X-linked Inhibitor Of Apoptosis Protein Gene Research Articles

Identification of Germline Non-coding Deletions in XIAP Gene Causing XIAP Deficiency Reveals a Key Promoter Sequence

X-linked inhibitor of apoptosis protein (XIAP) deficiency, also known as the X-linked lymphoproliferative syndrome of type 2 (XLP-2), is a rare immunodeficiency characterized by recurrent hemophagocytic lymphohistiocytosis, splenomegaly, and inflammatory bowel disease. Variants inXIAPincluding missense, non-sense, frameshift, and deletions of coding exons have been reported to cause XIAP deficiency. We studied three young boys with immunodeficiency displaying XLP-2-like clinical features. No genetic variation in the coding exons ofXIAPwas identified by whole-exome sequencing (WES), although the patients exhibited a complete loss of XIAP expression. Targeted next-generation sequencing (NGS) of the entire locus ofXIAPwas performed on DNA samples from the three patients. Molecular investigations were assessed by gene reporter expression assays in HEK cells and CRISPR-Cas9 genome editing in primary T cells. NGS ofXIAPidentified three distinct non-coding deletions in the patients that were predicted to be driven by repetitive DNA sequences. These deletions share a common region of 839bp that encompassed the first non-coding exon ofXIAPand contained regulatory elements and marks specific of an active promoter. Moreover, we showed that among the 839bp, the exon was transcriptionally active. Finally, deletion of the exon by CRISPR-Cas9 in primary cells reduced XIAP protein expression. These results identify a key promoter sequence contained in the first non-coding exon ofXIAP. Importantly, this study highlights that sequencing of the non-coding exons that are not currently captured by WES should be considered in the genetic diagnosis when no variation is found in coding exons.

XIAP inhibits gentamicin-induced hair cell damage and ototoxicity through the caspase-3/9 pathway

Gentamicin (GM), an aminoglycoside antibiotic, is one commonly used clinical drugs with ototoxic side effects. One of the most principal mechanisms of its ototoxicity is that GM can activate caspase-mediated cell death pathways in the cochlea. Since the anti-apoptotic protein known as X-linked Inhibitor of Apoptosis Protein (XIAP) has been reported to directly bind to activated caspase protein and inhibit their activities, we hypothesized that it might protect cochlea hair cells from GM ototoxicity. To evaluate this hypothesis, postnatal day 2–3 (P2-3) transgenic (TG) mice, in which XIAP gene is over-expressed under a pure C57BL/6J genetic background was constructed. We first extracted the cochlea tissue of normal mice and treated them with different concentrations of GM, and the number of hair cells were observed to determine the concentration of GM used in subsequent experiments. Next, we used Western Blot experiment to examine the effect of GM on XIAP protein expression in normal mouse cochlea, and then Western Blot and RT-PCR experiments were used to identify the transgenic mice. Finally, immunofluorescence assays were used to detect the effect of GM on the expression of caspase protein and verify the protective effect of XIAP. We found that GM at a concentration of 0.5 mM significantly affected the function of cochlea hair cells, up-regulating the expression of cleaved-caspase-3 and cleaved-caspase-9 protein but down-regulating XIAP protein. In the cochlea tissues of TG mice, this effect of GM was suppressed, and the destruction of hair cells was significantly reduced, and the cleaved-caspase-3 and cleaved-caspase-9 proteins were significantly suppressed. These results suggested that XIAP reduces GM-induced ototoxicity and caspase-3/9 pathway is associated with this process.

Epstein\u2013Barr virus-related hemophagocytic lymphohistiocytosis complicated with coronary artery dilation and acute renal injury in a boy with a novel X-linked inhibitor of apoptosis protein (XIAP) variant: a case report

BackgroundX-linked lymphoproliferative disease (XLP) is a rare inherited X-linked primary immunodeficiency diseases (PID). One such disease, X-linked inhibitor of apoptosis protein (XIAP) deficiency, is characterized by Epstein–Barr virus-related hemophagocytic lymphohistiocytosis (EBV-HLH). However, EBV-HLH with coronary artery dilation and acute renal injury (AKI) in children is unusual.Case presentationWe report the case of a young boy aged 17 months with a novel XIAP variant. He was initially diagnosed with EBV-HLH based on the HLH-2004 diagnostic criteria and the condition was accompanied by coronary artery dilation and acute renal injury. The comprehensive genetic analysis of peripheral blood-derived DNA revealed a hemizygous variant of the XIAP gene [c.116G > C(p.G39A)], which was inherited from his mother (heterozygous condition). After combined treatment with rituximab, intravenous immunoglobulin, corticosteroids, antiviral drugs, and mycophenolate mofetil (MMF) in addition to supportive therapy, his clinical manifestations and laboratory indexes were improved. The patient achieved complete remission with MMF treatment in the 8-month follow-up.ConclusionsWe report the [c.116G > C(p.G39A)] variant in the XIAP gene for the first time in a case of XLP-2 associated with EBV-HLH. For male patients with severe EBV-HLH, the possibility of XLP should be considered and molecular genetic testing should be used early in auxiliary diagnosis. Reports of EBV-HLH with coronary artery dilation and AKI in children are rare. In the patients with EBV-HLH, color Doppler echocardiography and urine tests should be monitored regularly. If necessary, renal biopsy can be performed to clarify the pathology. Treatment with rituximab, immunosuppressors and supportive therapy achieved a good effect, but long-term follow-up is required.

In vivo Study of siRNA Silencing XIAP Gene to Reverse Taxol-resistance in Human Ovarian Cancer Cells

To study the relationship between down-regulated expression of X linked inhibitor of apoptosis protein (XIAP) gene and the reversal effect of taxol-resistance by using siRNA interference technology in the taxol-resistant ovarian cancer. Randomly assigned the nude mice into six groups (6 in each group) . Group A: normal saline; Group B: taxol; Group C: siRNA-NC+normal saline; Group D: siRNA-NC+taxol; Group E: siRNA XIAP+normal saline; Group F: siRNA XIAP+taxol. Each group was dealt with the corresponding processing depending on the agreed protocol and the transplanted tumors had a multi-point injection with reagents related siRNA, one time every 3 days, 9 times (27 d) in total. Taxol (2 mg/kg) was used in the intraperitoneal injection, 0.2 mL every time, once a week, for four weeks. After 27 d of siRNA treatment, xenograft volumes and qualities were measured and the inhibitory rate was calculated; RNA expression levels and protein levels of XIAP gene in xenografts were detected respectively by real-time fluorescent quantitative PCR and Western blot. Apoptosis of the transplanted tumor cells was examined by TUNEL method. Among the six groups, the proliferation of transplanted tumor in Group F was the slowest, and the tumor inhibition rate was the highest compared with control Group A, followed by Group E, and the tumor inhibition rate was the lowest in Group C. Group F and E expressed the lowest XIAP mRNA and protein expressions ( P<0.05, vs. the other 4 groups) .The apoptosis rate was highest in Group F, followed by Group E, and lowest in Group A and C ( P<0.05). XIAP siRNA has synergy with taxol in taxol-resistant ovarian cancer cells.

Role of XIAP gene overexpressed bone marrow mesenchymal stem cells in the treatment of cerebral injury in rats with cerebral palsy

BackgroundThis study is performed to investigate the effects of adenovirus-mediated X-linked inhibitor of apoptosis protein (XIAP) overexpressed bone marrow mesenchymal stem cells (BMSCs) on brain injury in rats with cerebral palsy (CP).MethodsRat’s BMSCs were cultured and identified. The XIAP gene of BMSCs was modified by adenovirus expression vector Ad-XIAP-GFP. The rat model of CP with ischemia and anoxia was established by ligating the left common carotid artery and anoxia for 2 h, and BMSCs were intracerebroventricularly injected to the modeled rats. The mRNA and protein expression of XIAP in brain tissue of rats in each group was detected by RT-qPCR and western blot analysis. The neurobehavioral situation, content of acetylcholine (Ach), activity of acetylcholinesterase (AchE), brain pathological injury, apoptosis of brain nerve cells and the activation of astrocytes in CP rats were determined via a series of assays.ResultsRats with CP exhibited obvious abnormalities, increased Ach content, decreased AchE activity, obvious pathological damage, increased brain nerve cell apoptosis, as well as elevated activation of astrocyte. XIAP overexpressed BMSCs improved the neurobehavioral situation, decreased Ach content and increased AchE activity, attenuated brain pathological injury, inhibited apoptosis of brain nerve cells and the activation of astrocytes in CP rats.ConclusionOur study demonstrates that XIAP overexpressed BMSCs can inhibit the apoptosis of brain nerve cells and the activation of astrocytes, increase AchE activity, and inhibit Ach content, so as to lower the CP caused by cerebral ischemia and hypoxia in rats.

Inhibition of X-linked inhibitor of apoptosis protein enhances anti-tumor potency of pure total flavonoids on the growth of leukemic cells.

Flavonoids, a vast group of polyphenols widely distributed in plants, are known to possess a range of biological activities and potential anti-tumor effects. X-linked inhibitor of apoptosis protein (XIAP) promotes the progression of leukemia by preventing tumor cells undergoing apoptosis. The present study investigated the potential effects and underlying mechanisms of pure total flavonoids from Citrus paradisi Macfad (PTFC) on human U937 cells, and explored the effects of short hairpin (sh)RNA-mediated XIAP knockdown on the anti-cancer effects of PTFC. Western blotting was used to determine level of apoptosis-associated effectors following PTFC treatment. A lentiviral vector of RNA interference of XIAP gene was constructed to downregulate XIAP expression. MTT assay and flow cytometry were used to determine the effects of PTFC separately or combined with XIAP-shRNA on inhibition and apoptosis of U937 cells, respectively. Treatment with PTFC effectively inhibited leukemic cell proliferation in a dose- and time-dependent manner. PTFC induced apoptosis of U937 cells in a dose-dependent manner, at a particular concentration range, by decreasing XIAP expression levels and activating caspases-3, −7 and −9. PTFC treatment combined with XIAP-shRNA additionally demonstrated a marked increase in cell apoptosis, compared with PTFC or XIAP-shRNA alone (P<0.05). Therefore, these findings suggest that PTFC inhibits growth and induces apoptosis in U937 cells in vitro. Furthermore, suppression of XIAP expression enhances these effects.

The correlation between XIAP gene polymorphisms and esophageal squamous cell carcinoma susceptibility and prognosis in a Chinese population

ObjectiveThis study aims to explore the correlation between X-linked inhibitor of apoptosis protein (XIAP) gene polymorphisms (rs8371 and rs9856) with the susceptibility and prognosis of esophageal squamous cell carcinoma (ESCC), providing a potential treatment for ESCC. MethodA total of 170 ESCC patients (case group) and 191 healthy people (control group) were enrolled in our study. Genotyping was conducted on the basis of the ligase detection reaction (LDR). The expressions of XIAP polymorphisms were detected. The patients were followed up every three months until death or the last follow-up day. The overall survival (OS) and progression free survival (PFS) were recorded by Kaplan-Meier survival curve, and the relationship between XIAP gene polymorphism and risk and prognosis of ESCC was assessed by Cox multivariate analysis. ResultTT + CT genotype and T allele frequencies of XIAP rs8371 and rs9856 in the case group were significantly lower compared to those of the control group (all P < 0.05), suggesting that TT + CT genotype of XIAP rs8371 and rs9856 was associated with ESCC susceptibility. XIAP rs8371 and rs9856 polymorphisms were associated with tumor node metastasis (TNM) staging, depth of invasion and lymph node metastasis. The OS and PFS of TT + CT genotype carriers of rs8371 were longer than those of CC genotype carriers. Smoking, alcohol, TNM staging, depth of invasion, and lymph node metastasis were significantly associated with the OS and PFS in ESCC patients. Higher TNM staging, depth of invasion, and presence of lymph node metastasis were independent risk factors, while XIAP rs8371 was an independent protective factor for the prognosis of ESCC patients. ConclusionThe present study demonstrates that XIAP rs8371 and rs9856 are associated with susceptibility to ESCC, and rs8371 polymorphisms might serve as an indicator for improved clinical efficacy and prognosis of ESCC patients.

In Vitro Effects of Mesenchymal Stem Cells and Various Agents on Apoptosis of Glioblastoma Cells.

To investigate a new anti-tumor treatment method using stem cells transfected with specific genes and proteins that induce apoptosis in tumor cells. We used glioblastoma (GBM) cells and human adipose tissue-derived mesenchymal stem cells (ADMSCs) in this study. The AD-MSCs were transfected with the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). To overcome apoptosis resistance in tumor cells, we used suberoylanilide hydroxamic acid (SAHA) as the histone deacetylase inhibitor and embelin as the X-linked inhibitor of apoptosis protein (XIAP). In addition, we silenced the XIAP gene on GBM cells with the shXIAP plasmid. Following the determination of half-maximal effective concentration (EC50%) doses of SAHA and embelin, GBM cells were incubated with them for 24 hours. XIAP-silenced and XIAP-non-silenced GBM cells were cultured with TRAIL-nontransfected and TRAIL-transfected stem cells for 24 hours. Viability and cell cycle analysis of all groups were determined using annexin V/propidium iodide and cell cycle method via flow cytometry. TRAIL-transfected AD-MSCs, XIAP silencing, embelin, and SAHA induced apoptosis in GBM cells and decreased their proliferation, whereas TRAIL-non-tranfected AD-MSCs did not. Engineered stem cell therapies and molecular studies show promise in developing combination therapies for effective treatment of GBM.

Role of the 26S proteasome inhibitor b-AP15 on Jurkat cells and RS4; 11 cells

Objective To investigate the effect of 26S proteasome inhibitor b-AP15 on proliferation and apoptosis of Jurkat cells and RS4; 11 cells and to explore its possible mechanism. Methods Jurkat cells and RS4; 11 cells were cultured under asepsis condition. According to the presence of b - AP15 during culturing, Jurkat cells and RS4; 11 cells were divided into four groups: ①Jurkat cells experimental group, ②Jurkat cells control group, ③RS4; 11 cells experimental group, ④RS4; 11 cells control group. Jurkat cells experimental group and RS4; 11 cells experimental group were cultured with 0.05, 0.10, 0.50, 1.00, 5.00 μmol/L b-AP15 for 24 h and 48 h, while Jurkat cells control group and RS4; 11 cells control group were cultured synchronously using RPMI1640 medium. The cell growth curves of the four groups were analyzed by CCK-8. The cell apoptosis of the four groups were analyzed by Annexin V- allophycocyanin (APC)/7-aminoactinomycin D (7-AAD) double labeling flow cytometry. The cell cycle changes of the four groups were analyzed by flow cytometry.The mRNA expressions levels of B cell lymphoma/ leukemia-2 associated X protein (Bax) gene, B cell lymphoma/ leukemia (Bcl)-2 gene, X-linked inhibitor of apoptosis protein (XIAP) gene, cyclin-dependent kinase (CDK)1 gene and cysteine-aspartic proteases (caspase)-3 gene of the four groups were determined by real- time PCR. Results ①b-AP15 significantly inhibited the growth of Jurkat cells and RS4; 11 cells, and both the inhibitory effect were in a time-and dose-dependent manner. The IC50 values of Jurkat cells were(1.52±0.35)μmol/L and(0.54±0.01)μmol/L after culturing with b-AP15 for 24 h and 48 h. The IC50 values of RS4; 11 cells were (0.97±0.02)μmol/L and(0.08±0.03)μmol/L after culturing with b-AP15 for 24 h and 48 h. ②The apoptosis rates of Jurkat cells were significantly higher than the control groups after cultured with 1.5 μmol/L b-AP15 (P<0.001). The apoptosis rates of RS4; 11 cells significantly higher than the control groups after cultured with 1.0 μmol/L b-AP15 (P<0.001). Meanwhile, The apoptosis rates of both Jurkat cells and RS4; 11 cells cultured with b-AP15 for 48 h were significantly higher than these for 24 h (t=11.887, 7.449; P<0.001). ③ Compared with Jurkat cells control group, cell cycle of Jurkat cells experimental group cultured with 1.5 μmol/L b-AP15 for 24 h was arrested at G2/M period (t=10.672, P<0.05). At the same time, the proportion of S phase cells were reduced significantly (t=19.053, P<0.05). Compared with RS4; 11 cells control group, cell cycle of RS4; 11 cells experimental group cultured with 1.0 μmol/L b-AP15 for 24 h was also arrested at G2/M period (t=13.643, P<0.05). and the proportion of S phase cells were also reduced significantly(t=6.992, P<0.05). ④For Jurkat cells experimental group and RS4; 11 cells experimental group, the real-time PCR assay revealed that the relative mRNA expression levels of Bax gene, caspase-3 gene were increased significantly, while the relative mRNA expression levels of Bcl-2 gene, XIAP gene were reduced significantly. Meanwhile, mRNA expression level of the cell cycle related gene CDK1 was reduced significantly compared with their own control groups (P<0.05). The mRNA expression level of caspase-3 gene in RS4; 11 cells experimental group increased more significantly than that of Jurkat cells experimental group (P<0.05). Conclusion b-AP15 can inhibit proliferation and induce apoptosis of Jurkat cells and RS4; 11 cells, and the cell cycle of the two cells were both arrested at G2/M period. The mechanism of b-AP15 promoting apoptosis may be related with up-regulating the expression levels of Bax gene, caspase-3 gene and down-regulating the expression levels of Bcl-2 gene, XIAP gene. And the cell cycle arrested at G2/M period may be associated with down-regulating the expression levels of CDK1 gene. Key words: Protease inhibitor; Cell proliferation; Apoptosis; Cell cycle; Jurkat cells; RS4; 11 cells; b-AP15

Gene transfection mediated by polyethyleneimine-polyethylene glycol nanocarrier prevents cisplatin-induced spiral ganglion cell damage.

Polyethyleneimine-polyethylene glycol (PEI-PEG), a novel nanocarrier, has been used for transfection and gene therapy in a variety of cells. In our previous study, we successfully carried out PEI-PEG-mediated gene transfer in spiral ganglion cells. It remains unclear whether PEI-PEG could be used for gene therapy with X-linked inhibitor of apoptosis protein (XIAP) in the inner ear. In the present study, we performed PEI-PEG-mediated XIAP gene transfection in the cochlea of Sprague-Dawley rats, via scala tympani fenestration, before daily cisplatin injections. Auditory brainstem reflex tests demonstrated the protective effects of XIAP gene therapy on auditory function. Immunohistochemical staining revealed XIAP protein expression in the cytoplasm of cells in the spiral ganglion, the organ of Corti and the stria vascularis. Reverse transcription-PCR detected high levels of XIAP mRNA expression in the cochlea. The present findings suggest that PEI-PEG nanocarrier-mediated XIAP gene transfection results in XIAP expression in the cochlea, prevents damage to cochlear spiral ganglion cells, and protects hearing.

MiR-618 inhibits anaplastic thyroid cancer by repressing XIAP in one ATC cell line

X-linked inhibitor of apoptosis protein (XIAP) is a major factor in cancer growth and progression. Reduction of XIAP induces apoptosis of anaplastic thyroid cancer (ATC), which accounts for more than 50% of thyroid cancer mortality. MicroRNAs (miRNAs) are short non-coding RNA molecules, which modulate gene expression via interaction with mRNA by binding to the 3′-untranslated region (3′-UTR), playing a critical role in cell proliferation, migration, and invasion. In this study, we recruited the ATC cell line 8305C and normal human thyroid cell Nthy-ori 3-1, aiming to find the miRNA which could regulate XIAP and therefore inhibit the growth and invasion of ATC. We first used quantitative real-time PCR (qPCR) to reveal that XIAP mRNA expression was 4.6±0.56 folds (P=0.029) up-regulated in 8305C cells, compared with Nthy-ori 3-1 cells. Then miR-618, predicted to target XIAP directly, was detected 0.24±0.06 folds (P=0.019) down-regulated in 8305C cells. Next we used Luciferase assay showing that XIAP was a target gene of miR-618, which could repress the XIAP expression at both mRNA and protein levels. After that, CCK-8 assay was performed to show that over-expression of miR-618 could inhibit the growth of 8305C cells. Finally, we employed transwell method to prove that miR-618 could prevent the invasion and migration of 8305C cells. In conclusion, our collective data showed that over-expression of miR-618 could inhibit ATC cells by targeting XIAP gene.

Apoptosis of Purkinje and granular cells of the cerebellum following chronic ethanol intake.

Ethanol alters motricity, learning, cognition, and cellular metabolism in the cerebellum. We evaluated the effect of ethanol on apoptosis in Golgi, Purkinje, and granule cells of the cerebellum in adult rats. There were two groups of 20 rats: a control group that did not consume ethanol and an experimental group of UChA rats that consumed ethanol at 10% (<2 g ethanol/kg body weight/day). At 120 days old, rats were anesthetized and decapitated, and their cerebella were collected and fixed. Cerebellar sections were subjected to immunohistochemistry for terminal deoxynucleotide transferase dUTP nick end labeling (TUNEL), caspase-3, X-linked inhibitor of apoptosis protein (XIAP), and insulin-like growth factor 1-receptor (IGF-1R); real-time PCR (RT-PCR) to determine caspase-3, XIAP, and IGF-1R gene expression; and transmission electron microscopy (TEM). We identified fragmentation of DNA and an increase in caspase-3 protein and XIAP in Purkinje cells, whereas granule cells exhibited increased caspase-3 and XIAP. IGF-1R expression was unchanged. There was no significant difference in gene expression of caspase-3, XIAP, and IGF-1R. There were an increase in lipid droplets, a reduction in the cellular cytoplasm in electron-dense nuclei, and changes in the myelin sheath in the cerebellar cortex. In conclusion, our data demonstrated that ethanol induced apoptosis in the Purkinje and granule cells of the cerebellum of adult UChA rats.

Copolymer of poly(ethylene glycol) and poly(l-lysine) grafting polyethylenimine through a reducible disulfide linkage for siRNA delivery

siRNA therapy research has primarily focused on the synthesis and development of effective siRNA delivery vectors with easy biodegradability and low toxicity. In the present study, we synthesized a ternary copolymer mPEG-b-PLL-g-(ss-lPEI), denoted as PLI, by introducing disulfide bond linkages to graft low molecular weight linear polyethylenimine (lPEI) to the block copolymer of poly(L-lysine) (PLL) and poly(ethylene glycol) (PEG) for siRNA delivery. The PLL block and disulfide linkage rendered the carrier biodegradability, while lPEI grafting brought about the proton buffering capacity for lysosomal siRNA release and low cationic toxicity. Conjugation of a single chain monoclonal antibody (Herceptin) to the carrier as a targeting ligand for the Her2/neu receptor significantly increased the transfection activity of the copolymer/siRNA nanocomplex (i.e. the polyplex) in Skov-3, a human ovarian cancer cell line. Determination of gene expression at both the mRNA and protein levels demonstrated that Her2-targeted delivery of siRNA (XIAP siRNA) effectively downregulated the targeted XIAP (X-linked inhibitor of apoptosis protein) gene, resulting in enhanced cancer cell apoptosis and improved therapeutic efficacy in vitro and in vivo. The distinct features of low cytotoxicity, easy degradability, and high siRNA transfection efficiency make the copolymer a promising candidate for siRNA therapy in tumors.

Decreased Activation of the X‐linked inhibitor of Apoptosis Protein (XIAP) is Associated with Increased Apoptosis in the Preeclamptic Placenta

Normal placentation and placental development are critical for a successful pregnancy and mediate important steps such as implantation and delivery of nutrients to the fetus. Preeclampsia (PE) is characterized by an increase in placental apoptosis, however the mechanism is not conclusive. The X‐linked inhibitor of apoptosis protein (XIAP) protects against apoptosis. Our objective was to determine the level and localization of active XIAP protein in PE placenta compared to controls. Control and PE placental samples were collected shortly after delivery. Tissues were frozen in liquid Nitrogen for RNA and western blot studies. For immunohistochemical studies tissues were fixed and embedded in paraffin and stained for activated (phospho) XIAP. We observed: 1) a trend for a significant decrease in XIAP gene expression (60%) in the PE placenta as compared to controls; 2) No significant differences in XIAP protein expression; 3) a significant decreased in active (phospho) XIAP protein (30%; p&lt;0.05) in the placentas of PE women; and 4) decreased in active (phopspho) XIAP immunostaining in the syncitiotrophoblast layer of the PE placenta. Our data confirms that XIAP protein is expressed by the syncytiotrophoblasts in the human placenta during PE. We conclude that the decrease in activation of this protein during PE could potentially account for the increased apoptosis observed in this tissue at term. This result gives insights into pathways associated with the increased placental apoptosis and could be useful in the understanding of potential mechanisms that lead to placental insufficiency associated with this disease.

The Chinese Herb Isolate Isorhapontigenin Induces Apoptosis in Human Cancer Cells by Down-regulating Overexpression of Antiapoptotic Protein XIAP

Although the Chinese herb Gnetum cleistostachyum has been used as a remedy for cancers for hundred years, the active compounds and molecular mechanisms underlying its anti-cancer activity have not been explored. Recently a new derivative of stilbene compound, isorhapontigenin (ISO), was isolated from this Chinese herb. In the present study, we examined the potential of ISO in anti-cancer activity and the mechanisms involved in human cancer cell lines. We found that ISO exhibited significant inhibitory effects on human bladder cancer cell growth that was accompanied by marked apoptotic induction as well as down-regulation of the X-linked inhibitor of apoptosis protein (XIAP). Further studies have shown that ISO down-regulation of XIAP protein expression was only observed in endogenous XIAP, but not in constitutionally exogenously expressed XIAP in the same cells, excluding the possibility of ISO regulating XIAP expression at the level of protein degradation. We also identified that ISO down-regulated XIAP gene transcription via inhibition of Sp1 transactivation. There was no significant effect of ISO on apoptosis and colony formation of cells transfected with exogenous HA-tagged XIAP. Collectively, current studies, for the first time to the best of our knowledge, identify ISO as a major active compound for the anti-cancer activity of G. cleistostachyum by down-regulation of XIAP expression and induction of apoptosis through specific targeting of a SP1 pathway, and cast new light on the treatment of the cancer patients with XIAP overexpression.

Combination Therapy of Antiandrogen and XIAP Inhibitor for Treating Advanced Prostate Cancer

Overexpression of the androgen receptor (AR) and anti-apoptotic genes including X-linked inhibitor of apoptosis protein (XIAP) provide tumors with a proliferative advantage. Therefore, our objective was to determine whether novel antiandrogen (CBDIV17) and XIAP inhibitor based combination therapy can treat advanced prostate cancer. CBDIV17 and embelin-6g were synthesized and their effect on cell proliferation, apoptosis, cell cycle and AR and XIAP gene silencing determined. CBDIV17 was more potent than bicalutamide and inhibited proliferation of C4-2 and LNCaP cells, IC(50) for CBDIV17 was ≈ 12 μM and ≈ 21 μM in LNCaP and C4-2 cells, respectively, whereas bicalutamide had IC(50) of ≈ 46 μM in LNCaP cells and minimal effect in C4-2 cells. CBDIV17 induced apoptosis more effectively compared to bicalutamide and significantly inhibited DNA replication. Combination of CBDIV17 and embelin resulted in supra-additive antiproliferative and apoptotic effects. Embelin downregulated AR expression and decreased androgen-mediated AR phosphorylation at Ser(81). These hydrophobic drugs were solubilized using micelles prepared with polyethylene glycol-b-poly (carbonate-co-lactide) (PEG-b-p(CB-co-LA)) copolymer. Combination therapy inhibited prostate tumor growth more effectively compared to control or monotherapy in vivo. Our results demonstrated that CBDIV17 in combination with embelin can potentially treat advanced prostate cancer.

XIAP inhibition of β-cell apoptosis reduces the number of islets required to restore euglycemia in a syngeneic islet transplantation model

Clinical pancreatic islet transplantation has great promise as a treatment for type 1 diabetes but despite recent advances, it is still limited by the need for lifelong immunosuppression, restricted availability of donor islets, and uncertainty regarding long-term graft survival. Using a syngeneic, suboptimal islet transplantation model, we asked whether adenoviral overexpression of an anti-apoptotic protein, the X-linked inhibitor of apoptosis protein (XIAP) would protect transplanted islet cells from death and reduce the number of islets required for successful transplantation. Transplantation of 100 XIAP-expressing islets into the kidney capsule of syngeneic Balb/c mice restored euglycemia in 86% of recipients, where transplantation of 100 islets transduced with a control adenovirus expressing LacZ restored euglycemia in only 27% of recipients. Analysis of islet grafts by insulin/TUNEL double immunostaining revealed fewer apoptotic beta-cells in recipients of XIAP- compared with LacZ-expressing grafts (0.8±0.5 vs. 2.4±0.8 double-positive cells/graft), suggesting that XIAP enhances graft success by inhibiting β-cell apoptosis in the immediate post-transplant period. In summary, XIAP overexpression inhibits beta cell apoptosis in syngeneic islet transplants, thereby reducing the number of islets and decreasing the number of days required to restore euglycemia. These data raise the possibility that ex vivo XIAP gene transfer in islets prior to transplantation has the potential to increase the number of donor islets available for transplantation and may enhance graft function and long-term transplant success.

Effect of XIAP gene interference on proliferation and apoptosis of human QBC939 cells

Objective To study the inhibitory effects of antisense oligodeoxynucleotide (ASODN) on X-linked inhibitor of apoptosis protein (XIAP) gene expression in QBC939 cell line,and the effects on proliferation and apoptosis of QBC939 cells. Methods ASODN was transfected into cell line QBC939 by LipofectamineTM 2000,and transfection efficiency at the 12th,24th,and 48th h after transfec-tion was observed by fluorescence microscopy. Proliferation inhibition rate, XIAP mRNA expression,cell cycle and apoptosis rate of QBC939 cells treated with 1. 11 μmol/L ASODN were assayed by MTT,RT-PCR and flow cytometry (FCM). Results Transfection efficiency was about 95%. In contrast with the control group,the inhibition rate of cell proliferation was about (27.63 ±1.15)% (24 h) and (42.95 ± 1.07) % (48 h) respectively after treatment with 1.11 μmol/L ASODN. The gene expression level of XI-AP in ASODN-transfected group was decreased by 23% (P<0.05). The apoptosis rate and the percent-age of QBC939 cells in G_0/G_1 were increased by 27. 34% and 9. 94% in ASODN-transfected group as compared with control group (P<0. 05). Conclusion The liposomal transfection of ASODN can down-regulate the expression of XIAP gene, which can inhibit proliferation and induce apoptosis of QBC939 cells. Key words: Carcinoma of bile duct; Gene therapy; Apoptosis

Inhibiting XIAP expression by RNAi to inhibit proliferation and enhance radiosensitivity in laryngeal cancer cell line

Objectives X-linked inhibitor of apoptosis protein (XIAP) is a novel member of the inhibitors of apoptosis (IAPs) family. The overexpression of XIAP is asscociated with radioresistance of human malignancies. The purpose of the present study was to investigate the effect of shRNA-targeted XIAP on the proliferation, apoptosis and radiosensitivity of human laryngeal carcinoma cells (Hep-2). Methods A siRNA expression vector (pSilencer4.1-XIAPshRNA) was constructed and stably transfected into human laryngeal carcinoma cells (Hep-2). The downregulation of XIAP expression was evaluated by RT-PCR and Western blot analyses. Then, we investigated the effect of XIAP-shRNA on the proliferation, cell cycle changes and apoptosis in vitro of Hep-2 cells. Finally, the radiosensitivity of Hep-2 cells was investigated by clonogenic cell survival assay. Results We established stably transfected cell line (Hep-2/XIAPshRNA) in which the expression of XIAP gene was downregulated. The cell viability of Hep-2/XIAP-RNA cells was obviously decreased compared with that of untransfected Hep-2 cells. Morever, XIAP-shRNA induced cell arrest in the G 0/ G 1 phase of cell cycle by flow cytometry analysis. Results of TUNEL assay indicated that Hep-2 cells stably transfected pSilencer4.1-XIAP-shRNA showed obvious apoptosis characters. Furthermore, the downregulation of XIAP expression could lead to significant radiosensitivity enhancement in laryngeal carcinoma cells. Conclusions RNAi-mediated downregulation of XIAP expression can inhibit proliferation, induce apoptosis and diminish the radioresistance of laryngeal carcinoma cells, so combined therapy with XIAP inhibition and radiation may be a potential strategy for the treatment of laryngeal carcinoma.

Effects of apoptosis protein XIAP inhibitor on the apoptosis and sensitivity of chemotherapy in A549 cell.

X-linked inhibitor of apoptosis protein (XIAP) is a newly discovered inhibitor of apoptosis protein which prevents apoptosis by inhibiting the activation of caspase. After down-regulating XIAP gene expression in A549 cells, a non-small cell lung cancer (NSCLC) cell lines, we investigated the role of XIAP specific siRNA in apoptosis and chemotherapy sensitivity. The mRNA levels of XIAP gene in A549 cells were assessed using a semi-quantitative reverse transcriptase-PCR (RT-PCR). The expression vector of XIAP small interfering RNA (XIAP siRNAwas constructed and transfected into A549 cells. The transfection was proved effective by the fluorescence microscope. Cell proliferation and cell killing rate after chemotherapeutics treatment were investigated by MTT assay. The rate of apoptosis was detected by flow cytometry assay. XIAP siRNA construction was proved successful by enzyme digestion and DNA sequencing. The transfection efficiency in A549 cells from positive transfection group and negative transfection group had no differences. Compared to those in cell from control group, the level of XIAP mRNA expression was significantly decreased, the inhibition activity of Cisplatin was significantly higher in cells from positive transfection group. Proliferation of cells from positive transfection group was significantly inhibited after 24, 48, 72, 96 hours . The rates cell killing and apoptosis in cells from positive transfection group caused by Cisplatin were significantly higher compared to those cells from control group. The increased expression of XIAP in NSCLC can inhibit the apoptosis of NSCLC cells and result in NSCLC chemotherapy drug resistance. XIAP siRNA could inhibit the NSCLC cell growth specifically, down-regulation of XIAP gene expression promote apoptosis and increase the chemotherapy sensitivity of NSCLC. XIAP siRNA sequence might become a therapeutic target of NSCLC.