Utility of Inter-Frequency Amplitude Ratio of Vestibular-Evoked Myogenic Potentials in Identifying Meniere's Disease: A Systematic Review and Meta-Analysis.

OBJECTIVES: A recently devised parameter of vestibular-evoked myogenic potential (VEMP) based on the principles of frequency tuning is the inter-frequency amplitude ratio (IFAR). It refers to the ratio of the amplitude of 1000 Hz tone burst evoked VEMP to 500 Hz evoked tone burst. A pathology like Meniere's disease changes the frequency response and alters the frequency tuning of the otolith organs. Because IFAR is based on the principle of frequency tuning of VEMP, it is likely to help identify Meniere's disease. Few studies in the last decade have investigated the utility of IFAR in identifying Meniere's disease. However, a systematic review and a meta-analysis on IFAR in Meniere's disease are lacking. The present study investigates whether the IFAR of VEMP helps identify Meniere's disease and differentiates it from healthy ears and other vestibular pathologies. DESIGN: The present study is a systematic review and a meta-analysis. The studies investigating the IFAR of cervical and ocular VEMPs in Meniere's disease, healthy controls, and other vestibular pathologies were searched across research databases such as PubMed, Science Direct, and Scopus. The search strategy was developed using the PICO (population, intervention, comparison, and outcomes) format, and Medical Subject Headings (MeSH) terms and Boolean operators were employed. The systematic review was performed using the Rayyan software, whereas the Review Manager software was used to carry out the meta-analysis. A total of 16,605 articles were retrieved from the databases. After the duplicate removal, 2472 articles remained. These were eliminated using title screening, abstract screening, and full-length inspections. A total of nine articles were found eligible for quality assessment and meta-analysis, and the New Castle-Ottawa Scale was used for quality assessment. After the data extraction, 24 six articles were found to have the desired data format for the meta-analysis. RESULTS: The results showed significantly higher IFAR in the affected ears of individuals in the Meniere's disease group than in the control group's unaffected ears. There was no significant difference between the unaffected ears of individuals in the Meniere's disease group and the ears of the control group. The only study on Meniere's disease and benign paroxysmal positional vertigo found significantly larger ocular VEMP IFAR in ears with Meniere's disease than in benign paroxysmal positional vertigo. CONCLUSIONS: This systematic review and meta-analysis found IFAR efficient in differentiating Meniere's disease from healthy controls. We also found an enhanced IFAR as a potential marker for Meniere's disease. However, more investigations are needed to confirm the utility of an enhanced IFAR value in the exclusive identification of Meniere's disease.

Dendritic cells matured with recombinant human sperm associated antigen 9 (rhSPAG9) induce CD4+, CD8+ T cells and activate NK cells: a potential candidate molecule for immunotherapy in cervical cancer.

BACKGROUND: Dendritic cell (DC)-based immunotherapy is capable of activating the immune system and in particular tumor-specific cytotoxic T lymphocytes (CTLs) to eradicate the tumor. However, major limitations are the availability of autologous tumor cells as antigenic source and the selection of antigen that may have potential to activate both CD4+ and CD8+ T cells in immune-specific manner. Recently, we reported the expression of sperm associated antigen 9 (SPAG9) that is associated with various types of malignancies including cervical cancer. We examined the recombinant human SPAG9 (rhSPAG9) as an antigenic source for generating efficient DCs to stimulate CD4+ and CD8+ T cell responses for future DCs-based vaccine trials in cervical cancer patients. METHODS: Human monocytes derived DCs were pulsed with different concentrations (250 ng/ml to 1000 ng/ml) of recombinant human SPAG9 (rhSPAG9) and evaluated for their phenotypic and functional ability. The efficacy of DCs primed with 750 ng/ml of rhSPAG9 (SPDCs) was compared with DCs primed with autologous tumor lysates (TLDCs), to induce CD4+, CD8+ T cells and activating NK cells. In addition, we investigated the effect of the chemotherapeutic drug cisplatin on phenotypic and functional potential of SPDCs. RESULTS: Phenotypic and functional characterization of DCs pulsed with 750 ng/ml rhSPAG9 was found to be optimal and effective for priming DCs. SPDCs were also capable of stimulating allogeneic T cells similar to TLDCs. SPDCs showed a statistically insignificant increase in the expression of maturation marker CD83 and migration towards CCL19 and CCL21 compared with TLDCs (CD83; P = 0.4; migration; P = 0.2). In contrast, although TLDCs showed better proliferation and secretion of Th1 cytokines (IL12p40, IL12p70 and IFNγ) compared to SPDCs, this difference was not statistically significant (IL12p40, P = 0.06). Further we also observed that clinical dose of cisplatin (200 µM) treated SPDCs were able to stimulate the proliferation of cytotoxic T lymphocytes without increasing the FOXP3+ Tregs in autologous co-cultures. CONCLUSIONS: In summary, in order to overcome the limitation of the availability of autologous tumor cells as antigenic sources, our present strategy provides an insight to consider rhSPAG9 as a strong immunogen for DC-based immunotherapy for cervical cancer trials and warrants further studies. This is the first report to suggest that rhSPAG9 is an effective antigen for pulsing DCs that are capable of eliciting a potent Th1 response which, in turn, may help in decreasing the tumor burden when used along with a cisplatin based combinatorial regimen for therapeutic intervention.

Knockdown of A-kinase anchor protein 4 inhibits proliferation of triple-negative breast cancer cells in vitro and in vivo.

Triple-negative breast cancers are the most aggressive subtypes with poor prognosis due to lack of targeted cancer therapy. Recently, we reported an association of A-kinase anchor protein 4 expression with various clinico-pathological parameters of breast cancer patients. In this context, we examined the effect of knockdown of A-kinase anchor protein 4 on cell cycle, apoptosis, cellular proliferation, colony formation, migration, and invasion in triple-negative breast cancer cells. We also examined the synergistic cytotoxic effect of paclitaxel on A-kinase anchor protein 4 downregulated triple-negative breast cancer cells. Knockdown of A-kinase anchor protein 4 resulted in significant reduction in cellular growth and migratory abilities. Interestingly, we also observed enhanced cell death in A-kinase anchor protein 4 downregulated cells treated with paclitaxel. Knockdown of A-kinase anchor protein 4 in cell cycle resulted in G0/G1 phase arrest. Knockdown of A-kinase anchor protein 4 also led to increased reactive oxygen species generation as a result of upregulation of NOXA and CHOP. In addition, levels of cyclins, cyclin-dependent kinases, anti-apoptotic molecules, and mesenchymal markers were reduced in A-kinase anchor protein 4 downregulated cells. Moreover, downregulation of A-kinase anchor protein 4 also caused tumor growth reduction in in vivo studies. These data together suggest that A-kinase anchor protein 4 downregulation inhibits various malignant properties and enhances the cytotoxic effect of paclitaxel, and this combinatorial approach could be useful for triple-negative breast cancer treatment.

Sperm associated antigen 9 (SPAG9) a promising therapeutic target of ovarian carcinoma.

SPAG9 is a novel tumor associated antigen, expressed in variety of malignancies. However, its role in ovarian cancer remains unexplored. SPAG9 expression was validated in ovarian cancer cells by real time PCR and Western blot. SPAG9 involvement in cell cycle, DNA damage, apoptosis, paclitaxel sensitivity and epithelial- mesenchymal transition (EMT) was investigated employing RNA interference approach. Combinatorial effect of SPAG9 ablation and paclitaxel treatment was evaluated in in vitro. Quantitative PCR and Western blot analysis revealed SPAG9 expression in A10, SKOV-3 and Caov3 compared to normal ovarian epithelial cells. SPAG9 ablation resulted in reduced cellular proliferation, colony forming ability and enhanced cytotoxicity of chemotherapeutic agent paclitaxel. Effect of ablation of SPAG9 on cell cycle revealed S phase arrest and showed decreased expression of CDK1, CDK2, CDK4, CDK6, cyclin B1, cyclin D1, cyclin E and increased expression of tumor suppressor p21. Ablation of SPAG9 also resulted in increased apoptosis with increased expression of various pro- apoptotic molecules including BAD, BID, PUMA, caspase 3, caspase 7, caspase 8 and cytochrome C. Decreased expression of mesenchymal markers and increased expression of epithelial markers was found in SPAG9 ablated cells. Combinatorial effect of SPAG9 ablation and paclitaxel treatment was evaluated in in vitro assays which showed that ablation of SPAG9 resulted in increased paclitaxel sensitivity and caused enhanced cell death. In vivo ovarian cancer xenograft studies showed that ablation of SPAG9 resulted in significant reduction in tumor growth. Present study revealed therapeutic potential of SPAG9 in ovarian cancer.

Sperm-associated antigen 9 (SPAG9) promotes the survival and tumor growth of triple-negative breast cancer cells.

Recently, we demonstrated the association of sperm-associated antigen 9 (SPAG9) expression with breast cancer. Among breast cancer, 15 % of the cancers are diagnosed as triple-negative breast cancers (TNBC) based on hormone receptor status and represent an important clinical challenge because of lack of effective available targeted therapy. Therefore, in the present investigation, plasmid-based small hairpin (small hairpin RNA (shRNA)) approach was used to ablate SPAG9 in aggressive breast cancer cell line model (MDA-MB-231) in order to understand the role of SPAG9 at molecular level in apoptosis, cell cycle, and epithelial-to-mesenchymal transition (EMT) signaling. Our data in MDA-MB-231 cells showed that ablation of SPAG9 resulted in membrane blebbing, increased mitochondrial membrane potential, DNA fragmentation, phosphatidyl serine surface expression, and caspase activation. SPAG9 depletion also resulted in cell cycle arrest in G0-G1 phase and induced cellular senescence. In addition, in in vitro and in vivo xenograft studies, ablation of SPAG9 resulted in upregulation of p21 along with pro-apoptotic molecules such as BAK, BAX, BIM, BID, NOXA, AIF, Cyto-C, PARP1, APAF1, Caspase 3, and Caspase 9 and epithelial marker, E-cadherin. Also, SPAG9-depleted cells showed downregulation of cyclin B1, cyclin D1, cyclin E, CDK1, CDK4, CDK6, BCL2, Bcl-xL, XIAP, cIAP2, MCL1, GRP78, SLUG, SNAIL, TWIST, vimentin, N-cadherin, MMP2, MMP3, MMP9, SMA, and ß-catenin. Collectively, our data suggests that SPAG9 promotes tumor growth by inhibiting apoptosis, altering cell cycle, and enhancing EMT signaling in in vitro cells and in vivo mouse model. Hence, SPAG9 may be a potential novel target for therapeutic use in TNBC treatment.

Interrogation of a context-specific transcription factor network identifies novel regulators of pluripotency.

The predominant view of pluripotency regulation proposes a stable ground state with coordinated expression of key transcription factors (TFs) that prohibit differentiation. Another perspective suggests a more complexly regulated state involving competition between multiple lineage-specifying TFs that define pluripotency. These contrasting views were developed from extensive analyses of TFs in pluripotent cells in vitro. An experimentally validated, genome-wide repertoire of the regulatory interactions that control pluripotency within the in vivo cellular contexts is yet to be developed. To address this limitation, we assembled a TF interactome of adult human male germ cell tumors (GCTs) using the Algorithm for the Accurate Reconstruction of Cellular Pathways (ARACNe) to analyze gene expression profiles of 141 tumors comprising pluripotent and differentiated subsets. The network (GCT(Net)) comprised 1,305 TFs, and its ingenuity pathway analysis identified pluripotency and embryonal development as the top functional pathways. We experimentally validated GCT(Net) by functional (silencing) and biochemical (ChIP-seq) analysis of the core pluripotency regulatory TFs POU5F1, NANOG, and SOX2 in relation to their targets predicted by ARACNe. To define the extent of the in vivo pluripotency network in this system, we ranked all TFs in the GCT(Net) according to sharing of ARACNe-predicted targets with those of POU5F1 and NANOG using an odds-ratio analysis method. To validate this network, we silenced the top 10 TFs in the network in H9 embryonic stem cells. Silencing of each led to downregulation of pluripotency and induction of lineage; 7 of the 10 TFs were identified as pluripotency regulators for the first time.

Heat shock protein 70-2 (HSP70-2) is a novel therapeutic target for colorectal cancer and is associated with tumor growth.

BACKGROUND: Colorectal cancer (CRC) is the third leading cause of cancer related deaths worldwide both in men and women. Our recent studies have indicated an association of heat shock protein 70-2 (HSP70-2) with bladder urothelial carcinoma. In the present study, we investigated the association of HSP70-2 with various malignant properties of colorectal cancer cells and clinic-pathological features of CRC in clinical specimens. METHODS: HSP70-2 mRNA and protein was investigated expression by RT-PCR, immunohistochemistry, immunofluorescence, flow cytometry and Western blotting in CRC clinical specimens and COLO205 and HCT116 cell lines. Plasmid-based gene silencing approach was employed to study the association of HSP70-2 with various malignant properties of COLO205 and HCT116 cells in in vitro and with tumor progression in in vivo COLO205 human xenograft mice model. RESULTS: HSP70-2 expression was detected in 78 % of CRC patients irrespective of various stages and grades by RT-PCR and IHC. Our analysis further revealed that HSP70-2 expression was detected in both COLO205 and HCT116 cell lines. Ablation of HSP70-2 expression resulted in reduced cellular growth, colony forming ability, migratory and invasive ability of CRC cells. In addition, ablation of HSP70-2 expression showed significant reduction in tumor growth in COLO205 human xenograft in in vivo mouse model. CONCLUSION: Collectively, our results indicate that HSP70-2 is associated with CRC clinical specimens. In addition, down regulation of HSP70-2 expression reduces cellular proliferation and tumor growth indicating that HSP70-2 may be a potential therapeutic target for CRC treatment.

Optimization and Validation of a Harmonized Protocol for Generating Therapeutic-Grade Dendritic Cells in a Randomized Phase II Clinical Trial, Using Two Varied Antigenic Sources.

Autologous dendritic cell (DC)-based immunotherapy is a cell-based advanced therapy medicinal product (ATMP) that was first introduced more than three decades ago. In the current study, our objective was to establish a harmonized protocol using two varied antigenic sources and a good manufacturing practice (GMP)-compliant, manual method for generating clinical-grade DCs at a limited-resource academic setting. After obtaining ethical committee-approved informed consent, the recruited patients underwent leukapheresis, and single-batch DC production was carried out. Using responder-independent flow cytometric assays as quality control (QC) criteria, we propose a differentiation and maturation index (DI and MI, respectively), calculated with the QC cut-off and actual scores of each batch for comparison. Changes during cryopreservation and personnel variation were assessed periodically for up to two to three years. Using our harmonized batch production protocol, the average DI was 1.39 and MI was 1.25. Allogenic responder proliferation was observed in all patients, while IFN-gamma secretion, evaluated using flow cytometry, was detected in 10/36 patients and significantly correlated with CD8+ T cell proliferation (p value-0.0002). Tracking the viability and phenotype of cryopreserved MDCs showed a >90% viability for up to three years, while a mature DC phenotype was retained for up to one year. Our results confirm that the manual/semi-automated protocol was simple, consistent, and cost-effective, without the requirement for expensive equipment and without compromising on the quality of the final product.

Expression and humoral response of A-kinase anchor protein 4 in cervical cancer.

BACKGROUND: Cervical cancer is one of the major gynecologic cancers. In developing countries, because of a lack of medical support and infrastructure, cervical cancer is the leading cause of cancer-related deaths. Therefore, there is a need to identify novel biomarkers for cervical cancers. In this context, cancer-testis (CT) antigens represent a unique class of tumor antigens that have been shown to be associated with various solid tumors. These antigens have restricted expression in testis and no expression in somatic tissues. Because of their restricted expression, CT antigens are novel candidate molecules for early detection and diagnosis and immunotherapy. In the present study, novel CT antigen A-kinase anchor protein 4 (AKAP4) expression and humoral response were investigated in patients with cervical cancer. METHODS: In this study, 74 cervical cancer tissue specimens, which included different tumor stages (stage I [n = 35], stage II [n = 39]) and histologic grades (grade 1 [n = 17], grade 2 [n = 46], and grade 3[n = 11]) and 62 adjacent noncancerous tissue specimens were investigated for AKAP4 gene expression by using reverse transcriptase polymerase chain reaction and in situ RNA hybridization. Furthermore, AKAP4 protein expression was determined by immunohistochemistry. In addition, humoral response against purified recombinant AKAP4 protein was determined in available sera of 70 patients with cervical cancer by enzyme-linked immuno assay (ELISA). FINDINGS: Our study revealed that AKAP4 gene and protein expression was detected in 86% of total patients with cervical cancer. Based on the AKAP4 immunoreactivity score, most of stage I (n = 22/29) and stage II (n = 30/35) specimens revealed high AKAP4 expression (≥50% AKAP4-positive cells). A-kinase anchor protein 4 expression was significantly associated with early grades tumor specimens (P = 0.023). In addition, humoral response was detected in 53% of patients irrespective of stages, lymph node positivity, and grades. CONCLUSIONS: Collectively, our data indicate the putative role of AKAP4 in early tumorigenesis and may be implicated as a biomarker and immunotherapeutic target for cervical cancer.

Treatment efficacy of repositioning maneuvers in multiple canal benign paroxysmal positional vertigo: a systematic review and meta-analysis.

Introduction: Benign paroxysmal positional vertigo (BPPV) involving the posterior canal is more common than other canals; however, simultaneous involvement of multiple canals can be seen up to 20% of all BPPV cases. The diagnosis and management of multiple canal BPPV can be quite challenging due to the complexity of findings. Therefore, this systematic review and meta-analysis aimed at unveiling the most effective repositioning strategy for the treatment of multiple canal BPPV. Methods: A literature search through PubMed, Scopus, and Web of Science databases was conducted using search terms such as BPPV, multiple canals, bilateral BPPV, repositioning maneuvers etc. After duplicate removal, the retained articles underwent various stages of elimination by two independent reviewers, and a third reviewer resolved the discrepancy between them. Results: A total of 22 articles were included in the systematic review. These publications documented 5,196 patients diagnosed with BPPV, of which 513 had multiple canal BPPV. Of 295 individuals with multiple canal BPPV, 58.9% were effectively treated in 1 session, whereas 18.3 and 4.4% achieved a symptom-free state after two and three sessions, respectively. Failure of treatment using repositioning maneuvers was found in 18.4%. Possible implications: This study offers insight into the real world of BPPV management in single and multiple canal BPPV. It is evident that repositioning maneuvers provide rapid and long-lasting relief of BPPV in most single canal BPPV patients; however, multiple canal BPPV often requires repeated treatment, and the risk of recurrence is higher in this variety than the single canal BPPV.

Erratum: Heat shock protein 70-2 (HSP70-2) a novel cancer testis antigen that promotes growth of ovarian cancer.

[This corrects the article on p. 1252 in vol. 7, PMID: 28670489.].

Test-Retest Reliability of Video Head Impulse Test in Healthy Individuals and Individuals with Dizziness.

BACKGROUND: Video head impulse test (vHIT) is widely accepted as a test for the assessment of functional integrity of semicircular canals (SCCs). It allows for the evaluation of the functioning of all six SCCs independent of each other. It works on the principle of the vestibulo-ocular reflex (VOR). In individuals with vestibular pathologies, the VOR is impaired, and hence, the use of vHIT may provide vital information about the functional status of SCCs and the VOR pathway originating from them. PURPOSE: In the recent past, studies reported excellent test-retest reliability of vHIT in healthy individuals. However, these studies used analysis of variance or the nonparametric counterpart Wilcoxon signed-rank test, which are insufficient statistical methods for conclusions about test-retest reliability. Further, because vHIT assesses VOR function in individuals with vestibular pathologies, it is important to assess test-retest reliability in the pathological group as well. Therefore, the present study aimed to evaluate test-retest reliability of vHIT in healthy individuals and individuals with vestibular pathology. RESEARCH DESIGN: Repeated measures. STUDY SAMPLE: Twenty healthy individuals with no history of vestibular pathology and 20 individuals with known vestibular pathology were included. DATA COLLECTION AND ANALYSIS: Each participant underwent vHIT testing for all three SCCs of both sides on four different occasions. VOR gain and the presence of pathological saccades were noted and analyzed for each recording. RESULTS: Intraclass correlation coefficient (ICC) revealed excellent test-retest reliability for VOR gain in both groups (ICC ≥ 0.76). Kappa coefficient analysis for the presence of refixation saccades demonstrated moderate to excellent agreement between test sessions (K ≥ 0.63) for the lateral canal. For the anterior and posterior SCC, there was large variability between sessions for refixation saccades. CONCLUSIONS: This study provides evidence about test-retest reliability of VOR gain and refixation saccades assessed using vHIT in healthy individuals and individuals with vestibulopathies. These findings suggest that both measures are highly reliable and replicable across test sessions, except refixation saccades in vertical canals which varied between sessions in some individuals.

Sperm-associated antigen 9, a novel cancer testis antigen, is a potential target for immunotherapy in epithelial ovarian cancer.

PURPOSE: Cancer testis antigens are a group of tumor antigens with gene expression restricted to male germ cells in the testis and in various cancerous tissues. Recently, we reported a novel testis-specific sperm-associated antigen 9 (SPAG9) gene, a new member of the c-Jun NH(2)-terminal kinase-interacting protein family, having functional role in sperm-egg fusion and mitogen-activated protein kinase signaling pathway. National Center for Biotechnology Information Blast searches revealed SPAG9 nucleotide sequence similarities with expressed sequence tags of various cancerous tissues. In an effort to examine the clinical utility of SPAG9, we investigated the SPAG9 mRNA and protein expression in epithelial ovarian cancer (EOC). Humoral immune response to SPAG9 was also evaluated in EOC patients. EXPERIMENTAL DESIGN: We determined the expression profile of SPAG9 transcript by reverse transcription-PCR and RNA in situ hybridization and SPAG9 protein expression by immunohistochemistry in EOC specimens and human ovarian cancer cell lines. Using ELISA and Western blotting, we analyzed specific antibodies for SPAG9 in sera from patients with EOC. RESULTS: SPAG9 mRNA and protein expression was detected in 90% of EOC tissues and in all three human ovarian cancer cell lines. Specific SPAG9 antibodies were detected in 67% of EOC patients and not in sera from healthy individuals. CONCLUSIONS: Our findings indicate that SPAG9 is highly expressed in EOC and immunogenic in patients. Humoral immune response against SPAG9 in early stages of EOC suggests its important role in early diagnostics. These results collectively suggest that SPAG9, a novel member of cancer testis antigen family, could be a potential target for the development of diagnostic and therapeutic methods in EOC.

Crystallization and preliminary X-ray characterization of phosphoglucose isomerase from Mycobacterium tuberculosis H37Rv.

Phosphoglucose isomerase is a ubiquitous enzyme that catalyzes the isomerization of D-glucopyranose-6-phosphate to D-fructofuranose-6-phosphate. The present investigation reports the expression, purification, crystallization and preliminary crystallographic studies of the phosphoglucose isomerase from Mycobacterium tuberculosis H37Rv, which shares 46% sequence identity with that of its human host. The recombinant protein, which was prepared using an Escherichia coli expression system, was crystallized by the hanging-drop vapour-diffusion method. The crystals diffracted to a resolution of 2.8 A and belonged to the orthorhombic space group I2(1)2(1)2(1), with unit-cell parameters a = 109.0, b = 119.8, c = 138.9 A.

Role of A-Kinase anchor protein (AKAP4) in growth and survival of ovarian cancer cells.

Ovarian cancer represents one of the most common malignancies among women with very high mortality rate worldwide. A-kinase anchor protein 4 (AKAP4), a unique cancer testis (CT) antigen has been shown to be associated with various malignant properties of cancer cells. However, its involvement in various molecular pathways in ovarian cancer remains unknown. In present investigation, employing gene silencing approach, we examined the role of AKAP4 in cell cycle, apoptosis and epithelial-mesenchymal transition (EMT). Further, we also investigated the effect of ablation of AKAP4 on tumor growth in SCID mice ovarian cancer xenograft mouse model. Our results showed that ablation of AKAP4 resulted in increased reactive oxygen species (ROS) generation, DNA damage, cell cycle arrest and apoptosis in ovarian cancer cells. AKAP4 knockdown lead to degradation of protien kinase A (PKA) which was rescued by proteosome inhibitor MG-132. ROS quencher N-acetyl cysteine (NAC) treatment rescued cell cycle arrest and resumed cell division. Subsequently, increased expression of pro-apoptotic molecules and decreased expression of pro-survival/anti-apoptotic factors was observed. As a result of AKAP4 depletion, DNA damage response proteins p-γH2AX, p-ATM and p21 were upregulated. Also, knockdown of CREB resulted in similar findings. Further, PKA inhibitor (H89) and oxidative stress resulted in similar phenotype of ovarian cancer cells as observed in AKAP4 ablated cells. Collectively, for the first time our data showed the involvement of AKAP4 in PKA degradation and perturbed signaling through PKA-CREB axis in AKAP4 ablated ovarian cancer cells.

Heat shock protein 70-2 (HSP70-2) a novel cancer testis antigen that promotes growth of ovarian cancer.

Heat shock protein 70-2 (HSP70-2) is known to be involved in tumor progression. However, its molecular role and mechanism in epithelial ovarian cancer (EOC) remains unknown. In the present investigation, we examined the role of HSP70-2 in cell cycle, apoptosis and epithelial mesenchymal transition pathways in EOC cells in in vitro and in-vivo xenograft mouse model. To investigate the role of HSP70-2 in ovarian cancer, plasmid driven short hairpin RNA approach was used to examine HSP70-2 gene and protein expression in ovarian cancer cell line A-10 (origin: serous papillary cystadenocarcinoma), Caov-3 (origin: adenocarcinoma) and SKOV3 (origin: adenocarcinoma; derived from metastatic site: ascites) by RT-PCR, quantitative-PCR, immunohistochemistry and Western blotting. Light microscopy, scanning electron microscopy, viability tests, and flow cytometry were used to study the cellular proliferation, onset of senescence, colony forming ability and morphological features of cancer cells. Cell migration and invasion ability was evaluated by wound healing and Boyden chamber assays. Further, we studied the effect of HSP70-2 protein ablation on human ovarian xenograft mice model. At molecular level, various molecules involved in apoptosis, cell cycle and epithelial-mesenchymal-transition were also examined both in in-vitro and in-vivo xenograft mouse model. The knockdown of HSP70-2 expression by gene silencing resulted in the onset of apoptosis, senescence, reduced cellular growth and colony forming ability of EOC cells. Interestingly, the migration, invasion and wound healing abilities of cells were also significantly inhibited. In addition, the ablation of HSP70-2 resulted in the upregulation of cytochrome-C, caspase 3, caspase 7, caspase 9, APAF1, BAX, BIM, BAK, BAD, BID, PUMA, NOXA, p16, p21, Rb, E-cadherin, cytokeratin 18, EMA in these cells as well as in the xenograft tumor specimens. However, there was downregulation of PARP1, BCL-2, Bcl-xL, MCL-1, Survivin, XIAP, cIAP2, CDK1, CDK2, CDK4, CDK6, cyclin D1, cyclin E, cyclin A2, cyclin B1, p-Rb, N-cadherin, SNAIL, SLUG, VIMENTIN, SMA, MMP2, MMP3, MMP9 and TWIST in these samples. Furthermore, the xenograft studies showed significant reduction in the tumor growth. Our results suggest that HSP70-2 can promote cellular growth and invasion of EOC cells and therefore may be a potential therapeutic target in EOC.

Characterization of a novel human sperm-associated antigen 9 (SPAG9) having structural homology with c-Jun N-terminal kinase-interacting protein.

We report a novel SPAG9 (sperm-associated antigen 9) protein having structural homology with JNK (c-Jun N-terminal kinase)-interacting protein 3. SPAG9, a single copy gene mapped to the human chromosome 17q21.33 syntenic with location of mouse chromosome 11, was earlier shown to be expressed exclusively in testis [Shankar, Mohapatra and Suri (1998) Biochem. Biophys. Res. Commun. 243, 561-565]. The SPAG9 amino acid sequence analysis revealed identity with the JNK-binding domain and predicted coiled-coil, leucine zipper and transmembrane domains. The secondary structure analysis predicted an alpha-helical structure for SPAG9 that was confirmed by CD spectra. Microsequencing of higher-order aggregates of recombinant SPAG9 by tandem MS confirmed the amino acid sequence and mono atomic mass of 83.9 kDa. Transient expression of SPAG9 and its deletion mutants revealed that both leucine zipper with extended coiled-coil domains and transmembrane domain of SPAG9 were essential for dimerization and proper localization. Studies of MAPK (mitogenactivated protein kinase) interactions demonstrated that SPAG9 interacted with higher binding affinity to JNK3 and JNK2 compared with JNK1. No interaction was observed with p38alpha or extracellular-signal-regulated kinase pathways. Polyclonal antibodies raised against recombinant SPAG9 recognized native protein in human sperm extracts and localized specifically on the acrosomal compartment of intact human spermatozoa. Acrosome-reacted spermatozoa demonstrated SPAG9 immunofluorescence, indicating its retention on the equatorial segment after the acrosome reaction. Further, anti-SPAG9 antibodies inhibited the binding of human spermatozoa to intact human oocytes as well as to matched hemizona. This is the first report of sperm-associated JNK-binding protein that may have a role in spermatozoa-egg interaction.

A novel cancer testis antigen target A-kinase anchor protein (AKAP4) for the early diagnosis and immunotherapy of colon cancer.

Colorectal cancer (CRC) is mainly a disease of developed countries and a major cause of death worldwide. The present study was undertaken to investigate the association of novel cancer testis (CT) antigen, A-kinase anchor protein (AKAP4) with CRC. AKAP4 gene and protein was examined by RT-PCR, in situ hybridization and immunohistochemistry (IHC) in 200 clinical specimens of different stages and grades. In addition, humoral response against AKAP4 was detected by enzyme-linked immunosorbent assay and Western blotting in 172 available sera samples of CRC patients. We observed that majority of CRC patients demonstrated AKAP4 expression and elicited immune response. AKAP4 protein expression, based on immunoreactivity score (IRS) predicted presence of CRC with 84% sensitivity, 100% specificity, 100% of positive predictive value (PPV) and 83.33% negative predictive value (NPV). Humoral response against AKAP4 protein was generated in 82% of the CRC patients. Further, statistical analysis revealed that antibodies found against AKAP4 in CRC patients predicted presence of malignancy with 81.98% sensitivity, 100% specificity, 100% PPV, and 63.53% NPV. Collectively, our data suggests that the majority of CRC cases show significant difference of AKAP4 expression among stages and grades and also generated antibodies against AKAP4 protein. Therefore, AKAP4 may be potential candidate molecule for developing as a biomarker for early diagnosis and immunotherapy of CRC.

Mechanism and Role of SOX2 Repression in Seminoma: Relevance to Human Germline Specification.

Human male germ cell tumors (GCTs) are derived from primordial germ cells (PGCs). The master pluripotency regulator and neuroectodermal lineage effector transcription factor SOX2 is repressed in PGCs and the seminoma (SEM) subset of GCTs. The mechanism of SOX2 repression and its significance to GC and GCT development currently are not understood. Here, we show that SOX2 repression in SEM-derived TCam-2 cells is mediated by the Polycomb repressive complex (PcG) and the repressive H3K27me3 chromatin mark that are enriched at its promoter. Furthermore, SOX2 repression in TCam-2 cells can be abrogated by recruitment of the constitutively expressed H3K27 demethylase UTX to the SOX2 promoter through retinoid signaling, leading to expression of neuronal and other lineage genes. SOX17 has been shown to initiate human PGC specification, with its target PRDM1 suppressing mesendodermal genes. Our results are consistent with a role for SOX2 repression in normal germline development by suppressing neuroectodermal genes.

Heat shock protein 70-2 (HSP70-2) overexpression in breast cancer.

BACKGROUND: Breast cancer is one of the leading cause of cancer-related deaths in women worldwide and increasing rapidly in developing countries. In the present study, we investigated the potential role and association of HSP70-2 with breast cancer. METHODS: HSP70-2 expression was examined in 154 tumor and 103 adjacent non-cancerous tissue (ANCT) specimens and breast cancer cell lines (MCF7, BT-474, SK-BR-3 and MDA-MB-231) by RT-PCR, quantitative-PCR, immunohistochemistry, Western blotting, flow cytometry and indirect immunofluorescence. Plasmid driven short hairpin RNA approach was employed to validate the role of HSP70-2 in cellular proliferation, senescence, migration, invasion and tumor growth. Further, we studied the effect of HSP70-2 protein ablation on signaling cascades involved in apoptosis, cell cycle and Epithelial-Mesenchymal-Transition both in culture as well as in-vivo human breast xenograft mouse model. RESULTS: HSP70-2 expression was detected in majority of breast cancer patients (83 %) irrespective of various histotypes, stages and grades. HSP70-2 expression was also observed in all breast cancer cells (BT-474, MCF7, MDA-MB-231 and SK-BR-3) used in this study. Depletion of HSP70-2 in MDA-MB-231 and MCF7 cells resulted in a significant reduction in cellular growth, motility, onset of apoptosis, senescence, cell cycle arrest as well as reduction of tumor growth in the xenograft model. At molecular level, down-regulation of HSP70-2 resulted in reduced expression of cyclins, cyclin dependent kinases, anti-apoptotic molecules and mesenchymal markers and enhanced expression of CDK inhibitors, caspases, pro-apoptotic molecules and epithelial markers. CONCLUSIONS: HSP70-2 is over expressed in breast cancer patients and was involved in malignant properties of breast cancer. This suggests HSP70-2 may be potential candidate molecule for development of better breast cancer treatment.