X-linked Inhibitor Of Apoptosis Overexpression Research Articles

Nicotine promotes renal interstitial fibrosis via upregulation of XIAP in an alpha7-nAChR-dependent manner

Renal fibrosis, characterized by excessive accumulation of the extracellular matrix in the renal tubulointerstitium, can lead to chronic kidney disease (CKD), resulting in a heavy burden on families and society. Clinical studies have shown that smoking is closely associated with CKD deterioration in patients with diabetes, hypertension, polycystic kidney disease, and kidney transplantation. However, the mechanism of action of nicotine in renal fibrosis pathogenesis remains largely unknown. X-linked inhibitor of apoptosis (XIAP), a member of the inhibitor of apoptosis protein (IAP) family, is involved in apoptosis, necroptosis, autophagy, and immune response. Here, the upregulated expression of XIAP and α7 nicotine acetylcholine receptor (α7-nAChR) was determined in the kidneys of the CKD smoking group in human and animal studies. A significant positive correlation between XIAP and cotinine was observed. In addition, the nuclear translocation and transcriptional activity of SP1 were promoted when nicotine bound to α7-nAChR, resulting in XIAP overexpression and renal interstitial fibrosis progression. This phenotype can be reversed by the nicotine receptor subtype α7-nAChR antagonists methyllycaconitine. Our results revealed the complex underlying mechanism of nicotine in promoting renal fibrosis by altering SP1 nucleocytoplasmic translocation and regulating XIAP expression. These results provide novel insights into the pathogenesis and treatment of CKD.

BRCA mutations lead to XIAP overexpression and sensitise ovarian cancer to inhibitor of apoptosis (IAP) family inhibitors

BackgroundWe tested the hypothesis that inhibitor of apoptosis family (IAP) proteins may be altered in BRCA1-mutated ovarian cancers and that could affect the sensitivity to IAP inhibitors.MethodsThe levels of IAP proteins were evaluated in human cancers and cell lines. Cell lines were used to determine the effects of IAP inhibitors. The in vivo effects of treatments were evaluated in PDX mouse models.ResultsExpression of X-linked inhibitor of apoptosis (XIAP) is increased in BRCA1-mutated cancers and high levels are associated with improved patient outcomes after platinum chemotherapy. XIAP overexpression is mediated by NF-kB activation and is associated with an optimisation of PARP. BRCA1-mutated cell lines are particularly sensitive to IAP inhibitors due to an inhibitory effect on PARP. Both a BRCA1-mutated cell line with acquired resistance to PARP inhibitors and one with restored BRCA1 remain sensitive to IAP inhibitors. Treatment with IAP inhibitors restores the efficacy of PARP inhibition in these cell lines. The IAP inhibitor LCL161 alone and in combination with a PARP inhibitor, exhibited antitumour effects in PDX mouse models of resistant BRCA2 and 1-mutated ovarian cancer, respectively.ConclusionA clinical trial may be justified to further investigate the utility of IAP inhibitors.

Decreased OLA1 (Obg-Like ATPase-1) Expression Drives Ubiquitin-Proteasome Pathways to Downregulate Mitochondrial SOD2 (Superoxide Dismutase) in Persistent Pulmonary Hypertension of the Newborn.

Persistent pulmonary hypertension of the newborn (PPHN) is a failure of pulmonary vascular resistance to decline at birth rapidly. One principal mechanism implicated in PPHN development is mitochondrial oxidative stress. Expression and activity of mitochondrial SOD2 (superoxide dismutase) are decreased in PPHN; however, the mechanism remains unknown. Recently, OLA1 (Obg-like ATPase-1) was shown to act as a critical regulator of proteins controlling cell response to stress including Hsp70, an obligate chaperone for SOD2. Here, we investigated whether OLA1 is causally linked to PPHN. Compared with controls, SOD2 expression is reduced in distal-pulmonary arteries (PAs) from patients with PPHN and fetal-lamb models. Disruptions of the SOD2 gene reproduced PPHN phenotypes, manifested by elevated right ventricular systolic pressure, PA-endothelial cells apoptosis, and PA-smooth muscle cells proliferation. Analyses of SOD2 protein dynamics revealed higher ubiquitinated-SOD2 protein levels in PPHN-lambs, suggesting dysregulated protein ubiquitination. OLA1 controls multiple proteostatic mechanisms and is overexpressed in response to stress. We demonstrated that OLA1 acts as a molecular chaperone, and its activity is induced by stress. Strikingly, OLA1 expression is decreased in distal-PAs from PPHN-patients and fetal-lambs. OLA1 deficiency enhanced CHIP affinity for Hsp70-SOD2 complexes, facilitating SOD2 degradation. Consequently, mitochondrial H2O2 formation is impaired, leading to XIAP (X-linked inhibitor of apoptosis) overexpression that suppresses caspase activity in PA-smooth muscle cells, allowing them to survive and proliferate, contributing to PA remodeling. In-vivo, ola1-/- downregulated SOD2 expression, induced distal-PA remodeling, and right ventricular hypertrophy. We conclude that decreased OLA1 expression accounts for SOD2 downregulation and, therefore, a therapeutic target in PPHN treatments.

4-HNE Induces Apoptosis of Human Retinal Pigment Epithelial Cells by Modifying HSP70.

The role of heat shock protein 70 (HSP70) in apoptosis of human retinal pigment epithelial cells (ARPE-19) induced by 4-hydroxy-2-nonenal (4-HNE) was explored. Different concentrations of 4-HNE were used to stimulate ARPE-19 cells, and apoptosis was measured by flow cytometry. The expression of apoptotic-related proteins, HSP70, X-linked inhibitor-of-apoptosis (XIAP), Bcl-2, and Bax were quantified by Western blotting. HSP70 and XIAP overexpression plasmids, or their corresponding siRNAs were transfected into ARPE-19 cells using Lipofectamine™ 2000. Co-immunoprecipitation and Western blotting were used to detect the effect of 4-HNE on the expression of HSP70 and the binding level between 4-HNE and HSP70. The results showed that 4-HNE induced late apoptosis in ARPE-19 cells, accompanied by elevated levels of 4-HNE-modified HSP70, but it did not affect HSP70 protein expression. 4-HNE-modified HSP70 down-regulated the expression of the apoptosis inhibitory protein XIAP. Overexpression of HSP70 or XIAP inhibited 4-HNE-induced apoptosis of ARPE-19 cells. It was suggested that 4-HNE could promote XIAP degradation by modification of HSP70 to induce late apoptosis of human retinal pigment epithelial cells.

Survivin and XIAP expression in distinct tumor compartments of surgically resected gastric cancer: XIAP as a prognostic marker in diffuse and mixed type adenocarcinomas.

There is considerable evidence that the inhibitor of apoptosis protein (IAP) family serves a role in tumorigenesis. The most studied IAP family members, survivin and X-linked inhibitor of apoptosis (XIAP), have been demonstrated to serve as biomarkers in distinct tumor entities. Thus, the present study aimed to investigate the expression levels of both IAPs in the tumor center, invasion front and lymph node metastases of surgically resected gastric cancer (GC) specimens. Tissue microarrays containing samples from 201 primary GCs were analyzed. IAP expression was detected using immunohistochemistry in different tumor compartments, normal mucosa and lymph node metastases. In addition, the association between the expression levels of these proteins, and clinicopathological parameters and overall survival was investigated. High levels of survivin and XIAP were evident in GC, when compared with normal mucosa, and were correlated with intestinal-type and well-differentiated GC, as well as low International Union Against Cancer stages. Increased XIAP expression was detected in lymph node metastases as compared with corresponding primary tumors. XIAP overexpression was identified to be an independent negative prognostic marker in diffuse and mixed type GC. These results suggest a potential role of survivin and XIAP in the early phase of gastric carcinogenesis. In addition, increased XIAP expression in lymph node metastases supports the observation that IAPs serve an essential role in metastatic tumor disease. Since XIAP expression was identified to be associated with poor survival in diffuse and mixed type GC, XIAP may serve as a novel therapeutic target in these types of GC.

XIAP over-expression is an independent poor prognostic marker in Middle Eastern breast cancer and can be targeted to induce efficient apoptosis

BackgroundBreast cancer is the most common cancer in females and is ranked second in cancer-related deaths all over the world in women. Despite improvement in diagnosis, the survival rate of this disease has still not improved. X-linked Inhibitor of Apoptosis (XIAP) has been shown to be over-expressed in various cancers leading to poor overall survival. However, the role of XIAP in breast cancer from Middle Eastern region has not been fully explored.MethodsWe examined the expression of XIAP in more than 1000 Middle Eastern breast cancer cases by immunohistochemistry. Apoptosis was measured by flow cytometry. Protein expression was determined by western blotting. Finally, in vivo studies were performed on nude mice following xenografting and treatment with inhibitors.ResultsXIAP was found to be over-expressed in 29.5% of cases and directly associated with clinical parameters such as tumor size, extra nodal extension, triple negative breast cancer and poorly differentiated breast cancer subtype. In addition, XIAP over-expression was also significantly associated with PI3-kinase pathway protein; p-AKT, proliferative marker; Ki-67 and anti-apoptotic marker; PARP. XIAP over-expression in our cohort of breast cancer was an independent poor prognostic marker in multivariate analysis. Next, we investigated inhibition of XIAP using a specific inhibitor; embelin and found that embelin treatment led to inhibition of cell viability and induction of apoptosis in breast cancer cells. Finally, breast cancer cells treated with combination of embelin and PI3-kinase inhibitor; LY294002 synergistically induced apoptosis and caused tumor growth regression in vivo.ConclusionThese data suggest that XIAP may be playing an important role in the pathogenesis of breast cancer and can be therapeutically targeted either alone or in combination with PI3-kinase inhibition to induce efficient apoptosis in breast cancer cells.

Abstract 17: Xiap over-expression is a poor prognostic marker in breast cancer and can be targeted to induce efficient apoptosis

Abstract Breast cancer is the most common cancer in females and is ranked only second to lung cancer in cancer-related deaths all over the world in women. Despite improvement in diagnosis and management of breast cancer, the survival rate of this disease for stage 4 disease is still only 22%. X-linked Inhibitor of Apoptosis (XIAP) is a member of the inhibitor of apoptosis family of proteins that has been shown to be over-expressed in a variety of cancers including breast cancer leading to an overall poor survival. However, the role of XIAP in breast cancer from Middle Eastern region has not been fully explored. We therefore examined the expression of XIAP in a cohort of 964 breast cancer cases by immunohistochemistry and found that XIAP was over-expressed in 29.5% (284/964) of the cases and was found to be directly associated with clinical parameters such as tumor size (p = 0.0044), extra nodal extension (p = 0.0041) and poorly differentiated breast cancer (p<0.0001). In addition, XIAP over-expression was also significantly associated with activated AKT (p<0.0001), Ki-67 (p<0.0001) and PARP (p<0.0001). Finally, XIAP over-expression in our cohort of breast cancer was an independent poor prognostic marker in multivariate analysis. Next, we investigated inhibition of XIAP using a specific inhibitor; Embelin on a panel of breast cancer cell lines and found that Embelin treatment led to inhibition of cell viability and induction of apoptosis in breast cancer cells. In order to identify the exact pathway involved in XIAP inhibition, we treated breast cancer cells with different doses of Embelin and found that there was down-regulation of XIAP leading to activation of caspases-9, -3 and PARP. We also found that Embelin treatment caused cleavage of caspase-8 and truncation of Bid leading to activation of the mitochondrial apoptotic pathway and efficient apoptosis. Finally, as our clinical data demonstrated a significant association between XIAP over-expression and activation of AKT in breast cancer samples, we synergistically induced apoptosis in breast cancer cells using sub-optimal doses of Embelin and PI3-kinase inhibitor; LY294002. These data suggest that XIAP may be playing an important role in the pathogenesis of breast cancer and XIAP can be therapeutically targeted either alone or in combination with PI3-kinase inhibition to induce efficient apoptosis in breast cancer cells. Citation Format: Azhar R. Hussain, Maqbool Ahmed, Rong Bu, Shaham Beg, Alanood M. Alrashed, Roxanne Melosantos, Dahish S. Ajarim, Shahab Uddin, Khawla S. AlKuraya. Xiap over-expression is a poor prognostic marker in breast cancer and can be targeted to induce efficient apoptosis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 17. doi:10.1158/1538-7445.AM2015-17

Prosurvival function of the cellular apoptosis susceptibility/importin-α1 transport cycle is repressed by p53 in liver cancer.

Proteins of the karyopherin superfamily including importins and exportins represent an essential part of the nucleocytoplasmic transport machinery. However, the functional relevance and regulation of karyopherins in hepatocellular carcinoma (HCC) is poorly understood. Here we identified cellular apoptosis susceptibility (CAS, exportin-2) and its transport substrate importin-α1 (imp-α1) among significantly up-regulated transport factor genes in HCC. Disruption of the CAS/imp-α1 transport cycle by RNAi in HCC cell lines resulted in decreased tumor cell growth and increased apoptosis. The apoptotic phenotype upon CAS depletion could be recapitulated by direct knockdown of the X-linked inhibitor of apoptosis (XIAP) and partially reverted by XIAP overexpression. In addition, XIAP and CAS mRNA expression levels were correlated in HCC patient samples (r=0.463; P<0.01), supporting the in vivo relevance of our findings. Furthermore, quantitative mass spectrometry analyses of murine HCC samples (p53-/- versus p53+/+) indicated higher protein expression of CAS and imp-α1 in p53-/- tumors. Consistent with a role of p53 in regulating the CAS/imp-α1 transport cycle, we observed that both transport factors were repressed upon p53 induction in a p21-dependent manner. The CAS/imp-α1 transport cycle is linked to XIAP and is required to maintain tumor cell survival in HCC. Moreover, CAS and imp-α1 are targets of p53-mediated repression, which represents a novel aspect of p53's ability to control tumor cell growth in hepatocarcinogenesis.

Over-expression of X-Linked Inhibitor of Apoptosis Protein Modulates Multiple Aspects of Neuronal Ca2+ Signaling

X-linked inhibitor of apoptosis (XIAP) protects and preserves the function of neurons in both in vitro and in vivo models of excitotoxicity. Since calcium (Ca(2+)) overload is a pivotal event in excitotoxic neuronal cell death, we have determined whether XIAP over-expression influences Ca(2+)-signaling in primary cultures of mouse cortical neurons. Using cortical neuron cultures derived from wild-type (Wt) mice transiently transfected with XIAP or from transgenic mice that over-express XIAP, we show that XIAP opposes the rise in intracellular Ca(2+) concentration by a variety of triggers. Relative to control neurons, XIAP over-expression produced a slight, but significant, elevation of resting Ca(2+) concentrations. By contrast, the rise in intracellular Ca(2+) concentrations produced by N-methyl-D-aspartate receptor stimulation and voltage gated Ca(2+) channel activation were markedly attenuated by XIAP over-expression. The release of Ca(2+) from intracellular stores induced by the sarco/endoplasmic reticulum Ca(2+) ATPase inhibitor thapsigargin was also inhibited in neurons transiently transfected with XIAP. The pan-caspase inhibitor zVAD did not, however, diminish the rise in intracellular Ca(2+) concentrations elicited by L-glutamate suggesting that XIAP influences Ca(2+) signaling in a caspase-independent manner. Taken together, these findings demonstrate that the ability of XIAP to block excessive rises in intracellular Ca(2+) by a variety of triggers may contribute to the neuroprotective effects of this anti-apoptotic protein.

XIAP Is a Predictor of Cisplatin-Based Chemotherapy Response and Prognosis for Patients with Advanced Head and Neck Cancer

BackgroundApproximately 60–80% of patients with advanced head and neck squamous cell carcinoma (HNSCC) die within five years after diagnosis. Cisplatin-based chemotherapy is the most commonly used palliative treatment for these patients. To evaluate the prognostic value of X-linked inhibitor of apoptosis (XIAP) level as a potential biomarker in these patients, we investigated the relationship between XIAP expression and cisplatin response of these patients and their prognosis.Methodology/Principal FindingsSixty patients with advanced HNSCC were recruited in this study. Expression of XIAP was examined both before and after chemotherapy and was correlated with chemotherapy response, clinicopathology parameters and clinical outcomes of the patients. We found that XIAP was expressed in 17 (20.83%) of the 60 advanced HNSCC samples and the expression was significantly associated with cisplatin resistance (P = 0.036) and poor clinical outcome (P = 0.025). Cisplatin-based chemotherapy induced XIAP expression in those post-chemotherapy samples (P = 0.011), was further associated with poorer clinical outcome (P = 0.029). Multivariate analysis demonstrated that only alcohol consumption, lymph node metastasis and XIAP level were independently associated with the prognosis of advanced HNSCC patients. Inhibiting XIAP expression with siRNA in XIAP overexpressed HNSCC cells remarkably increased their sensitivity to cisplatin treatment to nearly a 3 fold difference.Conclusions/SignificanceOur results demonstrate that XIAP overexpression plays an important role in the disease course and cisplatin-resistance of advanced HNSCC. XIAP is a valuable predictor of cisplatin-response and prognosis for patients with advanced head and neck cancer. Down-regulation of XIAP might be a promising adjuvant therapy for those patients of advanced HNSCC.

AAV-Mediated Gene Transfer of Human X-Linked Inhibitor of Apoptosis Protects against Oxidative Cell Death in Human RPE Cells

To determine whether human X-linked inhibitor of apoptosis (XIAP) enhances the survival of cultured human retinal pigment epithelial cells exposed to H(2)O(2). ARPE-19 cells were exposed to H(2)O(2) to induce oxidative cell death. Intracellular reactive oxygen species (ROS) were measured using 2',7'-dichlorofluorescein diacetate. MTT assay was performed to quantify mitochondrial stress. Cell apoptosis was determined by TUNEL assay. Human XIAP was delivered with bicistronic expression of green fluorescent protein (GFP), using recombinant adeno-associated virus (AAV-XIAP-GFP). The null vector, containing identical sequences but without XIAP, was used as a control (AAV-NULL-GFP). Transduced cells underwent fluorescence-activated cell sorting. XIAP overexpression was examined by immunostaining and Western blot analysis. ARPE-19 cells exposed to 0.25 mM H(2)O(2) for 1 hour showed increased TUNEL staining compared with nonstressed cells (17 ± 1.4 vs. 1.8 ± 0.4 cells per 20 × field; P = 0.000006), accompanied by a significant increase in intracellular ROS (207 ± 46% vs. 100 ± 9.5%; P = 0.0002). The AAV-XIAP-GFP transduced cells had 11-fold higher XIAP expression than the AAV-NULL-GFP controls (1300 ± 126% vs. 120 ± 10%; P = 0.0006). XIAP over-expression significantly reduced the number of apoptotic cells after stress compared with the AAV-NULL-GFP controls (3.2 ± 0.6 vs. 18 ± 1.6 cells per 20 × field; P = 0.00003). Mitochondrial stress was reduced by AAV-XIAP-GFP, but did not reach a statistical significance (68 ± 3.5% vs. 74 ± 3.8%; P = 0.24). Overexpression of human XIAP protects ARPE-19 cells against H(2)O(2)-induced oxidative cell death by acting downstream on the apoptotic pathway. XIAP gene therapy using AAV may provide a means of reducing the effect of oxidative stress to RPE cells in age-related macular degeneration.

Effect of XIAP overexpression on sodium butyrate-induced apoptosis in recombinant Chinese hamster ovary cells producing erythropoietin

Previously, overexpression of X-linked inhibitor of apoptosis (XIAP), which is known to inhibit activities of caspase-3, -7, and -9 in CHO-K1 cells offered protection against Sindbis virus-induced apoptosis. In this study, the potential role of XIAP overexpression in recombinant CHO (rCHO) cells treated with sodium butyrate (NaBu), which can increase the specific productivity, was investigated by establishing erythropoietin (EPO)-producing rCHO cells with regulated XIAP overexpression (EPO-off-XIAP). The XIAP overexpression in EPO-off-XIAP was tightly regulated by doxycycline. The XIAP overexpression could simultaneously reduce the activation of caspase-3, -7, and -9 induced by NaBu addition. However, XIAP overexpression could not inhibit NaBu-induced apoptosis, as evidenced by DNA fragmentation. In addition, it also did not help the maintenance of the mitochondrial membrane potential in the presence of NaBu, suggesting that the release of mitochondrial proteins might induce caspase-independent apoptosis. As a result, XIAP overexpression did not affect cell growth and EPO production significantly. Taken together, XIAP overexpression, which was reported to inhibit Sindbis virus-induced apoptosis, could not inhibit the NaBu-induced apoptosis in rCHO cells.

BRAFV600E Mutation and X-Linked Inhibitor of Apoptosis Expression in Papillary Thyroid Carcinoma

The BRAF mutation V600E (BRAF(V600E)) is the most common genetic alteration in papillary thyroid carcinoma (PTC), while overexpression of X-linked inhibitor of apoptosis (XIAP) has been found in various tumors. Both of these events are implicated in carcinogenesis, tumor progression, recurrence, etc. There are few reports, however, of the BRAF(V600E) mutation and XIAP overexpression in PTC patients. The aim of this study was to investigate the frequency of the BRAF(V600E) mutation in PTC and its relationship to clinicopathological parameters and the expression of XIAP. Genomic DNA was extracted from 123 paraffin-embedded PTC tumor tissue samples and amplified for analysis of the V600E mutation in exon 15 of the BRAF gene by polymerase chain reaction. XIAP expression was examined by immunohistochemical methods in 46 PTCs, 18 benign nodular goiters, and 10 Hashimoto's thyroiditis samples. The BRAF(V600E) mutation was found in 34.1% of PTC, and was especially prevalent in the classic type. BRAF(V600E) was significantly correlated with younger age at diagnosis (p = 0.026), tumor size (p = 0.009), and histological variants (p = 0.024). There was a trend towards association with extrathyroidal invasion (p = 0.067). By logistic regression analysis, a significant relationship was found between tumor size and the BRAF(V600E) mutation (p = 0.03). XIAP was expressed in 82.6% of PTCs, which was a higher percentage than observed in the group of benign thyroid disorders (35.7%, p < 0.001). Neither the intensity (p = 0.611) nor the extent (p = 0.723) of XIAP staining was correlated with the presence of BRAF(V600E) in PTC patients. These data indicate that BRAF(V600E) is associated some of the aggressive clinicopathological features of PTC including younger age at diagnosis, larger tumor size, and classic histological type, as well as also extrathyroidal invasion. XIAP-positive staining was more prevalent in PTCs than in the benign thyroid disorders. Although BRAF(V600E) and XIAP expression are commonly seen in PTC, their presence together seems unrelated.

Effects on XIAP Retinal Detachment–Induced Photoreceptor Apoptosis

To evaluate the ability of X-linked inhibitor of apoptosis (XIAP) gene therapy to provide neuroprotection to cells of the outer nuclear layer (ONL) of the retina after retinal detachment. Subretinal injections of a recombinant adenoassociated virus (rAAV) encoding either XIAP or green fluorescent protein (GFP; injection control) were performed in the left eye of Brown Norway rats. Two weeks later, retinal detachments were created at the site of viral injection by delivering sodium hyaluronate into the subretinal space. Retinal tissue was harvested at 24 hours after retinal detachment and was analyzed for caspase 3 and 9 activity. Histologic analysis was conducted on samples taken at 3 days and 2 months after detachment to confirm the presence of XIAP or GFP expression and to assess levels of apoptosis and changes in retinal thickness. Caspase assays performed 24 hours after detachment confirmed an expected increase in caspase 3 and 9 activity in the detached regions of GFP-treated retinas, whereas XIAP-treated detached retinas behaved comparably to attached controls. TUNEL analysis of 3-day tissue samples showed fewer apoptotic cells in XIAP-treated detachments than in GFP-treated detachments. At 2 months after the detachment, histology and immunohistochemistry confirmed the preservation of the ONL at sites of XIAP overexpression, whereas the GFP-treated detached retinas had significantly deteriorated. The results suggest that XIAP confers structural neuroprotection of photoreceptors for at least 2 months after retinal detachment.

X-linked inhibitor of apoptosis protein is an important regulator of vascular endothelial growth factor-dependent bovine aortic endothelial cell survival.

Vascular endothelial growth factor (VEGF) is a critical regulator of endothelial cell biology and vascular function. Chronic VEGF treatment has been shown to inhibit tumor necrosis factor-induced apoptosis in endothelial cells. However, the mechanism for this cell survival is unclear. Interestingly, VEGF also enhances the expression of X-linked inhibitor of apoptosis (XIAP), a well-established antiapoptotic factor. XIAP has been shown to suppress apoptosis by blocking caspase activity in cancer cells, but it remains under studied in the endothelium. Therefore, we hypothesized that VEGF affects important endothelial functions, such as apoptosis and cell migration, by regulating XIAP expression and downstream caspase activity. To test this hypothesis, caspase activity, apoptosis, and cell migration were assessed following XIAP overexpression or depletion in bovine aortic endothelial cells. Much like VEGF treatment, ectopic expression of XIAP blocked tumor necrosis factor-induced apoptosis. Surprisingly, the mechanism was caspase-independent. In addition, XIAP-associated cell survival was the result of enhanced nitric oxide (NO) production, and XIAP was partially localized in caveolae. In these lipid rafts, XIAP interacted with a regulator of NO production, caveolin-1, via a binding motif (FtFgtwiY, where the bold letters represent aromatic amino acids) in the baculoviral IAP repeat-3 domain. Endothelial NO synthase binding to caveolin-1 was competitively inhibited by XIAP, suggesting that XIAP acts as a modulator of NO production by releasing endothelial NO synthase from caveolin-1. Further studies showed that endothelial cell migration was also controlled by XIAP-dependent NO. Taken together, these results suggest that XIAP plays a novel role in endothelial cells, interacting with caveolin-1 and acting as a regulator of vascular antiatherogenic function.

The X-Linked Inhibitor of Apoptosis Protein Enhances Survival of Murine Islet Allografts

Allotransplantation of pancreatic islets represents a promising approach to treat type 1 diabetes. Destruction of beta-cells in islet allografts involves multiple immune mechanisms that lead to activation of caspases and apoptotic cell death. The X-linked inhibitor of apoptosis (XIAP) inhibits apoptosis induced by a variety of triggers, primarily by preventing the activation of caspases. To determine whether XIAP would protect beta-cells from apoptosis, we used a recombinant adenovirus to overexpress XIAP in transformed murine beta-cells and in freshly isolated islets. In vitro cytokine-induced beta-cell death was decreased to baseline levels in XIAP-transduced MIN-6 and NIT-1 cell lines compared with controls. To evaluate the potential of XIAP overexpression to prevent in vivo allogeneic graft rejection, we transduced Balb/c islets ex vivo with XIAP before transplantation into CBA mice with streptozotocin-induced diabetes. We observed that almost all mice receiving allografts of XIAP-expressing islets maintained normoglycemia until the experiment was terminated (45-72 days posttransplant), whereas control mice receiving islets transduced with adenovirus expressing LacZ were hyperglycemic by approximately 17 days posttransplantation due to graft rejection. Immunohistochemistry revealed preservation of beta-cells and clearance of infiltrating immune cells in the XIAP-expressing islet grafts. The in vitro allogeneic response of splenocytes isolated from recipients of XIAP-expressing grafts 8 weeks posttransplant was similar to that seen in nonprimed allogeneic mice, suggesting that XIAP overexpression may lead to the acceptance of islet allografts in diabetic recipients. Long-term protection of islet allografts by XIAP overexpression may enhance the survival of islet transplants in diabetes.

Reactive Oxygen Species Regulate Caspase Activation in Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand–Resistant Human Colon Carcinoma Cell Lines

The effects of reactive oxygen species (ROS) on tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in solid cancers have yet to be clearly defined. In this study, we found that the classic uncoupler of oxidative phosphorylation, carbonyl cyanide m-chlorophenylhydrazone (CCCP), induced a reduction in DeltaPsim and generation of ROS. This uncoupling effect enhanced TRAIL-induced apoptosis in TRAIL-resistant human colon carcinoma cell lines (RKO, HT29, and HCT8). Sensitization was inhibited by benzyloxycarbonyl-valine-alanine-aspartate fluoromethylketone, indicating the requirement for caspase activation. CCCP per se did not induce apoptosis or release of proapoptotic factors from mitochondria. Generation of ROS by CCCP was responsible for TRAIL-induced Bax and caspase activation because scavenging ROS completely abrogated apical caspase-8 activation and further downstream events leading to cell death. Overexpression of Bcl-2 did not prevent the initial loss of DeltaPsim and ROS generation following CCCP treatment, but did prevent cell death following TRAIL and CCCP exposure. Uncoupling of mitochondria also facilitated TRAIL-induced release of proapoptotic factors. X-linked inhibitor of apoptosis overexpression abrogated TRAIL-induced apoptosis in the presence of CCCP and decreased initiator procaspase-8 processing, indicating that additional processing of caspase-8 required initiation of a mitochondrial amplification loop via effector caspases. Of interest, depletion of caspase-9 in RKO cells did not protect cells from TRAIL/CCCP-induced apoptosis, indicating that apoptosis occurred via a caspase-9-independent pathway. Data suggest that in the presence of mitochondrial-derived ROS, TRAIL induced mitochondrial release of Smac/DIABLO and inactivation of X-linked inhibitor of apoptosis through caspase-9-independent activation of caspase 3.