Alkoxy-functionalised dihydropyrimido[4,5-b]quinolinones enabling anti-proliferative and anti-invasive agents.

In this communication, we explored the synthesis of novel alkoxy-functionalised dihydropyrimido[4,5-b]quinolinones using a microwave-assisted multicomponent reaction. All the synthesized molecules were screened for anti-proliferative and anti-invasive activity against glioblastoma cells. 5c shows the most potent anti-proliferative activity with a half maximal effective concentration of less than 3 µM against primary patient-derived glioblastoma cells. 5c effectively inhibited invasion and tumor growth of 3D primary glioma cultures in a basement membrane matrix. This suggests that the novel compounds could inhibit both the proliferation and invasive spread of glioma and they were selected for further study.

Non-POU Domain-Containing Octomer-Binding (NONO) protein expression and stability promotes the tumorigenicity and activation of Akt/MAPK/ß-catenin pathways in human breast cancer cells.

Breast cancer is one of the most common cancers with a high mortality rate, underscoring the need to identify new therapeutic targets. Here we report that non-POU domain-containing octamer-binding (NONO) protein is overexpressed in breast cancer and validated the interaction of the WW domain of PIN1 with c-terminal threonine-proline (thr-pro) motifs of NONO. The interaction of NONO with PIN1 increases the stability of NONO by inhibiting its proteasomal degradation, and this identifies PIN1 as a positive regulator of NONO in promoting breast tumor development. Functionally, silencing of NONO inhibits the growth, survival, migration, invasion, epithelial to mesenchymal transition (EMT), and stemness of breast cancer cells in vitro. A human metastatic breast cancer cell xenograft was established in transparent zebrafish (Danio rerio) embryos to study the metastatic inability of NONO-silenced breast cancer cells in vivo. Mechanistically, NONO depletion promotes the expression of the PDL1 cell-surface protein in breast cancer cells. The identification of novel interactions of NONO with c-Jun and ß-catenin proteins and activation of the Akt/MAPK/ß-catenin signaling suggests that NONO is a novel regulator of Akt/MAPK/ß-catenin signaling pathways. Taken together, our results indicated an essential role of NONO in the tumorigenicity of breast cancer and could be a potential target for anti-cancerous drugs. Video Abstract.

Underrecognition of sleep-disordered breathing and other common health conditions in the West Virginia Medicaid population: a driver of poor health outcomes.

STUDY OBJECTIVES: To assess the prevalence rates of sleep-disordered breathing (SDB) in a high-risk and rural-dwelling Medicaid population with significant comorbidities. METHODS: Our study analyzed anonymized administrative claims data from West Virginia (WV) Medicaid. Claims data from 2019 were aggregated at the individual level to assess the overall prevalence of SDB and related conditions among adult Medicaid beneficiaries. The prevalence rate of SDB, specifically among individuals who had comorbid congestive heart failure, chronic obstructive pulmonary disease, or obesity, was determined. Finally, we compared our prevalence estimates from this Medicaid database with prevalence rates from national datasets including the Centers for Disease Control and Prevention's Behavioral Risk Factor Surveillance System. RESULTS: Of the total 413,757 Medicaid enrollees ≥ 18 years old analyzed, 36,433 had a diagnosis code of SDB for an overall prevalence of 8.8%. Based on national datasets and our study cohort characteristics, we conservatively estimated the prevalence of SDB in this WV Medicaid population to be 25%. For our secondary analyses, we determined the prevalence of SDB in specific disease cohorts of congestive heart failure (SDB prevalence 45%), chronic obstructive pulmonary disease (SDB prevalence 27%), and obesity (SDB prevalence 14%). CONCLUSIONS: Our analysis of WV Medicaid claims data indicates that SDB and other important medical conditions are underrecognized in this vulnerable, high-risk, primarily rural population. Interestingly, SDB was identified at high rates in the disease cohorts of interest. Our team believes SDB represents an ideal target/model for addressing the growing health disparities in the United States, which is a major concern for all stakeholders in health care. CITATION: Stansbury R, Strollo P, Pauly N, et al. Underrecognition of sleep-disordered breathing and other common health conditions in the West Virginia Medicaid population: a driver of poor health outcomes. J Clin Sleep Med. 2022;18(3):817-824.

p73 - NAV3 axis plays a critical role in suppression of colon cancer metastasis.

p73 is a member of the p53 tumor suppressor family, which transactivates p53-responsive genes and mediates DNA damage response. Recent evidences suggest that p73 exerts its tumor suppressor functions by suppressing metastasis, but the exact mechanism remains unknown. Here, we identify Navigator-3 (NAV3), a microtubule-binding protein, as a novel transcriptional target of p73, which gets upregulated by DNA damage in a p73-dependent manner and plays a vital role in p73-mediated inhibition of cancer cell invasion, migration, and metastasis. Induction of p73 in response to DNA damage leads to rapid increase in endogenous NAV3 mRNA and protein levels. Through bioinformatic analysis, we identified two p73-binding sites in NAV3 promoter. Consistent with this, p73 binding to NAV3 promoter was confirmed through luciferase, Chromatin Immunoprecipitation, and site-directed mutagenesis assays. Abrogation of NAV3 and p73 expression significantly increased the invasion and migration rate of colorectal cancer cells as confirmed by wound-healing, cell invasion, and cell migration assays. Also, knockdown of NAV3 decreased the expression of E-cadherin and increased the expression of other prominent mesenchymal markers such as N-cadherin, Snail, Vimentin, and Fibronectin. Immunohistochemistry analysis revealed the downregulation of both NAV3 and p73 expression in metastatic colon cancer tissues as compared to non-metastatic cancer tissues. Additionally, the expression pattern of NAV3 and p73 showed extensively significant correlation in both non-metastatic and metastatic human colon cancer tissue samples. Taken together, our study provide conclusive evidence that Navigator-3 is a direct transcriptional target of p73 and plays crucial role in response to genotoxic stress in p73-mediated inhibition of cancer cell invasion, migration, and metastasis.

Epigenetic deregulations of Wnt/ß-catenin and transforming growth factor beta-Smad pathways in esophageal cancer: Outcome of DNA methylation.

BACKGROUND: Promoter methylation of tumor suppressor genes (TSGs) is a well-reported portent in carcinogenesis; hence, it is worthy to investigate this in high-risk Northeast population of India. The study was designed to investigate methylation status of 94 TSGs in esophageal squamous cell carcinoma (ESCC). Further, the effect of OPCML promoter methylation on gene expression was analyzed by immunohistochemistry. Moreover, in silico protein-protein interactions were examined among 8 TSGs identified in the present study and 23 epigenetically regulated genes reported previously by our group in ESCC. MATERIALS AND METHODS: Methylation profiling was carried out by polymerase chain reaction array and OPCML protein expression was examined by tissue microarray-based immunohistochemistry. RESULTS: OPCML, NEUROG1, TERT, and WT1 genes were found hypermethylated and SCGB3A1, CDH1, THBS1, and VEGFA were hypomethylated in Grade 2 tumor. No significant change in OPCML expression was observed among control, Grade 1, and Grade 2 tumor. Conclusively, hypermethylation of the studied OPCML promoter in Grade 2 tumor produced no effect on expression. Unexpectedly, OPCML expression was downregulated in Grade 3 tumor in comparison to other groups signifying that downregulation of OPCML expression may lead to higher grade of tumor formation at the time of diagnosis of ESCC in patients. Significant interactions at protein level were found as VEGFA:PTK2, CTNNB1:CDH1, CTNNB1:VEGFA, CTNNB1:NEUROG1, CTNND2:CDH1, and CTNNB1:TERT. These interactions are pertinent to Wnt/ß-catenin and TGF-ß-Smad pathways. CONCLUSIONS: Deranged OPCML expression may lead to high-grade ESCC as well as epigenetically regulated genes, that is, CDH1, CTNNB1, CTNND2, THBS1, PTK2, WT1, OPCML, TGFB1, and SMAD4 may alter the Wnt/ß-catenin and TGF-ß-Smad pathways in ESCC. Further study of these genes could be useful to understand the molecular pathology of ESCC with respect to epithelial-mesenchymal transition (EMT) mediated by Wnt/ß-catenin and TGF-ß signaling pathways.

IL-8/CXCR1/2 signalling promotes tumor cell proliferation, invasion and vascular mimicry in glioblastoma.

BACKGROUND: Glioblastoma multiforme (GBM) is one of the lethal malignant tumors of the central nervous system. Despite advances made in understanding this complex disease, little has been achieved in improving clinical efficacy towards it. Factors such as chemokines play important role in shaping the tumor microenvironment which in turn plays a significant role in deciding course of tumor progression. In this study, we investigated the role of chemokine IL-8 in glioblastoma progression with particular emphasis on immunomodulation, cellular proliferation, invasion and vascular mimicry. METHODS: Role of IL-8 in GBM immunology was determined by correlating the expression of IL-8 by immunohistochemistry with other immune cell markers such as CD3 and CD68. Effect of high IL-8 expression on overall survival, the difference in expression level between different GBM subgroups and anatomic structures were analyzed using other databases. Two GBM cell lines -U-87MG and LN-18 were used to study the impact of targeting IL-8-CXCR1/2 signalling using neutralizing antibodies and pharmacological antagonist. Reverse transcriptase-polymerase chain reaction and immunocytochemistry were used to determine the expression of these axes. Impact on cell viability and proliferation was assessed by MTT, proliferation marker-ki-67 and clonogenic survival assays. Multicellular tumor spheroids generated from GBM cell lines were used to study invasion in matrigel. RESULTS: Weak Positive correlation was observed between IL-8 and CD3 as well as between IL-8 and CD68. High IL-8 expression in GBM patients was found to be associated with dismal survival. No significant difference in IL-8 expression between different molecular subgroups of GBM was observed. In vitro targeting of IL-8-CXCR1/2 signalling displayed a significant reduction in cell viability and proliferation, and spheroid invasion. Furthermore, the presence of CD34-/CXCR1+ vessels in GBM tissues showed the involvement of IL-8/CXCR1 in vascular mimicry structure formation. CONCLUSION: These results suggest a direct involvement of IL-8-CXCR1/2 axes in GBM progression by promoting both cell proliferation and invasion and indirectly by promoting neovascularization in the form of vascular mimicry.

Linear Regression Model to Identify the Factors Associated with Carbon Stock in Chure Forest of Nepal.

Use of woody plants for greenhouse gas mitigation has led to the demand for rapid cost-effective estimation of forest carbon stock and related factors. This study aims to assess the factors associated with carbon stock in Chure forest of Nepal. The data were obtained from Department of Forest Research and Survey (DFRS) of Nepal. A multiple linear regression model and then sum contrasts were used to observe the association between variables such as stem volume, diameter at breast height, altitude, districts, number of trees per plot, and ownership of the forest. 95% confidence interval (CI) plots were drawn for comparing the adjusted carbon stocks with each of the factors and with the overall carbon stock. The linear regression showed a good fit of the model (adjusted R2 = 83.75%) with the results that the stem volume (sv), diameter at breast height (dbh), and the number of trees per plot showed statistically significant (p value ≤ 0.05) positive association with carbon stock. The highest carbon stock was associated with sv more than 199 m3/ha, average dbh more than 43.3 cm/plot, and number of trees more than 20/plot, whereas the altitude, geographical location, and ownership had no statistical associations at all. The results can be of use to the government for enhancing carbon stock in Chure that supports both natural resource conservation and United Nations-Reducing Emission from Deforestation and Forest Degradation program to mitigate carbon emission issues.

Gene Expression Profiling of Chemokines and Their Receptors in Low and High Grade Astrocytoma

Background: Despite intense interest in molecular characterization and searches for novel therapeutic targets, the glioblastoma remains a formidable clinical challenge. Among many contributors to gliomagenesis, chemokines have drawn special attention due to their involvement in a plethora of biological processes and pathological conditions. In the present study we aimed to elucidate any pro-gliomagenic chemokine axis and probable roles in development of glioblastoma multiforme (GBM). Method: An array of 84 chemokines, chemokine receptors and related genes were studied by real time PCR with comparison between low grade astrocytoma (diffuse astrocytoma ­ grade II) and high grade astrocytoma (glioblastoma multiforme ­ grade IV). Gene ontology analysis and database mining were performed to funnel down the important axis in GBM followed by validation at the protein level by immunohistochemistry on tissue microarrays. Results: Gene expression and gene ontology analysis identified CXCL8 as an important chemokine which was more frequently up-regulated in GBM as compared to diffuse astrocytoma. Further we demonstrated localization of CXCL8 and its receptors in glioblastoma possibly affecting autocrine and paracrine signalling that promotes tumor cell proliferation and neovascularisation with vascular mimicry. Conclusion: From these results CXCL8 appears to be an important gliomagenic chemokine which may be involved in GBM growth by promoting tumor cell proliferation and neovascularization via vascular mimicry. Further in vitro and in vivo investigations are required to explore its potential candidature in anti-GBM therapy.

Immunohistochemical expression of chemokine receptor CXCR3 and its ligand CXCL10 in low-grade astrocytomas and glioblastoma multiforme: A tissue microarray-based comparison.

UNLABELLED: Glioblastoma multiforme (GBM) and diffuse astrocytoma (DA) are the most frequently encountered gliomas. Due to poor prognosis and limited success of the currently available treatment modalities there is a need to identify new therapeutic targets. Chemokines (CKs) regulate cellular functions like chemotaxis, angiogenesis, apoptosis, and cell cycle progression that play role in tumor growth. OBJECTIVE: To study comparative immunoexpression of CXCR3 and CXCL10 in DA and GBM using a high-throughput tissue microarray (TMA). MATERIALS AND METHODS: A TMA of 1.0 mm core diameter was made from formalin-fixed, paraffin-embedded donor blocks of 25 pilocytic astrocytomas (PA), 45 DA, and 75 GBM. Immunohistochemical staining for CXCR3 and CXCL10 was performed. RESULTS: Out of 145, 129 cores were suitable for immunohistochemical evaluation after processing and immunohistochemistry. Strong CXCR3 immunoexpression was observed in 72.7% cases of GBM as compared to 31.8% cases of DA. 50.7% of GBM and 24.5% of DA showed strong immunoexpression of CXCL10. Overall comparisons between DA and GBM for CXCR3 and CXCL10 showed statistically significant correlation between the two with P < 0.001 and P = 0.02, respectively. A positive correlation was observed between CXCR3 and MIB-1. Pearson's correlation coefficient r = 0.548 and 0.330 for DA and GBM, respectively with P < 0.01. CONCLUSION: GBM shows overexpression of CXCR3 and CXCL10 in comparison to DA, indicating that they play an important role in tumor growth and progression. Inhibition of this receptor-ligand axis may be a potential therapeutic target for arresting tumor growth and development of a glioblastoma.

Is CXCL10/CXCR3 axis overexpression a better indicator of leprosy type 1 reaction than inducible nitric oxide synthase?

BACKGROUND & OBJECTIVES: Leprosy type 1 reactions (T1R) are acute episodes of immune exacerbation that are a major cause of inflammation and nerve damage. T1R are diagnosed clinically and supported by histopathology. No laboratory marker is currently available that can accurately predict a T1R. Increased plasma and tissue expression of inducible nitric oxide synthase (i-NOS) and chemokine CXCL10 have been demonstrated in T1R. We studied the gene expression and immunoexpression of i-NOS, CXCL10 and its receptor CXCR3 in clinically and histopathologically confirmed patients with T1R and compared with non-reactional leprosy patients to understand which biomarker has better potential in distinguishing reaction from non-reaction. METHODS: Gene expression of i-NOS, CXCL10 and CXCR3 was studied in 30 skin biopsies obtained from patients with borderline tuberculoid (BT), mid-borderline (BB) and borderline lepromatous (BL) leprosy with and without T1R by real-time PCR. Further validation was done by immunohistochemical expression on 60 borderline leprosy biopsies with and without T1R. RESULTS: Of the 120 patients histopathological evaluation confirmed T1R in 65 (54.2%) patients. CXCR3 gene expression was significantly (P<0.05) higher in BT- and BB-T1R patients compared to those without T1R. The CXCL10 gene expression was significantly higher (P<0.05) in BB leprosy with T1R but the difference was not significant in patients with BT with or without T1R. Immunoexpression for CXCR3 was significant in both BB-T1R and BB (P<0.001) and BT and BT-T1R (P<0.001). Immunoexpression of CXL10 was significant only in differentiating BB from BB-T1R leprosy (P<0.01) and not the BT cases. i-NOS immunoexpression was not useful in differentiating reactional from non-reactional leprosy. INTERPRETATION & CONCLUSIONS: Both CXCL10 and CXCR3 appeared to be useful in differentiating T1R reaction in borderline leprosy while CXCR3 alone differentiated BT from BT-T1R. CXCR3 may be a potentially useful immunohistochemical marker to predict an impending T1R.

Immunohistochemical expression of IDH1 in gliomas: a tissue microarray-based approach.

BACKGROUND: Mutations in the gene encoding isocitrate dehydrogenase (IDH1) have been reported in gliomas. This study analyses a series of 184 glioma cases in a tissue microarray (TMA)-based approach to assess the frequency of R132H point mutations in formalin-fixed, paraffin-embedded tissue samples. MATERIALS AND METHODS: A total of 195 gliomas (30 pilocytic astrocytoma (PA), 45 diffuse astrocytoma [DA], 75 glioblastoma multiforme [GBM], 25 oligodendroglioma [OLIG] and 20 ependymoma [EPEN]). A TMA of core size 1.0 mm was constructed using a semi-automatic tissue arrayer. Immunohistochemical staining for IDH1, p53 and EGFR proteins was performed by the labeled sterptavidin avidin biotin LSAB method. RESULTS: The frequency of mutant IDH1 detection by immunohistochemistry on formalin-fixed, paraffin-embedded tissue was 15.8% in 29/184 tumors found suitable for evaluation. DA, OLIG and GBM showed IDH1 expression in 17/40 (42.5%), 5/22 (22.7%) and 7/72 (9.7%) cases, respectively. Of all the GBMs, prim-GBM showed immunoexpression in 1/7 (1.5%) while sec-GBM showed IDH1 expression in 6/7 (85.7%). PA and EPEN did not react with anti-IDH1 antibody. DA and GBM showed positive correlation with p53, but IDH1 and EGFR coexpression was rare. CONCLUSION: Monoclonal antibody to IDH1 (R132) is a useful and less-labor-intensive method to detect mutations in gliomas. IDH1 is a useful immunohistochemical marker to differentiate reactive gliosis from low-grade astrocytoma, has potential as an independent prognostic marker and also helps in distinguishing primary from secondary GBM. Its sensitivity and specificity need to be assessed by simultaneous sequencing and its validation on clinically annotated samples.