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Department of Medical Oncology (C.S.M., D.M., C.M., J.N., G.F., N.M.), Dana Farber Cancer Institute, and Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115; Department of Pathology (V.K.), School of Medicine, Aristotle University of Thessaloniki, 54621 Thessaloniki, Greece; Massachusetts Eye and Ear Infirmary (V.P.), Harvard Medical School, Boston, Massachusetts 02114; Department of Pathology (G.F., S.T.-B.), University of Athens, 11527 Athens, Greece; Thyroid Cancer Research Laboratory (K.B.A.), Veterans Affairs Medical Center, Lexington, Kentucky 40511; and Thyroid Oncology Program (K.B.A.), Division of Hematology/Oncology, Department of Internal Medicine, University of Kentucky, Lexington, Kentucky 40536
Address all correspondence and requests for reprints to: Constantine S. Mitsiades, M.D., Ph.D., Department of Medical Oncology, Dana Farber Cancer Institute, Mayer Building, Room M555, 44 Binney Street, Boston, Massachusetts 02115. E-mail: constantine_mitsiades{at}dfci.harvard.edu.
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Abstract |
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B degradation, which leads to inactivation of the transcriptional factor nuclear factor-B (NF-B). NF-B has been implicated in the pathophysiology of the most aggressive forms of thyroid carcinoma, i.e. medullary and anaplastic. Objective and Methods: We evaluated the effect of bortezomib on a panel of thyroid carcinoma cell lines, originating from papillary, follicular, anaplastic, and medullary carcinomas.
Results: Bortezomib induced apoptosis in medullary and anaplastic cell lines with IC50 values well within the range of clinically achievable concentrations and much lower than respective IC50 values for other solid malignancies. Bortezomib inhibited NF-B activity; increased p53, p21, and jun expression; and induced caspase-dependent apoptosis. Sensitivity of thyroid carcinoma cells to bortezomib was partially decreased by overexpression of Bcl-2 or treatment with IGF-I, whereas the combination of bortezomib with chemotherapy (doxorubicin) was synergistic.
Conclusions: These data provide both insights into the molecular mechanisms of antitumor activity of proteasome inhibitors and the rationale for future clinical trials of bortezomib, alone or in combination with conventional chemotherapy, to improve patient outcome in medullary and anaplastic thyroid carcinomas.
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Introduction |
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B (NF-B) inhibitor (IB), p53, c-myc, and c-Jun N-terminal kinase (JNK), are known substrates for proteasomal degradation (1). Proteasome inhibitors constitute a novel class of antitumor agents with preclinical evidence of activity against hematological malignancies and solid tumors (1). Specifically bortezomib, a boronic acid dipeptide proteasome inhibitor, is approved by the Food and Drug Administration for use in relapsed refractory multiple myeloma (MM) (2) and currently is being evaluated in a variety of other hematological and solid malignancies (3, 4).
Proteasome inhibition abrogates degradation and induces cytoplasmic accumulation of IB, which blocks the nuclear translocation and transcriptional activity of NF-B. This may contribute to the proapoptotic effects of bortezomib in malignancies such as MM, in which NF-B function is important for tumor cell proliferation and survival (5). Moreover, bortezomib sensitizes malignant cells to cytotoxic chemotherapeutic agents by down-regulating the NF-B-dependent expression of several inhibitors of apoptosis such as A1, cellular inhibitor of apoptosis protein-2, and X-linked inhibitor of apoptosis (XIAB) (6). Other NF-B-independent effects of bortezomib on MM cells include stabilization of p53 protein and up-regulation of p53 mRNA, stabilization of c-myc (7), phosphorylation and activation of c-Jun (7), and activation of the Fas pathway (7).
Thyroid cancer is the most prevalent endocrine malignancy, increasing in incidence each year, with nearly 26,000 new cases and 1,500 deaths in 2005 (8). Recently the transcription factor NF-B was implicated in the pathophysiology of both anaplastic (9) and medullary (10) carcinomas, suggesting that novel therapies targeting NF-B may be effective in these aggressive malignancies.
We evaluated the in vitro effect of bortezomib on thyroid carcinoma cells lines representing all histological types. We found that medullary and anaplastic thyroid carcinoma cell lines were sensitive to bortezomib at clinically achievable concentrations. Our studies therefore provide the framework for the use of proteasome inhibitor-based therapies in the treatment of aggressive thyroid carcinomas.
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Materials and Methods |
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The SW579 cell line, derived from a poorly differentiated human thyroid adenocarcinoma (poorly differentiated carcinoma with nuclear features of papillary carcinoma and squamous differentiation), the TT cell line, derived from a medullary thyroid carcinoma (MTC), the 6–23 (clone 6) rat medullary thyroid carcinoma cell line as well as the SW480 (colorectal), Caki-1 (renal), LNCaP (prostate), and SK-OV-3 (ovarian) cell lines were purchased from American Type Culture Collection (Manassas, VA). The papillary thyroid carcinoma cell line NPA, the follicular carcinoma cell line WRO, and the anaplastic thyroid carcinoma lines FRO and ARO were generous gifts of Dr. James A. Fagin (University of Cincinnati School of Medicine, Cincinnati, OH) (11, 12). The DRO81–1 (medullary), HRO85–1 (medullary), and DRO90–1 (anaplastic) cell lines were generous gifts of Dr. Guy J. F. Juillard (University of California, Los Angeles, School of Medicine, Los Angeles, CA). The BHT-101 anaplastic cell line was a gift from Dr. István Pályi (Research Center of Oncology, National Institute of Oncology, Budapest, Hungary). The anaplastic SW1736 cell line was developed by Drs. A. Leibowitz and W. M. McCombs III (Scott and White Memorial Hospital, Temple, TX) and provided to us by Dr. Nils-Erik Heldin (Uppsala University, Uppsala, Sweden). The anaplastic KAT18 cell line was originally established in the laboratory of one of us (K.B.A.) and has been described previously (13). The glioma cell lines U87 and LN827 were generous gifts of Dr. Andrew Kung (Dana Farber Cancer Institute, Boston, MA). All cells were grown in DMEM (BioWhittaker, Walkersville, MD) with 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% fetal bovine serum (Invitrogen, Carlsbad, CA), unless stated otherwise.
Reagents
Bortezomib [also known as PS-341, pyrazylCONH(CHPhe)CONH(CHisobutyl)B(OH)2; Millennium Pharmaceuticals, Cambridge, MA] was dissolved in dimethylsulfoxide (DMSO) and stored at –20 C until use. Bortezomib and control media contained less than 0.0005% DMSO. MG132 and caspase inhibitors were from Calbiochem (La Jolla, CA); 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and doxorubicin were from Sigma Chemical Co. (St. Louis, MO).
MTT colorimetric survival assay
Cell survival was examined using the MTT colorimetric assay, as previously described (7). Cell viability was expressed as a percentage of the value of untreated controls. All experiments were repeated at least three times, and each experimental condition was repeated at least in quadruplicate wells in each experiment. Data reported are average values ± SD of representative experiments.
Treatment of thyroid carcinoma cell lines with the NF-B inhibitor SN50
We next evaluated the role of NF-B in thyroid carcinoma cell survival by treating the TT, FRO, SW579, and WRO cells for 24 h in serum-free medium with the NF-B inhibitory peptide SN50 (BIOMOL, Plymouth Meeting, PA), which consists of the nuclear localization signal (NLS) sequence of p50 and inhibits the transportation of active NF-B into the nucleus (5). Cell viability was calculated with the MTT colorimetric assay and expressed as a percentage of the value of untreated controls. Data reported are average values ± SD.
Propidium iodide (PI) staining
Cells were incubated with or without 25 nM bortezomib in serum-free medium for 16 h and then harvested by scraping, permeabilized with 70% ethanol in PBS for 30 min at 4 C, washed with PBS, incubated with 0.5 ml of a 50 µg/ml PI solution containing 20 U/ml RNase-A (Roche Molecular Biochemicals, Indianapolis, IN) for 30 min, and analyzed by flow cytometry, as previously described (14).
Lactate dehydrogenase (LDH) release assay
Cells were preincubated with the pancaspase inhibitor Z-Val-Ala-Asp(OMe)-fluoromethylketone (ZVAD-FMK), the caspase-8 inhibitor Z-Ile-Glu(OMe)-Thr-Asp(OMe)-fluoromethylketone (IETD-FMK), the caspase-9 inhibitor Z-Leu-Glu(OMe)-His-Asp(OMe)-fluoromethylketone (LEHD-FMK), the caspase-3/-7 inhibitor Z-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-fluoromethylketone (DEVD-FMK), or the caspase-2 inhibitor Z-Val-Asp(OMe)-Val-Ala-Asp(OMe)-fluoromethylketone (Z-VDAD-FMK) (all used at 20 µM) for 1 h before exposure to bortezomib (50 nM for 36 h). Cell death was quantified by measuring the activity of LDH released from the cytosol of damaged cells into the culture supernatant, using the cytotoxicity detection kit (LDH) (Roche Molecular Biochemicals), according to the instructions of the manufacturer.
Caspase activity assays
Cells were treated with bortezomib (25 nM) for 0–16 h, washed in PBS, and harvested by centrifugation at 800 x g for 10 min at 4 C. Caspase-8 and -3 enzymatic activities were measured with respective ApoAlert caspase colorimetric assay kits (CLONTECH, Palo Alto, CA), normalized for protein content, and expressed in arbitrary units.
Quantification of NF-B activity in vitro
The DNA binding activity of NF-B was quantified by ELISA using the Trans-AM NF-B p65 transcription factor assay kit (Active Motif North America, Carlsbad, CA), according to the instructions of the manufacturer.
Immunoblotting analysis
Immunoblotting was performed as previously described (14). The antibodies used were: mouse monoclonal antibodies for Bcl-2, Bax, and tubulin and polyclonal antibodies for caspases-3 and -9 (Santa Cruz Biotechnology, Santa Cruz, CA); monoclonal antibody for caspase-8 and p53 and polyclonal antibodies for inhibitor of caspase-activated DNase (ICAD) (DFF45), phospho-c-Jun, and total c-Jun from Upstate Biotechnologies (Lake Placid, NY); monoclonal antibody for Noxa (Alexis Biochemicals, San Diego, CA); monoclonal antibody for poly(ADP-ribose) polymerase (PARP) from BIOMOL; polyclonal antiserum against cellular inhibitor of apoptosis protein-2 and XIAP (R&D Systems Inc., Minneapolis, MN); monoclonal antibody for p21 (Oncogene Research, Cambridge, MA); and polyclonal antiserum against phospho-IB (Ser32), total IB, Bid, and caspase-2 (Cell Signaling, Beverly, MA).
Immunohistochemistry for activated NF-B in carcinoma tissue sections
The anti-p65 antibody MAB3026 (formerly Roche 1697838) (Chemicon International, Temecula, CA) recognizes an epitope overlapping the NLS of the p65 subunit of the NF-B heterodimer (15). This epitope is masked by IB binding. Thus, this antibody selectively binds the IB-free, activated form of NFB, and we used it to evaluate the functional status of NF-B in 52 papillary, 10 follicular, 35 MTC, and five anaplastic formalin-fixed, paraffin-embedded thyroid carcinoma specimens (retrieved retrospectively from the files of the Pathology Department, University of Athens, and in accordance with the Declaration of Helsinki principles and institutional review board policies). Immunohistochemistry was performed and evaluated as previously described (16). The MAB3026 antibody was used at 1:50 dilution.
Effect of bortezomib in thyroid carcinoma cells overexpressing Bcl-2
To evaluate the role of the antiapoptotic molecule Bcl-2 in bortezomib-induced cell death, anaplastic carcinoma FRO cells were stably transfected with a vector carrying the Bcl-2 cDNA (Upstate Biotechnologies) or the empty (neo) vector using Lipofectamine 2000 (Invitrogen) according to the instructions of the manufacturer. Forty-eight hours later, the cells were incubated in growth medium containing G418 (500 µg/ml, Invitrogen) to select stable clones. Four stable clones were selected based on the overexpression of Bcl-2, which was confirmed by immunoblotting, and were subsequently treated with bortezomib (0–50 nM for 24 h).
Statistical analysis
To evaluate the differences across various experimental conditions in the viability experiments with bortezomib (e.g. with or without caspase inhibitors and with or without IGF-I), one-way ANOVA was performed and post hoc tests (Duncan and Dunnett’s T3 tests) served to evaluate differences between individual pairs of experimental conditions. The effect of bortezomib in FRO-neo cells vs. the various bcl-2-transfected clones was evaluated, across different doses of bortezomib, with two-way ANOVA (followed by Duncan and Dunnett’s T3 post hoc tests to evaluate differences between individual experimental conditions). IC50 values were calculated with the help of STATISTICA software (StatSoft, Tulsa, OK). The additive or synergistic nature of the interaction between bortezomib and doxorubicin was evaluated by isobologram analysis using the Calcusyn software program (Biosoft, Ferguson, MO). In all analyses, P < 0.05 was considered statistically significant.
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Results |
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We evaluated the response of a panel of thyroid and (for comparison) nonthyroid tumor cell lines to bortezomib. The medullary cell lines TT, DRO81–1, HRO85–1, and 6–23 were very sensitive to bortezomib-induced cell death (IC50 values in the range of 4.5–19 nM) (Fig. 1A
and Table 1). The anaplastic cell lines FRO, DRO90–1, and BHT101 were also very sensitive to bortezomib (IC50 values in the range of 10–19 nM) (Table 1). These concentrations are well within those achieved clinically in patients treated with bortezomib (4) and significantly lower than the IC50 values for the panel of nonthyroidal tumor cell lines treated under identical conditions in our study. The anaplastic cell lines ARO, SW1736, and KAT18 were less sensitive to a 24-h incubation, but exposure for 48 h to bortezomib resulted in cell death with IC50 values of 10, 16, and 30 nM, respectively. The other cell lines (papillary and follicular) were less sensitive than medullary and anaplastic lines (Fig. 1A).
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). Cell cycle analysis of bortezomib treatment of thyroid carcinoma cells
The cell cycle profile of bortezomib-treated TT (Fig. 1C) and FRO (supplemental Fig. 1A, published as supplemental data on The Endocrine Society’s Journals Online web site at http://jcem.endojournals.org) cells was evaluated by PI analysis with flow cytometry. Most cells treated with bortezomib (25 nM for 16 h in serum-free media) were detected in the sub-G1 region, indicating significant bortezomib-induced apoptosis.
Activation of NF-B in thyroid carcinoma specimens
The transcription factor NF-B has been implicated in the pathogenesis of thyroid carcinoma, and elevated baseline activity has been reported in several cell lines. FRO cells have been reported to have very high baseline NF-B activity, with ARO cells having lower activity and WRO cells having activity almost at the baseline level of normal, untransformed thyrocytes (17). Moreover, NF-B is constitutively active in MTC cells, including TT cells (10), due to Ret-induced phosphorylation, ubiquitination, and proteasomal degradation of IB. We evaluated the activation status of NF-B in papillary, follicular, medullary, and anaplastic carcinoma specimens and found activation of NF-B in 22 of 52 papillary, nine of 10 follicular, 25 of 35 medullary, and four of five anaplastic carcinomas. Immunostaining for NF-B was present in the cytoplasm, with a prominent perinuclear granular pattern and the nucleus of tumor cells, whereas normal thyroid follicular cells were negative (Fig. 2, A–D).
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B activity in thyroid carcinoma cells
Bortezomib increased both phosphorylated and total IB levels (Fig. 3A) and potently suppressed constitutive NF-B DNA binding activity in TT cells (Fig. 3B). We also treated thyroid carcinoma cell lines with the peptide SN50, which consists of the NLS sequence of p50 and inhibits the transportation of active NF-B into the nucleus, resulting in apoptosis in susceptible cells (5). TT and ARO cells were very sensitive to SN50-induced cell death, whereas the bortezomib-resistant follicular carcinoma cells, WRO, were also resistant to the proapoptotic effect of SN50. The SW579 cells, which have intermediate response to bortezomib, also had an intermediate response to SN50 (Fig. 3C). These findings suggest a correlation between sensitivity to bortezomib and response to SN50. Moreover, they correlate with the high constitutive activity of NF-B in TT and FRO cells (10, 17) vs. the low activity of NF-B in WRO cells (17).
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We investigated further the mechanism of bortezomib-induced growth arrest and apoptosis. We evaluated the levels of p53, a proteasome substrate (18), and p21 in bortezomib-treated TT cells. We found that bortezomib potently increased the protein levels of p53 and p21 (Fig. 4A). This pathway may contribute to bortezomib’s antigrowth and proapoptotic effect on TT cells. Similar results were obtained with the anaplastic FRO cells (supplemental Fig. 2, published on The Endocrine Society’s Journals Online Web site at http://jcem.endojournals.org).
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Bortezomib induces caspase cleavage in TT cells (Fig. 4A). Cleavage of the caspase substrates PARP and ICAD was also detected in bortezomib-treated cells, confirming the enzymatic activation of caspases (Fig. 4A). Similar results were obtained with the anaplastic FRO cells (supplemental Fig. 1B). The activation of caspases was also confirmed directly with an enzymatic method (Fig. 4, B and C). The pancaspase inhibitor ZVAD-FMK and the caspase-8 inhibitor IETD-FMK strongly suppressed bortezomib-induced apoptosis. The specific inhibitors of caspase-9 (LEHD-FMK) and caspase-3 (DEVD-FMK) were also partially protective (Fig. 4D). Overall, our data support a role for the caspase cascade as a mediator of bortezomib-induced apoptosis in thyroid carcinoma cells.
Bortezomib induces cleavage of Bid in thyroid carcinoma cells
We detected cleavage of Bid, which is a proapoptotic member of the Bcl-2 family that is proteolytically activated by caspase-8, and up-regulation of Noxa in our model (Fig. 5A), suggesting an implication of Bcl-2 family members in bortezomib-induced apoptosis of thyroid carcinoma cells. To functionally investigate this hypothesis, we overexpressed Bcl-2 in thyroid carcinoma cells. TT cells overexpress Bcl-2 at baseline, compared with all other cell lines in our panel (our unpublished observations). As a result, we chose as a model the FRO cell line, which expresses minimal levels of Bcl-2. Overexpression of Bcl-2 partially protected FRO cells from bortezomib-induced apoptosis (Fig. 5B).
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Treatment with bortezomib resulted in up-regulation of c-Jun protein and phospho-c-Jun levels (Fig. 5C). Similar results were obtained with the anaplastic FRO cells (supplemental Fig. 2).
IGF-I protects thyroid carcinoma cells from bortezomib
IGF-I is a potent growth and survival factor for many normal and neoplastic cells, including thyroid carcinoma cells (19). IGF-I can potently activate the Akt and NF-B pathway (20). We thus studied the effect of IGF-I on bortezomib-induced apoptosis in TT cells. We found that IGF-I suppressed bortezomib-induced cell death in TT cells (Fig. 5D).
Bortezomib sensitizes thyroid carcinoma cells to doxorubicin
We studied the effect of bortezomib on the response of thyroid carcinoma cells to the chemotherapeutic drug doxorubicin, which is frequently used for the treatment of aggressive thyroid carcinomas. We found that bortezomib and doxorubicin had a strong synergistic effect in all thyroid carcinoma cell lines tested (SW579, FRO, TT; Fig. 5E).
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Discussion |
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The similarly high sensitivity to bortezomib shared by medullary and anaplastic carcinoma cells (as well as MM cells) is, obviously, not due to a common histological origin but perhaps to their common feature of high baseline NF-B activity (Refs. 10 , 9 , and 5 , respectively). In the case of MTCs, the high baseline activity of NF-B has been attributed to Ret-induced phosphorylation, ubiquitination, and proteosomal degradation of IB, which allows the transcriptionally active NF-B dimer to enter the nucleus and bind to DNA (10). Constitutive activation of NF-B may also be associated with a more aggressive neoplastic phenotype and decreased susceptibility to apoptosis (5, 6). In our study, the sensitivity of thyroid carcinoma cells to bortezomib correlated with sensitivity to apoptosis induced by the NF-B inhibitor SN50. Furthermore, we detected immunohistochemically the presence of activated NF-B in the thyroid carcinoma specimens, in agreement with prior studies in medullary and anaplastic carcinomas (9, 10). Normal thyrocytes were negative for activated NF-B in our study. Bortezomib inhibits NF-B activity in both MM, as shown previously (7), and thyroid carcinoma cells, as shown in this study. Bortezomib-induced inhibition of proteasome-mediated IB degradation results in IB accumulation, as we found in our model, which would bind NF-B and sequester it in the cytoplasm, preventing its translocation to the nucleus, thus sensitizing the cell to apoptosis.
Bortezomib also strongly up-regulated the expression of the transcription factor c-Jun (both total and phosphorylated forms), indicating the activation of stress pathways, in TT (Fig. 5C) and FRO (supplemental Fig. 2) cells. We previously reported that bortezomib stabilizes JNK and increases c-Jun phosphorylation and DNA binding activity of the transcription factor activator protein-1 in MM cells (7). In thyroid carcinoma cells, inhibition of NF-B activates JNK (9, 17). These data implicate the JNK/activator protein-1 pathway in the stress response to proteasome and NF-B inhibition, as in the case of bortezomib treatment.
In our study, bortezomib increased p53 protein levels in MTC TT (Fig. 4A) and anaplastic FRO (supplemental Fig. 2) cells. FRO cells are known to lack p53 abnormalities in exons 5–8 but have markedly decreased p53 mRNA content (11) and undetectable expression of thyroglobulin (21) at baseline. p53 is an additional proteasome substrate that is involved in apoptosis. The guardian of the genome p53 transcriptionally activates the MDM2 gene, and the MDM2 protein itself functions as a negative regulator of p53 by binding to it and inducing its proteasome-mediated degradation (18). As a result, proteasome inhibition stabilizes p53 protein levels (18). Another explanation for p53 stabilization could be from bortezomib’s inhibitory effect on NF-B activity. IB kinase-1/2(–/–) mouse embryonic fibroblasts, which lack detectable NF-B activity, are more prone to apoptosis and p53 induction in response to doxorubicin, suggesting that NF-B promotes degradation of p53 and that inhibition of NF-B activity may promote p53 stabilization (22). These findings may support a p53/p21-mediated signaling pathway for growth arrest and apoptosis induced by proteasome inhibitors (18). It should be pointed out, however, that proteasome inhibitors are effective in inducing apoptosis even in tumor cells that lack functional p53. The p21 gene is a transcriptional target of p53, and the p21 protein is also degraded by the proteasome, thus suggesting two possible mechanisms for up-regulation of p21 protein levels (transcriptional and posttranslational).
Bortezomib induced caspase-dependent apoptosis in TT (Fig. 4A) and FRO (supplemental Fig. 1B) cells, in our study. The Bcl-2 family member Noxa, which has been implicated in mediating apoptosis induced by cellular stress, DNA-damage, and p53 activation (23), was also found to be up-regulated in TT (Fig. 5A) and FRO (supplemental Fig. 2) cells, in agreement with recent findings in bortezomib-treated melanoma cells (24, 25). Noxa localizes to the mitochondria, in which it interacts with the antiapoptotic Bcl-2 family members Bcl-2, Bcl-XL, and Mcl-1 (23), resulting in activation of caspase-9 (23). In agreement with such a role in our model, overexpression of Bcl-2 in thyroid carcinoma cells attenuated bortezomib-induced apoptosis in our study. Our study suggests that bortezomib-induced apoptosis in thyroid carcinomas is mediated by caspases and may be modulated by the mitochondria and the Bcl-2 family members.
The bortezomib-sensitivity of TT cells was reduced in the presence of IGF-I, suggesting that the IGF-I/Akt axis and the proteasome inhibition constitute two opposing forces, which tend to promote cell survival vs. apoptosis, respectively, and that, from a therapeutic standpoint, the antitumor activity of bortezomib can be enhanced by inhibition of IGF-I and its downstream signaling.
As part of its important antiapoptotic and prosurvival role, the transcription factor NF-B protects cells against DNA damage induced by irradiation and anticancer chemotherapy (5, 6). In agreement with its anti-NF-B activity, bortezomib has been demonstrated to sensitize tumor cells to sublethal concentrations of conventional DNA-damaging chemotherapeutics (6). Aggressive, poorly differentiated, anaplastic and medullary thyroid carcinomas are frequently treated with chemotherapeutic agents, such as doxorubicin, usually with poor outcome due to their intrinsic chemoresistance. We thus studied the effect of bortezomib on the response of thyroid carcinoma cells to doxorubicin. We found that bortezomib and doxorubicin had a strong synergistic effect in all thyroid carcinoma cell lines tested (papillary, anaplastic, medullary). This finding suggests that bortezomib could be incorporated in chemotherapy protocols and used as a chemosensitizer. This appears to be a very promising approach because a recent phase I trial of bortezomib combined with liposomal doxorubicin in patients with advanced hematologic malignancies showed that the combination was safely administered and had enhanced antitumor activity (26).
In summary, we investigated the effect of bortezomib treatment in a panel of thyroid carcinoma cells in vitro and defined apoptotic pathways triggered by this novel anticancer agent. Our study shows that MTCs and poorly differentiated/anaplastic carcinomas are likely to respond to bortezomib treatment. Combining bortezomib with conventional chemotherapeutic agents could yield a synergistic effect. These studies therefore not only shed light onto mechanisms of action of proteasome inhibitors against thyroid carcinoma cells but also suggest novel therapeutic strategies to improve clinical outcome in aggressive cases. We propose the clinical use of bortezomib, alone or in combination with conventional chemotherapy, for patients with MTC, poorly differentiated, and anaplastic carcinomas.
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Acknowledgments |
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Footnotes |
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First Published Online July 18, 2006
Abbreviations: DEVD-FMK, Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-fluoromethylketone; DMSO, dimethylsulfoxide; IB, NF-B inhibitor; ICAD, inhibitor of caspase-activated DNase; IETD-FMK, Ile-Glu(OMe)-Thr-Asp(OMe)-fluoromethylketone; JNK, c-Jun N-terminal kinase; LDH, lactate dehydrogenase; LEHD-FMK, Leu-Glu(OMe)-Hisl-Asp(OMe)-fluoromethylketone; MM, multiple myeloma; MTC, medullary thyroid carcinoma; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NF-B, nuclear factor-B; NLS, nuclear localization signal; PARP, poly(ADP-ribose) polymerase; PI, propidium iodide; XIAB, X-linked inhibitor of apoptosis; ZVAD-FMK, Z-Val-Ala-Asp(OMe)-fluoromethylketone.
Received November 11, 2005.
Accepted July 7, 2006.
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B blockade in multiple myeloma: therapeutic applications. Blood 99:4079–4086B in human thyroid carcinomas. J Biol Chem 279:54610–54619B is constitutively active in C-cell carcinoma and required for RET-induced transformation. Cancer Res 61:4526–4535B by a monoclonal antibody. Biol Chem Hoppe Seyler 376:9–16[Medline]B cascade potentiates the effect of a combination treatment of anaplastic thyroid cancer cells. J Clin Endocrinol Metab 89:410–418B and up-regulation of intracellular anti-apoptotic proteins via the IGF-1/Akt signaling in human multiple myeloma cells: therapeutic implications. Oncogene 21:5673–5683[CrossRef][Medline]B activation: a role for NFB in acquisition of resistance to chemotherapy. Cancer Cell 1:493–503[CrossRef][Medline]This article has been cited by other articles:
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  G. Riesco-Eizaguirre and P. Santisteban New insights in thyroid follicular cell biology and its impact in thyroid cancer therapy Endocr. Relat. Cancer, December 1, 2007; 14(4): 957 - 977. [Abstract] [Full Text] [PDF]
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 C. S. Mitsiades, P. Hayden, V. Kotoula, D. W. McMillin, C. McMullan, J. Negri, J. E. Delmore, V. Poulaki, and N. Mitsiades Bcl-2 Overexpression in Thyroid Carcinoma Cells Increases Sensitivity to Bcl-2 Homology 3 Domain Inhibition J. Clin. Endocrinol. Metab., December 1, 2007; 92(12): 4845 - 4852. [Abstract] [Full Text] [PDF]
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 V. Poulaki, C. S. Mitsiades, V. Kotoula, J. Negri, D. McMillin, J. W. Miller, and N. Mitsiades The Proteasome Inhibitor Bortezomib Induces Apoptosis in Human Retinoblastoma Cell Lines In Vitro Invest. Ophthalmol. Vis. Sci., October 1, 2007; 48(10): 4706 - 4719. [Abstract] [Full Text] [PDF]
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S.-C. J. Yeung, M. She, H. Yang, J. Pan, L. Sun, and D. Chaplin Combination Chemotherapy Including Combretastatin A4 Phosphate and Paclitaxel Is Effective against Anaplastic Thyroid Cancer in a Nude Mouse Xenograft Model J. Clin. Endocrinol. Metab., August 1, 2007; 92(8): 2902 - 2909. [Abstract] [Full Text] [PDF]
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C. Conticello, L. Adamo, R. Giuffrida, L. Vicari, A. Zeuner, A. Eramo, G. Anastasi, L. Memeo, D. Giuffrida, G. Iannolo, et al. Proteasome Inhibitors Synergize with Tumor Necrosis Factor-Related Apoptosis-Induced Ligand to Induce Anaplastic Thyroid Carcinoma Cell Death J. Clin. Endocrinol. Metab., May 1, 2007; 92(5): 1938 - 1942. [Abstract] [Full Text] [PDF]
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