Inhibition of heat shock protein (Hsp) 27 potentiates the suppressive effect of Hsp90 inhibitors in targeting breast cancer stem-like cells

Heat shock protein (Hsp) 90 is an ATP-dependent chaperone and its expression has been reported to be associated with poor prognosis of breast cancer. Cancer stem cells (CSCs) are particular subtypes of cells in cancer which have been demonstrated to be important to tumor initiation, drug resistance and metastasis. In breast cancer, breast CSCs (BCSCs) are identified as CD24-CD44 + cells or cells with high intracellular aldehyde dehydrogenase activity (ALDH+). Although the clinical trials of Hsp90 inhibitors in breast cancer therapy are ongoing, the BCSC targeting effect of them remains unclear. In the present study, we discovered that the expression of Hsp90α was increased in ALDH + human breast cancer cells. Geldanamycin (GA), a Hsp90 inhibitor, could suppress ALDH + breast cancer cells in a dose dependent manner. We are interesting in the insufficiently inhibitory effect of low dose GA treatment. It was correlated with the upregulation of Hsp27 and Hsp70. By co-treatment with HSP inhibitors, quercetin or KNK437 potentiated BCSCs, which determined with ALDH+ population or mammosphere cells, toward GA inhibition, as well as anti-proliferation and anti-migration effects of GA. With siRNA mediated gene silencing, we found that knockdown of Hsp27 could mimic the effect of HSP inhibitors to potentiate the BCSC targeting effect of GA. In conclusion, combination of HSP inhibitors with Hsp90 inhibitors could serve as a potential solution to prevent the drug resistance and avoid the toxicity of high dose of Hsp90 inhibitors in clinical application. Furthermore, Hsp27 may play a role in chemoresistant character of BCSCs.     

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.     

Apigenin-induced apoptosis of leukemia cells is mediated by a bimodal and differentially regulated residue-specific phosphorylation of heat-shock protein�C27

Apigenin, a natural plant flavonoid with antiproliferative activity, is emerging as a promising compound for cancer prevention and therapy, but its mechanism of action remains unclear. High expression of the small heat-shock protein-27 (Hsp27) in leukemia contributes to the resistance of these cells to cancer treatments. Changes in Hsp27 phosphorylation have been associated with heat and metabolic stress, but its role in flavonoid anticancer activity has not been investigated. In this study, we examined the effect of apigenin in the regulation of Hsp27 on leukemia. We showed that apigenin does not affect Hsp27 expression but induces a bimodal phosphorylation on Ser78 and Ser82. The phosphorylation at early times was regulated by p38. At later times, Hsp27 phosphorylation was dependent on p38 activity and for some residues on PKCδ. Silencing of p38 expression reduced apigenin-induced phosphorylation on Ser15, Ser78, and Ser82, whereas silencing of PKCδ expression reduced the phosphorylation on Ser15 and Ser82 without affecting Ser78. In addition, we found that apigenin-induced PKCδ activity is mediated by p38. We also showed that the phosphorylation of Hsp27 significantly increased the susceptibility of leukemia cells to apigenin-induced apoptosis. Together, these results identify a complex signaling network regulating the cytotoxic effect of apigenin through Hsp27 phosphorylation.     

Modulation of heat shock protein response in SH-SY5Y by mobile phone microwaves

AIM: To investigate putative biological damage caused by GSM mobile phone frequencies by assessing electromagnetic fields during mobile phone working. METHODS: Neuron-like cells, obtained by retinoicacid-induced differentiation of human neuroblastoma SH-SY5Y cells, were exposed for 2 h and 4 h to microwaves at 1800 MHz frequency bands. RESULTS: Cell stress response was evaluated by MTT assay as well as changes in the heat shock protein expression (Hsp20, Hsp27 and Hsp70) and caspase-3 activity levels, as biomarkers of apoptotic pathway. Under our experimental conditions, neither cell viability nor Hsp27 expression nor caspase-3 activity was significantly changed. Interestingly, a significant decrease in Hsp20 expression was observed at both times of exposure, whereas Hsp70 levels were significantly increased only after 4 h exposure. CONCLUSION: The modulation of the expression of Hsps in neuronal cells can be an early response to radiofrequency microwaves.     

Over-expression of ERp29 attenuates doxorubicin-induced cell apoptosis through up-regulation of Hsp27 in breast cancer cells

The endoplasmic reticulum protein 29 (ERp29) has a critical role in regulating protein folding, maturation and secretion. However, its role in carcinogenesis remains elusive. Recently, we reported that ERp29 is a novel tumor suppressor and regulates mesenchymal-epithelial transition in MDA-MB-231 breast cancer cells. Here, we investigated whether ERp29 plays a role in the response of breast cancer cells to chemotherapeutic agents. We found that expression of ERp29 increased the resistance to doxorubicin, but not cisplatin and paclitaxel, and decreased the doxorubicin-induced cell apoptosis in MDA-MB-231 cells, whereas knockdown of ERp29 in MCF-7 cells increased the doxorubicin cytotoxicity. A proteomics study identified up-regulation of Hsp27 and down-regulation of stathmin-1, galectin and prohibitin in the doxorubicin-resistant, ERp29 over-expressing MDA-MB-231 cells. Further, we demonstrated that ERp29 up-regulated expression of Hsp27 by down-regulating eukaryotic translational initiation factor 2α (eIF2α). When Hsp27 was knocked down by siRNA in the doxorubicin-resistant, ERp29 over-expressing MDA-MB-231 cells and parental MCF-7 cells, cell viability was significantly decreased and doxorubicin-induced cell apoptosis was enhanced. These results indicate that Hsp27 is involved in the ERp29-mediated resistance to doxorubicin. Therefore, targeting of Hsp27, with a combination of other chemotherapeutic agents, is a rational strategy in treating doxorubicin-resistant cancer cells.     

A Jurkat T cell variant resistant to death receptor-induced apoptosis. Correlation with heat shock protein (Hsp) 27 and 70 levels

Ligation of Fas induces an apoptotic program in Jurkat cells (Jd). We describe a Jurkat T cell variant (Jr) which shows total resistance to Fas-mediated apoptosis but which exhibits sensitivity to non-death-receptor pro-apoptotic stimuli such as staurosporine. Resistance to Fas-induced apoptosis in Jr cells is correlated with high expression of Hsps. A prior heat-shock increases Hsp27 and 70 expression and protects Jd and Jr cells from Fas- and staurosporine-induced apoptosis. Staurosporine, but not the anti-Fas antibody CH11, abrogates constitutive Hsp70 expression at 37 degrees C and staurosporine also inhibit Hsp27 expression in Jd and Jr cells at 42 degrees C. These data suggest that constitutive expression of Hsp27 inhibits Fas-mediated apoptosis, but only induced expression of Hsp70 can protect T cells from staurosporine-induced apoptosis. Thus, Hsp27 could play a role in the regulation of death receptor-mediated apoptosis, while Hsp70 could regulate mitochondrial-dependent cell death.     

Identification of Sestrin3 Involved in the In vitro Resistance of Colorectal Cancer Cells to Irinotecan

Irinotecan, an analogue of camptothecin, is frequently used as a single agent or in combination with other anticancer drugs for the treatment of colorectal cancer. However, the drug resistance of tumors is a major obstacle to successful cancer treatment. In this study, we established that cells acquire chronic resistance to irinotecan. We profiled their differential gene expression using microarray. After gene ontology (GO) and KEGG pathway analysis of the microarray data, we specifically investigated whether Sestrin3 could decrease irinotecan resistance. Our results revealed that Sestrin3 enhanced the anticancer effect of irinotecan in vitro in LoVo cells that had acquired resistance to irinotecan. Irinotecan-resistant LoVo cells showed lower reactive oxygen species (ROS) production than their irinotecan-sensitive parental cells. ROS production was increased by Sestrin3 knockdown in irinotecan-resistant LoVo cells. Our results indicate that Sestrin3 might be a good target to develop therapeutics that can help to overcome resistance to irinotecan.     

Over-expression of Hsp70 in BHK-21 cells engineered to produce recombinant factor VIII promotes resistance to apoptosis and enhances secretion

Production of coagulation factor VIII (FVIII) by recombinant cell lines is limited by its failure to reach or maintain the native conformation in the endoplasmic reticulum. This results in significant cytoplasmic degradation and/or aggregation of the misfolded product. The molecular chaperone Hsp70 was overexpressed in an attempt to increase the recombinant FVIII (rFVIII) secretion. The characteristics of increased Hsp70 expression were investigated by comparing a clone of BHK-21 cells expressing rFVIII (rBHK-21(host)) to a chaperone clone derived by transfection of the host clone with human Hsp70 (rBHK-21(Hsp70)) in small-scale batch cell cultures. To aid this investigation a number of fluorescence based cellular apoptosis assays were developed and optimized. These assays demonstrated sub-populations of rBHK-21(host) cells that were apoptotic in nature and were identified prior to the loss in plasma membrane integrity. Dual staining for intracellular rFVIII and caspase-3 activation showed a reduction in intracellular rFVIII in rBHK-21(host) cells that correlated with a significant increase in active caspase-3, suggesting that apoptosis was a factor limiting rFVIII secretion. In sharp contrast there was more intracellular rFVIII and less active caspase-3 in rBHK-21(Hsp70) cell cultures. Moreover when grown in batch culture, rBHK-21(Hsp70) cells released rFVIII of higher specific activity (active FVIII protein/total FVIII protein), suggesting improved product quality. Thus, increased expression of HSP70 led to an increased yield of a secreted recombinant protein by inhibition of apoptosis and promoting proper conformational maturation of rFVIII in sub-optimal bioreactor conditions.     

紫草素促进caspase-9活化诱导大肠癌细胞凋亡的分子机制研究

目的探讨紫草素对大肠癌（CRC）细胞LoVo的抗肿瘤作用及其机制。 方法以不同紫草素浓度梯度（0、2、4、6 μmol/L）处理CRC细胞LoVo 24 h，以4 μmol/L紫草素处理不同时间梯度（0、12、24、48 h）的CRC细胞LoVo。流式细胞技术结合Annexin V-FITC/PI双染色测定细胞凋亡率，Western blot检测细胞中caspase-9蛋白的表达及切割情况。多组间比较采用单因素方差分析，组间两两比较采用LSD-t检验。 结果与0 μmol/L处理CRC细胞LoVo 24 h比较，2、4、6 μmol/L的细胞凋亡率[（6.94±1.02）﹪比（10.61±1.12）﹪、（15.55± 1.35）﹪、（36.51±1.46）﹪]均升高；与4 μmol/L紫草素处理0 h的CRC细胞LoVo比较，12、24、48 h的细胞凋亡率[（1.33±0.59）﹪比（19.23±1.24）﹪、（22.24±1.41）﹪、（28.41±1.52）﹪]均升高，差异具有统计学意义（P均< 0.001）。当紫草素剂量≥2 μmol/L，处理时间≥12 h时，caspase-9蛋白表达上调并被诱导活化，而caspase-9抑制剂（Z-LEHD-FMK）预处理后，LoVo细胞凋亡率下降38.7﹪，差异有统计学意义（P < 0.05）。 结论紫草素可以通过caspase-9蛋白的表达及其切割活性诱导CRC细胞凋亡。     

Combined treatment with quercetin and imperatorin as a potent strategy for killing HeLa and Hep-2 cells

The aim of the present study was to assess the effect of quercetin and imperatorin administered separately and in combination on apoptosis and autophagy induction in human cervical carcinoma HeLa cells and laryngeal carcinoma Hep-2 cells cultured in vitro. Conducted MTT measurements proved that quercetin and imperatorin displayed a strong antiproliferative activity manifested in markedly reduction of HeLa and Hep-2 cells viability as a result of treatment with 50 μM of each compound. Further cell staining assays revealed that concentration mentioned above generated the highest percentage of apoptotic cells especially in the case of application of both drugs for 48 h. Simultaneous quercetin and imperatorin administration induced apoptosis remarkably stronger than treatment with single drugs. Experiments at the molecular level confirmed these results accompanied with the decreased Hsp27 and Hsp72 expression and, in addition, with increased caspases activity. Autophagy was not observed and no significant changes in the expression of beclin-1 were noticed. Additionally, experiments were performed on the above-mentioned cell lines with blocked Hsp27 and Hsp72 expression. In these cells, no significant changes in the sensitivity to apoptosis induction upon quercetin and imperatorin treatment were observed. The present study has provided evidence supporting the potential of the combination of quercetin and imperatorin drugs as a novel tool to be used in anticancer therapy. Our results have also demonstrated that blocking of the Hsp27 and Hsp72 gene expression is not enough to sensitize cancer cells to programmed cell death induction in HeLa and Hep-2 cells.     

Concomitant inhibition of HSP90, its mitochondrial localized homologue TRAP1 and HSP27 by green tea in pancreatic cancer HPAF-II cells

Pancreatic cancer is a deadly disease characterized by poor prognosis and patient survival. Green tea polyphenols have been shown to exhibit multiple antitumor activities in various cancers, but studies on the pancreatic cancer are very limited. To identify the cellular targets of green tea action, we exposed a green tea extract (GTE) to human pancreatic ductal adenocarcinoma HPAF-II cells and performed two-dimensional gel electrophoresis of the cell lysates. We identified 32 proteins with significantly altered expression levels. These proteins are involved in drug resistance, gene regulation, motility, detoxification and metabolism of cancer cells. In particular, we found GTE inhibited molecular chaperones heat-shock protein 90 (Hsp90), its mitochondrial localized homologue Hsp75 (tumor necrosis factor receptor-associated protein 1, or Trap1) and heat-shock protein 27 (Hsp27) concomitantly. Western blot analysis confirmed the inhibition of Hsp90, Hsp75 and Hsp27 by GTE, but increased phosphorylation of Ser78 of Hsp27. Furthermore, we showed that GTE inhibited Akt activation and the levels of mutant p53 protein, and induced apoptosis and growth suppression of the cells. Our study has identified multiple new molecular targets of GTE and provided further evidence on the anticancer activity of green tea in pancreatic cancer.     

Increased BNip3 and decreased mutant p53 in cisplatin-sensitive PDT-resistant HT29 cells

We have reported previously the isolation of three photodynamic therapy (PDT)-resistant human colon carcinoma HT29 cell lines that show increased expression of the Hsp27 and BNip3 protein and a decreased expression of the mutant p53 protein compared to parental HT29 cells. Since mutant p53 and increased expression of Hsp27 have been associated with resistance to various chemotherapeutic agents, whereas BNip3 is a potent inducer of apoptosis, we were interested in determining whether these PDT-resistant cells were cross-resistant to other cytotoxic agents. We report here that the PDT-resistant HT29 cell lines showed a significant increase in cisplatin sensitivity and an increase in both spontaneous and cisplatin-induced apoptosis compared to parental HT29 cells. In addition, the cisplatin sensitivity of the PDT-resistant HT29 variants and several other clonal variants of HT29 cells correlated with increased BNip3 and decreased mutant p53 protein levels, but not Hsp27 protein levels.     

NF-kappaB and Hsp70 are involved in the phospholipase Cgamma1 signaling pathway in colorectal cancer cells

The majority of deaths from colorectal cancer are due to tumor invasion and metastasis. Induced migration of tumor cell is generally considered to be one critical step in cancer progression to the invasive and metastatic stage. Phospholipase Cgamma1 (PLCgamma1) is a key molecular switch in the process. But, the mechanism and function of PLCgamma1 in colorectal cancer motility are unclear. We showed first in this report that epidermal growth factor (EGF) stimulated the phosphorylation of PLCgamma1 in human colorectal cancer cell line LoVo. Inhibition of PLCgamma1 with the pharmacologic agent U73122 decreased the migration of LoVo cells in a dose-dependent manner while EGF treatment reversed it partially. PLCgamma1 signaling pathway also upregulated the activity of NF-kappaB. Furthermore, expression of Hsp70 was increased by treatment with U73122 or pyrrolidine dithiocarbamate (PDTC), a NF-kappaB inhibitor. These data indicated that PLCgamma1 played a pivotal role in the migration of human colorectal cancer cell and first demonstrated that upregulation of NF-kappaB binding activity and downregulation of Hsp70 expression were PLCgamma1-dependent in LoVo cells.     

Deoxyribonucleic acid damage induced by doxorubicin in peripheral blood mononuclear cells: possible roles for the stress response and the deoxyribonucleic acid repair process

Doxorubicin is an antineoplastic drug widely used in cancer treatment. However, many tumors are intrinsically resistant to the drug or show drug resistance after an initial period of response. Among the different molecules implicated with doxorubicin resistance are the heat shock proteins (Hsps). At present we do not know with certainty the mechanism(s) involved in such resistance. In the present study, to advance our knowledge on the relationship between Hsps and drug resistance, we have used peripheral blood mononuclear cells obtained from healthy nonsmoker donors to evaluate the capacity of a preliminary heat shock to elicit the Hsp response and to establish the protection against the deoxyribonucleic acid (DNA) damage induced by doxorubicin. DNA damage and repair were determined using the alkaline comet assay. We also measured the expression of Hsp27, Hsp60, Hsp70, Hsp90, hMLH1, hMSH2, and proliferating cell nuclear antigen by immunocytochemistry. The damage induced by doxorubicin was more efficiently repaired when the cells were previously heat shocked followed by a resting period of 24 hours before drug exposure, as shown by (1) the increased number of undamaged cells (P < 0.05), (2) the increased DNA repair capacity (P < 0.05), and (3) the high expression of the mismatch repair (MMR) proteins hMLH1 and hMSH2 (P < 0.05). In addition, in the mentioned group of cells, we confirmed by Western blot high expression levels of Hsp27 and Hsp70. We also noted a nuclear translocation of Hsp27 and mainly of Hsp70. Furthermore, inducible Hsp70 was more expressed in the nucleus than Hsc70, showing a possible participation of Hsp70 in the DNA repair process mediated by the MMR system.     

Heat shock protein-27 protects human bronchial epithelial cells against oxidative stress-mediated apoptosis: possible implication in asthma

Inflammation of the human bronchial epithelium, as observed in asthmatics, is characterized by the selective death of the columnar epithelial cells, which desquamate from the basal cells. Tissue repair initiates from basal cells that resist inflammation. Here, we have evaluated the extent of apoptosis as well as the Hsp27 level of expression in epithelial cells from bronchial biopsy samples taken from normal and asthmatic subjects. Hsp27 is a chaperone whose expression protects against oxidative stress. We report that in asthmatic subjects the basal epithelium cells express a high level of Hsp27 but no apoptotic morphology. In contrast, apoptotic columnar cells are devoid of Hsp27 expression. Moreover, we observed a decreased resistance to hydrogen peroxide-induced apoptosis in human bronchial epithelial 16-HBE cells when they were genetically modified to express reduced levels of Hsp27.