Bioinformatics approach combined with experimental verification reveals OAS3 gene implicated in paclitaxel resistance in head and neck cancer.

BACKGROUND: This study aimed to identify a candidate gene associated with paclitaxel (PTX) resistance and to evaluate functionally its biological role in the PTX-resistant head and neck squamous cell carcinoma (HNSCC) cell lines and clinical specimens. METHODS: Microarray data series containing samples of different types of cancers resistant to PTX were analyzed and then a candidate gene associated with PTX resistance was identified using various bioinformatics tools. After the suppression of the target gene expression, changes in cell viability and colony-forming ability were evaluated in PTX-resistant FaDu and SCC-9 cell lines. RESULTS: Bioinformatics analyses of upregulated genes in PTX-resistant cancer cells indicated that OAS3 was associated with PTX resistance. The downregulation of OAS3 expression significantly reduced the viability and colony-forming capacity of PTX-resistant SCC-9 cells by inducing apoptosis and cell cycle arrest at G0/G1 phase. CONCLUSIONS: The therapeutic targeting of OAS3 may resensitize PTX-resistant HNSCC cells with high OAS3 expression to PTX treatment.

Process development for an effective COVID-19 vaccine candidate harboring recombinant SARS-CoV-2 delta plus receptor binding domain produced by Pichia pastoris.

Recombinant protein-based SARS-CoV-2 vaccines are needed to fill the vaccine equity gap. Because protein-subunit based vaccines are easier and cheaper to produce and do not require special storage/transportation conditions, they are suitable for low-/middle-income countries. Here, we report our vaccine development studies with the receptor binding domain of the SARS-CoV-2 Delta Plus strain (RBD-DP) which caused increased hospitalizations compared to other variants. First, we expressed RBD-DP in the Pichia pastoris yeast system and upscaled it to a 5-L fermenter for production. After three-step purification, we obtained RBD-DP with > 95% purity from a protein yield of > 1 g/L of supernatant. Several biophysical and biochemical characterizations were performed to confirm its identity, stability, and functionality. Then, it was formulated in different contents with Alum and CpG for mice immunization. After three doses of immunization, IgG titers from sera reached to > 106 and most importantly it showed high T-cell responses which are required for an effective vaccine to prevent severe COVID-19 disease. A live neutralization test was performed with both the Wuhan strain (B.1.1.7) and Delta strain (B.1.617.2) and it showed high neutralization antibody content for both strains. A challenge study with SARS-CoV-2 infected K18-hACE2 transgenic mice showed good immunoprotective activity with no viruses in the lungs and no lung inflammation for all immunized mice.

Identification of upregulated genes in glioblastoma and glioblastoma cancer stem cells using bioinformatics analysis.

Glioblastoma (GBM) is the most common malignant brain tumor among adults. Cancer stem cells (CSCs) are known to drive treatment resistance and recurrence. However, a few CSC markers have been identified as therapeutic targets for GBM. This study aimed to show highly coexpressed genes in GBM CSCs and TCGA GBM samples and to identify possible therapeutic targets for GBM. The gene expression profiles of GBM CSCs were obtained from Gene Expression Omnibus database. After the differentially upregulated genes were screened, functional enrichment analyses were performed using DAVID and Reactome databases. For upregulated genes, biological processes were mainly associated with the regulation of transcription. Subsequently, a protein-protein interaction network was constructed for upregulated genes through STRING, in which DUSP6, FGFR3, EGFR, SOX2, NES, and PLP1 were further identified as hub genes via MCC and MNC methods. Expression profiles of hub genes and their association with survival were examined in TCGA GBM dataset using GEPIA2 platform. The expression levels of four hub genes were found to be increased in TCGA GBM samples. Of these, DUSP6 and SOX2 had prognostic value for patients with GBM. Molecular compounds targeting DUSP6 were searched through PubChem database. (E/Z)-BCI and BCI were found to be inhibitors of DUSP6. The molecular docking was performed using Autodock vina 1.02. The compounds showed strong binding capacities by forming various interactions with the ERK2 binding domain of DUSP6. Hence, the current study unravels the potential of (E/Z)-BCI and BCI compounds as possible anti-cancer molecules for GBM treatment.

Bioinformatics analysis of recurrent deletion regions in neuroblastoma.

Neuroblastoma (NB) is the most common extra-cranial solid tumor in childhood. Very few genes in recurrent deletion regions have been identified as tumor suppressors for NB, and interactions among proteins encoded by genes in these regions have been overlooked. This study aims to identify possible tumor suppressor genes located within regions commonly deleted in NB tumors and to show possible interaction network of proteins encoded by genes in these regions by bioinformatics analysis. The genes localized in the recurrent deletion regions were identified using the Ensembl BioMart web-tool. GSE1824 microarray dataset was analyzed to determine downregulated differently expressed genes (dDEGs) selected from deletion regions using Orange Canvas software. The DAVID v6.8 tool and Reactome database were used to perform gene ontology and pathway enrichment analysis, respectively. The protein-protein interaction (PPI) network and sub-module analysis were conducted by STRING database and Cytoscape plugin MCODE software, respectively. Copy number variation status and mutations of hub genes were examined in TARGET neuroblastoma dataset using UCSC Xena platform. Biological processes of genes were specific to chromosomes. The 219 genes selected from these regions were found to be downregulated. A PPI network was constructed for dDEGs. Copy number losses and mutations were observed for hub genes. Hub genes identified in the current study may act as tumor suppressors in NB tumorigenesis. Disruption of protein-protein interaction network consisting of proteins encoded by genes on various recurrent deletion regions may give rise to NB by interrupting gene regulatory network orchestrating neural crest formation.

AZD4547 targets the FGFR/Akt/SOX2 axis to overcome paclitaxel resistance in head and neck cancer.

PURPOSE: Development of chemoresistance is one of the major obstacles to the treatment of head and neck squamous cell carcinoma (HNSCC). The PI3K/Akt pathway, involved in drug resistance, has been found to be overactivated in > 90% of HNSCCs. Aberrant activation of the FGF receptors (FGFRs) has been reported to cause overactivation of the PI3K/Akt pathway and to be associated with the maintenance of stem cell features, which is controlled via SOX2 expression. In this study, we aimed at investigating the potential of using AZD4547, an orally bioavailable FGFR inhibitor, to overcome taxol-resistance by targeting the FGFR/Akt/SOX2 axis in HNSCC. METHODS: We initially evaluated FGFR2 and SOX2 expression using in silico tools. We analyzed the FGFR/Akt/SOX2 axis in normal/tumor tissue pairs and in recombinant FGF2 treated HNSCC cells. Next, we explored the effects of AZD4547 alone and in combination with taxol on the proliferation, migration and colony forming capacities of parental/taxol-resistant cells using in vitro models. RESULTS: We found that the p-FGFR, p-AKT, p-GSK-3ß and SOX2 expression levels were higher in tumor tissues than in its corresponding normal tissues, and that AZD4547 effectively suppressed the expression of FGFR and its downstream targets in recombinant FGF2 treated HNSCC cells. We also found that AZD4547 diminished the viability, migration and colony forming capacity of HNSCC cells, and that co-treatment with taxol potentiated the impact of taxol on these cells. Finally, we found that AZD4547 inhibited the overexpressed FGFR/Akt/SOX2 axis and profoundly suppressed cancer-related phenotypes in taxol-resistant HNSCC cells. CONCLUSION: From our data we conclude that AZD4547 may increase the impact of taxol during HNSCC treatment. We suggest AZD4547 as a therapeutic agent to overcome taxol-resistance.

Combination of resveratrol and BIBR1532 inhibits proliferation of colon cancer cells by repressing expression of LncRNAs.

Colorectal cancer (CRC) is the third most common cancer worldwide. The development of tumor drug resistance is observed in the treatment of CRC. Combinations of anticancer agents are attracting considerable interest in order to overcome drug resistance in CRC. This study aims to investigate the effect of resveratrol and BIBR1532, either alone or in combination, on the cell viability as well as on expression of long non-coding RNAs (LncRNAs) for HT-29 colon adenocarcinoma cells. The cytotoxic effects of resveratrol and BIBR1532 on HT-29 cells were determined using WST-1 test. Flow cytometry was used to determine apoptotic cell death after treatments. Real-Time PCR was used to identify expression of LncRNAs after treatments. LncExpDB and GEPIA2 were used to evaluate expression profiles of LncRNAs, whose expression levels were decreased in HT-29 cells after treatments, in normal tissues and colon adenocarcinoma tumors. IC50 concentrations of BIBR1532 and resveratrol were found to be 50.81 µM at 48 h and 86.23 µM at 72 h, respectively. Combination index value was 1.07617. BIBR1532, resveratrol, or their combination reduced the cell viability of HT-29 cells. CCAT1, CRNDE, HOTAIR, PCAT1, PVT1, SNHG16 were down-regulated after treatments. In silico analysis revealed that LncRNAs whose expression levels were decreased after treatments were associated with CRC. Resveratrol, BIBR1532, or their combination may have anti-proliferative effect on colorectal cancer cells through repressing expression of LncRNAs that are involved in progression of CRC.

Temozolomide treatment combined with AZD3463 shows synergistic effect in glioblastoma cells.

Temozolomide (TMZ) is used in the standard therapy regimen for patients with glioblastoma (GBM). However, some GBM patients do not respond to TMZ therapy. The combining therapeutic agents in GBM treatment are attracting considerable interest due to TMZ resistance. This study aims to identify the combinatorial effect of TMZ and AZD3463 on the viability of the T98G GBM cells. The cytotoxic effects of compounds were determined by using WST-8 assay. Flow cytometry was used to determine apoptosis and cell cycle profiles after treatments. Real-time PCR was used to identify mRNA expression of genes in the PI3K/AKT signaling pathway after treatments. IC50 concentrations of TMZ and AZD3463 were found to be 1.54 mM and 529 nM after incubation for 48 h, respectively. The combination treatment showed a synergistic effect on reducing the viability of GBM cells. Each one of TMZ, AZD3463, and combination treatments induced apoptosis. Treatments, either alone or the combination of these agents, caused the cell cycle arrest in distinct phases. TMZ and AZD3463 treatments, either alone or in combination, downregulated mRNA expression of genes in the PI3K/AKT signaling pathway. The combination of TMZ with AZD3463 may increase the efficacy of single TMZ treatment in GBM cells due to decreased expression of genes in the PI3K/AKT signaling pathway that is responsible for drug resistance and intratumoral heterogeneity.

Alterations of cell cycle genes in cancer: unmasking the role of cancer stem cells.

The cell cycle is a complex and strictly controlled process, consisting of different phases. Cell cycle regulation depends on phase-specific transcriptions of cell cycle genes. The alterations of cell cycle genes can predispose normal cells to have a cancerous phenotype. Indeed, several mechanisms underlying the deregulation of the cell cycle have been identified in different types of cancer. Cancer stem cells (CSCs), a fraction of tumor cells, are selectively capable of initiating tumor development. However, the deregulation of the cell cycle progression in CSCs still remains incompletely understood. This review describes epigenetic alterations and aberrant transcriptional regulation of cell cycle genes in CSCs as well as cell cycle patterns of CSCs.

Genistein-induced mir-23b expression inhibits the growth of breast cancer cells.

AIM OF THE STUDY: Genistein, an isoflavonoid, plays roles in the inhibition of protein tyrosine kinase phosphorylation, induction of apoptosis, and cell differentiation in breast cancer. This study aims to induce cellular stress by exposing genistein to determine alterations of miRNA expression profiles in MCF-7 cells. MATERIAL AND METHODS: XTT assay and trypan blue dye exclusion assays were performed to examine the cytotoxic effects of genistein treatment. Expressions of miRNAs were quantified using Real-Time Online RT-PCR. RESULTS: The IC50 dose of genistein was 175 µM in MCF-7 cell, line and the cytotoxic effect of genistein was detected after 48 hours. miR-23b was found to be up-regulated 56.69 fold following the treatment of genistein. It was found that miR-23b was upregulated for MCF-7 breast cancer cells after genistein treatment. CONCLUSIONS: Up-regulated ex-expression of miR-23b might be a putative biomarker for use in the therapy of breast cancer patients. miR-23b up-regulation might be important in terms of response to genistein.

Regulation of URG4/URGCP and PPARα gene expressions after retinoic acid treatment in neuroblastoma cells.

Neuroblastoma (NB), originating from neural crest cells, is the most common extracranial tumor of childhood. Retinoic acid (RA) which is the biological active form of vitamin A regulates differentiation of NB cells, and RA derivatives have been used for NB treatment. PPARα (peroxisome proliferator-activated receptor) plays an important role in the oxidation of fatty acids, carcinogenesis, and differentiation. URG4/URGCP gene is a proto-oncogene and that overexpression of URG4/URGCP is associated with metastasis and tumor recurrence in osteosarcoma. It has been known that URG4/URGCP gene is an overexpressed gene in hepatocellular carcinoma and gastric cancers. This study aims to detect gene expression patterns of PPARα and URG4/URGCP genes in SH-SY5Y NB cell line after RA treatment. Expressions levels of PPARα and URG4/URGCP genes were analyzed after RA treatment for reducing differentiation in SH-SY5Y NB cell line. To induce differentiation, the cells were treated with 10 µM RA in the dark for 3-10 days. Gene expression of URG4/URGCP and PPARα genes were presented as the yield of polymerase chain reaction (PCR) products from target genes compared with the yield of PCR products from the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. SH-SY5Y cells possess small processes in an undifferentiated state, and after treatment with RA, the cells developed long neurites, resembling a neuronal phenotype. PPARα gene expression increased in RA-treated groups; URG4/URGCP gene expression decreased in SH-SY5Y cells after RA treatment compared with that in the control cells. NB cell differentiation might associate with PPARα and URG4/URGCP gene expression profile after RA treatment.

Assessment of genetic markers and glioblastoma stem-like cells in activation of dendritic cells.

Glioblastoma (GBM) is the most common and aggressive intraparenchymal primary brain tumor in adults. The principal reasons for the poor outcomes of GBM are the high rates of recurrence and resistance to chemotherapy. The aim of this study was to determine the role of tailored cellular therapy for GBM with a poor prognosis and compare the activity of dendritic cells (DCs) that have encountered GBM cells. Detecting the correlations between methylation and expression of MGMT and PTEN genes and GBM cancer stem cells (CSCs) markers after co-cultures with a mononuclear cell cocktail are also aims for this study. Allogenic umbilical cord blood (UCB)-derived DCs were labeled with the CD11a and CD123 for immature DCs, and CD80 and CD11c for mature DCs. CD34, CD45, and CD56 cells were isolated from allogenic UCB for using in DCs maturation. GBM CSCs were detected with CD133/1 and CD111 antibodies after co-culture studies. DC activation was carried out via GBM cells including CD133 and CD111 cells and a mononuclear cells cocktail including CD34, CD45, and CD56 natural killer cells. Real-time PCR was performed to detect the expression and promoter methylation status of PTEN and MGMT genes. The expression of CSCs markers was found in all GBM cases, and a statistically significant correlation was found among them after co-culture studies. The most pronounced affinity of DCs to GBM cells was observed at dilutions between 1/4 and 1/256 in co-cultures. There was a statistically significant correlation between cellularity and granularity ratios for CD123 and CD11c. PTEN and MGMT gene expression and methylation values were evaluated with respect to CSCs expression and no statistical significance was found. Activation of DCs might associate with CSCs and the mononuclear cells cocktail including CD34, CD45, and CD56 cells which were obtained from allogenic UCB.