甲状腺乳头状癌BRAFV600E基因突变与钠碘同向转运体表达的研究

目的:探讨甲状腺乳头状癌(PTC)患者中BRAFV600E基因突变及钠碘同向转运体(NIS)蛋白的表达及两者间的相关性.方法:收集2008-01-01—2011-01-01青岛大学医学院附属医院病理科PTC石蜡包埋组织30例,其中癌旁组织为正常组织(G正常组)15例,癌旁组织为结节性甲状腺肿组织(G结甲组)15例.对PTC组织行DNA提取、PCR扩增、基因测序检测BRAFV600E基因突变,同时采用免疫组化的方法分析NIS蛋白表达,结果以免疫组化评分(IHS)表示.结果:30例PTC均无BRAFV600E基因突变;30例PTC组织中NIS蛋白表达,从0～12分,G正常组IHS=(7.92±3.01)分,G结甲组IHS=(6.58±2.71)分,两组NIS差异无统计学1意义,t=1.11,P=0.95;BRAFV600E突变与NIS蛋白表达无相关性.结论:尚未发现癌旁不同的PTCBRAFV600E基因有突变及NIS蛋白表达的差异.BRAFV600E基因突变与NIS表达在PTC中的关系有待进一步研究.     

钠/碘转运体在甲状腺癌中的表达

目的:探讨钠/碘转运体(sodium iodide symporter,NIS)在甲状腺癌组织中的表达.方法:应用实时荧光定量PCR(real-time fluorescence quantitative-PCR, RFQ-PCR)和免疫组织化学法检测NIS mRNA和蛋白在18例正常甲状腺组织、27例结节性甲状腺肿组织和23例甲状腺癌组织中的表达.结果: 甲状腺癌组织中NIS mRNA的表达显著低于正常甲状腺组织和结节性甲状腺肿组织(P=0.002).免疫组织化学检测结果表明,甲状腺癌组织中NIS蛋白主要定位于细胞质,强阳性表达13例(56.6%)、阳性表达3例(13.0%),强阳性表达率显著高于正常甲状腺组织(P=0.010).与正常甲状腺组织相比,甲状腺癌组织中NIS mRNA表达下降并且NIS蛋白表达升高者17例(74%),显著高于结节性甲状腺肿组织(χ2=4.428,P=0.035).结论:甲状腺癌组织中NIS mRNA的表达下降而NIS蛋白的表达增强,推测NIS蛋白主要位于细胞质可能是NIS不能发挥正常的运输功能,导致甲状腺癌患者不能聚碘的重要原因之一.     

Low expression of sodium iodide symporter identifies aggressive thyroid tumors

A decreased radioiodine uptake is frequently detected in differentiated thyroid carcinomas (DTC) and is associated with high recurrence rate and reduced survival. We investigated the correlation between NIS mRNA expression levels in the primary tumor and patient outcome using a quantitative real-time RT-PCR method. NIS expression was decreased in 17 DTC (21.04+/-39.66 pg Eq) compared to four autoimmune thyroid disease (180.51+/-92.63 pg Eq) and 14 normal tissues (75.71+/-66.98 pg Eq) (p<0.0001). The 17 thyroid differentiated carcinoma patients were submitted to surgery complemented by radioiodine ablation and had at least 24 months of follow-up, under levothyroxine continued suppressive therapy. According to their outcome, we could characterize a group of papillary carcinoma patients with aggressive carcinomas, whose NIS mRNA levels were markedly lower than a group with non-aggressive carcinomas (0.62+/-0.79 versus 54.87+/-53.79; p<0.005). We suggest that the quantification of NIS mRNA relative levels in the primary tumor may predict poor outcome.     

全反式维甲酸对甲状腺癌细胞NIS基因表达及吸碘能力影响的实验研究

目的：探讨全反式维甲酸（ATRA）对甲状腺癌细胞株钠/碘同向转运体（NIS）基因表达、吸碘能力的影响,为ATRA用于放射性碘治疗甲状腺癌提供理论依据。方法：分别以不同浓度（10^-7mol/L、10^-6mol/L、10^-5mol/L、10^-4mol/L）的ATRA处理体外培养的甲状腺癌细胞株（FTC-133）,48h后利用半定量RT-PCR检测细胞NISmRNA表达,γ-计数仪检测细胞吸碘能力。结果：ARTA浓度在（0～10%-5）mol/L范围内,细胞NIS基因表达及吸碘能力随ARTA剂量的增加而增加（P〈0.05）。当ARTA浓度达10%-4mol/L时,增加与前一浓度相比无统计学意义（P〉0.05）。结论：ATRA可上调甲状腺癌FTC-133细胞NIS基因表达,增强其吸碘能力,而且这种作用在一定浓度范围内具有剂量依赖性。     

Low expression of sodium iodide symporter expression in aggressive variants of papillary thyroid carcinoma

Background

A decreased radioiodine uptake is associated with high recurrence rate and reduced survival in papillary thyroid carcinoma (PTC). Sodium iodide symporter (NIS) expression status in aggressive PTC variants is unknown.

Methods

NIS expression in conventional PTC and aggressive PTC variants such as tall cell variant (TCV) and diffuse sclerosing variant (DSPTC) was investigated using immunohistochemical detection.

Results

Patients having TCV and DSPTC were significantly more likely to have extrathyroidal extension and lymph nodes metastases (P < 0.05). Positive NIS staining was identified in 228 of 312 (73.1 %) patients with conventional PTC, 9 of 28 (32.1 %) patients with TCV and 12 of 30 (40.0 %) patients with DSPTC. NIS expression distinguished conventional PTC from TCV and DSPTC significantly (P < 0.01).

Conclusion

Due to their low NIS expression, TCV and DSPTC need higher cumulative doses of radioactive iodine therapy to improve their prognosis.     

Targeting sodium/iodide symporter gene expression for estrogen-regulated imaging and therapy in breast cancer

Expression of the sodium iodide symporter (hNIS) has been detected in breast cancer tissue, but frequently, not at the levels necessary to mediate (131)I accumulation. Transducing the hNIS gene into breast cancer cells with adenovirus could be a tractable strategy to render breast cancer susceptible to radioiodide therapy. We constructed the replication-incompetent virus, AdSERE, in which an estrogen-responsive promoter directs the expression of hNIS. In vitro, we demonstrate that AdSERE mediates hNIS expression and iodide uptake in ER+ breast cancer cells. In vivo, we show that AdSERE-infected ER+ tumors can be imaged due to tracer accumulation; in addition, AdSERE in combination with therapeutic doses of (131)I suppresses tumor growth.     

细胞因子对乳腺癌细胞钠碘转运体基因表达的调控

目的本实验通过三种细胞因子(肿瘤坏死因子-α,干扰素-γ和白细胞介素-6)对乳腺细胞钠碘转运体(NIS)基因表达的影响,探讨乳腺肿瘤组织NIS的表达特点和调控因素.方法采用乳腺癌细胞MCF-7和MB453进行培养,分别给予不同浓度的肿瘤坏死因子-α,干扰素-γ或白细胞介素-6刺激72 h.采用RT-PCR方法检测细胞中NIS mRNA表达情况.结果乳腺癌细胞在经过肿瘤坏死因子-α,干扰素-γ或白细胞介素-6刺激后,细胞中NIS的mRNA表达比对照组明显降低,并且与细胞因子浓度呈负相关.结论肿瘤坏死因子-α,干扰素-γ和白细胞介素-6对乳腺癌细胞MCF-7和MB453的NIS基因mRNA表达有抑制作用,这提示细胞因子可能通过调节乳腺癌NIS基因mRNA的表达,从而影响乳腺癌细胞对放射性碘治疗的敏感性.     

Breast cancer brain metastases express the sodium iodide symporter

Breast cancer brain metastases are on the rise and their treatment is hampered by the limited entry and efficacy of anticancer drugs in this sanctuary. The sodium iodide symporter, NIS, actively transports iodide across the plasma membrane and is exploited clinically to deliver radioactive iodide into cells. As in thyroid cancers, NIS is expressed in many breast cancers including primary and metastatic tumors. In this study NIS expression was analyzed for the first time in 28 cases of breast cancer brain metastases using a polyclonal anti-NIS antibody directed against the terminal C-peptide of human NIS gene and immunohistochemical methods. Twenty-five tumors (84%) in this retrospective series were estrogen/progesterone receptor-negative and 15 (53.6%) were HER2+. Overall 21 (75%) cases and 80% of HER2 positive metastases were NIS positive. While the predominant pattern of NIS immunoreactivity is intracellular, plasma membrane immunopositivity was detected at least focally in 23.8% of NIS-positive samples. Altogether, these findings indicate that NIS expression is prevalent in breast cancer brain metastases and could have a therapeutic role via the delivery of radioactive iodide and selective ablation of tumor cells.     

Rat sodium iodide symporter for radioiodide therapy of cancer.

Design and development of new approaches for targeted radiotherapy of cancer and improvement of therapeutic index by more local radiation therapy are very important issues. Adenovirus-mediated delivery of the sodium iodide symporter (NIS) gene to cancer cells is a powerful technique to concentrate lethal radiation in tumor cells and eradicate tumors with increased therapeutic index. A replication-defective adenoviral vector expressing the rat NIS gene (Ad-rNIS) was used for in vitro gene delivery and into human prostate cancer xenografts to study antitumor effect. Robust function of the rat symporter was detected in DU145, T47D, and HCT-15 human cancer cell lines transduced with Ad-rNIS. All three cancer cell lines successfully transferred functionally active rat symporter to the plasma membrane, resulting in very high levels of iodine-125 accumulation. Three-dimensional multicellular tumor spheroids derived from DU145 human prostate cancer cells were transduced with Ad-rNIS and incubated with (131)I for 24 hours. After treatment, spheroids rapidly decreased in size and disappeared within 10 days. In vivo data revealed an inhibition of tumor growth in athymic nude mice after intratumoral Ad-rNIS injection followed by (131)I administration. Eighty-eight percent of experimental mice survived >30 days, whereas control groups had only 18% survival >30 days. This is the first report that demonstrates the rat NIS gene can effectively induce growth arrest of human tumor xenografts after in vivo adenoviral gene delivery and (131)I administration. The data confirm our hypothesis that the rat NIS gene is an attractive suicide gene candidate for cancer treatment.     

Expression of sodium iodide symporter in metastatic and follicular human thyroid tissues

Active iodide uptake across the basal membrane mediated by human sodiumiodide symporter (hNIS) has been shown to be a process coupled with the flowof sodium. There is still controversy as to the amount of hNIS expressionpresent in different kinds of human thyroid cancer tissues. In this study, wepresent a 58-year-old women with follicular thyroid carcinoma with vertebraand skull metastases. ²⁰¹Tl and 5 mCi ¹³¹I scans clearlydemonstrated the metastatic lesions in the brain of this patient. Thyroid andmetastatic tissues were then obtained for this study, which is aimed atcomparing the iodide trapping ability in vivo and in vitro of hNIS, and then comparing their expression in both thyroid tissue andmetastatic tissues. Polyclonal antibodies to hNIS and competitive RT–PCRwere used to analyze the symporter protein and mRNA expressed in follicularhuman thyroid and metastatic tissues. Positive staining of the symporterprotein was performed in the follicular thyroid carcinomas, otherwise, themetastatic tissues could not have demonstrated the protein in the staining.Follicular thyroid carcinoma tissues from thyroid were revealed around 5 pghNIS expressed in follicular thyroid carcinoma tissues from the thyroid.Otherwise, there was almost an absence of hNIS expression in the metastatictissue. These discrepancies of the expression in hNIS in vivo and in vitrostudies need further investigation.     

Effect of ligands of nuclear hormone receptors on sodium/iodide symporter expression and activity in breast cancer cells

Iodide uptake by normal and cancerous thyroid cells is an active process mediated by the sodium/iodide symporter (NIS). Using quantitative real-time RT-PCR, we found that all 22 fresh human breast cancer samples had very low NIS expression similar to levels in untreated MCF-7 breast cancer cells. 9-cis retinoic acid (9-cis RA), a ligand for both retinoic acid receptor (RAR)/retinoic X receptor (RXR) heterodimers as well as RXR/RXR homodimers, markedly induced NIS mRNA expression in MCF-7 breast cancer cells in a dose- and time-dependent fashion, with maximal levels occurring at 12 h. All-trans retinoic acid, ATRA, a RAR specific ligand had a similar potency. Among eight breast cancer cell lines, three out of four estrogen receptor (ER)-positive and zero of four ER-negative cell lines responded to 9-cis RA by increasing their expression of NIS. Combining a RAR with a RXR selective ligand enhanced both NIS mRNA expression and iodide uptake in MCF-7 cells. Similarly, a ligand for proliferator-activated receptor (PPAR) when combined with 9-cis RA synergistically increased both NIS mRNA levels and iodide uptake in these MCF-7 cells. The iodide uptake was blocked by KClO₄. In conclusions, these findings suggest that selected combinations of NHR ligands should be examined in a limited trial to determine if their administration to patients allows the use of radioactive iodine for diagnosis and possibly treatment of metastatic breast cancer.     

PI3K activation is associated with intracellular sodium/iodide symporter protein expression in breast cancer

Background  

The sodium/iodide symporter (NIS) is a membrane glycoprotein mediating active iodide uptake in the thyroid gland and is the molecular basis for radioiodide imaging and therapeutic ablation of thyroid carcinomas. NIS is expressed in the lactating mammary gland and in many human breast tumors, raising interest in similar use for diagnosis and treatment. However, few human breast tumors have clinically evident iodide uptake ability. We previously identified PI3K signaling as important in NIS upregulation in transgenic mouse models of breast cancer, and the PI3K pathway is commonly activated in human breast cancer.     

Treatment of prostate cancer by radioiodine therapy after tissue-specific expression of the sodium iodide symporter

Causing prostate cancer cells to express functionally active sodium iodide symporter (NIS) by targeted NIS gene transfer might offer the possibility of radioiodine therapy of prostate cancer. Therefore, we investigated radioiodine accumulation and therapeutic effectiveness of 131I in NIS-transfected prostate cancer cells in vitro and in vivo. The human prostatic adenocarcinoma cell line LNCaP was stably transfected with NIS cDNA under the control of the prostate-specific antigen promoter. The stably transfected LNCaP cell line NP-1 showed perchlorate-sensitive, androgen-dependent iodide uptake in vitro that resulted in selective killing of these cells by 131I in an in vitro clonogenic assay. Xenografts were established in athymic nude mice and imaged using a gamma camera after i.p. injection of 500 microCi of 123I. In contrast to the NIS-negative control tumors (P-1) which showed no in vivo uptake of 123I, NP-1 tumors accumulated 25-30% of the total 123I administered with a biological half-life of 45 h. In addition, NIS protein expression in LNCaP cell xenografts was confirmed by Western blot analysis and immunohistochemistry. After a single i.p. application of a therapeutic 131I dose (3 mCi), significant tumor reduction was achieved in NP-1 tumors in the therapy group compared with P-1 tumors and tumors in the control group. In conclusion, a therapeutic effect of 131I has been demonstrated in prostate cancer cells after induction of tissue-specific iodide uptake activity by prostate-specific antigen promoter-directed NIS expression in vitro and in vivo. This study demonstrates the potential of NIS as a novel therapeutic gene for nonthyroidal cancers, in particular prostate cancer.     

Adenovirus-mediated transfer of the thyroid sodium/iodide symporter gene into tumors for a targeted radiotherapy

The Na+/I- symporter (NIS) present in the membranes of thyroid cells is responsible for the capacity of the thyroid to concentrate iodide. This allows treatment of thyroid cancers with 131I. We propose to enlarge this therapeutic strategy to nonthyroid tumors by using an adenoviral vector to deliver the NIS gene into the tumor cells. We constructed a recombinant adenovirus encoding the rat NIS gene under the control of the cytomegalovirus promoter (AdNIS). Infection of SiHa cells (human cervix tumor cells) with AdNIS resulted in perchlorate-sensitive 125I uptake by these cells to a level 125-225 times higher than that in noninfected cells. Similar results were obtained for other human tumor cell lines, including MCF7 and T-47D (mammary gland), DU 145 and PC-3 (prostate), A549 (lung), and HT-29 (colon), demonstrating that the AdNIS vector can function in tumor cells of various origins. In addition, AdNIS-infected tumor cells were selectively killed by exposure to 131I, as revealed by clonogenic assays. To assess the efficiency of this cancer gene therapy strategy in vivo, we injected the AdNIS vector in human tumors (SiHa or MCF7 cells) established s.c. in nude mice. Immunohistological analysis confirmed the expression of the NIS protein in the tumor. Three days after intratumoral injection, AdNIS-treated tumors could specifically accumulate 125I or 123I, as revealed by kinetics and imaging experiments. A quantitative analysis demonstrated that the uptake in AdNIS-injected tumors was 4-25 times higher than that in nontreated tumors. On average, 11% of the total amount of injected 125I could be recovered per gram of AdNIS-treated tumor tissue. Altogether, these data indicate that AdNIS is very efficient in triggering significant iodide uptake by a tumor, outlining the potential of this novel cancer gene therapy approach for a targeted radiotherapy.     

Systemic retinoic acid treatment induces sodium/iodide symporter expression and radioiodide uptake in mouse breast cancer models

Lactating breast tissue and some breast cancers express the sodium/iodide symporter (NIS) and concentrate iodide. We recently demonstrated that all-trans retinoic acid (tRA) induces both NIS gene expression and iodide accumulation in vitro in well-differentiated human breast cancer cells (MCF-7). In the present study, we investigated the in vivo efficacy and specificity of tRA-stimulated iodide accumulation in mouse breast cancer models. Immunodeficient mice with MCF-7 xenograft tumors were treated with systemic tRA for 5 days. Iodide accumulation in the xenograft tumors was markedly increased, approximately 15-fold greater than levels without treatment, and the effects were tRA dose dependent. Iodide accumulation in other organs was not significantly influenced by tRA treatment. Significant induction of NIS mRNA and protein in the xenograft tumors was observed after tRA treatment. Iodide accumulation and NIS mRNA expression were also selectively induced in breast cancer tissues in transgenic mice expressing the oncogene, polyoma virus middle T antigen. These data demonstrate selective induction of functional NIS in breast cancer by tRA. Treatment with short-term systemic retinoic acid, followed by radioiodide administration, is a potential tool in the diagnosis and treatment of some differentiated breast cancer.     

hNIS基因转导黑色素瘤细胞介导放射性碘摄取的实验研究

目的获取人钠/碘同向转运体(hNIS)基因cDNA,研究其转导黑色素瘤细胞在体外和体内能否介导放射性碘摄取,从而探索该策略用于黑色素瘤放射性碘治疗的可能性.方法运用逆转录聚合酶链反应从人甲状腺组织总RNA中扩增出hNIS基因cDNA,将其克隆至真核载体pc-DNA3中,电转化法分别将重组质粒pc-DNA3-hNIS及空质粒pc-DNA3转导黑色素瘤细胞(B16),分别建立了细胞系B16-A和B16-B.在体外培养条件下检测其对放射性碘的摄取及外流情况.继而将三种细胞系接种C57小鼠行131I显像和肿瘤摄取125I动态定量测定.结果成功克隆到hNIS基因,并建立了能稳定表达hNIS的新型细胞系B16-A.B16-A细胞的摄碘能力较B16细胞高17倍,较B16-B细胞高19倍.碘的外流过程迅速,T1/2eff仅10 min.体内实验发现B16-A细胞所形成肿瘤能摄取放射性碘,腹腔注射125I后1、2、4、12、24 h肿瘤组织的%ID/g平均为12.22.10.91、8.73、1.24、0.19,125I在肿瘤组织内的生物半衰期约为6 h.B16-A细胞系所成肿瘤摄碘量与对照组相比较,P＜0.01,差别具有高度统计意义.结论hNIS基因转导黑色素瘤细胞足以介导放射性碘摄取,但有效半衰期较短,难以产生足够的治疗剂量,有必要进一步研究如何增加放射性碘的摄取量及延长碘在细胞内的滞留时间以提高其放射生物学效应.