Research On The Status Of Retinoic Acid Signaling Pathways Of Anaplastic Thyroid Cancer On The Effects Of Retinoic Acid Response

Backgrounds and Objectives:Thyroid carcinoma is the most common malignant tumor of the endocrine system and head and neck cancer. Every year new cases of thyroid cancer accounts for 1%-5% of all cancer, the incidence of female is higher than that in male. The incidence of thyroid cancer increases with age. In recent years, it has aroused widespread concern that the incidence of thyroid cancer has sustained rapid growth.According to the pathological features, thyroid carcinoma divided into papillary thyroid carcinoma (PTC), follicular thyroid carcinoma (FTC), medullary thyroid carcinoma (MTC), anaplastic thyroid carcinoma (ATC), et al. PTC and FTC are differentiated thyroid cancer which prognosis is good. ATC is a rare, but with high degree of malignancy which prognosis is very poor. At present, the main treatment plan of thyroid cancer is surgical operation, postoperative radioiodine therapy and thyroid stimulating hormone suppression therapy. The conventional chemotherapy drugs have little effect on thyroid cancer. The differentiation degree of most PTC and FTC is high, with a good response to treatment. However, there are still some patients in which progress to PDTC (poorly differentiated thyroid cancer) with poor tumor histological differentiation, high degree of invasion, loss of iodine uptake capacity of lesion. ATC is one of the highest degree of human malignancy carcinoma, with most aggressive and lack of effective treatment. The mortality rate of ATC is high after diagnosis, and the survival time is short. Currently, due to poor response to conventional treatment, those patients are resulting in disease recurrence or death; therefore, it is importance and urgency for actively seeking new strategies to improve the curative effect, personalized treatment regimens and improving the survival rate of patients with PDTC or ATC.Retinoic acid, as the intermediate metabolite of vitamin A, is synthesized by sequential oxidation steps:after entering cytoplasm of cell, retinol is first converted to retinaldehyde by retinol dehydrogenase, which is then converted to retinoic acid by retinaldehyde dehydrogenases. Retinoic acid has different subtypes including all trans-retinoic acid (RA),9-cis retinoic acid and 13-cis retinoic acid. Wherein RA is clinically been used to promote differentiation for tumor-specific drug therapy. For example, RA can not only improve iodine uptake rate in some thyroid cancer, but also induce cell differentiation of tongue squamous cell carcinoma, and many evidence shows that RA have certain effect on many types of cancer, such as cervical cancer, skin cancer and renal cell carcinoma. However, it has been found that there are biological activity of RA only for special type of tumor cells, even different subtypes of the same tumor cells to retinoic acid sensitivity are not the same. The molecular mechanism has not been completely elucidated.The biological effects of RA accompany with transformation of tumor cells into differentiation and apoptosis and increased sensitivity of chemotherapeutic medicine. Liposolubility makes exogenous RA into cytoplasm through cell membrane. With the recently understanding on the RA signal pathways, it has found that there are two RA signal transduction pathways in cells, mediated by two different intracellular binding protein, cellular retinoic acid-binding pretein II (CRABP-II) and fat acid-binding protein (FABP5) transport RA to the nuclear receptor RARs or PPARβ/δ,respectively. Transcriptional activation of the nuclear receptor RARs by RA can induce cell apoptosis and inhibition of cell proliferation. In addition to functioning through RARs, RA can also activate another receptor peroxisome proliferators-activated receptor (PPAR), which promotes cell survival and hyperplasia.To our knowledge, so far there is no report about the status of two signal pathways of RA-CRABP-II-RAR and RA-FABP5-PPARβ/δ in thyroid carcinoma.It has been already proved that the development of cancer is related with multiple factors. Thus, the onefold principle can not meet the requirements about clinical cancer treatment. It has been of great significance to find effective drugs and rational regimen of medicine combination for increased sensitivity of tumor and reduce side effects and tumor tolerance produced by a single drug. In the past few years, epigenetic modification have been come to realize a key factor for regulate of gene transcription in carcinogenesis and development of tumor, so the impact of epigenetic drugs play an increasingly important role in academic research and clinical course of chemotherapy. Although there are variety mechanisms of epigenetic modification, DNA methylation is the most important and most widely studied.It is confirmed by many reports that demethylation drugs role can reverse tumor cell epigenetic regulation disorders, and effectively improve the sensitivity of tumour cells to chemical drugs. Decitabine (DAC) as the cytosine analogues, is a kind of demethylation drugs, has been approved by FDA for clinical treatment of myelodysplastic syndrome. There is existence of aberrant gene methylation in thyroid cancer as other malignancies, and our laboratory found that demethylation can be reversed CRABP-Ⅱ silence and make sensitivity to RA in some medulloblastoma cell lines.The aim of this thesis is to investigate the molecular mechanisms of RA signals in ATC. Therefore, RA relatively sensitive medulloblastoma cell lines MED3 cells as control cell. We observed the RA susceptibility in ATC and intracellular expression of retinoic acid binding protein in anaplastic thyroid cancer cell line KAT-18 and PDTC or ATC tissues. We analyzed the relations between the expression of retinoic acid signaling pathway components and RA sensitivity by molecular biology methods. By the application of demethylating agent decitabine alone or with RA, we explored whether it can alter the sensitivity of tumor on RA signal after change tumor cells epigenetic modification.Materials and Methods:Human anaplastic thyroid cancer cell lines KAT-18 was kindly provided by Professor Kenneth B. Ain, MD, Thyroid Cancer Molecular Endocrinology Research Laboratory, American Medical Center of University of Kentucky. Med-3 medulloblastoma cell line was kindly provided by the doctors in the Department of Neurosurgery, Kobe University School of Medicine, Japan. PDTC or ATC tissue samples were provided by Department of Pathology, the first affiliated hospital of Dalian Medical University and China Medical University; the normal control group was 20 cases of tumor adjacent tissues.The KAT-18 cells and the Med-3 medulloblastoma cells were cultured. Cells were routinely split every 3-4 days with trypsin-EDTA and harvested for subsequent experiments when closing to 80% of confluence. Thyroid cancer tissue microarrays were producted by conventional method.1) The chemo-sensitivities of human anaplastic thyroid cancer cell line KAT-18 and human medulloblastoma cell line Med-3 to RA were analyzed by HE staining, MTT and Hoechst staining for live cell.2) The transcriptional level of the retinoic acid signaling pathway components, CRABP-II, FABP5, nuclear receptors RARa and PPARβ/δ wewe detected in KAT-18 cells and Med-3 cells.3) The protein level of CRABP-II and FABP5, nuclear receptors RARa and PPARβ/δ were detected by Western blotting and ICC.4) The effect and cytotoxicity of rational concentration of decitabine were observed by microscope, HE staining, MTT and Hoechst staining for live cell.5) The retinoic acid sensitivity of KAT-18 cells was detected by HE staining and Hoechst staining for live cell after decitabine alone or with RA treatment.6) Posttreated by decitabine for 72 hours, KAT-18 cells were cultured with or without RA treatment for another 48 hours. The response of KAT-18 cells was analyzed by HE staining, Hoechst staining for live cell.7) RT-PCR was applied to detect the expression of CRABP-II, FABP5, RARa and PPARβ/δ after RA treatment, The groups of DAC or DAC combination with RA were also studied.8) By combination of human thyroid cancer tissue microarray and IHC, the expression of CRABP-II and FABP5 were analyzed in PDTC or ATC tissues. The experimental data were statistically analyzed by Prism 5 software (GraphPad Software, Inc., La Jolla, CA).Results:1. The study of retinoic acid signaling pathways state in PDTC or ATC tissuesThere are 29 cases of human thyroid carcinoma tissue in tissue microarrays which all specimen loss of normal structure and showed low differentiated or undifferentiated performance.1.1 The pattern of the expression of FABP5/CRABP-ⅡBased on the relative FABP5, CRABP-Ⅱ expression level, the expression of retinoic acid signaling pathway components can be divided into four types: FABP5(++)/CRABP-Ⅱ(+/-) expression pattern; FABP5(+)/CRABP-Ⅱ(+) equilibrium expression pattern; CRABP-Ⅱ(-)/FABP5(-) expression pattern; CRABP-Ⅱ(+)/FABP5 (-) expression pattern.1.2 The expression pattern of FABP5/CRABP-Ⅱ in PDTC or ATC tissuesIn this group of PDTC or ATC tissues, there was 37.9%(11/29) with FABP5(++)/ CRABP-Ⅱ(+/-) expression pattern; 41.4%(12/29) with FABP5(+)/CRABP-Ⅱ(+) equilibrium expression pattern; 6.9%(2/29) with CRABP-Ⅱ(-)/FABP5 (-) expression pattern; 13.8%(4/29) with CRABP-Ⅱ(+)/FABP5(-) expression pattern. The positive expression rate of FABP5 was 79.3%; the positive rate of CRABP-Ⅱ was65.5%.In 20 cases of normal thyroid tissue, the positive rate of CRABP-Ⅱ expression was 25%, FABP5 positive rate was 40%. There are no statistical significance of correlation between the expression of CRABP-Ⅱ and FABP5 in PDTC or ATC.2. The study on the effects of retinoic acid reaction with retinoic acid signaling pathways state in anaplastic thyroid cancer cell lines2.1. Morphological observation after RA treatment in anaplastic thyroid carcinoma KAT-18 cellsHE staining showed that the morphology and cell number of KAT-18 were no significant difference after treatment with 10μM RA,48h; KAT-18 cells were not sensitive to RA.2.2 MTT results and the mRNA level expression of CRABP-Ⅱ, FABP5, RARα and PPARβ/δ in KAT-18 cells after 10μM RA,48h treatmentThe MTT results were no significant difference between 1,5,10μM RA,48h treatment in KAT-18 cells. KAT-18 cells were not sensitive to RA, which is consistent with the results of HE staining. FABP5 expression was significantly stronger than CRABP-Ⅱ expression in mRNA level. The expression levels of CRABP-Ⅱ, FABP5, RARa and PPARβ/δ were almost no changes after 10μM RA,48h treatment.The MTT analysis showed there are no difference, compared with control group, in RA insensitive KAT-18 cells with FABP5(++)/CRABP-Ⅱ(+/-) expression pattern. However, MTT results between MED-3 cells with CRABP-Ⅱ(+)/FABP5(-) expression pattern and control group were significant difference (*, P<0.05).2.3 The protein expression of CRABP-Ⅱ, FABP5, RARa and PPARβ/δ in KAT-18 cells after 10μM RA,48h treatmentThe expression of FABP5 were almost no change, but the expression of CRABP-Ⅱ slightly increased after RA treatment by ICC. The expression of FABP5 was almost no change, but the expression of CRABP-Ⅱ slightly increased after RA treatment by western-blotting, those results were same as ICC. The expressions of RARa and PPARβ/δ were almost no change in protein level after RA treatment in KAT-18 cells.2.4 The expression of thyroid cancer differentiation markerWeak Tg and NIS expression was observed in control group and RA group in KAT-18 cells; however,Tg expression seem to be slightly enhanced after treated by 10μM RA.The expression of Tg and NIS ws clearly visible in normal thyroid tissue. 3. The study on the effects of retinoic acid response and retinoic acid signaling pathway status by decitabine combined with RA treatment in anaplastic thyroid cancer cell lines3.1 The observation of decitabine treatment in anaplastic thyroid cancer cell lines KAT-18HE staining showed that the morphology of KAT-18 cells slightly alter and the number of KAT-18 cells significantly reduced treatment by 25 μM DAC,72h; but the morphology of KAT-18 cells was no significant differences by 10μM RA,48h or 10μM DAC,72h+10μM RA,48h treatment.3.2 The results of Hoechst live cell stainingHoechst live cell staining showed that DAC alone or combined with RA treatment of KAT-18 cells by 10μM RA,48h; 5,10,25μM DAC,72h; 5,10,25μM DAC,72h+ 10μM RA,48h, the number of cells of DAC combined with RA groups in each view were slightly increased more than corresponding DAC alone groups. The number of cells of the DAC alone group (5,10,25 μM,72h) was significantly less than the control group (versus control group,*P<0.05).3.3 The mRNA level expression of retinoic acid signaling pathway componentsRT-PCR results showed that treatment by decitabine (5,10μM DAC,72h); DAC combined RA(10μM DAC,72h+10μM RA,48h) or 10μM RA,48h in KAT-18 cells, the expression of CRABP-II and FABP5 were no change in mRNA level after treatment; DAC or combined with RA treatment can not change expression ratio of FABP5/ CRABP-II in KAT-18 cells.Conclusion:1. In PDTC or ATC specimens,CRABP-II(+)/FABP5(-) expression pattern account for 13.8%, and the remaining 86.2% of the patients with anther CRABP-II/FABP5 expression patterns, which may not respond to RA signal. The 79.3% positive expression rate of FABP5 is significantly higher than that of normal thyroid tissues 40%; while 65.5% CRABP-II positive expression rate is significantly higher than that of 25% of normal thyroid tissues. There is no statistical significance of CRABP-II and FABP5 expression correlation in PDTC or ATC specimens.2. Human anaplastic thyroid cancer cell lines KAT-18 is not sensitive to RA. The expression of retinoic acid carrier is FABP5(++)/CRABP-II (+/-) expression pattern in KAT-18 cell.3. The treatment of RA can not change the expression of retinoic acid signaling pathway components in human anaplastic thyroid cancer cell lines KAT-18. The treatment of RA can not change low or no expression of Tg and NIS in human anaplastic thyroid cancer cell lines KAT-18.4. Demethylation agent decitabine can inhibit growth of human anaplastic thyroid cancer cell lines KAT-18. Decitabine alone or with RA can not change the retinoic acid sensitive and alter the ratio of FABP5/CRABP-Ⅱ expression in human anaplastic thyroid cancer cell lines KAT-18.5. When sequential treatment with decitabine and RA, the effects of combined with RA groups are less than decitabine alone groups, RA may have certain growth-promoting effect in KAT-18 cells. Therefore, it is apparent that the effects of RA-FABP5-PPARβ/δ proliferation signaling in RA-related therapeutic strategies on thyroid cancer should be taken into consideration.