Indomethacin promotes apoptosis in gastric cancer cells through concomitant degradation of Survivin and Aurora B kinase proteins.

Regular usage of nonsteroidal anti-inflammatory drugs (NSAIDs) is associated with reduced incidence of a variety of cancers. The molecular mechanisms underlying these chemopreventive effects remain poorly understood. This current investigation showed that in gastric cancer cells: (1) Indomethacin treatment enhanced the degradation of chromosomal passenger proteins, Survivin and Aurora B kinase; (2) Indomethacin treatment down-regulated Aurora B kinase activity in a cell cycle-independent fashion; (3) siRNA knockdown of Survivin level promoted Aurora B kinase protein degradation, and vice versa; (4) ectopic overexpression of Survivin blocked reduction of Aurora B kinase level and activity by indomethacin treatment, and vice versa; (5) siRNA knockdown of Aurora B kinase level and AZD1152 inhibition of its activity induced apoptosis, and overexpression of Aurora B kinase inhibited indomethacin-induced apoptosis; (6) indomethacin treatment reduced Aurora B kinase level, coinciding with reduction of Survivin level and induction of apoptosis, in KATO III and HT-29 cells, and in mouse gastric mucosa. A role for Aurora B kinase function in NSAID-induced apoptosis was not previously explored. Thus this report provides better understanding of the molecular mechanisms underlying the anti-cancer effect of NSAIDs by elucidating a significant role for Aurora B kinase in indomethacin-induced apoptosis.

Glioma big potassium channel expression in human cancers and possible T cell epitopes for their immunotherapy.

Big potassium (BK) ion channels have several spliced variants. One spliced variant initially described within human glioma cells is the glioma BK (gBK) channel. This isoform consists of 34 aa inserted into the intracellular region of the basic BK ion channel. PCR primers specific for this inserted region confirmed that human glioma cell lines and freshly resected surgical tissues from glioblastoma multiforme patients strongly expressed gBK mRNA. Normal human brain tissue very weakly expressed this transcript. An Ab specific for this gBK isoform confirmed that human glioma cells displayed this protein in the cell membrane, mitochondria, Golgi, and endoplasmic reticulum. Within the gBK region, two putative epitopes (gBK1 and gBK2) are predicted to bind to the HLA-A*0201 molecule. HLA-A*0201-restricted human CTLs were generated in vitro using gBK peptide-pulsed dendritic cells. Both gBK1 and gBK2 peptide-specific CTLs killed HLA-A2⁺/gBK⁺ gliomas, but they failed to kill non-HLA-A2-expressing but gBK⁺ target cells in cytolytic assays. T2 cells loaded with exogenous gBK peptides, but not with the influenza M1 control peptide, were only killed by their respective CTLs. The gBK-specific CTLs also killed a variety of other HLA-A*0201⁺ cancer cells that possess gBK, as well as HLA-A2⁺ HEK cells transfected with the gBK gene. Of clinical relevance, we found that T cells derived from glioblastoma multiforme patients that were sensitized to the gBK peptide could also kill target cells expressing gBK. This study shows that peptides derived from cancer-associated ion channels maybe useful targets for T cell-mediated immunotherapy.

Suppression of growth arrest and DNA damage-inducible 45alpha expression confers resistance to sulindac and indomethacin-induced gastric mucosal injury.

Nonsteroidal anti-inflammatory drugs (NSAIDs) such as sulindac and indomethacin are a major cause of gastric erosions and ulcers. Induction of apoptosis by NSAIDs is an important mechanism involved. Understanding how NSAIDs affect genes that regulate apoptosis is useful for designing therapeutic or preventive strategies and for evaluating the efficacy of safer drugs being developed. We investigated whether growth arrest and DNA damage-inducible 45alpha (GADD45alpha), a stress signal response gene involved in regulation of DNA repair and induction of apoptosis, plays a part in NSAID-induced gastric mucosal injury and apoptosis in vivo in mice and in vitro in cultured human AGS and rat RGM-1 gastric epithelial cells. Intraperitoneal administration of sulindac and indomethacin both resulted in up-regulation of GADD45alpha expression and induction of significant injury and apoptosis in gastric mucosa of wild-type mice. GADD45alpha(-/-) mice were markedly more resistant to both sulindac- and indomethacin-induced gastric mucosal injury and apoptosis than wild-type mice. Sulindac sulfide and indomethacin treatments also concentration-dependently increased GADD45alpha expression and apoptosis in AGS and RGM-1 cells. Antisense suppression of GADD45alpha expression significantly reduced sulindac and indomethacin-induced activation of caspase-9 and apoptosis in AGS cells. Pretreatments with exogenous prostaglandins and small interfering RNA suppression of cyclooxygenase (COX)-1 and -2 did not affect up-regulation of GADD45alpha by sulindac sulfide and indomethacin in AGS cells. These findings indicate that GADD45alpha up-regulation is a COX-independent mechanism that is required for induction of severe gastric mucosal apoptosis and injury by NSAIDs, probably via a capase-9-dependent pathway of programmed cell death.

Up-regulation of GADD45alpha expression by NSAIDs leads to apoptotic and necrotic colon cancer cell deaths.

Growth arrest and DNA damage inducible 45 alpha (GADD45alpha) is a central player in mediating apoptosis induced by a variety of stress stimuli and genotoxic agents. Regular usage of nonselective nonsteroidal anti-inflammatory drugs (NSAIDs) such as indomethacin and sulindac is associated with reduced risk for various cancers, including colon cancer. The role of GADD45alpha in NSAID-induced colon cancer cell cytotoxicity is unknown. In this study, we report that indomethacin and sulindac sulfide treatments up-regulate GADD45alpha mRNA expression and protein levels in colon cancer HT-29, RKO and Caco-2 cells. This up-regulation of GADD45alpha is accompanied by necrotic cell death and apoptosis. Anti-sense suppression of GADD45alpha expression inhibited indomethacin and sulindac sulfide-induced necrotic cell death and apoptosis. These findings confirm a role for GADD45alpha in NSAID-induced cytotoxicity, a mechanism for the anti-neoplastic effect of NSAIDs in colon tumorigenesis and cancer growth.

Selective cyclooxygenase (COX) inhibition causes damage to portal hypertensive gastric mucosa: roles of nitric oxide and NF-kappaB.

Portal hypertension (PHT) is associated with increased susceptibility of the gastric mucosa to injury by a variety of factors, including nonsteroidal anti-inflammatory drugs (NSAIDs) that nonselectively inhibit both isoforms of cyclooxygenase (COX-1 and -2). PHT gastric mucosa also has excessive nitric oxide (NO) production that contributes to the general increased susceptibility to injury. Using a rat model of PHT, we studied whether selective COX inhibition, which does not damage normal (normotensive) gastric mucosa, is sufficient to cause PHT gastric damage and, if so, whether and how excessive NO is involved. Indomethacin, a nonselective NSAID, caused 2.4-fold more gastric injury to PHT vs. normotensive sham-operated (SO) control rats. Neither NS-398 nor celecoxib, selective COX-2 inhibitors, caused gastric damage in either SO or PHT rats. SC-560, a selective COX-1 inhibitor, did not cause gastric damage in SO rats but dose-dependently caused gastric damage in PHT rats. There was a compensatory increase in COX-2 expression and activity in SC-560-treated SO rats but not SC-560-treated PHT rats. Partial inhibition of NO production restored gastric COX-2 expression and activity levels in SC-560-treated PHT rats to those of SC-560-treated SO rats, by a mechanism consistent with induction of NF-kappaB, and significantly reduced gastric damage. These studies indicate that, in contrast to normotensive gastric mucosa, inhibition of COX-1 alone is sufficient to cause PHT gastric damage as a result of excessive NO that prevents the induction of NF-kappaB and the compensatory increase in COX-2.

Nutrient availability alters the effect of autophagy on sulindac sulfide-induced colon cancer cell apoptosis.

Autophagy is a catabolic process by which a cell degrades its intracellular materials to replenish itself. Induction of autophagy under various cellular stress stimuli can lead to either cell survival or cell death via apoptotic and/or autophagic (nonapoptotic) pathways. The NSAID sulindac sulfide induces apoptosis in colon cancer cells. Here, we show that inhibition of autophagy under serum-deprived conditions resulted in significant reductions of sulindac sulfide-induced apoptosis in HT-29 colon cancer cells. In contrast, inhibition of autophagy under conditions where serum is available significantly increased sulindac sulfide-induced apoptosis in HT-29 cells. We previously showed that the apoptosis inhibitor, survivin, plays a role in regulating NSAID-induced apoptosis and autophagic cell death. Here, we show that survivin protein half-life is increased in the presence of autophagy inhibitors under serum-deprived conditions, but not under conditions when serum is available. Thus, the increased levels of survivin may be a factor contributing to inhibition of sulindac sulfide-induced apoptosis under serum-deprived conditions. These results suggest that whether a cell lives or dies due to autophagy induction depends on the balance of factors that regulate both autophagic and apoptotic processes.

Differential glioma-associated tumor antigen expression profiles of human glioma cells grown in hypoxia.

Human U251 and D54 glioma cells were tested for expression of 25 glioma-associated tumor antigen precursor proteins (TAPP) under hypoxic (1% O(2)) or normoxic (21% O(2)) conditions. Hypoxic glioma cell lines increased their mRNA expression for nine TAPP (Aim2, Art-4, EphA2, EZH2, Fosl1, PTH-rP, Sox 11, Whsc2 and YKL-40), as assessed by quantitative reverse transcriptase real-time/polymerase chain reaction (qRT-PCR). Increased differences with three hypoxic-induced TAPP: EZH2, Whsc2 and YKL-40 were shown at the protein levels by fluorescent antibody staining and quantitative electrophoretic analysis. Two TAPP (MRP3 and Trp1) were down-regulated by hypoxia in glioma cell lines. Growing the glioma cells under hypoxia for 13 days, followed by returning them back to normoxic conditions for 7 days, and restored the original normoxic TAPP profile. Thus, hypoxia was an environmental factor that stimulated the transient expression of these antigens. Intracranial xenografts grown in nude mice derived from U251 cells that had been cultured under neurosphere stem cell conditions showed increased expression of Whsc2 or YKL-40, demonstrating that these in vitro properties of glioma also occur in vivo. Whsc2-specific cytotoxic T lymphocytes killed the hypoxic U251 glioma cells better than normoxic glioma cells. The antigens expressed by hypoxic tumor cells may be a better source of starting tumor material for loading dendritic cells for novel immunotherapy of glioma using tumor-associated antigens.

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.

Roles of survivin isoforms in the chemopreventive actions of NSAIDS on colon cancer cells.

NSAIDs downregulate survivin (an apoptosis inhibitor), increase apoptosis and reduce growth of colon polyps and cancers. Recently, anti- and pro-apoptosis isoforms of survivin were identified. The roles of these isoforms in NSAID-induced colon cancer cell death have not been examined, and is the focus of this study. The anti-apoptosis isoforms, wild-type (WT) survivin and survivin-DeltaEx3, and the pro-apoptosis isoform, survivin-2b, were present in HT-29 and RKO cells. Indomethacin treatment significantly decreased WT survivin and survivin-DeltaEx3 (30.5+/-10.4% and 20.3+/-6.7%, respectively) but not survivin-2b mRNA in RKO cells. In HT-29 cells, all three isoform mRNAs were slightly decreased by indomethacin treatment. Consistently, indomethacin treatment dramatically reduced WT survivin protein in RKO but not HT-29 cells. Indomethacin treatment increased apoptosis and general cell death more significantly in RKO cells (75.7+/-1.1% cell death at 48 h) than in HT-29 cells (25.4+/-3.7% cell death at 48 h). Anti-sense suppression of survivin-2b mRNA increased resistance of both RKO and HT-29 cells to indomethacin. These data support a role for survivin isoforms in colon cancer cell apoptosis, and thus in prevention of colon cancer growth by NSAIDs.

Survivin: a novel target for indomethacin-induced gastric injury.

BACKGROUND & AIMS: Nonsteroidal anti-inflammatory drugs (NSAIDs) cause gastrointestinal erosions and ulcers. Apoptosis is one of the mechanisms. The role of survivin, an antiapoptosis protein, in NSAID-induced gastric injury is unknown. We examined the role of survivin in NSAID-induced gastric mucosal and gastric cell injury. METHODS: We examined: (1) the effects of indomethacin (nonselective NSAID), celecoxib and NS-398 (cyclooxygenase [COX]-2-selective NSAIDs), SC-560 (a COX-1-selective NSAID), and SC-560 plus celecoxib on survivin expression and extent of injury in rat gastric mucosa; (2) the effects of indomethacin, NS-398, SC-560, and SC-560 plus NS-398 on survivin expression and injury in gastric epithelial (RGM-1) cells; and (3) the effects of survivin suppression with small interfering RNA (siRNA) on RGM-1 cell integrity at baseline and following indomethacin injury. RESULTS: Indomethacin treatment dose-dependently reduced survivin protein levels and caused severe injury of gastric mucosa and RGM-1 cells. Suppression of survivin expression with siRNA in RGM-1 cells caused cell damage and increased susceptibility to injury by indomethacin. Celecoxib treatment caused exfoliation of the mucosal surface epithelium, but neither caused deep erosions or altered survivin expression. Neither NS-398 nor SC-560 treatment altered survivin levels or produced injury in vivo or in vitro. COX-1 and COX-2 inhibitor combination caused injury in vivo and in vitro but did not decrease survivin expression. CONCLUSIONS: (1) Indomethacin, but not selective COX-1 or COX-2 inhibitors alone or in combination, reduces survivin expression in gastric mucosal cells and (2) significant reduction of survivin precedes greater severity of gastric injury.

Survivin - an anti-apoptosis protein: its biological roles and implications for cancer and beyond.

Survivin is a protein that inhibits apoptosis and regulates cell division. Survivin contains a baculovirus inhibitor of apoptosis repeat (BIR) protein domain that classifies it as a member of the inhibitor of apoptosis protein (IAP) family. Survivin inhibits apoptosis, via its BIR domain, by either directly or indirectly interfering with the function of caspases. Survivin is also a chromosomal passenger protein that is required for cell division. Survivin is expressed in embryonic tissues as well as in the majority of human cancers, but is not expressed in most normal adult tissues. The cancer-specific expression of survivin, coupled with its importance in inhibiting cell death and in regulating cell division, makes it a useful diagnostic marker of cancer and a potential target for cancer treatment. Recently, there is emerging evidence that survivin is involved in tissue injury and its healing. Understanding the mechanism of survivin function can potentially allow for the development of therapeutic strategies for cancer and other diseases.

Survivin expression in the stomach: implications for mucosal integrity and protection.

Survivin, an apoptosis inhibitor, is highly expressed in a majority of human cancers and is required during embryonic development. Our present studies show that survivin is also expressed in normal gastric mucosa of adult humans and rats. In both human and rat gastric mucosa, survivin is expressed predominantly in the nuclei of mucosal surface epithelial cells. In rats, survivin is also detected in the nuclei of some neck cells, whereas in the humans, survivin is expressed in both nuclei and cytoplasm of chief and parietal cells. Furthermore, survivin is expressed at higher levels in the nuclei of cultured gastric mucosal epithelial cells than in gastric microvascular endothelial cells, which supports the expression pattern in intact tissues. Based on these expression studies, and the known role of survivin as an anti-apoptosis protein, survivin may play a role in maintaining gastric mucosal integrity and regulating cell renewal in the gastric mucosa.