Temozolomide increases heat shock proteins in extracellular vesicles released from glioblastoma cells

Background Glioblastoma (GBM) is the most malignant and the fastest-progressing type of primary brain tumours. Temozolomide (TMZ) is a chemotherapeutic drug for the treatment of GBM. Extracellular vesicles (EVs) have been recently confirmed to have a substantial role in the GBM, and their contents released from GBM cells have been considered a target for treatment. The purpose of this study is to evaluate the impact of TMZ on heat shock proteins (HSPs) derived from EVs originated from GBM cell lines (U87-MG and LN229) and the significance of EVs in response to chemotherapy in GBM. Methods and results NTA, ELISA, and immunoblotting were used to characterization studies of EVs and results showed that U87-MG cells released many EVs compared to LN229 cells. The effect of TMZ treatments on HSPs expression levels were assessed with immunoblotting and was found to be led to increases in HSF-1, Hsp90, Hsp70, Hsp60 and Hsp27 expression in GBM cells and their EV contents, which these increases are related to therapeutic resistance. What is more, in Real-time PCR studies showing which signalling pathways might be associated with these increases, it was observed that TMZ triggered the expression of RAD51 and MDM2 genes in cells and EV contents. More strikingly, we discover a correlation between EV and parental cells in regard of mRNA and protein level in both cell lines as a result of TMZ treatment. Conclusions Our data suggest of EVs in the treatment of GBM may have potential biomarkers that can be used to investigate the treatment response

Preparation and characterization of polymers based on PDMS and PEG-DMA as potential scaffold for cell growth

This work describes a soft lithographic method for the generation of patterned both biopolymer and silver with each covered on microscope glass. Because of their biocompatible nature and permeability to gases the biopolymers are convenient for cell culture studies. The microscope glass was covered by polyethylene glycol dimethyl acrylate (PEG-DMA), as biopolymer and patterned by the UV light passing through the printed photomask for the preparation of the PDMS stamps. PDMS stamps were originally fabricated in this work for pattern transfer. Ag and polymer covered on the microscope glass were patterned by using these PDMS stamps. The patterned Ag, PDMS mold and PEG-DMA biopolymer were used as scaffolds and cell growth experiments have been performed on these materials. The degree of cell viability was measured by seeding them with L929 mouse fibroblasts and the number of the cells was measured by neutral red uptake assay. An increase in the number of cells on the material surfaces was observed. The pattern and the cell growth properties were followed by optic microscope. The results obtained from the cell growth was subjected to student's t-test. (C) 2017 Elsevier B.V. All rights reserved

Investigation of the role of quercetin as a heat shock protein inhibitor on apoptosis in human breast cancer cells

High expression of heat shock proteins (Hsp) in breast cancer has been closely associated with tumor cell proliferation and thus a poor clinical outcome. Quercetin, a good Hsp inhibitor as a dietary flavonoid, possesses anticarcinogenic properties. Although there are many studies on the effects of quercetin on Hsp levels in human breast cancer cells, research on elucidation of its molecular mechanism continues. Herein, we aimed to investigate the effect of quercetin on Hsp levels and whether quercetin is a suitable therapeutic for two breast cancer cell lines (MCF-7 and MDA-MB-231) representing breast tumors which differed in hormone receptor, aggressiveness and treatment responses. To examine the response to high and low doses of quercetin, the cells were treated with three doses of quercetin (10, 25 and 100 mu M) determined by MTT. The effects of quercetin on Hsp levels, apoptosis and DNA damage were examined by western blot analysis, caspase activity assay, comet assay and microscopy in human breast cancer cells. Compared to MDA-MB231 cells, MCF-7 cells were more affected by quercetin treatments. Quercetin effectively suppressed the expression of Hsp27, Hsp70 and Hsp90. While quercetin did not induce DNA damage, it triggered apoptosis at high levels. Although an increase in NF-kappa B levels is observed in the cells exposed to quercetin, the net result is the anticancer effect in case of Hsp depletion and apoptosis induction. Taken together our findings suggested that quercetin can be an effective therapeutic agent for breast cancer therapy regardless of the presence or absence of hormone receptors

Chitosan/alginate crosslinked hydrogels: Preparation, characterization and application for cell growth purposes

Chitosan hydrogels may be formed by various mechanisms. In this study, we aimed to form hybrid polymer networks of chitosan with alginate using a crosslinker which enabled the covalent binding of the two macromolecules. The structural and thermal characterization of these hydrogels was performed by using Fourier transform infrared (FTIR) and differential scanning calorimeter (DSC). The morphological analysis of the crosslinked material was investigated by scanning electron microscopy (SEM) and a scanning probe microscope with atomic force microscope (AFM) attachment. The swelling properties of these gels were analyzed in water and in phosphate buffered saline (PBS) solution. The presence of alginate in a chitosan/alginate hydrogel was shown to support the hydrogel stability. Compared to chitosan/alginate (1/2) hydrogel prepared with 1 wt% DCC, the swelling of chitosan/alginate (1/2) hydrogels prepared with 3 wt% DCC was limited. To measure the degree of cell proliferation, the hydrogels were seeded with L929 mouse fibroblasts and cell numbers measured by neutral red uptake assay. The cell attachment was also followed by (SEM) photography. It was observed that chitosan/alginate (1/2) hydrogels with 1 wt% (DCC) provides a better environment for cell attachment and proliferation. This study presents functional hydrogel formation by crosslinked chitosan and alginate, a novel biomaterial which also supports cell growth. (C) 2013 Published by Elsevier B.V

Alginate/Polyoxyethylene and Alginate/Gelatin Hydrogels: Preparation, Characterization, and Application in Tissue Engineering

Hydrogels are attractive biomaterials for three-dimensional cell culture and tissue engineering applications. The preparation of hydrogels using alginate and gelatin provides cross-linked hydrophilic polymers that can swell but do not dissolve in water. In this work, we first reinforced pure alginate by using polyoxyethylene as a supporting material. In an alginate/PEO sample that contains 20 % polyoxyethylene, we obtained a stable hydrogel for cell culture experiments. We also prepared a stable alginate/gelatin hydrogel by cross-linking a periodate-oxidized alginate with another functional component such as gelatin. The hydrogels were found to have a high fluid uptake. In this work, preparation, characterization, swelling, and surface properties of these scaffold materials were described. Lyophilized scaffolds obtained from hydrogels were used for cell viability experiments, and the results were presented in detail

Chitosan/alginate crosslinked hydrogels: Preparation, characterization and application for cell growth purposes

Chitosan hydrogels may be formed by various mechanisms. In this study, we aimed to form hybrid polymer networks of chitosan with alginate using a crosslinker which enabled the covalent binding of the two macromolecules. The structural and thermal characterization of these hydrogels was performed by using Fourier transform infrared (FTIR) and differential scanning calorimeter (DSC). The morphological analysis of the crosslinked material was investigated by scanning electron microscopy (SEM) and a scanning probe microscope with atomic force microscope (AFM) attachment. The swelling properties of these gels were analyzed in water and in phosphate buffered saline (PBS) solution. The presence of alginate in a chitosan/alginate hydrogel was shown to support the hydrogel stability. Compared to chitosan/alginate (1/2) hydrogel prepared with 1 wt% DCC, the swelling of chitosan/alginate (1/2) hydrogels prepared with 3 wt% DCC was limited. To measure the degree of cell proliferation, the hydrogels were seeded with L929 mouse fibroblasts and cell numbers measured by neutral red uptake assay. The cell attachment was also followed by (SEM) photography. It was observed that chitosan/alginate (1/2) hydrogels with 1 wt% (DCC) provides a better environment for cell attachment and proliferation. This study presents functional hydrogel formation by crosslinked chitosan and alginate, a novel biomaterial which also supports cell growth. (C) 2013 Published by Elsevier B.V

Smooth Muscle Cells Isolated from Thoracic Aortic Aneurysms Exhibit Increased Genomic Damage, but Similar Tendency for Apoptosis

Aortic aneurysms (AA) are characterized by structural deterioration leading to progressive dilation. During the development of AA, two key structural changes are pronounced, one being degradation of extracellular matrix and the other loss of smooth muscle cells (SMCs) through apoptosis. Reactive oxygen species (ROS) are produced above physiological levels in dilated (aneurismal) part of the aorta compared to the nondilated part and they are known to be associated with both the extracellular matrix degradation and the loss of SMCs. In this study, we hypothesized that aneurismal SMCs are more prone to apoptosis and that at least some cells undergo apoptosis due to elevated ROS in the aortic wall. To test this hypothesis, we first isolated SMCs from thoracic aneurismal tissue and compared their apoptotic tendency with normal SMCs in response to H2O2, oxidized sterol, or UV treatment. Exposed cells exhibited morphological changes characteristic of apoptosis, such as cell shrinkage, membrane blebbing, chromatin condensation, and DNA fragmentation. Terminal deoxynucleotidyl transferased UTP nick end labeling (TUNEL) further confirmed the fragmentation of nuclear DNA in these cells. Vascular SMCs were analyzed for their micronuclei (MN) and binucleate (BN) frequency as indicators of genomic abnormality. These data were then compared to patient parameters, including age, gender, hypertension, or aortic diameter for existing correlations. While the tendency for apoptosis was not significantly different compared to normal cells, both the %MN and %BN were higher in aneurismal SMCs. The data suggest that there is increased DNA damage in TAA samples, which might play a pivotal role in disease development