| Background:Thyroid cancer is the most common cancer occurring in the endocrine system. In the recent years, thyroid cancer has become one of the malignant cancers whose incidence rate is most rapidly increasing and is higher in females than in males. Thyroid cancer can be classified into thyroid papillary carcinoma, thyroid follicular carcinoma, and thyroid anaplastic carcinoma originating from thyroid follicular epithelium and thyroid medullary carcinoma originating from parafollicular cells. Among the subtypes of thyroid cancer, thyroid papillary carcinoma and thyroid follicular carcinoma are traditionally defined as the differentiated thyroid cancers. Their incidence rates are higher than 90%, their disease progression is slower and the curative effect is quite good. While the undifferentiated thyroid cancer is rare, its death rate is extremely high. Among the differentiated thyroid cancers, a part of patients who have lower differentiating capability or the patients who originally have highly differentiated thyroid cancer become poorly differentiated cancer with the disease progression and the increase in the numbers of radioiodine 131I therapy. They are between the highly differentiated thyroid cancer and the undifferentiated thyroid cancer and known as poorly differentiated thyroid carcinoma. After treatment with thyroidectomy,131I radiotherapy and combined thyroid hormone therapy, the prognosis of a majority of patients with differentiated thyroid cancer is good. However, for those patients with poorly differentiated thyroid cancer or with undifferentiated thyroid cancer, the outcome of 131I radiotherapy is poor, especially radiotherapy for those patients with undifferentiated thyroid cancer, they totally can not uptake radioactive 131I. Thus, the outcomes of clinical radioiodine therapy and the long-term prognosis for this part of patients are extremely terrible. How to improve the iodine uptake rate of poorly differentiated and undifferentiated thyroid cancer is vital for their clinical efficacy and long-term prognosis.During the carcinogenic progression of thyroid cancer, when the abnormal changes such as gene mutations, gene translocation and methylation, occur, these changes can cause carcinogenesis through activation of the related signal transduction pathways, leading to abnormal cell proliferation and/or apoptosis. The most frequetly mutated genes include B-Raf proto-oncogene (BRAF), RAS and phosphatase and tensin homolog (PTEN) genes etc. and gene translocations such as RET/PTC translocation. The abnormal gene transcription and protein expression can induce somatic mutations through regulation of certain unique cellular signal transduction pathways, causes the abnormal cell proliferation and/or apoptosis, leading to carcinogenesis. During the carcinogenic progression of thyroid cancer, the more important signal transduction pathways involved include thyroid stimulating hormone receptor (TSHR), mitogen activated protein kinase pathway (MAPK), and phosphatidyinositol 3-kinase/protein kinase B (PI3K-AKT) pathway, Notch signaling pathways. The other signal pathways involved also include Janus kinase (JAK)-signal transducers and activators of transcription (STAT), NF-κB and WNT-catenin pathway etc. However, the studies on the mechanisms underlying the lower uptake rate of 1311 by thyroid cancer cells are not available.The basis for 131I radiotherapy for treatment of thyroid disease is that thyroid cells or thyroid cancer cells are capable of taking in iodine. The transport process of iodine in thyroid cells includes two steps:in the first step, iodine is transported into the thyroid cells from the basement membrane against the membrane electrochemical gradient through the mediation of sodium-iodine (Na+/I-) symportor (NIS). In the second step, the iodine is overflowed from apical cells into follicular cavity following the electrochemical gradient through the mediation of pendrin expression protein and human apical iodide transporter (hAIT) protein. This process requires the supply of energy by Na+/I+-ATPase, when Na+ flows following the electrochemical gradient, iodine ion is transported into thyroid cells in a ratio of I-:Na+of 1:2.NIS protein is mainly expressed in the basal membrane of thyroid follicle cells. It is also slightly expressed in other organs such as salivary gland, lacrimal gland, parasynan, submandibular gland, pancreatic gland, breast, heart/thymus, ovary, adrenal gland, rectum, and testicle etc.Currently, our understanding about the deficiency in iodine uptake of thyroid cancer cells is mainly focused in two aspects. The first aspect is the reduction or deficiency of NIS protein; and the second aspect is the retarded subcellular localization of NIS protein. Thyroid-stimulating hormone (TSH) is the main factor involved in regulating the expression of NIS protein. The regulation of NIS expression is related to its transcriptional factors. The transcriptional factors such as TSH, thyroid transcription factor-1 (TTF-1), paired box 8 (PAX-8), cAMP response element-binding protein (CREB), β-catenin, sterol regulatory element-binding proteins (SREBPs), pituitary tumor-transforming gene protein (PTTG), papillomavirus binding factor (PBF) and forkhead box E1 (FoxE1) are involved in regulation of the activities of NIS_PP and NIS far upstream enhancer (NUE) through different pathways, enhancing or inhibiting the expression and the subcellular localization of NIS protein. The post-translational modifications such as phosphorylation and glycosylation are also involved in these processes.Paired box gene 8 (PAX8) is a lineage-restricted transcriptional factor. This protein was firstly detected during the development process of mouse thyroid and subsequently found to be highly expressed in follicular carcinoma of thyroid. PAX8 can not only regulate the activities of the promoters of thyroid globulin, thyroid peroxidase and thyroid-stimulating hormone receptor genes and regulate the proliferation and differentiation of thyroid cells but also is an important factor involved in regulation of the activities of NIS gene promoter and its upstream enhancer (NUE). The expression of NIS is regulated by TSH but also requires the PAX8 to turn on the NIS gene promoter.Bortezomib is a new type of protease inhibitor. Its curative effects in the treatment of the intractable/recurrent myeloid tumor, lymphoma, other type of plasma cell diseases, acute myeloid leukemia and some solid tumors are noticeable. Among them, the mechanism underlying the curative effects of bortezomib in treatment of multiple myeloma (MM) is generally thought to be related to its inhibition of NF-B pathway. In tumor cells, when bortezomib specifically inhibits the activity of 26S subunit of proteasome. Thus, the degradation rate of the inhibitory factor of NF-B (IκB) is significantly reduced. The binding of IκB to NF-B can significantly inhibit the activity of NF-B and inhibit the expression of genes related to cell proliferation and differentiation, reduce the secretion of myeloma cell growth factors such as IL-6 etc. and the expression of adhesion factors, finally leading to apoptosis of tumor cells. It has been confirmed by previous studies that bortezomib can delay the proliferation of many types of cancer cells including multiple myeloma, induce apoptosis and inhibit angiogenesis and can also enhance the sensitivity of cancer cells toward chemotherapeutic agents, improve the tolerance of myeloma cells to dexamethasone, doxorubicin, and alkeran etc. Altmann et al. studied the anti-cancer effects of bortezomib on the undifferentiated thyroid cancer cells. They found that after treatment of these cancer cells with bortezomib, the expression levels of thyroid-related factors such as PAX8 and TTF-1 etc. were increased.However, there have been no reports of the studies confirming the curative effects of bortezomib on thyroid cancer. Thus, by taking PAX8 protein as the starting point, we attempted to treat various subtypes of thyroid cancer with bortezomib and to observe the effects of bortezomib on the expression of PAX8 protein and then observed the effects of regulation of PAX8 protein expression on the rate of iodine uptake rate of thyroid cancer, aiming to find out a new way for radioiodine therapy for thyroid cancer that display low rate of clinical iodine uptake or do not uptake iodine at all.Objectives:1. To observe the expression difference of PAX8 protein among different subtypes of thyroid cancer cells;2. To observe the effects of treatment of various subtypes of thryroid cancer cells with bortezomib on the expression of PAX8 protein and to investigate the role of bortezomib in stimulating differentiation of thyroid cancer cells;3. To observe the expression difference of NIS protein among different types of thyroid cancer cells;4. To observe the expression status of NIS protein on cell membrane of various subtypes of thyroid cancer cells after being treated with bortezomib;5. To test whether or not the iodine uptake rate is changed in various subtypes of thyroid cancer cells after being treated with bortezomib and to investigate whether treatment of these thyroid cancer cells with bortezomib can cause stimulating effects on the differentiation of these thyroid cancer cells, aiming to find out a new way for clinical radioiodine therapy for various subtypes of thyroid cancer.Methods:1. Cell grouping and culture1.1 Subculture of cells:After being digested with trypsin, the cells were sub-cultured in 6-well plate at concentration of 3x105 cells/well.2mL of complete culture medium was added to each well and then cultured for 24h. After the cells had attached to the wall, the cell culture medium was replaced with medium containing low level (2%) of fetal bovine serum (FBS) and the cells were starved for 24h,1.2 Preparation of Different Treatment Media:10% FBS and 1% penicillin-streptomycin and 2 mM/L glutamate were added to sterile RPMI 1640 culture medium. Different concentrations of bortezomib (28,56,112 and 196 and 28.0 ng/mL) were add to the culture media, respectively, and mixed well.1.3 Treatment of Cells with Bortezomib:The culture medium was removed with pipette and the cells were washed with PBS twice. The fresh media containing bortezomib at the doses described above were added to the cells, respectively, and incubated at 37℃,5% CO2 and saturated humidity for 24,48 and 72 h respectively.2. Screening of the Optimal Dose and Timing for the Action of Bortezomib Various subtypes of thyroid cancer cells were treated with bortezomib at 28,56,112, 196 and 280 (ng/mL) for 24 and 48 h, respectively. The activities of these cancer cells being treated with bortezomib were examined with Cell Counting Kit-8 (CCK-8) assay to determine the optimal dose and the optimal timing for the action of bortezomib.3. The expression levels of PAX8 protein in different subtypes of thyroid cancer cells before and after being treated with bortezomib were examined with Western blot.4. The expression levels of NIS protein in in different subtypes of thyroid cancer cells before and after being treated with bortezomib were examined with Western blot.5. The iodine uptake rate in various subtypes of thyroid cancer cells were assayed with 125I to judge whether or not bortezomib can stimulate the differentiation of thyroid cancer cells and can enhance the capability of iodine uptake rate.The experimental data were treated with SPSS 19.0 statistical software. The measurement data were expressed as mean±standard deviation (SEM). The comparison between two groups was conducted by using student’s t-test. The multiple-group comparison was done with One-way ANOVA. The inspection criteria α=0.05. When the P<0.05, the difference between groups was regarded as statistically significant.Results:1. At 24 h after being treated with bortezomib at the indicated doses, the activities of various subtypes of cancer cells were examined. The survival rates of these cells were determined as follows:for thyroid papillary carcinoma cell line B-CPAP, control group (98%),28 ng/mL (94%),56ng/mL (86%),112ng/mL (80%),196ng/mL (76%) and 280ng/mL (69%); for poorly differentiated thyroid tumor cell line WRO 82-1, control group (98.3%),28 ng/mL (94%),56ng/mL (88%),112ng/mL (84%), 196ng/mL (77%)and 280ng/mL (71%); for undifferentiated thyroid cancer cell line ARO, control group (99.7%),28ng/mL (95%),56ng/mL(89%),112ng/mL (84%), 196ng/mL (78%) and 280 ng/mL (74%). At 48 h after being treated with bortezomib at the indicated doses, the survival rates of these cells were as follows:for thyroid papillary carcinoma cell line B-CPAP, the survival rates were as follows:control group (76%),28ng/mL (71%),56ng/mL (62%),112ng/mL (48%),196ng/mL (42%) and 280ng/mL (36%); for the poorly differentiated thyroid cancer cell line WRO 82-1: the survival rates were:control group (78.7%),28ng/mL (74%),56ng/mL (65%), 112ng/mL (53%),196ng/mL (44%),280ng/mL (39%); For undifferentiated thyroid cancer cell line ARO, the survival rates were control group (81.7%),28ng/mL (77%), 56ng/mL (68%),112ng/mL (54%),196ng/mL (47%) and 280 ng/mL (43%). The corresponding IC50 concentration was 112ng/mL. These results indicate that bortezomib causes a dose-dependent effect on reduction of the survival rates of the subtypes of thyroid cancer cells tested. Thus, this concentration was selected to conduct the subsequent experiments. There was significant difference in survival rates of various subtypes of thyroid cancer cells the treated with bortezomib between for 24h and 48h. Thus,48h treatment was selected to conduct the subsequent experiments.2. Western blot was used to examine the expression levels of PAX8 protein in various subtypes of thyroid cancer cells before being treated with bortezomib. The expression levels of PAX8 protein in these cells are different, the expression in normal thyroid cell (Nthy-ori 3-1) is highest, the expression in papillary thyroid cancer cells (B-CPAP) take second place, the expression in poorly differentiated thyroid cancer cells (WRO 82-1) and undifferentiated thyroid cancer cells (ARO) express the lowest, and high expression of the former than the latter.3. Western blot was used to examine the changes in expression levels of PAX8 protein in various subtypes of thyroid cancer cells before and after being treated with bortezomib for 48h. The expression levels of PAX8 protein in these cells after being treated with bortezomib at the dose of 112 (ng/mL) for 48h were significantly increased as compared to those before treatment.4. Western blot was used to examine the expression levels of NIS protein in various subtypes of thyroid cancer cells before being treated with bortezomib. The expression levels of NIS protein in these cells are different, the expression in normal thyroid cell (Nthy-ori 3-1) is highest, the expression in papillary thyroid cancer cells (B-CPAP) take second place, the expression in poorly differentiated thyroid cancer cells (WRO 82-1) and undifferentiated thyroid cancer cells (ARO) express the lowest, and high expression of the former than the latter.5. Western blot was used to examine the changes in expression levels of NIS in cellular membrane of various subtypes of thyroid cancer cells before and after being treated with bortezomib for 48h. The expression levels of NIS on cellular membrane of various subtypes of thyroid cancer cells after being treated with bortezomib at the dose of 112 ng/mL for 48h were not significantly increased as compared to that before treatment.6.125I was used to measure the iodine uptake rate of various subtypes of thyroid cancer. After being treated with bortezomib at 112 ng/mL for 48h, the iodine uptake rates of various bortezomib-treated groups were not significantly changed as compared to that of the controlled group.Conclusions:1. Among different subtypes of thyroid cells tested, there existed differences in the expression levels of PAX8 protein. Among them, the expression level of PAX8 protein in the normal thyroid cells was the highest one, followed by that in papillary carcinoma of thyroid gland. The expression levels of PAX8 protein in poorly differentiated and undifferentiated thyroid cancer cells were the lowest ones and the expression levels in the former was higher than that of the later.2. After being treated with bortezomib at 112 ng/mL for 48h, the expression levels of PAX8 in various subtypes of thyroid cancer cells were all higher than those before being treated.3. Among different subtypes of thyroid cells tested, there existed differences in the expression levels of NIS protein. Among them, the expression level of NIS protein in the normal thyroid cells was the highest one, followed by that in papillary carcinoma of thyroid gland. The expression levels of NIS protein in poorly differentiated and undifferentiated thyroid cancer cells were the lowest ones and the expression levels in the former was higher than that of the later.4. After being treated with bortezomib at 112 ng/mL for 48h, the expression levels of NIS protein in various subtypes of thyroid cancer cells were not changed significantly as compared to those before treatment.5. After being treated with bortezomib at 112 ng/mL for 48h, the iodine uptake rates of various subtypes of thyroid cancer cells were not significantly different from those before treatment. |
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