Inhibitor Of Apoptotic Protein Research Articles

LCL161 increases paclitaxel-induced apoptosis by degrading cIAP1 and cIAP2 in NSCLC.

BackgroundLCL161, a novel Smac mimetic, is known to have anti-tumor activity and improve chemosensitivity in various cancers. However, the function and mechanisms of the combination of LCL161 and paclitaxel in non-small cell lung cancer (NSCLC) remain unknown.MethodsCellular inhibitor of apoptotic protein 1 and 2 (cIAP1&2) expression in NSCLC tissues and adjacent non-tumor tissues were assessed by immunohistochemistry. The correlations between cIAP1&2 expression and clinicopathological characteristics, prognosis were analyzed. Cell viability and apoptosis were measured by MTT assays and Flow cytometry. Western blot and co-immunoprecipitation assay were performed to measure the protein expression and interaction in NF-kB pathway. siRNA-mediated gene silencing and caspases activity assays were applied to demonstrate the role and mechanisms of cIAP1&2 and RIP1 in lung cancer cell apoptosis. Mouse xenograft NSCLC models were used in vivo to determine the therapeutic efficacy of LCL161 alone or in combination with paclitaxel.ResultsThe expression of cIAP1 and cIAP2 in Non-small cell lung cancer (NSCLC) tumors was significantly higher than that in adjacent normal tissues. cIAP1 was highly expressed in patients with late TNM stage NSCLC and a poor prognosis. Positivity for both cIAP1 and cIAP2 was an independent prognostic factor that indicated a poorer prognosis in NSCLC patients. LCL161, an IAP inhibitor, cooperated with paclitaxel to reduce cell viability and induce apoptosis in NSCLC cells. Molecular studies revealed that paclitaxel increased TNFα expression, thereby leading to the recruitment of various factors and the formation of the TRADD-TRAF2-RIP1-cIAP complex. LCL161 degraded cIAP1&2 and released RIP1 from the complex. Subsequently, RIP1 was stabilized and bound to caspase-8 and FADD, thereby forming the caspase-8/RIP1/FADD complex, which activated caspase-8, caspase-3 and ultimately lead to apoptosis. In nude mouse xenograft experiments, the combination of LCL161 and paclitaxel degraded cIAP1,2, activated caspase-3 and inhibited tumor growth with few toxic effects.ConclusionThus, LCL161 could be a useful agent for the treatment of NSCLC in combination with paclitaxel.

Abstract A16: Role of Hsp90alpha-1, a splice variant of Hsp90alpha, in cancer

Abstract Molecular chaperone complexes, such as heat shock proteins 90 (Hsp90) and its co-chaperones, are essential for folding nascent polypeptides into their biologically active structures and for the refolding of aggregated and denatured proteins that occur upon cellular stress including cancer. Hsp90 exists as four isoforms in humans. Among them, Hsp90alpha is induced under stressful conditions, detected in the cytoplasm, nucleus and is secreted into extracellular space under metastatic conditions. Hsp90alpha is phosphorylated by DNA-PK at the nucleus under apoptotic conditions and localizes at the edge of the nucleus. Intrigued by the fact that Hsp90alpha might play a role in metastasis as well as in stress resistance, the mechanism of action of Hsp90alpha was investigated. Here we report that Hsp90alpha-1, a splice variant of Hsp90alpha, is up-regulated in cancer cells, predominantly associates with co-chaperone Aha1 in-vivo at the filopodia like extrusion of cell. The Hsp90alpha-1/Aha1 complex was partially disrupted by C- terminal inhibitors in-vivo conditions; and Hsp90alpha-1 and Aha1 redistributed to the cytoplasm from the filopodia following the treatment with the C- terminal inhibitors. Gene knock down analysis reveals that several P-Akt pathway and MAP Kinase pathway proteins were Hsp90alpha-1 dependent. P-Akt also co-localized with the Hsp90alpha-1/Aha1 complex at the filopodia region and suggests that Hsp90alpha-1 plays critical role in maturing/folding proteins that are important for establishing and maintaining cell polarity which is perturbed by the C- terminal inhibitors. Under apoptotic conditions the Hsp90alpha-1/Aha1 complex is present in the cytoplasm and nucleus and Hsp90alpha-1 is phosphorylated. Survivin, being a Hsp90alpha-1 dependent client protein, binds with the Hsp90alpha-1/Aha1 complex at normal and apoptotic conditions confirms that Survivin play dual role in mediating metastases as well as an Inhibitor of Apoptotic Protein (IAP). Citation Format: Suman Ghosh, Laura B. Peterson, George Vielhauer, Brian S. J Blagg. Role of Hsp90alpha-1, a splice variant of Hsp90alpha, in cancer. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr A16.

Fucoidan derived from Undaria pinnatifida induces apoptosis in human hepatocellular carcinoma SMMC-7721 cells via the ROS-mediated mitochondrial pathway.

Fucoidans, fucose-enriched sulfated polysaccharides isolated from brown algae and marine invertebrates, have been shown to exert anticancer activity in several types of human cancer, including leukemia and breast cancer and in lung adenocarcinoma cells. In the present study, the anticancer activity of the fucoidan extracted from the brown seaweed Undaria pinnatifida was investigated in human hepatocellular carcinoma SMMC-7721 cells, and the underlying mechanisms of action were investigated. SMMC-7721 cells exposed to fucoidan displayed growth inhibition and several typical features of apoptotic cells, such as chromatin condensation and marginalization, a decrease in the number of mitochondria, and in mitochondrial swelling and vacuolation. Fucoidan-induced cell death was associated with depletion of reduced glutathione (GSH), accumulation of high intracellular levels of reactive oxygen species (ROS), and accompanied by damage to the mitochondrial ultrastructure, depolarization of the mitochondrial membrane potential (MMP, Δψm) and caspase activation. Moreover, fucoidan led to altered expression of factors related to apoptosis, including downregulating Livin and XIAP mRNA, which are members of the inhibitor of apoptotic protein (IAP) family, and increased the Bax-to-Bcl-2 ratio. These findings suggest that fucoidan isolated from U. pinnatifida induced apoptosis in SMMC-7721 cells via the ROS-mediated mitochondrial pathway.

Clinical analyses of 81 infertile and 20 testicular tumor men with suspected X-chromosome-linked gene anomalies in Japan

A total of 101 cases of 81 infertile men and 20 men with testicular tumors were analyzed clinically for suspected X-chromosome-linked gene abnormalities. Among the 81 infertile men analyzed by testicular biopsy, 11 Klinefelter syndrome (KS) cases showed Leydig cell nodular hyperplasia with azoospermia. They had low levels of testosterone (131 ± 81 ng/dl) and high levels of luteinizing hormone (21 ± 7 mIU/ml) and follicle-stimulating hormone (36 ± 11 mIU/ml). From Leydig cell hyperplasia combined with atrophic tubules, X-linked androgen receptor dysfunctions in Sertoli and Leydig cells were judged in the KS cases. One azoospermia case of non-KS had primary yolk sac tumor of the mediastinum and liver. The removed yolk sac tumor showed complete necrosis after bleomycin, etoposide, and cisplatin (BEP) chemotherapy, which indicated cisplatin-sensitive apoptosis. X-linked inhibitor of apoptotic protein (XIAP) must exhibit an apoptosis-inducible mutation (XIAP-S87A) in the non-KS case. Subsequently, 20 testicular tumor cases were classified into ten with seminoma, eight with mixed germ cell tumors (MGCT), one with mature teratoma, and one with diffuse large B cell lymphoma. Although among the eight MGCT cases, six cases had high levels of α-fetoprotein (68–43647 ng/ml), their blood levels of human chorionic gonadotropin-β were <0.1 to 23 ng/ml with a good prognosis. Radiological examinations of the 20 cases showed nodular, hemorrhagic, or cystic changes in lung, liver, or periarterial lymph nodes, but no metastatic changes in bone or brain. Besides cisplatin-induced acute renal failure in one of the two seminoma cases treated with BEP chemotherapy, the two cases showed chronic renal failure (CRF) due to glomerular disturbance, but three treated MGCT cases did not. Their mutated humanin in spermatocytes, which located in X-chromosome and bound to podocytes, might have aggravated the CRF. Infertility and testis-related tumors were caused by X-chromosome-linked gene anomalies.

Survivin acts as anti‐apoptotic factor during the development of bovine pre‐implantation embryos

Survivin, an inhibitor of apoptotic protein containing a single baculoviral inhibit apoptotic protein repeat domain, is a bifunctional protein that suppresses apoptosis and regulates cell division. Thus, we used double stranded RNA (dsRNA) interference to manipulate survivin expression in bovine embryos and analyze its role in blocking apoptosis and facilitating development of pre-implantation embryos. In vitro fertilized embryos (1-cell) were injected with survivin dsRNA, and expression of survivin mRNA was evaluated by real-time quantitative RT-PCR. To analyze survivin protein expression, we performed immunocytochemistry using a rabbit anti-bovine suvivin antibody. Expression levels of survivin mRNA and protein were decreased in the dsRNA group compared to the sham group. Rates of in vitro blastocyst development were lower in the survivin dsRNA-injected group than in the sham-injected group. Also, the total cell number seen in blastocysts was decreased in the dsRNA group. TUNEL assays of DNA fragmentation indicated an increased apoptotic index in the dsRNA group compared to the sham group. These results indicate that survivin is important for optimal development of bovine blastocysts and confirm that survivin expression suppresses apoptosis of pre-implantation embryos.

Mutational analysis of proapoptotic ARTS P-loop domain in common human cancers

Mounting evidence indicates that deregulation of apoptosis is involved in mechanisms of cancer development. ARTS is released from mitochondria into the cytosol during apoptosis, promoting caspase activation by neutralizing the inhibition of inhibitor of apoptotic protein (IAP) on caspases. ARTS contains a P-loop GTP-binding domain, which is essential for the apoptotic function of ARTS. The aim of this study was to ascertain whether a genetic alteration of ARTS gene is involved in the development of human cancers possibly by inactivating the apoptotic function of ART. We analyzed the coding region of the P-loop domain of human ARTS gene for the detection of somatic mutations in 100 gastric carcinomas, 100 non-small cell lung cancers, and 69 hepatocellular carcinomas using a polymerase chain reaction (PCR)-based single strand conformation polymorphism (SSCP). However, there was no mutation in the P-loop coding region in the cancers. The data presented here suggest that ARTS P-loop is not frequently mutated in gastric, lung, and hepatocellular carcinomas, and that apoptosis deregulation in cancers is not dependent on the mutation of ARTS gene.

Autoimmune response to anti-apoptotic protein survivin and its association with antibodies to p53 and c-myc in cancer detection

Survivin, an inhibitor of apoptotic protein, is over-expressed in many cancers but not in normal differentiated adult tissues. Recently, antibodies to survivin have been demonstrated in patients with lung and colorectal cancer. Whether antibodies to survivin can be used as a marker for the diagnosis of cancer, and how antibody to survivin is related to antibodies against tumor suppressor protein p53 and oncoprotein c-myc remains to be evaluated. In the present study, the full-length recombinant proteins survivin, p53 and c-myc, were expressed and used as antigens in enzyme-linked immunosorbent assay (ELISA) and Western blot for the detection of antibodies to these three proteins. Sera from 1137 patients with 11 different types of cancer were analyzed. Antibodies to survivin were detected in 8.4% (96/1137), with a significant difference from the control groups consisting of normal individuals and autoimmune disease patients ( p < 0.05). Of 1137 cancer sera, 546 were also tested for the presence of antibodies to p53 and c-myc. Frequencies of antibodies to p53 and c-myc were 11.5 and 12.3%, respectively. Although antibodies to either one of three antigens do not reach levels of sensitivity, which could become routinely useful in diagnosis, it appears that there are different patterns of antibody frequency in individual cancer type. The results also indicated that when the presence of antibody to any one of these three antigens was considered, the cumulative frequency was increased to 27.3% (149/546) for the total group of cancer patients. It became apparent from our data that the combination of antibodies might acquire higher sensitivity.