HSP27-overexpressing Cells Research Articles

Resveratrol protects HaCaT cells from ultraviolet B-induced photoaging via upregulation of HSP27 and modulation of mitochondrial caspase-dependent apoptotic pathway

The skin is the outermost protective barrier between the internal and external environment in humans. Chronic exposure to ultraviolet (UV) radiation is a major cause of photoaging. Evidence suggests that resveratrol suppresses UVB-induced photoaging. In this study, we aimed to investigate the protective effects of resveratrol against UVB-induced photoaging in HaCaT cells and to determine the underlying mechanisms. Apoptosis of normal or HSP27-overexpressing HaCaT cells in the presence of UVB was analyzed by flow cytometry. The mRNA and protein expression levels were measured by quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting. Resveratrol inhibited UVB-induced apoptosis by upregulating the expression of HSP27, reducing the production of proapoptotic proteins such as p65, Bax, and cleaved caspase-3, and promoting the expression of anti-apoptotic protein Bcl-2. However, UVB irradiation on HaCaT cells pretreated with resveratrol led to the upregulation of Bax, downregulation of Bcl-2, and promotion of p65 and caspase-3 activation after silencing of HSP27 gene. These findings suggest that the inhibition of HSP27 expression can partially reverse the anti-apoptotic effect of resveratrol and confirm that resveratrol can regulate HSP27 and thus control p65 and caspase-3 activation. In summary, resveratrol plays a role in photoprotection by upregulating HSP27 expression, increasing Bcl-2/Bax ratio, and inhibiting caspase-3 activity and p65 expression.

Heat shock protein 27 (HSPB1) suppresses the PDGF-BB-induced migration of osteoblasts.

Heat shock protein 27 (HSP27/HSPB1), one of the small heat shock proteins, is constitutively expressed in various tissues. HSP27 and its phosphorylation state participate in the regulation of multiple physiological and pathophysiological cell functions. However, the exact roles of HSP27 in osteoblasts remain unclear. In the present study, we investigated the role of HSP27 in the platelet-derived growth factor-BB (PDGF-BB)-stimulated migration of osteoblast-like MC3T3-E1 cells. PDGF-BB by itself barely upregulated the expression of HSP27 protein, but stimulated the phosphorylation of HSP27 in these cells. The PDGF-BB-induced cell migration was significantly downregulated by HSP27 overexpression. The PDGF-BB-induced migrated cell numbers of the wild-type HSP27-overexpressing cells and the phospho-mimic HSP27-overexpressing (3D) cells were less than those of the unphosphorylatable HSP27-overexpressing (3A) cells. PD98059, an inhibitor of MEK1/2, SB203580, an inhibitor of p38 mitogen-activated protein kinase, and SP600125, an inhibitor of stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) reduced the PDGF-BB-induced migration of these cells, whereas Akt inhibitor or rapamycin, an inhibitor of upstream kinase of p70 S6 kinase (mTOR), barely affected the migration. However, the PDGF-BB-induced phosphorylation of p44/p42 MAPK, p38 MAPK and SAPK/JNK was not affected by HSP27 overexpression. There were no significant differences in the phosphorylation of p44/p42 MAPK, p38 MAP kinase or SAPK/JNK between the 3D cells and the 3A cells. These results strongly suggest that HSP27 functions as a negative regulator in the PDGF-BB-stimulated migration of osteoblasts, and the suppressive effect is amplified by the phosphorylation state of HSP27.

Cytoprotective Effect of Heat Shock Protein 27 Against Lipopolysaccharide-Induced Apoptosis of Renal Epithelial HK-2 Cells

Background: In response to various stimuli, heat shock protein 27 (Hsp27) functions as an anti-apoptotic or/and anti-inflammatory factor which confers a survival advantage to cells. This study was aimed to explore whether Hsp27 also has a cytoprotective role in human renal tubular epithelial cells, and to evaluate its potential in treating septic acute kidney injury (septic AKI). Methods: HK-2 cells were subjected to different concentrations (0-10 µg/mL) of lipopolysaccharide (LPS) for various times (0-24 h) to establish a septic AKI model in vitro. Before LPS administration, HK-2 cells were transfected either with vectors or siRNA against Hsp27, and the changes in cell viability and apoptotic cells rate were assessed using CCK-8 and flow cytometry. The expression changes in apoptosis-related proteins, proinflammatory cytokines and chemokine, as well as main factors in NF-κB and JNK pathways were mainly determined by Western blotting. Besides, the relationship between Hsp27 and Bcl-2 was detected by co-immunoprecipitation. Results: LPS remarkably damaged HK-2 cells by reduction of cell viability, induction of apoptosis, and stimulation of proinflammatory cytokines and chemokine release. Hsp27 overexpression significantly impaired LPS-induced damage in HK-2 cells. Hsp27 overexpression couldn’t alter the mRNA level of Bcl-2, but could interact with Bcl-2 at an endogenous level. Both NF-κB and JNK pathways were activated by LPS, while were blocked in Hsp27-overexpressing cells. Conclusion: Hsp27 overexpression conferred a survival advantage to LPS-injured HK-2 cells by controlling cell viability, apoptosis and inflammation, possibly via interaction with Bcl-2 and modulation of NF-κB and JNK pathways.

Overcoming HSP27-mediated resistance by altered dimerization of HSP27 using small molecules.

Heat shock protein 27 (HSP27, HSPB1) is an anti-apoptotic protein characterized for its tumorigenic and metastatic properties, and now referenced as a major therapeutic target in many types of cancer. The biochemical properties of HSP27 rely on a structural oligomeric and dynamic organization that is important for its chaperone activity. Down-regulation by small interfering RNA or inhibition with a dominant-negative mutant efficiently counteracts the anti-apoptotic and protective properties of HSP27. However, unlike other HSPs such as HSP90 and HSP70, small molecule approaches for neutralization of HSP27 are not well established because of the absence of an ATP binding domain. Previously, we found that a small molecule, zerumbone (ZER), induced altered dimerization of HSP27 by cross linking the cysteine residues required to build a large oligomer, led to sensitization in combination with radiation. In this study, we identified another small molecule, a xanthone compound, more capable of altering dimeric HSP27 than ZER and yielding sensitization in human lung cancer cells when combined with HSP90 inhibitors or standard anticancer modalities such as irradiation and cytotoxic anticancer drugs. Therefore, altered dimerization of HSP27 represents a good strategy for anticancer therapy in HSP27-overexpressing cancer cells.

Prolonged Survival of Transplanted Osteoblastic Cells Does Not Directly Accelerate the Healing of Calvarial Bone Defects.

Considering the increased interest in cell-based bone regeneration, it is necessary to reveal the fate of transplanted cells and their substantive roles in bone regeneration. The aim of this study was to analyze the fate of transplanted cells and the effect of osteogenic cell transplantation on calvarial bone defect healing. An anti-apoptotic protein, heat shock protein (HSP) 27, was overexpressed in osteoblasts. Then, the treated osteoblasts were transplanted to calvarial bone defect and their fate was analyzed to evaluate the significance of transplanted cell survival. Transient overexpression of Hsp27 rescued MC3T3-E1 osteoblastic cells from H2 O2 -induced apoptosis without affecting osteoblastic differentiation in culture. Transplantation of Hsp27-overexpressing cells, encapsulated in collagen gel, showed higher proliferative activity, and fewer apoptotic cells in comparison with control cells. After 4-week of transplantation, both control cell- and Hsp27 overexpressed cell-transplanted groups showed significantly higher new bone formation in comparison with cell-free gel-transplantation group. Interestingly, the prolonged survival of transplanted osteoblastic cells by Hsp27 did not provide additional effect on bone healing. The transplanted cells in collagen gel survived for up to 4-week but did not differentiate into bone-forming osteoblasts. In conclusion, cell-containing collagen gel accelerated calvarial bone defect healing in comparison with cell-free collagen gel. However, prolonged survival of transplanted cells by Hsp27 overexpression did not provide additional effect. These results strongly indicate that cell transplantation-based bone regeneration cannot be explained only by the increment of osteogenic cells. Further studies are needed to elucidate the practical roles of transplanted cells that will potentiate successful bone regeneration. J. Cell. Physiol. 231: 1974-1982, 2016. © 2016 Wiley Periodicals, Inc.

Hsp27 Inhibition with OGX-427 Sensitizes Non-Small Cell Lung Cancer Cells to Erlotinib and Chemotherapy.

Non-small cell lung cancer (NSCLC) is the most frequent cause of death from cancer worldwide. Despite the availability of active chemotherapy regimens and EGFR tyrosine kinase inhibitors, all advanced patients develop recurrent disease after first-line therapy. Although Hsp27 is a stress-induced chaperone that promotes acquired resistance in several cancers, its relationship to treatment resistance in NSCLC has not been defined. Understanding adaptive responses of acquired resistance will help guide new strategies to control NSCLC. Hsp27 levels were evaluated in an HCC827 erlotinib-resistant-derived cell line (HCC-827Resistant), and sensitivity to erlotinib was examined in Hsp27-overexpressing A549 cells. The role of Hsp27 in both erlotinib and cytotoxic treatment resistance was evaluated in HCC-827 and A549 NSCLC cells using the Hsp27 antisense drug OGX-427. The effect of OGX-427 in combination with erlotinib was also assessed in mice bearing A549 xenografts. Hsp27 is induced by erlotinib and protects NSCLC cells from treatment-induced apoptosis, whereas OGX-427 sensitizes NSCLC cells to erlotinib. Interestingly, increased resistance to erlotinib was observed when Hsp27 was increased either in HCC827 erlotinib-resistant or overexpressing A549 cells. Combining OGX-427 with erlotinib significantly enhanced antitumor effects in vitro and delayed A549 xenograft growth in vivo. OGX-427 also significantly enhanced the activity of cytotoxic drugs used for NSCLC. These data indicate that treatment-induced Hsp27 contributes to the development of resistance, and provides preclinical proof-of-principle that inhibition of stress adaptive pathways mediated by Hsp27 enhances the activity of erlotinib and chemotherapeutics.

Overexpression of heat shock protein 27 (HSP27) increases gemcitabine sensitivity in pancreatic cancer cells through S-phase arrest and apoptosis.

We previously established a role for HSP27 as a predictive marker for therapeutic response towards gemcitabine in pancreatic cancer. Here, we investigate the underlying mechanisms of HSP27-mediated gemcitabine sensitivity. Utilizing a pancreatic cancer cell model with stable HSP27 overexpression, cell cycle arrest and apoptosis induction were analysed by flow cytometry, nuclear staining, immunoblotting and mitochondrial staining. Drug sensitivity studies were performed by proliferation assays. Hyperthermia was simulated using mild heat shock at 41.8°C. Upon gemcitabine treatment, HSP27-overexpressing cells displayed an early S-phase arrest subsequently followed by a strongly increased sub-G1 fraction. Apoptosis was characterized by PARP-, CASPASE 3-, CASPASE 8-, CASPASE 9- and BIM- activation along with a mitochondrial membrane potential loss. It was reversible through chemical caspase inhibition. Importantly, gemcitabine sensitivity and PARP cleavage were also elicited by heat shock-induced HSP27 overexpression, although to a smaller extent, in a panel of pancreatic cancer cell lines. Finally, HSP27-overexpressing pancreatic cancer cells displayed an increased sensitivity also towards death receptor-targeting agents, suggesting another pro-apoptotic role of HSP27 along the extrinsic apoptosis pathway. Taken together, in contrast to the well-established anti-apoptotic properties of HSP27 in cancer, our study reveals novel pro-apoptotic functions of HSP27—mediated through both the intrinsic and the extrinsic apoptotic pathways—at least in pancreatic cancer cells. HSP27 could represent a predictive marker of therapeutic response towards specific drug classes in pancreatic cancer and provides a novel molecular rationale for current clinical trials applying the combination of gemcitabine with regional hyperthermia in pancreatic cancer patients.

Unphosphorylated heat shock protein 27 suppresses fibroblast growth factor-2-stimulated vascular endothelial growth factor release in osteoblasts

Heat shock protein 27 (HSP27) also known as heat shock protein β1 (HSPB1) is a member of the family of small heat shock proteins ubiquitously expressed in all tissues. It has previously been demonstrated that HSP27 regulated the synthesis of osteocalcin and interleukin‑6 in osteoblast‑like MC3T3‑E1 cells. In the present study, the effect of HSP27 on basic fibroblast growth factor (FGF‑2)‑stimulated vascular endothelial growth factor (VEGF) synthesis in MC3T3‑E1 cells, was observed. The levels of VEGF release stimulated by FGF‑2 in the HSP27‑overexpressing MC3T3‑E1 cells were significantly lower compared with those in the control cells. In addition, the levels of VEGF release stimulated by FGF-2 in the phosphomimic HSP27-overexpressing cells were significantly higher compared with those in the non‑phosphorylatable HSP27‑overexpressing cells. Furthermore, no significant differences were observed in the FGF‑2‑induced phosphorylation levels of p44/p42 mitogen‑activated protein (MAP) kinase, p38 MAP kinase, stress‑activated protein kinase/c‑Jun N‑terminal kinase (SAPK/JNK) or p70 S6 kinase among the four types of transfected cells. These results suggested that unphosphorylated HSP27 attenuated the FGF‑2‑stimulated VEGF synthesis in osteoblasts.

Role of HSP27 in tumor necrosis factor-α-stimulated interleukin-6 synthesis in osteoblasts

Heat shock protein 27 (HSP27) is known to act as a molecular chaperone. We have recently reported that HSP27 regulates osteocalcin synthesis in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the role of HSP27 in tumor necrosis factor-α (TNF-α)-stimulated interleukin-6 (IL-6) synthesis in MC3T3-E1 cells. The levels of IL-6 release and IL-6 mRNA stimulated by TNF-α in MC3T3-E1 cells transfected with HSP27 was significantly higher than those in the control cells. In addition, the levels of secreted IL-6 and IL-6 mRNA in the phospho-mimic HSP27-overexpressing cells were significantly higher than those in the non-phosphoryl-atable HSP27-overexpressing cells. Furthermore, we observed no significant differences in the phosphorylation levels of IκB/NFκB, Akt, and p44/p42 mitogen-activated protein kinase among the 4 types of transfected cells. Therefore, these results strongly suggest that HSP27 enhances TNF-α-stimulated IL-6 synthesis, and that the phosphorylation status of HSP27 is related to IL-6 synthesis in osteoblasts.

Regulation by Heat Shock Protein 27 of Osteocalcin Synthesis in Osteoblasts

We have previously reported that various stimuli, including sphingosine 1-phosphate, are able to induce heat shock protein (HSP) 27 in osteoblast-like MC3T3-E1 cells. However, the precise role of HSP27 in bone metabolism has not been satisfactory clarified. In this study, we investigated the effect of HSP27 on osteocalcin synthesis induced by bone morphogenetic protein (BMP)-4 or T₃ in these cells. In MC3T3-E1 cells, pretreatment with sphingosine 1-phosphate, sodium arsenite, or heat stress caused the attenuation of osteocalcin synthesis induced by BMP-4 or T₃ with concurrent HSP27 induction. To further investigate the effect of HSP27, we established stable HSP27-transfected cells. The osteocalcin synthesis was significantly reduced in the stable HSP27-transfected MC3T3-E1 cells and normal human osteoblasts compared with empty-vector transfected cells. On the other hand, anisomycin, a p38 MAPK activator, caused the phosphorylation of HSP27 in both sphingosine 1-phosphate-stimulated untransfected MC3T3-E1 cells and HSP27-transfected MC3T3-E1 cells. An immunofluorescence microscopy study showed that the phosphorylated HSP27 induced by anisomycin concentrated perinuclearly in these cells, in which it colocalized with the endoplasmic reticulum. We also established stable mutant-HSP27-transfected cells. Osteocalcin synthesis induced by either BMP-4 or T₃ was markedly suppressed in the nonphosphorylatable HSP27-overexpressing MC3T3-E1 cells compared with the phosphomimic HSP27-overexpressing cells. In contrast, the matrix mineralization was more obvious in nonphosphorylatable HSP27-overexpressing cells than that in phosphomimic HSP27-overexpressing cells. Taken together, these results strongly suggest that unphosphorylated HSP27 has an inhibitory effect on osteocalcin synthesis, but has a stimulatory effect on mineralization, in osteoblasts.

Heat Shock Protein 27-Targeted Heptapeptide of the PKCΔ Catalytic V5 Region Sensitizes Tumors With Radio- and Chemoresistance

Previous data suggest that the PKCδ catalytic V5 (PKCδ-V5) heptapeptide (HEPT) (FEQFLDI) binds HSP27 and blocks HSP27-mediated radio- or chemoresistance. Here we investigated further the in vivo function of the PKCδ-V5 HEPT. Labeling of HEPT with Cy5.5 or fluorescein isothiocyanate was performed to evaluate in vitro or in vivo distribution of HEPT. A clonogenic survival assay, flow cytometry, and Western blotting of cleaved caspase-3 were performed to determine in vitro sensitization effects of HEPT plus ionizing radiation (IR) versus IR alone or those of HEPT plus cisplatin(Cis) versus Cis alone. A nude mouse xenografting system was also applied to detect in vivo sensitizing effects of HEPT. HEPT efficiently bound to HSP27 and showed sensitization after combined treatment with IR versus treatment with Cis alone in NCI-H1299 lung carcinoma cells, with higher HSP27 expression, which was similar to that of combined treatment with IR or with Cis alone in NCI-H460 lung carcinoma cells with lower HSP27 expression. In vivo image analysis using Cy5.5-labeled HEPT showed that HEPT was retained in HSP27-overexpressing cancer cells after xenografting to nude mice. Combined treatment of HEPT with IR versus that with Cis alone in xenografted mice showed that HEPT increased radio- or chemosensitization in NCI-H1299 cells compared to that in mice xenografted with NCI-H460 cells. The novel PKCδ-V5 HEPT may help overcome HSP27-mediated radio- or chemoresistance.

Sulforaphane Activates Heat Shock Response and Enhances Proteasome Activity through Up-regulation of Hsp27

It is conceivable that stimulating proteasome activity for rapid removal of misfolded and oxidized proteins is a promising strategy to prevent and alleviate aging-related diseases. Sulforaphane (SFN), an effective cancer preventive agent derived from cruciferous vegetables, has been shown to enhance proteasome activities in mammalian cells and to reduce the level of oxidized proteins and amyloid β-induced cytotoxicity. Here, we report that SFN activates heat shock transcription factor 1-mediated heat shock response. Specifically, SFN-induced expression of heat shock protein 27 (Hsp27) underlies SFN-stimulated proteasome activity. SFN-induced proteasome activity was significantly enhanced in Hsp27-overexpressing cells but absent in Hsp27-silenced cells. The role of Hsp27 in regulating proteasome activity was further confirmed in isogenic REG cells, in which SFN-induced proteasome activation was only observed in cells stably overexpressing Hsp27, but not in the Hsp27-free parental cells. Finally, we demonstrated that phosphorylation of Hsp27 is irrelevant to SFN-induced proteasome activation. This study provides a novel mechanism underlying SFN-induced proteasome activity. This is the first report to show that heat shock response by SFN, in addition to the antioxidant response mediated by the Keap1-Nrf2 pathway, may contribute to cytoprotection.

The role of the cysteine residue in the chaperone and anti‐apoptotic functions of human Hsp27

The small heat shock protein Hsp27 is a molecular chaperone and an anti-apoptotic protein. Human Hsp27 has one cysteine residue at position 137. We investigated the role of this cysteine residue in the chaperone and anti-apoptotic functions of Hsp27 by mutating the cysteine residue to an alanine (Hsp27(C137A)) and comparing it to wild-type protein (Hsp27(WT)). Both proteins were multi-subunit oligomers, but subunits of Hsp27(WT) were disulfide-linked unlike those of Hsp27(C137A), which were monomeric. Hsp27(C137A) was indistinguishable from Hsp27(WT) with regard to its secondary structure, surface hydrophobicity, oligomeric size and chaperone function. S-thiolation and reductive methylation of the cysteine residue had no apparent effect on the chaperone function of Hsp27(WT). The anti-apoptotic function of Hsp27(C137A) and Hsp27(WT) was studied by overexpressing them in CHO cells. No difference in the caspase-3 or -9 activity was observed in staurosporine-treated cells. The rate of apoptosis between Hsp27(C137A) and Hsp27(WT) overexpressing cells was similar whether the cells were treated with staurosporine or etoposide. However, the mutant protein was less protective relative to the wild-type protein in preventing caspase-3 and caspase-9 activation and apoptosis induced by 1 mM H(2)O(2) in CHO and HeLa cells. These data demonstrate that in human Hsp27, disulfide formation by the lone cysteine does not affect its chaperone function and anti-apoptotic function against chemical toxicants. However, oxidation of the lone cysteine in Hsp27 might at least partially affect the anti-apoptotic function against oxidative stress.

HSP27 regulates cell adhesion and invasion via modulation of focal adhesion kinase and MMP-2 expression

Heat-shock protein 27 (HSP27), a member of the small heat-shock protein family, is a molecule involved in cellular protection in response to a variety of stresses such as heat shock, toxicants, and oxidative stress. HSP27 is also known to modulate cell functions via interaction with the actin cytoskeleton. To elucidate the functions of HSP27 in adhesion and invasion in more detail, we examined NIH3T3 cells overexpressing HSP27. HSP27 overexpression affected FAK phosphorylation and focal adhesion formation, depending on integrin-mediated actin cytoskeleton polymerization. In addition, the HSP27-overexpressing cells showed a retarded cell migration and invasion in wound-healing assays. Such HSP27-mediated retarded wound healing was correlated with reduced matrix metalloproteinase-2 (MMP-2) expression. The transcription factor for MMP-2 expression, signal transducer and activator or transcription 3 (STAT3), was correspondingly less phosphorylated. When a phosphomimetic form of HSP27 was transiently transfected, migration and invasion were similarly decreased via the regulation of the FAK/STAT3/MMP-2 signaling pathway, whereas a non-phosphorylatable form of HSP27 blocked HSP27-mediated phenotypes probably due to a dominant-negative effect on phosphorylation of endogenous HSP27. Altogether, our results suggest that HSP27 can enhance cell adhesion and modulate cell migration and invasion via the coordination of FAK-dependent actin organization and STAT3-dependent MMP-2 expression, and that phosphorylation of HSP27 is indispensable to regulate this signal pathway.

Heat Shock Protein 27 Protects L929 Cells from Cisplatin-Induced Apoptosis by Enhancing Akt Activation and Abating Suppression of Thioredoxin Reductase Activity

Heat shock protein 27 (Hsp27) is up-regulated in multiple malignancies and implicated in cisplatin resistance. It is attempted to know how Hsp27 endues cell with cisplatin resistance by interfering with upstream of both apoptosis signal-regulating kinase 1 (ASK1)/p38 mitogen-activated protein kinase-activated apoptotic signaling and serine/threonine kinase Akt-dependent survival signaling. The mouse L929 cells stably transfected with human Hsp27 or its dominant-negative mutant and the human cervical cancer HeLa cells transfected with Hsp27 siRNA were used. The cisplatin-induced apoptosis and activation of ASK1, p38, and Akt were compared in control cells, cells overexpressing Hsp27, and cells with their endogenous Hsp27 knocked down. Hsp27 effectively protected the cells from cisplatin-induced DNA fragmentation. The p38 inhibitors obviously decreased whereas Akt inhibitors markedly increased the apoptotic fraction in cisplatin-treated cells. Overexpression of Hsp27 doubly enhanced the drug-induced Akt activation while substantially depressing the drug-induced activation of ASK1 and p38. Knockdown of the endogenous Hsp27 in HeLa cells resulted in the effects opposite to that observed in the Hsp27-overexpressing cells. Enhancement of Akt activation is associated with complex formation between Akt and Hsp27, whereas depression of ASK1/p38 activation is attributed to a reversion of the drug-induced inhibition of thioredoxin reductase activity and subsequent oxidation of thioredoxin. Hsp27 endues cells with cisplatin resistance via depression of the drug-induced ASK1/p38 activation and enhancement of the drug-induced Akt activation. This study revealed the intervention of Hsp27 in upstream of both ASK1/p38 apoptotic signaling and phosphatidylinositol 3-kinase/Akt survival signaling. Therefore, the inhibition of Hsp27 may be a novel strategy of cancer chemotherapy.

Overexpression of human 27 kDa heat shock protein in laryngeal cancer cells confers chemoresistance associated with cell growth delay

Among the family of heat shock proteins (HSPs), HSP70 and HSP27 have been implicated in tumorigenesis and chemoresistance, probably via the prevention of apoptosis. HSP27 levels are frequently increased in large populations of tumors of the head and neck, but the mechanism of its chemoresistance is not yet fully understood. In the present study, the role of HSP27 in the resistance to cytotoxic stress was studied in Hep-2 human laryngeal cancer cells. We established a Hep-2 cell line overexpressing HSP27 and examined whether the expression of HSP27 provides resistance to heat shock and several cytotoxic agents using a MTT colorimetic assay. Cell cycle progression was assessed by flow cytometry and fluorescence staining was performed for F-actin filaments. HSP27 overexpression induced cellular resistance to heat shock at 45 degrees C for 1 h as well as against several cytotoxic agents, including cisplatin, staurosporin and H(2)O(2). However, no difference in sensitivity to irradiation or serum starvation was found. Moreover, HSP27 overexpressing Hep-2 cells showed a delayed cell growth, compared to control cells. To determine if the decreased cell proliferation in HSP27 overexpressing cells contributed to chemoresistance, control Hep-2 cells were synchronized at the late G1 phase by treatment with mimosine. The synchronized Hep-2 cells were resistant to cisplatin and H(2)O(2), but not to irradiation or serum starvation, correlating the protection effect shown in HSP27 overexpressing cells. These results suggest that the overexpression of HSP27 in Hep-2 cells confers chemoresistance which is associated with the delay in cell growth. We also propose that the stabilization of F-actin observed in Hep-2/hsp27 cells is partly related to the delay in cell cycle progression, by showing that the induction of actin polymerization in Hep-2/neo cells results in the retardation of cell growth as well as a cytoprotective effect as observed in Hep-2/hsp27.

Heat shock protein 27 interacts with vimentin and prevents insolubilization of vimentin subunits induced by cadmium

Vimentin is an intermediate filament that regulates cell attachment and subcellular organization. In this study, vimentin filaments were morphologically altered, and its soluble subunits were rapidly reduced via cadmium chloride treatment. Cadmium chloride stimulated three major mitogen-activated protein kinases (MAPKs): extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38, and led apoptotic pathway via caspase-9 and caspase-3 activations. In order to determine whether MAPKs were involved in this cadmium-induced soluble vimentin disappearance, we applied MAPK-specific inhibitors (PD98059, SP600125, SB203580). These inhibitors did not abolish the cadmium-induced soluble vimentin disappearance. Caspase and proteosome degradation pathway were also not involved in soluble vimentin disappearance. When we observed vimentin levels in soluble and insoluble fractions, soluble vimentin subunits shifted to an insoluble fraction. As we discovered that heat-shock protein 27 (HSP27) was colocalized and physically associated with vimentin in unstressed cells, the roles of HSP27 with regard to vimentin were assessed. HSP27-overexpressing cells prevented morphological alterations of the vimentin filaments, as well as reductions of soluble vimentin, in the cadmium-treated cells. Moreover, HSP27 antisense oligonucleotide augmented these cadmium-induced changes in vimentin. These findings indicate that HSP27 prevents disruption of the vimentin intermediate filament networks and soluble vimentin disappearance, by virtue of its physical interaction with vimentin in cadmium-treated SK-N-SH cells.

Up-regulation of Hsp27 Plays a Role in the Resistance of Human Colon Carcinoma HT29 Cells to Photooxidative Stress¶

The Photofrin-resistant cell line (HT29-P14) was used in the present study to investigate the mechanism(s) involved in Photofrin-mediated photodynamic therapy (PDT). We compared gene expression profiles between the resistant cell line and its parental cell line (HT29) using DNA microarray analysis. A significant up-regulation of small heat shock protein 27 (Hsp27) was found in HT29-P14 cells. The elevated Hsp27 level may play an important role in the resistance of HT29-P14 to Photofrin-PDT. To test this hypothesis, we stably transfected HT29 cells with human Hsp27 complementary DNA. The potential role of Hsp27 in the resistance to PDT was examined in Hsp27-overexpressing cells. Stable trasnfected cells (H13) showed an increased survival after Photofrin-PDT, suggesting that the up-regulation of Hsp27 is related to the induced resistance to Photofrin-PDT. Phosphorylation of Hsp27 has been suggested to play an important role in cytoprotection. We have examined the phosphorylation activity of Hsp27 among the parental and resistant cells, as well as the overexpression cells. An elevated level of Hsp27 resulted in an increased ability of phosphorylation in both resistant and overexpressing cells after PDT. The activation of the phosphorylation of Hsp27 induced by PDT was not mediated by the p38 mitogen-activated protein kinase. These data suggest that Hsp27 may play an important role in mediating the adaptive response to Photofrin-PDT-induced oxidative stress and that the pathways leading to Hsp27 phosphorylation may contribute to the resistance of the cells to photooxidative damage.

Overexpression of Hsp27 affects the metastatic phenotype of human melanoma cells in vitro.

Overexpression of the small heat shock protein Hsp27 has been shown by us to inhibit the in vitro proliferation rate and to delay tumor development of a human melanoma cell line (A375) in nude mice. We hypothesized that Hsp27 may influence the neoplastic phenotype. In the present study Hsp27 transfectants from this cell line were analyzed for various cellular aspects associated with the metastatic process. We found that Hsp27-overexpressing clones exhibited an altered cellular morphology as compared with control transfected cells. The Hsp27-positive cells tended to develop an epithelial-like phenotype growing in clusters and were characterized by a loss of transcytoplasmic stressfibers. In parallel, Hsp27-expressing cells lost the ability to form colonies in soft agar. The invasive potential was studied in vitro by the use of a reconstituted extracellular matrix-coated filter (Matrigel). Compared with controls, Hsp27-overexpressing cells showed decreased cell invasiveness through Matrigel. A correlation between invasion and activation of matrix metalloproteinases (MMPs) has been shown in several cell models. Secretion of MMPs (MMP-2 and MMP-9) was studied by gelatin-substrate zymogram analysis, as well as by a sensitive gelatinase activity assay. The Hsp27-transfected A375 melanoma cell line showed decreased secretion of MMP-2 and MMP-9 as compared with the control transfected cells. Integrins are adhesion receptors and function in cell invasion by mediating cell movement on matrix molecules and by regulating the expression of MMPs. Both fluorescence-activated cell sorter analysis and immunofluorescence analysis revealed a loss of alpha(v)beta3 integrin in Hsp27-transfected cell colonies. Our results demonstrate that Hsp27 overexpression has a profound impact on several parameters regulating the invasive and metastatic potential of melanoma cells in vitro.

Hsp27-Induced MMP-9 Expression Is Influenced by the Src Tyrosine Protein Kinase Yes

The small heat shock protein hsp27 is associated with aggressive tumor behavior in certain subsets of breast cancer patients. Previously we demonstrated that hsp27 overexpression in breast cancer cells increased in vitro and in vivo invasiveness, suggesting that hsp27 influences the metastatic process. To investigate this role for hsp27, we have utilized MDA-MB-231 breast cancer cells that overexpress hsp27 and cDNA expression array technology. We demonstrate that hsp27 overexpression up-regulates MMP-9 expression and activity and down-regulates Yes expression. Furthermore, our results suggest that Yes may be involved in regulating MMP-9 expression, as well as in vitro invasion. Reconstitution of Yes expression by transfection into hsp27-overexpressing cells decreased MMP-9 expression, and increased in vitro invasiveness, abrogating the phenotype conferred by hsp27 overexpression. Therefore, our results provide a new potential mechanism by which hsp27 affects the metastatic cascade—through regulation of MMP-9 and Yes expression.