Diosgenin Induces Apoptosis In Igf-1Stimulated Human Thyrocytes Through Two Caspase-Dependent Pathways

Introduction:Graves'disease (GD) is an autoimmune disorder that is caused by circulating anti-thyroid-stimulating hormone receptor autoantibodies (TRAb) and characterized by hyperthyroidism and diffuse goiter. Numerous studies have indicated that the prognosis of GD is closely related to the extent of the thyroid goiter:GD patients with large goiter have low rates of remission and high relapse rates.The imbalance between the proliferation and apoptosis of thyrocytes is a major cause of goiter formation in GD. Many study hanve demonstrated that the expression of anti-apoptotic protein such as BCL-2, FLIP increased and the proapoptotic protein such as FAS, FASL, Bax decreased in thyroid of GD patient. Insulin-like growth factor-1(IGF-1) is a well-known hypertrophic and antiapoptotic factor for a number of cell types, including thyrocytes. Recently, it has been demonstrated that the expressions of IGF-1and its receptor are increased in the hyperplastic thyroid tissues of GD patients. Our previous study showed that the serum level of IGF-1was positively correlated with the size of the goiter in GD patients. These findings indicate an important role for IGF-1in the formation of goiters associated with GD. Further investigations into the mechanisms of action of IGF-1have shown that IGF-1prompted thyrocyte proliferation via upregulation of cyclin D and inhibited thyrocyte apoptosis via upregulation of Fas-associated death domain-like interleukin-1-converting enzyme (FLICE)-like inhibitory protein (FLIP), a apoptosis-blocking protein. Three therapies including antithyroid drugs, radioiodine and thyroidectomy were used for GD treatment. However, the remission rate of GD is low and there are many side effects in the therapies. It is important to study the mechanism of goiter in GD and find a new therapy for the goiter treatment in GD.Diosgenin, a steroid sapogenin existing in some natural plants such as Rhizoma Dioscoreae Nippponicae, Dioscorea species (yams) and Fenugreek, is often used to produce steroidal hormones as raw material in pharmaceutical industry. In recent years, the antitumor effect of diosgenin has been further studied via its mutiple pathways in many cell lines. Diosgenin inhibited proliferation and induced apoptosis through decreasing the expression of3-hydroxy-3-methylglutaryl Coenzyme A (HMG-CoA) reductase in HCT-116human colon carcinoma cells, reregulating Ca2+homeostasis and mitochondrial apoptotic factor in human leukemia K562cells, and suppressing fatty acid synthase expression and modulating Akt, mTOR and JNK phosphorylation in HER2-overexpressing breast cancer cells. Moreover, diosgenin played its apoptotic role not only in tumor cell lines but also in hyperplastic normal cell lines such as human rheumatoid arthritis fibroblast-like synoviocytes (FLS). Yet the proapoptotic effect of diosgenin has not been focused on thyroid diseases.As we have known, apoptosis is a programmed cell death process, which is controled by extracellular and intracellular cell signal pathways. In extracellular cell signal way, the combination of death receptor and death receptor ligand results in the formation of the death-inducing signaling complex (DISC), which contains the Fas-associated death domain protein (FADD) and caspase-8, and then caspase-8is self-activated. FLIP can block the activation of caspase-8via competitively binding with FADD and preventing DISC formation. In intracellular pathway, apoptotic factors dissipate the membrane potential of mitochondria (MMP) and cytochrome c is released from mitochondria. Once cytochrome c is released, it binds with apoptotic protease activating factor-1(Apaf-1) and ATP, which then bind to caspase-9to create a protein complex known as an apoptosome. The apoptosome cleaves the caspase-9to its active form. Bcl-2family proteins, such as Bcl-2and Bax, influence mitochondria apoptosis by changing the MMP. The activated caspase-8and caspase-9finally activates the dead effector caspase-3.In a word, the imbalance of proliferation and apoptosis in thyrocytes make an important role in goiter information. In this work, we determined the effects of diosgenin on thyrocyte proliferation and apoptosis in the presence of IGF-1. Our results showed that diosgenin induced the apoptosis of human thyrocytes. The probable mechanisms may involve the downregulation of FLIP expression, the induction of ROS production and the modulation of the Bcl-2/Bax ratio, which can collectively lead to the activation of both the intrinsic and extrinsic caspase-dependent pathways. These findings suggest that diosgenin may be useful treatment of goiter associated with GD.Objective:1. The antiproliferative and apoptotic effect of diosgenin in IGF-1-stimulated human thyrocytes were detected through MTT, fluorescence staining and flow cytometry.2. The proapoptotic mechanisms of diosgenin in IGF-1-stimulated human thyrocytes were detected by western blot and ROS.Method:1. Primary human thyrocytes isolation and cultureNormal thyroid tissue was obtained as surgical waste from the distal end of thyroid from patients who had undergone subtotal thyroidectomy due to nodular goiter or thyroid adenoma at Shandong Provincial Hospital. Each normal specimen was confirmed by a pathologist. In brief, fresh thyroid tissue was divided and minced into small pieces (1mm3). Following multiple washes with D-Hanks'solution, the tissue was digested using0.25%collagenase I (Sigma) and0.125%trypsin (Sigma) at37℃in a water bath for40-60min. The mixture was filtered through a metallic mesh and centrifuged. Thyroid cells were collected and cultured in DMEM/F12K (1:1) complete medium including10%newborn calf serum (NBS, Sigma), TSH (Thyroid Stimulating Hormone,2mU/mL, Sigma), penicillin-streptomycin (Sigma). Cells were incubated in5%CO2atmosphere at37℃2. Cell treatment and groupingAfter3-5days when primary thyrocyte monolayer reached70-80%confluence, the complete medium was replaced with starved medium DMEM/F12K (1:1) supplemented with0.2%NBS, and no TSH for24hours. Then the cells were pretreated with or without IGF-1(100ng/ml, sigma) for24h and then supplemented with different concentrations of diosgenin for indicated times.3. Method(1) The antiproliferative and apoptotic effect were detected by MTT, fluorescence staining and flow cytometry.(2) The expression of apoptotic signaling proteins were detected by western blot.(3) The generation of ROS was detected by flow cytometry.Result:1. Diosgenin inhibits the proliferation of thyrocytes induced by IGF-1100ng/ml IGF-1prompted the proliferation of thyrocytes significantly and was about1.6-fold of the group without IGF-1(p<0.05). Diosgenin inhibited the proliferation of thyrocytes induced by IGF-1in a dose-and time-dependent manner (p<0.05).2. The morphological change of apoptosis induced by diosgenin in IGF-1-treated primary human thyrocytesAfter staining with Hoechst33342and PI, most control thyrocytes showed normal nuclear morphology lightly stained with Hoechst33342(blue) and fewer apoptotic cells with condensation of nuclei. The thyrocytes pretreated with IGF-1displayed increased cell numbers and less apoptosis compared with the control. When the cells were exposed to20μmol/1diosgenin, partial cells revealed apoptotic morphology with shrinkage and condensation of nuclei. The early apoptotic cells were deeply stained with Hoechst33342only and the later were double stained with Hoechst33342and PI. Cells treated with40μmol/1diosgenin revealed not only apoptotic cells, but also necrotic cells which were stained with PI only. The morphology change of thyrocytes induced by diosgenin was dose dependent.3. Diosgenin elevates apoptotic proportion in IGF-1-treated primary human thyrocytes Our results showed that the total proportions of early and late apoptosis (annexin V-positive/PI-negative and annexin V-positive/PI-positive cells) in IGF-1pretreated cells was lower than that in the control group with the ratio from6.70%to7.53%(p<0.05). Yet, cells treated with20μmol/1diosgenin displayed that the apoptotic rate was significantly enhanced to27.83%compared with IGF-1pretreated cells (p<0.05). Moreover,40μmol/1diosgenin treated thyrocytes presented a dose dependent apoptotic effect with the apoptotic rate increasing to34.41%(p<0.05).4. Diosgenin activated caspase-8and-9as well as caspase-3in IGF-1-treated primary human thyrocytesTo detect whether the apoptotic effect of diosgenin is through activation of caspase cascades, we assayed the protein level of caspase-8, caspase-9and caspase-3by western blot. The untreated thyrocytes express full form of caspase-8, caspase-9and caspase-3and revealed the active forms of them weakly. After pretreated with IGF-1, the ultimately active forms of caspase-8, caspase-9and caspase-3were decreased compared with the untreated thyrocytes, indicating that IGF-1prohibiting the activation of caspase cascades. Upon treatment with diosgenin (20and40μmol/1), caspase-8, caspase-9and caspase-3were completely cleaved to their final fragments (18kd,17kd and17kd) with dose dependent.5. Diosgenin downregulated the expression of FLIP protein through inhibting phosphatidylinositol3-kinase (PI3K) signal pathway in primary human thyrocytes exposed to IGF-1To evaluate the role of PI3K cell signal way in diosgenin induced apoptosis, we used a specific phosphatidylinositol3-kinase (PI3K) inhibitor, LY294002. Tyrocytes were preincubated with10ng/ml LY294002or not for1hour, then, exposed to IGF-1or not for24hour, and then treated with40μmol/1diosgenin for48h. Protein level of FLIP and caspase-8were assayed by western blot. We observed that IGF-1increased the basal expression of FLIP, while decreased the protein level of cleaved caspase-8. By pretreating with LY294002for lh, the effects of IGF-1were blocked with the degradation of FLIP and the increase of active caspase-8. After treatment with diosgenin, the protein level of FLIP were also decreased in IGF-1preincubated thyrocytes and the trend was similar to LY29004treatment. Furthermore, Cells combined with LY294002and diosgenin showed stronger inhibiton of FLIP than their treatment respectively. Diosgenin activated caspase-8and forming a18kd active fragment.6. Diosgenin regulated the balance of bcl-2and bax and activated caspase9Caspase-9, bcl-2, and bax were assayed by western blot. Our results showed IGF-1inhibited the activation of caspase-9and decreased the terminal cleavage of caspase-9campared with the control group. Following treatment with diosgenin, the full length caspase-9was cleaved into its terminal form (P17). Bcl-2and Bax were a couple of antagonistic factor in mitochondrial passageway. By pretreatment with IGF-1, the antiapoptotic factor bcl-2was increased while the proapoptotic protein bax was decreased. In contrast, after exposing to diosgenin for48h, the level of bcl-2was notablely lowered and the level of bax was elevated.7. Diosgenin activated ROSThe generation of ROS was detected by flow cytometry. The amount of ROS was lower in100ng/ml IGF-1pretreated thyrocytes than in untreated cells. In contrast, following treatment with40μmol/1diosgenin, the generation of ROS was enhanced compared with IGF-1incubated cells. In a word, diosgenin prompted mitochondria apoptosis by generating ROS.Conclusin:1. Diosgenin induces apoptosis in IGF-1-stimulated human thyrocytes.2. Diosgenin activated the death performer caspase3.3.Diosgenin downregulation expression FLIP proteins through inhibting phosphatidylinositol3-kinase (PI3K) signal pathway4. Diosgenin regulated the balance of bcl-2and bax and activated caspase9.5. diosgenin prompted mitochondria apoptosis by generating ROS.