Biological significance of aminopeptidase N/CD13 in thyroid carcinomas

Aminopeptidase N (APN)/CD13 is a transmembrane ectopeptidase expressed on a wide variety of cells. However, the precise function of APN/CD13 in tumor cells and the relationship of APN/CD13 to thyroid cancer remain unclear. In our study, we quantified the expression of APN/CD13 and additionally dipeptidyl peptidase IV (DPIV)/CD26 in thyroid carcinoma cell lines and in tissues of patients with thyroid carcinomas. Undifferentiated anaplastic thyroid carcinomas expressed more APN/CD13 than differentiated thyroid carcinomas. DPIV/CD26 showed an opposite expression pattern. We detected higher levels of DPIV/CD26 in follicular thyroid carcinomas (FTCs) and papillary thyroid carcinomas than in undifferentiated anaplastic thyroid carcinomas. In the undifferentiated thyroid carcinoma cell line 1736, APN/CD13 mRNA expression could be increased by epidermal growth factor, basic fibroblast growth factor, interleukin-6, and tumor necrosis factor alpha. FTC-133 cells stably transfected with an expression vector for APN-enhanced green fluorescent protein showed a higher migration rate than FTC-133 cells transfected with the enhanced green fluorescent protein-control plasmid. Overexpression of APN/CD13 in stably transfected cells is associated with down-regulation of N-myc down-regulated gene (NDRG)-1, melanoma-associated antigen ME491/CD63, and DPIV/CD26 gene expression. Inhibition of APN/CD13 mRNA expression by small interfering RNA induced NDRG-1, ME491/CD63, and DPIV/CD26 mRNA expression in cells of the undifferentiated thyroid carcinoma cell line C643. We conclude that APN/CD13-associated down-regulation of NDRG-1, ME491/CD63, and DPIV/CD26 in thyroid carcinoma cells is an important step of tumor progression to more malignant phenotypes, and we underline the important role of APN/CD13 as mediator in a multimolecular process regulating cell migration.     

Growth and function of thirty-four human benign and malignant thyroid xenografts in untreated nude mice

Tissue was taken from 16 patients with benign thyroid lesions (10 nontoxic nodular colloid goiter, two follicular adenoma, one autonomous adenoma, one iodine-induced thyrotoxicosis, 2 Graves' disease) and 18 patients with malignant thyroid tumors [seven papillary, five follicular, five undifferentiated (anaplastic), and one medullalry carcinoma] and was xenotransplanted into the flanks of 124 syngeneic female BALB/c-nu/nu mice 6 weeks of age. Subsequently, without any further treatment, serum levels of thyroglobulin (TG), T3, T4, and thyroid-stimulating hormone were determined by radioimmunoassay at 4 or 5 weeks posttransplantation and at the end of the experimental time period of 4 months. All animals were autopsied. The grafts were examined by light microscopy and TG immunohistochemistry. Morphologically, the grafts of benign and malignant thyroid tumors showed features overall identical to the original tissue. Conversely, nontoxic nodular colloid goiter and Graves' disease grafts revealed a transformation to normofollicular structures. All benign thyroid grafts showed a stationary growth, as did most differentiated thyroid carcinomas. Tumor take rates in differentiated and in medullary carcinoma were 15%, and in undifferentiated carcinomas, 100%. In the cancer grafts, a correlation between resting phase (period until progressive tumor growth) and survival time of the corresponding patients was disclosed. All patients whose tumors were not taken by nude mice are still alive and show no signs of progressive tumor growth at 9 to 34 months after surgery. All but one patient with tumors revealing positive tumor take died within 3 months (resting phase, 3 weeks) or one year (resting phase, 7 to 14 weeks) after surgery. Integrity of hormonal function in benign and malignant xenografts at 4 months posttransplantation could be shown by significantly higher T3 and T4 serum concentrations in animals with benign thyroid tissues (T3, 1.69 +/- 0.13 nmol/liter; T4, 45.69 +/- 2.09 nmol/liter; S.E.) as compared to controls without grafted tissue [T3, 1.29 +/- 0.10 nmol/liter (p less than 0.05); T4, 33.39 +/- 2.71 nmol/liter (p less than 0.05)] and by increased TG serum concentrations in animals receiving benign (TG, 2.70 +/- 1.39 ng/ml) or malignant (e.g., TG in follicular carcinoma, 34.44 +/- 13.83 ng/ml; controls, 0.30 +/- 0.02 ng/ml) thyroid tissue. Thus, we conclude that benign and malignant thyroid xenografts in the nude mouse maintain full morphological and, regarding T3, T4, and TG serum levels, functional integrity for at least 4 months after transplantation.     

Overexpression of human tumor-associated antigen,RCAS1, is significantly linked to dedifferentiation of thyroid carcinoma

OBJECTIVE: Counterattack by RCAS1 on carcinoma to cytotoxic T cells and natural killer (NK) cells has been suggested as a contribution to carcinoma progression, because RCAS1 can inhibit their proliferation and induce apoptosis. In this study, we examined RCAS1 expression in various thyroid neoplasms in order to clarify its clinical significance. METHODS: We studied RCAS1 expression by means of immunohistochemistry using a mouse monoclonal antibody against RCAS1 for normal thyroid epithelium, follicular adenoma, follicular carcinoma, papillary carcinoma and undifferentiated (anaplastic) carcinoma. RESULTS: Normal epithelium and follicular adenoma did not express or only faintly expressed RCAS1. In thyroid carcinomas. RCAS1 overexpression was more frequently observed in anaplastic (undifferentiated) carcinomas than papillary (p < 0.0001) and follicular carcinomas (p = 0.0018). In follicular carcinoma, the widely invasive type more frequently overexpressed RCAS1 than the minimally invasive type (p = 0.0488). Furthermore, the incidences of RCAS1 overexpression increased with carcinoma dedifferentiation (p < 0.0001). CONCLUSION: These results suggest that RCAS1 may contribute to the progression of thyroid carcinoma with high biological aggressiveness.     

曲古抑菌素A诱导甲状腺鳞癌SW579细胞株凋亡的实验研究

目的:观察曲古抑菌素A(trichostatin A,TSA)对甲状腺鳞癌SW579细胞株生长增殖和凋亡的影响,并探讨其作用机制.方法:四甲基偶氮唑蓝(MTT)法检测培养细胞的增殖情况;吖啶橙染色后荧光显微镜下观察细胞凋亡情况;流式细胞术检测细胞凋亡率;RT-PCR方法检测细胞凋亡相关基因 caspase-3 mRNA表达.结果:TSA明显抑制SW579细胞生长,且呈剂量依赖性.吖啶橙染色后荧光显微镜下观察发现,经TSA处理的各组细胞均可见染色质浓集和边集,核膜出芽及凋亡小体等现象.流式细胞仪分析细胞凋亡率随TSA浓度的升高而升高(P＜0.05) .RT-PCR方法检测发现细胞凋亡相关基因 caspase-3 mRNA表达水平上调.结论:不同浓度的TSA可抑制甲状腺鳞癌SW579细胞增殖,促进细胞凋亡,且呈剂量依赖性;其诱导细胞凋亡的作用机制可能与上调 caspase-3 基因表达,激活 caspase 途径有关.     

Divergent effects of cytokines on human leukocyte antigen-DR antigen expression of neoplastic and non-neoplastic human thyroid cells.

Apparently complex modulatory effects of alpha-interferon (alpha-IFN), tumor necrosis factor (TNF), and epidermal growth factor (EGF) have been found in neoplastic human thyroid cells, which could possibly affect the final outcome in neoplastic disease. This was achieved by examining the influence of alpha-IFN, TNF, and EGF alone and in combination, on human leukocyte antigen-DR (DR) antigen expression and viability of neoplastic and non-neoplastic human thyroid cells in culture. alpha-IFN-induced DR antigen expression on non-neoplastic human thyroid cells, whereas TNF-alpha or EGF alone were ineffective. The addition of the same TNF-alpha concentrations (10 to 100 ng/ml) to alpha-IFN enhanced the expression of DR antigens compared with the effect of alpha-IFN alone. However, EGF inhibited alpha-IFN-induced DR on the same cells and at the same concentrations (10 to 500 ng/ml) at which the growth factor alone was ineffective. In contrast to the common pattern of cytokine effects on DR expression of all nonmalignant thyroid cell lines, neoplastic thyroid cell lines showed divergent responses to alpha-IFN, TNF-alpha, and EGF. In three malignant thyroid cell lines that were DR negative (follicular carcinoma WRO 82-1 and NRO 87-1 cell lines, and anaplastic carcinoma ARO 81-1), DR antigen could be induced by alpha-IFN and enhanced by TNF-alpha, whereas EGF was ineffective. In a fourth cell line (an anaplastic carcinoma SW1736) alpha-IFN, TNF-alpha, and EGF alone were capable of inducing DR, and a combination of either TNF-alpha and EGF with alpha-IFN potentiated DR induction. In a fifth neoplastic cell line (papillary carcinoma, NPA) that constitutively expressed surface DR, its expression was inhibited by both alpha-IFN and TNF-alpha and was not affected by EGF.     

曲古抑菌素A诱导甲状腺鳞癌SW579细胞株凋亡的实验研究

目的：观察曲古抑菌素A（trichostatin A,TSA）对甲状腺鳞癌SW579细胞株生长增殖和凋亡的影响,并探讨其作用机制。方法：四甲基偶氮唑蓝（MTT）法检测培养细胞的增殖情况;吖啶橙染色后荧光显微镜下观察细胞凋亡情况;流式细胞术检测细胞凋亡率;RT—PCR方法检测细胞凋亡相关基因caspase-3 mRNA表达。结果：TSA明显抑制SW579细胞生长,且呈剂量依赖性。吖啶橙染色后荧光显微镜下观察发现,经TSA处理的各组细胞均可见染色质浓集和边集,核膜出芽及凋亡小体等现象。流式细胞仪分析细胞凋亡率随TSA浓度的升高而升高（F〈0．05）。RT—PCR方法检测发现细胞凋亡相关基因caspase-3 mRNA表达水平上调。结论：不同浓度的TSA可抑制甲状腺鳞癌SW579细胞增殖,促进细胞凋亡,且呈剂量依赖性;其诱导细胞凋亡的作用机制可能与上调caspase-3基因表达,激活caspase途径有关。     

Antitumor effects of peroxisome proliferator activate receptor gamma ligands on anaplastic thyroid carcinoma

Anaplastic thyroid carcinoma is an aggressive neoplasm and resistant to all sorts of treatment due to its rapid growth and invasive potential. Peroxisome proliferator-activated receptor gamma (PPARgamma) is a nuclear receptor modulating variety of biological properties, such as regulating of adipogenesis, inhibition of cancer cell proliferation or differentiation of tumor cells. The purpose of this study was to evaluate the possibility for the therapeutic effect of PPARgamma ligands against anaplastic thyroid tumor in vitro. Expressions of the PPARc gene and protein were examined in 5 human anaplastic carcinoma cell lines (MSA, IAA, ROA, K119 and KOA-2). We next evaluated the effects of PPARgamma ligands (Thiazolidinedione, Prostaglandin J2 and RS1303) on proliferation, differentiation, apoptosis and invasion. Five cell lines showed higher level of the PPARc gene and protein expression than papillary thyroid carcinoma. PPARgamma ligands inhibited cell proliferation by inducing apoptosis instead of differentiation in dose-dependent manner. PPARgamma ligands also down regulated the invasive potential of 5 cell lines. The inhibitory effect of proliferation or invasion was prominent in 3 cell lines, which exhibited higher expression level of the PPARc gene or protein. Our results indicated that PPARgamma ligands modify malignant potential of anaplastic carcinoma cell lines altering growth or invasive properties, suggesting that PPARgamma could be potentially the novel molecular target for human thyroid anaplastic carcinoma.     

Expression of galectin-1 in normal human thyroid gland and in differentiated and poorly differentiated thyroid tumors

We previously reported that galectin-l gene expression increases up to 100-fold in oncogene-transformed rat thyroid cells compared with their normal counterparts and that the relative mRNA levels correlate with the degree of malignancy. In the present study we investigated whether galectin-l is differentially expressed in human thyroid neoplasms, which range from well-differentiated tumors to undifferentiated ana-plastic carcinomas. We analyzed 74 human thyroid specimens of neoplastic, hyperproliferative and normal tissues and several tumor cell lines. Galectin-l mRNA and protein levels were higher in 6 thyroid carcinoma-derived cell lines than in normal thyroid primary cultures and adenoma cells. Galectin-l mRNA levels increased in 28/40 papillary carcinomas and in 6/7 anaplastic carcinomas compared with normal or hyperplastic thyroid. Conversely, galectin-l expression was unaffected in follicular carcinomas and benign adenomas. Immunohistochemi-cal analysis of normal thyroid and papillary carcinoma sections revealed a higher content of galectin-l protein in neoplastic follicular cells than in normal cells. © 1995 Wiley-Liss, Inc.     

人绒毛膜促性腺激素对甲状腺乳头状癌的细胞增殖及周期的影响

目的 研究人绒毛膜促性腺激素（hCG）对甲状腺乳头状癌细胞增殖活力及细胞周期的影 响。方法 选用甲状腺乳头状癌细胞株IHH-4为研究对象,采用细胞增殖活性实验MTT（四甲基偶氮 唑盐比色法）及流式细胞术检测不同浓度、不同作用时间hCG对IHH-4细胞增殖活力、细胞周期的影 响。结果 hCG能刺激IHH-4细胞的活力,浓度为100 IU/ml时最为明显;在100 IU/ml的hCG作用48 h 后, IHH-4细胞G2M+S期的百分比为67.1%,显著高于对照组。结论 人绒毛膜促性腺激素对甲状腺 乳头状癌细胞的生长活力有促进作用,并能加快其细胞周期。     

Glycerol enhances CDDP-induced growth inhibition of thyroid anaplastic carcinoma tumor carrying mutated p53 gene

To increase the chemo-sensitivity of anaplastic thyroid carcinoma, we examined the effects of glycerol on the tumor growth after CDDP treatment. The cultured cells of an anaplastic thyroid carcinoma cell line (8305c) carrying a mutated p53 gene (mp53) were transplanted into the thighs of nude mice. Tumor growth was evaluated until 24 days after intraperitoneal injection of CDDP and/or pre-injection of glycerol to the tumor. We treated the mice with half the tumor volume of glycerol (1.2 M) and/or CDDP at 6 mg/kg (BW) either of which hardly inhibited tumor growth by itself. When we treated the mice with the combination of glycerol and CDDP at these concentrations, however, a clear delay of the tumor growth was observed. We also immunohistochemically analyzed the effects of glycerol on the induction of caspase-3 activity and apoptosis. Cells positive for cleavage to active caspase-3 and 85 kDa PARP, and apoptosis were hardly observed in the tumors when they were treated with glycerol or CDDP alone. In contrast, when they were treated with CDDP combined with glycerol, such positive cells were significantly increased. It has been shown that glycerol synergistically enhanced the effects of CDDP as a tumor suppressive agent through the induction of caspase-3-mediated apoptosis in 8305c tumors. Therefore, glycerol might be useful for chemotherapy in patients with mp53 cancer cells.     

Cannabinoid 2 receptor induction by IL-12 and its potential as a therapeutic target for the treatment of anaplastic thyroid carcinoma

Anaplastic thyroid carcinoma is the most aggressive type of thyroid malignancies. Previously, we demonstrated that tumorigenicity of anaplastic thyroid carcinoma cell line ARO was significantly reduced following interleukin (IL)-12 gene transfer. We suspected that tumor target structure in ARO/IL-12 cells might be changed and such a change may make them more susceptible to be killed through mechanisms apart from natural killer-dependent pathway. To identify genes involved, we examined gene expression profile of ARO and ARO/IL-12 by microarray analysis of 3757 genes. The most highly expressed gene was cannabinoid receptor 2 (CB2), which was expressed eightfold higher in ARO/IL-12 cells than ARO cells. CB2 agonist JWH133 and mixed CB1/CB2 agonist WIN-55,212-2 could induce significantly higher rate of apoptosis in ARO/IL-12 than ARO cells. Similar results were obtained when ARO cells were transfected with CB2 transgene (ARO/CB2). A considerable regression of thyroid tumors generated by inoculation of ARO/CB2 cells was observed in nude mice following local administration of JWH133. We also demonstrated significant increase in the induction of apoptosis in ARO/IL12 and ARO/CB2 cells following incubation with 15 nM paclitaxel, indicating that tumor cells were sensitized to chemotherapy. These data suggest that CB2 overexpression may contribute to the regression of human anaplastic thyroid tumor in nude mice following IL-12 gene transfer. Given that cannabinoids have shown antitumor effects in many types of cancer models, CB2 may be a viable therapeutic target for the treatment of anaplastic thyroid carcinoma.     

Thyroid carcinoma cells are resistant to FAS-mediated apoptosis but sensitive to tumor necrosis factor-related apoptosis-inducing ligand

Fas (APO-1/CD95) is a transmembrane protein of the tumor necrosis factor (TNF)/nerve growth factor receptor superfamily that induces apoptosis in susceptible normal and neoplastic cells upon cross-linking by its ligand (FasL). TNF-related apoptosis-inducing ligand (TRAIL) is a more recently identified member of the TNF superfamily that has been shown to selectively kill neoplastic cells by engaging two cell-surface receptors, DR4 and DR5. Two additional TRAIL receptors (DcR1 and DcR2) do not transmit an apoptotic signal and have been proposed to confer protection from TRAIL-induced apoptosis. We addressed the expression of Fas, DR4, and DR5 in thyroid carcinoma cell lines and in 31 thyroid carcinoma specimens by Western blot analysis and immunohistochemistry, respectively, and tested the sensitivity of thyroid carcinoma cell lines to Fas- and TRAIL-induced apoptosis. Fas was found to be expressed in most thyroid carcinoma cell lines and tissue specimens. Although cross-linking of Fas did not induce apoptosis in thyroid carcinoma cell lines, Fas-mediated apoptosis did occur in the presence of the protein synthesis inhibitor cycloheximide, suggesting the presence of a short-lived inhibitor of the Fas pathway in these cells. Cross-linking of Fas failed to induce recruitment and activation of caspase 8, whereas transfection of a constitutively active caspase 8 construct effectively killed the SW579 papillary carcinoma cell line, arguing that the action of the putative inhibitor occurs upstream of caspase 8. By contrast, recombinant TRAIL induced apoptosis in 10 of 12 thyroid carcinoma cell lines tested, by activating caspase-10 at the receptor level and triggering a caspase-mediated apoptotic cascade. Resistance to TRAIL did not correlate with DcR1 or DcR2 protein expression and was overcome by protein synthesis inhibition in 50% of the resistant cell lines. One medullary carcinoma cell line was resistant to Fas-and TRAIL-induced apoptosis, even in the presence of cycloheximide, and to transfection of constitutively active caspase-8, suggesting a different regulation of the apoptotic pathway. Our observations indicate that TRAIL effectively kills carcinomas that originate from the follicular epithelium of the thyroid gland, by inducing caspase-mediated apoptosis, and may provide a potentially potent therapeutic reagent against thyroid cancer.     

Phase I clinical and pharmacokinetic study of plitidepsin as a 1-hour weekly intravenous infusion in patients with advanced solid tumors.

PURPOSE: Plitidepsin, given as a 1-hour weekly i.v. infusion for 3 consecutive weeks during a 4-week treatment cycle, was investigated in patients with solid tumors to determine the maximum tolerated dose and the recommended dose (RD) using this administration schedule. EXPERIMENTAL DESIGN: Consecutive cohorts of patients with metastatic solid tumors or non-Hodgkin's lymphomas were to be treated at escalating doses of plitidepsin in a conventional phase I study including pharmacokinetic analyses of plitidepsin in plasma, whole blood, and blood cell pellets. RESULTS: Forty-nine patients with solid tumors were enrolled, and 48 were treated with plitidepsin (doses from 0.133 to 3.6 mg/m2/week). Dose-limiting toxicities (defining 3.6 mg/m2/week as the maximum tolerated dose) included myalgia, increased creatine phosphokinase levels, and sustained grade 3/4 increases of hepatic enzyme levels. The RD was established at 3.2 mg/m2/week. The most common toxicities were fatigue, vomiting/nausea, anorexia, injection site reaction, and pain, mostly of mild or moderate severity. Muscular toxicity manifested by mild-moderate myalgia, weakness, and/or creatine phosphokinase elevations occurred in approximately 25% of patients and seemed to be dose related. Transient transaminase elevations were frequent but achieved grade 3 or 4 in only approximately 10% of patients. Plitidepsin lacked significant hematologic toxicity. No complete or partial tumor responses were observed; however, five patients had disease stabilization (including one patient with medullary thyroid carcinoma with an unconfirmed partial response and one patient with renal carcinoma with major tumor shrinkage in lung metastases). Pharmacokinetic results for the RD indicated a long plasma half-life give value (16.8 +/- 7.7 hour) and a high volume of distribution value (525.2 +/- 219.3 L). CONCLUSIONS: The recommended dose for plitidepsin given as a weekly 1-hour schedule was 3.2 mg/m2/week. Muscular and liver toxicity were dose limiting at 3.6 mg/m2/week. Additional evaluation of this dose dense schedule is warranted.     

Primary localized non-Hodgkin's lymphoma of the thyroid: a retrospective clinicopathological review

Primary localized non-Hodgkin's lymphomas (NHL) of the thyroid are rare. The data presented are derived from 819 consecutive patients with NHL and six patients with anaplastic thyroid carcinomas of small cell type investigated and treated at our department between 1970 and 1981. The present analyses are based on the 19 patients, who were found to have localized primary thyroid lymphomas. Four of these patients were initially considered to have undifferentiated small cell carcinomas of the thyroid but revealed to be lymphomas at re-examination supplemented with immunohistopathologic staining. Prognosis has been evaluated with regard to initial stage, histopathology according to the Kiel classification and therapy. Median follow-up was 5 years. The crude survival was 77% 5 years after diagnosis. This was not significantly less (4.2%) than the overall-survival in an age- and sex-matched population, despite the majority of patients having tumours which were locally advanced, often with spread to regional lymph nodes, and in many cases a histology showing a high-grade malignancy according to Kiel classification. The excellent prognosis in the current study compared to other studies is probably mainly attributed to more extensive staging procedures. The biologic behaviour supports the hypothesis that these lymphomas represent lymphomas of mucosa associated lymphoid tissue (MALT). According to present results anaplastic thyroid carcinoma of small cell type must be extremely rare.     

甲状腺未分化癌

甲状腺未分化癌较少见。我们从１９７７年～１９９０年共收治甲状腺癌３７２例，未分化癌仅１１例，占３％。病理类型分为梭形细胞巨细胞型和小细胞型。此型癌以恶性程度高为特点，治疗很困难。作者采用手术＋放疗＋化疗提高了疗效，本组生存５年者３例，占２７．３％；存活２年３例；１／２以上的病人存活超过２年。     

In vivo and microarray analysis of rexinoid-responsive anaplastic thyroid carcinoma.

PURPOSE: Anaplastic thyroid carcinoma is rare, yet lethal despite aggressive therapy. Molecular targeting may be beneficial using the rexinoid LGD1069, a retinoid X receptor-selective agonist, as a novel treatment. In this report, we describe the efficacy of LGD1069 in anaplastic thyroid carcinoma in vitro and assess the in vivo treatment effects on a responsive cancer. Additionally, we explore potential mediators of the rexinoid effect on a responsive anaplastic thyroid cancer using comparative microarray analysis. EXPERIMENTAL DESIGN: Anaplastic thyroid cancer cell lines DRO, ARO, and FRO were treated with LGD1069 in vitro. Responsive DRO xenograft tumors were treated with control chow or chow containing a low dose (30 mg/kg/d) or a high dose (100 mg/kg/d) of LGD1069. Comparative microarray analysis of DRO cells treated with LGD1069 compared with volume-equivalent control was assessed after 24 h of treatment to evaluate early gene expression changes. RESULTS: DRO xenograft tumor growth was inhibited by LGD1069 treatment in a dose-dependent manner. Comparative microarray analysis showed that 80 genes had a significant increase in expression and 29 genes had a decrease in expression after 24 h of treatment with LGD1069. Expression of angiopoietin-like 4 (ANGPTL4) mRNA was increased 6.5-fold. A trend towards an increase in ANGPTL4 mRNA (not statistically significant) was seen in treated tumors in vivo and this correlated with decreased tumor vascularity and increased necrosis. CONCLUSIONS: LGD1069 therapy decreases proliferation in an anaplastic thyroid cancer cell line that expresses retinoid X receptor-gamma, and this effect is confirmed with decreased tumor size in vivo in a nude mouse model. ANGPTL4 is increased in DRO in response to LGD1069 and may be a potential mediator of the effects of rexinoid treatment.