乳头状甲状腺癌的发病机制及干预实验研究
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摘要
目的
甲状腺癌是比较少见的由特异性染色体异位、嵌合、基因融合及突变而引起的上皮细胞恶性肿瘤。其中乳头状甲状腺癌(PTC)为分化型甲状腺癌,占甲状腺癌的80%。尽管包括PTC在内的分化型甲状腺癌恶性度较低并且高度可愈,但是此类肿瘤在初次治疗后的复发率很高,这是由于甲状腺特异性蛋白包括基底膜上的钠／碘协同转运体(NIS)和顶膜上的Pendrin蛋白及促甲状腺激素受体(TSHR)的表达的缺失或功能发生异常。有研究表明DNA甲基化可能是导致肿瘤组织中某种蛋白表达减少或功能异常的一个原因,最终使放射性碘治疗的无效。
最近研究发现的BRAF基因与PTC关系较为密切,MarineN等在对320例甲状腺肿瘤及6个未分化癌细胞株进行突变检测发现在低分化和未分化癌中也有BRAF基因突变。随后,还有不少学者比较了各种甲状腺癌中BRAF基因的突变率发现PTC中BRAF基因的突变率可高达83%,未分化癌中的突变率达35%,其它类型甲状腺癌中均未见突变,提示散发性成年人PTC的BRAF基因突变率高,具有BRAF基因T1799A突变热点。综上提示BRAF基因突变可能与PTC的转移与预后密切相关。
BRAF基因突变可能还影响到对甲状腺癌的治疗策略。甲状腺癌对常规化疗药物不敏感,且后者副作用大。因此,研制新型抗癌药物就显得尤为重要。最近发现在我国传统中草药莪术的根茎中可以提取一种不具细胞毒性的抗肿瘤药物p-榄香烯,它的低毒性和抗肿瘤的广谱性得到了医生和患者的广泛接受。但目前β-榄香烯对甲状腺癌的治疗效应及机制研究尚浅。
本研究的目的是在PTC中与碘代谢密切相关的TSHR和NIS的蛋白表达及TSHR和NIS基因甲基化和BRAF基因T1799A突变,分析遗传学改变与TSHR和NIS蛋白的表达关系。进一步研究在体内和体外情况下p-榄香烯对人乳头状甲状腺癌IHH-4细胞系和未分化甲状腺癌ASH-3细胞系的效应和作用机制。
方法
一、研究对象
研究病例来自2005年至2006年在滨州人民医院行甲状腺全切术的PTC患者(n=60),其中男性12例,女性48例,年龄26-66岁,平均年龄44岁。
人类乳头状甲状腺癌IHH-4细胞系
人类未分化甲状腺癌ASH-3细胞系
6周龄雄性BALB/c裸鼠
二、研究方法
1、甲状腺乳头状癌病例标本中BRAF基因T1799A突变检测
提取癌组织切片中的基因组DNA,应用聚合酶链反应(PCR)对标本DNA BRAF基因的第15外显子进行扩增后,对基因测序。
2、甲状腺乳头状癌病例标本中的TSHR和NIS蛋白表达和甲基化检测
TSHR和NIS蛋白表达直接进行免疫组化染色；甲基化分析是提取癌组织切片中的基因组DNA, DNA重亚硫酸盐处理,针对TSHR和NIS基因甲基化的特异性PCR。
3、p-榄香烯对人类乳头状甲状腺癌IHH-4细胞系和未分化甲状腺癌ASH-3细胞系增殖及对细胞周期的影响检测,IHH-4细胞系细胞周期调节蛋白水平检测。
增殖能力的研究在体外是通过MTT比色分析方法进行测定的。应用流式细胞仪对IHH-4和ASH-3细胞系细胞周期进行分析。使用western印记分析法检测细胞周期调节蛋白。
4、p-榄香烯诱导人类乳头状甲状腺癌IHH-4细胞系的凋亡及凋亡调节蛋白水平和活性检测。
使用TUNEL检测法分析判断p-榄香烯对IHH-4细胞系诱导凋亡作用,选用抗caspases-8、caspases-9和Bcl-2的抗体做免疫印记分析凋亡蛋白的水平和活性。
5、Matrigel侵袭实验
在体外情况下β-榄香烯对人类乳头状甲状腺癌IHH-4细胞系移动性的作用。
6、p-榄香烯对荷瘤BALB/c裸鼠肿瘤的影响检测
包括对荷瘤BALB/c裸鼠肿瘤体积、生存率的检测,及对体内的细胞周期调节蛋白及VEGF检测。
三、统计学处理
分类数据采用频率和百分数表示,连续变量应用均数±标准差表示。分类变量的统计应用x2检验,连续变量的统计应用t检验。p值采用双侧检验,以p<0.05为有统计学差异。采用SPSS软件(11.5版本),进行数据分析。
结果
一、PTC中TSHR、NIS基因甲基化和蛋白表达及其与BRAF基因T1799A突变情况
1、PTC标本中BRAF基因T1799A突变状况
60例PTC标本中,BRAF基因T1799A突变率为65%(n=39)。突变与临床病理特征未见显著关联。20例癌对侧非癌甲状腺组织中,未检出BRAF基因突变。
2、PTC标本中TSHR和NIS蛋白的表达
癌对侧的甲状腺组织(阳性对照)中,TSHR阳性颗粒分布于约70%的甲状腺滤泡上皮细胞的胞浆和胞膜上,显色程度中到强。全部被检测的PTC标本中,均可见到TSHR阳性颗粒显色：57%(n=34)的标本TSHR呈现散在或弥漫性的胞浆淡染,35%(n=21)可见到与癌对侧组织染色强度和分布类似的阳性显色,8%(n=5)的标本染色强度高于癌对侧组织。
癌对侧的甲状腺组织(阳性对照)中,NIS阳性颗粒分布于约20%的甲状腺滤泡上皮细胞的胞浆和胞膜上,显色程度强。全部被检测的PTC标本中,除1例外均可见到NIS阳性颗粒显色,但显色强度较阳性对照明显为弱,仅有5%(n=3)的标本可见到与癌对侧组织染色强度和分布类似的阳性显色。
3、PTC标本中的TSHR和NIS基因甲基化状况
60例PTC标本中,TSHR和NIS基因甲基化的发生率分别为43%(n=26)和27%(n=16)。上述基因的甲基化与某些临床病理特征有相关的趋势,但没有统计学意义。20例癌对侧非癌甲状腺组织中,未检出上述基因甲基化。
二、p-榄香烯对PTC作用机制的体外研究
1、p-榄香烯对IHH-4及ASH-3细胞增殖及对细胞周期调节蛋白水平影响。
β-榄香烯对ASH-3和IHH-4细胞具有显著的抑制细胞增殖的作用,在20μg/ml到60μg/ml的浓度范围内其抑制作用呈现明显的剂量依赖性关系。应用流式细胞仪对IHH-4和ASH-3细胞的细胞周期进行了分析。结果显示,用30、40和50μg／ml的β-榄香烯处理IHH-4细胞后,处于细胞周期G1期中的细胞比例明显增加。而经过β-榄香烯处理后的ASH-3细胞G1期细胞数量没有显著增加。细胞周期中G1期是受细胞周期调节蛋白E、CDK2和CDK6等的调节。实验结果显示,用不同浓度的β-榄香烯处理IHH-4细胞系后,细胞中cyclin E、CDK2和CDK6的表达水平下降。
2、p-榄香烯对IHH-4细胞凋亡的诱导作用及凋亡调节蛋白水平和活性的影响。
结果显示,未经处理的对照细胞很少发生凋亡,而随着β-榄香烯浓度的增加,凋亡细胞数量也随之增加。因此,同未经处理的对照细胞相比,β-榄香烯以剂量依赖的方式在48小时处引起了凋亡的IHH-4细胞显著增加。凋亡调节蛋白水平及活性的实验结果显示,Bcl-2的表达降低,caspases-9裂解蛋白的水平以剂量依赖的方式升高。另外,caspases-8水平未见改变。
3、p-榄香烯对IHH-4细胞移动性的作用。
Matrigel侵袭实验结果显示,虽然我们在细胞增殖实验中已经证实,在极低浓度(10μg/ml)下,p-榄香烯对IHH-4细胞的增殖无明显抑制作用,但在此浓度下p-榄香烯仍然对IHH-4细胞的移动性具有明显的抑制作用。
三、β-榄香烯对PTC作用机制的动物实验
1、β-榄香烯对荷瘤BALB/c裸鼠肿瘤体积的影响
结果显示β-榄香烯治疗组荷瘤裸鼠的肿瘤体积明显小于空白乳剂组(p＜0.05),说明在体内情况下β-榄香烯对PTC的生长有明显抑制作用。
2、β-榄香烯对荷瘤BALB/c裸鼠生存率的影响
从给药后第7天开始,β-榄香烯治疗组(50mg/kg)的荷瘤BALB/c裸鼠的生存率明显高于空白乳剂组(p<0.01),说明在体内情况下β-榄香烯通过抑制PTC的生长,来提高荷瘤裸鼠的生存率。
3、β-榄香烯在体内情况下对细胞周期调节蛋白及VEGF的影响
经β-榄香烯治疗(50mg/kg)后,肿瘤组织中的cyclin E和CDK6的表达水平下降,提示在体内情况下β-榄香烯也能通过调控cyclin E和CDK6的表达,导致IHH-4细胞阻滞于细胞周期的G1期。此外,经β-榄香烯治疗后,VEGF蛋白的表达水平下降,提示肿瘤组织中的血管新生受到抑制。
结论
1、免疫组织化学染色提示PTC组织中TSHR和NIS蛋白表达异常,与相应基因的甲基化发生有关,亦与BRAF基因T1799A突变的发生有关。提示：PTC中相应基因的甲基化和BRAF基因突变可能是TSHR和NIS蛋白表达减弱并分布异常的原因。TSHR和NIS基因甲基化与BRAF基因突变相关,可能是BRAF基因突变导致肿瘤恶性程度增高的内在机制之一
2、β-榄香烯对PTC具有明显的治疗作用,其作用机制是通过阻滞细胞周期、诱导细胞凋亡和抑制肿瘤细胞侵袭而实现的。
Objective
Thyroid cancer is relatively rare in the specific chromosome entopic, chimerical, gene fusion and mutation caused by epithelial cell tumors. Papillary thyroid cancer (PTC) is differentiated thyroid cancer, accounting for thyroid cancer 80%. While including the PTC, differentiated thyroid cancer's malignancy is low and highly more cure, but these tumors in the initial treatment of the recurrence rate is high, this is because the thyroid-specific proteins, including the basement membrane Na+/I-symporter (NIS) and the top membrane Pendrin protein and thyrotrophic receptor (TSHR) expression in the absence or functional abnormalities. Research has shown that DNA methylation may lead to tumor tissues a protein to reduce or abnormal function of a cause, and finally to radioactive iodine treatment invalid.
Recent research discovered BRAF gene thyroid papillary closer relationship, Marine N etc. on 320 cases thyroid tumor and 6 undifferentiated cancer strains mutation detection found poorly differentiated and undifferentiated carcinoma also BRAF gene mutation. Subsequently, many scholars comparison various thyroid carcinoma BRAF gene mutation rate, found PTC BRAF genes mutation rate up 83%, undifferentiated carcinoma mutations rate 35%, other types of thyroid cancer have no mutation, indicating sporadic adult PTC of the BRAF gene mutation rate, with BRAF T1799A mutation hot spots. Summing prompted BRAF gene mutation may papillary thyroid cancer metastasis prognosis.
BRAF gene mutation may also affect the thyroid cancer treatment strategies. Thyroid cancer to conventional chemotherapy is not sensitive, and which side effects. Therefore, development of new anticancer drugs is particularly important.
Elemene, an extract from the ginger plant Rhizoma zeodaria, is a novel anticancer drug. The extract of elemene is a mixture ofα-,β-andδ-elemene, withβ-elemene as the main component, which accounts for 60～72% of the three isoforms.β-elemene has a broad-spectrum antitumor effect in many types of cancer, it has low toxicity and is therefore well tolerated and accepted by cancer patients. Recent studies showed thatβ-elemene-inhibited cell proliferation and trigger apoptosis in glioma cells and leukemia cells, and the apoptosis. However, the anti-cancer effect and mechanism of (3-elemene on human thyroid cancer remains unknown.
The purpose of this study is PTC with iodine metabolism in the closely related TSHR and NIS protein expression and the TSHR and NIS gene methylation and BRAF T1799A mutation analysis of genetic changes and the TSHR and NIS protein expression in. Further research in vivo and in vitroβ-elemene cases of human papillary thyroid cancer cell lines IHH-4 and anaplastic thyroid cancer cell lines ASH-3 effect and its mechanism.
Methods
1、Subject
Case studies from 2005 to 2006 in the People's Hospital of Bin zhou thyroidectomy for PTC hysterectomy patients (n= 60), male 12, female 48 cases, aged 26-66 years, mean age 44 years
Human papillary thyroid cancer IHH-4 cell lines and anaplastic thyroid cancer cell lines ASH-3 were obtained from Health Science Research Resources Bank (Osaka, Japan).6 weeks old male BALB/c nude mice were purchased from Wei Tong Company (Beijing, China).
2、Methods
Cases of papillary thyroid carcinoma specimens BRAF T1799A mutation, cancer tissue sections extracted genomic DNA, polymerase chain reaction (PCR) on samples of DNA BRAF gene exon 15 were amplified, and sequenced.
Specimens of papillary thyroid cancer cases in the TSHR and NIS protein expression and methylation detection, TSHR and NIS protein expression in immunohistochemical directly; methylation analysis was extracted cancer tissue sections of genomic DNA, DNA bisulfite treatment, for TSHR and NIS methylation specific PCR.
Cell viability or the effects ofβ-elemene on cell proliferation in IHH-4 cells and ASH-3 cells were assessed with an MTT-based colorimetric cell proliferation assay following the manufacturer's instructions (Sigma). Flow cytometry onβ-elemene treated IHH-4 and ASH-3 cell line cell cycle were analyzed. After stained with PI dye, the cells were analyzed for cell cycle perturbation using a FACSCalibur (Becton-Dickinson, San Diego, Calif.), and the CellQuest Pro software (Becton-Dickinson) and the ModFit LT software (Verity Software House, Topsham, Maine) were used to determine the distribution of cells in the various cell cycle compartments:G1, S and G2-M.
β-elemene induces apoptosis in human papillary thyroid carcinoma IHH-4 cell levels and activity and apoptosis detection. We detectedβ-elemene inducing apoptosis in IHH-4 cells using the TUNEL assay and the apoptosis regulatory protein, caspases-8, caspases-9 and Bcl-2 by immunoblotting analysis.
We detected invasiveness of the IHH-4 cells using in vitro invasion assay. Cells (5×104 per well) were placed in the upper chamber of a 24-well Transwell apparatus containing Matrigel membranes (BD Biosciences, San Jose, CA), and the lower chamber was filled with 750μL DMEM supplemented with 0.1% bovine serum albumin and fibronectin (10μg/mL; Roche) as a chemoattractant. After incubation for 36 hours at 37℃, cells that had migrated to the lower surface of each membrane were stained with the use of a Diff-Quik kit (International Reagents, Kobe, Japan) and counted. Each experiment was done with triplicate wells and repeated thrice.
Human cancer xenograft models were established using the methods reported previously. When 80% confluence reached cultured cells in monolayer were trypsinized and harvested by centrifugation. The IHH-4 cellsβ-elemene on tumor-bearing BALB/c nude mice of tumor detection, including the tumor-bearing BALB/c nude mice tumor volume, survival rate of detection, and cell cycle regulation in vivo detection of protein and VEGF.
3、Statistical analysis
Disaggregated data by frequency and percentage that applied continuous variables mean±standard deviation. Classification variables in Statistics x2 test, continuous variables t test statistical applications. p value using two-sided test, with p<0.05 as significantly different. Using SPSS software (11.5 version) for data analysis.
Results
1. TSHR, NIS gene methylation and protein expression and T1799A BRAF mutation in PTC
The prevalence of the T1799A BRAF mutation in this series of PTC was 65% (39 of 60). Mutation and clinic pathologic features has no significant association. 20 cases of carcinoma of the contra lateral non-cancerous thyroid tissues, BRAF mutations were not detected.
Samples of TSHR and NIS protein expression in a contra lateral thyroid cancer (positive control) in, TSHR-positive particles were distributed in about 70% of thyroid follicular epithelial cell cytoplasm and membrane, the degree of moderate to strong color. All were detected in the PTC samples, positive particles can be seen TSHR color: 57% (n= 34) of the specimens showed TSHR scattered or diffuse cytoplasm staining, 35% (n= 21) can be seen with cancer of the lateral organizations, similar staining intensity and distribution of positive color,8% (n= 5) tumor specimens staining intensity was higher than the contra lateral tissue. Contra lateral thyroid cancer (positive control) in, NIS-positive particles were distributed in about 20% of thyroid follicular epithelial cell cytoplasm and membrane, the color degree of intensity. All samples were detected in PTC, in addition to NIS 1 case, positive particles can be seen the color, but color intensity than the positive control significantly as the weak, and only 5% (n= 3) of the specimens can be seen opposite staining with cancer similar intensity and distribution of the positive color.
The prevalence of the TSHR, and NIS gene methylation in this series of PTC was 43% (26 of 60) and 27%(16 of 60). The gene methylation and clinic pathological features some of the trends are related, but not statistically significant.20 cases of carcinoma of the contra lateral non-cancerous thyroid tissue, methylation of these genes were not detected.
2、The mechanism ofβ-elemene anti-cancer ability on human thyroid cancer in vitro
Our studies shownβ-elemene inhibited growth of thyroid cancer IHH-4 and ASH-3 cells and affected the expression of G1 cell cycle regulatory proteins.β-elemene significantly inhibited ASH-3 and IHH-4 cells cell proliferation, and this inhibition is dose-dependent. The cell cycle ofβ-elemene treated IHH-4 and ASH-3 cell lines were analyzed by Flow cytometry. Our results shownβ-elemene (30,40 and 50μg/ml) arrested IHH-4 cells at G1 phase. On the other hand, afterβ-elemene treatment, the ASH-3 cells population in G1 phase did not increase significantly. G1 cell cycle phase is regulated by cell cycle regulatory protein E, CDK2 and CDK6 and other adjustments. As shown in the experiments, the levels of cyclin E, CDK2 and CDK6 were decreased after exposure of IHH-4 cells to different concentrations ofβ-elemene.
β-elemene induces apoptosis in human papillary thyroid carcinoma IHH-4 cells. The TUNEL technique is classically used to detect apoptotic cells in culture. Here the induction of apoptosis followingβ-elemene treatment was confirmed by the TUNEL assay. As can be seen, few apoptotic cells were detected in the untreated control cells. In contrast, apoptosis in the IHH-4 cells treated withβ-elemene was increased as measured by the TUNEL assay. Quantitative analysis of the sections revealed thatβ-elemene induced a significant increase in apoptotic IHH-4 cells at 48 h in a dose-dependent manner, compared with the controls.
The results shown in untreated control cells, little apoptosis, and as the concentration ofβ-elemene increased number of apoptotic cells also increased. Therefore, the same compared to untreated control cells,β-elemene showed a dose-dependent manner in the office 48 hours caused apoptosis IHH-4 cells were significantly increased. The expression level of apoptosis regulation protein were detected:Bcl-2 expression decreased, caspases-9 cleavage protein levels increased in a dose dependent manner. In addition, caspases-8 levels did not change.
We detected the invasiveness of human papillary thyroid cancer IHH-4 cell lines afterβ-elemene treatment. Matrigel invasion assay showed that, although experiments in cell proliferation has been confirmed in the very low concentration (10μg/ml), 10μg/mlβ-elemene has no effect on growth of IHH-4 cell lines, but at this concentration, the invasiveness of IHH-4 cells were significantly inhibited.
3、Effects of elemene on the growth of IHH-4 cells transplanted tumors in vivo
β-elemene on tumor-bearing BALB/c nude mice tumor volume of the results showed thatβ-elemene treatment group in nude tumor volume was significantly smaller than the blank emulsion group (p<0.05). This result explained that P-elemene inhibited growth of PTC in vivo.
β-elemene on tumor-bearing BALB/c nude mice survival rate. From the 7th day after administration,β-elemene treatment group (50mg/kg) in tumor-bearing BALB/c nude mice survival rate was significantly higher than the blank emulsion group (p<0.01). This results suggested thatβ-elemene inhibite the growth of PTC, and then improve the survival rate of nude mice.
β-elemene in vivo cases, the cell cycle regulatory proteins and the impact of VEGF. Afterβ-elemene treatment (50mg/kg), the expression of cycle E CDK6 (the cell cycle regulatory proteins) were decreased. Our studies suggesting that in vivoβ-elemene inhibite the tumor growth through regulating the expression of cell cycle regulatory proteins cyclin E and CDK6, leading to IHH-4 cell line cell cycle arrest in G1 phase.In addition, theβ-elemene treatment, VEGF protein level decreased, suggesting that tumor angiogenesis is also inhibited.
Conclusions
1、Immunohistochemical staining showed PTC tissue abnormalities TSHR and NIS protein expression, and the corresponding gene methylation was also related to the occurrence of BRAF T1799A mutation. Tip:PTC in the corresponding gene methylation and BRAF mutation may be the TSHR and NIS expression was decreased and the reasons for abnormal distribution. TSHR and NIS gene methylation and BRAF mutations, BRAF mutations may lead to increased malignant tumor of the internal mechanism.
2、Elemene inhibits the growth of IHH-4 cells and trigger apoptosis in vitro and in vivo. These data provide useful information for further clinical study on the treatment of human thyroid cancer byβ-elemene.
甲状腺癌是比较少见的由特异性染色体异位、嵌合、基因融合及突变而引起的上皮细胞恶性肿瘤。其中乳头状甲状腺癌(PTC)为分化型甲状腺癌,占甲状腺癌的80%。尽管包括PTC在内的分化型甲状腺癌恶性度较低并且高度可愈,但是此类肿瘤在初次治疗后的复发率很高,这是由于甲状腺特异性蛋白包括基底膜上的钠／碘协同转运体(NIS)和顶膜上的Pendrin蛋白及促甲状腺激素受体(TSHR)的表达的缺失或功能发生异常。有研究表明DNA甲基化可能是导致肿瘤组织中某种蛋白表达减少或功能异常的一个原因,最终使放射性碘治疗的无效。
最近研究发现的BRAF基因与PTC关系较为密切,MarineN等在对320例甲状腺肿瘤及6个未分化癌细胞株进行突变检测发现在低分化和未分化癌中也有BRAF基因突变。随后,还有不少学者比较了各种甲状腺癌中BRAF基因的突变率发现PTC中BRAF基因的突变率可高达83%,未分化癌中的突变率达35%,其它类型甲状腺癌中均未见突变,提示散发性成年人PTC的BRAF基因突变率高,具有BRAF基因T1799A突变热点。综上提示BRAF基因突变可能与PTC的转移与预后密切相关。
BRAF基因突变可能还影响到对甲状腺癌的治疗策略。甲状腺癌对常规化疗药物不敏感,且后者副作用大。因此,研制新型抗癌药物就显得尤为重要。最近发现在我国传统中草药莪术的根茎中可以提取一种不具细胞毒性的抗肿瘤药物p-榄香烯,它的低毒性和抗肿瘤的广谱性得到了医生和患者的广泛接受。但目前β-榄香烯对甲状腺癌的治疗效应及机制研究尚浅。
本研究的目的是在PTC中与碘代谢密切相关的TSHR和NIS的蛋白表达及TSHR和NIS基因甲基化和BRAF基因T1799A突变,分析遗传学改变与TSHR和NIS蛋白的表达关系。进一步研究在体内和体外情况下p-榄香烯对人乳头状甲状腺癌IHH-4细胞系和未分化甲状腺癌ASH-3细胞系的效应和作用机制。
方法
一、研究对象
研究病例来自2005年至2006年在滨州人民医院行甲状腺全切术的PTC患者(n=60),其中男性12例,女性48例,年龄26-66岁,平均年龄44岁。
人类乳头状甲状腺癌IHH-4细胞系
人类未分化甲状腺癌ASH-3细胞系
6周龄雄性BALB/c裸鼠
二、研究方法
1、甲状腺乳头状癌病例标本中BRAF基因T1799A突变检测
提取癌组织切片中的基因组DNA,应用聚合酶链反应(PCR)对标本DNA BRAF基因的第15外显子进行扩增后,对基因测序。
2、甲状腺乳头状癌病例标本中的TSHR和NIS蛋白表达和甲基化检测
TSHR和NIS蛋白表达直接进行免疫组化染色；甲基化分析是提取癌组织切片中的基因组DNA, DNA重亚硫酸盐处理,针对TSHR和NIS基因甲基化的特异性PCR。
3、p-榄香烯对人类乳头状甲状腺癌IHH-4细胞系和未分化甲状腺癌ASH-3细胞系增殖及对细胞周期的影响检测,IHH-4细胞系细胞周期调节蛋白水平检测。
增殖能力的研究在体外是通过MTT比色分析方法进行测定的。应用流式细胞仪对IHH-4和ASH-3细胞系细胞周期进行分析。使用western印记分析法检测细胞周期调节蛋白。
4、p-榄香烯诱导人类乳头状甲状腺癌IHH-4细胞系的凋亡及凋亡调节蛋白水平和活性检测。
使用TUNEL检测法分析判断p-榄香烯对IHH-4细胞系诱导凋亡作用,选用抗caspases-8、caspases-9和Bcl-2的抗体做免疫印记分析凋亡蛋白的水平和活性。
5、Matrigel侵袭实验
在体外情况下β-榄香烯对人类乳头状甲状腺癌IHH-4细胞系移动性的作用。
6、p-榄香烯对荷瘤BALB/c裸鼠肿瘤的影响检测
包括对荷瘤BALB/c裸鼠肿瘤体积、生存率的检测,及对体内的细胞周期调节蛋白及VEGF检测。
三、统计学处理
分类数据采用频率和百分数表示,连续变量应用均数±标准差表示。分类变量的统计应用x2检验,连续变量的统计应用t检验。p值采用双侧检验,以p<0.05为有统计学差异。采用SPSS软件(11.5版本),进行数据分析。
结果
一、PTC中TSHR、NIS基因甲基化和蛋白表达及其与BRAF基因T1799A突变情况
1、PTC标本中BRAF基因T1799A突变状况
60例PTC标本中,BRAF基因T1799A突变率为65%(n=39)。突变与临床病理特征未见显著关联。20例癌对侧非癌甲状腺组织中,未检出BRAF基因突变。
2、PTC标本中TSHR和NIS蛋白的表达
癌对侧的甲状腺组织(阳性对照)中,TSHR阳性颗粒分布于约70%的甲状腺滤泡上皮细胞的胞浆和胞膜上,显色程度中到强。全部被检测的PTC标本中,均可见到TSHR阳性颗粒显色：57%(n=34)的标本TSHR呈现散在或弥漫性的胞浆淡染,35%(n=21)可见到与癌对侧组织染色强度和分布类似的阳性显色,8%(n=5)的标本染色强度高于癌对侧组织。
癌对侧的甲状腺组织(阳性对照)中,NIS阳性颗粒分布于约20%的甲状腺滤泡上皮细胞的胞浆和胞膜上,显色程度强。全部被检测的PTC标本中,除1例外均可见到NIS阳性颗粒显色,但显色强度较阳性对照明显为弱,仅有5%(n=3)的标本可见到与癌对侧组织染色强度和分布类似的阳性显色。
3、PTC标本中的TSHR和NIS基因甲基化状况
60例PTC标本中,TSHR和NIS基因甲基化的发生率分别为43%(n=26)和27%(n=16)。上述基因的甲基化与某些临床病理特征有相关的趋势,但没有统计学意义。20例癌对侧非癌甲状腺组织中,未检出上述基因甲基化。
二、p-榄香烯对PTC作用机制的体外研究
1、p-榄香烯对IHH-4及ASH-3细胞增殖及对细胞周期调节蛋白水平影响。
β-榄香烯对ASH-3和IHH-4细胞具有显著的抑制细胞增殖的作用,在20μg/ml到60μg/ml的浓度范围内其抑制作用呈现明显的剂量依赖性关系。应用流式细胞仪对IHH-4和ASH-3细胞的细胞周期进行了分析。结果显示,用30、40和50μg／ml的β-榄香烯处理IHH-4细胞后,处于细胞周期G1期中的细胞比例明显增加。而经过β-榄香烯处理后的ASH-3细胞G1期细胞数量没有显著增加。细胞周期中G1期是受细胞周期调节蛋白E、CDK2和CDK6等的调节。实验结果显示,用不同浓度的β-榄香烯处理IHH-4细胞系后,细胞中cyclin E、CDK2和CDK6的表达水平下降。
2、p-榄香烯对IHH-4细胞凋亡的诱导作用及凋亡调节蛋白水平和活性的影响。
结果显示,未经处理的对照细胞很少发生凋亡,而随着β-榄香烯浓度的增加,凋亡细胞数量也随之增加。因此,同未经处理的对照细胞相比,β-榄香烯以剂量依赖的方式在48小时处引起了凋亡的IHH-4细胞显著增加。凋亡调节蛋白水平及活性的实验结果显示,Bcl-2的表达降低,caspases-9裂解蛋白的水平以剂量依赖的方式升高。另外,caspases-8水平未见改变。
3、p-榄香烯对IHH-4细胞移动性的作用。
Matrigel侵袭实验结果显示,虽然我们在细胞增殖实验中已经证实,在极低浓度(10μg/ml)下,p-榄香烯对IHH-4细胞的增殖无明显抑制作用,但在此浓度下p-榄香烯仍然对IHH-4细胞的移动性具有明显的抑制作用。
三、β-榄香烯对PTC作用机制的动物实验
1、β-榄香烯对荷瘤BALB/c裸鼠肿瘤体积的影响
结果显示β-榄香烯治疗组荷瘤裸鼠的肿瘤体积明显小于空白乳剂组(p＜0.05),说明在体内情况下β-榄香烯对PTC的生长有明显抑制作用。
2、β-榄香烯对荷瘤BALB/c裸鼠生存率的影响
从给药后第7天开始,β-榄香烯治疗组(50mg/kg)的荷瘤BALB/c裸鼠的生存率明显高于空白乳剂组(p<0.01),说明在体内情况下β-榄香烯通过抑制PTC的生长,来提高荷瘤裸鼠的生存率。
3、β-榄香烯在体内情况下对细胞周期调节蛋白及VEGF的影响
经β-榄香烯治疗(50mg/kg)后,肿瘤组织中的cyclin E和CDK6的表达水平下降,提示在体内情况下β-榄香烯也能通过调控cyclin E和CDK6的表达,导致IHH-4细胞阻滞于细胞周期的G1期。此外,经β-榄香烯治疗后,VEGF蛋白的表达水平下降,提示肿瘤组织中的血管新生受到抑制。
结论
1、免疫组织化学染色提示PTC组织中TSHR和NIS蛋白表达异常,与相应基因的甲基化发生有关,亦与BRAF基因T1799A突变的发生有关。提示：PTC中相应基因的甲基化和BRAF基因突变可能是TSHR和NIS蛋白表达减弱并分布异常的原因。TSHR和NIS基因甲基化与BRAF基因突变相关,可能是BRAF基因突变导致肿瘤恶性程度增高的内在机制之一
2、β-榄香烯对PTC具有明显的治疗作用,其作用机制是通过阻滞细胞周期、诱导细胞凋亡和抑制肿瘤细胞侵袭而实现的。
Objective
Thyroid cancer is relatively rare in the specific chromosome entopic, chimerical, gene fusion and mutation caused by epithelial cell tumors. Papillary thyroid cancer (PTC) is differentiated thyroid cancer, accounting for thyroid cancer 80%. While including the PTC, differentiated thyroid cancer's malignancy is low and highly more cure, but these tumors in the initial treatment of the recurrence rate is high, this is because the thyroid-specific proteins, including the basement membrane Na+/I-symporter (NIS) and the top membrane Pendrin protein and thyrotrophic receptor (TSHR) expression in the absence or functional abnormalities. Research has shown that DNA methylation may lead to tumor tissues a protein to reduce or abnormal function of a cause, and finally to radioactive iodine treatment invalid.
Recent research discovered BRAF gene thyroid papillary closer relationship, Marine N etc. on 320 cases thyroid tumor and 6 undifferentiated cancer strains mutation detection found poorly differentiated and undifferentiated carcinoma also BRAF gene mutation. Subsequently, many scholars comparison various thyroid carcinoma BRAF gene mutation rate, found PTC BRAF genes mutation rate up 83%, undifferentiated carcinoma mutations rate 35%, other types of thyroid cancer have no mutation, indicating sporadic adult PTC of the BRAF gene mutation rate, with BRAF T1799A mutation hot spots. Summing prompted BRAF gene mutation may papillary thyroid cancer metastasis prognosis.
BRAF gene mutation may also affect the thyroid cancer treatment strategies. Thyroid cancer to conventional chemotherapy is not sensitive, and which side effects. Therefore, development of new anticancer drugs is particularly important.
Elemene, an extract from the ginger plant Rhizoma zeodaria, is a novel anticancer drug. The extract of elemene is a mixture ofα-,β-andδ-elemene, withβ-elemene as the main component, which accounts for 60～72% of the three isoforms.β-elemene has a broad-spectrum antitumor effect in many types of cancer, it has low toxicity and is therefore well tolerated and accepted by cancer patients. Recent studies showed thatβ-elemene-inhibited cell proliferation and trigger apoptosis in glioma cells and leukemia cells, and the apoptosis. However, the anti-cancer effect and mechanism of (3-elemene on human thyroid cancer remains unknown.
The purpose of this study is PTC with iodine metabolism in the closely related TSHR and NIS protein expression and the TSHR and NIS gene methylation and BRAF T1799A mutation analysis of genetic changes and the TSHR and NIS protein expression in. Further research in vivo and in vitroβ-elemene cases of human papillary thyroid cancer cell lines IHH-4 and anaplastic thyroid cancer cell lines ASH-3 effect and its mechanism.
Methods
1、Subject
Case studies from 2005 to 2006 in the People's Hospital of Bin zhou thyroidectomy for PTC hysterectomy patients (n= 60), male 12, female 48 cases, aged 26-66 years, mean age 44 years
Human papillary thyroid cancer IHH-4 cell lines and anaplastic thyroid cancer cell lines ASH-3 were obtained from Health Science Research Resources Bank (Osaka, Japan).6 weeks old male BALB/c nude mice were purchased from Wei Tong Company (Beijing, China).
2、Methods
Cases of papillary thyroid carcinoma specimens BRAF T1799A mutation, cancer tissue sections extracted genomic DNA, polymerase chain reaction (PCR) on samples of DNA BRAF gene exon 15 were amplified, and sequenced.
Specimens of papillary thyroid cancer cases in the TSHR and NIS protein expression and methylation detection, TSHR and NIS protein expression in immunohistochemical directly; methylation analysis was extracted cancer tissue sections of genomic DNA, DNA bisulfite treatment, for TSHR and NIS methylation specific PCR.
Cell viability or the effects ofβ-elemene on cell proliferation in IHH-4 cells and ASH-3 cells were assessed with an MTT-based colorimetric cell proliferation assay following the manufacturer's instructions (Sigma). Flow cytometry onβ-elemene treated IHH-4 and ASH-3 cell line cell cycle were analyzed. After stained with PI dye, the cells were analyzed for cell cycle perturbation using a FACSCalibur (Becton-Dickinson, San Diego, Calif.), and the CellQuest Pro software (Becton-Dickinson) and the ModFit LT software (Verity Software House, Topsham, Maine) were used to determine the distribution of cells in the various cell cycle compartments:G1, S and G2-M.
β-elemene induces apoptosis in human papillary thyroid carcinoma IHH-4 cell levels and activity and apoptosis detection. We detectedβ-elemene inducing apoptosis in IHH-4 cells using the TUNEL assay and the apoptosis regulatory protein, caspases-8, caspases-9 and Bcl-2 by immunoblotting analysis.
We detected invasiveness of the IHH-4 cells using in vitro invasion assay. Cells (5×104 per well) were placed in the upper chamber of a 24-well Transwell apparatus containing Matrigel membranes (BD Biosciences, San Jose, CA), and the lower chamber was filled with 750μL DMEM supplemented with 0.1% bovine serum albumin and fibronectin (10μg/mL; Roche) as a chemoattractant. After incubation for 36 hours at 37℃, cells that had migrated to the lower surface of each membrane were stained with the use of a Diff-Quik kit (International Reagents, Kobe, Japan) and counted. Each experiment was done with triplicate wells and repeated thrice.
Human cancer xenograft models were established using the methods reported previously. When 80% confluence reached cultured cells in monolayer were trypsinized and harvested by centrifugation. The IHH-4 cellsβ-elemene on tumor-bearing BALB/c nude mice of tumor detection, including the tumor-bearing BALB/c nude mice tumor volume, survival rate of detection, and cell cycle regulation in vivo detection of protein and VEGF.
3、Statistical analysis
Disaggregated data by frequency and percentage that applied continuous variables mean±standard deviation. Classification variables in Statistics x2 test, continuous variables t test statistical applications. p value using two-sided test, with p<0.05 as significantly different. Using SPSS software (11.5 version) for data analysis.
Results
1. TSHR, NIS gene methylation and protein expression and T1799A BRAF mutation in PTC
The prevalence of the T1799A BRAF mutation in this series of PTC was 65% (39 of 60). Mutation and clinic pathologic features has no significant association. 20 cases of carcinoma of the contra lateral non-cancerous thyroid tissues, BRAF mutations were not detected.
Samples of TSHR and NIS protein expression in a contra lateral thyroid cancer (positive control) in, TSHR-positive particles were distributed in about 70% of thyroid follicular epithelial cell cytoplasm and membrane, the degree of moderate to strong color. All were detected in the PTC samples, positive particles can be seen TSHR color: 57% (n= 34) of the specimens showed TSHR scattered or diffuse cytoplasm staining, 35% (n= 21) can be seen with cancer of the lateral organizations, similar staining intensity and distribution of positive color,8% (n= 5) tumor specimens staining intensity was higher than the contra lateral tissue. Contra lateral thyroid cancer (positive control) in, NIS-positive particles were distributed in about 20% of thyroid follicular epithelial cell cytoplasm and membrane, the color degree of intensity. All samples were detected in PTC, in addition to NIS 1 case, positive particles can be seen the color, but color intensity than the positive control significantly as the weak, and only 5% (n= 3) of the specimens can be seen opposite staining with cancer similar intensity and distribution of the positive color.
The prevalence of the TSHR, and NIS gene methylation in this series of PTC was 43% (26 of 60) and 27%(16 of 60). The gene methylation and clinic pathological features some of the trends are related, but not statistically significant.20 cases of carcinoma of the contra lateral non-cancerous thyroid tissue, methylation of these genes were not detected.
2、The mechanism ofβ-elemene anti-cancer ability on human thyroid cancer in vitro
Our studies shownβ-elemene inhibited growth of thyroid cancer IHH-4 and ASH-3 cells and affected the expression of G1 cell cycle regulatory proteins.β-elemene significantly inhibited ASH-3 and IHH-4 cells cell proliferation, and this inhibition is dose-dependent. The cell cycle ofβ-elemene treated IHH-4 and ASH-3 cell lines were analyzed by Flow cytometry. Our results shownβ-elemene (30,40 and 50μg/ml) arrested IHH-4 cells at G1 phase. On the other hand, afterβ-elemene treatment, the ASH-3 cells population in G1 phase did not increase significantly. G1 cell cycle phase is regulated by cell cycle regulatory protein E, CDK2 and CDK6 and other adjustments. As shown in the experiments, the levels of cyclin E, CDK2 and CDK6 were decreased after exposure of IHH-4 cells to different concentrations ofβ-elemene.
β-elemene induces apoptosis in human papillary thyroid carcinoma IHH-4 cells. The TUNEL technique is classically used to detect apoptotic cells in culture. Here the induction of apoptosis followingβ-elemene treatment was confirmed by the TUNEL assay. As can be seen, few apoptotic cells were detected in the untreated control cells. In contrast, apoptosis in the IHH-4 cells treated withβ-elemene was increased as measured by the TUNEL assay. Quantitative analysis of the sections revealed thatβ-elemene induced a significant increase in apoptotic IHH-4 cells at 48 h in a dose-dependent manner, compared with the controls.
The results shown in untreated control cells, little apoptosis, and as the concentration ofβ-elemene increased number of apoptotic cells also increased. Therefore, the same compared to untreated control cells,β-elemene showed a dose-dependent manner in the office 48 hours caused apoptosis IHH-4 cells were significantly increased. The expression level of apoptosis regulation protein were detected:Bcl-2 expression decreased, caspases-9 cleavage protein levels increased in a dose dependent manner. In addition, caspases-8 levels did not change.
We detected the invasiveness of human papillary thyroid cancer IHH-4 cell lines afterβ-elemene treatment. Matrigel invasion assay showed that, although experiments in cell proliferation has been confirmed in the very low concentration (10μg/ml), 10μg/mlβ-elemene has no effect on growth of IHH-4 cell lines, but at this concentration, the invasiveness of IHH-4 cells were significantly inhibited.
3、Effects of elemene on the growth of IHH-4 cells transplanted tumors in vivo
β-elemene on tumor-bearing BALB/c nude mice tumor volume of the results showed thatβ-elemene treatment group in nude tumor volume was significantly smaller than the blank emulsion group (p<0.05). This result explained that P-elemene inhibited growth of PTC in vivo.
β-elemene on tumor-bearing BALB/c nude mice survival rate. From the 7th day after administration,β-elemene treatment group (50mg/kg) in tumor-bearing BALB/c nude mice survival rate was significantly higher than the blank emulsion group (p<0.01). This results suggested thatβ-elemene inhibite the growth of PTC, and then improve the survival rate of nude mice.
β-elemene in vivo cases, the cell cycle regulatory proteins and the impact of VEGF. Afterβ-elemene treatment (50mg/kg), the expression of cycle E CDK6 (the cell cycle regulatory proteins) were decreased. Our studies suggesting that in vivoβ-elemene inhibite the tumor growth through regulating the expression of cell cycle regulatory proteins cyclin E and CDK6, leading to IHH-4 cell line cell cycle arrest in G1 phase.In addition, theβ-elemene treatment, VEGF protein level decreased, suggesting that tumor angiogenesis is also inhibited.
Conclusions
1、Immunohistochemical staining showed PTC tissue abnormalities TSHR and NIS protein expression, and the corresponding gene methylation was also related to the occurrence of BRAF T1799A mutation. Tip:PTC in the corresponding gene methylation and BRAF mutation may be the TSHR and NIS expression was decreased and the reasons for abnormal distribution. TSHR and NIS gene methylation and BRAF mutations, BRAF mutations may lead to increased malignant tumor of the internal mechanism.
2、Elemene inhibits the growth of IHH-4 cells and trigger apoptosis in vitro and in vivo. These data provide useful information for further clinical study on the treatment of human thyroid cancer byβ-elemene.
引文
1 Ries LAG, Melbert D, Krapcho M, Stinchcomb DG, Howlader N, Horner MJ,Mariotto A, Miller BA, Feuer EJ, Altekruse SF, Lewis DR, Clegg L, Eisner MP,Reichman M, Edwards BK, eds.2008 SEER cancer statistics review,1975-2005, National Cancer Institute (Bethesda, MD).
2 Leenhardt L, Grosclaude P, Cherie-Challine L 2004 Increased incidence of thyroid carcinoma in France:a true epidemic or thyroid nodule management effects? Report from the French thyroid cancer committee. Thyroid 14:1056-1060
3 Davies L, WelchHG2006 Increasing incidence of thyroid cancer in the United States, 1973-2002. JAMA 295:2164-2167
4 GoodmanMT,Yoshizawa CN, KolonelLN1988 Descriptive epidemiology of thyroid cancer in Hawaii. Cancer 61:1272-1281
5 Hrafnkelsson J, Jonasson JG, Sigurdsson G, Sigvaldason H, Tulinius H 1988 Thyroid cancer in Iceland 1955-1984. Acta Endocrinol (Copenh) 118:566-572
6 Kuijpens JL, Coebergh JW, van der Heijden LH, Kruis H, Ribot JG, de Rooij HA 1994 Thyroid cancer in Southeastern Netherlands,1970-1989:trends in incidence, treatment and survival. Ned Tijdschr Geneeskd 138:464-468
7 Teng W, Shan Z, Teng X, Guan H, Li Y, Teng D, Jin Y, Yu X, Fan C, Chong W,Yang F, Dai H,YuY, Li J, Chen Y, Zhao D, Shi X,HuF,MaoJ,GuX, Yang R, Tong Y, Wang W, Gao T, Li C 2006 Effect of iodine intake on thyroid diseases in China. N Engl J Med 354:2783-2793
8 Williams ED, Doniach I, Bjarnason O, Michie W 1977 Thyroid cancer in an iodide rich area:a histopathological study. Cancer 39:215-222
9 Williams ED 1979 The aetiology of thyroid tumours. Clin Endocrinol Metab 8:193-207
10 Harach HR, Williams ED 1995 Thyroid cancer and thyroiditis in the goitrous region of Salta, Argentina, before and after iodine prophylaxis. Clin Endocrinol (Oxf) 43:701-706
11 Gomez Segovia I, Gallowitsch HJ, Kresnik E, Kumnig G, Igerc I, Matschnig S, Stronegger WJ, Lind P 2004 Descriptive epidemiology of thyroid carcinoma in Carinthia, Austria:1984-2001. Histopathologic features and tumor classification of 734 cases under elevated general iodination of table salt since 1990:population-based age-stratified analysis on thyroid carcinoma incidence. Thyroid 14:277-286
12 Farahati J, Geling M, Ma"der U, Mo" rtl M, Luster M, Mu" ller JG, Flentje M, Reiners C 2004 Changing trends of incidence and prognosis of thyroid carcinoma in lower Franconia, Germany, from 1981-1995. Thyroid 14:141-147
13 Lind P, Langsteger W, Molnar M, Gallowitsch HJ, Mikosch P, Gomez I 1998 Epidemiology of thyroid diseases in iodine sufficiency. Thyroid 8:1179-1183
14 Yamashita H, Noguchi S, Murakami N, Kato R, Adachi M, Inoue S, Kato S, Nakayama I 1990 Effects of dietary iodine on chemical induction of thyroid carcinoma. Acta Pathol Jpn 40:705-712
15 Xing M 2007 BRAF mutation in papillary thyroid cancer:pathogenic role, molecular bases, and clinical implications. Endocr Rev 28:742-762
16 Knauf JA, Ma X, Smith EP, Zhang L, Mitsutake N, Liao XH, Refetoff S, Nikiforov YE, Fagin JA 2005 Targeted expression of BRAFV600E in thyroid cells of transgenic mice results in papillary thyroid cancers that undergo dedifferentiation. Cancer Res 65:4238-4245
17 Zhao J, Chen Z, Maberly G 1998 Iodine-rich drinking water of natural origin in China. Lancet 352:2024
18 Guan H, Li C, Li Y, Fan C, Teng Y, Shan Z, TengW2005 High iodine intake is a risk factor of post-partum thyroiditis:result of a survey from Shenyang, China. J Endocrinol Invest 28:876-881
19 Yu J, Liu S, Su X, Zhang S 2004 Results of water iodine in the 2002 national iodine deficiency disorders surveillance. Chin J Endocrinol 23:223-224
20 Wang J, Shao M, An Z, Li P, Shao X 1995 Iodine contents in drinking water and urine in residence living in Qingdao. Zhong Guo Di Fang Bing Xue Za Zhi (Chin J Endemiol) 14:384-385
21 Guo X, Zhai L, Liu Y, Wang X 2005 Study on the present status of the areas with high iodine concentration in drinking water and edible salt at household levels in Ohio of Yellow River. Wei Sheng Yan Jiu (J Hygiene Res) 34:695-697
22 Guo X, Liu Y, Zhai L, Wang X, Huang J, Liu C, Bian J, Qin Q, Chen Z 2005 Study on the present status of the areas with high iodine concentration in drinking water and edible salt at household levels in southwest of Shandong Province, China. Chin J Epidemiol 26:745
23 Li P, Li S, Zhang J, Jiang F, Li Z 2003 Results of iodine deficiency disorder surveillance in Qingdao. Chin J Ctrl Endem Dis 18:69
24 Su XH, Liu SJ, Shen HM 2007 National iodine deficiency disorder surveillance:a sum up of data in 2005 and an analysis. Chin J Endemiol 26:67-69
25 XingM,WestraWH,Tufano RP, Cohen Y, Rosenbaum E, Rhoden KJ, Carson KA, Vasko V, Larin A, Tallini G, Tolaney S, Holt EH, Hui P, Umbricht CB, Basaria S, Ewertz M, Tufaro AP, Califano JA, RingelMD,Zeiger MA, Sidransky D, Ladenson PW 2005 BRAF mutation predicts a poorer clinical prognosis for papillary thyroid cancer. J Clin Endocrinol Metab 90:6373-6379
26 Frasca F, Nucera C, Pellegriti G, Gangemi P, Attard M, Stella M, Loda M, Vella V, Giordano C, Trimarchi F, Mazzon E, Belfiore A, VigneriR2008 BRAF(V600E) mutation and the biology of papillary thyroid cancer. Endocr Relat Cancer 15:191-205
27 Smyth P, Finn S, Cahill S, O'Regan E, Flavin R, O'Leary JJ, Sheils O 2005 Ret/PTC and BRAF act as distinct molecular, time-dependent triggers in a sporadic Irish cohort of papillary thyroid carcinoma. Int J Surg Pathol 13:1-8
28 Hollowell JG, Staehling NW, Hannon WH, Flanders DW, Gunter EW, Maberly GF, Braverman LE, Pino S, Miller DT, Garbe PL, DeLozier DM, Jackson RJ 1998 Iodine nutrition in the United States:trends and public health implications:iodine excretion data from National Health and Nutrition Examination Surveys Ⅰ and Ⅲ (1971-74 and 1988-1994). J Clin Endocrinol Metab 83:3401-3408
29 Bosetti C, Kolonel L, Negri E, Ron E, Franceschi S, Dal Maso L, Galanti MR,Mark SD, Preston-Martin S, McTiernan A, Land C, Jin F, Wingren G, Hallquist A, Glattre E, Lund E, Levi F, Linos D, La Vecchia C 2001 A pooled analysis of case-control studies of thyroid cancer. VI. Fish and shellfish consumption. Cancer Causes Control 12:375-382
1 Hegedus L. Clinical practice. The thyroid nodule. N Engl J Med,2004,351:1764-1771.
2 Sherman SI. Thyroid carcinoma. Lancet,2003,361:501-511.
3 Jemal A, Murray T, Ward E, et al. Cancer statistics 2005. CA Cancer J Clin,2005,55:10-30.
4 Hodgson NC, Button J, Solorzano CC. Thyroid cancer:Is the incidence still increasing? Ann Surg Oncol,2004,11:1093-1097.
5 Davies L, Gilbert Welch H. Increasing Incidence of Thyroid Cancer in the United States, 1973-2002. JAMA,2006,295:2164-2167.
6 钱碧云,陈可欣,何敏,等。天津市区甲状腺癌流行状况调查。中国肿瘤临床,2005,32：218-221。
7 关海霞,单忠艳,米小轶,等。普遍食盐碘化前后甲状腺癌发病变化的11年病理资料分析。中国医科大学学报,2006,35：284-85。
8 Zhao J, Li QQ, Zou B, et al.In vitro combination characterization of the new anticancer plant drug beta-elemene with taxanes against human lung carcinoma [J]. Int J Oncol,2007,31 (2):241-252.
9 Tao L, Zhou L, Zheng L, et al. Elemene displays anti-cancer ability on laryngeal cancer cells in vitro and in vivo [J]. Cancer Chemother Pharmacol,2006,58:24-34.
10 Zhou H., Shen J., Hou J., Qiu Y. and Luo Q. (2003) Experimental study on apoptosis induced by elemene in glioma cells (in Chinese). Ai Zheng.22:959-963
11 Yang H., Wang X., Yu L. and Zheng S. (1999) Study on the anticancer mechanism of elemene (in Chinese). Chin. Clin. Cancer 26:4-7
12 Wang G, Li X, Huang F, et al.Antitumor effect of β-elemene in non-small-cell lung cancer cells is mediated via induction of cell cycle arrest and apoptotic cell death[J]. Cell Mol Life Sci, 2005,62:881-893.
13 LiX, Wang G, Zhao J, et al.Antiproliferative effect of β-elemene in chemoresistant ovarian carcinoma cells is mediated through arrest of the cell cycle at the G2-M phase[J].Cell Mol Life Sci,2005,62:894-904.
14 Zou L., Liu W. and Yu L. (2001) beta-elemene induces apoptosis of K562 leukemia cells (in Chinese). Zhonghua Zhong Liu Za Zhi 23:196-198
15 Morgan D. (1995) Principles of CDK regulation. Nature 374
16 Sherr C. (1996) Cancer cell cycles. Science 274:1672-1677
17 Dynlacht B. (1997) Regulation of transcription by proteins that control the cell cycle. Nature 389:149-152
18 Ashkenazi A. and Dixit V. (1999) Apoptosis control by death and decoy receptors. Curr. Opin. Cell Biol.11:255-260
19 Li P., Nijhawan D., Budihardjo I., Srinivasula S., Ahmad M., Alnemri E. et al. (1997) Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell 91:479-489
20 Liu X., Kim C., Yang J., Jemmerson R. and Wang X. (1996) Induction of apoptotic program in cell-free extracts:requirement for dATP and cytochrome c. Cell 86:147-157
21 Earnshaw W., Martins L. and Kaufmann S. (1999) Mammalian caspases:structure, activation, substrates, and functions during apoptosis. Annu. Rev. Biochem.68:383-424
22 Thornberry N. and Lazebnik Y. (1998) Caspases:enemies within. Science 281:1312-1316
23 Gross A., McDonnell J. and Korsmeyer S. (1999) BCL-2 family members and the mitochondria in apoptosis. Genes Dev.13:1899-1911
24 Kroemer G. (1997) The proto-oncogene Bcl-2 and its role in regulating apoptosis. Nat. Med.3: 614-620
25 Kluck R., Bossy-Wetzel E., Green D. and Newmeyer D. (1997) The release of cytochrome c from mitochondria:a primary site for Bcl-2 regulation of apoptosis. Science 275:1132-1136
1 Mazzaferri EL, Jhiang SM Long-term impact of initial surgical and medical therapy on papillary and follicular thyroid cancer. Am J Med 1994;97:418-428.
2 Xing M BRAF Mutation in Papillary Thyroid Cancer:Pathogenic Role, Molecular Bases, and Clinical ImplicationsEndocrine Reviews 2007;28(7):742-762.
3 Xing M BRAF mutation in thyroid cancer. Endocr Relat Cancer 2005;12:245-262.
4 Keelawat S, Poumsuk U Association between different variants of papillary thyroid carcinoma and risk-group according to AMES (age, metastasis, extent and size) classification system. J Med Assoc Thai 2006;89:484-489.
5 Michels JJ, Jacques M, Henry-Amar M, Bardet S. Prevalence and prognostic significance of tall cell variant of papillary thyroid carcinoma. Hum Pathol 2007;38:212-219.
6 MacCorkle RA, Tan TH Mitogen-activated protein kinases in cell-cycle control. Cell Biochem Biophys 2005;43:451-461
7 Mercer KE, Pritchard CA Raf proteins and cancer:B-Raf is identified as a mutational target. Biochim Biophys Acta 2003;1653:25-40
8 Rodriguez-Viciana P, Tetsu O, Oda K, Okada J, Rauen K, McCormick F Cancer targets in the Ras pathway. Cold Spring Harb Symp Quant Biol 2005;70:461-467.
9 Sebolt-Leopold JS, Herrera R Targeting the mitogen-activated protein kinase cascade to treat cancer. Nat Rev Cancer 2004;4:937-947
10 Dhomen N, Marais RNew insight into BRAF mutations in cancer. Curr Opin Genet Dev 2007; 17:31-39
11 Garnett MJ, Marais R Guilty as charged:B-RAF is a human oncogene. Cancer Cell 2004; 6:313-319.
12 Xing M BRAF Mutation in Papillary Thyroid Microcarcinoma:the Promise of Better Risk ManagementAnn Surg Oncol.2009;16(4):801-803.
13 Shuntaro I,M ik iharu F, Yo sh to U, et al. Braf, a new member of the raf fam ily, is act ivated by DNA rearrangment. Mo lecular Celluar Biology,1988; 8:2651
14 尹香利,张国昌.BRAF原癌基因与甲状腺癌关系的研究进展.陕西医学杂志,2006；35(3)：324-327.
15 Davies H, B ignell GR, Cox C, et al. M utat ions of the BRA F gene in human cancer. N ature, 2002; 417:949
16 Marine N,Nikifo rova Edna T,M ano j G, et al. BRAF mutations in thyro id tumo rs are restricted to papillary carcinomas and anap last icorpoorly different iated carcinomas arising from pap illary carcinomas. J Clin Endocrino 1M etab,2003; 88 (11):5399
17 Kim KH, Kang DW, Kim SH, Seong IO & Kang DY Mutations of the BRAF gene in papillary thyroid carcinoma in a Korean population. Yonsei Medical Journal 2004;45 818-821.
18 Soares P, Trovisco V, Rocha AS, Feijao T, Rebocho AP, Fonseca E, Vieira de Castro I, Cameselle-Teijeiro J,Cardoso-Oliveira M & Sobrinho-Simoes M BRAF mutations typical of papillary thyroid carcinoma are morefrequently detected in undifferentiated than in insular and insular-like poorly differentiated carcinomas. Virchows Archiv 2004;444 572-576.
19 N amba H, N akash ima M, Hayash i T, et al, Clinical implication of hot spot BRAF mutation, V599E, in papillary thyroid cancers. J Clin Endocrineo Metab 2003; 88:4393.
2 Leenhardt L, Grosclaude P, Cherie-Challine L 2004 Increased incidence of thyroid carcinoma in France:a true epidemic or thyroid nodule management effects? Report from the French thyroid cancer committee. Thyroid 14:1056-1060
3 Davies L, WelchHG2006 Increasing incidence of thyroid cancer in the United States, 1973-2002. JAMA 295:2164-2167
4 GoodmanMT,Yoshizawa CN, KolonelLN1988 Descriptive epidemiology of thyroid cancer in Hawaii. Cancer 61:1272-1281
5 Hrafnkelsson J, Jonasson JG, Sigurdsson G, Sigvaldason H, Tulinius H 1988 Thyroid cancer in Iceland 1955-1984. Acta Endocrinol (Copenh) 118:566-572
6 Kuijpens JL, Coebergh JW, van der Heijden LH, Kruis H, Ribot JG, de Rooij HA 1994 Thyroid cancer in Southeastern Netherlands,1970-1989:trends in incidence, treatment and survival. Ned Tijdschr Geneeskd 138:464-468
7 Teng W, Shan Z, Teng X, Guan H, Li Y, Teng D, Jin Y, Yu X, Fan C, Chong W,Yang F, Dai H,YuY, Li J, Chen Y, Zhao D, Shi X,HuF,MaoJ,GuX, Yang R, Tong Y, Wang W, Gao T, Li C 2006 Effect of iodine intake on thyroid diseases in China. N Engl J Med 354:2783-2793
8 Williams ED, Doniach I, Bjarnason O, Michie W 1977 Thyroid cancer in an iodide rich area:a histopathological study. Cancer 39:215-222
9 Williams ED 1979 The aetiology of thyroid tumours. Clin Endocrinol Metab 8:193-207
10 Harach HR, Williams ED 1995 Thyroid cancer and thyroiditis in the goitrous region of Salta, Argentina, before and after iodine prophylaxis. Clin Endocrinol (Oxf) 43:701-706
11 Gomez Segovia I, Gallowitsch HJ, Kresnik E, Kumnig G, Igerc I, Matschnig S, Stronegger WJ, Lind P 2004 Descriptive epidemiology of thyroid carcinoma in Carinthia, Austria:1984-2001. Histopathologic features and tumor classification of 734 cases under elevated general iodination of table salt since 1990:population-based age-stratified analysis on thyroid carcinoma incidence. Thyroid 14:277-286
12 Farahati J, Geling M, Ma"der U, Mo" rtl M, Luster M, Mu" ller JG, Flentje M, Reiners C 2004 Changing trends of incidence and prognosis of thyroid carcinoma in lower Franconia, Germany, from 1981-1995. Thyroid 14:141-147
13 Lind P, Langsteger W, Molnar M, Gallowitsch HJ, Mikosch P, Gomez I 1998 Epidemiology of thyroid diseases in iodine sufficiency. Thyroid 8:1179-1183
14 Yamashita H, Noguchi S, Murakami N, Kato R, Adachi M, Inoue S, Kato S, Nakayama I 1990 Effects of dietary iodine on chemical induction of thyroid carcinoma. Acta Pathol Jpn 40:705-712
15 Xing M 2007 BRAF mutation in papillary thyroid cancer:pathogenic role, molecular bases, and clinical implications. Endocr Rev 28:742-762
16 Knauf JA, Ma X, Smith EP, Zhang L, Mitsutake N, Liao XH, Refetoff S, Nikiforov YE, Fagin JA 2005 Targeted expression of BRAFV600E in thyroid cells of transgenic mice results in papillary thyroid cancers that undergo dedifferentiation. Cancer Res 65:4238-4245
17 Zhao J, Chen Z, Maberly G 1998 Iodine-rich drinking water of natural origin in China. Lancet 352:2024
18 Guan H, Li C, Li Y, Fan C, Teng Y, Shan Z, TengW2005 High iodine intake is a risk factor of post-partum thyroiditis:result of a survey from Shenyang, China. J Endocrinol Invest 28:876-881
19 Yu J, Liu S, Su X, Zhang S 2004 Results of water iodine in the 2002 national iodine deficiency disorders surveillance. Chin J Endocrinol 23:223-224
20 Wang J, Shao M, An Z, Li P, Shao X 1995 Iodine contents in drinking water and urine in residence living in Qingdao. Zhong Guo Di Fang Bing Xue Za Zhi (Chin J Endemiol) 14:384-385
21 Guo X, Zhai L, Liu Y, Wang X 2005 Study on the present status of the areas with high iodine concentration in drinking water and edible salt at household levels in Ohio of Yellow River. Wei Sheng Yan Jiu (J Hygiene Res) 34:695-697
22 Guo X, Liu Y, Zhai L, Wang X, Huang J, Liu C, Bian J, Qin Q, Chen Z 2005 Study on the present status of the areas with high iodine concentration in drinking water and edible salt at household levels in southwest of Shandong Province, China. Chin J Epidemiol 26:745
23 Li P, Li S, Zhang J, Jiang F, Li Z 2003 Results of iodine deficiency disorder surveillance in Qingdao. Chin J Ctrl Endem Dis 18:69
24 Su XH, Liu SJ, Shen HM 2007 National iodine deficiency disorder surveillance:a sum up of data in 2005 and an analysis. Chin J Endemiol 26:67-69
25 XingM,WestraWH,Tufano RP, Cohen Y, Rosenbaum E, Rhoden KJ, Carson KA, Vasko V, Larin A, Tallini G, Tolaney S, Holt EH, Hui P, Umbricht CB, Basaria S, Ewertz M, Tufaro AP, Califano JA, RingelMD,Zeiger MA, Sidransky D, Ladenson PW 2005 BRAF mutation predicts a poorer clinical prognosis for papillary thyroid cancer. J Clin Endocrinol Metab 90:6373-6379
26 Frasca F, Nucera C, Pellegriti G, Gangemi P, Attard M, Stella M, Loda M, Vella V, Giordano C, Trimarchi F, Mazzon E, Belfiore A, VigneriR2008 BRAF(V600E) mutation and the biology of papillary thyroid cancer. Endocr Relat Cancer 15:191-205
27 Smyth P, Finn S, Cahill S, O'Regan E, Flavin R, O'Leary JJ, Sheils O 2005 Ret/PTC and BRAF act as distinct molecular, time-dependent triggers in a sporadic Irish cohort of papillary thyroid carcinoma. Int J Surg Pathol 13:1-8
28 Hollowell JG, Staehling NW, Hannon WH, Flanders DW, Gunter EW, Maberly GF, Braverman LE, Pino S, Miller DT, Garbe PL, DeLozier DM, Jackson RJ 1998 Iodine nutrition in the United States:trends and public health implications:iodine excretion data from National Health and Nutrition Examination Surveys Ⅰ and Ⅲ (1971-74 and 1988-1994). J Clin Endocrinol Metab 83:3401-3408
29 Bosetti C, Kolonel L, Negri E, Ron E, Franceschi S, Dal Maso L, Galanti MR,Mark SD, Preston-Martin S, McTiernan A, Land C, Jin F, Wingren G, Hallquist A, Glattre E, Lund E, Levi F, Linos D, La Vecchia C 2001 A pooled analysis of case-control studies of thyroid cancer. VI. Fish and shellfish consumption. Cancer Causes Control 12:375-382
1 Hegedus L. Clinical practice. The thyroid nodule. N Engl J Med,2004,351:1764-1771.
2 Sherman SI. Thyroid carcinoma. Lancet,2003,361:501-511.
3 Jemal A, Murray T, Ward E, et al. Cancer statistics 2005. CA Cancer J Clin,2005,55:10-30.
4 Hodgson NC, Button J, Solorzano CC. Thyroid cancer:Is the incidence still increasing? Ann Surg Oncol,2004,11:1093-1097.
5 Davies L, Gilbert Welch H. Increasing Incidence of Thyroid Cancer in the United States, 1973-2002. JAMA,2006,295:2164-2167.
6 钱碧云,陈可欣,何敏,等。天津市区甲状腺癌流行状况调查。中国肿瘤临床,2005,32：218-221。
7 关海霞,单忠艳,米小轶,等。普遍食盐碘化前后甲状腺癌发病变化的11年病理资料分析。中国医科大学学报,2006,35：284-85。
8 Zhao J, Li QQ, Zou B, et al.In vitro combination characterization of the new anticancer plant drug beta-elemene with taxanes against human lung carcinoma [J]. Int J Oncol,2007,31 (2):241-252.
9 Tao L, Zhou L, Zheng L, et al. Elemene displays anti-cancer ability on laryngeal cancer cells in vitro and in vivo [J]. Cancer Chemother Pharmacol,2006,58:24-34.
10 Zhou H., Shen J., Hou J., Qiu Y. and Luo Q. (2003) Experimental study on apoptosis induced by elemene in glioma cells (in Chinese). Ai Zheng.22:959-963
11 Yang H., Wang X., Yu L. and Zheng S. (1999) Study on the anticancer mechanism of elemene (in Chinese). Chin. Clin. Cancer 26:4-7
12 Wang G, Li X, Huang F, et al.Antitumor effect of β-elemene in non-small-cell lung cancer cells is mediated via induction of cell cycle arrest and apoptotic cell death[J]. Cell Mol Life Sci, 2005,62:881-893.
13 LiX, Wang G, Zhao J, et al.Antiproliferative effect of β-elemene in chemoresistant ovarian carcinoma cells is mediated through arrest of the cell cycle at the G2-M phase[J].Cell Mol Life Sci,2005,62:894-904.
14 Zou L., Liu W. and Yu L. (2001) beta-elemene induces apoptosis of K562 leukemia cells (in Chinese). Zhonghua Zhong Liu Za Zhi 23:196-198
15 Morgan D. (1995) Principles of CDK regulation. Nature 374
16 Sherr C. (1996) Cancer cell cycles. Science 274:1672-1677
17 Dynlacht B. (1997) Regulation of transcription by proteins that control the cell cycle. Nature 389:149-152
18 Ashkenazi A. and Dixit V. (1999) Apoptosis control by death and decoy receptors. Curr. Opin. Cell Biol.11:255-260
19 Li P., Nijhawan D., Budihardjo I., Srinivasula S., Ahmad M., Alnemri E. et al. (1997) Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell 91:479-489
20 Liu X., Kim C., Yang J., Jemmerson R. and Wang X. (1996) Induction of apoptotic program in cell-free extracts:requirement for dATP and cytochrome c. Cell 86:147-157
21 Earnshaw W., Martins L. and Kaufmann S. (1999) Mammalian caspases:structure, activation, substrates, and functions during apoptosis. Annu. Rev. Biochem.68:383-424
22 Thornberry N. and Lazebnik Y. (1998) Caspases:enemies within. Science 281:1312-1316
23 Gross A., McDonnell J. and Korsmeyer S. (1999) BCL-2 family members and the mitochondria in apoptosis. Genes Dev.13:1899-1911
24 Kroemer G. (1997) The proto-oncogene Bcl-2 and its role in regulating apoptosis. Nat. Med.3: 614-620
25 Kluck R., Bossy-Wetzel E., Green D. and Newmeyer D. (1997) The release of cytochrome c from mitochondria:a primary site for Bcl-2 regulation of apoptosis. Science 275:1132-1136
1 Mazzaferri EL, Jhiang SM Long-term impact of initial surgical and medical therapy on papillary and follicular thyroid cancer. Am J Med 1994;97:418-428.
2 Xing M BRAF Mutation in Papillary Thyroid Cancer:Pathogenic Role, Molecular Bases, and Clinical ImplicationsEndocrine Reviews 2007;28(7):742-762.
3 Xing M BRAF mutation in thyroid cancer. Endocr Relat Cancer 2005;12:245-262.
4 Keelawat S, Poumsuk U Association between different variants of papillary thyroid carcinoma and risk-group according to AMES (age, metastasis, extent and size) classification system. J Med Assoc Thai 2006;89:484-489.
5 Michels JJ, Jacques M, Henry-Amar M, Bardet S. Prevalence and prognostic significance of tall cell variant of papillary thyroid carcinoma. Hum Pathol 2007;38:212-219.
6 MacCorkle RA, Tan TH Mitogen-activated protein kinases in cell-cycle control. Cell Biochem Biophys 2005;43:451-461
7 Mercer KE, Pritchard CA Raf proteins and cancer:B-Raf is identified as a mutational target. Biochim Biophys Acta 2003;1653:25-40
8 Rodriguez-Viciana P, Tetsu O, Oda K, Okada J, Rauen K, McCormick F Cancer targets in the Ras pathway. Cold Spring Harb Symp Quant Biol 2005;70:461-467.
9 Sebolt-Leopold JS, Herrera R Targeting the mitogen-activated protein kinase cascade to treat cancer. Nat Rev Cancer 2004;4:937-947
10 Dhomen N, Marais RNew insight into BRAF mutations in cancer. Curr Opin Genet Dev 2007; 17:31-39
11 Garnett MJ, Marais R Guilty as charged:B-RAF is a human oncogene. Cancer Cell 2004; 6:313-319.
12 Xing M BRAF Mutation in Papillary Thyroid Microcarcinoma:the Promise of Better Risk ManagementAnn Surg Oncol.2009;16(4):801-803.
13 Shuntaro I,M ik iharu F, Yo sh to U, et al. Braf, a new member of the raf fam ily, is act ivated by DNA rearrangment. Mo lecular Celluar Biology,1988; 8:2651
14 尹香利,张国昌.BRAF原癌基因与甲状腺癌关系的研究进展.陕西医学杂志,2006；35(3)：324-327.
15 Davies H, B ignell GR, Cox C, et al. M utat ions of the BRA F gene in human cancer. N ature, 2002; 417:949
16 Marine N,Nikifo rova Edna T,M ano j G, et al. BRAF mutations in thyro id tumo rs are restricted to papillary carcinomas and anap last icorpoorly different iated carcinomas arising from pap illary carcinomas. J Clin Endocrino 1M etab,2003; 88 (11):5399
17 Kim KH, Kang DW, Kim SH, Seong IO & Kang DY Mutations of the BRAF gene in papillary thyroid carcinoma in a Korean population. Yonsei Medical Journal 2004;45 818-821.
18 Soares P, Trovisco V, Rocha AS, Feijao T, Rebocho AP, Fonseca E, Vieira de Castro I, Cameselle-Teijeiro J,Cardoso-Oliveira M & Sobrinho-Simoes M BRAF mutations typical of papillary thyroid carcinoma are morefrequently detected in undifferentiated than in insular and insular-like poorly differentiated carcinomas. Virchows Archiv 2004;444 572-576.
19 N amba H, N akash ima M, Hayash i T, et al, Clinical implication of hot spot BRAF mutation, V599E, in papillary thyroid cancers. J Clin Endocrineo Metab 2003; 88:4393.