Sulindac Sulfide Treatment Research Articles

Novel thrombospondin-1 transcript exhibits distinctive expression and activity in thyroid tumorigenesis.

Thrombospondin 1 (TSP1) is known for its cell-specific functions in cancer progression, such as proliferation and migration. It contains 22 exons that may potentially produce several different transcripts. Here, we identified TSP1V as a novel TSP1-splicing variant produced by intron retention (IR) in human thyroid cancer cells and tissues. We observed that TSP1V functionally inhibited tumorigenesis contrary to TSP1 wild-type, as identified in vivo and in vitro. These activities of TSP1V are caused by inhibiting phospho-Smad and phospho-focal adhesion kinase. Reverse transcription polymerase chain reaction and minigene experiments revealed that some phytochemicals/non-steroidal anti-inflammatory drugs enhanced IR. We further found that RNA-binding motif protein 5 (RBM5) suppressed IR induced by sulindac sulfide treatment. Additionally, sulindac sulfide reduced phospho-RBM5 levels in a time-dependent manner. Furthermore, trans-chalcone demethylated TSP1V, thereby preventing methyl-CpG-binding protein 2 binding to TSP1V gene. In addition, TSP1V levels were significantly lower in patients with differentiated thyroid carcinoma than in those with benign thyroid nodule, indicating its potential application as a diagnostic biomarker in tumor progression.

Cyclin G2, a novel target of sulindac to inhibit cell cycle progression in colorectal cancer

Sulindac has shown significant clinical benefit in preventing colorectal cancer progression, but its mechanism of action has not been fully elucidated. We have found that sulindac sulfide (SS) is able to inhibit cell cycle progression in human colorectal cancer cells, particularly through G1 arrest. To understand the underlying mechanisms of sulindac inhibitory activity, we have demonstrated that Cyclin G2 up-regulation upon SS treatment can substantially delay cell cycle progression by enhancing the transcriptional activity of FOXO3a in human colorectal tumor cells. MiR-182, an oncogenic microRNA known to inhibit FOXO3a gene expression, is also involved in the suppressive effect of SS on cell cycle progression. This process begins with the down-regulation of miR-182, followed by the enhancement of FOXO3a transcriptional activity and the up-regulation of Cyclin G2. To further determine the clinical utility of this axis, we analyzed the expression of miR-182/FOXO3a/Cyclin G2 in human colorectal tumor samples. Our results show not only that there are significant differences in miR-182/FOXO3a/Cyclin G2 between tumors and normal tissues, but also that the synergetic effect of miR-182 and FOXO3a is associated with predicting tumor progression. Our study demonstrates a novel mechanistic axis consisting of miR-182/FOXO3a/Cyclin G2 that mediates sulindac inhibition of cell cycle progression.

SULINDAC SULFIDE INDUCES APOPTOSIS OF BREAST CANCER CELLS ACTIVATED BY CO-CULTURE MODEL: A MECHANISM MEDIATED BY RAS SIGNALING PATHWAY.

Macrophage–epithelial interactions play a crucial role in breast cancer progression and metastasis. Tumor associated macrophages (TAMs) provide the tumor microenvironment with multiple inflammatory mediators that stimulate several signaling pathways which participate in survival and growth of cancer cells, thus they have become an attractive target for chemotherapeutic agents. Sulindac Sulfide is one of NSAIDs and its anticancer activity in prevention of tumor incidence and progression has been documented in several types of cancer. The aim of this study was to test the effects of media conditioned by U937 human monocytes (U937-CM) as well as the effect of Sulindac Sulfide on the survival oncogenic expression and apoptosis of MCF-7 cells. The results showed that U937-CM enhanced MCF-7 survival and proliferation through significant increase in Ras expression which resulted in upregulation of anti-apoptotic protein Bcl-2 and down regulation of tumor suppressor Par-4. Sulindac Sulfide treatment inhibited the growth of induced cells by inhibition of Ras expression and its downstream signaling. Suppression of Ras is accompanied by activation of apoptotic machinery through down regulation of Bcl-2 and upregulation of Par-4 that resulted in significant activation of caspase-3.The current data demonstrate that Sulindac Sulfide succeeded in suppressing the paracrine effect of TAMs on breast cancer cells suggesting the promising role in breast cancer treatment.

Abstract 4790: Let-7 mediates sulindac inhibition of tumor cell transformation

Abstract Cell transformation is the process in which normal cells acquire the properties of cancer. Sulindac Sulfide (SS) is one of non-steroidal anti-inflammatory drugs (NSAIDs), and its anticancer activity in prevention of tumor occurrence and progression has been documented in various human cancers. In this study, we observed the anti-transformative activity of SS in NIH/3T3 cells by using a two-stage transformation assay induced by the carcinogens, 3-methylcholanthrene (MCA) and 2-O-tetradecanoylphorbol-13-acetate (TPA) in which foci formation is used to characterize the transformed cells. We found that SS could inhibit foci formation by attenuation of TPA’s promotive effect. K-Ras was upregulated in the transformed cells, while SS treatment could selectively inhibit the growth of transformed cell in which K-Ras was suppressed significantly. Loss-of-function study demonstrated that knockdown of K-Ras in NIH/3T3 cells could significantly reduce foci formation, which support that K-Ras might be one of the key targets involved in SS inhibiting tumor cell transformation. When studying the mechanism by which SS regulates K-Ras, we triggered tumor suppressor miRNA, let-7b, which was previously reported to target K-Ras and repress its expression through the post-transcriptional or translational modulation. We found that let-7b could be upregulated in the transformed cells by SS treatment. However, the inhibitory effect of SS on foci formation was significantly attenuated when let-7b was knocked down by CRISPR/Cas9. In parallel, K-Ras maintained static expression in the transformed cells, regardless of SS treatment. Meanwhile, the overexpression of endogenous let-7b can significantly inhibit tumor transformation. In addition, we demonstrated that SS could suppress the expression of Lin28 that interacts with let-7 through a documented feedback loop. Given our previous studies reporting that SS was able to inhibit the transcriptional activity of NF-κB that is involved in regulation of Lin28 expression, our study demonstrates that the NF-κB/Lin28/Let-7/K-Ras axis addresses a new mechanism responsible for anti-transformative activity of SS. These results will provide novel insights into understanding the mechanisms involved in tumorigenesis and benefiting identification of new targets for drug development in prevention of tumor progression. Citation Format: Zhipin Liang, Bin Yi, Hongyou Zhao, Ruixia Ma, Yaguang Xi. Let-7 mediates sulindac inhibition of tumor cell transformation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4790. doi:10.1158/1538-7445.AM2017-4790

Abstract 1480: Mir-182 is involved in sulindac anticancer activity in colon cancer

Abstract Nonsteroidal anti-inflammatory drugs (NSAIDs) display promising antineoplastic activity in many human solid tumors including colorectal cancer. Previous studies reported that sulindac sulfide (SS) can inhibit the growth of tumor cells through cyclooxygenase-2 (COX-2) dependent or independent pathways. Obviously, COX-2 independent pathway involves a low toxic property to support the clinical potential for using sulindac as a chemoprevention drug. However, the molecular mechanisms responsible for COX-2 independent pathway have not been completely elucidated. In this project, we employed two human colon cancer cell lines, referred to HCT116 and HT29. HCT116 cells are characterized for low COX-2 expression, while HT29 cells show relatively high COX-2 expression. We found that SS could unbiasedly inhibit the growth of HCT116 and HT29 cells by arresting cells in G1/G2 phases. CyclinG2 was found to be upregulated in response to SS treatment. FOXO3a has been reported to regulate CyclinG2 expression at the transcriptional level. Our results demonstrate that SS could also upregulate FOXO3a. By using the loss-of-function strategy, we found that SS could not efficiently upregulate CyclinG2 and lead to cell cycle arrest in cells with FOXO3a knockdown. In addition, we studied the mechanism that could be involved in SS regulation of FOXO3a. Our previous studies reported that a panel of miRNAs could be altered by SS treatment in both colon and breast cancer cells. In the downregulated miRNA list, we found that miR-182 could potentially target FOXO3a. By using luciferase assay, we validated the direct regulation of miR-182 on the expression of FOXO3a. When miR-182 was downregulated, SS could neither efficiently upregulate the expression of FOXO3a nor inhibit cell growth as it did in control cells. We gained highly consistent results in HT29 and HT29 cells with COX-2 knockdown. Therefore, our study demonstrates a pathway consisting of miR-182/ FOXO3a/ CyclinG2 as a novel mechanism responsible for SS anticancer activity in colon cancer, which may imply a COX-2 independent pathway. Citation Format: Hongyou Zhao, Bin Yi, Zhipin Liang, Ruixia Ma, Yaguang Xi. Mir-182 is involved in sulindac anticancer activity in colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1480. doi:10.1158/1538-7445.AM2017-1480

Abstract B31: Sulindac inhibition of tumor cell transformation

Abstract Cell transformation is the process in which normal cells acquire the properties of cancer. Sulindac Sulfide (SS) is one of non-steroidal anti-inflammatory drugs (NSAIDs), and its anticancer activity in prevention of tumor occurrence and progression has been documented in various human cancers. In this study, we observed the anti-transformative activity of SS in NIH/3T3 cells by using a two-stage transformation assay induced by the carcinogens, 3-methylcholanthrene (MCA) and 2-O-tetradecanoylphorbol-13-acetate (TPA) in which foci formation is used to characterize the transformed cells. We found that SS could inhibit foci formation by attenuation of TPA's promotive effect. K-Ras was upregulated in the transformed cells, while SS treatment could selectively inhibit the growth of transformed cell in which K-Ras was suppressed significantly. Loss-of-function study demonstrated that knockdown of K-Ras in NIH/3T3 cells could significantly reduce foci formation, which support that K-Ras might be one of the key targets involved in SS inhibiting tumor cell transformation. When studying the mechanism by which SS regulates K-Ras, we triggered two tumor suppressor miRNAs, let-7b and let-7g, which were previously reported to target K-Ras and repress its expression through the post-transcriptional or translational modulation. We found that let-7b and let-7g could be upregulated in the transformed cells by SS treatment. However, the inhibitory effect of SS on foci formation was significantly attenuated when let-7b and let-7g were knocked down by CRISPR/cas9. In parallel, K-Ras maintained static expression in the transformed cells, regardless of SS treatment. These results suggest that upregulation of let-7b and let-7g is the key step for SS to inhibit tumor transformation. In addition, we demonstrated that SS could suppress the expression of Lin28 that interacts with let-7 through a documented feedback loop. Given our previous studies reporting that SS was able to inhibit the transcriptional activity of NF-kappaB that is involved in regulation of Lin28 expression, our study demonstrates that the NF-kappaB/Lin28/Let-7/K-Ras axis addresses a new mechanism responsible for anti-transformative activity of SS. These results will provide novel insights into understanding the mechanisms involved in tumorigenesis and benefiting identification of new targets for drug development in prevention of tumor progression. This study is supported by the NIH/NCI R01 Grant (1R01CA192395) and the American Cancer Society Research Scholar Grant (RSG-13-265-01-RMC). Citation Format: Zhipin Liang, Xiangling Feng, Hong Chang, Bin Yi, Ruixia Ma, Yaguang Xi. Sulindac inhibition of tumor cell transformation. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Cancer Cell Cycle - Tumor Progression and Therapeutic Response; Feb 28-Mar 2, 2016; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(11_Suppl):Abstract nr B31.

HIF1α deficiency reduces inflammation in a mouse model of proximal colon cancer.

ABSTRACTHypoxia-inducible factor 1α (HIF1α) is a transcription factor that regulates the adaptation of cells to hypoxic microenvironments, for example inside solid tumours. Stabilisation of HIF1α can also occur in normoxic conditions in inflamed tissue or as a result of inactivating mutations in negative regulators of HIF1α. Aberrant overexpression of HIF1α in many different cancers has led to intensive efforts to develop HIF1α-targeted therapies. However, the role of HIF1α is still poorly understood in chronic inflammation that predisposes the colon to carcinogenesis. We have previously reported that the transcription of HIF1α is upregulated and that the protein is stabilised in inflammatory lesions that are caused by the non-steroidal anti-inflammatory drug (NSAID) sulindac in the mouse proximal colon. Here, we exploited this side effect of long-term sulindac administration to analyse the role of HIF1α in colon inflammation using mice with a Villin-Cre-induced deletion of Hif1α exon 2 in the intestinal epithelium (Hif1αΔIEC). We also analysed the effect of sulindac sulfide on the aryl hydrocarbon receptor (AHR) pathway in vitro in colon cancer cells. Most sulindac-treated mice developed visible lesions, resembling the appearance of flat adenomas in the human colon, surrounded by macroscopically normal mucosa. Hif1αΔIEC mice still developed lesions but they were smaller than in the Hif1α-floxed siblings (Hif1αF/F). Microscopically, Hif1αΔIEC mice had significantly less severe colon inflammation than Hif1αF/F mice. Molecular analysis showed reduced MIF expression and increased E-cadherin mRNA expression in the colon of sulindac-treated Hif1αΔIEC mice. However, immunohistochemistry analysis revealed a defect of E-cadherin protein expression in sulindac-treated Hif1αΔIEC mice. Sulindac sulfide treatment in vitro upregulated Hif1α, c-JUN and IL8 expression through the AHR pathway. Taken together, HIF1α expression augments inflammation in the proximal colon of sulindac-treated mice, and AHR activation by sulindac might lead to the reduction of E-cadherin protein levels through the mitogen-activated protein kinase (MAPK) pathway.

Abstract B01: Nonsteroidal anti-inflammatory drugs selectively kill colon cancer cells through a synthetic lethal interaction

Abstract Colorectal tumorigenesis is initiated by genetic alterations in the APC tumor suppressor pathway. Nonsteroidal anti-inflammatory drugs (NSAIDs) can inhibit tumorigenesis by eliminating APC-deficient early pre-malignant cells. However, how and why NSAIDs selectively kill APC-deficient cells while leaving normal cells unharmed has remained elusive. We demonstrate that the NSAID sulindac sulfide initiates death receptor signaling in both colon cancer cells and NCM356 normal colonic epithelial cells, but only induces apoptosis in colon cancer cells and APC-deficient NCM356 cells. Loss of APC leads to down-regulation of c-FLIP, largely due to increased expression of c-Myc. Sulindac sulfide treatment further enhanced down-regulation of c-FLIP. Loss of c-FLIP allows death receptor signaling to progress through caspase-8 and BID cleavage, leading to amplified apoptotic signaling via the mitochondria. Depleting APC in NCM356 cells recapitulates this pathway, and is sufficient to lead to sulindac sulfide-induced apoptosis. Based on these data, we conclude that NSAIDs selectively kill colon cancer cells through a synthetic lethal interaction between death receptor signaling and APC deficiency. These findings potentially open a door to development of more effective colon cancer prevention and treatment strategies. Citation Format: Brian Leibowitz, Mary P. Moyer, Jian Yu, Lin Zhang. Nonsteroidal anti-inflammatory drugs selectively kill colon cancer cells through a synthetic lethal interaction. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr B01.

Abstract LB-172: MicroRNAs mediate the inhibition of tumor cell invasion by sulindac

Abstract A large body of evidence indicates that nonsteroidal anti-inflammatory drugs (NSAIDs) have strong antineoplastic properties against a broad range of cancer types in early or late stage animal models of tumorigenesis. However, their use in humans for chemoprevention is limited by toxicities resulting from cyclooxygenase (COX) inhibition and the suppression of physiologically important prostaglandins. The anticancer properties of NSAIDs are generally attributed to the suppression of tumor cell proliferation and induction of apoptosis by inhibiting cyclooxygenase (COX), although numerous studies suggest that a COX-independent mechanism may either contribute to or be fully responsible for their activity. The effect of NSAIDs on tumor cell motility and invasion has not been well studied, despite clinical studies showing that certain NSAIDs can significantly increase the survival of patients with metastatic disease. Our results show that the NSAID, sulindac sulfide (SS) can potently inhibit the motility of human breast (MDA-MB-231 and SUM1315) and colorectal (HCT116 and HT29) tumor cells by a mechanism that is distinct from its tumor cell growth and COX inhibitory activity. To study the molecular basis for this activity, we investigated the involvement of microRNA (miRNA). A total of 132 miRNAs were found to be altered in response to SS treatment including miR-10b, miR-17, miR-21, and miR-9, which have been previously implicated in tumor invasion and metastasis. We confirmed that these miRNAs can stimulate tumor cell invasion and show that SS can attenuate their invasive effects (individual and cooperative) by down-regulating their expression. Employing luciferase and chromatin immunoprecipitation (ChIP) assays, NF-κB was found to bind the promoters of all four miRNAs to regulate their expression at the transcriptional level. Results from the immunofluorescence and Western Blotting assays show that SS can inhibit the translocation of NF-κB to the nucleus by decreasing the phosphorylation of IKKβ and IκB, which leads to down-regulation of these miRNAs. Analysis of the promoter sequences of the miRNAs suppressed by SS revealed that 81 of 115 sequences contained NF-κB binding sites. Our results demonstrate that SS can inhibit tumor cell invasion by suppressing NF-κB mediated transcription of miRNAs, which illustrate the mechanistic basis of this novel anticancer property of sulindac on tumor progression and metastasis. We expect these results are capable of supporting future clinical trials of sulindac in patients with metastatic disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-172. doi:1538-7445.AM2012-LB-172

Sulindac inhibits tumor cell invasion by suppressing NF-κB-mediated transcription of microRNAs

Nonsteroidal anti-inflammatory drugs (NSAIDs) have been widely reported to display strong efficacy for cancer chemoprevention, although their mechanism of action is poorly understood. The most well documented effects of NSAIDs include inhibition of tumor cell proliferation and induction of apoptosis, but their effect on tumor cell invasion has not been well studied. Here we show that the NSAID, sulindac sulfide (SS) can potently inhibit the invasion of human MDA-MB-231 breast and HCT116 colon tumor cells in vitro at concentrations less than those required to inhibit tumor cell growth. To study the molecular basis for this activity, we investigated the involvement of microRNA (miRNA). A total of 132 miRNAs were found to be altered in response to SS treatment including miR-10b, miR-17, miR-21, and miR-9, which have been previously implicated in tumor invasion and metastasis. We confirmed that these miRNA can stimulate tumor cell invasion and show that SS can attenuate their invasive effects by down-regulating their expression. Employing luciferase and chromatin immunoprecipitation assays, NF-κB was found to bind the promoters of all four miRNAs to suppress their expression at the transcriptional level. We show that SS can inhibit the translocation of NF-κB to the nucleus by decreasing the phosphorylation of IKKβ and IκB. Analysis of the promoter sequences of the miRNAs suppressed by SS revealed that 81 of 115 sequences contained NF-κB binding sites. These results show that SS can inhibit tumor cell invasion by suppressing NF-κB mediated transcription of miRNAs.

Sulindac sulfide induces autophagic death in gastric epithelial cells via Survivin down-regulation: A mechanism of NSAIDs-induced gastric injury

Sulindac sulfide, a nonsteroidal anti-inflammatory drug (NSAID), has anti-tumorigenic and anti-inflammatory activities, but causes gastric mucosal damage. NSAIDs cause gastric injury in part by down-regulation of Survivin, an apoptosis inhibitor, resulting in apoptosis induction. Autophagy is a process that promotes cellular health by destroying unwanted cellular materials. Excessive autophagy induction could lead to a non-apoptotic cell death (autophagic cell death). The present study showed that sulindac sulfide at a physiological concentration also induces autophagic death in human gastric epithelial AGS and rat gastric epithelial RGM-1 cells, and that Survivin down-regulation is a mechanism involved: Sulindac sulfide treatment increased LC3b-II and APG7 levels and cytosolic vacuole formation, indications of autophagy induction, in AGS and RGM-1 cells. Sulindac sulfide treatment induced AGS and RGM-1 cell death, which was significantly reduced by pretreatment with the autophagy inhibitors 3-methyladenine and chloroquine, indicating that sulindac sulfide induced autophagic cell death. Stable overexpression of Survivin in RGM-1 cells did not inhibit the induction of LC3b-II levels or vacuole formation by sulindac sulfide, but significantly reduced the resulting cell death, suggesting that Survivin may inhibit autophagic cell death downstream of LC3b-II induction and vacuole formation. Indeed, siRNA depletion of LC3b in AGS cells inhibited the down-regulation of Survivin levels and the induction of cell death by sulindac sulfide, confirming that down-regulation of Survivin occurs in the autophagy pathway downstream of LC3b-II induction by sulindac sulfide. Induction of Survivin-dependent autophagic cell death is a novel mechanism by which sulindac sulfide induces gastric mucosal injury.

Alternative splicing of Kruppel‐like factor 4 plays a pivotal role in human tumorigenesis

Most human genes undergo alternative splicing, and many abnormal splicing processes are associated with disease including cancer. However, the relationship between alternative splicing and tumorigenesis is not well‐understood. Here, we found that novel Kruppel‐like factor 4 (KLF4) splicing variants produced by exon skipping could play a role during tumorigenesis in several human cancer cell lines. The human KLF4 gene produces at least three splicing variants, and one of them, small KLF4 (KLF4S), antagonizes large KLF4 (KLF4L) activity. To further investigate KLF4 splicing mechanism, we developed the KLF4 minigene and mutation of the 3′ splice site in the KLF4 intron 2 plays a pivotal role in the KLF4 splicing ratio. Interestingly, sulindac sulfide treatment restored KLF4L transcript and phosphorylation of the RNA binding motif 5 (RBM5) was decreased in HCT‐116 cells. Furthermore, mutation of the RBM5 phosphorylation site (S69A) decreased the KLF4 splicing event. Over‐expression and RBM5 siRNA also changed the KLF4 splicing ratio. Our data suggest that phosphorylation of RBM5 and the cis‐acting element in the 3′ splice site regulate alternative splicing of KLF4, which is altered during tumorigenesis.

Generation of Aβ38 and Aβ42 Is Independently and Differentially Affected by Familial Alzheimer Disease-associated Presenilin Mutations and γ-Secretase Modulation

Alzheimer disease amyloid beta-peptide (Abeta) is generated via proteolytic processing of the beta-amyloid precursor protein by beta- and gamma-secretase. Gamma-secretase can be blocked by selective inhibitors but can also be modulated by a subset of non-steroidal anti-inflammatory drugs, including sulindac sulfide. These drugs selectively reduce the generation of the aggregation-prone 42-amino acid Abeta(42) and concomitantly increase the levels of the rather benign Abeta(38). Here we show that Abeta(42) and Abeta(38) generation occur independently from each other. The amount of Abeta(42) produced by cells expressing 10 different familial Alzheimer disease (FAD)-associated mutations in presenilin (PS) 1, the catalytic subunit of gamma-secretase, appeared to correlate with the respective age of onset in patients. However, Abeta(38) levels did not show a negative correlation with the age of onset. Modulation of gamma-secretase activity by sulindac sulfide reduced Abeta(42) in the case of wild type PS1 and two FAD-associated PS1 mutations (M146L and A285V). The remaining eight PS1 FAD mutants showed either no reduction of Abeta(42) or only rather subtle effects. Strikingly, even the mutations that showed no effect on Abeta(42) levels allowed a robust increase of Abeta(38) upon treatment with sulindac sulfide. Similar observations were made for fenofibrate, a compound known to increase Abeta(42) and to decrease Abeta(38). For mutants that predominantly produce Abeta(42), the ability of fenofibrate to further increase Abeta(42) levels became diminished, whereas Abeta(38) levels were altered to varying extents for all mutants analyzed. Thus, we conclude that Abeta(38) and Abeta(42) production do not depend on each other. Using an independent non-steroidal anti-inflammatory drug derivative, we obtained similar results for PS1 as well as for PS2. These in vitro results were confirmed by in vivo experiments in transgenic mice expressing the PS2 N141I FAD mutant. Our findings therefore have strong implications on the selection of transgenic mouse models used for screening of the Abeta(42)-lowering capacity of gamma-secretase modulators. Furthermore, human patients with certain PS mutations may not respond to gamma-secretase modulators.

Growth compensatory role of sulindac sulfide-induced thrombospondin-1 linked with ERK1/2 and RhoA GTPase signaling pathways

Previously, we reported that non-steroidal anti-inflammatory drugs (NSAIDs) suppress cellular invasion which was mediated by thrombospondin-1 (TSP-1). As the extending study of the previous observation, we investigated the effect of NSAID-induced TSP-1 on the cellular growth and its related signaling transduction of the TSP-1 production. Among diverse NSAIDs, sulindac sulfide was most potent of inducing the human TSP-1 protein expression. Functionally, induced TSP-1 expression was associated with the growth-compensatory action of NSAID. TSP-1 expression was also elevated by mitogenic signals of ERK1/2 and RhoA GTPase pathway which had also growth-promotive capability after sulindac sulfide treatment. These findings suggest the possible mechanism through which tumor cells can survive the chemopreventive action of NSAIDs or the normal epithelium can reconstitute after NSAID-mediated ulceration in a compensatory way.

Insensitivity to Aβ42-lowering Nonsteroidal Anti-inflammatory Drugs and γ-Secretase Inhibitors Is Common among Aggressive Presenilin-1 Mutations

Abeta42-lowering nonsteroidal anti-inflammatory drugs (NSAIDs) constitute the founding members of a new class of gamma-secretase modulators that avoid side effects of pan-gamma-secretase inhibitors on NOTCH processing and function, holding promise as potential disease-modifying agents for Alzheimer disease (AD). These modulators are active in cell-free gamma-secretase assays indicating that they directly target the gamma-secretase complex. Additional support for this hypothesis was provided by the observation that certain mutations in presenilin-1 (PS1) associated with early-onset familial AD (FAD) change the cellular drug response to Abeta42-lowering NSAIDs. Of particular interest is the PS1-DeltaExon9 mutation, which provokes a pathogenic increase in the Abeta42/Abeta40 ratio and dramatically reduces the cellular response to the Abeta42-lowering NSAID sulindac sulfide. This FAD PS1 mutant is unusual as a splice-site mutation results in deletion of amino acids Thr(291)-Ser(319) including the endoproteolytic cleavage site of PS1, and an additional amino acid exchange (S290C) at the exon 8/10 splice junction. By genetic dissection of the PS1-DeltaExon9 mutation, we now demonstrate that a synergistic effect of the S290C mutation and the lack of endoproteolytic cleavage is sufficient to elevate the Abeta42/Abeta40 ratio and that the attenuated response to sulindac sulfide results partially from the deficiency in endoproteolysis. Importantly, a wider screen revealed that a diminished response to Abeta42-lowering NSAIDs is common among aggressive FAD PS1 mutations. Surprisingly, these mutations were also partially unresponsive to gamma-secretase inhibitors of different structural classes. This was confirmed in a mouse model with transgenic expression of the PS1-L166P mutation, in which the potent gamma-secretase inhibitor LY-411575 failed to reduce brain levels of soluble Abeta42. In summary, these findings highlight the importance of genetic background in drug discovery efforts aimed at gamma-secretase, suggesting that certain AD mouse models harboring aggressive PS mutations may not be informative in assessing in vivo effects of gamma-secretase modulators and inhibitors.

인간 대장암 세포주에서 sulindac sulfide 처리에 의해 차별적으로 발현되는 유전자 군의 분석

본 연구에서는 NSAID계 약물인 sulindac, sulindac sulfone, 그리고 sulindac sulfide 처리에 의한 암세포 생존율에 미치는 영향을 확인하기 위하여, 인간 대장암 세포주인 HCTl16에 각각 10 <TEX>${\mu}M$</TEX>의 NSAID들을 처리하였다. 처리한약물 중 sulindac sulfide에 의한 암세포 생존율이 가장 높게 감소하는 것으로 MTS assay 결과 확인되었다. 또한 sulindac sulfide의 처리 농도가 증가됨에 따라 세포 생존율이 감소하는 것으로 확인되었다. Sulindac sulfide의 처리에 따른 이러한 암 세포 사멸의 분자생물학적 기전을 이해하기 위하여, oligo DNA microarray 실험을 수행하였다. 그 결과, 10 <TEX>${\mu}M$</TEX>의 sulindac sulfide의 처리에 의해 2배 이상 발현이 증가되는 유전자가 23개 확인되었고, 반대로 2배 이상 발현이 감소되는 유전자가 33개 확인되었다. 증가되는 유전자중 3개(NAG-1, DDIT3, PCK2)를 선택하여, RT-PCR과 real-time PCR을 수행하였다. 그 결과 두 실험 모두 DNA microarray 실험결과와 동일하게 발현이 증가되었다. 이 중 sulindac, sulindac sulfone, sulindac sulfide에 의 한 NAG-1 유전자의 발현변화를 RT-PCR과 real-time PCR 방법으로 확인한 결과, sulindac sulfide에 의한 암 억제유전자인 NAG-1의 발현이 가장 많이 발현되었다. 이러한 연구결과는 세포생존율 결과와 비교하였을 때, NAG-1의 높은 발현과 암 세포 생존율의 감소가 관련이 있음을 간접적으로 시사한다. 따라서 이들 연구결과는 sulindac sulfide에 의한 화학적 암 예방법의 분자생물학적 기전을 이해하는데 도움을 줄 것으로 생각한다. To investigate whether sulindac, sulindac sulfone, and sulindac sulfide could affect cancer cell viabilities, human colorectal HCTl16 cells were treated with 10 <TEX>${\mu}M$</TEX> of each NSAID. Among treated NSAms, sulindac sulfide dramatically decreased the cell viabilities detected by MTS and the cytotoxic effect showed dose-dependent manner. To understand the molecular mechanism of cell death in response to sulindac sulfide treatment, we performed oligo DNA microarray analysis. We found that 23 genes were up-regulated more than 2 folds, whereas 33 genes were down-regulated more than 2 folds by treatment of 10 <TEX>${\mu}M$</TEX> sulindac sulfide. Among the up-regulated genes, we selected 3 genes (NAG-1, DDIT3, PCK2) and performed RT-PCR and quantitative real-time PCR to cofirm microarray data. The results of RT-PCR and real-time PCR were highly accorded with those of microarray experiment. As NAG-1 is well-known gene as tumor suppressor, we detected changes of NAG-1 expression by 10 <TEX>${\mu}M$</TEX> of sulindac, sulindac sulfone, and sulindac sulfide. The results of RT-PCR and quantitacve real-time PCR indicated that sulindac sulfide was the strongest inducer of NAG-1 among treated NSAIDS. This result implies that induction of NAG-1 by sulindac sulfide plays important role in cell death of colorectal cancer. Overall, we speculate that these results may be helpful in understanding the molecular mechanism of the cancer chemoprevention by sulindac sulfide in human colorectal cancer.

Relative non‐steroidal anti‐inflammatory drug (NSAID) antiproliferative activity is mediated through p21‐induced G1 arrest and E2F inhibition

This study was performed to compare the relative antineoplastic activity of 10 different non-steroidal anti-inflammatory drugs (NSAIDs) in clinical use, and to investigate the underlying mechanisms of this activity in a squamous cell carcinoma of the head and neck model (SCCHN). A standard 5-day MTT assay was used to calculate IC(50) values in UM-SCC-1 cells for 10 NSAIDs, including celecoxib, rofecoxib, sulindac sulfide, sulindac sulfone, indomethacin, ketoprofen, flurbiprofen, naproxen, piroxicam, and aspirin. Celecoxib, a COX-2 specific inhibitor, was by far the most potent NSAID, with an IC(50) of 39.9 +/- 1.1 microM, followed by sulindac sulfide (116.5 +/- 2.34 microM). Celecoxib and sulindac sulfide also induced more activation of caspase-3 than any other NSAID. Cell cycle analysis showed that celecoxib and sulindac sulfide both induced a 3-fold increase in G(1) phase distribution, and this correlated with strong induction of p21(waf1/cip1), inhibition of cyclin D1, and hypophosphorylation of Rb. Celecoxib and sulindac sulfide treatment induced strong downstream inhibition of E2F transactivating activity as determined by a luciferase reporter assay. These data demonstrate the wide range of activity of various NSAID agents, and reveal a mechanism of action through cell cycle inhibition and induction of apoptosis.

O1-02-07: Different actions of dipeptidic compounds on presenilins define the enzyme specificity of γ-secretase inhibitor

accumulates as full-length protein. Objectives: Three possibilities are being tested that could explain the attenuated A 42-response of PS1Exon9. (i) The attenuated A 42-response is caused by lack of endoproteolytic cleavage, or (ii) is specifically associated with the S290C mutation, or (iii) resides in the 291-319 deletion or its conformational effects on regions flanking exon 9. Methods: CHO cell lines with stable overexpression of WT-PS1 or various PS-1 mutants are being compared in their response to the A 42-lowering NSAID sulindac sulfide. Results: CHO cells overexpressing WT-PS1 or mutants PS1Exon9, S290C, M292D and 291-319 have been created. M292D suppresses endoproteolytic cleavage but is otherwise fully functional in -secretase cleavage. Surprisingly, we found that the S290C mutation alone did not cause increased A 42 production indicating that the pathogenic effect of PS1Exon9 is a consequence of the combined action of the S290C mutation and the 291-319 deletion. Accordingly, the S290C mutation behaved similar to WT-PS1 and was not responsible for the blunted A 42-response of PS1Exon9 after sulindac sulfide treatment. Ongoing studies further aim to clarify whether lack of endoproteolysis or the 291-319 deletion underlie the attenuated A 42-response of PS1Exon9. Conclusions: These studies should resolve the molecular basis for the attenuated A 42-response of the PS1Exon9 mutation. They may further lead to definition of a critical region within PS1 that is required for the A 42-lowering activity of certain NSAIDs.

Both PPARγ and PPARδ influence sulindac sulfide-mediated p21WAF1/CIP1 upregulation in a human prostate epithelial cell line

Nonsteroidal anti-inflammatory drugs (NSAIDs) including sulindac sulfide are known to exert cancer chemopreventative activity in a range of cell lines. This activity has been shown to involve the upregulation of the cyclin-dependent kinase inhibitor p21WAF1/CIP1. It is also known that NSAIDs can act as peroxisome proliferator-activated receptor (PPAR) agonists and antagonists. In this study, we show that sulindac sulfide acts both as a PPARgamma agonist and a PPARdelta antagonist in an immortalized prostatic epithelial cell line (PNT1A). We utilized siRNA technology to show that PPARgamma is required for both growth inhibition and p21WAF1/CIP1 upregulation in response to sulindac sulfide treatment in PNT1A cells. In addition, the overexpression of PPARdelta partially rescued these cells from growth inhibition and also dramatically inhibited sulindac sulfide-mediated p21WAF1/CIP1 upregulation. Together these data identify a novel link between PPARgamma/PPARdelta/p21WAF1/CIP1 and the cancer chemo-preventative properties of NSAIDs.

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.