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Department of Surgery, University of California, Mount Zion Medical Center (J.-W.P., M.W.Y., M.G.W., M.L., O.H.C.), San Francisco, California 94143-1674; University of California Comprehensive Cancer Center (W.C.H.), San Francisco, California 94115; and Surgical Services, Veterans Affairs Medical Center (Q.H.C.), San Francisco, California 94121
Address all correspondence and requests for reprints to: Orlo H. Clark, M.D., University of California/Mount Zion Medical Center (Surgery), 1600 Divisadero Street, San Francisco, California 94115. E-mail: clarko{at}surgery.ucsf.edu.
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Introduction |
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p53 mutations are thought to be a late step in thyroid tumorigenesis and also appear to play an important role in the dedifferentiation of thyroid carcinomas to poorly DTC or anaplastic thyroid cancer. p53 mutations are thought to be associated with reduced chemosensitivity and radiosensitivity of cancer cells. Recently, several chemotherapeutic agents that do not depend upon the p53 pathway for their cytotoxic mechanisms have been introduced as novel anticancer drugs for cancers with mutant p53. One of these drugs is the benzoquinoid ansamycin antibiotic, geldanamycin, which was isolated from Streptomyces hygroscopicus var. geldanus and works via the dissociation of heat shock protein 90 (HSP90).
HSP90 is one of the most abundant chaperone proteins in the cytosol of eukaryotic cells. It helps newly synthesized proteins to make stable conformations (maturation) or to translocate them to their ultimate locations (3, 4). HSP90 under normal physiological conditions is essential for fundamental cellular processes such as hormone signaling, cell cycle control, proliferation, and differentiation (5). It also plays an important role in stress such as heat shock and is associated with mitogen- or growth factor-stimulated cascade. HSP90 expression is higher in tumors than in normal tissues (6, 7, 8, 9). HSP90 may therefore play a critical role in tumor cell growth and/or survival. Although HSP90 is essential for both normal cells and cancer cells, cancer cells appear to be more sensitive to inhibition of this chaperone’s activity. Several oncoproteins, such as Raf-1, erbB2, and mutant p53 proteins, are reported to be substrates for HSP90 (10, 11, 12).
Geldanamycin specifically binds to HSP90 and its homolog, glucose-regulated protein 94 (GRP94), and it therefore affects the stability and steady state level of these oncoproteins (11, 13, 14). The NCI-screened drug sensitivity of geldanamycin in 60 tumor cell lines in vitro, but none was thyroid cancer derived. They reported that geldanamycin might be a promising anticancer drug effective at nanomolar concentrations (15). The purpose of this investigation was to evaluate effects of geldanamycin on cell proliferation, oncoprotein expression, and invasion in human thyroid cancer cell lines in vitro.
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Materials and Methods |
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The following reagents and materials were used: DMEM/Ham’s F-12 medium, L-glutamine, 1x trypsin/EDTA solution, 1x PBS from Cellgro Mediatech (Newark, DE); penicillin-streptomycin, fetal calf serum (FCS), and fungizone from Irvine Scientific (Santa Ana, CA); anti-Raf-1 mouse monoclonal antibody from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); anti-p53 mouse monoclonal antibody from Oncogene Research Products (Boston, MA); annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit from Oncogene Research Products (Boston, MA); propidium iodide (PI) from Sigma-Aldrich Corp. (St. Louis, MO); anti-epidermal growth factor (anti-EGF) receptor (EGFR) mouse monoclonal antibody from Oncogene Research Products; streptavidin-horseradish peroxidase conjugate (Zymed Laboratories, Inc., San Francisco, CA); FITC-conjugated antimouse immunoglobulin G (IgG) heavy and light chain goat antibody from Calbiochem (San Diego, CA); and geldanamycin from Sigma-Aldrich Corp. (St. Louis, MO). A primary stock solution of geldanamycin was dissolved in dimethylsulfoxide at a concentration of 5 mM. Geldanamycin was reconstituted in 4H medium, and its final concentration of dimethylsulfoxide in the medium was less than 0.02% (vol/vol) in the treatment range (10–1000 nM).
Cell lines and culture conditions
All three follicular thyroid cancer (FTC) cell lines were established from a single patient and were provided by Dr. Peter Goretzki (Dusseldorf, Germany): FTC-133 from a primary thyroid tumor, FTC-236 from a lymph node metastasis, and FTC-238 from a lung metastasis (16). A papillary thyroid cancer cell line (TPC-1) was provided by Dr. Nabuo Satoh (Kanazawa University, Kanazawa, Japan), and the Hürthle cell carcinoma cell line (XTC-1) was established in our laboratory (17). An anaplastic thyroid cancer cell line (ARO82–1) was provided by Dr. Guy J. F. Juillard (University of California School of Medicine, Los Angeles, CA).
The cell lines were maintained in DMEM/Ham’s 12 supplemented with 10% FCS, penicillin (10,000 U/ml), streptomycin (50µg/ml), fungizone (250 mg/ml), TSH (10 mU/ml), glutamine (12.5 mg/liter), and insulin (0.01 mg/ml) in a standard humidified incubator at 37 C in a 5% CO2/95% O2 atmosphere. All experiments were performed in serum-free 4H medium containing DMEM/Ham’s 12 supplemented with transferrin (5 µg/ml), somatostatin (10 ng/ml), glycyl-L-histidyl acetate (2 ng/ml), and hydrocortisone (0.36 ng/ml) by a modified Ambesi-Impiombato method (18). The culture media were changed to 4H medium 24 h before conducting experiments.
Proliferation assay
Growth experiments were performed in a 96-well plate in hexaplicate. Cells at 85–100% confluence were harvested with 1x trypsin/EDTA solution and seeded into a 96-well plate at 3–5 x 103 cells/well depending upon growth rate and maintained in 200 µl 4H medium in a humidified incubator. After 24 h, the cells were incubated with different concentrations of geldanamycin, and the medium was changed daily. Colorometric dimethyl-thiazol-diphenyltetrazolium bromide (MTT) proliferation assays were performed at 0, 2, 4, and 6 d after treatment as previously described (19). MTT (400 µg/ml) was added to each well and incubated for 3 h. It was solubilized with 0.04 N HCl/iso-propanol/3% sodium dodecyl sulfate and incubated for 1 h. The ODs in the 96-well plates were determined using an ELISA microplate reader (Molecular Devices, Sunnyvale, CA) at 595 nm/620 nm.
Clonogenic assay
FTC-133 and ARO82–1 cells were grown in 6-well plates (Costar, Cambridge, MA) in DMEM with 10% FCS until 70–80% confluence was achieved. The media were then removed and replaced with 4H (serum-deprived medium). The next day culture media were replaced with 4H medium containing geldanamycin. After 24 h the drug was removed. The control cells were counted and plated at 1000 viable cells/well in 6-well plates. Geldanamycin-treated cells were plated by the same number of viable cells or the same volume of cell suspension in the same dilution as that used for the controls. Triplicate plates were incubated for 7 d. After a 7-d incubation, the clones were fixed in methanol and stained with 0.1% (wt/vol) crystal violet. Colonies of more than 50 cells were counted. The cloning efficiency of control cells was between 30–40% for both cell lines.
Western blotting
Cells were lysed in protein lysis buffer. The protein concentration was determined with the bicinchoninic acid reagent. The protein (25 µg) was subjected to electrophoresis on 8% polyacrylamide gels, transferred to nitrocellulose by electroblotting, and probed with anti-p53 mouse monoclonal antibodies (Oncogene Science, Inc., Cambridge, MA), or anti-Raf-1 mouse monoclonal antibodies (Santa Cruz Biotechnology, Inc.), as previously described (12). Aliquots of equal amounts of protein (25 µg) from different treatment groups of FTC-133 cells were mixed with the sample buffer and boiled for 3 min at 95 C. Samples were immediately cooled on ice and subjected to SDS-PAGE on 8% gels (Bio-Rad Laboratories, Inc., Hercules, CA). The proteins were transferred to a nitrocellulose membrane. Nonspecific binding was blocked using 5% nonfat dry milk in 10 mM Tris-NaCl buffer overnight. To demonstrate depletion of Raf-1, the blot was incubated for 1 h with anti-Raf-1 antibody diluted 1:200. To determine the levels of p53, the blot was incubated with anti-p53 mouse monoclonal antibodies diluted 1:1,000. After three washes in Tris-NaCl buffer, the membranes were incubated for 1 h with horseradish peroxidase-conjugated goat antimouse IgG antibodies (Sigma-Aldrich Corp.) diluted 1:1,000 for Raf-1 blots and 1:15,000 for p53 blots. After three additional washes, the blots were incubated for 1 min with enhanced chemiluminescence detection solution, and signal levels were determined using radiography. The immunoreactive bands were quantified by digital densitometric imaging.
Apoptosis assay
Apoptosis was detected using an annexin-V-FITC binding assay kit (Oncogene Research Products, Inc.) following the manufacturer’s instructions. In the beginning stages of apoptosis, phosphatidylserine is translocated from the inner to the outer surface of the plasma membrane. Annexin V binds to this apoptotic marker. The thyroid cancer cell line (FTC-133) was incubated with and without geldanamycin to assess the effect of geldanamycin on cell death by apoptosis. After 4 d of exposure to geldanamycin, FTC-133 cells (1 x 106) were harvested and washed twice with ice-cold PBS. The cell pellet was resuspended in 1 ml of 1x binding buffer [10 mM HEPES/NaOH (pH 7.4), 140 mM NaCl, and 2.5 mMCaCl2]. To 500 µl of this cell suspension, 1.25 µl annexin V-FITC were added, and cells were incubated for 15 min in the dark. After removal of supernatant containing unbound annexin V-FITC by centrifugation, cells were resuspended in 500 µl of 1x binding buffer, and 10 µl PI (30 µg/ml stock) were added for dead cell discrimination. Flow cytometric analysis for apoptosis was performed on a FACScan (BD Biosciences, Franklin Lakes, NJ). Gating and event threshold were performed on single cells by forward and orthogonal light scatter. Live cells were determined by PI exclusion. Early apoptotic fraction was determined by annexin V-positive and PI-negative stain.
Cell cycle analysis
After treatment with or without geldanamycin, cells (1 x 106) were harvested and washed with PBS. Cell pellets were resuspended with cold 70% ethanol in PBS and incubated for at least 2 h at 4 C. After washing with PBS, ribonuclease A was added to the cell suspension to make a final concentration of 100 mU/ml. After a 30-min incubation, cells were washed with PBS and resuspended with PBS. Ten microliters of propidium iodide (PI) (20 µg/ml stock) were added, and samples were stored at 4 C.
Flow cytometric analysis for cell cycle was performed on a FACScan (BD Biosciences). Data files were generated for 10,000 events (cells) or more per sample using a positive PI threshold and gating on single cells. Doublets, cell clumps, and debris were excluded by PI fluorescence pulse width and pulse area measurements using CellQuest software (BD Biosciences). Cell cycle analysis on the gated PI distribution was performed using the peak deconvolution algorithms in Modfit software (Verity Software House, Inc.).
Immunofluorescence labeling for surface EGFR
Indirect immunofluorescence labeling for surface EGFR was performed with anti-EGFR mouse monoclonal antibody (Oncogene Research Products, Inc.) and FITC-conjugated antimouse IgG heavy and light chain goat antibody (Calbiochem) (20).
After treatment with or without geldanamycin, FTC-133 cells (1 x 106) were harvested and washed twice with ice-cold PBS. After a 30-min incubation with anti-EGFR antibody, cells were washed twice with 0.1% sodium azide in PBS. To label immunofluorescence, cells were incubated with FITC-conjugated antimouse goat antibody for 30 min and then washed twice with 0.1% sodium azide in PBS. After removal of supernatant by centrifugation, 10 µl PI (30 µg/ml stock) were added to exclude late apoptotic or dead cells.
Matrigel invasion assay
We modified the originally described Boyden chamber method by using a Transwell polycarbonate membrane (Costar, Cambridge, MA) (21, 22). Matrigel diluted in 4H medium was solidified onto a Transwell polycarbonate membrane. FTC-133 cells (2 x 105 cells/well) suspended in 4H medium with a combination of EGF and geldanamycin were added to the top of the well. Wells were incubated at 37 C in humidified air containing 5% CO2 for 72 h. The cells on the upper side of the polycarbonate membrane were wiped off, and the remaining cells that traversed the Matrigel and spread on the lower surface of the filter were collected separately. An MTT assay was then performed on harvested cells. The relative invasive rate was calculated as the percent OD of the cells from the top of the membrane to the overall OD from the total cells. The Matrigel assay was performed in quadruplicate.
Statistical analysis
The t test for paired data, ANOVA for multiple group comparison (control plus multiple geldanamycin treatment concentrations), and Kolmogorov-Smirnov test for flow cytometric data (EGFR expression) were used, with a statistically significant result defined as P < 0.05.
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Results |
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Geldanamycin inhibited the growth of thyroid cancer cell lines, except for FTC-238, at a concentration between 10–50 nM. The antiproliferative effect of geldanamycin was time and dose dependent. This treatment appeared to be cytotoxic, especially in FTC-133 and FTC-236, because the cell viability after 6-d treatment with 50 nM geldanamycin was less than 10% of the control value (Fig. 1A
). Growth inhibition was first evident after 24–48 h of treatment. In a highly sensitive cell line such as FTC-133, 20 nM or higher concentrations of geldanamycin induced a significant cytotoxic effect (Fig. 1B). The ARO82–1 cell line was relatively resistant to geldanamycin, as a concentration of 100 nM or higher was required to achieve a significant antiproliferative effect (Fig. 1C).
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We chose FTC-133 and ARO82–1 cell lines to compare the changes in clonogenicity after treatment of geldanamycin because the drug sensitivities of these two cell lines were different. After 24 h of exposure to geldanamycin at varying concentrations, we harvested cells and plated them in six-well plates in triplicate. The cloning efficiency of control cells was between 40% and 50% for both cell lines. When we plated the same volume of harvested cell suspension (the same dilution), both of these cell lines showed dose-dependent decreases in clonogenicity (Fig. 2). ARO82–1 cells, however, required higher dose of geldanamycin to achieve the same degree of inhibition of clonogenicity. When we plated the same number of viable cells using trypan blue exclusion, FTC-133 cells showed a similar dose-dependent decrease, but ARO82–1 cells did not.
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After 4 d of treatment with geldanamycin, a dose-dependent increase in the apoptotic cell fraction in FTC-133 cells was identified (Fig. 3). After 4 d of exposure to geldanamycin, the early apoptotic cell population characterized by PI-negative and annexin V-positive cells was 4.0% in controls, 7.9% in 12.5 nM geldanamycin-treated cells, 20.4% in 25 nM geldanamycin-treated cells, and 39.7% in 50 nM geldanamycin-treated cells, respectively. There was also a dose-dependent increase in the dead or late apoptotic cell population characterized by PI-positive and annexin V-positive cells.
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). The average of G2/M phase fractions after 2 d of treatment in three different experiments was 7.2 ± 1.3% (mean ± SD) in controls, 6.7 ± 2.1% in 12.5 nM geldanamycin-treated cells, 9.2 ± 3.9% in 25 nM geldanamycin-treated cells, and 15.6 ± 4.5% in 50 nM geldanamycin-treated cells, respectively. Increases in G2/M phase fraction were statistically significant only after treatment with 50 nM geldanamycin (P < 0.05). The average of S phase fractions was 15.1 ± 3.1% in controls, 11.8 ± 0.7% in 12.5 nM geldanamycin-treated cells, 13.2 ± 2.8% in 25 nM geldanamycin-treated cells, and 18.9 ± 0.2% in 50 nM geldanamycin-treated cells, respectively.
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We treated FTC-133 cells with geldanamycin to evaluate its effect on oncoproteins, c-Raf-1, and mutant p53. We previously reported the p53 mutational status of FTC-133; that is, FTC-133 has a mutation in exon 8 of p53 and overexpression of p53 by immunocytochemical staining (23). After 3 d of treatment with geldanamycin, protein expressions of c-Raf-1 and mutant p53 were down-regulated in a dose-dependent manner (Fig. 5). Protein expression of c-Raf-1 was markedly decreased by treatment with 25 nM geldanamycin. Densitometric analysis showed 25% and 45% decreases in mutant p53 expression at concentrations of 100 and 200 nM, respectively.
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Flow cytometric analysis showed that geldanamycin treatment down-regulated cell surface expression of EGFR in the FTC-133 cell line in dose-dependent manner (Fig. 6). The percent invasion of FTC-133 cells after 72 h of incubation was 13.5 ± 2.5% (mean ± SE) in control conditions, 20.2 ± 2.5% after treatment with EGF (10 ng/ml), and 13.0 ± 0.4% after treatment with both EGF (10 ng/ml) and geldanamycin of 10 nM (P < 0.05; Fig. 7).
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Discussion |
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and HSP90ß) and a homolog (GRP94) in the endoplasmic reticulum (24). HSP90 is located primarily in the cytoplasm, but its nuclear import rate is rapidly increased by various stresses, including heat shock. The expression of HSP90 is up-regulated by stimulation of serum, mitogens, or growth factors and is 2- to 10-fold higher in tumor cells than in normal cells (6, 8, 9). HSP90 levels correlate with survival and proliferation of cancer cells. HSP90 interacts with many signaling proteins, such as steroid receptors; mutated p53; a wide variety of tyrosine and serine/threonine kinases, including members of the Src family; p185erbB-2; and c-Raf-1. It also associates with cell cycle regulators such as cyclin-dependent kinases (Cdk4 and Cdk6) and the cell cycle-associated kinase Wee1 (24). Clinically, the presence of anti-HSP90 antibodies has been reported to correlate with pathological stage and survival in patients with breast cancer and with chemosensitivity in patients with osteosarcoma (25, 26). HSP90, therefore, certainly plays an important role in cancer cell growth and survival. The benzoquinone ansamycin antibiotic geldanamycin and herbimycin A are specific inhibitors of HSP90 and its homolog GRP94. Stebbins et al. (27) and Bergerat et al. (28) reported that HSP90 has an ATP-binding motif, and ATP binding causes a conformational change (29). Geldanamycin blocks HSP90 chaperone complex formation by binding to this site and induces destabilization of substrate proteins (30).
Inhibition of HSP90 by geldanamycin induces antiproliferative effects in many human cancer cell lines in vitro with variable sensitivity (15). To our knowledge, the effect of geldanamycin in thyroid cancers has not been investigated. In the present investigation geldanamycin induced antiproliferative effects in all thyroid cancer cell lines from differentiated and undifferentiated tumors. FTC cell lines (FTC-133 and FTC-236) were more sensitive to geldanamycin than the others. An anaplastic thyroid cancer cell line, ARO-82-1, was the most resistant of the thyroid cancer cell lines investigated. The 50% inhibitory dose on the fifth day of treatment was less than 20 nM in FTC-133 and less than 100 nM in ARO-82-1 cell lines, respectively. Geldanamycin also reduced clonogenicity in both FTC-133 and ARO-82-1 cells. ARO-82-1 cells required higher dose of geldanamycin to achieve the same degree of inhibition of clonogenicity.
Recently, Srethapakdi et al. (31) reported that treatment with herbimycin A induced G1 arrest in tumor cell lines with a functional retinoblastoma gene product (RB), but not in those lacking functional RB. Hostein et al. (32), however, reported G2/M arrest in colon cancer cell lines independent of RB status. In our cell cycle analysis in FTC-133 cells, the fraction of G2/M phase was increased after treatment of geldanamycin. This finding is consistent with Hostein’s results. FTC-133 is known to have a p53 mutation, but the exact mechanism of G2/M arrest remains unknown. Apoptotic assay using an annexin V detection kit showed a dose-dependent increase in apoptotic cell population in FTC-133 cells. The antiproliferative effect by geldanamycin in FTC-133 can therefore be explained by G2/M arrest and apoptotic cell death.
Antitumor effects of geldanamycin can be partly explained by the inhibition of oncoproteins, such as v-Src, mutant p53 proteins, Raf-1, p185erbB2 proteins, and EGFR. Geldanamycin can inhibit their conformational changes and stabilization by HSP90 (10, 11, 12).
Herbimycin A was reported to induce the reversal of the transformed phenotype by v-Src (33, 34). HSP90 participates in the maturation and translocation of v-Src. The effect of herbimycin on phenotype change is not immediate, but takes 16–24 h (14). In the present investigation, the antiproliferative effect of geldanamycin required at least 24 h in most thyroid cancer cell lines. The relationship between v-Src and thyroid cancer growth and invasion has not been investigated to our knowledge.
p53 mutations are rare in DTC, but are more common in poorly differentiated thyroid cancers, anaplastic thyroid cancers, and thyroid cancer cell lines (23). Mutated p53 proteins have different functions in different tissues. It usually functions as a recessive negative (loss of the wild-type p53 on one allele with mutation in the remaining allele), but rarely as a dominant negative on wild-type p53. In our investigation, geldanamycin down-regulated mutated p53 protein expression in FTC-133 cells 3 d after treatment at a concentration of more than 100 nM. It is, however, difficult to expect obvious changes by down-regulation of p53, because a high concentration of geldanamycin was required to induce down-regulation of p53, and the p53 mutation is thought to function as a recessive negative in FTC-133 cells (23). The exact role of mutated p53 and its response to down-regulation are unknown.
Activation of the ras oncogene is thought to be a relatively common and early genetic event in thyroid carcinogenesis. GTP-bound ras complexes with Raf kinase. Through the multistep phosphorylation cascade, it induces changes in transcriptional activity for mitogenesis and maintenance of cell survival (35) (36). Among three Raf isoforms, Raf-1 plays an important role in the regulation of proliferation, differentiation, and apoptosis (37). Inhibition of Raf-1 by antisense treatment in vitro induced potent antiproliferative effects in tumor cell lines (38). Raf-1 can, therefore, be an attractive target for novel cancer treatment. Depletion of Raf-1 protooncogene by treatment with geldanamycin induces apoptosis (39). Inhibition of HSP90-Raf-1 kinase complex formation decreases Raf-1 stability, blocks MAPK activation, and prevents its trafficking to the membrane (40, 41). In the present investigation, significant down-regulation of Raf-1 in FTC-133 cells was induced by treatment with geldanamycin at a concentration of 25 nM for 3 d; it, therefore, appears to be one of the mechanisms of cell death induced by geldanamycin in thyroid cancer cells. To our knowledge, Raf-1 antisense has not been reported in thyroid cancer cell lines.
Geldanamycin and herbimycin A inhibit normal maturation of the EGFR, EGFR precursor, IGF receptor, platelet-derived growth factor receptor, and p185erbB2 receptor (5, 42, 43). Benzoquinone ansamycins destabilize these proteins and also prevent trafficking to the plasma membrane. Although p185erbB2 has been studied in detail in many breast and ovarian cancers and is associated with poor prognosis and drug resistance, its prognostic relationship is not clear in thyroid cancer. The effects of EGF in thyroid cancer are, however, relatively well studied. EGF stimulated the growth and invasion of FTC-133 thyroid cancer cells in vitro (44). Although EGF and EGFR were widely expressed in normal and abnormal thyroid tissues, the degree of EGFR expression was reported as a prognostic factor in thyroid cancer (45, 46). Geldanamycin has been reported to inhibit EGFR signaling by both accelerated degradation and intracellular retention of the EGFR (41, 43, 47). In our experiment we confirmed that EGF increased cancer cell invasion, and geldanamycin reduced EGF-induced invasion partly by down-regulation of surface expression of EGFR in thyroid cancer cells.
Most cancers have multiple genetic aberrations and altered signal transduction pathways. Targeting one of these altered pathways unfortunately has usually failed to achieve successful control of cancer cell growth, because cancer cells can use alternative pathway. Targeting HSP90 may be an effective form of cancer chemotherapy, because multiple oncoproteins can be targeted at the same time in thyroid cancer cells, as our investigation demonstrates. Geldanamycin is a novel anticancer drug based on differential dependence of HSP90 between cancer cells and normal cells; unfortunately, it may have significant side effects due to inhibition of diverse substrates for HSP90. However, continued characterization of the ansamycin-binding site on HSP90 may make it possible to develop more substrate- or tissue-specific HSP90 inhibitors. In fact, the geldanamycin analog, 17-allylamino-17-desmethoxygeldanamycin, is currently in a Phase I clinical trial at the NCI (48). Structurally different HSP90 binding drugs, such as radicicol, were also introduced as another class of HSP90 inhibitor. Several hybrid forms, such as geldanamycin-testosterone hybrids and immunoconjugates of geldanamycin and anti-HER2 monoclonal antibodies, were developed to increase tissue specificity (49, 50).
In conclusion, geldanamycin inhibited thyroid cancer growth and reduced clonogenicity in human thyroid cancer cell lines in vitro. Geldanamycin down-regulated c-Raf-1 and mutant p53 protein expressions in a FTC cell line. Apoptosis appears to be the mechanism of antiproliferative effects of geldanamycin. Geldanamycin also down-regulated EGFR and inhibited EGF-stimulated invasion. Although clinical data to date are very limited, our investigation suggests that targeting HSP90 might be a promising new alternative treatment for treatment of patients who have thyroid cancer that fails to respond to conventional treatment.
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Acknowledgments |
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Footnotes |
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Received March 4, 2002.
Accepted March 19, 2003.
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-stimulated invasion and growth of follicular thyroid cancer cells can be blocked by antagonism to the EGF receptor and tyrosine kinase in vitro. Eur J Endocrinol 132:229–235This article has been cited by other articles:
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