Effects Of Diosgenin On Cell Proliferation Induced By IGF-1 In Primary Throcytes

Background:Graves' disease (GD) is a kind of common autoimmune thyroid disease. Diffuse thyroid goiter is one of the characteristic features of GD. The bigger the thyroid volumes are, the longer the period of medical treatment for GD is. So it is very important to study the mechanism of thyroid goiter formation to find effective therapeutic targets for this disease.Previous studies also showed that growth factors, especially insulin-like growth factor-1 (IGF-1), plays important roles in thyroid cell growth and human thyroid diseases. IGF-1 is an important hypertrophic factor for thyroid cells. It regulates cell proliferation and a vast variety of differentiated cell functions. Our previous research showed that in patients with untreated GD, there was a significant increase in serum IGF-1 levels, and localized IGF-1 expression in thyroid tissues was higher in those patients compared with healthy subjects. Meanwhile, thyroid volumes were positively correlated with IGF-1 levels in untreated patients. In addition, IGF-1 promoted cell cycle progression and up-regulated cyclin Dl expression by stimulating the PI3K/ NF-?B pathway in thyroid cells. These findings indicated that IGF-1 plays a key role in the formation of thyroid goiters.Diosgenin is a steroidal sapogenin belonging to the group of triterpenes. It is found in several plants including Dioscorea species (yams), fenugreek and Costus speciosus. So far the benefits of diosgenin were limited to preclinical studies demonstrating efficacy against skin aging, hyperglycemia, hypercholesterolemia, and hypertriacylglycerolemia. A recent study has demonstrated that diosgenin controlled hypercholesterolemia in rats fed a high-cholesterol supplemented diet by improving the lipid profile as well as by modulating oxidative stress. An interesting biological activity of diosgenin was demonstrated by Turchan-Cholewo who concluded that diosgenin might have potential therapeutic effect against an increased risk of developing dementia in opiate abusers with HIV infection. All these studies showed that diosgenin had various biologic activities, but the effects of diosgenin on human primary thyrocytes have never been reported.The role of cyclins in proliferative thyroid diseases also get great attention. Cyclin D1 is a G1 phase cell cycle protein. Accumulation of this kind of protein by stimulation of extracellular signals promotes cell proliferation. Cyclin D1 can be combined with CDK4 and CDK6, which can make Rb phosphorylated and progress G1 phase to S phase. It has been reported that in thyroid papillary carcinomas, the expression of cyclin D1 was elevated, which was thought to be a contributory factor to thyroid tumorigenesis. While cyclin B1 regulates a G2 checkpoint and promotes cell entry into M phase. Cyclin B1 is produced in S phase and located in cytoplasma in G2 phase. Cyclin B1 can be combined with p34cdc2 to Mitosis-Promoting Factor(MPF), which promotes cell entry into M phase. But in normal thyroid cells, informations regarding the relationship between cyclin D1?cyclin B1 induced by IGF-1 and cell proliferation are uncertain.In addition, IGF-1 may play a important role in Graves'disease. The aim of the present study was to evaluate the possible effects of diosgenin on cell proliferation induced by IGF-1 in primary human thyroid cells. And this makes a possible alternative therapy for goiter patients.Objectives:1. The effects of IGF-1 in different concentration on cell proliferation in primary human thyroid cells.2. The effects of diosgenin on cell proliferation induced by IGF-1 in primary human thyroid cells. 3. IGF-1 and diosgenin possibly control the cyclin D1 and cyclin B1 protein expression to affect the cell progress.Methods:1. Cell culture:The specimens were obtained from normal thyroid tissue of euthyroid patients operated on for benign follicular nodules. The tissue was incubated with Ca2+- and Mg2+-free Hanks' salt solution containing type I collagenase, trypsin (0.25%),0.75 mg/ml heat-inactivated dialyzed chicken serum, in a shaking water bath at 37?for 40-60 minutes. The supernatants were collected, combined, and washed in DMEM/F12 culture medium for 2-3 times, then centrifuged. Thyrocytes were suspended in DMEM/F12 culture medium containing 10% newborn calf serum (NBS), TSH (Thyroid Stimulating Hormone,2 mU/ml), penicillin-streptomycin (100 U/ml), fungizone (2?g/ml). Cells were plated in Falcon 25-cm2 tissue culture flasks for primary culture with DMEM/F12 medium and incubated at 37?in humidified air atmosphere containing 5% CO2. After 24 h the supernatant containing no adherent cells was removed. The primary thyroid cells formed a confluent monolayer within 3-5 d; and the cells were treated when they reached 70-80% confluence.2. Identification of primary huaman thyroid cells:Primary human thyrocytes were seeded on a glass slide. The cells were assessed by immunofluorescence. Nuclei were stained by DAPI, and membranes were specially stained by anti-TSHR antibody.3. Cell proliferation assay:The effects of IGF-1 and diosgenin on cell proliferation were measured by 3-(4,5-dimefhylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and 5-ethynyl-20-deoxyuridine (EdU) incorporation assay using a MTT cell proliferation assay kit and EdU assay kit, respectively.4. Cell cycle analysis was performed by using FACS:Briefly, after starvation overnight, IGF-1 was added and treated for 24h,then diosgenin was added and treated for another 24h. Cells harvested by trypsinization were washed with PBS and stained with propidium iodide (PI) using Coulter DNA PREPTM Reagents kit. Samples were analyzed by FACS on a Coulter Elite ESP using standard filter sets. Results were expressed graphically and quantified as the fraction of cells in Go/G1, S, or G2/M phase using Coulter cytologic software. 5. The protein expression of cyclin D1 and cyclin B1 was determined by Western blot analysis:After treatment cells were harvested, centrifuged, washed with PBS and lysed for 20 min on ice in RIPA buffer containing 1×PBS,1% NP-40,0.1% SDS,5 mM EDTA,0.5% sodium dexyccholate,1 mM sodium orthovanadate, 1mM PMSF. Cells were then centrifugated at 12000 rpm for 10 min at 4?. The resulting supernatants (whole cell lysates) were collected and frozen at-80?or used immediately. Protein concentrations were determined by BCA protein assay (Pierce, USA).40?g of each sample was heated for 30 min at 60?, then analyzed by 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted onto nitrocellulose membranes. Membranes were block-ed in 5% nonfat milk for 1 h and then incubated with the specific primary antibody overnight at 4?, washed and incubated with appropriate horseradish peroxidase conjugated secondary antibody. After that, immune complexes were detected using the ECL method, and immunoreactive bands were quantified using Alphalmager 2200. Values were corrected with the absorbency of the internal control (?-actin).Results:1. ImmunofluorescencePrimary human thyrocytes were identified by expression of TSH receptor using immunofluorescence. Nuclei were stained by DAPI, and membranes were specially stained by anti-TSHR antibody. The immunofluorescent photograph confirmed that the monolayer, irregular phenotype cells which aggregated to islands were human thyrocytes.2. Effects of diosgenin and IGF-1 on cell viability (MTT)We found that 100 ng/ml IGF-1 significantly increased cell viability (p< 0.001). However, there was no dose-dependent or time-dependent effect of IGF-1 on cell viability in primary human thyrocytes.20?mol/L diosgenin began to markedly inhibit cell viability (p< 0.01). And there were dose-dependent and time-dependent effects of diosgenin on cell viability. Importantly, when human thyrocytes were exposed to diosgenin in the present of IGF-1, the inhibition effect of diosgenin on the cell viability was significant (p< 0.01) and presented a dose-and time-dependent effect. 3. Effects of diosgenin and IGF-1 on cell proliferation (EdU)We found that the number of EdU+ cells (EdU-labeled replicating cells) was increased after treated with IGF-1, while the number was significantly reduced in diosgenin treatment group. More importantly, the number of EdU+ cells was also significantly decreased after treated with both diosgenin and IGF-1, compared with that of the control.4. Effects of diosgenin and IGF-1 on cell cycle (FACS);Compared with the control group, the proportion of cells treated by IGF-1 in G0/G1 phase was decreased, and the proportion in the S phase was increased. However, the proportion in G2/M phase was not changed. When the cells were exposed to 25?mol/L diosgenin and IGF-1100 ng/ml together, the proportion of the cells in G0/G1 phase was increased, while S phase decreased. And the proportion in G2/M phase was still not changed.5. Effects of diosgenin and IGF-1 on cyclin D1 and cyclin B1 expressionThe results showed that IGF-1 treatment for 6 h and 12 h increased the cyclin D1 protein level. However, after 24 h treatment with IGF-1, the cyclin D1 protein level began to decrease. Consequently, the cyclin D1 protein level was markedly decreased after 48 h treatment compared with the control. On the other hand, Diosgenin treatment for 6 h alone had no apparent effects on cyclin D1 protein expression, whereas diosgenin treatment for 12 h and 48 h decreased the cyclin D1 protein expression. Interestingly, diosgenin treatment in the present of IGF-1 could dramatically decrease the cyclin Dl expression. However, the cyclin B1 protein level was not significantly changed in each group.Conclusions:In summary, this study showed that IGF-1 enhanced proliferation and promoted cell cycle progression to S, whereas diosgenin reduced cell proliferation induced by IGF-1 and rendered G0/G1 arrest by decreasing cyclin D1 expression in primary human thyrocytes. The effect of diosgenin on cell proliferation in present of IGF-1 in primary human thyrocytes was much more similar to that of diosgenin alone, and this implicated that diosgenin and IGF-1 might have a same signal pathway on proliferation and cell cycle in primary human thyrocytes.