Elucidating the Role of miRNA-326 Modulating Hedgehog Signaling in Pancreatic Carcinoma.

BACKGROUND AND AIM: Pancreatic ductal adenocarcinoma (PDAC) is one of the lethal malignancies worldwide characterized by poor prognosis. MicroRNAs (miRNAs) function as the key regulators in carcinogenesis and may act as noninvasive biomarkers in various malignancies including PDAC. The present study aimed to elucidate the role of miR-326, a known modulator of hedgehog (Hh) pathway in PDAC. MATERIALS AND METHODS: miR-326 circulating levels were assessed in 105 PDAC patients, 31 with chronic pancreatitis (CP) and 36 healthy controls by quantitative Polymerase chain reaction. The expression of miR-326 and smoothened (SMO) was checked in surgical PDAC tissue. SMO protein expression was analyzed by immunohistochemistry in different groups. Finally, the role of miR-326 as a modulator of Hh pathway was assessed in vitro. RESULTS: Our results demonstrate that miR-326 is downregulated in both blood and tissue of PDAC patients as compared with controls. In contrast, the target gene/protein expression of SMO is upregulated in PDAC. Moreover, the tumor stromal expression of SMO was found to be clinically associated with lymph-node metastasis and vascular encasement in PDAC. Overexpression of miR-326 in Panc1 cell line was found to induce downregulation of SMO suggesting the tumor suppressor role of miR-326 in PDAC. CONCLUSIONS: Taken together, miR-326 acts as a tumor suppressor in PDAC by modulating Hh pathway. It may be a promising target for the development of efficient drug therapies for the treatment of PDAC.

NFκB (RelA) mediates transactivation of hnRNPD in oral cancer cells.

Heterogeneous Ribonucleoprotein D (hnRNPD) is an RNA binding protein involved in post-transcriptional regulation of multiple mediators of carcinogenesis. We previously demonstrated a strong association of hnRNPD over expression with poor outcome in Oral Squamous Cell Carcinoma (OSCC). However, hitherto the precise molecular mechanism of its overexpression in oral cancer was not clear. Therefore, in an attempt to elucidate the transcriptional regulation of hnRNPD expression, we cloned 1406 bp of 5' flanking region of human hnRNPD gene along with 257 bp of its first exon upstream to promoterless luciferase reporter gene in pGL3-Basic. Transfection of the resulting construct in SCC-4 cells yielded 1271 fold higher luciferase activity over parent vector. By promoter deletion analysis, we identified a canonical TATA box containing 126 bp core promoter region that retained ~ 58% activity of the full length promoter. In silico analysis revealed the presence of four putative NFκB binding motifs in the promoter. Sequential deletion of these motifs from the full-length promoter reporter construct coupled with luciferase assays revealed an 82% decrease in promoter activity after deletion of the first (-1358/-1347) motif and 99% reduction after the deletion of second motif (-1052/-1041). In-vivo binding of NFκB (RelA) to these two motifs in SCC-4 cells was confirmed by ChIP assays. Site directed mutagenesis of even one of these two motifs completely abolished promoter activity, while mutagenesis of the remaining two motifs had marginal effect on the same. Consistent with these findings, treatment of SCC-4 cells with PDTC, a known inhibitor of NFκB dramatically reduced the levels hnRNPD mRNA and protein. Finally, the expression of hnRNPD and NFκB in clinical specimen from 37 oral cancer patients was assessed and subjected to Spearmen's Correlation analysis which revealed a strong positive correlation between the two. Thus, results of the present study for the first time convincingly demonstrate NFκB (RelA) mediated transcriptional upregulation of hnRNPD expression in oral cancer.

Pleiotropic role of PARP1: an overview.

Poly (ADP-ribose) polymerase 1 (PARP1) protein is encoded by the PARP1 gene located on chromosome 1 (1q42.12) in human cells. It plays a crucial role in post-translational modification by adding poly (ADP-ribose) (PAR) groups to various proteins and PARP1 itself by utilizing nicotinamide adenine dinucleotide (NAD +) as a substrate. Since the discovery of PARP1, its role in DNA repair and cell death has been its identity. This is evident from an overwhelmingly high number of scientific reports in this regard. However, PARP1 also plays critical roles in inflammation, metabolism, tumor development and progression, chromatin modification and transcription, mRNA stability, and alternative splicing. In the present study, we attempted to compile all the scattered scientific information about this molecule, including the structure and multifunctional role of PARP1 in cancer and non-cancer diseases, along with PARP1 inhibitors (PARPis). Furthermore, for the first time, we have classified PARP1-mediated cell death for ease of understanding its role in cell death pathways.

Panel of serum miRNAs as potential non-invasive biomarkers for pancreatic ductal adenocarcinoma.

Early-stage diagnosis of pancreatic ductal adenocarcinoma (PDAC) is difficult due to non-specific symptoms. Circulating miRNAs in body fluids have been emerging as potential non-invasive biomarkers for diagnosis of many cancers. Thus, this study aimed to assess a panel of miRNAs for their ability to differentiate PDAC from chronic pancreatitis (CP), a benign inflammatory condition of the pancreas. Next-generation sequencing was performed to identify miRNAs present in 60 FFPE tissue samples (27 PDAC, 23 CP and 10 normal pancreatic tissues). Four up-regulated miRNAs (miR-215-5p, miR-122-5p, miR-192-5p, and miR-181a-2-3p) and four down-regulated miRNAs (miR-30b-5p, miR-216b-5p, miR-320b, and miR-214-5p) in PDAC compared to CP were selected based on next-generation sequencing results. The levels of these 8 differentially expressed miRNAs were measured by qRT-PCR in 125 serum samples (50 PDAC, 50 CP, and 25 healthy controls (HC)). The results showed significant upregulation of miR-215-5p, miR-122-5p, and miR-192-5p in PDAC serum samples. In contrast, levels of miR-30b-5p and miR-320b were significantly lower in PDAC as compared to CP and HC. ROC analysis showed that these 5 miRNAs can distinguish PDAC from both CP and HC. Hence, this panel can serve as a non-invasive biomarker for the early detection of PDAC.

Influence of different forms and materials (zirconia or titanium) of abutments in peri-implant soft-tissue healing using matrix metalloproteinase-8: A randomized pilot study.

STATEMENT OF PROBLEM: It is unclear how pathogenic bacteria adhere to different implant materials and whether biomarker matrix metalloproteinase-8 (MMP-8) level provides a reliable method of evaluating the connective tissue status of peri-implant tissues. PURPOSE: The purpose of this pilot clinical study was to evaluate peri-implant connective tissue response by assessing the MMP-8 levels in peri-implant crevicular fluid around titanium and zirconia abutments. MATERIAL AND METHODS: The study was designed as a prospective, within-subject comparison with left-right randomization low. Twelve participants with partial edentulism were selected according to inclusion and exclusion criteria. Peri-implant sulcal fluid sampling and pocket probing depths were assessed at 1, 3, and 12 months after placing the abutments. The MMP-8 protein level of the peri-implant sulcal fluid was determined by MMP-8-specific sandwich enzyme-linked immunosorbent assay system. The independent t test or Wilcoxon test was used to compare MMP-8 levels and probing depth assessment between the zirconia and titanium groups at different time points (1, 3, and 12 months). Repeated measures ANOVA was used for within-group comparison of the MMP-8 levels at 3 time points (α=.05). RESULTS: At 1 and 3 months, the titanium abutments showed significantly higher MMP-8 levels and probing depths than the zirconia abutments (P<.05), but no significant differences were found at 12 months for either variable (P>.05). CONCLUSIONS: This study suggests the presence of more remodeling and/or inflammatory phenomena around titanium implant abutments than around zirconia abutments of a different design during the early stages but not at 1 year.

End Binding 1 (EB1) overexpression in oral lesions and cancer: A biomarker of tumor progression and poor prognosis.

INTRODUCTION: Oral squamous cell carcinoma (OSCC) patients are at high risk of loco-regional recurrence and despite the improvement in treatment strategy, 5-year survival rates are about 50%. Identification of patients at high risk of recurrence may enable rigorous personalized post-treatment management. In an earlier proteomics study we observed overexpression of End Binding Protein (EB1) in OSCC. In the present study we investigated the diagnostic and prognostic significance of alterations in expression of EB1 in oral cancer. METHODS: In this retrospective study, the expression of EB1 protein was evaluated in 259 OSCCs, 41 dysplasia, 166 hyperplasia and 126 normal tissues using immunohistochemistry and correlated with clinical-pathological parameters and prognosis of OSCC patients over a follow-up period of up to 91months. RESULTS: Significantly higher expression of cytoplasmic EB1 was observed in hyperplasia [p<0.001, OR=7.2, 95% CI=4.1-12.8], dysplasia (p<0.001, OR=21.8, CI=8.8-50.2) and OSCCs (p<0.001, OR=10.1, CI=5.8-17.4) in comparison with normal mucosa. Univariate analysis revealed cytoplasmic EB1 association with tumor grade, tumor size and recurrence of the disease. Kaplan Meier survival analysis of EB1 expression showed significantly reduced disease free survival (DFS) (p=0.003). Notably, OSCC patients showing cytoplasmic EB1 overexpression demonstrated significantly reduced DFS (p=0.004, HR=2.1). CONCLUSION: EB1 overexpression is an early event in oral tumorigenesis and cytoplasmic EB1 accumulation is associated with poor prognosis and tumor recurrence in OSCC patients.

Nuclear heterogeneous nuclear ribonucleoprotein D is associated with poor prognosis and interactome analysis reveals its novel binding partners in oral cancer.

BACKGROUND: Post-transcriptional regulation by heterogeneous ribonucleoproteins (hnRNPs) is an important regulatory paradigm in cancer development. Our proteomic analysis revealed hnRNPD overexpression in oral dysplasia as compared with normal mucosa; its role in oral carcinogenesis remains unknown. Here in we determined the hnRNPD associated protein networks and its clinical significance in oral squamous cell carcinoma (OSCC). METHODS: Immunoprecipitation (IP) followed by tandem mass spectrometry was used to identify the binding partners of hnRNPD in oral cancer cell lines. Ingenuity pathway analysis (IPA) was carried out to unravel the protein interaction networks associated with hnRNPD and key interactions were confirmed by co-IP-western blotting. hnRNPD expression was analyzed in 183 OSCCs, 44 oral dysplasia and 106 normal tissues using immunohistochemistry (IHC) and correlated with clinico-pathological parameters and follow up data over a period of 91 months. Kaplan-Meier survival and Cox-multivariate-regression analyses were used to evaluate the prognostic significance of hnRNPD in OSCC. RESULTS: We identified 345 binding partners of hnRNPD in oral cancer cells. IPA unraveled novel protein-protein interaction networks associated with hnRNPD and suggested its involvement in multiple cellular processes: DNA repair, replication, chromatin remodeling, cellular proliferation, RNA splicing and stability, thereby directing the fate of oral cancer cells. Protein-protein interactions of hnRNPD with 14-3-3ζ, hnRNPK and S100A9 were confirmed using co-IP-western blotting. IHC analysis showed significant overexpression of nuclear hnRNPD in oral dysplasia [p = 0.001, Odds ratio (OR) = 5.1, 95% CI = 2.1-11.1) and OSCCs (p = 0.001, OR = 8.1, 95% CI = 4.5-14.4) in comparison with normal mucosa. OSCC patients showing nuclear hnRNPD overexpression had significantly reduced recurrence free survival [p = 0.026, Hazard ratio = 1.95, 95% CI = 1.0-3.5] by Kaplan-Meier survival and Cox-multivariate-regression analyses and has potential to define a high-risk subgroup among OSCC patients with nodal negative disease. CONCLUSIONS: Our findings suggest novel functions of hnRNPD in cellular proliferation and survival, besides RNA splicing and stability in oral cancer. Association of nuclear hnRNPD with poor prognosis in OSCC patients taken together with its associated protein networks in oral cancer warrant future studies designed to explore its potential as a plausible novel target for molecular therapeutics.

Prognostic significance of cytoplasmic S100A2 overexpression in oral cancer patients.

BACKGROUND: Oral squamous cell carcinoma (OSCC) patients are at high risk of loco-regional recurrence and 5-year survival rates are about 50%. Identification of patients at high risk of recurrence will enable rigorous personalized post-treatment management. Most novel biomarkers have failed translation for clinical use because of their limited successful validation in external patient cohorts. The aim of this study was to determine the prognostic significance of alterations in sub-cellular expression of S100A2, a pro-tumorigenic calcium binding protein, identified as a candidate biomarker in our proteomic analysis in OSCC and validation of its clinical utility in an external cohort. METHODS: In a retrospective study, immunohistochemical analysis of S100A2 was carried out in 235 Indian OSCC (Test set) and 129 normal oral tissues, correlated with clinicopathological parameters and disease outcome over 122 months for OSCC patients following the REMARK criteria. The findings were validated in an external cohort (Validation set 115 Canadian OSCC and 51 normal tissues) and data analyzed using the R package. RESULTS: Significant increase in cytoplasmic and decrease in nuclear S100A2 expression was observed in OSCC in comparison with normal tissues. Cox multivariable regression analysis internally and externally validated cytoplasmic S100A2 association with tumor recurrence. Kaplan Meier analysis of patients stratified to high and low risk groups showed significantly different recurrence free survival (Test set- log rank test, p = 0.005, median survival 16 and 69 months respectively and Validation set - p < 0.00001, median survival 9.4 and 59.9 months respectively); 86% and 81% of patients who had recurrence were correctly stratified into the high risk group. Seventy percent and 81% patients stratified into low risk group did not show cancer recurrence within 1 year in Test and Validation sets. CONCLUSIONS: Our study provided clinical evidence for the potential of cytoplasmic S100A2 overexpression as a predictor of recurrence risk in OSCC patients. A unique translational aspect of our study is validation of S100A2 as prognostic marker in two independent cohorts (Canadian and Indian) suggesting this protein is likely to find widespread utility in clinical practice for identifying oral cancer patients at high risk of disease recurrence.

The rational design of specific peptide inhibitor against p38α MAPK at allosteric-site: a therapeutic modality for HNSCC.

p38α is a significant target for drug designing against cancer. The overproduction of p38α MAPK promotes tumorigenesis in head and neck squamous cell carcinoma (HNSCC). The ATP binding and an allosteric site referred as DFG are the key sites of the p38α mitogen activated protein kinase (MAPK) exploited for the design of inhibitors. This study demonstrated design of peptide inhibitor on the basis of allosteric site using Glide molecular docking software and the biochemical analysis of the best modeled peptide. The best fitted tetrapeptide (FWCS) in the allosteric site inhibited the pure recombinant and serum p38α of HNSCC patients by 74 and 72%, respectively. The potency of the peptide was demonstrated by its IC50 (4.6 nM) and KD (3.41×10-10 M) values, determined by ELISA and by surface plasmon resonance (SPR) technology, respectively. The cell viability of oral cancer i.e. KB cell line was reduced in dose dependent manner by 60 and 97% by the treatment of peptide and the IC50 was 600 and 210 µM after 24 and 72 h incubation, respectively. Our result provides an insight for the development of a proficient small peptide as a promising anticancer agent targeting DFG site of p38α kinase.

siRNA-mediated downregulation of TC21 sensitizes esophageal cancer cells to cisplatin.

AIM: To determine the functional significance of TC21 in esophageal squamous cell carcinoma (ESCC). METHODS: TC21 siRNA transfection was carried out using Hyperfectamine to knock down TC21, and transcripts were analyzed by reverse transcription-polymerase chain reaction and protein by Western blotting. We demonstrated the effect of TC21 downregulation of cell signaling in esophageal cancer cells by assessing the phosphorylation status of its downstream targets, phosphoinositide 3-kinase (PI3K), phosphatase and tensin homolog (PTEN), protein kinase B (pAkt), nuclear factor-κB (NF-κB) and cyclinD1 using specific antibodies. Cell survival analysis after cisplatin treatment was carried out by cell viability assay and cell cycle analysis using flow cytometry. RESULTS: TC21 knockdown in human ESCC cell line TE13 cells, showed only a marginal increase (14.2%) in cell death compared with control cells. The expressions of the signaling proteins PI3K and pAkt, transcription factor NF-κB, and cell cycle protein cyclin D1 were markedly decreased in response to TC21 downregulation, whereas the level of pPTEN, an antagonist of PI3K, was increased. In addition, we evaluated the potential of TC21 as a putative target for sensitizing ESCC cells to the chemotherapeutic agent cisplatin. Increased cell death (38.4%) was observed in cells treated with cisplatin after TC21 knockdown compared with cells which were treated with cisplatin alone (20% cell death). CONCLUSION: Results suggest that TC21 mediates its effects via the PI3K-Akt pathway, NF-κB and cyclin D1, and enhances chemoresistance in esophageal cancer cells.

Prevalence, distribution and functional significance of the -237C to T polymorphism in the IL-12Rß2 promoter in Indian tuberculosis patients.

Cytokine/cytokine receptor gene polymorphisms related to structure/expression could impact immune response. Hence, the -237 polymorphic site in the 5' promoter region of the IL-12Rß2 (SNP ID: rs11810249) gene associated with the AP-4 transcription motif GAGCTG, was examined. Amplicons encompassing the polymorphism were generated from 46 pulmonary tuberculosis patients, 35 family contacts and 28 miscellaneous volunteers and sequenced. The C allele predominated among patients, (93.4%, 43/46), and in all volunteers and contacts screened, but the T allele was exclusively limited to patients, (6.5%, 3/46). The functional impact of this polymorphism on transcriptional activity was assessed by Luciferase-reporter and electrophoretic mobility shift assays (EMSA). Luciferase-reporter assays showed a significant reduction in transcriptional efficiency with T compared to C allele. The reduction in transcriptional efficiency with the T allele construct (pGIL-12Rb2-T), in U-87MG, THP-1 and Jurkat cell lines, were 53, 37.6, and 49.8% respectively, compared to the C allele construct (pGIL-12Rb2-C). Similarly, densitometric analysis of the EMSA assay showed reduced binding of the AP-4 transcription factor, to T compared to the C nucleotide probe. Reduced mRNA expression in all patients (3/3) harboring the T allele was seen, whereas individuals with the C allele exhibited high mRNA expression (17/25; 68%, p = 0.05). These observations were in agreement with the in vitro assessment of the promoter activity by Luciferase-reporter and EMSA assays. The reduced expression of IL-12Rß2 transcripts in 8 patients despite having the C allele was attributed to the predominant over expression of the suppressors (IL-4 and GATA-3) and reduced expression of enhancers (IFN-α) of IL-12Rß2 transcripts. The 17 high IL-12Rß2 mRNA expressers had significantly elevated IFN-α mRNA levels compared to low expressers and volunteers. Notwithstanding the presence of high levels of IL-12Rß2 mRNA in these patients elevated IFN-α expression could modulate their immune responses to Mycobacterium tuberculosis.

Structure based design and synthesis of peptide inhibitor of human LOX-12: in vitro and in vivo analysis of a novel therapeutic agent for breast cancer.

Human breast cancer cell proliferation involves a complex interaction between growth factors, steroid hormones and peptide hormones. The interaction of growth factors, such as epidermal growth factor (EGF), with their receptors on breast cancer cells can lead to the hydrolysis of phospholipids and release of fatty acid such as arachidonic acid, which can be further metabolized by cyclooxygenase (COX) and lipoxygenase (LOX) pathways to produce prostaglandins. The high concentration of prostaglandins has been associated with chronic inflammatory diseases and several types of human cancers. This is due to the over expression COX, LOX and other inflammatory enzymes. Ten peptides were designed and synthesized by solid phase peptide synthesis and analyzed in vitro for enzyme inhibition. Out of these peptides, YWCS had shown significant inhibitory effects. The dissociation constant (K(D)) was determined by surface plasmon resonance (SPR) analysis and was found to be 3.39 × 10(-8) M and 8.6 × 10(-8) M for YWCS and baicalein (positive control), respectively. The kinetic constant Ki was 72.45 × 10(-7) M as determined by kinetic assay. The peptide significantly reduced the cell viability of estrogen positive MCF-7 and estrogen negative MDA-MB-231 cell line with the half maximal concentration (IC(50)) of 75 µM and 400 µM, respectively. The peptide also induced 49.8% and 20.8% apoptosis in breast cancer cells MCF-7 and MDA-MB-231, respectively. The YWCS was also found to be least hemolytic at a concentration of 358 µM. In vivo studies had shown that the peptide significantly inhibits tumor growth in mice (p<0.017). This peptide can be used as a lead compound and complement for ongoing efforts to develop differentiation therapies for breast cancer.

Loss of DLC1 is an independent prognostic factor in patients with oral squamous cell carcinoma.

Deleted in liver cancer (DLC1), a Rho GTPase-activating protein, was observed to be differentially expressed in oral squamous cell carcinoma in comparison with normal tissues using tissue proteomics. In the current study, we investigated the clinical significance of loss of DLC1 expression in different stages of development of oral squamous cell carcinoma to determine its potential as a biomarker for oral dysplasia and prognosis of oral squamous cell carcinoma. Immunohistochemical analysis of DLC1 expression was carried out in oral squamous cell carcinoma patients (n=214), dysplasia (n=51), hyperplastic squamous mucosa (n=45), and histologically normal oral tissues (n=80), and correlated with clinicopathological parameters and disease prognosis over 91 months for oral squamous cell carcinomas. Loss of DLC1 expression was observed in oral squamous cell carcinoma (64%), oral dysplasia (31%), hyperplastic squamous mucosa (22%), and normal mucosa (16%). Significant loss of DLC1 expression was observed in oral squamous cell carcinomas as compared with dysplasia (P<0.001, odds ratio=3.8, 95% CI=2.0-7.3), suggesting it may be an important event involved in cancer progression. Among oral squamous cell carcinomas, the loss of DLC1 expression was significantly associated with poor prognosis (P=0.021, hazards ratio (HR)=1.8, 95% CI=1.1-2.9). Multivariate analysis revealed loss of DLC1 (P=0.023, HR=2.1, 95% CI=1.2-3.9) and histopathological grade (P=0.015, HR=1.7, 95% CI=1.1-2.7) to be independent predictors for disease-free survival in oral squamous cell carcinoma patients in comparison with known prognostic factors, viz. tumor stage, nodal status, and overall stage. Loss of DLC1 expression emerged as an important biomarker for predicting patients diagnosed with oral dysplasia at high risk of transformation upon future validation in longitudinal studies. Loss of DLC1 expression is a poor prognostic marker for oral squamous cell carcinoma patients.

Overexpression of prothymosin alpha predicts poor disease outcome in head and neck cancer.

BACKGROUND: In our recent study, tissue proteomic analysis of oral pre-malignant lesions (OPLs) and normal oral mucosa led to the identification of a panel of biomarkers, including prothymosin alpha (PTMA), to distinguish OPLs from histologically normal oral tissues. This study aimed to determine the clinical significance of PTMA overexpression in oral squamous cell hyperplasia, dysplasia and head and neck squamous cell carcinoma (HNSCC). METHODOLOGY: Immunohistochemistry of PTMA protein was performed in HNSCCs (n = 100), squamous cell hyperplasia (n = 116), dysplasia (n = 50) and histologically normal oral tissues (n = 100). Statistical analysis was carried out to determine the association of PTMA overexpression with clinicopathological parameters and disease prognosis over 7 years for HNSCC patients. RESULTS: Our immunohistochemical analysis demonstrated significant overexpression of nuclear PTMA in squamous cell hyperplasia (63.8%), dysplasia (50%) and HNSCC (61%) in comparison with oral normal mucosa (p(trend)<0.001). Chi-square analysis showed significant association of nuclear PTMA with advanced tumor stages (III+IV). Kaplan Meier survival analysis indicated reduced disease free survival (DFS) in HNSCC patients (p<0.001; median survival 11 months). Notably, Cox-multivariate analysis revealed nuclear PTMA as an independent predictor of poor prognosis of HNSCC patients (p<0.001, Hazard's ratio, HR = 5.2, 95% CI = 2.3-11.8) in comparison with the histological grade, T-stage, nodal status and tumor stage. CONCLUSIONS: Nuclear PTMA may serve as prognostic marker in HNSCC to determine the subset of patients that are likely to show recurrence of the disease.

Guggulsterone targets smokeless tobacco induced PI3K/Akt pathway in head and neck cancer cells.

BACKGROUND: Epidemiological association of head and neck cancer with smokeless tobacco (ST) emphasizes the need to unravel the molecular mechanisms implicated in cancer development, and identify pharmacologically safe agents for early intervention and prevention of disease recurrence. Guggulsterone (GS), a biosafe nutraceutical, inhibits the PI3K/Akt pathway that plays a critical role in HNSCC development. However, the potential of GS to suppress ST and nicotine (major component of ST) induced HNSCC remains unexplored. We hypothesized GS can abrogate the effects of ST and nicotine on apoptosis in HNSCC cells, in part by activation of PI3K/Akt pathway and its downstream targets, Bax and Bad. METHODS AND RESULTS: Our results showed ST and nicotine treatment resulted in activation of PI3K, PDK1, Akt, and its downstream proteins--Raf, GSK3ß and pS6 while GS induced a time dependent decrease in activation of PI3K/Akt pathway. ST and nicotine treatment also resulted in induction of Bad and Bax phosphorylation, increased the association of Bad with 14-3-3ζresulting in its sequestration in the cytoplasm of head and neck cancer cells, thus blocking its pro-apoptotic function. Notably, GS pre-treatment inhibited ST/nicotine induced activation of PI3K/Akt pathway, and inhibited the Akt mediated phosphorylation of Bax and Bad. CONCLUSIONS: In conclusion, GS treatment not only inhibited proliferation, but also induced apoptosis by abrogating the effects of ST/nicotine on PI3K/Akt pathway in head and neck cancer cells. These findings provide a rationale for designing future studies to evaluate the chemopreventive potential of GS in ST/nicotine associated head and neck cancer.

Nuclear and cytoplasmic accumulation of Ep-ICD is frequently detected in human epithelial cancers.

BACKGROUND: We previously demonstrated that nuclear and cytoplasmic accumulation of the intracellular domain (Ep-ICD) of epithelial cell adhesion molecule (EpCAM) accompanied by a reciprocal reduction of its extracellular domain (EpEx), occurs in aggressive thyroid cancers. This study was designed to determine whether similar accumulation of Ep-ICD is a common event in other epithelial cancers. METHODOLOGY AND RESULTS: Ten epithelial cancers were immunohistochemically analyzed using Ep-ICD and EpEx domain-specific antibodies. The subcellular localization of EpEx and Ep-ICD in the human colon adenocarcinoma cell line CX-1 was observed using immunofluorescence. Nuclear and cytoplasmic Ep-ICD expression was increased in cancers of the breast (31 of 38 tissues, 82%), prostate (40 of 49 tissues, 82%), head and neck (37 of 57 tissues, 65%) and esophagus (17 of 46 tissues, 37%) compared to their corresponding normal tissues that showed membrane localization of the protein. Importantly, Ep-ICD was not detected in the nuclei of epithelial cells in most normal tissues. High nuclear and cytoplasmic Ep-ICD accumulation also occurred in the other six epithelial cancer types analyzed - lung, colon, liver, bladder, pancreatic, and ovarian. A concomitant reduction in membrane EpEx expression was observed in a subset of all cancer types. Receiver operating characteristic curve analysis revealed nuclear Ep-ICD distinguished breast cancers with 82% sensitivity and 100% specificity and prostate cancers with 82% sensitivity and 78% specificity. Similar findings were observed for cytoplasmic accumulation of Ep-ICD in these cancers. We provide clinical evidence of increased nuclear and cytoplasmic Ep-ICD accumulation and a reduction in membranous EpEx in these cancers. CONCLUSIONS: Increased nuclear and cytoplasmic Ep-ICD was observed in all epithelial cancers analyzed and distinguished them from normal tissues with high-sensitivity, specificity, and AUC. Development of a robust high throughput assay for Ep-ICD will facilitate the determination of its diagnostic, prognostic and therapeutic relevance in epithelial cancers.

Nuclear S100A7 is associated with poor prognosis in head and neck cancer.

BACKGROUND: Tissue proteomic analysis of head and neck squamous cell carcinoma (HNSCC) and normal oral mucosa using iTRAQ (isobaric tag for relative and absolute quantitation) labeling and liquid chromatography-mass spectrometry, led to the identification of a panel of biomarkers including S100A7. In the multi-step process of head and neck tumorigenesis, the presence of dysplastic areas in the epithelium is proposed to be associated with a likely progression to cancer; however there are no established biomarkers to predict their potential of malignant transformation. This study aimed to determine the clinical significance of S100A7 overexpression in HNSCC. METHODOLOGY: Immunohistochemical analysis of S100A7 expression in HNSCC (100 cases), oral lesions (166 cases) and 100 histologically normal tissues was carried out and correlated with clinicopathological parameters and disease prognosis over 7 years for HNSCC patients. Overexpression of S100A7 protein was significant in oral lesions (squamous cell hyperplasia/dysplasia) and sustained in HNSCC in comparison with oral normal mucosa (p(trend)<0.001). Significant increase in nuclear S100A7 was observed in HNSCC as compared to dysplastic lesions (p = 0.005) and associated with well differentiated squamous cell carcinoma (p = 0.031). Notably, nuclear accumulation of S100A7 also emerged as an independent predictor of reduced disease free survival (p = 0.006, Hazard ratio (HR = 7.6), 95% CI = 1.3-5.1) in multivariate analysis underscoring its relevance as a poor prognosticator of HNSCC patients. CONCLUSIONS: Our study demonstrated nuclear accumulation of S100A7 may serve as predictor of poor prognosis in HNSCC patients. Further, increased nuclear accumulation of S100A7 in HNSCC as compared to dysplastic lesions warrants a large-scale longitudinal study of patients with dysplasia to evaluate its potential as a determinant of increased risk of transformation of oral premalignant lesions.

Presence and significance of a R110W mutation in the DNA-binding domain of GCM2 gene in patients with isolated hypoparathyroidism and their family members.

OBJECTIVE: Glial cells missing 2 (GCM2) gene encodes a parathyroid-specific transcription factor. We assessed GCM2 gene sequence in patients with isolated hypoparathyroidism (IH). DESIGN: Case-control study. METHODS: Complete DNA sequencing of the GCM2 gene including its exons, promoter, and 5' and 3' UTRs was performed in 24/101 patients with IH. PCR-restriction fragment length polymorphism was used to detect a novel R110W mutation in all 101 IH patients and 655 healthy controls. Significance of the mutation was assessed by electrophoretic mobility shift assay (EMSA) and nuclear localization on transfection. RESULTS: A heterozygous R110W mutation was present in DNA-binding domain in 11/101 patients (10.9%) and absent in 655 controls (P<10(-7)). Four of 13 nonaffected first-degree relatives for five of these index cases had R110W mutation. Four heterozygous single nucleotide polymorphisms were found in the 5' region. One of the 11 patients with R110W also had T370M change in compound heterozygous form. Mutant R110W and T370M GCM2 proteins showed decreased binding with GCM recognition elements on EMSA indicating loss of function. Both wild-type and R110W mutant GCM2 proteins showed nuclear localization. CONCLUSIONS: The present study indicates a significant association of R110W variant with IH. Absence of effect of heterozygous R110W mutation on DNA binding and presence of the same mutation in asymptomatic family members indicate that additional genetic (akin to T370M change) or nongenetic factors might contribute to the expression of diseases in IH. Alternatively, it is possible that association of R110W with IH could be due to linkage disequilibrium with the unidentified relevant genes in IH.

Antimalarial evaluation of copper(II) nanohybrid solids: inhibition of plasmepsin II, a hemoglobin-degrading malarial aspartic protease from Plasmodium falciparum.

A new class of copper(II) nanohybrid solids, LCu(CH(3)COO)(2) and LCuCl(2), have been synthesized and characterized by transmission electron microscopy, dynamic light scattering, and IR spectroscopy, and have been found to be capped by a bis(benzimidazole) diamide ligand (L). The particle sizes of these nanohybrid solids were found to be in the ranges 5-10 and 60-70 nm, respectively. These nanohybrid solids were evaluated for their in vitro antimalarial activity against a chloroquine-sensitive isolate of Plasmodium falciparum (MRC 2). The interactions between these nanohybrid solids and plasmepsin II (an aspartic protease and a plausible novel target for antimalarial drug development), which is believed to be essential for hemoglobin degradation by the parasite, have been assayed by UV-vis spectroscopy and inhibition kinetics using Lineweaver-Burk plots. Our results suggest that these two compounds have antimalarial activities, and the IC(50) values (0.025-0.032 microg/ml) are similar to the IC(50) value of the standard drug chloroquine used in the bioassay. Lineweaver-Burk plots for inhibition of plasmepsin II by LCu(CH(3)COO)(2) and LCuCl(2) show that the inhibition is competitive with respect to the substrate. The inhibition constants of LCu(CH(3)COO)(2) and LCuCl(2) were found to be 10 and 13 microM, respectively. The IC(50) values for inhibition of plasmepsin II by LCu(CH(3)COO)(2) and LCuCl(2) were found to be 14 and 17 microM, respectively. Copper(II) metal capped by a benzimidazole group, which resembles the histidine group of copper proteins (galactose oxidase, beta-hydroxylase), could provide a suitable anchoring site on the nanosurface and thus could be useful for inhibition of target enzymes via binding to the S1/S3 pocket of the enzyme hydrophobically. Both copper(II) nanohybrid solids were found to be nontoxic against human hepatocellular carcinoma cells and were highly selective for plasmepsin II versus human cathepsin D. The pivotal mechanism of antimalarial activity of these compounds via plasmepsin II inhibition in the P. falciparum malaria parasite is demonstrated.

Heterogeneous ribonucleoprotein K is a marker of oral leukoplakia and correlates with poor prognosis of squamous cell carcinoma.

Oral leukoplakia is a heterogeneous lesion with risk of cancer development; there are no biomarkers to predict its potential of malignant transformation. Tissue proteomic analysis of oral leukoplakia using iTRAQ labeling liquid chromatography-mass spectrometry showed overexpression of heterogeneous ribonucleoprotein K (hnRNP K), a transformation-related RNA-binding protein, in leukoplakia in comparison with normal tissue. Herein, we investigated the clinical significance of hnRNP K in identification of oral leukoplakic lesions in early stages and as a prognostic marker in head-and-neck/oral squamous cell carcinomas (HNOSCCs). Immunohistochemical analysis of hnRNP K was performed in 100 HNOSCCs, 199 leukoplakias and 55 nonmalignant tissues and correlated with clinicopathologic parameters and disease prognosis over 6 years for HNOSCCs. hnRNP K nuclear expression increased from normal tissues to leukoplakia, and frank malignancy (p < 0.001). Cytoplasmic hnRNP K increased significantly from leukoplakia to HNOSCCs (p < 0.001) and was associated with poor prognosis of HNOSCCs (p = 0.011) by Kaplan-Meier analysis. The most important finding of our follow-up study is that cytoplasmic hnRNP K is an independent predictor of disease recurrence in HNOSCC patients. In conclusion, nuclear hnRNP K may serve as a potential marker for early diagnosis, whereas its cytoplasmic accumulation can help to identify a subgroup of HNOSCC patients with poor prognosis, suggesting its putative utility in clinical management of HNOSCC.