Hsp27, Hsp60, Hsp70, or Hsp90 depletion enhances the antitumor effects of resveratrol via oxidative and ER stress response in human glioblastoma cells.

Therapeutic resistance of gliomas is still a crucial issue and closely related to induced heat shock response (HSR). Resveratrol (RSV) is a promising experimental agent for glioblastoma (GB) therapy. However, the role of heat shock protein (Hsp)27, Hsp60, Hsp70, and Hsp90 on the therapeutic efficacy of RSV remains unclear in gliomas. Herein, small interfering (si)RNA transfection was performed to block Hsp expressions. RSV treatments reduced glioma cells' viability dose- and time-dependent while keeping HEK-293 normal cells alive. Furthermore, a low dose of RSV (15 µM/48 h) offered protection against oxidative stress and apoptosis due to Hsp depletion in healthy cells. On the contrary, in glioma cells, RSV (15 µM/48 h) increased ROS (reactive oxygen species) production, led to autophagy and induced endoplasmic reticulum (ER) stress and apoptosis, and reduced 2D- and 3D-clonogenic survival. Hsp27, Hsp60, Hsp70, or Hsp90 depletion also resulted in cell death through ER stress response and ROS burst. Remarkably, the heat shock response (increased HSF1 levels) due to Hsp depletion was attenuated by RSV in glioma cells. Collectively, our data show that these Hsp silencings make glioma cells more sensitive to RSV treatment, indicating that these Hsps are potential therapeutic targets for GB treatment.

Acutely increased aquaporin-4 exhibits more potent protective effects in the cortex against single and repeated isoflurane-induced neurotoxicity in the developing rat brain.

Damage to hippocampus, cerebellum, and cortex associated with cognitive functions due to anesthetic-induced toxicity early in life may cause cognitive decline later. Aquaporin 4 (AQP4), a key protein in waste clearance pathway of brain, is involved in synaptic plasticity and neurocognition. We investigated the effects of single and repeated isoflurane (Iso) anesthesia on AQP4 levels and brain damage. Postnatal-day (P)7 Wistar albino rats were randomly assigned to Iso or Control (C) groups. For single-exposure, pups were exposed to 1.5% Iso in 30% oxygenated-air for 3-h at P7 (Iso1). For repeated-exposure, pups were exposed to Iso for 3 days, 3-h each day, at 1-day intervals (P7 + 9 + 11) starting at P7 (Iso3). C1 and C3 groups received only 30% oxygenated-air. Based on HE-staining and immunoblotting (Bax/Bcl-2, cleaved-caspase3 and PARP1) analyses, Iso exposures caused a higher degree of apoptosis in hippocampus. Anesthesia increased 4-hydroxynonenal (4HNE), oxidative stress marker; the highest ROS accumulation was determined in cerebellum. Increased inflammation (TNF-α, NF-κB) was detected. Multiple Iso-exposures caused more significant damage than single exposure. Moreover, 4HNE and TNF-α contributed synergistically to Iso-induced neurotoxicity. After anesthesia, higher expression of AQP4 was detected in cortex than hippocampus and cerebellum. There was an inverse correlation between increased AQP4 levels and apoptosis/ROS/inflammation. Correlation analysis indicated that AQP4 had a more substantial protective profile against oxidative stress than apoptosis. Remarkably, acutely increased AQP4 against Iso exhibited a more potent neuroprotective effect in cortex, especially frontal cortex. These findings promote further research to understand better the mechanisms underlying anesthesia-induced toxicity in the developing brain.

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.

Acute Changes in Myocardial Expression of Heat Shock Proteins and Apoptotic Response Following Blood, delNido, or Custodiol Cardioplegia in Infants Undergoing Open-Heart Surgery.

Stress caused by cardioplegic ischemic arrest was shown to alter the expression levels of heat shock proteins (Hsp), but little is known about their effects, particularly on pediatric hearts. This study aimed to investigate whether myocardial cellular stress and apoptotic response changes due to different cardioplegia (CP) solutions during cardiopulmonary bypass (CPB) in infants and to determine their influence on surgical/clinical outcomes. Therefore, twenty-seven infants for surgical closure of ventricular septal defect were randomly assigned to a CP solution: normothermic blood (BCP), delNido (dNCP), and Custodiol (CCP). Hsp levels and apoptosis were determined by immunoblotting in cardiac tissue from the right atrium before and after CP, and their correlations with cardiac parameters were evaluated. No significant change was observed in Hsp27 levels. Hsp60, Hsp70, and Hsp90 levels decreased significantly in the BCP-group but increased markedly in the CCP-group. Decreased Hsp60 and increased Hsp70 expression were detected in dNCP-group. Importantly, apoptosis was not observed in dNCP- and CCP-groups, whereas marked increases in cleaved caspase-3 and -8 were determined after BCP. Serum cardiac troponin-I (cTn-I), myocardial injury marker, was markedly lower in the BCP- and dNCP-groups than CCP. Additionally, Hsp60, Hsp70, and Hsp90 levels were positively correlated with aortic cross-clamp time, total perfusion time, and cTn-I release. Our findings show that dNCP provides the most effective myocardial preservation in pediatric open-heart surgery and indicate that an increase in Hsp70 expression may be associated with a cardioprotective effect, while an increase in Hsp60 and Hsp90 levels may be an indicator of myocardial damage during CPB.

Long non-coding RNA NKILA regulates expression of HSP90α, NF-κB and ß-catenin proteins in the MCF-7 breast cancer cell line.

Non-coding RNAs are increasingly being investigated and have shown great potential for diagnosis, prognosis and treatment of cancer. Thus, we have investigated a possible regulatory mechanism between NF-κB suppressor-NKILA, and HSP90, NF-κB, and ß-catenin molecules in MCF-7 breast cancer cells. HSP90 is an important stress protein and together with ß-catenin and NF-κB molecules can be responsible for cancer cell development. However, there is no comprehensive data available on the novel molecule NKILA unlike for HSP90, ß-catenin and NF-κB alone. Therefore, we suggest there might be a correlation between NKILA and these proteins. To investigate the NKILA role on HSP90, NF-κB and ß-catenin proteins we inhibited the NKILA by using transfection in MCF-7 breast cancer cells. NKILA-siRNA transfected cells were incubated for 5 h. Then, cells were collected and proteins were extracted to be separated by SDS-PAGE. The aforementioned proteins of siRNA transfected group were evaluated by quantification and comparison of their relative expression levels with the control group by immunoblotting. Results showed, HSP90 and NF-κB/p105, NF-κB/p65 and NF-κB/p50 subunits significantly increased while the level of ß-catenin decreased after NKILA inhibition. For the first time we have demonstrated that HSP90 and expression levels of beta-catenin are associated with NKILA levels which may be closely related to the canonical NF-κB pathway in MCF-7 cells. These novel findings may have significant implications in cancer cells development and possibly present important hints for the future studies of the cancer cell targeted therapy.

The calcimimetic R-568 attenuates subarachnoid hemorrhage-induced vasospasm through PI3K/Akt/eNOS signaling pathway in the rat model.

Cerebral vasospasm (CVS) causes mortality and morbidity in patients after subarachnoid hemorrhage (SAH). The mechanism and adequate treatment of CVS are still elusive. R-568 is a calcimimetic agent known to exert a vasodilating effect. However, there is no report on its vasodilator effect against SAH-induced vasospasm. In the present study, we investigated the therapeutic effect of R-568 on the SAH-induced CVS model in rats. Seventy-two adult male Sprague-Dawley rats were divided into 8 groups: sham surgery; SAH only; SAH + Vehicle, SAH + R-568; SAH + R-568 + Wortmannin (the PI3K inhibitor); SAH + Wortmannin; SAH + R-568 + Calhex-231 (a calcilytic agent); SAH + Calhex-231. SAH was induced by blood (0.3 mL) given by intracisternal injection. R-568 (20 µM) was administered intracisternal immediately prior to experimental SAH. Basilar arteries (BAs) were obtained to evaluate PI3K/Akt/eNOS pathway (immunoblotting) and morphological changes 48 h after SAH. Perimeters of BAs were decreased by 24.1% in the SAH group compared to the control group and the wall thickness was increased by 75.3%. With R-568 treatment, those percentages were 9.6% and 29.6%, respectively, indicating that vasospasm was considerably improved when compared with the SAH group (P < 0.001 in both). While p-PI3K/PI3K and p-Akt/Akt ratio and eNOS protein expression were markedly decreased in the SAH rats, treatment with R-568 resulted in a significant increase in these levels. The beneficial effects of R-568 were partially blocked in the presence of Calhex-231 and completely blocked in the presence of Wortmannin. Herein, we found that treatment with R-568 would attenuate SAH-induced CVS through the PI3K/Akt/eNOS pathway and demonstrate therapeutic promise in CVS treatment following SAH.

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 µM) 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-κB 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.

Folic acid-modified methotrexate-conjugated gold nanoparticles as nano-sized trojans for drug delivery to folate receptor-positive cancer cells.

Methotrexate (MTX), an analog of folic acid (FA), is a drug widely used in cancer treatment. To prevent its potential toxicity and enhance therapeutic efficacy, targeted drug delivery systems, especially nanotechnology-folate platforms, are a central strategy. Gold nanoparticles (AuNPs) are promising candidates to be used as drug delivery systems because of their small particle sizes and their inertness for the body. In this study, glutathione (GSH)-coated FA-modified spherical AuNPs (5.6 nm) were successfully synthesized, and the anticancer activity of novel MTX-loaded (MTX/Au-GSH-FA) NPs (11 nm) was examined. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) results showed that MTX/AuNPs possess spherical morphology, nanoscaled particle size, narrow size distribution, and good stability. In vitro studies showed that cytotoxicity of MTX/Au-GSH-FA to folate receptor-positive (FR+) human brain (U-87 MG) and cervical (HeLa) cancer cells enhanced significantly (∼3 and ∼10 fold, respectively) compared to free MTX while there was no significant effect in FR-negative human cell lines A549 (lung carcinoma), PC3 (prostate carcinoma), HEK-293 (healthy embryonic kidney). Moreover, the receptor specificity of the conjugate was shown by fluorescent microscopic imaging. In conclusion, these results indicate that the synthesized novel MTX/Au-GSH-FA NP complex seems to be a good candidate for effective and targeted delivery in FR+ cancer therapy.

Resveratrol and siRNA in combination reduces Hsp27 expression and induces caspase-3 activity in human glioblastoma cells.

GBM cells can easily gain resistance to conventional therapy, and therefore treatment of glioblastoma multiforme (GBM) is difficult. One of the hallmark proteins known to be responsible for this resistance is heat shock protein 27 (Hsp27) which has a key role in the cell survival. Resveratrol, a natural compound, exhibits antitumor effects against GBM, but there are no reports regarding its effect on Hsp27 expression in gliomas. The aim of the present study was to asses the effect of resveratrol on Hsp27 expression and apoptosis in non-transfected and transfected U-87 MG human glioblastoma cells. In order to block the Hsp27 expression, siRNA transfection was performed. Non-transfected and transfected cells were treated with either 10 or 15 µM resveratrol. The effects of resveratrol were compared with quercetin, a well-known Hsp27 inhibitor. Resveratrol was found to induce apoptosis more effectively than quercetin. Our data showed that resveratrol induces dose- and time-dependent cell death. We also determined that silencing of Hsp27 with siRNA makes the cells more vulnerable to apoptosis upon resveratrol treatment. The highest effect was observed in the 15 µM resveratrol and 25 nM siRNA combination group (suppressed Hsp27 expression by 93.4% and induced apoptosis by 101.2%). This study is the first report showing that resveratrol reduces Hsp27 levels, and siRNA-mediated Hsp27 silencing enhances the therapeutic effects of resveratrol in glioma cells. Our results suggest that resveratrol administration in combination with Hsp27 silencing has a potential to be used as a candidate for GBM treatment.

Rosmarinic acid and siRNA combined therapy represses Hsp27 (HSPB1) expression and induces apoptosis in human glioma cells.

High expression of Hsp27 in glioma cells has been closely associated with tumor cell proliferation and apoptosis inhibition. The aim of the present study was to asses the effects of rosmarinic acid (RA) on Hsp27 expression and apoptosis in non-transfected and transfected human U-87 MG cells. The effect of rosmarinic acid was compared to quercetin, which is known to be a good Hsp27 inhibitor. In order to block the expression of Hsp27 gene (HSPB1), transfection with specific siRNAs was performed. Western blotting technique was used to assess the Hsp27 expression, and caspase-3 colorimetric activity assay was performed to determine apoptosis induction. According to the results, it was found that RA and quercetin effectively silenced Hsp27 and both agents induced apoptosis by activating the caspase-3 pathway. Eighty and 215 µM RA decreased the level of Hsp27 by 28.8 and 46.7% and induced apoptosis by 30 and 54%, respectively. For the first time, we reported that rosmarinic acid has the ability to trigger caspase-3 induced apoptosis in human glioma cells. As a result of siRNA transfection, the Hsp27 gene was silenced by ~ 50% but did not cause a statistically significant change in caspase-3 activation. It was also observed that apoptosis was induced at a higher level as a result of Hsp27 siRNA and subsequent quercetin or RA treatment. siRNA transfection and 215 µM RA treatment suppressed Hsp27 expression level by 90.5% and increased caspase-3 activity by 58%. Herein, we demonstrated that RA administered with siRNA seems to be a potent combination for glioblastoma therapy.

Involvement of dying beta cell originated messenger molecules in differentiation of pancreatic mesenchymal stem cells under glucotoxic and glucolipotoxic conditions.

Beta cell mass regulation represents a critical issue for understanding and treatment of diabetes. The most important process in the development of diabetes is beta cell death, generally induced by glucotoxicity or glucolipotoxicity, and the regeneration mechanism of new beta cells that will replace dead beta cells is still not fully understood. The aim of this study was to investigate the generation mechanism of new beta cells by considering the compensation phase of type 2 diabetes mellitus. In this study, pancreatic islet derived mesenchymal stem cells (PI-MSCs) were isolated from adult rats and characterized. Then, beta cells isolated from rats were co-cultured with PI-MSCs and they were exposed to glucotoxicity, lipotoxicity and glucolipotoxicity conditions for 72 hr. As the results apoptotic and necrotic cell death were increased in both PI-MSCs and beta cells especially by the exposure of glucotoxic and glucolipotoxic conditions to the co-culture systems. Glucotoxicity induced-differentiated beta cells were functional due to their capability of insulin secretion in response to rising glucose concentrations. Moreover, beta cell proliferation was induced in the glucotoxicity-treated co-culture system whereas suppressed in lipotoxicity or glucolipotoxicity-treated co-culture systems. In addition, 11 novel proteins, that may release from dead beta cells and have the ability to stimulate PI-MSCs in the direction of differentiation, were determined in media of glucotoxicity or glucolipotoxicity-treated co-culture systems. In conclusion, these molecules were considered as important for understanding cellular mechanism of beta cell differentiation and diabetes. Thus, they may be potential targets for diagnosis and cellular or therapeutic treatment of diabetes.

Effect of α-tocopheryl succinate on the molecular damage induced by indomethacin in C6 glioma cells.

Indomethacin is a member of the non-steroidal anti-inflammatory drug (NSAID) class, which has great potential for use in the treatment of glioma. However, it induces the generation of reactive oxygen species (ROS) and causes molecular damage while inducing its effects. Vitamin E is widely used in the complementary therapy of cancers. The main goal of the present study was to investigate the effects of α-tocopheryl succinate (α-TOS) against the oxidative damage induced by indomethacin in C6 glioma cells. Cells were treated with 10 µM α-TOS alone or in combination with 200 µM indomethacin for two days. The intracellular ROS level, molecular damage as revealed by lipid peroxidation and protein carbonyl formation, and the COX activity in C6 glioma cells were measured. Treatment of the cells with α-TOS and indomethacin, alone or in combination, caused the levels of ROS generation and protein damage to increase, but protected against lipid peroxidation and reduced COX activity.

Increased eNOS levels in hereditary angioedema.

BACKGROUND: Hereditary angio-edema (HAE), characterized by recurrent episodes of angioedema involving the skin and the mucosa of the upper respiratory or the gastrointestinal tracts, results from heterozygosity for deficiency of the serine proteinase inhibitor (serpin), C1 inhibitor (C1-INH). OBJECTIVE: In this study, serum inflammatory cytokine levels and circulating endothelial cells collected from HAE patients during both acute attacks and asymptomatic periods were evaluated. METHOD: Twenty-four patients with Type I and 1 patient with Type II HAE in an asymptomatic period (Group I), 8 patients with Type I HAE during a mild to moderate acute attack (Group II) and 20 healthy subjects (13 females, mean age: 32.1±8.2years) were included. Serum IL-6, IL-8, IL-1ß, TNF-α, vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS) levels were detected by ELISA. Circulating endothelial cells (CECs) and circulating endothelial progenitors (CEPs) were evaluated using Fluorescence Activated Cell Sorting (FACS). RESULTS: Serum eNOS levels of HAE patients were significantly higher than healthy subjects (p<0.006) while mean TNF-α levels in Group I were slightly lower (p<0.03) than Group II. There were no differences in terms of other inflammatory cytokines between the control subjects and HAE patients who were either in an asymptomatic period or experiencing an acute attack. CECs and CEPs were also similar. CONCLUSION: These results suggest that an inflammatory response is not necessary to trigger HAE attacks. On the other hand, increased eNOS levels might reflect a sustained hyperpermeability state in HAE patients.

Comparison of antioxidant capacity, protein profile and carbohydrate content of whey protein fractions.

Whey is used as an additive in food industry and a dietary supplement in nutrition. Here we report a comparative analysis of antioxidant potential of whey and its fractions. Fractions were obtained by size exclusion chromatography, before and after enzymatic digestion with pepsin or trypsin. Superoxide radical scavenging, lipid peroxidation inhibition and cupric ion reducing activities of different fractions were checked. Peptides were detected by SDS-PAGE and GC-MS was used to determine carbohydrate content of the fractions. All samples showed antioxidant activity and the second fraction of the trypsin hydrolysate showed the highest superoxide radical scavenging activity. CUPRAC value of this fraction was two-times higher than that of whey filtrate. The first fraction of the pepsin hydrolysate was the most effective inhibitor of lipid peroxidation. Each sample exhibited a different polypeptide profile. Different percentages of carbohydrates were identified in whey filtrate and in all second fractions, where galactose was the major component.

Viscum album L. Extracts Protects HeLa Cells against Nuclear and Mitochondrial DNA Damage.

Viscum album L. is a semiparasitic plant grown on trees and widely used for the treatment of many diseases in traditional and complementary therapy. It is well known that some activities of Viscum album extracts are varied depending on the host trees, such as antioxidant, apoptosis-inducing, anticancer activities of the plant. The aim of the present study is to examine the comparative effects of methanolic extracts of V. album grown on three different host trees (locust tree, lime tree, and hedge maple tree) on H(2)O(2)-induced DNA damage in HeLa cells. Oxidative damage in mitochondrial DNA and two nuclear regions was assessed by QPCR assay. The cells were pretreated with methanolic extracts (10 µg/mL) for 48 h, followed by the treatment with 750 µM H(2)O(2) for 1 hour. DNA damage was significantly induced by H(2)O(2) while it was inhibited by V. album extracts. All extracts completely protected against nuclear DNA damage. While the extract from lime tree or white locust tree entirely inhibited mitochondrial DNA damage, that from hedge maple tree inhibited by only 50%. These results suggest that methanolic extracts of V. album can prevent oxidative DNA damage, and the activity is dependent on the host tree.

Antioxidant and cytotoxic activities of Aphanes arvensis extracts.

The objectives of this study were to examine the free radical scavenging activity and the protective effects against macromolecular oxidation as well as the cytotoxic activity of Aphanes arvensis aqueous and methanolic extracts. Free radical scavenging activity was determined by DPPH method. The methanolic extract showed a scavenging activity nearly equivalent to Trolox and Vitamin C and has an IC(50) value of 4.54 microg/mL. Total antioxidant capacity was determined by CUPRAC method. The antioxidant capacity of aqueous and methanolic extract was 0.792 and 1.550 mmol TE/g DWE, respectively. The protective effect of A. arvensis extracts against lipid peroxidation was evaluated using a liposome oxidation system. The methanolic extract was more active than the aqueous extract. The aqueous extract possessed protective effect against protein oxidation in a dose dependent manner. Both extracts showed inhibitory effect on DNA oxidation as measured by plasmid relaxation assay. Results presented here indicate that A. arvensis possess strong antioxidant activity and protective effects with very little cytotoxic effect, and they can therefore be used as a natural additive in food, cosmetic and pharmaceutical industries.