Investigating the Effects of HMGB1 Overexpression on Colorectal Cancer Cell Migration via Oncolytic Herpes simplex Virus Type 1 (oHSV-1).

Background: Colorectal Cancer (CRC) represents a significant global health challenge, and its progression, resistance to therapy, and metastasis are strongly influenced by the tumor microenvironment, including factors like hypoxia. This study explores the impact of High Mobility Group Box 1 (HMGB1) overexpression on CRC cell migration, while identifying potential genes associated with this process. Methods: To explore this, we developed oncolytic virotherapy, resulting in HSVHMGB1, an oncolytic Herpes simplex virus that expresses HMGB1. HMGB1 is known its role in cancer progression, particularly in the context of cancer cell migration. Results: Contrary to expectations, our scratch assays indicated that HSV-HMGB1 did not significantly induce migration in CRC cells, suggesting that HMGB1 might not directly contribute to this process. Employing microarray analysis, we investigated gene expression changes linked to CRC cell migration, leading to construction of a Protein-Protein Interaction (PPI) network. This network revealed the presence of hub proteins, including as NDRG1, LGALS1, and ANGPTL4, which are recognized for their roles in cancer cell migration. The differential expression of these genes under hypoxic conditions was further validated using quantitative RT-PCR, aligning with the findings from our microarray data. Conclusion: Our findings emphasize the complex regulation of CRC cell migration, and provides valuable insights into potential molecular mechanisms and pathways. These findings have implications for further research into cancer progression and the development of therapeutic strategies.

In silico Identification of Hypoxic Signature followed by reverse transcription-quantitative PCR Validation in Cancer Cell Lines

Background: Hypoxic tumor microenvironment is one of the important impediments for conventional cancer therapy. This study aimed to computationally identify hypoxia-related messenger RNA (mRNA) signatures in nine hypoxic-conditioned cancer cell lines and investigate their role during hypoxia. Methods: Nine RNA sequencing (RNA-Seq) expression data sets were retrieved from the Gene Expression Omnibus database. Differentially expressed genes (DEGs) were identified in each cancer cell line. Then 23 common DEGs were selected by comparing the gene lists across the nine cancer cell lines. Reverse transcription-quantitative PCR (qRT-PCR) was performed to validate the identified DEGs. Results: By comparing the data sets, GAPDH, LRP1, ALDOA, EFEMP2, PLOD2, CA9, EGLN3, HK, PDK1, KDM3A, UBC, and P4HA1 were identified as hub genes. In addition, miR-335-5p, miR-122-5p, miR-6807-5p, miR-1915-3p, miR-6764-5p, miR-92-3p, miR-23b-3p, miR-615-3p, miR-124-3p, miR-484, and miR-455-3p were determined as common micro RNAs. Four DEGs were selected for mRNA expression validation in cancer cells under normoxic and hypoxic conditions with qRT-PCR. The results also showed that the expression levels determined by qRT-PCR were consistent with RNA-Seq data. Conclusion: The identified protein-protein interaction network of common DEGs could serve as potential hypoxia biomarkers and might be helpful for improving therapeutic strategies.

Modifying oncolytic virotherapy to overcome the barrier of the hypoxic tumor microenvironment. Where do we stand?

Viruses are completely dependent on host cell machinery for their reproduction. As a result, factors that influence the state of cells, such as signaling pathways and gene expression, could determine the outcome of viral pathogenicity. One of the important factors influencing cells or the outcome of viral infection is the level of oxygen. Recently, oncolytic virotherapy has attracted attention as a promising approach to improving cancer treatment. However, it was shown that tumor cells are mostly less oxygenated compared with their normal counterparts, which might affect the outcome of oncolytic virotherapy. Therefore, knowing how oncolytic viruses could cope with stressful environments, particularly hypoxic environments, might be essential for improving oncolytic virotherapy.

Cell type-specific response of colon cancer tumor cell lines to oncolytic HSV-1 virotherapy in hypoxia.

BACKGROUND: Novel strategies are required since the hypoxic tumor microenvironment is one of the important impediments for conventional cancer therapy. High mobility group box 1 (HMGB1) protein can block aerobic respiration in cancer cells. We hypothesized that HMGB1could also kill the colorectal cancer cells during hypoxia. METHODS: In this study, we developed oncolytic herpes simplex virus type 1 expressing HMGB1 protein (HSV-HMGB1) and investigated the cytotoxic effect of HSV-HMGB1 and its parental virus (HSV-ble) on three colorectal cancer cells (HCT116, SW480, and HT29) under normoxic (20% oxygen) and hypoxic (1% oxygen) conditions. We further identified potential autophagy- related genes in HT29 cells by retrieving mRNA expression microarray datasets from the Gene Expression Omnibus database. These genes were then detected in HT29 cells infected with HSV-HMGB1 and HSV-ble during normoxia and hypoxia by Real-Time quantitative PCR (qRT-PCR). RESULTS: The cytotoxic effect of HSV-HMGB1 was significantly higher than that of HSV-ble during normoxia; however, during hypoxia, HSV-HMGB1 enhanced the viability of HT29 cells at MOI 0.1. Analyzing the cell death pathway revealed that HSV-HMGB1 induced autophagy in HT29 cells under hypoxic conditions. CONCLUSION: In conclusion, it appears that oncolytic virotherapy is cell context-dependent. Therefore, understanding the cancer cells' characteristics, microenvironment, and cell signaling are essential to improve the therapeutic strategies.

Cytotoxic effect of dual fluorescent-labeled oncolytic herpes simplex virus type 1 on mouse tumorigenic cell lines.

The increasing incidences of cancer at the global scale have recently resulted in the invention of various biotechnology approaches among which the oncolytic virotherapy is a new strategy for the treatment of multiple tumors. Herpes simplex virus (HSV) based vectors are one of the most studied oncolytic agents, worldwide. Moreover, syngeneic animal models are the principal parts of the oncolytic virotherapies investigation. The effects of a dual fluorescent γ34.5 deleted vector-HSV-GR- on three mouse tumor cell lines were studied in this work. We previously generated a dual fluorescent labeled oncolytic HSV-HSV-GR- (both copies of γ34.5 were inactivated by insertion of two distinct fluorescent dyes, GFP and mCherry) in our laboratory; subsequently, they were used as oncolytic viruses. The three 4T1, TC-1, and CT26 cell lines were infected with HSV-GR. The infection efficacy and the elimination potency of HSV-GR were analyzed by photomicrography and flow cytometry methods. HSV-GR showed a significant efficiency to infect the cell lines examined. Flow cytometry analyses demonstrated that HSV-GR infected 89.3%, 86.1%, and 92.4% of 4T1, TC-1, and CT26 cells, respectively. Moreover, propidium iodide (PI) staining of infected cells indicated that HSV-GR could kill 27.9%, 21.2%, and 21.3% of 4T1, TC-1, and CT26 cells, respectively. Interestingly, HSV-GR infected cells were capable of expressing both GFP and mCherry at the same time. The promising effects of the oncolytic virus HSV-GR in the mouse syngeneic tumor cell system have shed more light on the therapeutic potential of this anti-cancer approach.

Detection of ESBL- and AmpC-producing E. coli isolates from urinary tract infections.

BACKGROUND: Extended-spectrum ß-lactamases (ESBLs) and AmpC enzymes have been observed in virtually all species of the family Enterobacteriaceae. The ß-lactamase producing bacteria cause many serious infections, including urinary tract infections. These enzymes are predominantly plasmid mediated. There are no recommended guidelines for detection of this resistance mechanism and there is a need to address this issue as much as the detection of ESBLs. This study was undertaken to characterize ESBL and AmpC producers among Escherichia coli by polymerase chain reaction (PCR), which were initially screened by phenotypic method. MATERIALS AND METHODS: A total of 90 isolates of E. coli were recovered from the urinary tract during a 7-month period, and were screened for ESBLs and AmpC production by disk diffusion test using cefoxitin (30 µg) disks and confirmed by combined disk diffusion test using phenyl boronic acid. The presence of genes encoding CIT, FOX, and TEM was detected by PCR. RESULTS: On disk diffusion test, 59 of 90 isolates were resistant to third generation of cephalosporins; of these 37 (62.7%) and 3 (5%) were ESBL and AmpC producers, respectively. PCR showed that 29 (49.1%) and 3 (5%) were positive for blaT EM and bla CMY-2, respectively. CONCLUSION: ESBL- and AmpC-producing E. coli isolates cause significant resistance to cephalosporin. There is a need for a correct and reliable phenotypic test to identify AmpC ß-lactamases and to discriminate between AmpC and ESBL producers. This work showed that boronic acid can differentiate ESBL enzymes from AmpC enzymes.

Crimean-Congo Hemorrhagic Fever.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a member of the Bunyaviridae family and Nairovirus genus. The viral genome consists of 3 RNA segments of 12 kb (L), 6.8 kb (M), and 3 kb (S). Crimean-Congo hemorrhagic fever (CCHF) is the most widespread tickborne viral infection worldwide: it has been reported in many regions of Africa, the Middle East, and Asia. The geographical distribution of CCHFV corresponds most closely with the distribution of members of the tick genera, and Hyalomma ticks are the principal source of human infection. In contrast to human infection, CCHFV infection is asymptomatic in all species. Treatment options for CCHF are limited; immunotherapy and ribavirin are effective in the treatment of CCHF; the efficacy of ribavirin in the treatment of CCHF has not yet been proven. This article reviews the history, epidemiology, clinical symptoms, pathogenesis, diagnosis, and treatment of CCHFV, as well as the development of a vaccine against it.

Prevalence and molecular characterization of AmpC-producing clinical isolates of Escherichia coli from southeastern Iran.

BACKGROUND AND OBJECTIVE: AmpC in Escherichia coli is noninducible but is regulated by promoter and attenuator mechanisms and can be expressed at high levels as a result of a mutation. This study was undertaken to characterize the AmpC hyperproducing clinical isolates of E. coli. METHODS: E. coli isolates recovered from three major hospitals in Zahedan, South Eastern Iran, were selected on the basis of resistance phenotype to the third-generation cephalosporins and cefoxitin. Phenyl boronic acid as an inhibitor and cefoxitin were used to confirm the overexpression of AmpC. The presence of genes encoding ACC, FOX, MOX, DHA, CIT, and EBC was detected using multiplex PCR. The existence of mutations in the regulatory region of the chromosomal ampC gene was assessed using PCR and sequencing. RESULTS: Thirteen of 392 E. coli isolates were selected as high-level AmpC producers. Eleven of the 13 isolates contained the blaCMY-2 gene; 12 of the 13 AmpC hyperproducing strains harbored changes in the promoter/attenuator region, which could explain the increased expression of the chromosome-encoded AmpC enzyme. In 10 of the 13 strains, we found both chromosomal- and plasmid-mediated mechanisms responsible for AmpC production. INTERPRETATION AND CONCLUSIONS: AmpC hyperproducing E. coli isolates exhibit significant resistance to cephalosporins. This work showed that strains hyperproducing chromosomal AmpC could be as frequent as strains with plasmid-mediated AmpC hyperproduction.