Non-Detection of HCMV Total Genomic DNA in Human Glioma Cells Genome.

AIM: To demonstrate if the human cytomegalovirus (HCMV) genome, that is involved in the pathogenesis of gliomas, is part of the genomic DNA of glioma cells or not. MATERIAL AND METHODS: The study included U87MG glioblastoma cell culture and tumor samples from glioma patients. The genomic DNA of tumor samples and U87MG cells were extracted and real-time quantitative PCR was used to assess the presence of the human cytomegalovirus genomic DNA. RESULTS: Consequently, HCMV positivity was not detected in the tumor and cell line genomic DNA under the aforementioned experimental conditions. CONCLUSION: We found that the genomic DNA of all the samples was negative for HCMV genomic DNA. Thus, HCMV could not be detected in human glioma tumors and we put forward that HCMV genomic DNA was not incorporated into the genomic DNA of glioma cells. Thus, total viral DNA is not involved in the pathogenesis of glioma; however, small viral particles or specific genes might be incorporated into the genomic DNA of glioma cells, leading to cancer development. This prompts further studies for verification.

Tracing 2D Growth of Pancreatic Tumoroids Using the Combination of Image Processing Techniques and Mini-Opto Tomography Imaging System.

Objectives: In this study, we aimed to trace the 2D growth development of tumoroids produced with MIA PaCa-2 pancreatic cancer cells at different time points. Methods We cultured 3 different tumoroids with 0.5%, 0.8%, and 1.5% agarose concentrations and calculated the growth rate of the tumoroids with their images acquired at 9 imaging time points by mini-Opto tomography imaging system applying image processing techniques. We used the metrics contrast-to-noise ratio (CNR), peak signal-to-noise ratio (PSNR), and mean squared error (MSE) to analyze the distinguishability of the tumoroid structure from its surroundings, quantitatively. Additionally, we calculated the increase of the radius, the perimeter, and the area of 3 tumoroids over a time period. Results In the quantitative assessment, the bilateral and Gaussian filters gave the highest CNR values (ie, Gaussian filter: at each of 9 imaging time points in range of 1.715 to 15.142 for image set-1). The median filter gave the highest values in PSNR in the range of 43.108 to 47.904 for image set-2 and gave the lowest values in MSE in the range of 0.604 to 2.599 for image set-3. The areas of tumoroids with 0.5%, 0.8%, and 1.5% agarose concentrations were 1.014 mm2, 1.047 mm2, and 0.530 mm2 in the imaging time point-1 and 33.535 mm2, 4.538 mm2, and 2.017 mm2 in the imaging time point-9. The tumoroids with 0.5%, 0.8%, and 1.5% agarose concentrations grew up to times of 33.07, 4.33, and 3.80 in area size over this period, respectively. Conclusions The growth rate and the widest borders of the different tumoroids in a time interval could be detected automatically and successfully. This study that combines the image processing techniques with mini-Opto tomography imaging system ensured significant results in observing the tumoroid's growth rate and enlarging border over time, which is very critical to provide an emerging methodology in vitro cancer studies.

An alternative approach to tracing the volumic proliferation development of an entire tumor spheroid in 3D through a mini-Opto tomography platform.

Microscopy, which is listed among the major in-situ imaging applications, allows to derive information from a biological sample on the existing architectural structures of cells and tissues and their changes over time. Large biological samples such as tumor spheroids cannot be imaged within one field of view, regional imaging in different areas and subsequent stitching are required to attain the full picture. Microscopy is not typically used to produce full-size visualization of tumor spheroids measuring a few millimeters in size. In this study, we propose a 3D volume imaging technique for tracing the growth of an entire tumor spheroid measuring up to 10 mm using a miniaturized optical (mini-Opto) tomography platform. We performed a primary analysis of the 3D imaging for the MIA PaCa-2 pancreatic tumoroid employing its 2D images produced with the mini-Opto tomography from different angles ranging from -25 ° to +25 ° at six different three-day-apart time points of consecutive image acquisition. These 2D images were reconstructed by using a 3D image reconstruction algorithm that we developed based on the algebraic reconstruction technique (ART). We were able to reconstruct the 3D images of the tumoroid to achieve 800 × 800-pixel 50-layer images at resolutions of 5-25 µm. We also created its 3D visuals to understand more clearly how its volume changed and how it looked over weeks. The volume of the tumor was calculated to be 6.761 mm3 at the first imaging time point and 46.899 mm3 15 days after the first (at the sixth time point), which is 6.94 times larger in volume. The mini-Opto tomography can be considered more advantageous than commercial microscopy because it is portable, more cost-effective, and easier to use, and enables full-size visualization of biological samples measuring a few millimeters in size.

The concurrent effect of acyclovir and rosemary on glioblastoma cell culture.

Human cytomegalovirus (HCMV) is a beta herpesvirus which large amount of people in world has interacted with. Recent studies indicated that CMV DNA is associated with several cancer types including "Glioblastoma (GBM)" which is the most common and aggressive type of primary brain cancer. In clinical studies it was shown that several antiviral medicines prolonged life span of glioblastoma patients. One of them is Acyclovir (ACV) which is a type of nucleoside analog, used to cure viral infections and might be a potential treatment supplement for Glioblastoma. In this study we aimed to investigate if ACV had cytotoxic effect on glioblastoma cell line U87 MG and also the effect of ACV on healthy cells. Furthermore it was aimed to search the effect of Rosmarinus Officinalis also known as rosemary which is an aromatic, perennial plant concurrent with ACV on glioblastoma and healthy cells.

The Effect of Ascorbic Acid over the Etoposide- and Temozolomide-Mediated Cytotoxicity in Glioblastoma Cell Culture: A Molecular Study.

AIM: Glioblastoma (GBM) is one of the lethal central nervous system tumors. One of the widely used chemical agents for the treatment of glioblastoma is temozolomide. It is an orally administered, deoxyribonucleic acid (DNA) alkylating agent. DNA alkylation triggers the death of tumor cells. However, some tumor cells are able to repair this type of DNA damage and thus lower the therapeutic effect of temozolomide. Laboratory and clinical studies indicate that temozolomide"s anticancer effects might be strengthened when combined with other chemotherapeutic agents like etoposide or antioxidant agents like ascorbic acid. In this study, we aimed to evaluate the cytotoxic and oxidative stress effects of ascorbic acid (1000 ?M), temozolomide (100 ?M) and etoposide (25 ?M) agents alone and in dual and triple combinations in a glioblastoma U87 MG cell culture. MATERIAL AND METHODS: The cytotoxic and oxidative stress effects were investigated by the 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) and liquid chromatography tandem-mass spectrometry (LC-MS/MS) analysis methods. RESULTS: Cytotoxicity tests showed that etoposide, temozolomide, "etoposide+ascorbic acid", "temozolomide+ascorbic acid", "temozolomide+etoposide" and "temozolomide+etoposide+ascorbic acid" combinations have anti-proliferative effects. The maximum anti-proliferation response was observed in the "temozolomide+etoposide+ascorbic acid"-added group. Similarly LCMS/ MS analyses showed that minimum oxidative DNA damage occurred in the "temozolomide+etoposide+ascorbic acid"-added group. CONCLUSION: Ascorbic acid decreases the cytotoxic and genotoxic effect of etoposide and etoposide-temozolomide combination but it has no meaningful effect on temozolomide"s toxicity.

The Effect of Rosmarinus Officinalis and Chemotherapeutic Etoposide on Glioblastoma (U87 MG) Cell Culture.

AIM: To investigate whether high dose toxicities of etoposide can be overcome when used in combination with a natural compound named Rosmarinus Officinalis for glioblastoma (GBM). MATERIAL AND METHODS: The impact of Rosmarinus Officinalis in combination with etoposide on GBM U87 MG cells and Mouse Embryonic Fibroblast (MEF) cells was investigated. Both neutral red and 3-(4, 5-Dimethylthiazol-2-Yl)-2, 5-Diphenyltetrazolium Bromide (MTT) assays were employed to gauge cell viability. RESULTS: We observed that increased quantities of Rosmarinus Officinalis induced MEF cell proliferation while it inhibited the survival of GBM cells. Our results indicate that Rosmarinus Officinalis did not affect the cytotoxicity of etoposide on GBM cell cultures. In contrast, in the MEF cell cultures, Rosmarinus Officinalis induced proliferation and diminished the impact of etoposide. CONCLUSION: Rosmarinus Officinalis offers hope for developing new cancer treatment strategies. However, further studies are needed to verify these results.

Effect of crosslinking methods on the structure and biocompatibility of polyvinyl alcohol/gelatin cryogels.

In this study, polyvinyl alcohol (PVA) and gelatin based cryogels were prepared by crosslinking chemically or physically for tissue engineering applications. Different PVA/Gelatin ratios (100:0, 90:10, 70:30, 50:50) and crosslinking methods have been used to prepare cryogels; chemical and physical structure of the prepared matrices were analysed by FTIR and SEM; swelling and degradation profiles were followed. Chemical and physical crosslinking was obtained by using glutaraldehyde as crosslinker and by applying freeze thawing cycle, respectively. Gelatin concentration and crosslinking method had significant effect on the pore size, swelling ratio and degradation profiles of the cryogels. Biocompatibility of the cryogels were also investigated by MTT assay. SEM was used to investigate the cell morphology on the scaffolds. The MTT assay findings prove that physically crosslinked PVA/Gelatin scaffolds are more biocompatible and enhance more the adhesion and proliferation of mouse embryonic fibroblast cells (MEF) than chemically crosslinked PVA/Gelatin scaffolds. The overall results demonstrated that, the PVA/Gelatin cyrogels as a suitable biomaterial for tissue engineering applications and crosslinking methods affect the architecture and characteristic properties of the cryogels.

Beta-catenin mutations are not observed in chronic myeloid leukemia.

AIMS AND BACKGROUND: Studies reporting activated Wnt signaling in all stages of chronic myeloid leukemia (CML) have demonstrated that deregulation of the pathway plays a role in the pathogenesis of this disease. Several reports have suggested mechanisms for the deregulated Wnt signaling and beta-catenin stabilization observed in CML. One possible mechanism for beta-catenin stabilization could be the acquisition of mutations at its N-terminal domain, especially in the third exon where it is marked via phosphorylation for degradation. We sought to determine whether mutations in the third exon of the beta-catenin gene are responsible for the observed Wnt activation in CML. MATERIAL AND METHODS: We screened bone marrow specimens from 33 patients with CML in the chronic phase and also examined the K562 cell line for beta-catenin mutations. RESULTS: None of the patients nor the K562 cell line were found to carry mutations. CONCLUSION: Beta-catenin amino-terminal mutations are not observed or very rare and therefore are not the underlying mechanism of activated Wnt signaling in CML.