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 mu M 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 mu M resveratrol and 25nM 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 mu M 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 similar to 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 transfcction and 215 mu M 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

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

Therapeutic Drugs and Natural Products: The Effect of Suppressing Heat Shock Proteins (Hsp) in Brain Tumors

Brain tumors are common in the population, and approximately 17,000 new patients are diagnosed every year. Although there are advancements in conventional treatment, including surgery, radiotherapy and chemotherapy, they have limited effectiveness for the patients with brain tumors. However, with recent developments of tumor biology, novel approaches discovering new agents or their combinations have been proposed. Heat shock proteins (HSP) serve as molecular chaperones and they play a prominent role in protein homeostasis. Expression level of HSP enhance under the stressful conditions and they protect the cells by assisting of protein folding. In addition, HSP have attracted a great interest as a potential anticancer target and the development of HSP inhibitors demonstrating treatment is promising for cancer. Several therapeutic drugs and natural products that target signaling pathways associated with HSP are being developed and the efficacy of these agents in brain tumors is investigated. Consequently, our chapter puts emphasis on application of HSP in the treatment of brain tumors and the potential of therapeutic drugs and/or natural products in these tumors. It is hoped that approaches discussed in this chapter will overcome the existing limitations in the treatment with brain tumors and lead to a better prognosis for the patients

Suppression of HSP70 Expression by Quercetin and Its Therapeutic Potential Against Cancer

Heat shock response is one of several survival pathways that protects cells against harsh conditions. This response mechanism, which is evolutionarily protected in all organisms, enhances the expression of heat shock proteins (HSP) that show protective properties for cells under stress conditions. High expression of many HSP is observed in cancer, and their functions aides the advancement of disease. It is known that overexpression of HSP70, a member of HSP family, in cancerous cells has been closely associated with tumor cell proliferation, apoptosis inhibition, enhanced migration and metastasis and drug resistance promotion. Therefore, targeting HSP70 in cancer treatment is very important. One of the best-studied inhibitors known for HSP70 is quercetin that is widely distributed flavonoid in the plant kingdom. Several in vivo and in vitro studies have reported the efficacy of quercetin in reducing elevated HSP70 levels in cancer therapy. It has become a focal point as an anticancer agent because of the induction of apoptosis in many different cancer cells. In this chapter, we reviewed the role of HSP70 in different cancer types and the suppressive effect of quercetin on expression of HSP70 family members. Moreover, we emphasized molecular mechanisms targeted by quercetin in cancer and its relationship to Hsp70

Can Hsp Targeted Gene Therapy Be a New Hope for Gliomas?

Gliomas from glial cells of the brain are highly aggressive neoplasms. Traditional treatment has very limited effectiveness, and the prognosis is still poor. One of the major obstacles to therapy of gliomas is the resistance to treatment and the increased heat shock protein (Hsp) levels have a great effect on this. Overexpression of many Hsp, especially Hsp27, Hsp70 and Hsp90, is observed in gliomas. There is a relationship between Hsp expression levels and the tumor grade, and their functions aides the advancement of cancer. Therefore, it has become important to target Hsp in glioma treatment. Today, the disciplines of gene therapy (GT) have been experiencing a revolutionary growth in the field of cancer treatment. Developments in the field of genome-editing have made the GT methods a powerful potential tool for downregulation of Hsp and the breakdown of gliomas' treatment resistance. In this respect, the GT appears to be a promising innovative approach against gliomas. In this chapter, we reviewed the role of overexpressed Hsp in gliomas and the importance of the GT methods for silencing of them. We emphasized the principles, expectations and limitations of the methods, and highlighted the potential of the GT methods for Hsp targeted treatment of gliomas

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 vitrostudies 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 (similar to 3 and similar to 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

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

Mild Hypothermia via External Cooling Improves Lung Function and Alleviates Pulmonary Inflammatory Response and Damage in Two-Hit Rabbit Model of Acute Lung Injury

Objectives Targeted temperature management (TTM) with therapeutic hypothermia (TH) has an organ-protective effect by mainly reducing inflammatory response. Here, our objective was to determine, for the first time, whether mild TH with external cooling, a simple and inexpensive method, could be safe or even beneficial in two-hit rabbit model of acute lung injury/acute respiratory distress syndrome (ALI/ARDS). Methods Twenty-two New Zealand rabbits (6-month-old) were randomly divided into healthy control (HC) with conventional ventilation, but without injury, model group (ALI), and hypothermia group with external cooling (ALI-HT). After induction of ALI/ARDS through mild lung-lavages followed by non-protective ventilation, mild hypothermia was started in ALI-HT group (body temperature of 33-34 degrees C). All rabbits were conventionally ventilated for an additional 6-h by recording respiratory parameters. Finally, lung histopathology and inflammatory response were evaluated. Results Hypothermia was associated with higher oxygen saturation, resulting in partial improvement in the P/F ratio (PaO2/FiO(2)), oxygenation index, mean airway pressure, and PaCO2, but did not affect lactate levels. The ALI-HT group had lower histopathological injury scores (hyperemia, edema, emphysema, atelectasis, and PMN infiltration). Further, tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-6 and -8 levels in lung tissue and serum samples markedly reduced due to hypothermia. Conclusion Mild TH with external cooling reduced lung inflammation and damage, whereas it resulted in partial improvement in gas exchanges. Our findings highlight that body temperature control may be a potentially supportive therapeutic option for regulating cytokine production and respiratory parameters in ALI/ARDS