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Purpose : Retinoblastoma (RB) is an intraocular malignancy that often occurs in childhood. Circular RNAs (circRNAs) play crucial roles in regulating the malignant phenotypes of various tumors. This study aimed to explore the role and potential mechanism of circ_0000527 in RB. Methods: The levels of circ_0000527, microRNA-98-5p (miR-98-5p) and X-linked inhibitor of apoptosis (XIAP) were determined by qRT-PCR or western blot. Cell proliferation was detected by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) and colony formation assays. Cell apoptosis, migration and invasion were evaluated by flow cytometry, transwell and scratch assays. Xenograft assay was conducted to analyze tumor growth in vivo . The binding relationship between miR-98-5p and circ_0000527 or XIAP was verified by dual-luciferase reporter and RNA immunoprecipitation (RIP) assays. Results: Circ_0000527 and XIAP levels were increased, while miR-98-5p level was reduced in RB tissues and cells. Silencing of circ_0000527 suppressed the proliferation, migration and invasion of RB cells and promoted apoptosis. In addition, knockdown of circ_0000527 inhibited tumor growth in xenograft mice. Besides, circ_0000527 sponged miR-98-5p to regulate RB cell progression, and miR-98-5p targeted XIAP to mediate RB cell development. Moreover, circ_0000527 modulated XIAP expression via sequestering miR-98-5p. Conclusion: Circ_0000527 facilitated RB progression via regulating miR-98-5p/XIAP axis, which provided a promising therapeutic target for RB.

Apoptosis is an interactive and dynamic biological process involved in all phases of embryogenesis. If apoptosis dominates the trophoblast cell growth process, it will result in adverse pregnancy outcomes. X-linked inhibitor of apoptosis protein (XIAP) is a potent caspase inhibitor and an important barrier to apoptotic cell death. MicroRNAs involve in posttranscriptional gene expression regulation and apoptosis. Online sequence alignment analysis showed that there was a putative binding site of miR-371a-5p on the 3'-untranslated region (UTR) of XIAP. Thirty chorionic villi samples were collected to examine the expression of miR-371a-5p and XIAP. The dual-luciferase reporter assay was applied to determine the relationship between miR-371a-5p and XIAP by human placental choriocarcinoma cells (JEG-3) cells in vitro. After 48-hour transfection of mimics and inhibitor by JEG-3 cells in vitro, Western blotting was used to, respectively, detect the protein expression levels of XIAP and caspase-3. Flow cytometry was used to validate the apoptosis ratio of transfection. The expression of miR-371a-5p and XIAP in recurrent pregnancy loss was greatly decreased. The results from the luciferase reporter assay strongly suggested binding of the XIAP 3'-UTR by miR-371a-5p. Apoptosis percentage of miR-371a-5p mimic was significantly greater than that of normal control. However, apoptosis percentage of miR-371a-5p inhibitor was significantly lower than that of normal control. A significant decrease in luciferase activity was observed in miR-371a-5p mimics-transfected JEG-3 cells compared with controls. These findings provide the evidence that miR-371a-5p is one of the regulating factors according to apoptosis pathway of XIAP-caspase-3 and may be involved in the pathogenesis of recurrent pregnancy loss.

Prostate cancer (PCa) is one of the major men's malignancies with high mortality worldwide. Circular RNAs (circRNAs) have been shown to serve as essential regulators in human cancers. CircRNA can exert their functions by cooperating with their host genes. In the present study, microarray analysis revealed an upregulated mRNA in PCa samples. X-linked inhibitor of apoptosis protein (XIAP), a key regulator in the progression of human cancers. Through bioinformatics analysis, we determined that XIAP is a host gene for circRNA0005276. Therefore, this study focused on the interaction between circ0005276 and XIAP as well as their functions in PCa progression. The upregulation of XIAP and circ0005276 was determined in PCa tissues and cell lines. Moreover, we confirmed the positive regulation of circ0005276 on XIAP expression. Functionally, we validated that circ0005276 and XIAP promoted cell proliferation, migration and epithelial-mesenchymal transition. Mechanistically, we verified that circ0005276 interacted with FUS binding protein (FUS) so as to activate the transcription of XIAP. Rescue assays were conducted to determine the crucial role of XIAP in circ0005276 and FUS-mediated PCa cellular processes. Collectively, our study revealed the mechanism and function of circ0005276 and its host gene XIAP in PCa progression.

Dexmedetomidine (Dex) is an anesthetic widely used in lumbar discectomy, but its effect on chondrocytes remains unclear. Dex is speculated to promote cartilage degeneration by activating α-2 adrenergic receptor. However, the antioxidative and anti-inflammatory effects of Dex implied the potential chondrocyte protective effect under stress conditions. The present study aimed to determine the effect of Dex on chondrocytes under non-stress and stress conditions. Chondrocytes were isolated from human annulus fibrosus (AF) tissues and oxidative stress was induced by treatment with 1 mM hydrogen peroxide (H 2 O 2 ). Chondrocytes were treated with Dex alone or in combination with H 2 O 2 Treatment with Dex alone decreased mRNA expression of COL2A1 and increased that of MMP-3 and MMP-13 , thus contributing to cartilage degeneration. However, Dex prevented H 2 O 2 -induced death and degeneration of chondrocytes partly by enhancing antioxidant capacity. Mechanistically, Dex attenuated H 2 O 2 -mediated activation of NF-κB and NACHT, LRR, and PYD domains-containing protein 3 (NLRP3), both of which play key roles in inflammation and inflammatory damage. Dex inactivated NLRP3 through the suppression of NF-κB and JNK signals. Co-treatment with Dex and H 2 O 2 increased protein level of XIAP (X-linked inhibitor-of-apoptosis, an anti-apoptosis protein), compared with H 2 O 2 treatment alone. H 2 O 2 treatment increased the expression of neural precursor cell expressed developmentally down-regulated protein 4 (NEDD4) that is a ubiquitin ligase targeting XIAP. However, Dex decreased the amount of NEDD4 adhering to XIAP, thus protecting XIAP protein from NEDD4-mediated ubiquitination and degradation. Given that surgery inevitably causes oxidative stress and inflammation, the protective effect of Dex on chondrocytes during oxidative stress is noteworthy and warrants further study., (© 2019 The Author(s).)

We aimed to explore the efficacy of active caspase-3 and X-chromosome linked inhibitor of apoptosis protein (XIAP) as diagnostic markers for breast cancer. Furthermore, we examined the relationship between the examined parameters and clinicopathological factors. The current study involved 96 patients diagnosed with breast cancer and 40 patients had benign breast diseases. The expression of active caspase-3 was analyzed by both ELISA and Western blot, whereas the expression of XIAP was determined by ELISA in cell lysates. Active caspase-3 was significantly downregulated, while XIAP was markedly upregulated in patients with breast cancer in comparison to benign group. A significant negative correlation was observed between active caspase-3 and XIAP in breast cancer patients. Low active caspase-3 expression was associated with high grade, whereas, the high XIAP level was correlated with poorly differentiated tumors and late tumor stages. The sensitivity and specificity were 73.96% and 80.0% for active caspase-3, and, 70.83% and 82.5% for XIAP. A combination of active caspase-3 and XIAP provided a promising sensitivity of 88.54% and specificity of 90.0%. Our data indicate that active caspase-3 and XIAP could be substantial diagnostic markers for breast cancer patients.

Physiological stress conditions attenuate global mRNA translation via modifications of key eukaryotic initiation factors. However, non-canonical translation initiation mechanisms allow cap-independent translation of certain mRNAs. We have previously demonstrated that eIF5B promotes cap-independent translation of the mRNA encoding the antiapoptotic factor, XIAP, during cellular stress. Here, we show that depletion of eIF5B sensitizes glioblastoma multiforme cells to TRAIL-induced apoptosis by a pathway involving caspases-8, -9, and -7, with no significant effect on cell cycle progression. eIF5B promotes evasion of apoptosis by promoting the translation of several IRES-containing mRNAs, encoding the antiapoptotic proteins XIAP, Bcl-xL, cIAP1, and c-FLIP S . We also show that eIF5B promotes translation of nuclear factor erythroid 2-related factor 2 and suggest that reactive oxygen species contribute to increased apoptosis under conditions of eIF5B depletion. Finally, eIF5B depletion leads to decreased activation of the canonical NF-κB pathway. Taken together, our data suggest that eIF5B represents a regulatory node, allowing cancer cells to evade apoptosis by promoting the translation of pro-survival proteins from IRES-containing mRNAs.

Our recent studies demonstrate that X-linked inhibitor of apoptosis protein (XIAP) is essential for regulating colorectal cancer invasion. Here, we discovered that RhoGDIβ was a key XIAP downstream effector mediating bladder cancer (BC) invasion in vitro and in vivo. We found that both XIAP and RhoGDIβ expressions were consistently elevated in BCs of N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN)-treated mice in comparison to bladder tissues from vehicle-treated mice and human BCs in comparison to the paired adjacent normal bladder tissues. Knockdown of XIAP attenuated RhoGDIβ expression and reduced cancer cell invasion, whereas RhoGDIβ expression was attenuated in BBN-treated urothelium of RING-deletion knockin mice. Mechanistically, XIAP stabilized RhoGDIβ mRNA by its positively regulating nucleolin mRNA stability via Erks-dependent manner. Moreover, ectopic expression of GFP-RhoGDIβ in T24T(shXIAP) cells restored its lung metastasis in nude mice. Our results demonstrate that XIAP-regulated Erks/nucleolin/RhoGDIβ axis promoted BC invasion and lung metastasis., (© 2017 UICC.)

Introduction: Due to Plasmodium resistance to antimalarial drugs, it is important to find new therapeutic alternatives for malaria treatment and control. Based on the knowledge of Colombian indigenous communities, we collected extracts of plants with potential antimalarial effects from the middle Vaupés region., Objective: To evaluate the mutagenic and genotoxic effects, as well as the gene expression of Rad51C, Xiap, P53 and Nrf2 induced by four ethanolic extracts with antimalarial activity (R001, T002, T015 and T028)., Materials and Methods: We evaluated four ethanolic extracts with antimalarial activity using the Ames test to assess mutagenicity, and the comet assay on HepG2 cells to determine the genotoxicicity. We also evaluated the expression of Rad51C, Xiap, P53 and Nrf2 from HepG2 cells stimulated with the four extracts., Results: None of the four extracts was mutagenic in Salmonella typhimurium TA98 strain in the presence and absence of S9 metabolic activity. Extracts R001, T015 and T028 were weakly mutagenic on the TA100 strain in the presence of S9, with mutagenic indexes (MI) of 1.58, 1.53 and 1.61, respectively. The T015 strain showed the same behavior without S9 with an MI of 1.36. The results of the comet assay showed that the four extracts produced category 1 or 2 damage, with comets between 36.7 and 51.48 μm in length. However, the genetic damage index suggested that most of the cells were affected by the treatments. Regarding gene expression, extracts R001 and T028 induced an overexpression of genes Xiap and P53 with an 1.84 to 3.99 fold-change compared with untreated cells., Conclusions: These results revealed that the T002 extract was the safest as it had antimalarial activity and was not cytotoxic on HepG2 cells. Moreover, it was not mutagenic and it only produced category 1 damage on the DNA. Also, the extract did not induce a change in the expression of the tested genes.

Background/aim: The aim of the study was to evaluate the anti-tumor mechanism of Z-360, a gastrin/cholecystokinin-2 receptor (CCK2R) antagonist, in MIA PaCa-2 cells and in a subcutaneous xenograft mice model., Materials and Methods: The anti-tumor effects of Z-360 and/or gemcitabine were monitored using a MIA PaCa-2 xenograft model. The effect of Z-360 on apoptosis in the model was examined by TUNEL staining and real-time PCR analysis and the effect in MIA PaCa-2 cells stably expressing human CCK2R was also evaluated by caspase-3/7 activity., Results: In this xenograft model, Z-360 significantly reduced the tumor weight, increased TUNEL-positive cells and suppressed the expression of anti-apoptosis factors such as survivin, XIAP and Mcl-1, and these effects of Z-360 combined with gemcitabine were more effective. Furthermore, gastrin-17 and gastrin-34 inhibited apoptosis in vitro and Z-360 dose-dependently abrogated this effect., Conclusion: These results suggest that Z-360 exerts an anti-tumor effect through a reduction in anti-apoptosis factors by blocking CCK2R., (Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

A recent study showned that complementary medicine is gradually gaining wide acceptance. In the present study, the herbal mixture extract (H3) composed of 3 oriental herbal plants was investigated for anticancer activity in vitro and in vivo. H3 inhibited PANC1 cell growth by promoting G0/G1 arrest (11% increase) and apoptotic cell death (9% increase). H3 also suppressed stem cell-like side population cells (4% decrease) and migration activity (24% decrease). In contrast, gemcitabine decreased side population cells and migration activity by 3 and 11%, respectively. These effects of H3 and gemcitabine were further studied by examining the expression of apoptosis-associated genes (CXCR4, JAK2 and XIAP) and stem cell-associated genes (ABCG2, POU5F1 and SOX2). We also found that H3 suppressed tumor growth by 46% in a PANC1‑xenograft model, while gemcitabine caused a 36% decrease. The antitumor effects of H3 were confirmed by western blot analysis for COX-2 and cytochrome c expression. Furthermore, necrotic cell death and erythrocyte-containing cavities were detected in tumor tissue by hematoxylin and eosin (H&E) staining. Notably, the combinatorial therapy (H3 and gemcitabine) increased tumor growth compared to that in the control. In conclusion, the present study shows that H3 has promise as a therapeutic agent against pancreatic cancer and its cancer stem cells.

Recently, several studies have shown that piperlongumine (PL) can selectively kill cancer cells by targeting reactive oxygen species (ROS). However, the potential therapeutic effects and detailed mechanism of PL in gastric cancer are still not clear. In the current report, we found that PL significantly suppressed gastric cancer both in vitro and in vivo. PL obviously increased ROS generation in gastric cancer cells. Anti-oxidant glutathione (GSH) and N-acetyl-L-cysteine (NAC) can abrogate PL-induced gastric cancer cell death and proliferation inhibition. GADD45α was induced in PL-treated cancer cells and led to G2/M phase arrest, whereas genetic depletion of GADD45α by small interfering RNAs (siRNAs) could partly reverse PL-induced cell cycle arrest in gastric cancer cells. Interestingly, we also found that PL treatment decreased the expression of telomerase reverse transcriptase (TERT) gene, which plays an essential role in cancer initiation and progression. Our findings thus revealed a potential anti-tumor effect of PL on gastric cancer cells and may have therapeutic implications.

The discovery of the TRAIL protein and its death receptors DR4/5 changed the horizon of cancer research because TRAIL specifically kills cancer cells. However, the validity of TRAIL-based cancer therapies has yet to be established, as most cancer cells are TRAIL-resistant. In this report, we demonstrate that TRAIL-resistance of many cancer cell lines can be overcome after siRNA- or rocaglamide-mediated downregulation of c-FLIP expression and simultaneous inhibition of IAPs activity using AT406, a pan-antagonist of IAPs. Combined triple actions of the TRAIL, the IAPs inhibitor, AT406, and the c-FLIP expression inhibitor, rocaglamide (ART), markedly improve TRAIL-induced apoptotic effects in most solid cancer cell lines through the activation of an extrinsic apoptosis pathway. Furthermore, this ART combination does not harm normal cells. Among the 18 TRAIL-resistant cancer cell lines used, 15 cell lines become sensitive or highly sensitive to ART, and two out of three glioma cell lines exhibit high resistance to ART treatment due to very low levels of procaspase-8. This study provides a rationale for the development of TRAIL-induced apoptosis-based cancer therapies.

Background: Cholangiocarcinoma is characterized by late diagnosis and a poor survival rate. MicroRNAs have been involved in the pathogenesis of different cancer types, including cholangiocarcinoma. Our aim was to identify novel microRNAs regulating cholangiocarcinoma cell growth in vitro and in vivo., Methods: A functional microRNA library screen was performed in human cholangiocarcinoma cells to identify microRNAs that regulate cholangiocarcinoma cell growth. Real-time PCR analysis evaluated miR-9 and XIAP mRNA levels in cholangiocarcinoma cells and tumors., Results: The screen identified 21 microRNAs that regulated >50 % cholangiocarcinoma cell growth. MiR-410 was identified as the top suppressor of growth, while its overexpression significantly inhibited the invasion and colony formation ability of cholangiocarcinoma cells. Bioinformatics analysis revealed that microRNA-410 exerts its effects through the direct regulation of the X-linked inhibitor of apoptosis protein (XIAP). Furthermore, overexpression of miR-410 significantly reduced cholangiocarcinoma tumor growth in a xenograft mouse model through induction of apoptosis. In addition, we identified an inverse relationship between miR-410 and XIAP mRNA levels in human cholangiocarcinomas., Conclusions: Taken together, our study revealed a novel microRNA signaling pathway involved in cholangiocarcinoma and suggests that manipulation of the miR-410/XIAP pathway could have a therapeutic potential for cholangiocarcinoma.

Purpose: To investigate effect of embelin on proliferation, apoptosis and gene expression profile changes in breast cancer cells., Methods: Cell viability was determined by MTT assay and apoptosis assayed using flow cytometry. Differential expression of 84 genes commonly involved in breast cancer carcinogenesis was assessed by real-time PCR using the Human Breast Cancer RT(2) Profiler PCR Array., Results: MCF-7 and MDA-MB-231 cells were treated with embelin (0-25μM) for 24 and 96h. Embelin exhibited time and dose dependence in both cell lines and was more potent in inhibiting MDA-MB-231 cell proliferation compared to MCF-7 cells. IC50 for embelin in MDA-MB-231 cells was ∼4.45μM and 3.28μM at 24h and 96h, respectively. In contrast, IC50 for embelin in MCF-7 cells was ∼6.04μM and 4.51μM at 24h and 96h, respectively. Embelin (50μM) induced apoptosis and activated caspase 3 activity in both cell lines when exposed for 72h. Treatment of MDA-MB-231 cells with embelin (10μM) for 24h resulted in significant differential expression of 27 genes commonly involved in breast cancer carcinogenesis., Conclusions: Our findings show that embelin inhibits cell proliferation, induces apoptosis and alters expression of breast cancer focused genes in MCF-7 and MDA-MB-231 cells. Based on RT(2)-PCR array analysis, embelin down-regulated expression of pivotal oncogenes. This knowledge could be beneficial in the development of effective embelin-based therapies for treating breast cancer., (Published by Elsevier Urban & Partner Sp. z o.o.)

In this study, we showed that the levels of EZH2 in bone marrow mononuclear cells (BMMNCs) isolated from individuals with chronic myeloid leukemia (CML) (n=12) were significantly greater than those in BMMNCs isolated from healthy volunteers (n=6) as well as individuals with Philadelphia chromosome-negative myeloproliferative neoplasms. Lentiviral transduction of the BCR/ABL gene in Ba/F3 cells increased EZH2 levels in parallel with phosphorylation of STAT5. Notably, chromatin immunoprecipitation assays showed that STAT5A bound to a promoter region of the EZH2 gene, resulting in an increase in the transcriptional activity of EZH2 in leukemia cells. Importantly, downregulation of EZH2 by short hairpin RNAs (shRNAs) inhibited the expression of XIAP and increased the miR-219 levels associated with a decrease in hypermethylation of miR-219-1 CpG islands. Moreover, overexpression of miR-219 decreased the levels of XIAP in CML cells. Since the 3'-untranslated region (3'-UTR) of XIAP contains miR219-5p-complementary binding site, miR-219 might modulate the expression of XIAP through binding of miR-219 on the 3'-UTR of XIAP. Taken together, BCR/ABL positively regulates the expression of EZH2 via STAT5 signaling. EZH2 modulates epigenetic changes at DNA methylated regions encoding miR-219 and downregulates the level of miR-219, resulting in upregulation of XIAP., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Development of an efficient treatment for triple-negative breast cancer is an urgent issues. Compounds from plant extracts are a potential source of novel cancer treatment. Isolinderalactone, a kind of sesquiterpenoids compound, was purified from the root of Lindera strychnifolia and Neolitsea daibuensis and shows anti-inflammatory and anticancer capacity. In the present study, isolinderalactone induced apoptosis in MDA-MB-231 cells which is a kind of triple-negative breast cancer cell line through induction of an intrinsic mitochondria-mediated and caspase-independent cell death. Treatment of isolinderalactone increased the protein level of the suppressor of cytokine signaling 3 (SCOS3), decreased phosphorylation of the signal transducer and activator of transcription 3 (STAT3), and suppressed expression of the down-stream genes of the X-linked inhibitor of apoptosis protein in MDA-MB-231 cells. Our results further showed that the level of SOCS3 expression was induced by isolinderalactone due to inhibiting the microRNA hsa-miR-30c-5p (miR-30c) expression. In addition, intraperitoneal injection of isolinderalactone induced apoptosis in a xenograft breast tumor while it did not significantly affect the histology of liver, kidney and lung of the treated mice. In conclusion, isolinderalactone induces apoptosis in MDA-MB‑231 cells and suppresses STAT3 signaling pathway through regulation of SOCS3 and miR-30c. It may become a novel treatment for triple-negative breast cancer in the future.

Epithelial ovarian cancer (EOC) accounts for 90% of all ovarian cancer, which is the third most common gynaecological malignancy worldwide. Dysregulation of miRNAs is involved in the development of different types of EOC. The present study was designed to investigate the role of abnormal expression of miR-215 in the development of EOC and to elucidate the possible molecular mechanisms. mRNA expression of miR-215 was significantly decreased in EOC tissues and cell lines. Upregulation of miR-215 inhibited cell proliferation, promoted apoptosis and increased sensitivity to chemotherapy drugs in EOC cells. In contrast, downregulation of miR-215 increased cell proliferation, inhibited apoptosis and decreased sensitivity to chemotherapy drugs in EOC cells. In addition, the X-chromosome-linked inhibitor of apoptosis (XIAP) expression was significantly increased in EOC tissues and cell lines. Downregulation of XIAP inhibited cell proliferation, promoted apoptosis and increased sensitivity to chemotherapy drugs in EOC cells. Upregulation of miR-215 notably inhibited the expression of XIAP. Moreover, overexpression of XIAP significantly inhibited miR-215-exerted decrease of proliferation, increase of apoptosis and increase of sensitivity to chemotherapy drugs. In conclusion, we identified miR-215 as a potential tumor suppressor in patients with EOC downregulating expression of the oncogenic regulator XIAP. The data demonstrate that miR-215/XIAP pathway may serve as novel therapeutic targets and prognostic markers in patients with EOC.

The signal transducers and activators of transcription 3 (STAT3) signaling pathway plays critical roles in the pathogenesis and progression of various human cancers, including non-small cell lung cancer (NSCLC). In this study, we aimed to evaluate the therapeutic potential of physalin A, a bioactive withanolide derived from Physalis alkekengi var. francheti used in traditional Chinese medicine, was evaluated in human NSCLC cells. Its and determined whether it effect oninhibited both constitutive and induced STAT3 activity, through repressing the phosphorylation levels of JAK2 and JAK3, resulting in anti-proliferation and pro-apoptotic effects on NSCLC cells was also determined, and. theThe antitumor effects of physalin A were also validated usingin an in vivo mouse xenograft models of NSCLC cells. Physalin A had anti-proliferative and pro-apoptotic effects in NSCLC cells with constitutively activated STAT3; it also suppressed both constitutive and induced STAT3 activity by modulating the phosphorylation of JAK2 and JAK3. Furthermore, physalin A abrogated the nuclear translocation and transcriptional activity of STAT3, thereby decreasing the expression levels of STAT3, its target genes, such as Bcl-2 and XIAP. Knockdown of STAT3 expression by small interfering RNA (siRNA) significantly enhanced the pro-apoptotic effects of physalin A in NSCLC cells. Moreover, physalin A significantly suppressed tumor xenograft growth. Thus, as an inhibitor of JAK2/3-STAT3 signaling, physalin A, has potent anti-tumor activities, which may facilitate the development of a therapeutic strategy for treating NSCLC.

Objectives: SL4, a chalcone-based compound, exhibits clearly inhibitory effects on HIF-1 and has been shown to effectively suppress tumour invasion and angiogenesis in vitro and in vivo. Here, studies were conducted to determine SL4's anti-apoptotic effects and its underlying mechanisms, in human cancer cells., Materials and Methods: Cytotoxicity, apoptotic induction and its involved mechanisms of SL4 were investigated using normal cells, cancer cells and mouse xenograft models. The role of reactive oxygen species (ROS) and mitogen-activated protein kinase (MAPK) signalling in SL4-induced apoptosis was explored by manipulating specific scavenger or signalling inhibitors, in cultured cells., Results: SL4 significantly inhibited cell population growth of human cancer cell lines but exhibited lower cytotoxicity against normal cells. In addition, SL4 effectively induced apoptosis of Hep3B and MDA-MB-435 cells by activating procaspase-8, -9 and -3, and down-regulating expression levels of XIAP, but did not affect HIF-1 apoptosis-related targets, Survivin and Bcl-XL. Further study showed that SL4 also reduced mitochondrial membrane potential and promoted generation of ROS. ROS generation and apoptotic induction by SL4 were blocked by NAC, a scavenger of ROS, suggesting SL4-induced apoptosis via ROS accumulation. We also found that MAPKs, JNK and p38, but not ERK1/2, to be critical mediators in SL4-induced apoptosis. SP600125 and SB203580, specific inhibitors of JNK kinase and p38 kinase, significantly retarded apoptosis induced by SL4. Moreover, anti-oxidant NAC blocked activation of JNK and p38 induced by SL4, indicating that ROS may act as upstream signalling of JNK and p38 activation. It is noteworthy that animal studies revealed dramatic reduction (49%) in tumour volume after 11 days SL4 treatment., Conclusions: These data demonstrate that SL4 induced apoptosis in human cancer cells through activation of the ROS/MAPK signalling pathway, suggesting that it may be a novel lead compound, as a cancer drug candidate, with polypharmacological characteristics., (© 2015 John Wiley & Sons Ltd.)

Background: The expression status of apoptotic regulators, such as caspases and inhibitors of apoptosis proteins (IAPs), could reflect the aggressiveness of tumors and, therefore, could be useful as prognostic markers. We explored the associations between tumor expression of caspases and IAPs and clinicopathological features of prostate cancer--clinical and pathological T stage, Gleason score, preoperative serum PSA levels, perineural invasion, lymph node involvement, surgical margin status and overall survival--and evaluated its capability to predict biochemical progression after radical prostatectomy., Methods: Protein expression of caspases (procaspase-8, cleaved caspase-8, procaspase-3, cleaved caspase-3, caspase-7 and procaspase-9) and IAPs (cIAP1/2, cIAP2, NAIP, Survivin and XIAP) was analyzed by immunohistochemistry in radical prostatectomy samples from 84 prostate cancer patients. Spearman's test, Kaplan-Meier curves, and univariate and multivariate Cox proportional hazard regression analysis were performed., Results: cIAP1/2, cIAP2, Survivin, procaspase-8, cleaved caspase-8, procaspase-3 and caspase-7 expression correlated with at least one clinicopathological feature of the disease. Patients negative for XIAP, procaspase-3 or cleaved caspase-3 had a significantly worse prognosis. Of note, XIAP, procaspase-3 and cleaved caspase-3 were predictors of biochemical progression independent of Gleason score and pathological T stage., Conclusions: Our results indicate that alterations in the expression of IAPs and caspases contribute to the malignant behavior of prostate tumors and suggest that tumor expression of XIAP, procaspase-3 and cleaved caspase-3 may help to identify prostate cancer patients at risk of progression.

Estrogen (E2) has been suggested to have a protective role in attenuating hepatocellular carcinoma (HCC) development. miRNAs have great potential as biomarkers and therapeutic agents owing to their ability to control gene expression. However, little is known about the mechanism underlying the protective role of E2 in hepatocarcinogenesis and the effects of E2 on apoptotic miRNAs expression. Using miRNA PCR array, we found more than 2-fold alteration was observed in 25 upregulated and 10 downregulated apoptotic miRNAs in E2-treated cells. Among these miRNAs, we found expression of miR-23a was related to p53 functional status in the male-derived liver cell-lines. We demonstrated that E2 via ERα transcriptionally activated miR-23a and p53 expression, and thus enhanced p53 activation of miR-23a expression. Moreover, miR-23a expression correlated inversely with the expression of target gene X-linked inhibitor of apoptosis protein (XIAP), but positively with the caspase-3/7 activity. Decreasing of XIAP might contribute to caspase-3 activity and cell apoptosis. Taken together, our findings reveal a novel E2-signaling mechanism in regulating miRNAs expression for controlling apoptosis in liver cells. Delineating the role of E2 in regulating the activation of p53 and miR-23a, expression in HCC is crucial to the understanding of the sex difference observed in HCC.

Background: Although Imatinib mesylate has revolutionized the treatment of chronic myeloid leukemia, some patients develop resistance with progression of leukemia. Alternative or additional targeting of signalling pathways deregulated in Bcr-Abl-driven chronic myeloid leukemia may provide a feasible option for improving clinical response and overcoming resistance., Results: In this study, we investigate ability of CR8 isomers (R-CR8 and S-CR8) and MR4, three derivatives of the cyclin-dependent kinases (CDKs) inhibitor Roscovitine, to exert anti-leukemic activities against chronic myeloid leukemia in vitro and then, we decipher their mechanisms of action. We show that these CDKs inhibitors are potent inducers of growth arrest and apoptosis of both Imatinib-sensitive and -resistant chronic myeloid leukemia cell lines. CR8 and MR4 induce dose-dependent apoptosis through mitochondrial pathway and further caspases 8/10 and 9 activation via down-regulation of short-lived survival and anti-apoptotic factors Mcl-1, XIAP and survivin which are strongly implicated in survival of Bcr-Abl transformed cells., Conclusions: These results suggest that CDK inhibitors may constitute a complementary approach to treat chronic myeloid leukemia.

Background: The mainstay of treatment in rectal cancer is neoadjuvant radio chemotherapy prior to surgery, in an attempt to downstage the tumour, allowing for more complete removal during surgery. In 40 % of cases however, this neoadjuvant radio chemotherapy fails to achieve tumour regression, partly due insufficient apoptosis signaling. X-linked Inhibitor of Apoptosis Protein (XIAP) is an anti-apoptotic protein that has been reported to contribute to disease progression and chemotherapy resistance., Methods: We obtained rectal biopsy normal and matched tumour tissue from 29 rectal cancer patients with varying degrees of tumour regression, and using Western blot, examined anti-apoptotic XIAP and pro-apoptotic Smac protein levels in these tissues, with the aim to examine whether disturbed XIAP/Smac levels may be an indicator of neoadjuvant radio chemotherapy resistance. Expression of inhibitor of apoptosis proteins cIAP-1 and cIAP-2 was also examined., Results: We found that levels of XIAP increased in accordance with the degree of radio chemotherapy resistance of the tissue. Levels of this protein were also significantly higher in tumour tissue, compared to matched normal tissue in highly resistant tissue. In contrast, Smac protein levels did not increase with radio chemotherapy resistance, and the protein was similarly expressed in normal and tumour tissue, indicating a shift in the balance of these proteins. Post treatment surgical resection tissue was available for 8 patients. When we compared matched tissue pre- and post- radio chemotherapy we found that XIAP levels increased significantly during treatment in both normal and tumour tissue, while Smac levels did not change. cIAP-1 and cIAP-2 levels were not differentially expressed in varying degrees of radio chemotherapy resistance, and neoadjuvant therapy did not alter expression of these proteins., Conclusion: These data indicate that disturbance of the XIAP/Smac balance may be a driver of radio chemotherapy resistance, and hence high levels of XIAP may be a useful indicator of neoadjuvant radio chemotherapy resistance in rectal cancer. Moreover, as XIAP levels increase with radio chemotherapy it is possible that a subset of more resistant tumour cells survive this treatment and may be resistant to further adjuvant treatment. Patients with resistant tumours highly expressing XIAP may benefit from alternative treatment strategies, such as Smac mimetics post neoadjuvant radio chemotherapy.

Pancreatic beta cell death is a hallmark of type 1 (T1D) and type 2 (T2D) diabetes, but the molecular mechanisms underlying this aspect of diabetic pathology are poorly understood. Here we report that expression of the microRNA (miR)-200 family is strongly induced in islets of diabetic mice and that beta cell-specific overexpression of miR-200 in mice is sufficient to induce beta cell apoptosis and lethal T2D. Conversely, mir-200 ablation in mice reduces beta cell apoptosis and ameliorates T2D. We show that miR-200 negatively regulates a conserved anti-apoptotic and stress-resistance network that includes the essential beta cell chaperone Dnajc3 (also known as p58IPK) and the caspase inhibitor Xiap. We also observed that mir-200 dosage positively controls activation of the tumor suppressor Trp53 and thereby creates a pro-apoptotic gene-expression signature found in islets of diabetic mice. Consequently, miR-200-induced T2D is suppressed by interfering with the signaling of Trp53 and Bax, a proapoptotic member of the B cell lymphoma 2 protein family. Our results reveal a crucial role for the miR-200 family in beta cell survival and the pathophysiology of diabetes.

X-linked inhibitor of apoptosis protein (XIAP) has been shown to be highly expressed in lung cancer, but not in normal lung tissue, which makes it an attractive target for lung cancer treatment. Celecoxib (CXB), a cyclooxygenase-2 inhibitor, is in wide clinical use for the treatment and prevention of non-small cell lung cancer (NSCLC). Therefore, in our study, we combined short hairpin RNA (shRNA) targeted to XIAP (XIAP-shRNA) with CXB and tested the effects of this combination on lung cancer cells to identify more effective therapeutics against lung cancer. An XIAP-shRNA plasmid was constructed and transfected into the A549 NSCLC cell line. The cells were then treated with CXB and XIAP-shRNA alone or in combination for indicated time periods, and the treatments were assessed for their effects on cell proliferation, apoptosis, migration, invasion and receptor signaling using the MTT, TUNEL, wound healing and Matrigel invasion assays and western blotting, respectively. In addition, an NSCLC xenograft model was prepared to observe tumor growth. It was found that both CXB and XIAP‑shRNA significantly inhibited cell proliferation, migration and invasion, and induced cell apoptosis in vitro, as well as suppressed tumor growth in vivo. Moreover, the combination of the agents significantly enhanced these effects compared to the single agent treatments. We also found that the combination treatment significantly suppressed constitutive phosphorylation of PI3K and AKT, which may contribute to the inhibition of tumor growth. These findings suggest that the combination of XIAP‑shRNA and CXB is a promising drug candidate for the treatment of NSCLC.

X-chromosome-linked inhibitor of apoptosis protein (XIAP) is an important member of the inhibitors of apoptosis (IAP) family. It has been shown that XIAP promotes the invasion, metastasis, growth and survival of malignant cells, and confers resistance to some chemotherapeutic drugs in various types of cancer. However, little is known regarding its detailed role in osteosarcoma (OS). In the present study, we first investigated the expression of XIAP in OS tissues, and an increased expression of XIAP in OS tissues compared to adjacent non-tumor tissue was identified. Additionally, its expression level correlated with key pathological characteristics including clinical stage, tumor size and metastasis. Subsequently, small interfering RNA (siRNA) was used to block XIAP expression to evaluate the effect of XIAP siRNA on cell proliferation, colony formation, cell cycle, apoptosis, tumorigenicity, and the combined effects of doxorubicin or cisplatin in OS cell lines (MG63 cells). Downregulation of XIAP expression using the RNA silencing approach efficiently decreased cell proliferation and colony formation, and induced cell apoptosis and cell cycle in the G0/G1 stage. In addition, this downregulation inhibited tumor growth in a nude murine model. The resuls also showed that treatment with XIAP-shRNA in combination with doxorubicin or cisplatin enhanced chemosensitivity. These results suggested that XIAP may aid the diagnosis of OS and may be an effective strategy for gene therapy of this disease.

Studies have indicated that hydroxycamptothecin (HCPT) induces apoptosis of fibroblasts. In this study, we investigated the apoptotic gene expression profiling in HCPT-treated -human Tenon's capsule fibroblasts (HTCFs) and identify the most implicated gene in apoptotic signaling of HTCFs by HCPT. Method HTCFs were incubated with HCPT at 0, 0.25 and 4 mg/L for 24, 48 and 72 h, respectively. Anti-proliferative effects were measured by MTT assay. Apoptosis was determined using the Annexin V-FITC/PI apoptosis detection kit and apoptotic cells were identified by flow cytometry. A PCR array was employed to analyze the most implicated apoptotic genes during HCPT-induced apoptosis in HTCFs. Results from our studies showed that HCPT induced apoptosis in HTCFs in a concentration-and time-dependent manner. The apoptotic HTCFs was increased by 9.38% with a multiplicity at 4 mg/L HCPT. PCR array demonstrated remarkable changes in 88 apoptotic genes, including 9 up-regulated genes and 36 down-regulated genes. HCPT treatment induced the upregulation of CHOP and downregulation of XIAP in HTCFs. To conclude, our results support HCPT induced the apoptosis of HTCFs, involving the activation of mitochondrial and endoplasmic reticulum stresses as well as the downregulation expression of XIAP.

MicroRNA (miR)-216a expression is significantly downregulated in human pancreatic cancer, however, the underlying mechanism remains unknown. In the present study, we aimed to identify and characterize the direct target gene and potential function of miR-216a in pancreatic cancer cells. Bioinformatics analysis and dual-luciferase reporter gene assay showed that Janus kinase 2 (JAK2) was a direct target gene of miR-216a. Quantitative polymerase chain reaction and western blot analysis demonstrated that miR-216a decreased the mRNA and protein levels of JAK2 in pancreatic cancer cells. Phosphorylation of the signal transducer and activator of transcription 3 (STAT3) was also downregulated by miR-216a, whereas the anti-miR-216a treatment had an opposite effect. Treatment of pancreatic cancer cells with miR-216a significantly inhibited cell growth and promoted cell apoptosis. In addition, the downstream genes of JAK2/STAT3, survivin and X-linked inhibitor of apoptosis protein, which are anti‑apoptotic genes, were also decreased by miR-216a. Moreover, miR-216a overexpression markedly inhibited the JAK2/STAT3 signaling pathway and xenograft tumor growth in vivo. Compared with miR-216a treatment, anti-miR-216a treatment exhibited opposite effects throughout the entire experiment, confirming the inhibitory effect of miR-216a on pancreatic cancer by regulating the JAK2/STAT3 signaling pathway. The results provided evidence that miR-216a targeting JAK2 negatively regulated the development of pancreatic cancer cells and may be used to develop a miRNA-based therapeutic strategy against pancreatic cancer.

Polyinosine-polycytidylic acid [pIC] is a synthetic dsRNA that acts as an immune agonist of TLR3 and RLR to activate dendritic and natural killer cells that can kill tumor cells. pIC can also trigger apoptosis in pancreatic ductal adenocarcinoma cells (PDAC) but its mechanism of action is obscure. In this study, we investigated the potential therapeutic activity of a formulation of pIC with polyethylenimine ([pIC](PEI)) in PDAC and investigated its mechanism of action. [pIC](PEI) stimulated apoptosis in PDAC cells without affecting normal pancreatic epithelial cells. Mechanistically, [pIC](PEI) repressed XIAP and survivin expression and activated an immune response by inducing MDA-5, RIG-I, and NOXA. Phosphorylation of AKT was inhibited by [pIC](PEI) in PDAC, and this event was critical for stimulating apoptosis through XIAP and survivin degradation. In vivo administration of [pIC](PEI) inhibited tumor growth via AKT-mediated XIAP degradation in both subcutaneous and quasi-orthotopic models of PDAC. Taken together, these results offer a preclinical proof-of-concept for the evaluation of [pIC](PEI) as an immunochemotherapy to treat pancreatic cancer., (©2014 American Association for Cancer Research.)

Increased trophoblast apoptosis has been implicated in pregnancies complicated by fetal growth restriction and preeclampsia (EC). We investigated placenta growth factor (PLGF) signaling during trophoblast apoptosis in culture and X-linked inhibitor of apoptosis protein (XIAP) and apoptosis inducible factor (AIF) in the preeclamptic placenta at term was determined. Primary trophoblasts were isolated and serum starved to induce apoptosis. Placenta growth factor was added and apoptosis markers were determined. Term preeclamptic placentae were homogenized and the levels of XIAP and XAF1 protein were assessed. In the absence of serum, primary cultures of term trophoblast showed a 5-fold increase in apoptosis as determined by annexin V binding. The increase in apoptosis induced by serum deprivation was caspase-independent and could be significantly reduced (p < 0.02) with the addition of 10 ng/ml rh PLGF to the media. In addition, PLGF mediated increased protein expression of the anti-apoptotic XIAP as well as decreased expression of the pro-apoptotic AIF in the primary trophoblast. In preeclamptic placenta, we determined the concomitant decrease in XIAP RNA as well as decreased expression of the phosphorylated XIAP protein. These results were coupled with increased levels of the pro-apoptotic protein XAF1. Our results suggest that PLGF protects trophoblast from caspase independent apoptosis in culture by increasing XIAP production and deceasing AIF. Also, our data suggests that decreased activation of XIAP and increased XAF1 could be factors associated with the increased placental apoptosis observed in the preeclamptic placenta at term.

Previous studies suggest that tazarotene, a new member of the acetylenic class of RARβ/γ selective retinoids which is approved to treat a variety of skin diseases, exhibits an anti-proliferative effect in human basal cell carcinoma (BCC) by triggering caspase-dependent apoptosis. However, the detailed molecular mechanisms underlying the anti-tumor activity of tazarotene are poorly understood. This study aims at investigating the molecular mechanisms of tazarotene-induced apoptosis in human BCC cells. Our results are the first to demonstrate that tazarotene induces mitochondria-dependent cleavage of caspase-9 and -3 and PARP in BCC cells by producing reactive oxygen species (ROS) and activating caspase-8 through both ROS and death receptor signaling. These events are accompanied by a decrease in BCL-2 and BCL-xl anti-apoptotic proteins as well as by survivin and XIAP, two IAP family members. Furthermore, our results presented for the first time that tazarotene triggers a convergence of the intrinsic and extrinsic apoptotic pathways via the caspase-8-truncated Bid signaling pathway. Collectively, these data provide insights into the molecular mechanisms underlying tazarotene-induced apoptosis in human BCC cells, suggesting that this compound is a potential anti-skin cancer drug.

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., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)

Objective: Oral squamous cell carcinoma (OSCC) is a prevalent cancer, especially in developing countries. Anthracyclines and their anthraquinone derivatives, such as doxorubicin, exhibit a cell growth inhibitory effect and have been used as anti-cancer drugs for many years. However, the cardiotoxicity of anthracycline antibiotics is a major concern in their clinical application. NSC745885 is a novel compound synthesized from 1,2-diaminoanthraquinone, which subsequently reacts with thionyl chloride and triethylamine. The present study aimed to investigate the anti-oral cancer potential and the safety of NSC745885., Methods: We investigated the anti-cancer potential of NSC745885 in oral squamous carcinoma cell lines and in an in vivo oral cancer xenograft mouse model. The expression of apoptotic related genes were evaluated by real-time RT-PCR and western bloting, and the in vivo assessment of apoptotic marker were measured by immunohistochemical staining. The anti-tumor efficiency and safety between doxorubicin and NSC745885 were also compared., Results: Our results demonstrated that NSC745885 exhibits anti-oral cancer activity through the induction of apoptosis in cancer cells and in tumor-bearing mice, and this treatment did not induce marked toxicity in experimental mice. This compound also exhibits a comparable anti-tumor efficiency and a higher safety in experimental mice when compared to doxorubicin., Conclusions: The data of this study provide evidence for NSC745885 as a potential novel therapeutic drug for the treatment of human OSCC.

The Axl receptor tyrosine kinase (RTK), along with Tyro 3 and Mer, belongs to the TAM subfamily that promotes survival, stimulates proliferation and/or inhibits apoptosis. In various types of human cancer, including breast, lung and prostate cancer, Axl expression is increased and correlates with an advanced clinical stage. In this study, we examined whether apigenin has an effect on Axl expression, which in turn can affect cell proliferation. The treatment of the non‑small cell lung cancer (NSCLC) cells, A549 and H460, with apigenin decreased Axl mRNA and protein expression in a dose‑dependent manner. Axl promoter activity was also inhibited by apigenin, indicating that apigenin suppressed Axl expression at the transcriptional level. Upon treatment with apigenin, the viability of both the A549 and H460 cells was gradually decreased and the anti-proliferative effects were further confirmed by the dose‑dependent decrease in the clonogenic ability of the apigenin‑treated cells. Subsequently, we found that the viability and clonogenic ability of the cells treated with apigenin was less or more affected by transfection of the cells with a Axl-expressing plasmid or Axl targeting siRNA, compared to transfection with the empty vector or control siRNA, respectively. In addition, apigenin increased the expression of p21, a cyclin-dependent kinase inhibitor, but reduced the expression of X-linked inhibitor of apoptosis protein (XIAP). These cell cycle arrest and pro-apoptotic effects of apigenin were also attenuated or augmented by the up- or downregulation of Axl expression, respectively, which suggests that Axl is a novel target of apigenin through which it exerts its inhibitory effects on cell proliferation. Taken together, our data indicate that apigenin downregulates Axl expression, which subsequently results in the inhibition of NSCLC cell proliferation through the increase and decrease of p21 and XIAP expression, respectively.

Neuroblastomas (NBs) are a clinically heterogeneous group of extra cranial pediatric tumors. Patients with high-risk, metastatic NBs have a long-term survival rate of below 40%, and are often resistant to current therapeutic modalities. Due to toxic side effects associated with radiation and chemotherapies, development of new agents is warranted to overcome resistance and effectively treat this disease in clinic. CARP-1 functional mimetics (CFMs) are an emerging class of small molecule compounds that inhibit growth of diverse cancer cell types. Here we investigated NB inhibitory potential of CFMs and the molecular mechanisms involved. CFM-1, -4, and -5 inhibited NB cell growth, in vitro, independent of their p53 and MYCN status. CFM-4 and -5 induced apoptosis in NB cells in part by activating pro-apoptotic stress-activated kinases (SAPKs) p38 and JNK, stimulating CARP-1 expression and cleavage of PARP1, while promoting loss of the oncogenes C and N-myc as well as mitotic cyclin B1. Treatments of NB cells with CFM-4 or -5 also resulted in loss of Inhibitory κB (IκB) α and β proteins. Micro-RNA profiling revealed upregulation of XIAP-targeting miR513a-3p in CFM-4-treated NB, mesothelioma, and breast cancer cells. Moreover, exposure of NB and breast cancer cells to CFM-4 or -5 resulted in diminished expression of anti-apoptotic XIAP1, cIAP1, and Survivin proteins. Expression of anti-miR513a-5p or miR513a-5p mimic, however, interfered with or enhanced, respectively, the breast cancer cell growth inhibition by CFM-4. CFMs also impacted biological properties of the NB cells by blocking their abilities to migrate, form colonies in suspension, and invade through the matrix-coated membranes. Our studies indicate anti-NB properties of CFM-4 and 5, and suggest that these CFMs and/or their future analogs have potential as anti-NB agents.

Background: X-chromosome-linked inhibitor of apoptosis (XIAP) is one of the anti-apoptotic proteins leading to chemoresistance in several cancers. The aim of this study is to evaluate the impact of XIAP expression upon ovarian clear cell carcinoma (CCC) that has a platinum-resistant phenotype., Methods: Tissue microarrays made from 90 CCC patients were analysed for immunohistochemical expression levels of XIAP, c-Met, p-Akt and Bcl-XL. In addition, CCC cell lines were evaluated whether XIAP silencing could modulate sensitivity to platinum agent in vitro., Results: High XIAP expression was observed in 30 (33%) of 90 CCC cases, and was associated with c-Met (<0.01) and Bcl-XL (<0.01) expression. Cases with high XIAP expression had lower response rate to primary platinum-based chemotherapy (10% vs 65%, P=0.02). In stages II-IV tumours, high XIAP expression was related with worse progression-free survival (PFS, P=0.02). Furthermore, high XIAP expression was identified as an independent worse prognostic factor for PFS and overall survival. Finally, downregulation of XIAP using XIAP-specific small interfering RNA increased sensitivity to cisplatin in human cancer cells derived from CCC., Conclusions: X-chromosome-linked inhibitor of apoptosis expression was correlated with chemoresistance of primary chemotherapy, and identified as a prognostic marker for CCC. X-chromosome-linked inhibitor of apoptosis could be a candidate for new therapeutic target in CCC.

Evidence suggests that solar ultraviolet B (UVB) radiation inhibits growth and/or induces apoptosis of human skin cells. However, mechanisms underlying the UVB-induced anti-survival and pro-apoptotic effects on human skin cells remain unclear. In this study, we investigated the effect of UVB radiation on survival and apoptosis of HaCaT human keratinocytes and determined possible molecular, cellular and signaling mechanisms including cross-regulation, which are responsible for the UVB's anti-survival and/or pro-apoptotic effects. The results showed that UVB radiation at 400 mJ/cm² for 8 h largely decreased cell survival and induced DNA fragmentation, an index of apoptosis, in HaCaT human keratinocytes. On a mechanistic level, UVB radiation triggered the activation of caspase-9, cleavage of poly(ADP-ribose) polymerase, and downregulation of myeloid cell leukemia-1 (Mcl-1), human inhibitor of apoptosis protein-1 (HIAP-1), X-linked IAP (XIAP), and protein kinase B (PKB), but did not affect the expression of B-cell lymphoma-2 in HaCaT cells. UVB radiation also upregulated the expression of glucose-regulated protein 78 (GRP78), an endoplasmic reticulum (ER) stress marker, in HaCaT cells. Of note, results of pharmacological inhibition studies have demonstrated that pretreatment with z-VAD-fmk, a pan-caspase inhibitor strongly attenuated UVB-induced apoptosis, the activation of caspase-9, downregulation of Mcl-1, XIAP and PKB (but not HIAP-1), and upregulation of GRP78, while pretreatment with GF109203 or GO6983, pan-PKC inhibitors, substantially blocked the UVB-induced reduction of cell survival, activation of caspase-9, downregulation of HIAP-1, XIAP, and PKB (but not Mcl-1), and GRP78 upregulation in HaCaT cells. Collectively, these results demonstrated that UVB has strong anti-survival and pro-apoptotic effects on HaCaT cells and the effects were largely mediated via the activation of caspase-9 and protein kinase Cs, which subsequently downregulated PKB, XIAP, HIAP-1 and Mcl-1, and triggered ER stress.

Although receptor-interacting protein 1 (RIP1) is well known as a key mediator in cell survival and death signaling, whether RIP1 directly contributes to chemotherapy response in cancer has not been determined. In this report, we found that, in human lung cancer cells, knockdown of RIP1 substantially increased cytotoxicity induced by the frontline anticancer therapeutic drug cisplatin, which has been associated with robust cellular reactive oxygen species (ROS) accumulation and enhanced apoptosis. Scavenging ROS dramatically protected RIP1 knockdown cells against cisplatin-induced cytotoxicity. Furthermore, we found that, in RIP1 knockdown cells, the expression of the hydrogen peroxide-reducing enzyme catalase was dramatically reduced, which was associated with increased miR-146a expression. Inhibition of microRNA-146a restored catalase expression, suppressed ROS induction, and protected against cytotoxicity in cisplatin-treated RIP1 knockdown cells, suggesting that RIP1 maintains catalase expression to restrain ROS levels in therapy response in cancer cells. Additionally, cisplatin significantly triggered the proteasomal degradation of cellular inhibitor of apoptosis protein 1 and 2 (c-IAP1 and c-IAP2), and X-linked inhibitor of apoptosis (XIAP) in a ROS-dependent manner, and in RIP1 knockdown cells, ectopic expression of c-IAP2 attenuated cisplatin-induced cytotoxicity. Thus, our results establish a chemoresistant role for RIP1 that maintains inhibitor of apoptosis protein (IAP) expression by release of microRNA-146a-mediated catalase suppression, where intervention within this pathway may be exploited for chemosensitization.

Pseudolaric acid B (PAB) is a diterpene acid isolated from the root and trunk bark of Pseudolarix kaempferi Gordon (Pinaceae). Recent studies have reported that PAB exhibits cytotoxic effects in several cancer cell lines. In the present study, we assessed its antitumor activity and molecular mechanisms in HO-8910 and A2780 ovarian cancer cells in vitro. We found that PAB reduced cell viability and induced apoptosis in a dose- and time-dependent manner in HO-8910 and A2780 human ovarian cancer cells. The induction of apoptosis was also accompanied by the regulation of Bcl-2 and XIAP family proteins, cytochrome c and Apaf-1. Moreover, we observed that PAB treatment resulted in the activation of caspase-3 and -9, which may partly explain the anticancer activity of PAB. Collectively, the present study for the first time suggests that PAB enhances apoptosis of HO-8910 and A2780 cells through regulation of Bcl-2 and IAP family proteins. Moreover, the triggering of caspase-3 and -9 activation mediated apoptotic induction. Our results suggest that PAB may be a new therapeutic option for the treatment of ovarian cancers.

Although cisplatin (CDDP) is a key drug in the treatment of esophageal squamous cell carcinoma (ESCC), acquired chemoresistance remains a major problem. Combination therapy may represent one strategy to overcome this resistance. Heat shock protein 90 (HSP90) is known to be overexpressed in several types of cancer cells, and its inhibition by small molecules, either alone or in combination, has shown promise in the treatment of solid malignancies. In the present study, we evaluated the synergistic effects of combining CDDP with the HSP90 inhibitor 17-N-allylamino-17-demethoxy geldanamycin (17-AAG) on two CDDP-resistant human esophageal squamous cancer cell lines, KYSE30 and KYSE150. The results obtained demonstrated the synergistic inhibitory effects of CDDP and 17-AAG on the growth of KYSE30 and KYSE150 cells. Cell growth and cell number were more effectively reduced by the combined treatment with CDDP and 17-AAG than by the treatment with either CDDP or 17-AAG alone. Western blotting revealed that the combined action of CDDP and 17-AAG cleaved poly (ADP-ribose) polymerase (PARP) and caspase-3, which demonstrated that the reduction in both cell growth and cell number was mediated by apoptosis. Time-course experiments showed that reduction in X-linked inhibitor of apoptosis protein (XIAP) and phosphorylated Akt were concomitant with apoptosis. The results of the present study demonstrate that 17-AAG synergizes with CDDP and induces apoptosis in CDDP-resistant ESCC cell lines, and also that modulation of the Akt/XIAP pathway may underlie this synergistic effect. Combination therapy with CDDP and an HSP90 inhibitor may represent a promising strategy to overcome CDDP resistance in ESCC.

Background: Lung cancer is the leading cause of cancer-related mortality worldwide, with near 1.4 million deaths each year. NSCLC accounts for nearly 85% of all case of lung cancer. MiRNAs play important roles in regulation of gene expression at the post-transcriptional level. MiRNAs profiles may predict prognosis and disease recurrence in early-stage NSCLC. Our previous study proved that over-expression of ubiquitin specific peptidase 14 (USP14), a deubiquitinating enzyme, was associated with favorable prognosis in NSCLC patients and promoted tumor cells proliferation. Here, we tried to identify which miRNAs targeted USP14, and the roles of these miRNAs in NSCLC., Methods: MiR-4782-3p and its potential targeted genes were identified by bioinformatics algorithm. Dual luciferase reporter assay system was used to analyze the interaction between miR-4782-3p and targeted genes. Cell proliferation was assayed by MTT and BdU assay. MiRNAs and mRNA expression were assayed by qRT-PCR. USP14 protein level was assayed by Western blot. The role of miR-4782-3p in patients survival was revealed by Kaplan-Meier plot of overall survival., Results: Up-expression of miR-4782-3p in NSCLC cells decreased the USP14 expression. Down-expression of miR-4782-3p increased USP14 expression. In NSCLC specimen, Negative correlation between USP14 mRNA level and miR-4782-3p level was identified. Higher miR-4782-3p expression is associated with longer survival. USP14, ZEB2, XIAP overexpression reversed the inhibitory effect of miR-4782-3p., Conclusions: High expression of miR-4782-3p was associated with favorable prognosis in NSCLC patients. MiR-4782-3p inhibited cell proliferation in NSCLC by targeting USP14, ZEB2 and XIAP., (© 2014 S. Karger AG, Basel.)

To investigate the expressions of casein kinase II β (CK2β) and X-Linked inhibitor of apoptosis protein (XIAP) in cholangiocarcinoma (CCA) and evaluated their correlations with major clinicopathologic features and patients' survival. Fifty CCA specimens and 20 normal liver tissues were included in the study. Immunohistochemical staining was used to determine the expression levels of CK2β, XIAP in normal and CCA tissues. The relationships of CK2β and XIAP expressions with clinicopathologic parameters and clinical outcome were evaluated. High immunostaining of CK2β and XIAP were observed in 66% (33/50) and 68% (34/50) of CCA tissues, which were significantly higher than that of normal liver tissues 0% (0/20) and 25% (5/20). The high expression of CK2β was significantly associated with TNM stage (P = 0.036), histological grade (P = 0.020) and high serum CEA level (P = 0.010), while high expression of XIAP was only associated with TNM stage (P = 0.014) and high serum CEA level (P = 0.001). By univariant analysis, patients with high expression of CK2β and XIAP demonstrate significantly poorer overall survival (P = 0.003 vs P = 0.018). Cox regression model showed that positive expression of CK2βis an independent factor of prognosis (P = 0.004). The expressions of CK2β and XIAP in CCA tissues showed strong correlations with the tumor progression, CK2β may be applied as a potential prognostic marker for CCA.

The development and characterization of effective anticancer drugs against colorectal cancer (CRC) is of urgent need since it is the second most common cause of cancer death. The study was designed to evaluate the effects of two IGF-1R antagonists, MK-0646, a recombinant fully humanized monoclonal antibody and OSI-906, a small molecule tyrosine kinase inhibitor on CRC cells. Xenograft study was performed on IGF-1R-dependent CRC cell lines for analyzing the antitumor activity of MK-0646 and OSI-906. Tumor proliferation and apoptosis were assessed using Ki67 and TUNEL assays, respectively. We also performed in vitro characterization of MK-0646 and OSI-906 treatment on CRC cells to identify mechanisms associated with drug-induced cell death. Exposure of the GEO and CBS tumor xenografts to MK-0646 or OSI-906 led to a decrease in tumor growth. TUNEL analysis showed an increase of approximately 45-55% in apoptotic cells in both MK-0646 and OSI-906 treated tumor samples. We report the novel finding that treatment with IGF-1R antagonists led to downregulation of X-linked inhibitor of apoptosis (XIAP) protein involved in cell survival and inhibition of cell death. In conclusion, IGF-1R antagonists (MK-0646 and OSI-906) demonstrated single agent inhibition of subcutaneous CRC xenograft growth. This was coupled to pro-apoptotic effects resulting in downregulation of XIAP and inhibition of cell survival. We report a novel mechanism by which MK-0646 and OSI-906 elicits cell death in vivo and in vitro. Moreover, these results indicate that MK-0646 and OSI-906 may be potential anticancer candidates for the treatment of patients with IGF-1R-dependent CRC.

Renal cell carcinoma (RCC) is resistant to traditional cancer therapies such as radiation therapy and chemotherapy. The use of targeted therapies has improved the clinical outcomes of patients with metastatic RCC. However, most patients acquire resistance against targeted therapies over time. We report that the combination of the novel histone deacetylase (HDAC) inhibitor OBP-801, also known as YM753 and the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 synergistically inhibits cell growth and induces apoptosis in RCC cells. This combination activated caspase-3, -8 and -9 and the pan-caspase inhibitor zVAD-fmk significantly reduced the apoptotic response to the treatment with OBP-801 and LY294002. Moreover, the combined treatment induced intracellular reactive oxygen species (ROS) and the radical scavenger N-acetyl-L-cysteine (NAC) blocked the intracellular ROS and apoptosis induced by OBP-801 and LY294002. The co-treatment with OBP-801 and LY294002 markedly decreased survivin and the X-linked inhibitor of apoptosis protein (XIAP) protein levels, but Bcl-2 family members were not altered by the OBP-801/LY294002 co-treatment. These alterations were restored by NAC treatment. The transient transfection of survivin and XIAP reduced the apoptotic response to the OBP-801/LY294002 co-treatment. Additionally, OBP-801 was significantly more effective than SAHA, another HDAC inhibitor, in the combination with LY294002 against 786-O cells. Taken together, these results strongly suggest the combination of OBP-801 and LY294002 to be a promising treatment for RCC.

This article presents a case report for a child presenting with mixed clinical features of autoimmune lymphoproliferative syndrome (ALPS), familial hemophagocytic lymphohistiocytosis (FHL), and X-linked lymphoproliferative (XLP) disease. From 6 months, he exhibited splenomegaly and lymphoadenopathy and from 4 years, he showed recurrent severe autoimmune hemocytopenia and sepsislike bouts of fever, from which he eventually died at the age of 12. Intriguingly, the patient carried mutations in FAS, XIAP, and UNC13D genes, which are involved in ALPS, XLP disease, and FHL, respectively. These mutations were inherited from the mother, who had rheumatoid arthritis but no signs of ALPS. A role for other modifying genes was suggested by the finding that the healthy father exhibited defective Fas function, without mutation of the FAS gene, and had transmitted to the patient an osteopontin (OPN) gene variant previously associated with ALPS. Therefore, several genes might influence the disease outcome in this family. In vitro analyses revealed that the FAS and the XIAP mutations decreased expression of the corresponding proteins, and the UNC13D mutation decreased granule secretion and Munc interaction with Rab-27a. These findings suggest that overlap may exist between ALPS, FHL, and XLP disease, in accordance with the notion that FHL and XLP disease are due to defective natural killer (NK)/NK T-cell function, which involves Fas. Therefore, we propose that NK cell defects should be evaluated in patients with ALPS-like characteristics, and hematopoietic stem cell transplantation should be considered in individuals with severe refractory cytopenia and FHL-like manifestations.

β-Elemene is a promising new plant-derived drug with broad-spectrum anticancer activity. It also increases cisplatin cytotoxicity and enhances cisplatin sensitivity in resistant human carcinoma cells. However, little is known about the mechanism of its action. To explore the potential therapeutic application of β-elemene as a drug-resistance modulator, this study investigated the underlying mechanism of β-elemene activity in cisplatin-resistant ovarian cancer cells. β-Elemene enhanced cisplatin sensitivity to a much greater extent in chemoresistant A2780/CP70 and MCAS human ovarian carcinoma cells compared to the chemosensitive parental cell line A2780. The dose-modifying factors for cisplatin were between 35 and 60 for A2780/CP70 cells and between 1.6 and 2.5 for A2780 cells. In the cisplatin-resistant ovarian carcinoma cells, β-elemene abrogated cisplatin‑induced expression of excision repair cross-complementation group‑1 (ERCC-1), a marker gene in the nucleotide excision repair pathway that repairs cisplatin-caused DNA damage. In addition, β-elemene not only reduced the level of X-linked inhibitor of apoptosis protein (XIAP), but also downregulated cisplatin-mediated XIAP expression in chemoresistant cells. Furthermore, β-elemene blocked the cisplatin-stimulated increase in the level of phosphorylated c-Jun NH2-terminal kinase (JNK) in these cells. These novel findings suggest that the β-elemene enhancement of cisplatin sensitivity in human chemoresistant ovarian cancer cells is mediated at least in part through the impairment of DNA repair activity and the activation of apoptotic signaling pathways, thereby making resistant ovarian cancer cells susceptible to cisplatin-induced cell death.

Significant changes of copper homeostasis were triggered by lipopolysaccharides, which result in systemic inflammatory response and contribute to hepatic injury. Administration of lipopolysaccharides resulted in the increase of plasma "free" copper and total copper concentrations, whereas, the decrease of "free" copper and total copper contents in liver tissue. Copper-associated proteins were detected and showed a down-regulation of X-linked inhibitor of apoptosis protein, and up-regulation of copper metabolism domain containing 1 and copper transporter 1. The alteration of these proteins would lower the apoptotic threshold. Meanwhile, the increasing of circulation copper might cause oxidative injury through Fenton reaction and contribute to tissue injury. Our findings underscored the possibility that these changes in systemic copper homeostasis might provide a novel insight of the characteristic of the acute phase of inflammatory response and the underlying influence on tissue injury.

Malignant pleural mesothelioma (MPM) is a highly aggressive and conventional treatment-resistant tumor with a dismal prognosis. Among the three histological subtypes of MPM, the epithelioid is the most common type. Numb is considered as a tumor suppressor playing a critical role in controlling asymmetric cell division, maintenance of stem cell compartments, ubiquitination of specific substrates and regulating Notch-, Hedgehog- and TP53-activated pathways. The present study was designed to analyze the role of Numb in epithelioid MPM. We investigated the expression of Numb in 39 epithelioid MPM and 22 normal pleural tissues by immunohistochemistry. Furthermore, we overexpressed Numb in NCI-H2452, an epithelioid human MPM cell line, and investigated the effect of Numb overexpression on the proliferation, apoptosis and sensitivity to cisplatin in cells. The expression of Numb was significantly lower in MPM compared to the control group and Numb had an inverse correlation with the ki-67 labeling index. Loss of Numb expression was associated with poor prognosis in epithelioid MPM. Overexpression of Numb in NCI-H2452 cells significantly inhibited proliferation, promoted apoptosis and enhanced sensitivity to cisplatin. Moreover, Numb overexpression activated caspase-9 and caspase-3 through release of cytochrome c as well as downregulation of XIAP and survivin. We speculate that cytochrome c/caspase signaling is a possible mechanism through which Numb enhances the apoptosis of NCI-H2452 cells. These results suggest that Numb may be involved in epithelioid MPM development, and its upregulation may confer sensitivity to cisplatin, suggesting potential therapeutic options for MPM.

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.

Increased expression of COX-2 has been linked to inflammation and carcinogenesis. Constitutive expression of COX-2 protects hepatocytes from several pro-apoptotic stimuli. Increased hepatic apoptosis has been observed in experimental models of diabetes. Our present aim was to analyze the role of COX-2 as a regulator of apoptosis in diabetic mouse liver. Mice of C57BL/6 strain wild type (Wt) and transgenic in COX-2 (hCOX-2 Tg) were separated into Control (vehicle) and SID (streptozotocin induced diabetes, 200 mg/kg body weight, i.p.). Seven days post-injection, Wt diabetic animals showed a decrease in PI3K activity and P-Akt levels, an increase of P-JNK, P-p38, pro-apoptotic Bad and Bax, release of cytochrome c and activities of caspases-3 and -9, leading to an increased apoptotic index. This situation was improved in diabetic COX-2 Tg. In addition, SID COX-2 Tg showed increased expression of anti-apoptotic Mcl-1 and XIAP. Pro-apoptotic state in the liver of diabetic animals was improved by over-expression of COX-2. We also analyzed the roles of high glucose-induced apoptosis and hCOX-2 in vitro. Non-transfected and hCOX-2-transfected cells were cultured at 5 and 25 mM of glucose by 72 h. At 25 mM there was an increase in apoptosis in non-transfected cells versus those exposed to 5 mM. This increase was partly prevented in transfected cells at 25 mM. Moreover, the protective effect observed in hCOX-2-transfected cells was suppressed by addition of DFU (COX-2 selective inhibitor), and mimicked by addition of PGE(2) in non-transfected cells. Taken together, these results demonstrate that hyperglycemia-induced hepatic apoptosis is protected by hCOX-2 expression., (Copyright © 2012 Wiley Periodicals, Inc.)