全反式维甲酸对各种人甲状腺肿瘤细胞株增殖、摄碘及甲状腺特异基因表达的影响

对4种甲状腺癌细胞株予全反式维甲酸处理,四甲基偶氮唑盐法检测细胞增殖,同位素法测定摄碘功能,半定量RT-PCR检测甲状腺特异基因及维甲酸受体表达.结果 显示全反式维甲酸可抑制FTC-133细胞增殖,促进其摄碘及甲状腺特异基因的表达,但对C643、HTH74及XTC.UC1细胞无影响,提示不同甲状腺肿瘤细胞对全反式维甲酸的反应性不相同.     

Beneficial effects of retinoic acid on extracellular matrix degradation and attachment behaviour in follicular thyroid carcinoma cell lines

The prognosis of patients with metastasised follicular thyroid carcinoma (FTC) is limited, necessitating the search for new treatment options. Beneficial effects of retinoids have been suggested in thyroid cancer and the present study was performed to investigate the effects of retinoic acid (RA) on important determinants of metastatic behaviour in FTC: the disengagement of tumour cells from the primary tumour and the degradation of extracellular matrix, focusing on the role of the plasmin activation system and the integrin and E-cadherin families of attachment molecules. Three FTC cell lines were studied: FTC-133, derived from the primary tumour; and FTC-236 and FTC-238, derived from metastases. FTC cell lines were cultured with 0.1, 1 and 10 microM 13-cis-RA or with the solvent DMSO for 1 and 5 days. Extracellular matrix degradation by these cell lines was studied by assessing the 48-h release of radioactivity from (35)S-methionine labelled extracellular matrix proteins synthesised by the MC3T3 cell line coated onto plastic. The involvement of constituents of the plasmin activation system was investigated by semi-quantitative RT-PCR and zymography. Attachment to extracellular matrix was studied by determining the number of adhering FTC cells to extracellular matrix coated onto plastic, 3 h after seeding. The involvement of attachment molecules was studied by RT-PCR with primers for integrin subclasses and E-cadherin and immunofluorescence for E-cadherin. Five days culturing with 10 microM RA reduced the degradation of extracellular matrix significantly in all cell lines: FTC-133 by 35%, FTC-236 by 74% and FTC-238 by 31%. Zymography revealed diminished activity of urokinase type plasminogen activator (uPA) in FTC-236 and FTC-238, but not in FTC-133 cultured with RA. mRNA expression of the uPA receptor was diminished in FTC-236. In the attachment assay, 10 microM RA for 5 days increased the number of adherent cells to extracellular matrix significantly by 91% in FTC-133, 64% in FTC-236 and 87% in FTC-238. No effects of RA on integrin or E-cadherin mRNA expression were observed. Immunofluorescence, however, revealed enhanced organisation of E-cadherin along the cell membrane by RA treatment. In conclusion, the present study demonstrates beneficial effects of RA on important determinants of metastatic behaviour in FTC cell lines, e.g. decreased degradation of extracellular matrix which may in part be explained by effects on the plasmin activation system and enhanced attachment to extracellular matrix. These findings may add to the explanations for beneficial effects of retinoids in thyroid cancer.     

Retinoic acid and retinoid X receptors are differentially expressed in thyroid cancer and thyroid carcinoma cell lines and predict response to treatment with retinoids

Therapy for patients with advanced thyroid carcinoma is limited. Clinical and in vitro studies suggest that some patients with advanced thyroid cancer may respond to therapy with retinoic acid. mRNA expression of the six retinoic acid (RAR) and retinoid X receptor (RXR) isoforms (RARalpha, -beta, -gamma and RXRalpha, -beta, -gamma) was measured in four human thyroid cell lines, and protein expression was subsequently measured in 10 thyroid cancer cell lines. Two isoforms, RARbeta and RXRgamma, were differentially expressed in the four cell lines. Comparison of 10 thyroid tumors and matched normal thyroid tissue confirmed differential tumor expression of RARbeta and RXRgamma and lack of the RXRgamma isoform in normal thyroid tissue. Cell lines expressing both RARbeta and RXRgamma demonstrated significant growth suppression when treated with retinoids, whereas cell lines lacking these isoforms were unaffected. Expression of RARbeta, the isoform associated with suppression of tumor growth in other cancer types, was not affected by treatment with retinoids in the thyroid cancer cell lines. LG346 increased apoptosis and decreased cells in the S-phase in an anaplastic carcinoma cell line, suggesting that this retinoid causes growth suppression of these cells by multiple mechanisms. In summary, we identified the RARbeta and RXRgamma isoform to be differentially expressed in thyroid cancer cell lines and tumor tissue. These isoforms seem to predict response to retinoid therapy in thyroid cancer cell lines.     

Retinoid activation of retinoic acid receptor but not retinoid X receptor is sufficient to rescue lethal defect in retinoic acid synthesis

Two isomers of retinoic acid (RA) may be necessary as ligands for retinoid signaling: all-trans-RA for RA receptors (RARs) and 9-cis-RA for retinoid X receptors (RXRs). This was explored by using retinaldehyde dehydrogenase (Raldh)2-/- mouse embryos lacking mesodermal RA synthesis that display early growth arrest unless rescued by all-trans-RA administration. Because isomerization of all-trans-RA to 9-cis-RA can occur, it is unclear whether both ligands are needed for rescue. We show here that an RAR-specific ligand can rescue Raldh2-/- embryos as efficiently as all-trans-RA, whereas an RXR-specific ligand has no effect. Further, whereas all-trans-RA was detected in embryos, 9-cis-RA was undetectable unless a supraphysiological dose of all-trans-RA was administered, revealing that 9-cis-RA is of pharmacological but not physiological significance. Because 9-cis-RA is undetectable and unnecessary for Raldh2-/- rescue, and others have shown that 4-oxo-RA is unnecessary for mouse development, all-trans-RA emerges as the only ligand clearly necessary for retinoid receptor signaling.     

Human adrenocortical carcinoma cell lines

The human adrenal cortex secretes mineralocorticoids, glucocorticoids and adrenal androgens. These steroids are produced from unique cell types located within the three distinct zones of the adrenal cortex. Disruption of adrenal steroid production results in a variety of diseases that can lead to hypertension, metabolic syndrome, infertility and androgen excess. The adrenal cortex is also a common site for the development of adenomas, and rarely the site for the development of carcinomas. The adenomas can lead to diseases associated with adrenal steroid excess, while the carcinomas are particularly aggressive and have a poor prognosis. In vitro cell culture models provide important tools to examine molecular and cellular mechanisms controlling both the normal and pathologic function of the adrenal cortex. Herein, we discuss currently available human adrenocortical carcinoma cell lines and their use as model systems for adrenal studies.     

维甲酸诱导失分化型甲状腺癌的观察

应用全反式维甲酸(ATRA)诱导失分化型甲状腺癌(dDTC)患者,5例患者(5/9,55.6%)ATRA诱导有效;5例患者(5/8,62.5%)131I治疗获益.3例患者(3/12,25.O%)因神经系统的不良反应,不能耐受而放弃ATRA诱导.ATRA是诱导dDTC分化的有效方法,可以提高dDTC病灶的摄碘能力,但使用中应关注ATRA的不良反应.     

Retinoic acid receptor beta2 re-expression and growth inhibition in thyroid carcinoma cell lines after 5-aza-2'-deoxycytidine treatment

The treatment of both undifferentiated and de-differentiated thyroid tumors, which are unresponsive to radioiodine, represents one of the biggest challenges for thyroidologists. The aim of the present study was to investigate in vitro the methylation status of retinoic acid receptors (RAR)beta2 promoter and the effect of the demethylating agent 5-aza-2'-deoxycytidine (5-Aza-CdR) on 5 human thyroid cancer cell lines. The methylation status of RARbeta2 promoter was analyzed by methylation-specific PCR. The effect of 5-Aza-CdR on cell growth and apoptosis was evaluated by cell counting, enzymelinked immunosorbent assay tests and fluorescence-activated cell sorting analysis, while the effect on the expression of RAR and thyroid-specific genes was measured by qualitative and quantitative RT-PCR. Methylation of RARbeta2 promoter was present only in ARO cells. 5-Aza-CdR determined growth inhibition in all cell lines, probably due to apoptosis in WRO, NPA, and ARO cells, and to inhibition of DNA synthesis in TT cells. Treatment with 5-Aza-CdR induced the expression of RARbeta mRNA in ARO and FRO cells, a slight increase of the expression of Tg, TPO and thyroid trancription factor 1 (TTF-1) mRNA and the new expression of low levels of NIS in TT cells. A significant increase of TTF-1 mRNA in FRO cells was also observed. In this study we demonstrated that RARbeta2 promoter was methylated in ARO cell line. However, the 5-Aza-CdR treatment induced RARbetamRNA expression not only in ARO but also in FRO and TT cell lines, whose RARbeta2 promoter was unmethylated. A significant reduction of cell growth, but not cell re-differentiation, was also observed after 5-Aza-CdR treatment.     

维甲酸对人肝癌细胞凋亡的影响及临床意义

目的：观察全反式维甲酸（ATRA）对肝癌细胞凋亡的作用并探讨ATR的作用机制。方法：在肝癌细胞株SMMC－7721细胞加入ATRA,使终浓度为0．5μg/ml,对照组加等剂量的0．02％DMSO,培养4d,ATRA对SMMC－7721细胞的影响通过苏木精－伊红染色,在光镜下进行细胞形态学观察;通过细胞计数绘制生长曲线及计算细胞生长抑制率,流式细胞术分析不同DNA含量的细胞分布,计算细胞凋亡百分率,凋亡相关基因Fas,p53,Bcl-2蛋白表达的检测亦采用流式细胞仪检测。结果：苏木精－伊红染色可见较多细胞核碎裂,胞浆浓缩,染色质深染,聚集于核膜下,部分细胞膜突起呈小泡样,小泡脱落形成凋亡小体,而对照组无明显的形态学改变,作用4d生细胞生长抑制率为47．5％,与对照组比较,差异显著（P＜0．05）,流式细胞仪分析在G1期前出现亚二倍体凋亡峰;凋亡细胞百分比为16．0％,与对照的6．9％比较有显著性差异（P＜0．01）。观察发现,ATRA对SMMC－771细胞Fax,P53的表达明显增加,并下调Bcl-2的表达,结论：0．5μg/ml的ATRA对肝癌细胞的生长有显著抑制效果,并可见癌细胞凋亡形态改变,ATRA促进SMMC－721肝癌细胞凋亡的机制之一可能是通过调控Fas,P53及Bcl-2的表达。     

Effect of retinoic acid on cell proliferation kinetics and retinoic acid receptor expression of colorectal mucosa

AIM: To investigate the effect of retinoic acid (RA) on cell proliferation kinetics and retinoic acid receptor (RAR)expression of colorectal mucosa.METHODS:One hundred sixty healthy male Wistar rats were randomly divided into 4 groups. Rats in groups Ⅰ and Ⅱ were subcutaneously injected with dimethylhydrazine (DMH) (20 mg/kg, once a week,) for 7 to 13 weeks, while groups Ⅲ and Ⅳ were injected with normal saline. Rats in groups Ⅱ and Ⅲ were also treated with RA (50 mg/kg,every day, orally) from 7th to 15th week, thus group Ⅳ was used as a control. The rats were killed in different batches.The expressions of proliferating cell nuclear antigen (PCNA),nucleolar organizer region-associated protein (AgNOR) and RAR were detected.RESULTS: The incidence of colorectal carcinoma was different between groupsⅠ(100 %) and Ⅱ (15 %) (P＜0.01).The PCNA indices and mean AgNOR count in group Ⅱ were significantly lower than those in group Ⅰ(F=5.418 and 4.243,P＜0.01). The PCNA indices and mean AgNOR count in groups Ⅰ and Ⅱ were significantly higher than those in the groups Ⅲ and Ⅳ (in which carcinogen was not used) (F=5.927and 4.348, P＜0.01). There was a tendency in group Ⅰ that the longer the induction with DMH the higher PCNA index and AgNOR count expressed (F=7.634 and 6.826, P＜0.05).However, there was no such tendency in groups Ⅱ, Ⅲ and Ⅳ(F=1.662 and 1.984, P＞0.05). The levels of RAR in normal and cancerous tissues in groups treated with RA were significantly higher than those in groups not treated with RA (F=6.343 and 6.024, P＜0.05).CONCLUSION: RA decreases the incidence of colorectal carcinoma induced by DMH. Coiorectal cancer tissue is associated with abnormal expression of PCNA, AgNOR and RAR. RA inhibits the expression of PCNA and AgNOR, and increases RAR concentration in colorectal tissues.     

Downregulation of retinoic acid receptor-β2expression is linked to aberrant methylation in esophageal squamous cell carcinoma cell lines

AIM: To study the role of hypermethylation in the loss ofretinoic acid receptorβ2(RARβ2) in esophageal squamous cell carcinoma (ESCC).METHODS: The role of hypermethylation in RAR,β2 gene silencing in 6 ESCC cell lines was determined by methylationspecific PCR (MSP), and its methylation status was compared with RARβ2 mRNA expression by RT-PCR. The MSP results were confirmed by bisulfite sequencing of RARβ2promoter regions. RESULTS: Methylation was detected in 4 of the 6 cell lines, and the expression of RARβ2was markedly downregulated in 3 of the 4 methylated cell lines. The expression of RARβ2was restored in one RARβ2-downregulated cell line with the partial demethylation of promoter region of RARβ after 5aza-2'-deoxycytidine (5-aza-dc) treatment.CONCLUSION: The methylation of the 5' region may play an important role in the downregulation of RARβ2 in someESCC cell lines, suggesting that multiple mechanisms contribute to the loss of RARβ2expression in ESCC cell lines. This study may have clinical applications for treatment and prevention of ESCC.     

Ito cell expression of a nuclear retinoic acid receptor.

Although it has been suggested that retinoids regulate Ito cell proliferation and collagen synthesis, little is known about the ability of Ito cells to respond to retinoids in vivo. Because retinoids may mediate their molecular effects through nuclear receptors, Ito cells were examined for the presence of one of these receptors, nuclear retinoic acid receptor-beta. The modulation of nuclear retinoic acid receptor-beta expression was also studied during cell culture and hepatic fibrogenesis. Northern hybridization analysis revealed that Ito cells freshly isolated from normal rat liver contained nuclear retinoic acid receptor-beta messenger RNA at levels significantly higher than those found in other hepatic cell types. Ito cells also contained messenger RNA for two other nuclear retinoic acid receptors, nuclear retinoic acid receptor-alpha and nuclear retinoic acid receptor-gamma. Using an antibody to human nuclear retinoic acid receptor-beta, the nuclear presence of this receptor was demonstrated in normal Ito cells. In contrast, Ito cells cultured for at least 7 days had no detectable messenger RNA or nuclear staining for nuclear retinoic acid receptor-beta despite a 20 +/- 5-fold increase in the messenger RNA level of another retinoid binding protein, cellular retinol binding protein. Analysis of Ito cells isolated from rats with carbon tetrachloride-induced hepatic fibrosis revealed an 81% +/- 3% decrease in nuclear retinoic acid receptor-beta messenger RNA levels in these cells when compared with normal Ito cells. No difference in the messenger RNA levels of cellular retinol binding protein was found in Ito cells isolated from either normal or fibrotic liver.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ubiquitous receptor: a receptor that modulates gene activation by retinoic acid and thyroid hormone receptors.

The cDNA for a member of the nuclear receptor family was cloned and named ubiquitous receptor (UR), since UR protein and mRNA are detected in many cell types. Rat UR/human retinoid X receptor alpha (hRXR alpha) heterodimers bound preferentially to double-stranded oligonucleotide direct repeats having the consensus half-site sequence AGGTCA and 4-nt spacing (DR-4). Coexpression of UR in COS-1 cells inhibited the stimulation of chloramphenicol acetyltransferase (CAT) reporter gene expression by hRXR alpha and human retinoic acid receptor alpha in the presence of all-trans-retinoic acid when DR-4 (but not DR-5) was present upstream of the promoter of a CAT reporter gene (DR-4-CAT). UR expression also inhibited the activation of a DR-4-CAT reporter gene by hRXR alpha and 9-cis-retinoic acid or by thyroid hormone receptor beta in the presence of thyroid hormone. However, in the absence of 9-cis-retinoic acid, UR in combination with hRXR alpha stimulation DR-4-CAT expression. Coexpression of thyroid hormone receptor markedly reduced this stimulation in the absence of thyroid hormone. UR may play an important role in normal growth and differentiation by modulating gene activation in retinoic acid and thyroid hormone signaling pathways.     

Oxyphilic and non-oxyphilic thyroid carcinoma cell lines differ in expressing apoptosis-related genes

Oxyphilic tumors of the thyroid are characterized by mitochondrion-rich cells and extensive DNA fragmentation. In order to clarify if a different expression of apoptosis-related genes could be responsible for DNA fragmentation in oxyphilic cell tumors, two thyroid follicular carcinoma-derived cell lines, having oxyphilic (XTC.UC1) and non-oxyphilic (WRO) features, were compared applying a gene array technique. Under basal culture conditions, several pro-apoptotic genes [caspases 3 and 10, Fas and the tumor necrosis factor-related apoptosis-inducing ligand (trail) genes] were switched on in oxyphilic, but not in non-oxyphilic cells. No difference in the mitochondrial apoptosis-related genes (bax, bad, bcl family etc.) was observed. Using the ISEL technique, the extent of DNA fragmentation did not differ under basal conditions in the two cell lines. Conversely, following an oxidative pro-apoptotic stress (6-h methylene blue treatment and light exposure), XTC.UC1 cells showed an extensive DNA fragmentation (up to 70% of cells), dramatically exceeding that observed in WRO cells (up to 20% of cells). In contrast, the oxidative stimulus induced a remarkable apoptosis gene activation in non-oxyphilic WRO cells only. These results suggest that oxyphilic cells may have a unique silent activation of a pro-apoptotic phenotype, which could be responsible for DNA instability and lead to cell death as the consequence of an increased sensitivity to ischemic stresses, as frequently observed in vivo.     

维甲酸对甲状腺癌细胞钠/碘转运体基因的调节作用

以RTPCR法检测维甲酸对甲状腺癌细胞钠/碘转运体(NIS)基因表达的影响,发现在5～50μmol/L的剂量区间,维甲酸能促进NIS基因表达,提示维甲酸在甲状腺癌及其转移灶的治疗中可能起到重要作用。     

Multifaceted suppression of aggressive behavior of thyroid carcinoma by all-trans retinoic acid induced re-differentiation

Since all-trans retinoic acid (ATRA) has shown promising results in differentiation therapy, the present study was designed to investigate the effects of ATRA on thyroid carcinoma and to evaluate the effectiveness of ATRA in redifferentiation induction of thyroid carcinoma. Therefore, we investigated cell growth rate, morphological and nuclear: cytoplasmic ratio, adherent-dependent growth, response to chemotherapy drug following differentiation, T3 and T4 measurement, and critical genes expression pattern. Papillary cell line showed more growth inhibition by ATRA, in addition, mesenchymal and spindle-shape of 8305C cells changed to polygonal. Additionally, high nuclear: cytoplasmic ratio of anaplastic decreased significantly. Redifferentiation significantly suppressed the anchorage-dependent growth in the both cell lines in a dose-dependent manner, potentiated the arsenic trioxide (ATO) effects in anaplastic and papillary cell lines. Furthermore, reduction in the expression of stemness, and invasion related genes was observed in the both cell lines. Altogether, ATRA treatment could hold the aggressive behavior of thyroid carcinoma in restraint and/or potentiate the effect of chemotherapy drug ATO.     

维甲酸诱导肺腺癌细胞凋亡的实验研究

目的　探讨诱导分化剂全反式维甲酸（ＡＴＲＡ）对人肺腺癌细胞株Ａ５４９ 和ＳＰＣＡ１ 的影响。　方法　实验分为不同浓度的维甲酸用药组及对照组,进行氚标记胸腺嘧啶脱氧核苷（３ＨＴｄＲ）掺入细胞生长测定,电子显微镜下观察细胞核形态及用流式细胞仪测定细胞ＤＮＡ 含量,分别做凋亡细胞的定性和定量分析。　结果　ＡＴＲＡ 在１０ μｍ ｏｌ／Ｌ浓度时即对肺癌细胞Ａ５４９ 和ＳＰＣＡ１的生长有明显的抑制作用,且呈剂量依赖性。此种抑制是通过诱导细胞凋亡达到的。Ａ５４９ 和ＳＰＣＡ１凋亡细胞率在５０ μｍ ｏｌ／Ｌ药物浓度分别为３５１８％ 和１９２０％ 。　结论　ＡＴＲＡ 在体外可以诱导肺腺癌细胞的凋亡,为临床上通过诱导细胞分化促进和触发肿瘤细胞凋亡,从而为预防和治疗肿瘤提供了理论依据。