Jennie L. Williams

Nitric Oxide-Donating Nonsteroidal Anti-Inflammatory Drugs Inhibit the Growth of Various Cultured Human Cancer Cells: Evidence of a Tissue Type-Independent Effect

The novel nitric oxide (NO)-donating nonsteroidal anti-inflammatory drugs (NO-NSAIDs), which are safer than their NSAID counterparts, inhibit the growth of colon cancer cells with far greater potency than traditional NSAIDs. We examined whether NO-NSAIDs inhibit the growth of cancer cells arising from other human tissues. Human pancreatic, colon, prostate, lung, and tongue cancer cell lines were treated with NO-aspirin, -sulindac, -ibuprofen, and -indomethacin or their traditional counterparts. We determined IC50 values, cell proliferation, apoptosis, cell cycle, cyclooxygenase (COX) protein levels, and morphological changes (light and electron microscopy). All NO-NSAIDs inhibited the growth of all cancer cell lines studied. The potency of NO-NSAIDs was 11- to 6000-fold greater than that of their counterparts (except for the effect of sulindac on lung cancer cells). NO-aspirin was consistently the most potent NO-NSAID in all cell lines tested (except for the lung cancer cell line), sometimes in excess of 100-fold over the other three NO-NSAIDs. NO-NSAIDs inhibited cell proliferation, induced apoptosis, and altered cell cycle phase distribution (G2/M to G0/G1 block). All altered cellular morphology, whereas NO-aspirin induced nuclear disintegration (“atypical” cells) established by electron microscopy. NO-aspirin showed similar effects on two pancreatic cancer cell lines, BxPC-3 (expresses COX) and MIA PaCa-2 (no COX expression), suggesting a COX-independent effect. NO-NSAIDs showed a tissue-type-independent effect. Their pleiotropic effects involve cell renewal, cell death, and cell cycle phase transitions. These results raise the possibility that NO-NSAIDs possess chemopreventive and/or chemotherapeutic activity against a wide variety of human cancers.

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.

Parallel comparison of Illumina RNA-Seq and Affymetrix microarray platforms on transcriptomic profiles generated from 5-aza-deoxy-cytidine treated HT-29 colon cancer cells and simulated datasets

BackgroundHigh throughput parallel sequencing, RNA-Seq, has recently emerged as an appealing alternative to microarray in identifying differentially expressed genes (DEG) between biological groups. However, there still exists considerable discrepancy on gene expression measurements and DEG results between the two platforms. The objective of this study was to compare parallel paired-end RNA-Seq and microarray data generated on 5-azadeoxy-cytidine (5-Aza) treated HT-29 colon cancer cells with an additional simulation study.MethodsWe first performed general correlation analysis comparing gene expression profiles on both platforms. An Errors-In-Variables (EIV) regression model was subsequently applied to assess proportional and fixed biases between the two technologies. Then several existing algorithms, designed for DEG identification in RNA-Seq and microarray data, were applied to compare the cross-platform overlaps with respect to DEG lists, which were further validated using qRT-PCR assays on selected genes. Functional analyses were subsequently conducted using Ingenuity Pathway Analysis (IPA).ResultsPearson and Spearman correlation coefficients between the RNA-Seq and microarray data each exceeded 0.80, with 66%~68% overlap of genes on both platforms. The EIV regression model indicated the existence of both fixed and proportional biases between the two platforms. The DESeq and baySeq algorithms (RNA-Seq) and the SAM and eBayes algorithms (microarray) achieved the highest cross-platform overlap rate in DEG results from both experimental and simulated datasets. DESeq method exhibited a better control on the false discovery rate than baySeq on the simulated dataset although it performed slightly inferior to baySeq in the sensitivity test. RNA-Seq and qRT-PCR, but not microarray data, confirmed the expected reversal of SPARC gene suppression after treating HT-29 cells with 5-Aza. Thirty-three IPA canonical pathways were identified by both microarray and RNA-Seq data, 152 pathways by RNA-Seq data only, and none by microarray data only.ConclusionsThese results suggest that RNA-Seq has advantages over microarray in identification of DEGs with the most consistent results generated from DESeq and SAM methods. The EIV regression model reveals both fixed and proportional biases between RNA-Seq and microarray. This may explain in part the lower cross-platform overlap in DEG lists compared to those in detectable genes.

NO-donating NSAIDs and cancer : An overview with a note on whether NO is required for their action

Nitric oxide-donating nonsteroidal anti-inflammatory drugs (NO-NSAIDs) consist of a conventional NSAID to which an NO-releasing moiety is attached covalently, often via a spacer molecule. NO-NSAIDs represent an emerging class of compounds with chemopreventive properties against a variety of cancers, demonstrated in preclinical models including cell culture systems and animal tumor models; their potential efficacy in humans has not been assessed. Their mechanism of action appears complex and involves the generation of reactive oxygen species, suppression of microsatellite instability in mismatch repair-deficient cells, and modulation of several signaling cascades that culminate in inhibited cell renewal and enhanced apoptosis. NO, long appreciated to be able to protect from and also promote cancer, is released form NO-NSAIDs and constitutes their defining property. Existing data are consistent with the notion that NO may mediate their anticancer effect. In addition there is evidence that long-term administration of NO-donating compounds is not associated with increased incidence of colon cancer. Whether NO release is required for the anticancer effect of NO-NSAIDs has being questioned by recent data indicating that, at least in the case of NO-aspirin, the NO-releasing moiety may serve as a leaving group while the spacer actually being the moiety responsible for its pharmacological action. Regardless of mechanistic issues, these compounds promise to contribute to the control of cancer.

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.

Regulation of peroxisome proliferator‐activated receptor‐β/δ by the APC/β‐CATENIN pathway and nonsteroidal antiinflammatory drugs

Studies indicate that peroxisome proliferator‐activated receptor‐β/δ (PPARβ/δ) can either attenuate or potentiate colon cancer. One hypothesis suggests that PPARβ/δ is upregulated by the adenomatous polyposis coli (APC)/β‐CATENIN pathway and a related hypothesis suggests that PPARβ/δ is downregulated by nonsteroidal antiinflammatory drugs (NSAIDs). The present study examined these possibilities using in vivo and in vitro models. While APC/β‐CATENIN‐dependent expression of CYCLIN D1 was observed in vivo and in vitro, expression of PPARβ/δ was not different in colon or intestinal polyps from wild‐type or Apcmin heterozygous mice or in human colon cancer cell lines with mutations in APC and/or β‐CATENIN. No difference in the level of PPARβ/δ was found in colon from wild‐type or Apcmin heterozygous mice following treatment with NO‐donating aspirin (NO‐ASA). NSAIDs inhibited cell growth in RKO (wild‐type APC) and DLD1 (mutant APC) human colon cancer cell lines but expression of PPARβ/δ was not downregulated in these cell lines in response to a broad concentration range of celecoxib, indomethacin, NS‐398, or nimesulide. However, indomethacin caused an increase in PPARβ/δ mRNA and protein that was accompanied with increased expression of a known PPARβ/δ target gene. Interestingly, expression of PPARα was also increased in the human colon cancer cell lines by several NSAIDs at the highest concentration examined. Results from these studies provide additional evidence indicating that PPARβ/δ is not upregulated by the APC/β‐CATENIN pathway. Further, these studies suggest that increased PPARβ/δ and/or PPARα by NSAIDs in human colon cancer cell lines could contribute to the mechanisms underlying the chemopreventive effects of NSAIDs. Mol. Carcinog.

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.

Functional characterization of peroxisome proliferator‐activated receptor‐β/δ expression in colon cancer

This study critically examined the role of PPARβ/δ in colon cancer models. Expression of PPARβ/δ mRNA and protein was lower and expression of CYCLIN D1 protein higher in human colon adenocarcinomas compared to matched non‐transformed tissue. Similar results were observed in colon tumors from Apc+/Min‐FCCC mice compared to control tissue. Dietary administration of sulindac to Apc+/Min‐FCCC mice had no influence on expression of PPARβ/δ in normal colon tissue or colon tumors. Cleaved poly (ADP‐ribose) polymerase (PARP) was either increased or unchanged, while expression of 14‐3‐3ε was not influenced in human colon cancer cell lines cultured with the PPARβ/δ ligand GW0742 under conditions known to increase apoptosis. While DLD1 cells exhibited fewer early apoptotic cells after ligand activation of PPARβ/δ following treatment with hydrogen peroxide, this change was associated with an increase in late apoptotic/necrotic cells, but not an increase in viable cells. Stable over‐expression of PPARβ/δ in human colon cancer cell lines enhanced ligand activation of PPARβ/δ and inhibition of clonogenicity in HT29 cells. These studies are the most quantitative to date to demonstrate that expression of PPARβ/δ is lower in human and Apc+/Min‐FCCC mouse colon tumors than in corresponding normal tissue, consistent with the finding that increasing expression and activation of PPARβ/δ in human colon cancer cell lines inhibits clonogenicity. Because ligand‐induced attenuation of early apoptosis can be associated with more late, apoptotic/necrotic cells, but not more viable cells, these studies illustrate why more comprehensive analysis of PPARβ/δ‐dependent modulation of apoptosis is required in the future.

Protein nitration and nitrosylation by NO-donating aspirin in colon cancer cells: Relevance to its mechanism of action

Nitric oxide-donating aspirin (NO-ASA) is a promising agent for cancer prevention. Although studied extensively, its molecular targets and mechanism of action are still unclear. S-nitrosylation of signaling proteins is emerging as an important regulatory mechanism by NO. Here, we examined whether S-nitrosylation of the NF-κB, p53, and Wnt signaling proteins by NO-ASA might explain, in part, its mechanism of action in colon cancer. NO-ASA releases significant amounts of NO detected intracellularly in HCT116 and HT-29 colon cells. Using a modified biotin switch assay we demonstrated that NO-ASA S-nitrosylates the signaling proteins p53, β-catenin, and NF-κB, in colon cancer cells in a time- and concentration-dependent manner. NO-ASA suppresses NF-κB binding to its cognate DNA oligonucleotide, which occurs without changes in the nuclear levels of the NF-κB subunits p65 and p50 and is reversed by dithiothreitol that reduces -S-NO to -SH. In addition to S-nitrosylation, we documented both in vitro and in vivo widespread nitration of tyrosine residues of cellular proteins in response to NO-ASA. Our results suggest that the increased intracellular NO levels following treatment with NO-ASA modulate cell signaling by chemically modifying key protein members of signaling cascades. We speculate that S-nitrosylation and tyrosine nitration are responsible, at least in part, for the inhibitory growth effect of NO-ASA on cancer cell growth and that this may represent a general mechanism of action of NO-releasing agents.

NO-donating aspirin inhibits angiogenesis by suppressing VEGF expression in HT-29 human colon cancer mouse xenografts

The inhibitory effect of NO-donating aspirin (NO-ASA) on colon cancer has been demonstrated in vivo and in vitro but its mechanism is still obscure. We investigated the effect of NO-ASA on angiogenesis. Four groups of athymic mice (N = 12) bearing subcutaneous xenotransplants of HT-29 human colon cancer cells were injected intratumorally twice a week for 3 weeks with vehicle or m-NO-ASA or p-NO-ASA; the fourth group received no injections. The necrotic area of tumors, expressed as percentage of total area, was similar in the non-injected and vehicle-injected groups (51.8 +/- 2.8 versus 52.2 +/- 4.1, P > 0.05; mean +/- SEM for these and subsequent values). Compared with the vehicle group, the necrotic area of tumors was higher in the m-NO-ASA-treated (61.0 +/- 2.7, P < 0.02) and p-NO-ASA (65.8 +/- 2.4, P < 0.001)-treated groups. NO-ASA decreased microvessel density: vehicle = 11.7 +/- 0.8; m-NO-ASA = 7.8 +/- 0.6 (P = 0.0003 versus vehicle) and p-NO-ASA 6.2 +/- 0.7 (P = 0.0001 versus vehicle). The expression of vascular endothelial growth factor (VEGF) was significantly reduced in response to NO-ASA, with the p- isomer being more potent than the m-. NO-ASA altered the spatial distribution of VGEF expression, with 16.7% of the vehicle-treated xenografts displaying diminished VEGF in the inner region of the area between necrosis and the outer perimeter of the tumor, compared with those treated with m- (58.3%) or p-NO-ASA (75%, P < 0.01 for both versus control). Our findings indicate that NO-ASA suppresses the expression of VEGF, which leads to suppressed angiogenesis. The antiangiogenic activity of NO-ASA may be part of its antineoplastic effect.