mainly of the gastrointestinal tract. Given these limitations, we synthesized phospho-sulindac (P-S; OXT-328), a novel sulindac derivative. METHODS Here, we evaluated the safety and efficacy of P-S in preclinical models, including its mechanism of action with human colon cancer cell (HCCC) lines and animal tumor models. RESULTS (1) Compared with sulindac, P-S is much more potent in inhibiting the growth of cultured HCCCs and more efficacious in preventing the growth of HT-29 xenografts in nude mice. P-S also prevents the growth of intestinal tumors in Apc/Min mice. (2) In combination with difluoromethylornithine (DFMO), P-S reduced tumor multiplicity in Apc/Min mice by 90%. (3) P-S is much safer than sulindac as evidenced by its in vitro toxicologic evaluation and animal toxicity studies. Mechanistically, P-S increases the intracellular levels of reactive oxygen and nitrogen species, which are key early mediators of its chemopreventive effect. Moreover, P-S induces spermidine/spermine N(1)-acetyltransferase enzymatic activity, and together with DFMO it reduces polyamine levels in vitro and in vivo. CONCLUSIONS P-S displays considerable safety and efficacy, two pharmacologic properties that are essential for a potential cancer chemopreventive agent, and thus merits further evaluation.

NO-donating aspirin inhibits the activation of NF-κB in human cancer cell lines and Min mice

Nitric oxide-donating aspirin (NO-ASA) is a promising agent for the control of cancer, whose mechanism of action remains unclear. NF-kappaB is an important signaling molecule in the pathogenesis of cancer. We studied in several human colon (HT-29, HCT-15, LoVo, HCT116 and SW-480), pancreatic (BxPC-3, MIA PaCa-2) and breast (MDA-MB-231 and MCF-7) cancer cell lines, the effect of NO-ASA on NF-kappaB activation, determined by electrophoretic mobility shift assays, immunofluorescence and western blot analyses of nuclear proteins. NO-ASA inhibited NF-kappaB activation, as early as 30 min and with IC(50)s ranging between 0.83 and 64 microM. Such inhibition was also observed at NO-ASA concentrations that had an insignificant or marginal effect on cell growth. The effect of NO-ASA on NF-kappaB binding to DNA was significantly correlated with its effect on cell growth (P < 0.05) indicating that the growth inhibitory effect of NO-ASA may be mediated by its effect on NF-kappaB. Compared with control, NO-ASA decreased NF-kappaB activation in intestinal epithelial cells of APC(min+/-) mice by 38.4% (P < 0.01). Western blot and immunofluorescence analyses revealed that the nuclear levels of the p50 and p65 NF-kappaB subunits were virtually unaffected, suggesting an inhibitory mechanism different from suppressed subunit translocation into the nucleus. Inhibition of NF-kappaB activation by NO-ASA may account, at least in part, for its chemopreventive efficacy.

Targeting Mitochondrial STAT3 with the Novel Phospho-Valproic Acid (MDC-1112) Inhibits Pancreatic Cancer Growth in Mice

New agents are needed to treat pancreatic cancer, one of the most lethal human malignancies. We synthesized phospho-valproic acid, a novel valproic acid derivative, (P-V; MDC-1112) and evaluated its efficacy in the control of pancreatic cancer. P-V inhibited the growth of human pancreatic cancer xenografts in mice by 60%–97%, and 100% when combined with cimetidine. The dominant molecular target of P-V was STAT3. P-V inhibited the phosphorylation of JAK2 and Src, and the Hsp90-STAT3 association, suppressing the activating phosphorylation of STAT3, which in turn reduced the expression of STAT3-dependent proteins Bcl-xL, Mcl-1 and survivin. P-V also reduced STAT3 levels in the mitochondria by preventing its translocation from the cytosol, and enhanced the mitochondrial levels of reactive oxygen species, which triggered apoptosis. Inhibition of mitochondrial STAT3 by P-V was required for its anticancer effect; mitochondrial STAT3 overexpression rescued animals from the tumor growth inhibition by P-V. Our results indicate that P-V is a promising candidate drug against pancreatic cancer and establish mitochondrial STAT3 as its key molecular target.

Phospho-sulindac (OXT-328) combined with difluoromethylornithine prevents colon cancer in mice

The nonsteroidal anti-inflammatory drug (NSAID) sulindac and the ornithine decarboxylase (ODC) antagonist difluoromethylornithine (DFMO), individually and together, are effective inhibitors of colon carcinogenesis. However, chronic use of sulindac is associated with significant side effects. We evaluated the chemopreventive efficacy of phospho-sulindac (P-S, OXT-328), an apparently safe derivative of sulindac, together with DFMO, in HT-29 human colon cancer xenografts. Nude mice were divided into four groups as follows: group 1 received vehicle (corn oil); group 2 received P-S (100 mg/kg/d) by oral gavage; group 3 received DFMO (2% in drinking water); and group 4 received P-S (100 mg/kg/d) by gavage plus DFMO (2% in drinking water; P-S/DFMO). Eighteen days after implantation, compared with controls, tumor volume was inhibited 65.9% by P-S, 52.9% by DFMO, and 70.9% by P-S/DFMO (P < 0.01 for all). P-S/DFMO reduced cell proliferation 27.1% and increased apoptosis 38.9% compared with controls (P < 0.05 for both). Compared with controls, P-S reduced the levels of thioredoxin-1 (Trx-1) and thioredoxin reductase (TrxR), whereas DFMO reduced polyamine content (putrescine and spermidine) and TrxR levels. Importantly, P-S/DFMO decreased putrescine and spermidine levels and the expression of Trx-1, TrxR, and cyclooxygenase (COX) 2. Of these molecular targets, TrxR most consistently correlated with tumor growth. Study results show that P-S/DFMO is an efficacious drug combination for colon cancer prevention and also show the safety of P-S, which may overcome the limiting side effects of conventional sulindac. P-S/DFMO has an intricate mechanism of action extending beyond polyamines and including the thioredoxin system, an emerging regulator of chemoprevention. P-S/DFMO merits further evaluation. Cancer Prev Res; 4(7); 1052–60. ©2011 AACR.

Chemotherapeutic Properties of Phospho-Nonsteroidal Anti-Inflammatory Drugs, a New Class of Anticancer Compounds

Nonsteroidal anti-inflammatory drugs (NSAID) exhibit antineoplastic properties, but conventional NSAIDs do not fully meet safety and efficacy criteria for use as anticancer agents. In this study, we evaluated the chemotherapeutic efficacy of 5 novel phospho-NSAIDs, each of which includes in addition to the NSAID moiety a diethylphosphate linked through a butane moiety. All 5 compounds inhibited the growth of human breast, colon, and pancreatic cancer cell lines with micromolar potency. In vivo investigations confirmed the antitumor activity of phospho-aspirin (PA) and phospho-sulindac (PS) in inhibiting tumor growth in established human xenograft models, in which cell proliferation was suppressed and apoptosis enhanced in the absence of detectable animal toxicity. Notably, all of the phospho-NSAIDs tested induced reactive oxygen and nitrogen species in cultured cells, with PA and PS inducing detectable levels of oxidative stress in vivo that were associated positively with apoptosis and negatively with proliferation. Potentially explaining these effects, all of the phospho-NSAIDs tested also inhibited the thioredoxin system and the redox sensitive transcription factor NF-κB. Taken together, our findings show the strong anticancer efficacy and promising safety of phospho-NSAIDs in preclinical models of breast, colon, and pancreatic cancer, suggesting further evaluation as anticancer agents.

Differential expression of miRNAs in colon cancer between African and Caucasian Americans: Implications for cancer racial health disparities

Colorectal cancer (CRC) incidence and mortality are higher in African Americans (AAs) than in Caucasian Americans (CAs) and microRNAs (miRNAs) have been found to be dysregulated in colonic and other neoplasias. The aim of this exploratory study was to identify candidate miRNAs that could contribute to potential biological differences between AA and CA colon cancers. Total RNA was isolated from tumor and paired adjacent normal colon tissue from 30 AA and 31 CA colon cancer patients archived at Stony Brook University (SBU) and Washington University (WU)-St. Louis Medical Center. miRNA profiles were determined by probing human genome-wide miRNA arrays with RNA isolated from each sample. Using repeated measures analysis of variance (RANOVA), miRNAs were selected that exhibited significant (p<0.05) interactions between race and tumor or significant (fold change >1.5, p<0.05) main effects of race and/or tumor. Quantitative polymerase chain reaction (q-PCR) was used to confirm miRNAs identified by microarray analysis. Candidate miRNA targets were analyzed using immunohistochemistry. RANOVA results indicated that miR-182, miR152, miR-204, miR-222 and miR-202 exhibited significant race and tumor main effects. Of these miRNAs, q-PCR analysis confirmed that miR-182 was upregulated in AA vs. CA tumors and exhibited significant race:tumor interaction. Immunohistochemical analysis revealed that the levels of FOXO1 and FOXO3A, two potential miR-182 targets, are reduced in AA tumors. miRNAs may play a role in the differences between AA and CA colon cancer. Specifically, differences in miRNA expression levels of miR-182 may contribute to decreased survival in AA colon cancer patients.

MC-12, an annexin A1-based peptide, is effective in the treatment of experimental colitis.

Annexin A1 (ANXA1) inhibits NF-κB, a key regulator of inflammation, the common pathophysiological mechanism of inflammatory bowel diseases (IBD). MC-12, an ANXA1-based tripeptide, suppresses NF-κB activation. Here, we determined the efficacy of MC-12 in the control of IBD. Mice with colitis induced by dextran sodium sulfate (DSS) or 2,4,6-trinitro benzene sulfonic acid (TNBS) were treated with various doses of MC-12 administered intraperitoneally, orally or intrarectally. We determined colon length and the histological score of colitis, and assayed: in colon tissue the levels of TNF-α, IFN-γ, IL-1β, IL-6 and IL-10 by RT-PCR; prostaglandin E2 (PGE2), cytoplasmic phospholipase A2 (cPLA2) and myeloperoxidase by immunoassay; and COX-2 and NF- κB by immunohistochemistry; and in serum the levels of various cytokines by immunoassay. In both models MC-12: reversed dose-dependently colonic inflammation; inhibited by up to 47% myeloperoxidase activity; had a minimal effect on cytoplasmic phospholipase A2; reduced significantly the induced levels of TNF-α, IFN-γ, IL-1β, IL-6 and IL-10, returning them to baseline. DSS and TNBS markedly activated NF-κB in colonic epithelial cells and MC-12 decreased this effect by 85.8% and 72.5%, respectively. MC-12 had a similar effect in cultured NCM460 normal colon epithelial cells. Finally, MC-12 suppressed the induction of COX-2 expression, the level of PGE2 in the colon and PGE2 metabolite in serum. In conclusion, MC-12, representing a novel class of short peptide inhibitors of NF-κB, has a strong effect against colitis in two preclinical models recapitulating features of human IBD. Its mechanism of action is complex and includes pronounced inhibition of NF-κB. MC-12 merits further development as an agent for the control of IBD.

Phosphosulindac (OXT‐328) Selectively Targets Breast Cancer Stem Cells In Vitro and in Human Breast Cancer Xenografts

Pharmacological targeting of breast cancer stem cells (CSCs) is highly promising for the treatment of breast cancer, as the small population of CSCs appears responsible for tumor initiation and progression and also for resistance to conventional treatment. Here we report that the novel phosphosulindac (OXT‐328, PS) selectively and effectively eliminates breast CSCs both in vitro and in vivo. PS reduced cell proliferation and induced apoptosis in various breast CSCs. Breast CSCs are resistant to conventional cancer drugs but are sensitive to PS. Long‐term treatment of mixtures of cultured breast CSCs and breast cancer cells with PS preferentially eliminated the CSCs. PS impaired the ability of CSCs to form mammospheres and markedly suppressed the expression of CSC‐related genes. More importantly, PS prevented by half (p =.06) the formation of tumors initiated by CSCs in immunodeficient mice, and inhibited by 83% (p <.05) the growth of already formed breast cancer xenografts, reducing the proportion of CSCs in them. PS suppressed the Wnt/β‐catenin pathway by stimulating the degradation of β‐catenin and its relocalization to the cell membrane and also blocked the epithelial–mesenchymal transition and the generation of breast CSCs. These results indicate that PS has a strong inhibitory effect against breast cancer, acting, at least in part, by targeting CSCs through a signaling mechanism involving Wnt signaling. STEM Cells2012;30:2065–2075

The metabolism and pharmacokinetics of phospho-sulindac (OXT-328) and the effect of difluoromethylornithine

BACKGROUND AND PURPOSE Phospho‐sulindac (PS; OXT‐328) prevents colon cancer in mice, especially when combined with difluoromethylornithine (DFMO). Here, we explored its metabolism and pharmacokinetics.