Papillary Thyroid Cancer Pathogenesis And Intervention In Experimental Study

ObjectiveThyroid 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.Methods1,SubjectCase 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 yearsHuman 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,MethodsCases 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 analysisDisaggregated 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. Results1. TSHR, NIS gene methylation and protein expression and T1799A BRAF mutation in PTCThe 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 vitroOur 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 vivoβ-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.Conclusions1,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.