Antiproliferative and apoptotic activities of tosylcyclonovobiocic acids as potent heat shock protein 90 inhibitors in human cancer cells

We evaluated whether inhibition of heat shock protein 90 (hsp90) function by novobiocin derivatives could induce the degradation of signal transducers that drive cancer cell growth and thereby promote apoptosis. Removal of the noviose moiety in novobiocin and introduction of a tosyl substituent at C-4 or C-7 coumarin nucleus provided derivatives 4TCNA and 7TCNA which compared favourably with novobiocin in MCF-7 breast cancer cells. Here we extend the antiproliferative and apoptotic properties of these analogues to a panel of cancer cell lines. Destabilization of hsp90 client proteins Raf-1, HER2, and cdk4 suggests inhibition of hsp90 chaperoning function. In HT29 colon and IGROV1 ovarian cancer cells, the growth inhibiting effect of 4TCNA and 7TCNA was consistent with the stimulation of cell death as assessed by the processing and activation of caspase 9, 8, 7 and 3 and the subsequent cleavage of poly(ADP-ribose) polymerase (PARP). In Ishikawa endometrial adenocarcinoma cells, 4TCNA also promoted apoptosis and the processing of PARP. These derivatives impacting multiple pathways involved in the neoplastic process may represent promising drugs for cancer therapy.     

Heat shock proteins: role in thermotolerance, drug resistance, and relationship to DNA topoisomerases

The events following a mild heat exposure to cells that appear to be closely linked are: enhanced synthesis of hsp's and thermotolerance. In some cases, thermotolerance is also associated with drug resistance. We have recently examined the role that DNA topoisomerase II may play in the induction of these phenomena. VM-26 was found to both initiate hsp synthesis and to cause thermotolerance. Furthermore, the permanent heat resistant or transient thermotolerant cells were more resistant to VM-26 than controls. These results suggest that topoisomerase II, or more likely topoisomerase II-DNA complexes, are affected by heat or by VM-26 in a phenomenologically overlapping manner. Elevated levels of hsp's apparently protect cells against the cytotoxic action of both heat and VM-26.     

Hsp27 overexpression inhibits doxorubicin–induced apoptosis in human breast cancer cells

Previously we demonstrated that heat shock protein 27 (hsp27) overexpression confers resistance to the chemotherapeutic agent doxorubicin in MDA–MB–231 breast cancer cells. Since induction of apoptosis is one underlying mechanism of chemotherapeutic drug action, we investigated the effect of hsp27 overexpression on doxorubicin–induced apoptosis, finding that hsp27 protects MDA–MB–231 cells from apoptosis. We also examined expression of the doxorubicin target, topoisomerase II (topo II), in control and hsp27–overexpressing stable transfectants, as topo II expression is important for both drug sensitivity and the initiation of apoptosis by doxorubicin. The relative levels of both topo II and were higher in the controls than the hsp27–overexpressing clones, suggesting that the apoptotic protective effect of hsp27 overexpression in MDA–MB–231 cells is associated with altered topo II expression.abstract     

Cell-free 27 kDa heat shock protein (hsp27) and hsp27-cytochrome c complexes in the cervix of women with ovarian or endometrial cancer

Antibodies to the 27 kDa heat shock protein (hsp27) are present in some women with ovarian and endometrial cancers but not in women with nonmalignant conditions or healthy women. The appearance of these antibodies suggests that the corresponding protein (hsp27) may be present in an extracellular form in gynecologic cancer patients. Synthesis of hsp27 is upregulated in gynecologic cancers and inhibits induction of apoptosis. We now report the detection of hsp27 as well as hsp27-cytochrome c complexes in cell-free endocervical or posterior vaginal specimens from women with endometrial or ovarian cancer. Specimens were obtained with a cotton swab from 209 consecutive patients seen by a gynecologic oncologist. After removal of cellular components, aliquots of supernatants were assayed by ELISA for hsp27, using cytochrome c bound to microtiter plate wells, and for hsp27-cytochrome c complexes, using antibodies to cytochrome c and hsp27. Among 47 women with ovarian cancer, 38.3% were positive for hsp27 and 27.7% had hsp27-cytochrome c complexes. Similarly, among 52 women with endometrial cancer, 34.6% were hsp27-positive and 30.8% had hsp27-cytochrome c complexes. In contrast to the women with ovarian or endometrial cancer, of the 86 women with benign diagnoses only, 10.5% had cervical hsp27 (p < 0.002) and 8.1% had hsp27-cytochrome c complexes (p < 0.004). Among ovarian cancer patients, hsp27 was identified in 44.0% of the 25 women with active disease as opposed to 17.6% of the 17 patients in remission (p < 0.05). In women with stage 1-2 active ovarian cancer, 8 of 10 (80.0%) were hsp27-positive as opposed to 3 of 14 (21.4%) stage 3-4 patients (p < 0.01). For hsp27-cytochrome c complexes, 50% of ovarian cancer patients with active stage 1-2 disease as opposed to 21.4% with stage 3-4 disease were positive. Among women with endometrial cancer, only 10 of the 52 patients had active disease and 44 were in stage 1-2. For this malignancy, there was no relation between detection of hsp27 or hsp27-cytochrome c and active disease or cancer stage. Our results suggest that cell-free hsp27 and hsp27-cytochrome c complexes can be detected in the lower genital tract of women with ovarian and endometrial cancers. Identification of these biomarkers may be beneficial in the early diagnosis of these malignancies.     

Mu3 opiate receptor expression in lung and lung carcinoma: ligand binding and coupling to nitric oxide release

The integral roles of heat shock proteins (hsps) in the cell cycle and in multistep processes leading to tumorigenesis have been implied. We examined the expression of hsp90alpha, hsp90beta and cyclin D1 in human breast cancer. Levels of mRNAs coding for hsp90alpha and cyclin D1 were significantly higher in cancer tissues than in non-cancer tissues. Moreover, there was a close relationship between the extent of the two mRNA levels, suggesting that increased expression of hsp90alpha, an isoform of the hsp90 family, is associated with the proliferation of human breast cancer. Hsp90beta was expressed in cancer cells, but not associated with cell proliferation.     

Identification of heat shock protein 90 and other proteins as tumour antigens by serological screening of an ovarian carcinoma expression library

Serological screening of recombinant cDNA expression libraries has been widely used for the identification of tumour antigens in various cancer types. Identification of tumour antigens in ovarian cancer may facilitate the development of vaccine-based therapies and of disease biomarkers. The purpose of our investigation is to identify tumour antigens in ovarian cancer by using the serological analysis of recombinant cDNA expression libraries method. A recombinant ovarian carcinoma cDNA expression library was screened with ascites fluid, pooled from five ovarian cancer patients. Twelve tumour antigens encoded by known genes were isolated, including ribosomal protein S18, heat shock protein 90, JK-recombination signal binding protein, ribonucleoprotein H1, RAN binding protein 7, TG-interacting factor, eukaryotic translation initiation factor p40 subunit, human amyloid precursor protein-binding protein 1, ribosomal protein L8, CDC23, IQ motif containing GTPase activating protein 1, and ribosomal protein L3. Heat shock protein 90 was chosen for further investigation. The prevalence of hsp90 autoantibodies in ovarian cancer was determined with immunoassay. Sera from 22 normal females, 32 from ovarian cancer (22 stage III/IV, 10 stage I/II), 37 colorectal cancer, 13 breast cancer, 10 lung cancer, 20 benign gynaecologic diseases, and 10 benign breast lesions were screened. Seven (32%) stage III/IV ovarian cancer, 1 (10%) stage I/II ovarian cancer, 1 (3%) colorectal cancer, 1 (8%) breast cancer, and 1 (5%) benign gynaecologic disease sera were found to contain hsp90 autoantibodies. These data support the view that hsp90 autoantibodies are frequently found in late stage ovarian cancer. Hsp90 may, therefore, represent a novel biomarker for ovarian cancer and a candidate ovarian cancer vaccine target.     

Response of human breast cancer cells to heat shock and chemotherapeutic drugs.

Previous studies have shown that certain chemotherapeutic drugs are less effective on tumor cells when cells have been previously exposed to hyperthermia. In the present study, we have evaluated whether specific modifications in heat shock protein (hsp) expression are associated with resistance to anticancer drugs. RNA levels for hsp90, hsp70, and hsp27 were studied by Northern and slot blots, while proteins were studied by two-dimensional gel electrophoresis, in MCF-7/BK and MDA-MB-231 breast cancer cells. The sensitivities of these cells to doxorubicin, colchicine, 5-fluorouracil, cisplatin, actinomycin D, and methotrexate were tested by clonogenic assays. These techniques were applied to both cell lines before (control) and after heat shock. The study revealed that elevated hsp70 and hsp27 levels were associated with doxorubicin resistance. In addition, the presence of phosphorylated hsp27 isoforms was also associated with doxorubicin resistance. The study showed that elevated hsps were not associated with multidrug resistance. Heat shock did not induce P170 glycoprotein mRNA overexpression or resistance to the other drugs tested. We also found that the level of doxorubicin protection conferred by the overexpression of hsp was lower than that obtained in cells expressing a multidrug resistance phenotype (MDA-A1R cells). In these cells, heat shock did not confer additional doxorubicin resistance and hsp27 phosphorylation was deficient. Our studies suggest that specific hsps are associated with doxorubicin resistance in certain human breast cancer cells and that this mechanism seems to be independent of the multidrug resistance system.     

Stereospecific antitumor activity of radicicol oxime derivatives

PURPOSE: Radicicol is a novel hsp90 antagonist, distinct from the chemically unrelated benzoquinone ansamycin compounds, geldanamycin and herbimycin. Both geldanamycin and radicicol bind in the aminoterminal nucleotide-binding pocket of hsp90, destabilizing the hsp90 client proteins, many of which are essential for tumor cell growth. We describe here antitumor activity of a novel oxime derivative of radicicol, KF58333. We also investigated the mechanism of antitumor activity of KF58333 in comparison with its oxime isomer KF58332. METHODS: Antiproliferative activities were determined in a panel of breast cancer cell lines in vitro. We also examined inhibition of hsp90 function and apoptosis induction in erbB2-overexpressing human breast carcinoma KPL-4 cells in vitro. Direct binding activity to hsp90 was assessed by hsp90-binding assays using geldanamycin or radicicol beads. In animal studies, we investigated plasma concentrations of these compounds after i.v. injection in BALB/c mice and antitumor activity against KPL-4 cells transplanted into nude mice. Inhibition of hsp90 function and induction of apoptosis in vivo were investigated using tumor specimens from drug-treated animals. RESULTS: KF58333 showed potent antiproliferative activity against all breast cancer cell lines tested in vitro, and was more potent than its stereoisomer KF58332. These results are consistent with the ability of KF58333 to deplete hsp90 client proteins and the induction of apoptosis in KPL-4 cells in vitro. Interestingly, KF58333, but not KF58332, showed significant in vivo antitumor activity accompanied by induction of apoptosis in KPL-4 human breast cancer xenografts. Although the plasma concentrations of these compounds were equivalent, KF58333, but not KF58332, depleted hsp90 client proteins such as erbB2, raf-1 and Akt in the tumor specimen recovered from nude mice. CONCLUSIONS: These results suggest that inhibition of hsp90 function, which causes depletion of hsp90 client proteins in tumor, contributes to the antitumor activity of KF58333, and that the stereochemistry of the oxime moiety is important for the biological activity of radicicol oxime derivatives.     

hsp27基因对鼻咽癌细胞增殖的作用及其机制

目的探讨hsp27基因对鼻咽癌细胞增殖的影响及其机制。方法利用实时定量PCR检测NP460(正常鼻咽部上皮细胞株）、CNE2(低分化鼻咽癌细胞株）及S18(CNE2的高转移亚克隆）的hsp27转录水平,利用慢病毒转染技术在hsp27转录水平较低的CNE2中过表达hsp27,利用小干扰RNA技术抑制S18细胞内的hsp27,用MTT技术检测过表达或抑制hsp27后细胞增殖速率的变化,同时检测转录因子NF-κB的转录水平的变化,分析hsp27影响鼻咽癌细胞增殖速率的可能机制。结果(1）三种细胞株中的内源性hsp27水平为S18>CNE2>NP460,差异具有统计学意义。(2）在CNE2细胞中过表达hsp27基因后,细胞的增殖速率明显加快,NF-κB转录水平明显上调。(3）抑制S18细胞内的hsp27基因后,细胞的增殖速率明显减缓,NF-κB转录水平明显下调。结论hsp27基因可能通过调节NF-κB信号通路而对鼻咽癌细胞发挥明显的促增殖作用,可设计针对hsp27基因的抗鼻咽癌分子治疗策略。     

Tyrosine kinase inhibitor emodin suppresses growth of HER-2/neu-overexpressing breast cancer cells in athymic mice and sensitizes these cells to the inhibitory effect of paclitaxel.

Overexpression of the HER-2/neu proto-oncogene, which encodes the tyrosine kinase receptor p185neu, has been observed in tumors from breast cancer patients. We demonstrated previously that emodin, a tyrosine kinase inhibitor, suppresses tyrosine kinase activity in HER-2/neu-overexpressing breast cancer cells and preferentially represses transformation phenotypes of these cells in vitro. In the present study, we examined whether emodin can inhibit the growth of HER-2/neu-overexpressing tumors in mice and whether emodin can sensitize these tumors to paclitaxel, a commonly used chemotherapeutic agent for breast cancer patients. We found that emodin significantly inhibited tumor growth and prolonged survival in mice bearing HER-2/neu-overexpressing human breast cancer cells. Furthermore, the combination of emodin and paclitaxel synergistically inhibited the anchorage-dependent and -independent growth of HER-2/neu-overexpressing breast cancer cells in vitro and synergistically inhibited tumor growth and prolonged survival in athymic mice bearing s.c. xenografts of human tumor cells expressing high levels of p185neu. Both immunohistochemical staining and Western blot analysis showed that emodin decreases tyrosine phosphorylation of HER-2/neu in tumor tissue. Taken together, our results suggest that the tyrosine kinase activity of HER-2/neu is required for tumor growth and chemoresistance and that tyrosine kinase inhibitors such as emodin can inhibit the growth of HER-2/neu-overexpressing tumors in mice and also sensitize these tumors to paclitaxel. The results may have important implications in chemotherapy for HER-2/neu-overexpressing breast tumors.     

Heat shock protein 105 is overexpressed in a variety of human tumors

We earlier reported that heat shock protein 105 (hsp105), which we identified by serological analyses of recombinant cDNA expression libraries (SEREX), was overexpressed in human colon and pancreatic adenocarcinoma. We went on to examine hsp105 expression in various colorectal cancers and adenomas, using immunohistochemical analysis. The 44 of 53 patients with colorectal cancers (83.0%) and only 2 of 21 (9.5%) with colorectal adenomas had an evident overexpression of hsp105, which means that overexpression of hsp105 is a late event in the colorectal adenoma-carcinoma sequence. Subsequently, we asked if hsp105 was overexpressed in other human tumors. During immunohistochemical studies, we discovered that overexpression of hsp105 occurred not only in cases of colorectal and pancreatic adenocarcinoma, but also thyroid, esophageal, breast, and bladder carcinoma and islet cell tumor, gastric malignant lymphoma, pheochromocytoma, and seminoma. On the other hand, hsp105 was evidently overexpressed only in the testis in human adult normal tissues. Thus, hsp105 is a useful marker of a variety of human tumors and hsp105 may prove to be a target molecule for designing anti-tumor immunotherapy.     

Flow cytometric measurements of heat shock proteins

A fixation and immunofluorescence staining procedure for measurements of heat shock proteins (hsp) by flow cytometry is reported. Three fixatives were compared: 80% methanol at —20°C for 1 h, 70% ethanol at 0°C for 1 h, and 3% paraformaldehyde at 4°C for 1 h followed by 0·2% NP-40. Cells fixed with methanol showed strongest immunofluorescence and lowest nonspecific fluorescence. The level of hsp 70 as a function of time after heating followed the same kinetics as the development of thermotolerance reported by others. The level of hsp 70 increased with increasing heat dose up to a maximum heat dose, and above this heat dose a decrease in the level of hsp 70 was observed. Correlated measurements of the level of hsp 70 and DNA showed that hsp 70 was found in all phases of the cell cycle. The level of hsp 70 increased about two-fold in unheated cells throughout the cell cycle. The increase in G₂ + M cells compared with G₁ cells was lower in cells heated at 45°C for 20 min followed by incubation at 37°C for 24 h before fixation and staining, than in unheated cells. The results show that flow cytometry provides a rapid and quantitative technique for measuring hsp. Correlated measurements of hsp and other cellular parameters might also be obtained.     

Antibodies to heat-shock protein 27 are associated with improved survival in patients with breast cancer.

The overexpression of the heat-shock proteins hsp90, hsp70 and hsp27 in human mammary carcinomas has previously been shown to correlate with reduced overall survival. Moreover, antibodies to hsp90 were detectable in the serum of a large proportion of breast cancer patients but they were not found in normal controls. High antibody levels also correlated with reduced survival. Here, we show that antibodies to hsp27 were also detectable in the sera from breast cancer patients but not from normal controls, whereas antibodies to hsp70 were detectable in approximately one-third of both groups. The presence of antibodies to hsp27 was correlated with an improved rather than a reduced survival, particularly beyond the first 5 years. Hence, the overexpression of hsps in breast cancer cells does not provoke a generalized immune response to all the hsps. Moreover, the presence of antibodies to different hsps has distinct associations with survival. These effects are discussed in terms of the mechanisms that provoke an immune response to the hsps and the protective/non-protective effects of such a response.     

Cancer stem cells in the human mammary gland and regulation of their differentiation by estrogen

The identification and characterization of normal and breast cancer stem cells have provided a new vision of breast tumorigenesis. Cancer stem cells may be responsible for breast tumor initiation, progression and development of resistance to therapy. Most breast cancers express the estrogen receptor, and several studies have linked long-term estrogen exposure to enhanced breast cancer risk; however, estrogen receptor-positive tumors usually present a better prognosis than estrogen receptor-negative ones. The finding that estrogen reduces the pool of human breast stem cells may explain the more differentiated phenotype observed in estrogen receptor-positive tumors. In this article, our current understanding of the complex role of estrogen in human breast stem cells is discussed in the context of breast malignancy.     

Differential expression of steroid receptors,hsp27, and pS2 in a series of drug resistant human breast tumor cell lines derived following exposure to antitumor drugs or to fractionated X-irradiation

Summary This study examined whether levels of estrogen receptor (ER), progesterone receptor (PR), and expression of estrogen regulated pS2 and/or heat shock protein (hsp) 27 were associated with drug resistance in a series of MCF-7 sublines expressing modest (i.e. 3- to 14-fold), yet clinically relevant, levels of resistance to vincristine (VCR). These sublines were variously derived following pulsed exposures to VCR, to fractionated X-irradiation, or to alternating drug and X-ray treatments. This selection procedure more closely reflects the clinical treatment of breast tumors than the use of continuous drug exposures. The drug-selected sublines exhibited the classical multidrug resistance phenotype (MDR) characterized by cross-resistance to vinblastine (VLB), etoposide (VP-16), and Adriamycin (ADR), overexpression of P-glycoprotein (Pgp), impaired accumulation of [³H]-VCR and of Rhodamine-123 (Rh 123), and altered activities of certain drug detoxification enzymes. This classic MDR phenotype was associated with a lack of mitogenic response to estrogen or antiestrogen, related to loss of detectable ER and PR; consistent with these data, neither pS2 nor hsp27 expression was detectable. In contrast, X-ray-pretreated VCR-resistant cells (MCF/DXR-10) cells exhibited a distinctive resistance phenotype proving cross-resistant to VLB and VP-16 but not to ADR, and Pgp overexpression was not detectable. Furthermore, these VCR-resistant DXR-10 cells retained parental levels of ER and PR, exhibited sensitivity to estrogen and 4-hydroxytamoxifen, and expressed detectable levels of pS2 and hsp27. Comparable characteristics to these MCF-7/DXR-10 cells were also identified in a similarly-derived X-ray-pretreated VCR-resistant subline of the ZR-75-1 human breast tumor cell line. These data therefore indicate that functional ER are frequently, but not invariably, modified in tumor cells which express resistance to multiple drugs.     

Ectopic expression of Hsp70 confers resistance and silencing its expression sensitizes human colon cancer cells to curcumin-induced apoptosis

We have shown earlier that heat shock renders human colon cancer cells resistant to curcumin-induced apoptosis, but the contribution of individual heat shock proteins (hsps) to this resistance has not been tested. High expression of hsp27 and hsp70 in breast, endometrial and gastric cancers has been associated with metastasis, poor prognosis and resistance to chemo- or radiotherapy. In this study, SW480 cells were transfected with hsp70 cDNA in either the sense or antisense orientation and stable clones were selected and tested for their sensitivity to curcumin. The cells were protected from curcumin-induced cell death by hsp70 while cells harboring antisense hsp70 (Ashsp70) were highly sensitive to curcumin. Curcumin-induced nuclear condensation was less in hsp70 but more in Ashsp70 cells when compared with control vector-transfected cells. Loss of mitochondrial transmembrane potential induced by curcumin was further accelerated by antisense hsp70 expression and hsp70 restored it partly. Ashsp70 cells released more cytochrome c, AIF and Smac from mitochondria upon curcumin treatment than control cells. hsp70 partly prevented the release of AIF but not the other proteins. Activation of caspases 3 and 9 induced by curcumin was also inhibited by hsp70, whereas more activation could be seen in Ashsp70 cells, although caspase 8 activation was unaffected by changes in hsp70 expression. Curcumin-induced cleavage of PARP and DFF45 was inhibited by hsp70 but enhanced in Ashsp70 cells. The present study demonstrates the potential of hsp70 in protecting SW480 cells from curcumin-induced apoptosis and highlights that silencing the expression of hsp70 is an effective approach to augment curcumin-based therapy in cancers that are resistant due to hsp70 expression.     

Hsp27 overexpression inhibits doxorubicin-induced apoptosis in human breast cancer cells

Previously we demonstrated that heat shock protein 27 (hsp27) overexpression confers resistance to the chemotherapeutic agent doxorubicin in MDA-MB-231 breast cancer cells. Since induction of apoptosis is one underlying mechanism of chemotherapeutic drug action, we investigated the effect of hsp27 overexpression on doxorubicin-induced apoptosis, finding that hsp27 protects MDA-MB-231 cells from apoptosis. We also examined expression of the doxorubicin target, topoisomerase II (topo II), in control and hsp27-overexpressing stable transfectants, as topo II expression is important for both drug sensitivity and the initiation of apoptosis by doxorubicin. The relative levels of both topo IIalpha and beta were higher in the controls than the hsp27-overexpressing clones, suggesting that the apoptotic protective effect of hsp27 overexpression in MDA-MB-231 cells is associated with altered topo II expression.