The Impact of Obesity on Health Care Utilization and Expenditures in a Medicare Supplement Population.

Objective: Obesity is a contributor to increased chronic conditions resulting in higher utilization of medical services among broad populations of older adults. The objective of this study was to evaluate the magnitude of the impact of weight on health care use patterns among Medicare Supplement insureds. Method: We estimated the impact of weight as a function of body mass index (BMI) on health care utilization and expenditures using propensity weighted multivariate regression models. The outcomes were controlled initially for demographics and socioeconomics and then additionally for chronic conditions and health status. Results: Among the 9,484 survey respondents, 22.9% were obese. Those categorized as obese were significantly more likely to incur inpatient admissions and orthopedic procedures. Annualized health care expenditures were US$1,496 higher for obese compared with normal weight. The excess utilization and expenditures associated with obesity were explained by chronic conditions and poor health status. Conclusion: Obesity-related expenditures associated with medical management are largely preventable and may benefit from interventions that target lifestyle behaviors and weight management among older adults.

Obese older adults report high satisfaction and positive experiences with care.

BACKGROUND: Obese, older adults often have multiple chronic conditions resulting in multiple health care encounters. However, their satisfaction and experiences with care are not well understood. The objective of this study was to examine the independent impact of obesity on patient satisfaction and experiences with care in adults 65 years of age and older with Medigap insurance. METHODS: Surveys were mailed to 53,286 randomly chosen adults with an AARP® Medicare Supplement Insurance Plan insured by UnitedHealthcare Insurance Company (for New York residents, UnitedHealthcare Insurance Company of New York) in 10 states. Following adjustment for non-response bias, multivariate regression modeling was used to adjust for demographic, socioeconomic and health status differences to estimate the independent impact of weight on satisfaction and experiences with care. Outcome variables included four global and four composite measures of satisfaction and experiences with care. RESULTS: 21.4% of the respondents were obese. Relative to normal weight, obesity was significantly associated with higher patient satisfaction and better experiences with care in seven of the eight ratings measured. CONCLUSIONS: Obese individuals were more satisfied and had better experiences with care. Obese individuals had more office visits and discussions about nutrition, exercise and medical checks. This may have led to increased attentiveness to care, explaining the increase in satisfaction and better experiences with care. Given the high level of satisfaction and experiences with care in older, obese adults, opportunities exist for clinicians to address weight concerns in this population.

Barriers to managing coronary artery disease among older women.

Numerous barriers to managing coronary artery disease (CAD) among older women are reported in the literature; however, few studies adjust for demographic and health status differences. A survey assessing barriers and other factors was distributed to a stratified random sampling of older women with CAD. Factor analysis and multiple logistic regression procedures were used to estimate the impact of these issues on receiving a CAD-related office visit. The most problematic barriers included denial and low health literacy. Efforts to promote patient awareness of heart health and better communication between patients and clinicians may alleviate these barriers.

The impact of pain on physical and mental quality of life in adults 65 and older.

Pain is a frequent and debilitating problem among older adults, decreasing quality of life (QOL) both physically and mentally. The burden of arthritis, sciatica, and back pain on QOL was estimated using ordinary least squares regression techniques to estimate the impact of each of these types of pain on QOL, controlling for patient demographic, socioeconomic, and health status characteristics. For individuals with arthritis, sciatica, and back pain, the adjusted average physical component scores were 4.19, 1.39, and 6.75 points lower, respectively (all p < 0.0001), than those without pain. Adjusted average mental component scores were 1.33, 0.47, and 2.93 points lower (all p < 0.01) for individuals with arthritis, sciatica, and back pain, respectively. The impact of pain on QOL was greater than that for many other commonly treated medical conditions. Clinicians should discuss pain with their patients to maximize their QOL.

Why do older adult women fall short of the recommended follow-up care for coronary artery disease?

BACKGROUND: Many women with coronary artery disease (CAD), commonly referred to as coronary heart disease, do not receive an annual office visit to manage their disease. We set out to determine what barriers factor into women not receiving an office visit to manage their disease. METHODS: A purposive sample of 26 eligible women (≥65 years of age) diagnosed with CAD completed in-depth, qualitative interviews. Systematic analysis of the content of interviews was performed on transcripts from these interviews. Participants with an AARP Medicare Supplement Insurance Plan insured by UnitedHealthcare insurance company that did not receive an annual office visit were eligible. In addition, we surveyed 100 physicians to obtain their thoughts about why women may not schedule at least one annual visit to manage their CAD. RESULTS: The most common barriers identified were skepticism of heart problems, having to take the initiative to schedule the appointment, and dealing with seemingly more pressing health problems. Many of these barriers identified were substantiated in a survey of physicians that treat women with CAD, but the relative rankings of the importance of these problems differed somewhat. CONCLUSIONS: Many women were skeptical about their heart health and often lacked the initiative to schedule a follow-up appointment. Most agreed that they would make an appointment if contacted by their doctor's office. Many of these women were receptive to the idea of receiving educational information by mail. Active involvement by doctors' offices to schedule appointments may help improve care, as might mail-based reminders.

The prevalence of hearing impairment and its burden on the quality of life among adults with Medicare Supplement Insurance.

PURPOSE: To estimate the impact of hearing impairment on quality of life from a physical and mental standpoint. METHODS: Data were obtained from the Health Update Survey, which contains questions on demographics, comorbid conditions, and the Veterans RAND 12-item health status/quality of life survey. It was fielded on a random sample of 15,000 adults with an AARP(®) Medicare Supplement plan, insured by UnitedHealthcare. Respondents were divided into those with hearing impairments and others, based on their response to a survey question. Univariate and multivariate analyses were conducted to estimate the likelihood of hearing impairment and its impact on quality of life while controlling for respondent demographics and comorbid conditions. RESULTS: Of the 5,515 eligible respondents, 10.4% reported having hearing impairments. The strongest predictor of hearing impairment was older age, while those with 4 years of college or more were least likely to have hearing impairments. Those with hearing impairments averaged significantly lower physical component and mental component scores, exceeding those of numerous other chronic conditions. CONCLUSIONS: This is the first known study of hearing impairment among those with Medigap coverage. Hearing impairment was strongly associated with lower quality of life from both a physical and mental health standpoint.

The burden of depressive symptoms and various chronic conditions and health concerns on the quality of life among those with Medicare Supplement Insurance.

OBJECTIVE: This study aims to estimate the burden of currently having depressive symptoms on quality of life independently and in combination with various chronic conditions/health concerns among adults with Medicare Supplement Insurance (i.e., Medigap) coverage. METHODS: Data were obtained from the Health Update Survey (HUS). The HUS contains questions on demographics, comorbid conditions, and all of the questions on the Veterans RAND 12-item (VR-12) health status/quality of life survey. The survey was mailed to a random sample of 30,000 insureds from 10 states between 2008 and 2009. On the basis of relevant questions from the survey, respondents were categorized into two groups: those currently suffering from current depressive symptoms (N = 2283) and those who never had depressive symptoms (N = 7058). Multiple regression modeling was used to test the impact on quality of life of depressive symptoms independently or as an interaction of depressive symptoms with various chronic conditions/health concerns. RESULTS: Depressive symptoms were common, with an estimated prevalence of 24.4%. The greatest impact of depressive symptoms in combination with various chronic conditions/health concerns on quality of life was on the ability to handle emotional roles, bodily pain, social functioning, and ability to handle physical roles. Most of the significant interactions between depressive symptoms and various chronic conditions/health concerns were demonstrated for those chronic conditions contributing to functional impairment (e.g., difficulty walking, falls, chronic pain, and diabetes). CONCLUSIONS: Although depressive symptoms independently reduced quality of life, having depressive symptoms in addition to other chronic conditions/health concerns had a greater impact on quality of life.

The dietary compounds resveratrol and genistein induce activating transcription factor 3 while suppressing inhibitor of DNA binding/differentiation-1.

Various chemopreventive compounds alter gene expression, possibly explaining their biological activity. One gene induced by a variety of chemopreventive compounds is the one coding for the transcription factor activating transcription factor 3 (ATF3). In this study, we performed microarray analysis on mRNA isolated from human colorectal cancer cells overexpressing ATF3 to ascertain the biological activity of this gene in cancer. As a result, 64 genes were induced or repressed. One gene identified by microarray analysis as repressed by overexpression of ATF3 was inhibitor of DNA binding/differentiation-1 (Id1). Id1 is important to cell growth and proliferation and therefore may represent an important downstream target of ATF3 responsible for the biological activity of ATF3. Id1 interacts with ATF3, thereby sequestering its activity, making it an ideal candidate for further study. The induction of ATF3 and repression of Id1 in these cells were confirmed at the mRNA and protein levels by semiquantitative real-time reverse transcription-polymerase chain reaction and western blot analysis, respectively. To determine if the repression of Id1 seen following microarray analysis of these cells occurred following treatment with dietary compounds with known chemotherapeutic activity, human colorectal cancer cells were treated with resveratrol and genistein, and their expression was determined. As a result, ATF3 was induced, and Id1 was repressed, by these compounds and by sulindac sulfide, a positive control, at the mRNA and protein level. Further work is needed to determine the molecular mechanism(s) responsible for the regulation of Id1 and to determine if biological activity of ATF3 overexpression is mediated by repression of Id1 by these compounds.

Postmarketing surveillance of serious adverse events associated with the use of rofecoxib from 1999-2002.

OBJECTIVE: The authors performed a postmarketing database analysis to evaluate the incidence of cardiovascular and other serious adverse events (SAEs) reported to the US Food and Drug Administration's Adverse Event Reporting System (AERS) involving the use of rofecoxib. RESEARCH DESIGN AND METHODS: The authors studied all adverse events involving rofecoxib reported to the AERS from inception of the drug until 2002. An emphasis was placed on cardiovascular and other SAEs of interest categorized using the high level group terms hemorrhage, edema, thrombosis, embolism, and death. RESULTS: There were 31,024 reports of SAEs associated with the use of rofecoxib, which was considered primary suspect in 97.8% of these reports. There were 3915, 3677, 1653, 1917, and 233 reports of hemorrhage, edema, death, thrombosis, and embolism, respectively. Relative to the overall population in this dataset, reports of hemorrhage, death, thrombosis, and embolism consisted of a greater proportion of males. The mean age for patients that reported hemorrhage, death, and thrombosis was older, whereas the mean age for embolism and edema was younger than the overall population in this dataset. Aspirin was the most commonly reported concomitant medication (7.4% of reports) followed by acetaminophen (5.4%). Reports containing concomitant use of anti-coagulants, Cox-1, and nonselective inhibitors (each p < 0.001) but not Cox-2 inhibitors were associated with increased age while only anti-coagulants and Cox-1 inhibitors (each p < 0.001) were associated with more males. Reports containing concomitant use of an anti-coagulant or an NSAID accounted for a disproportionate incidence of hemorrhage, edema, embolism, and death. Most notably, the odds of a hemorrhagic event for those reporting concomitant use of an anti-coagulant, Cox-1, non-selective, or Cox-2 inhibitor was 3.05 (p < 0.001), 3.07 (p < 0.001), 2.07 (p < 0.001), and 1.18 (p < 0.001), respectively. Some weaknesses of this type of analysis are the retrospective nature of such a study, the inability to find causality, and that the data can contains multiple reports from any one individual. CONCLUSIONS: It can be postulated that in addition to the risk of heart attack and stroke, rofecoxib users were at increased risk of hemorrhage, in addition to other thrombotic and embolic adverse events, which was exacerbated in those taking blood thinners or NSAIDs.

Identification and functional characterization of polymorphisms in human cyclooxygenase-1 (PTGS1).

OBJECTIVE: Cyclooxygenase-1 (COX-1, PTGS1) catalyzes the conversion of arachidonic acid to prostaglandin H2, which is subsequently metabolized to various biologically active prostaglandins. We sought to identify and characterize the functional relevance of genetic polymorphisms in PTGS1. METHODS: Sequence variations in human PTGS1 were identified by resequencing 92 healthy individuals (24 African, 24 Asian, 24 European/Caucasian, and 20 anonymous). Using site-directed mutagenesis and a baculovirus/insect cell expression system, recombinant wild-type COX-1 and the R8W, P17L, R53H, R78W, K185T, G230S, L237M, and V481I variant proteins were expressed. COX-1 metabolic activity was evaluated in vitro using an oxygen consumption assay under basal conditions and in the presence of indomethacin. RESULTS: Forty-five variants were identified, including seven nonsynonymous polymorphisms encoding amino acid substitutions in the COX-1 protein. The R53H (35+/-5%), R78W (36+/-4%), K185T (59+/-6%), G230S (57+/-4%), and L237M (51+/-3%) variant proteins had significantly lower metabolic activity relative to wild-type (100+/-7%), while no significant differences were observed with the R8W (104+/-10%), P17L (113+/-7%), and V481I (121+/-10%) variants. Inhibition studies with indomethacin demonstrated that the P17L and G230S variants had significantly lower IC50 values compared to wild-type, suggesting these variants significantly increase COX-1 sensitivity to indomethacin inhibition. Consistent with the metabolic activity data, protein modeling suggested the G230S variant may disrupt the active conformation of COX-1. CONCLUSIONS: Our findings demonstrate that several genetic variants in human COX-1 significantly alter basal COX-1-mediated arachidonic acid metabolism and indomethacin-mediated inhibition of COX-1 activity in vitro. Future studies characterizing the functional impact of these variants in vivo are warranted.

Suppression of tumor cell invasion by cyclooxygenase inhibitors is mediated by thrombospondin-1 via the early growth response gene Egr-1.

Cyclooxygenase (COX) inhibitors have antitumorigenic activity and increase the expression of the early growth response gene Egr-1, a tumor suppressor gene and transcription factor. In this study, we have investigated the gene regulatory and anti-invasive activity of two traditional nonsteroidal anti-inflammatory drugs (NSAID), sulindac sulfide and indomethacin. These compounds inhibited tumor cell invasion and induced Egr-1 expression in lung adenocarcinoma A549 cells. Overexpression of Egr-1 reduced cellular invasion in the Matrigel system, whereas suppression of Egr-1 by small interference RNA (siRNA) attenuated the inhibition of Matrigel invasion by these compounds, indicating that Egr-1 is responsible for the decrease in invasion reported following treatment with NSAIDs. Egr-1-overexpressing cells were analyzed for genes involved in invasion and metastasis. Thrombospondin-1 (TSP-1) an antiangiogenic and anti-invasion protein was up-regulated by Egr-1 overexpression, which was confirmed following treatment with sulindac sulfide. Furthermore, the induction of TSP-1 by sulindac sulfide was blocked by Egr-1 siRNA. When TSP-1 was sequestered by the addition of anti-TSP-1 antibody, the inhibition of invasion by sulindac sulfide was attenuated, indicating that TSP-1 is involved in the inhibition of invasion by NSAIDs. We used the Min mouse model to determine if sulindac sulfide would increase Egr-1 and TSP-1 in vivo, because this model is widely used to study the effects of NSAIDs on tumor formation. Treatment of Min mice with concentrations of sulindac sulfide that inhibit tumor formation increased the expression of Egr-1 and TSP-1 in colonic tissues and in the polyps of these mice. This is the first report suggesting that COX inhibitors suppress tumor cell invasion via TSP-1, which occurs downstream of Egr-1.

Overexpression of 15-lipoxygenase-1 induces growth arrest through phosphorylation of p53 in human colorectal cancer cells.

To investigate the function of 15-lipoxygenase-1 (15-LOX-1) in human colorectal cancer, we overexpressed 15-LOX-1 in HCT-116 human colorectal cancer cells. Clones expressing the highest levels of 15-LOX-1 displayed reduced viability compared with the HCT-116-Vector control cells. Further, by cell cycle gene array analyses, the cyclin-dependent kinase inhibitor p21WAF1/CIP1 and MDM2 genes were up-regulated in 15-LOX-1-overexpressing cells. The induction of p21(WAF1/CIP1) and MDM2 were linked to activation of p53 by 15-LOX-1, as there was a dramatic induction of phosphorylated p53 (Ser15) in 15-LOX-1-overesxpressing cells. However, the 15-LOX-1 metabolites 13(S)-hydroxyoctadecadienoic acid and 15(S)-hydroxyeicosatetraenoic acid failed to induce phosphorylation of p53 at Ser15, and the 15-LOX-1 inhibitor PD146176 did not inhibit the phosphorylation of p53 at Ser15 in 15-LOX-1-overexpressing cells. Nonetheless, the growth-inhibitory effects of 15-LOX-1 were p53 dependent, as 15-LOX-1 overexpression had no effect on cell growth in p53 (-/-) HCT-116 cells. Finally, treatment of HCT-116-15-LOX-1 cells with different kinase inhibitors suggested that the effects of 15-LOX-1 on p53 phosphorylation and activation were due to effects on DNA-dependent protein kinase. Collectively, these findings suggest a new mechanism to explain the biological activity of 15-LOX-1, where 15-LOX plays a stoichiometric role in activating a DNA-dependent protein kinase-dependent pathway that leads to p53-dependent growth arrest.

Transcriptional regulation of activating transcription factor 3 involves the early growth response-1 gene.

Previously, our laboratory identified activating transcription factor 3 (ATF3) as up-regulated by nonsteroidal anti-inflammatory drugs using microarray analysis of mRNA from human colorectal cancer cells treated with sulindac sulfide. ATF3 is a transcription factor involved in cell growth, apoptosis, and invasion and is induced by a variety of anticancer and dietary compounds. However, the regulation of ATF3 by anticancer agents is not known. The promoter of ATF3 contains several transcription factor binding sites. We identified three putative Egr-1 binding sites in the promoter of ATF3 and report for the first time that the molecular mechanism responsible for the transcriptional regulation of ATF3 by two divergent pharmaceutical compounds, sulindac sulfide and troglitazone, involved the early growth response gene-1 (Egr-1). For example, overexpression of Egr-1 protein induced ATF3 mRNA 3.5-fold and transcriptional activity of an ATF3 promoter construct more than 20-fold. ATF3 and Egr-1 mRNA and protein and ATF3 promoter activity were induced by these compounds, whereas induction of ATF3 by these compounds was blocked by Egr-1 small interfering RNA. Sulindac sulfide and troglitazone regulated ATF3 promoter activity, which was suppressed when the two Egr-1 sites were mutated. These compounds induced phosphorylation of extracellular signal-regulated kinase1/2 (Erk1/2), whereas a dominant-negative inhibitor of mitogen-activate protein kinase kinase (MEK) 1 blocked the induction of ATF3. The MEK1/2 inhibitor PD98059 (2'-amino-3'-methoxyflavone) blocked the induction of ATF3 and Egr-1 mRNA expression and ATF3 promoter activity by these compounds. Therefore, this is a novel first report demonstrating that the expression of ATF3 occurs via Egr-1 downstream of Erk1/2.

The anti-invasive activity of cyclooxygenase inhibitors is regulated by the transcription factor ATF3 (activating transcription factor 3).

We previously showed that nonsteroidal anti-inflammatory drugs (NSAID) such as sulindac sulfide, which has chemopreventive activity, modulate the expression of several genes detected by microarray analysis. Activating transcription factor 3 (ATF3) was selected for further study because it is a transcription factor involved in cell proliferation, apoptosis, and invasion, and its expression is repressed in human colorectal tumors as compared with normal adjacent tissue. In this report, we show that ATF3 mRNA and protein expression are up-regulated in HCT-116 human colorectal cancer cells following treatment with NSAIDs, troglitazone, diallyl disulfide, and resveratrol. To ascertain the biological significance of ATF3, we overexpressed full-length ATF3 protein in the sense and antisense orientations. Overexpression of ATF3 in the sense orientation decreased focus formation in vitro and reduced the size of mouse tumor xenografts by 54% in vivo. Conversely, overexpression of antisense ATF3 was protumorigenic in vitro, however, not in vivo. ATF3 in the sense orientation did not modulate apoptosis, indicating another mechanism is involved. With microarray analysis, several genes relating to invasion and metastasis were identified by ATF3 overexpression and were confirmed by real-time reverse transcription-PCR, and several of these genes were modulated by sulindac sulfide, which inhibited invasion in these cells. Furthermore, overexpression of ATF3 inhibited invasion to a similar degree as sulindac sulfide treatment, whereas antisense ATF3 increased invasion. In conclusion, ATF3 represents a novel mechanism in which NSAIDs exert their anti-invasive activity, thereby linking ATF3 and its gene regulatory activity to the biological activity of these compounds.

The cyclooxygenase inhibitor indomethacin modulates gene expression and represses the extracellular matrix protein laminin gamma1 in human glioblastoma cells.

The induction of cyclooxygenase-2 (COX-2) expression is associated with more aggressive gliomas and poor survival. Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit COX activity and have antitumorigenic properties. In this report, our initial aim was to determine if indomethacin would alter gene expression as measured by suppression subtractive hybridization (SSH). Three up-regulated and four down-regulated genes by indomethacin treatment were identified. Laminin gamma1, an extracellular matrix molecule, was the most significantly repressed gene. The repression of laminin gamma1 by indomethacin was confirmed by Northern and Western blot analyses and occurred in a concentration- and time-dependent manner at the protein level. Among several NSAIDs tested, only sulindac sulfide and indomethacin suppressed laminin gamma1 protein expression, and this repression was observed in both COX-expressing and -deficient cell lines, suggesting that laminin gamma1 repression by COX inhibitors was independent of COX. Indomethacin, at a concentration that represses laminin gamma1, inhibited glioblastoma cell invasion that was partially restored with additional human laminin protein containing gamma1 chain. The repression of laminin gamma1 by NSAIDs may be related to attenuation of invasion of brain tumors. These findings are important in understanding the chemopreventive activity of some NSAIDs and could be relevant for designing therapeutic strategies against glioblastoma.

Neutrophils augment recovery of porcine ischemia-injured ileal mucosa by an IL-1beta- and COX-2-dependent mechanism.

Polymorphonuclear neutrophils (PMNs) play a critical role in intestinal mucosal injury and repair. To study effects of PMNs on acutely injured mucosa, we applied PMNs isolated from circulation or peritoneal fluid from animals with chemically induced peritonitis to ischemia-injured porcine ileal mucosa. In preliminary experiments, PMNs enhanced recovery of transepithelial electrical resistance (TER), and this action was inhibited by pretreatment with the nonselective cyclooxygenase (COX) inhibitor indomethacin. Because COX-2 is upregulated by inflammatory mediators such as IL-1beta, which is released by PMNs, we postulated that PMNs enhance recovery of ischemia-injured mucosa by a pathway involving IL-1beta and COX-2. Application of 5 x 10(6) PMNs to the serosal surface of ischemia-injured mucosa significantly enhanced recovery of TER (P < 0.05), an effect that was inhibited by the selective COX-2 inhibitor NS-398 (5 microM) and by an IL-1beta receptor antagonist (0.1 mg/ml). Addition of 10 ng/ml IL-1beta to the serosal surface of injured tissues caused a significant increase in TER (P < 0.05) that was inhibited by pretreatment with NS-398. Western blot analysis of mucosal homogenates revealed dramatic upregulation of COX-2 in response to IL-1beta or peritoneal PMNs, and the latter was inhibited by an IL-1beta receptor antagonist. Real-time PCR revealed that increased mRNA COX-2 expression preceded increased COX-2 protein expression in response to IL-1beta. We concluded that PMNs augment recovery of TER in ischemia-injured ileal mucosa via IL-1beta-dependent upregulation of COX-2.

15-Lipoxygenase-1 has anti-tumorigenic effects in colorectal cancer.

The localization of 15-lipoxygenase-1 (15-LO-1) in human colorectal carcinoma and normal adjacent tissue was examined using immunohistochemistry. In normal tissues, 15-LO-1 was strongly localized in the mucosal epithelium. Conversely, in tumor tissues, staining for 15-LO-1 was dispersed throughout the tissue, weak in neoplastic epithelium, and strong in stromal inflammatory cells. The addition of 50 microM 13(S)-hydroxyeicosatetraenoic acid (HODE), resulted in decreased cell proliferation after 72 h, but lower concentrations (5 or 10 microM) had no effect compared to vehicle treated Caco-2 cells. In addition, 13(S)-HODE had no effect on apoptosis or differentiation of the Caco-2 cells. Microarray analyses of RNA from Caco-2 cells treated with 5 microM 13(S)-HODE revealed changes in 17 genes. HCT-116 colorectal cells were stably transfected with 15-LO-1. In athymic nude mice, transplantable tumors derived from 15-LO-1 HCT-116 cells were smaller than tumors derived from vector HCT-116 cells. These data demonstrate that 13(S)-HODE induces changes in gene expression and has anti-tumorigenic effects.

Gene modulation by Cox-1 and Cox-2 specific inhibitors in human colorectal carcinoma cancer cells.

Cox-1 and Cox-2 specific inhibitors exert chemo-preventative activity. However, the exact mechanisms for this activity remain unclear. Increasing evidence suggests that non-steroidal anti-inflammatory drugs regulate gene expression, which may be responsible, in part, for this activity. In this study, human colorectal carcinoma HCT-116 cells were treated with the Cox-1 specific inhibitor SC-560 and the Cox-2 specific inhibitor SC-58125 to evaluate their ability to induce apoptosis, inhibit cell proliferation, inhibit growth on soft agar and modulate gene expression. The Cox-1 specific inhibitor, SC-560 significantly induced apoptosis and inhibited the growth of HCT-116 cells on soft agar, an in vitro assay for tumorigenicity. SC-58125 moderately induced apoptosis and inhibited growth on soft agar at higher concentrations than were required for SC-560. Previously, we reported that the potent chemo-preventative drug sulindac sulfide altered the expression of eight genes including several transcription factors that may be linked to this drug's chemo-preventative activity. HCT-116 cells were treated with various concentrations of SC-560 or SC-58125 and changes in the expression of these eight genes were determined by real-time reverse transcription- polymerase chain reaction. SC-560 modulated mRNA expression of the eight genes studied. In contrast, SC-58125 required approximately 5-10-fold higher concentrations to achieve similar degrees of gene modulation in six of eight genes. Changes in protein expression by SC-560 also occurred for five of these genes with antibodies available (NAG-1, ATF3, C/EBPbeta, MAD2 and MSX1). In conclusion, this is the first report to suggest that like sulindac sulfide, the Cox-1 specific inhibitor SC-560 appears to elicit chemo-preventative activity by altering gene expression, while the chemo-preventative effects of SC-58125 are complex and probably work through these and other mechanisms, such as the inhibition of Cox-2.

Gene modulation by the cyclooxygenase inhibitor, sulindac sulfide, in human colorectal carcinoma cells: possible link to apoptosis.

The mechanisms underlying the anti-tumorigenic properties of cyclooxygenase inhibitors are not well understood. One novel hypothesis is alterations in gene expression. To test this hypothesis sulindac sulfide, which is used to treat familial adenomatous polyposis, was selected to detect gene modulation in human colorectal cells at physiological concentrations with microarray analysis. At micromolar concentrations, sulindac sulfide stimulated apoptosis and inhibited the growth of colorectal cancer cells on soft agar. Sulindac sulfide (10 microm) altered the expression of 65 genes in SW-480 colorectal cancer cells, which express cyclooxygenase-1 but little cyclooxygenase-2. A more detailed study of 11 genes revealed that their expression was altered in a time- and dose-dependent manner as measured by real-time RT-PCR. Northern analysis confirmed the expression of 9 of these genes, and Western analysis supported the conclusion that sulindac sulfide altered the expression of these proteins. Cyclooxygenase-deficient HCT-116 cells were more responsive to sulindac sulfide-induced gene expression than SW-480 cells. However, this response was diminished in HCT-116 cells overexpressing cyclooxygenase-1 compared with normal HCT-116 cells suggesting the presence of cyclooxygenase attenuates this response. However, prostaglandin E2, the main product of cyclooxygenase, only suppressed the sulindac sulfide-induced expression of two genes, with little known biological function while it modulated the expression of two more. The most likely explanation for this finding is the metabolism of sulindac sulfide to inactive metabolites by the peroxidase activity of cyclooxygenase. In conclusion, this is the first report showing sulindac sulfide, independent of cyclooxygenase, altered the expression of several genes possibly linked to its anti-tumorigenic and pro-apoptotic activity.

Diallyl disulfide (DADS) induces the antitumorigenic NSAID-activated gene (NAG-1) by a p53-dependent mechanism in human colorectal HCT 116 cells.

Garlic is appealing as an anti-carcinogenic agent due to its ability to induce apoptosis in vitro and inhibit the formation and growth of tumors in animals in vivo. Diallyl disulfide (DADS) is a constituent of garlic that suppresses neoplastic cell growth and induces apoptosis. We examined the effects of DADS on various cancer cell lines to better understand its effect on apoptosis and apoptosis-related genes. The nonsteroidal anti-inflammatory drug (NSAID)-activated gene (NAG-1) has proapoptotic and antitumorigenic activities and is upregulated by anticancer agents such as NSAIDs. In this study, human colorectal HCT-116 (wild-type p53), HCT-15 (p53 mutant) and human prostate PC-3 (p53 mutant) cells were exposed to DADS. DADS inhibited cell proliferation in all cell lines albeit to a lesser extent in HCT-15 and PC-3 cells at 11.5 and 23 micromol/L. In HCT-116 cells, DADS induced p53 and NAG-1 in a dose-dependent manner and the induction of p53 preceded that of NAG-1. In HCT-116 cells, NAG-1 protein expression was increased 2.4-fold +/- 0.6 at 4.6 micromol/L and 6.1-fold +/- 1.7 at 23 micromol/L DADS, whereas p53 was induced 1.5-fold +/- 0.1 and 2.3-fold +/- 0.4. DADS did not induce NAG-1 or p53 in p53 mutant cell lines; however, NAG-1 expression was induced by sulindac sulfide. HCT-116 cells treated with 4.6 and 23 micromol/L DADS resulted in a 1.9- and 2.9-fold increase in apoptosis, respectively. In contrast, 23 micromol/L DADS induced apoptosis only 1.8-fold in HCT-15 cells and not at all in PC-3 cells. Thus, DADS-induced apoptosis and NAG-1 protein expression appear to occur via p53.