Study Of Heat Shock Protein 90 On Proliferation And Iodine Uptake Ability Of Anaplastic Thyroid Cancer Cell

Background and purposeAnaplastic thyroid cancer (ATC) has both high malignancy and poor prognosis, and it has the potency of early infiltration in adjacent tissues and distant metastasis. Although external beam radiotherapy can temporarily remit the situation, easy relapse does occur. In addition, chemotherapy have little effect on ATC. It was reported that heat shock protein 90 (HSP90), as a molecular chaperone, plays an important role in tumor development and is becoming a new target for cancer therapy. 17-Allylamino-17-demethoxy geldanamycin (17-AAG) is a specific inhibitor of HSP90, which can block tumor survival signaling pathway in the body. This in vitro study was designed to investigate the effects of 17-AAG on proliferation and apoptosis of ATC cell, and to further analyze its possible mechanism.Sodium/iodide symporter (NIS), which resides on thyroid follicular cell membrane, promotes active transport of iodine. After transfection of NIS full DNA into ATC cells, NIS expression can be induced, and thus can promote tumor cell uptaking iodine. However, tumor cells can not organificate iodine, resulting in the rapid efflux of intracellular iodine. The purpose of this study is to understand the effect of 17-AAG on iodine uptake kinetics of the NIS-transfected ATC cells, and to provide experimental basis for combining genetic engineering technique and radionuclide therapy with the aim of clinical applications.Method1. Human ATC cell line FRO was cultured and passaged. In the 96 well plate, different concentrations (0.1 to 10μM) of 17-AAG were added, after incubation of 24, 48 and 72 hours, MTT assay was performed to measure the rate of growth inhibition of different concentration at different time points. 2. Different groups of FRO cells (incubated with 0.1μM, 1μM and 10μM of the 17-AAG for 48 hours) were collected. After DNA staining with PI, flow cytometry was performed to test the proportion of cells in different stages of cell cycle. After Annexin-V-FITC and PI double staining, flow cytometry was performed to detected apoptosis, then the proportion of apoptotic cells was calculated.3. Total protein was extracted from FRO cells which were treated with 1μM of 17-AAG for 48 hours. Then Western-blot was performed to examine the protein expressive level changes of HSP90, cancer proteins of pAkt, Raf-1 and etc. before and after 17-AAG treatment.4. Alkaline lysis method was used to extract the recombinant plasmid, namely pcDNA3.1-hNIS, which was then identified by restriction enzyme method.5. Lipofection method was used to transfect the above plasmid into FRO cells.24 hours after transfection, transfection efficiency was determined by transient 125I uptake ability test.6. G418 resistance selection to obtain a stable cell line hNIS-FRO.7. After introduction of 125I into the medium of hNIS-FRO cells,125I influx and efflux experiments were performed. Then time-radioactivity curve was drawn, and the results were compared with the untransfected cells.8.125I influx and efflux experiments were used to compare hNIS-FRO cells treated with 1μM 17-AAG for 24 hours and untreated control cells.Results1. After 24,48 and 72 hours treatment of different concentrations of 17-AAG, cell growth inhibition rates of ATC showed phenomenon of dose-dependency and time-dependency. Inhibition rates of 72 hours treatment of 0.1,0.2,0.5,1,2,5 and 10 μM of 17-AAG were 35.9%,40.8%,54.2%,63.9%,68.3%,73.6% and 78.1% respectively.2.48 hours after 17-AAG treatment, with the increase of drug concentration, G0/G1 phase cells increased, S phase and G2/M phase cells decreased, and cell cycle arrest showed dose-dependency. Control group cells and cells treated with 0.1,1 and 10μM of 17-AAG for 48 hours demonstrated significant difference in terms of apoptotic rate.3. Compared with the control group, after treatment of 1μM 17-AAG for 48 hours, FRO cells showed upregulate of HSP90 expression, yet cancer protein expressions of pAkt, Raf-1 were significantly reduced.4. Digestion products of recombinant plasmid showed two bands of 5502 bp and 1922 bp, which confirmed that the plasmid was indeed pcDNA3.1-hNIS.5.24 hours after pcDNA3.1-hNIS transfection into FRO cells,125I transient uptake ability increased 2.53 times in comparison with the control group.6. Final concentration after G418 resistance screening was determined to be 700μg/ml. Untransfected FRO showed no G418 resistant.7. After the introduction of 125I into the medium of hNIS-FRO cells, uptake ability increased significantly. At 60 minute time point, the 125I uptake rate was about 11.54 times of the control group. However, when 125I was removed from the medium,125I rapidly effluxed. At 30 minute time point, retention rate of 125I in the hNIS-FRO cells was only 9.32% of the initial amount.8. In hNIS-FRO cells,24 hours after the treatment of 1μM 17-AAG,125I was introduced into the medium. During the time course of 20 to 60 minutes,17-AAG treated hNIS-FRO cells demonstrated increased uptake abilities of different extents. 20 to 60 minutes after 125I was removed from the medium, retention rate of 125I in the hNIS-FRO cells was significantly increased as compared with the control group, and 125I efflux was reduced.30 minutes after the treatment of 17-AAG,125I retention rate was 32.69%,3.51 times of the control group.Conclusion1.17-AAG can inhibit the proliferation of ATC cells, and this inhibition showed dose-dependency and time-dependency.2. After the treatment of 17-AAG, G0/G1 phase ATC cells increased and the apoptotic rate increased, these effects also demonstrated dose-dependency. This confirmed that 17-AAG can not only inhibit tumor cell proliferation, but can also induce apoptosis.3. After the treatment of 17-AAG, HSP90 expression was upregulated, yet cancer proteins pAkt, Raf-1 were downregulated. This confirmed that the anti-proliferation effect of p17-AAG may be related with cell cycle arrest in G0/G1 phase and blockage of the signaling pathways in the tumor cells.4. Stable NIS-transfected ATC cells with greatly increased uptake ability of iodine can be obtained, which was completely dependent on NIS gene expression. However, this method can not overcome the limitation of rapid efflux of the uptaked iodine in tumor cells.5. The iodine uptake ability can be enhanced in NIS-transfected ATC cells after the treatment of 17-AAG, and more importantly iodine efflux can be significantly delayed. As a result, intracellular iodine retention rate increased.