Prognostic significance of heat shock protein 27 (HSP27) in patients with oral squamous cell carcinoma

Heat shock proteins (HSPs) have been defined as proteins induced by heat shock and other environmental and pathophysiologic stress. Heat shock protein 27 (HSP27) is one of the small heat shock proteins. HSP27 is implicated in protein-protein interactions such as folding, translocation, and prevention of inappropriate protein aggregation. Many of their functions suggest that they play important roles in cancers. Archival tissues from 40 patients with oral squamous cell carcinoma who received primary surgical resection were examined for HSP27 by immunohistochemistry and correlated with clinical stage, lymph node metastasis, histological grade and survival period. HSP27 expression was positive staining (+) in 20 (50%), weak or negative staining (-) in 20 (50%) of total 40 cases. There was no correlation between HSP27 expression and clinical stage, lymph node metastasis and histological grade. However, when compared with clinicopathological features, the expression of HSP27 correlated inversely with survival period. This study suggests that the expression of HSP27 is frequently promoted in patients with oral squamous cell carcinoma and should be considered an independent prognostic factor of oral squamous cell carcinoma patients.     

Geldanamycin and 17-allylamino-17-demethoxygeldanamycin potentiate the in vitro and in vivo radiation response of cervical tumor cells via the heat shock protein 90-mediated intracellular signaling and cytotoxicity

Ansamycin antibiotics inhibit function of the heat shock protein (HSP) 90, causing selective degradation of several intracellular proteins regulating such processes as proliferation, cell cycle regulation, and prosurvival signaling cascades. HSP90 has been identified previously as a molecular target for anticancer agents, including ionizing radiation (IR). Therefore, we hypothesized that the ansamycin geldanamycin and its 17-allylamino-17-demethoxy analog (17-AAG), which inhibit HSP90, would enhance tumor cell susceptibility to the cytotoxicity of IR. Treatment of two human cervical carcinoma cell lines (HeLa and SiHa) with geldanamycin and 17-AAG resulted in cytotoxicity and, when combined with IR, enhanced the radiation response, each effect with a temporal range from 6 to 48 h after drug exposure. In addition, mouse in vivo models using 17-AAG at clinically achievable concentrations yielded results that paralleled the in vitro radiosensitization studies of both single and fractioned courses of irradiation. The increase in IR-induced cell death appears to be attributable to a combination of both programmed and nonprogrammed cell death. We also measured total levels of several prosurvival and apoptotic signaling proteins. Akt1, extracellular signal-regulated kinase-1, Glut-1, HER-2/neu, Lyn, cAMP-dependent protein kinase, Raf-1, and vascular endothelial growth factor expression were down-regulated in 17-AAG-treated cells, identifying these factors as molecular markers and potential therapeutic targets. Finally, a series of immortalized and human papillomavirus-transformed cell lines were used to demonstrate that the radiosensitizing effects of 17-AAG were limited to transformed cells, suggesting a possible differential cytotoxic effect. This work shows that altered HSP90 function induces significant tumor cytotoxicity and radiosensitization, suggesting a potential therapeutic utility.     

Heat stress: A risk factor for skin carcinogenesis

Recent evidence suggests that heat stress may also be a risk factor of skin carcinogenesis. Heat stress causes activation of heat shock proteins (HSPs), chaperone proteins which prevent cells from undergoing apoptosis and ensuring their cellular function. However, HSPs recruitment may also have deleterious effects particularly if the cells rescued from apoptosis carry oncogenic mutations. We hypothesise that exposures to both heat and UV induce skin cancer(s) by concomitant expression of HSPs and oncogenic mutant proteins. Here we review studies demonstrating that heat stress-activated heat shock proteins such as HSP72 and HSP90 can influence signalling pathways such as MAPK, JNK and p53, which are all involved in regulating cell proliferation, survival and apoptosis.     

Heat shock protein expression in testis and bladder cancer cell lines exhibiting differential sensitivity to heat.

Testis cancer cells are more sensitive than bladder and most other cancer cells to chemotherapeutic drugs both in the clinic and in vitro. In this study we show that they are also more sensitive than bladder cancer cells to heat. Since heat and drug sensitivity may be related to the ability of a cell to mount a stress response, constitutive and induced levels of heat shock proteins (HSPs) in three testis and three bladder human cancer cell lines were measured using Western blotting and scanning densitometry. No correlation between constitutive levels of HSP 90 or HSP 73/72 and cellular heat sensitivity was found. However, HSP 27 levels were much lower in the testis tumour cells, suggesting that low HSP 27 expression might contribute to heat sensitivity. Protein synthesis studies using [35S]methionine indicated that, for the same heat shocks, the kinetics of synthesis and decay of HSP 90 and HSP 73/72 in 833K (the most heat sensitive testis cells) was similar to or greater than that in HT1376 (the most heat-resistant bladder cells). Both 833K and HT1376 developed thermotolerance, and this followed an increase in synthesis of HSPs. These results indicate that, although there are differences in the constitutive levels of HSPs between testis and bladder cancer cells, both cell types are capable of mounting an induced heat shock response and can develop a similar degree of thermotolerance.     

Inhibition of telomerase activity enhances hyperthermia-mediated radiosensitization

Hyperthermia is a potent sensitizer of cell killing by ionizing radiation (IR); however, hyperthermia also induces heat shock protein 70 (HSP70) synthesis and HSP70 expression is associated with radioresistance. Because HSP70 interacts with the telomerase complex and expression of the telomerase catalytic unit (hTERT) extends the life span of the human cells, we determined if heat shock influences telomerase activity and whether telomerase inhibition enhances heat-mediated IR-induced cell killing. In the present study, we show that moderate hyperthermia (43 degrees C) enhances telomerase activity. Inhibition of telomerase activity with human telomerase RNA-targeted antisense agents, and in particular GRN163L, results in enhanced hyperthermia-mediated IR-induced cell killing, and ectopic expression of catalytic unit of telomerase (TERT) decreased hyperthermia-mediated IR-induced cell killing. The increased cell killing by heat and IR exposure in telomerase-inhibited cells correlates with delayed appearance and disappearance of gamma-H2AX foci as well as decreased chromosome repair. These results suggest that inactivation of telomerase before combined hyperthermia and radiotherapy could improve tumor killing.     

Thermotolerance and sensitivity of human cancer cells to cisplatin and doxorubicin

Testis tumour cells are more sensitive than most other types of cancer cell to heat, radiation and chemotherapeutic drugs, both in the clinic and in vitro. Since heat shock proteins (HSP) can protect cells from the cytotoxic effects of stress, we studied their role in the sensitivity of testis tumour cells to the frequently used cancer chemotherapeutic drugs, cisplatin and doxorubicin. Clonogenic assays of 3 testis tumour cell lines (833K, GCT27, GH) and 3 bladder cancer cell lines (RT112, HT1376, MGH-U1) following a 1 h exposure to the two drugs confirmed that the testis tumour cell lines were inherently more sensitive. Having shown previously that heat shock proteins were upregulated in both cell types following a heat shock, in this study no induction of HSP was seen following treatment for 1 h with cisplatin or doxorubicin (at concentrations reducing colony forming ability by 50%) in either cell type. In contrast, chronic exposure to cisplatin (at concentrations on the threshold of cytotoxicity), but not doxorubicin, resulted in upregulation of HSP 27, but not HSP73/72, in both cell types. Testis and bladder cancer cells with heat-induced increases in HSP were thermotolerant, and this was associated with increased resistance to doxorubicin, but not cisplatin.     

Heat shock proteins in breast cancer progression–A suitable case for treatment?

Heat shock proteins (HSP) and heat shock factor 1 (HSF1), key factors in the heat shock response (HSR) have been implicated in the etiology of breast cancer. At least two members of the HSP family, Hsp27 and Hsp70 undergo significant increases in cellular concentration during the transformation of mammary cells. These changes result in HSP-mediated inhibition of tumour cell inactivation through blockade of the apoptosis and replicative senescence pathways. The increases in HSP thus mediate two of the common hallmarks of cancer and favour cell birth over cell death. In addition, Hsp90 plays a role in facilitating transformation by stabilising the mutated and over-expressed oncoproteins found in breast tumours, and permitting the activation of growth stimulatory and transforming pathways in the absence of growth factors. HSF1 appears to play a similar role as a facilitator of transformation in mammary carcinoma. Induction of some facets of the HSR in breast cancer cells therefore leads to growth stimulation and inhibits cell death. Pharmacological targeting of HSP and HSF1 is therefore indicated and in the case of Hsp90, inhibitory drugs are undergoing clinical trial for treatment of breast carcinoma and other cancers.     

LY294002, an inhibitor of PI-3K, enhances heat sensitivity independently of p53 status in human lung cancer cells

The aim of this study was to ascertain whether LY294002, an inhibitor of PI-3K, enhances heat sensitivity in human cancer cells regardless of their p53 status. Colony formation assays showed that LY294002 enhanced heat sensitivity in two human lung cancer cell lines; H1299/wild-type p53 (wtp53) and H1299/mutated p53 (mp53) cells. These cell lines have identical genetic backgrounds except for their p53 status. LY294002 suppressed the heat-induced accumulation of heat shock protein 27 (hsp27) and heat shock protein 72 (hsp72) in these cell lines. Heat-induced apoptosis was observed more frequently in H1299/wtp53 cells than in H1299/mp53 cells, and was enhanced by LY294002 in both cell lines. In addition, both the heat-induced phosphorylation of Akt and the accumulation of survivin were suppressed by LY294002. These results suggest that LY294002 inhibits anti-apoptosis signaling through hsp27 and hsp72 as well as cell survival signaling through Akt and survivin. LY294002 appears to be an attractive candidate for a p53-independent heat sensitizer in hyperthermic cancer therapy.     

Heat shock protein 72 expression allows permissive replication of oncolytic adenovirus dl1520 (ONYX-015) in rat glioblastoma cells

In this study we have made novel observations with regards to potentiation of the tumoricidal activity of the oncolytic adenovirus, dl1520 (ONYX-015) in rat glioblastoma cell lines expressing heat shock protein 72 (HSP72) due to permissive virus replication. ONYX-015 is a conditionally replicating adenovirus that is deleted for the E1B 55 kDA gene product whose normal function is to interact with cell-cycle regulatory proteins to permit virus replication. However, many murine and rodent cell lines are not permissive for adenovirus replication. Previously, it has been reported that the heat shock response is necessary for adenovirus replication and that induction of heat shock proteins is mediated by E1 region gene products. Therefore, we hypothesized that HSP72 expression may allow for permissive replication of ONYX-015 in previously non-permissive cells. Rat glioma cell lines 9L and RT2 were transfected with a plasmids expressing HSP72 or GFP. After infection with ONYX-015, no tumoricidal activity is observed in GFP expressing cell lines despite adequate transduction. In contrast, HSP72 transfected cells show cytopathic effects by 72 hours and greater than 75% loss of viability by 96 hours. Burst assays show active virus replication in the HSP72 expressing cell lines. Therefore, 9L-HSP72 and RT2-HSP72 are ideal models to evaluate the efficacy of ONYX-015 in an immunocompetent rat model. Our study has implications for creating rodent tumor models for pre-clinical studies with E1 region deleted conditionally replicating adenovirus.     

HSP27高表达与人白血病多药耐药细胞K562/VCR的关系

背景与目的:白血病细胞多药耐药(multidrug resistance,MDR)是白血病化疗失败的常见原因,虽然已有研究揭示了一些肿瘤MDR机制,但目前仍然不能完全解释MDR现象。本文旨在应用蛋白质组学方法筛选白血病耐药相关蛋白,并研究其与白血病MDR的关系,为进一步阐明白血病MDR发生的分子机制提供理论依据。方法:二维聚丙烯酰胺凝胶电泳(two-dimensional electrophoresis,2-DE)技术分离白血病细胞K562和人白血病耐药细胞K562/VCR的总蛋白,用基质辅助激光解吸电离飞行时间质谱(matrix assisted laser desorption/ionization-time offlight-mass spectrometry,MALDI-TOF-MS)对差异表达的蛋白质点进行鉴定。应用反义核酸技术将分离鉴定差异表达蛋白的反义核酸转染至耐药细胞,Western blot检测转染后差异蛋白的表达情况,MTT法检测转染后细胞存活率,流式细胞仪检测转染后细胞凋亡率。结果:在K562/VCR细胞与K562细胞中鉴定出一差异表达蛋白点,并用质谱分析证实其为热休克蛋白27(heat shock protein27,HSP27)。用HSP27反义核酸转染K562/VCR细胞,在不同浓度长春新碱作用下,HSP27反义核酸转染的K562/VCR细胞的存活率较对照错义核酸转染组明显降低(P<0.05),流式细胞仪显示细胞凋亡率为16.37%,明显高于对照组(P<0.05)。结论:HSP27在K562/VCR细胞中高表达,其表达抑制后,K562/VCR细胞对长春新碱的敏感性增强。     

Heat shock proteins in hepatocellular carcinoma: Molecular mechanism and therapeutic potential

Heat shock proteins (HSPs) are highly conserved proteins, which are expressed at low levels under normal conditions, but significantly induced in response to cellular stresses. As molecular chaperones, HSPs play crucial roles in protein homeostasis, apoptosis, invasion and cellular signaling transduction. The induction of HSPs is an important part of heat shock response, which could help cancer cells to adapt to stress conditions. Because of the constant stress condition in tumor microenvironment, HSPs overexpression is widely reported in many human cancers. In light of the significance of HSPs for cancer cells to survive and obtain invasive phenotype under stress condition, HSPs are often associated with poor prognosis and treatment resistance in many types of human cancers. It has been described that upregulation of HSPs may serve as diagnostic and prognostic markers in hepatocellular carcinoma (HCC). Targeting HSPs with specific inhibitor alone or in combination with chemotherapy regimens holds promise for the improvement of outcomes for HCC patients. In this review, we summarize the expression profiles, functions and molecular mechanisms of HSPs (HSP27, HSP70 and HSP90) as well as a HSP‐like protein (clusterin) in HCC. In addition, we address progression and challenges in targeting these HSPs as novel therapeutic strategies in HCC.     

Prognostic significance of heat shock proteins 27 and 70 in patients with squamous cell carcinoma of the esophagus

BACKGROUND: Heat shock proteins (HSPs) first were defined as proteins induced by heat shock and other environmental and pathophysiologic stresses and are implicated in protein-protein interactions such as folding, translocation, and prevention of inappropriate protein aggregation. Many of their functions suggest that they play important roles in cancer. METHODS: Immunohistochemical study for HSP 27 and HSP 70 was performed on buffered formalin fixed, paraffin embedded sections of 102 esophageal squamous cell carcinoma specimens using monoclonal anti-HSP 27 antibody and anti-HSP 70 antibody. RESULTS: Normal squamous cells expressed both HSP 27 and HSP 70 with the exception of the basal layer. In cancerous tissue, expression of HSP 27 was evaluated as positive (+) (39 cases; 38%), reduced (+/-) (53 cases; 52%), or negative (-) (10 cases; 10%) and expression of HSP 70 was evaluated as (+) (14 cases; 14%), (+/-) (57 cases; 56%), or (-) (31 cases; 30%). There was a strong correlation between the expression of HSP 27 and HSP 70 (P < 0.0001). When compared with clinicopathologic features, expression of both HSP 27 and HSP 70 correlated negatively with lymph node metastases (P < 0.05), but not with depth of invasion or histologic grade. The reduction of the HSPs was associated significantly with poor postoperative survival (P < 0.0001). In addition, multivariate analysis revealed that HSP 27 (-) was the strongest prognostic factor among the clinicopathologic features. CONCLUSIONS: This study suggests that the expression of HSP 27 and HSP 70 frequently is reduced in patients with esophageal squamous cell carcinoma and therefore should be considered an independent prognostic factor of this disease.     

Heat shock protein 27, a novel regulator of 5-fluorouracil resistance in colon cancer

The resistance of colon cancer to 5-fluorouracil (5-FU) is a critical issue, and the cause of this resistance cannot always be explained based on the known molecules. Heat shock protein 27 (HSP27) mRNA expression has recently been shown to be correlated with 5-FU resistance in 5-FU-resistant cell lines. In this study, we attempted to elucidate the functional mechanism of HSP27 in 5-FU resistance in colon cancer. HSP27 protein levels in several human colon cancer cell lines (LoVo, HCT15, WiDr, HCT116, HT-29 and SW480) were determined by immunoblot and densitometry analysis. The in vitro growth inhibition rates (IR) of the cell lines at various concentrations of 5-FU were assessed by MTT assay. The degree of 5-FU resistance was estimated as the drug concentration inducing 50% IR (IC50). The HSP27 protein level and IC50 were significantly correlated in these cell lines (p=0.010). The effect of HSP27 overexpression on IC50 was evaluated in LoVo cells. HSP27 transfectants significantly increased IC50 and reduced HSP27 resistance. The effect of HSP27 down-regulation by HSP27 siRNA on IC50 was confirmed in HCT15 cells. HSP27 siRNA suppressed HSP27 protein levels and reduced IC50 in a dose-dependent manner. These data indicated that HSP27 is closely connected with 5-FU resistance in colon cancer and suggested that HSP27 levels predicted 5-FU resistance. HSP27 down-regulation overcame 5-FU resistance and HSP27 may be a clinical target in patients with 5-FU-resistant colon cancer.     

Heat-shock protein expression in cisplatin-sensitive and -resistant human tumor cells

It has been suggested that the expression of certain heat-shock proteins (HSPs) may be prognostic markers in several tumor types. Since HSPs may be involved in determining cellular sensitivity to chemotherapeutic drugs, the possible relation between HSP expression and cisplatin (cDDP) sensitivity was studied. Three human germ-cell tumor cell lines, 1 human small-cell lung carcinoma (SCLC) cell line and 3 human colon carcinoma cell lines were used as a model for differences in intrinsic cDDP sensitivity. The constitutive expression of a panel of HSPs was studied by immunoblotting. No correlation was found between expression of HSP90, HSP73, HSP72, HSP60 and HSP27 and the extent of intrinsic cDDP sensitivity when all cell lines studied were considered. However, for the 3 cell lines derived from germ-cell carcinomas, HSP27 expression was inversely related to cDDP sensitivity, i.e. decreased HSP27 levels were associated with decreased sensitivity. Constitutive HSP expression was also studied in 2 sets of human cell lines with in vitro acquired cDDP resistance. In both resistant cell lines, decreased expression of HSP27 (as determined by Western blotting) was found as compared to the sensitive parent cell lines. Thus, acquired resistance to cDDP was also accompanied by decreased HSP27 expression. Interestingly, when basal HSP27 mRNA levels were measured in the SCLC cell line (GLC₄) and its subline with acquired resistance (GLC₄-cDDP), no significant differences were detected. Continuous cDDP incubation increased HSP27 levels and induced HSP27 phosphorylation in GLC₄ cells, but not in the resistant subline. Thus, although no general relationships between HSP expression and cDDP sensitivity are apparent, high HSP27 expression in vitro relates to high sensitivity to cDDP treatment in some tumor types. This is in accordance with reported clinical data on high HSP27 levels in tumors correlating with good prognosis. © 1996 Wiley-Liss, Inc.     

热休克蛋白 HSP27 抑制食管鳞癌细胞的侵袭转移能力简

目的：探讨HSP27分子的表达水平与食管鳞癌高、低转移潜能细胞系侵袭转移能力的关系。方法：选用EC9706-H、EC9706-L和EC109-H、EC109-L两对食管鳞癌高、低转移潜能细胞系,利用细胞划痕实验检测不同细胞侵袭转移能力的差异;利用Western blot检测HSP27在不同转移潜能细胞中的表达水平;利用慢病毒转染技术上调HSP27低表达细胞中HSP27的表达,采用细胞划痕实验检测其侵袭转移能力的变化。结果：细胞划痕实验证实,EC9706-H细胞和EC109-H细胞的体外侵袭转移能力高于EC9706-L细胞和EC109-L细胞;Western blot实验结果显示,HSP27在EC9706-H细胞和EC109-H细胞中呈低表达,在EC9706-L细胞和EC109-L细胞中呈高表达;通过慢病毒转染技术上调EC9706-H细胞和EC109-H细胞中HSP27的表达,二者的侵袭转移能力明显降低。结论：HSP27与食管鳞癌的侵袭转移能力呈负相关,即HSP27高表达的食管鳞癌细胞其侵袭转移能力受到抑制。     

热休克蛋白27与肿瘤关系的研究进展

热休克蛋白27（HSP27）是一种重要的小分子热休克蛋白。它与肿瘤的关系非常密切,与肿瘤细胞的耐药性、照射敏感性及肿瘤患者的预后直接相关,现对此作一简要综述。     

HSP90 inhibitor 17AAG causes apoptosis in ATRA-resistant acute promyelocytic leukemia cells

The effects of a novel heat shock protein inhibitor, 17AAG, on established APL cell lines (NB4 and R1) were analyzed. 17AAG induces apoptosis in APL cell lines both sensitive (NB4) and resistant (R1) to ATRA after 72 h of incubation. Apoptosis occurs by a mechanism different than ATRA-mediated response, as the cells do not undergo differentiation before apoptosis. Analysis of bax and bcl-2 shows that pro-apoptotic (bax) and anti-apoptotic (bcl-2) proteins are decreased in expression after incubation with 17AAG. We believe this data supports potential clinical use of agents that target HSP90 in APL patients failing conventional therapy.     

Radioresistant human lung adenocarcinoma cells that survived multiple fractions of ionizing radiation are sensitive to HSP90 inhibition

Despite the common usage of radiotherapy for the treatment of NSCLC, outcomes for these cancers when treated with ionizing radiation (IR) are still unsatisfactory. A better understanding of the mechanisms underlying resistance to IR is needed to design approaches to eliminate the radioresistant cells and prevent tumor recurrence and metastases. Using multiple fractions of IR we generated radioresistant cells from T2821 and T2851 human lung adenocarcinoma cells. The radioresistant phenotypes present in T2821/R and T2851/R cells include multiple changes in DNA repair genes and proteins expression, upregulation of EMT markers, alterations of cell cycle distribution, upregulation of PI3K/AKT signaling and elevated production of growth factors, cytokines, important for lung cancer progression, such as IL-6, PDGFB and SDF-1 (CXCL12). In addition to being radioresistant these cells were also found to be resistant to cisplatin.HSP90 is a molecular chaperone involved in stabilization and function of multiple client proteins implicated in NSCLC cell survival and radioresistance. We examined the effect of ganetespib, a novel HSP90 inhibitor, on T2821/R and T2851/R cell survival, migration and radioresistance. Our data indicates that ganetespib has cytotoxic activity against parental T2821 and T2851 cells and radioresistant T2821/R and T2851/R lung tumor cells. Ganetespib does not affect proliferation of normal human lung fibroblasts. Combining IR with ganetespib completely abrogates clonogenic survival of radioresistant cells.Our data show that HSP90 inhibition can potentiate the effect of radiotherapy and eliminate radioresistant and cisplatin -resistant residual cells, thus it may aid in reducing NSCLC tumor recurrence after fractionated radiotherapy.     

热休克蛋白27在乳腺癌发生发展中的作用

乳腺癌已经成为继肺癌之后威胁人类生命的第二大癌症。热休克蛋白27(heat shock protein 27, HSP27)是真核生物细胞内普遍存在的一种小分子热休克蛋白, 作为一种ATP非依赖性分子伴侣, 当细胞受到各种生理和环境上的刺激出现非致命性的细胞损伤时发挥保护细胞的功能。近年来的研究发现乳腺癌细胞中存在着HSP27的持续高水平表达, 且被认为与乳腺癌的发生、发展、预后以及化疗耐药的发生有密切的关系。肿瘤的发生、增殖、侵袭和转移是一个极其复杂的多步骤动态过程, 深入研究HSP27在乳腺癌发生、发展中的作用机制, 将有望为乳腺癌的防治提供新思路。      

HSP27在头颈鳞癌中的研究进展

HSP27是小分子量热休克蛋白(small heat stress proteirs,sHSP)家族中的重要一员,其重要的生物学功能是保护细胞免受各种应激因素的损伤.此外,HSP27也可参与细胞的增殖、分化及细胞凋亡的信号转导通路调节等.近年来,HSP27与临床疾病尤其是与肿瘤的关系日益受到重视,本文就HSP27在头颈鳞癌中的研究进展作一综述.