Shikonin Induced Medullary Thyroid Carcinoma Tt Cell Apoptosis And Autophagy Experimental Research

Objective:The purposes of this study were evaluating the shikonin on human thyroid carcinoma TT cell apoptosis, and researching the mitochondrial signal transduction pathway for shikonin inducing human thyroid carcinoma TT cell during apoptosis by the changes of mitochondrial membrane potential (MMP) in vitro.Methods:①Applying the MTT method to measure the cell proliferation inhibited by shikonin on human thyroid carcinoma TT cell;②Applying flow cytometry to test cell apoptosis of human thyroid carcinoma TT cell induced by shikonin;③Testing the cell apoptosis by flow cytometry with Annexin V-PE conjugated with7-AAD staining.④Observing the morphological features of cell apoptosis by electron microscope.⑤Testing the mitochondrial membrane potential changes by flow cytometry;⑥Analyzing the influence of shikonin on the expression of Bcl-2family proteins and enzyme caspases expression by Western-blot;⑦Analyzing the influence of shikonin on the expression of Bcl-2family proteins and enzyme caspases expression by RT-PCR.Results:①shikonin inhibited the proliferation of TT cell in different concentrations for6-48hours treatment in a dose-and time-dependent manner(P<0.05)②The TT cell apoptosis flow cytometry image of control and shikonin treated for24hours. After treated with shikonin2μg/ml and4μg/ml for24hours, there were appearance sub-diploid peaks. The cell treated with shikonin over2μg/ml would induce typical appearance change--the apoptosis peak (sub-diploid peak), and increased in a dose-and time-dependent manner and significant in statistics compared to the control group(P<0.01). But there was no cell cycles arrest.③In order to further proving the sub-diploid peak was apoptosis peak, we used Annexin V-PE conjugated with7-AAD staining evaluating cell apoptosis. Compared with control group, when TT cell treated with2μg shikonin for24hours, the number of early AnnexinV-PE+/7-AAD-(apoptosis cell) increased from0.1%to8.03%. The proportion of AnnexinV-PE +/7-AAD+(necrotic cells) was increased from0.1%to11.33%in control group. After treated with shikonin for48hours, the number of AnnexinV-PE+/7-AAD-(apoptotic cells) increased24.99%; the proportion of AnnexinV-PE+/7-AAD+(necrotic cells) was increased14.19%. After treated with shikonin for72hours, the number of AnnexinV-PE+/7-AAD-(apoptotic cells) increased6.1%; the proportion of AnnexinV-PE+/7-AAD+(necrotic cells) was increased68.73%.The difference between groups are significant (P<0.01);④Electron microscope showed tipical morphological changes characteristics of apoptosis treated by shikonin, including cell shrinkage, disappearance of microvilli, chromatin condensation containing a half-moon of condensed chromatin and appearance of membrance blebbing and many apoptotic bodies.⑤Mitochondrial membrane potential induced by shikonin is decreased in a dose-dependent manner.⑥Abnormal mitochondria membrane swelling and rupture, were observed by electron microscopy.⑦Western blotting and RT-PCR showed that shikonin influenced the expression of Bax and Bcl-2. The data also showed that shikonin changed the levels of Bax and Bcl-2expression, suggesting a profound increase of Bax protein and decrease of Bcl-2protein in a dose-dependent manner. When the TT cell treated with2μg/ml and4μg/ml shikonin, we found that caspase-3and caspase-9were activated by shikonin in a dose-dependent manner.Conclusions:Shikonin is able to inhibit the proliferation of human thyroid carcinoma TT cell, it's dual dependence between inhibition dose and time. The shikonin over2μg/ml can induce the apoptosis of TT cell, and the apoptosis and necrosis rate increased by drug concentration. The main reason is inducing the necrosis of TT cell. Processing TT cell by2μg/ml, the typical form of necrosis cell can be seen by transmission electron microscope:the cells are shrinking and crimping, the cytomembrane are complete, the karyon getting small or collapse, then dispersion in endochylema; the chromatin are concentrated and marginalized, looking like crescent karyon, and the apoptosis bodies being seen. The mechanism of shikonin-induced apoptosis through the mitochondrial pathway could be:shikonin can enhance pro-apoptotic Bcl-2family protein-bax protein, reduce the anti-apoptosis protein Bcl-2,thus the ratio of Bax/Bcl-2ratio was increased,which caused a decrease of mitochondrial membrane potential, subsequent activating caspase-9and caspase-3, led to cell apoptosis in the end. Objective:To evaluate the influence of shikonin on human medullary thyroid carcinoma TT cells autophagy in vitro.Methods:①Observing the morphological features of cell autophagy by electron microscope;②Analyzing the influence of shikonin on the autophagy markers expression of Beclin1and LC3proteins;③Analyzing the LC3-Ⅱ points focal by fluorescence microscope, and find out the incidence of autophagy.Results:①Electron microscope found that autophagy changed TT cell when it treated with4μg/ml shikonin. The TT cell showed typical autophagy cyst with double round or oval shape structures.②Compared with control group, the Beclin1gene expression was significantly lower in shikonin treated group, and the expression of p62decreased in a dose-dependent manner. The expression of autophagy marker protein LC3-Ⅱ increased.③When the TT cell trasinfected with GFP-LC3plasmid for24hours, and the treated with shikonin for24hours, there were a large amounts of LC3-Ⅱ confocal points in shikonin treated groups by fluorescence microscope.Conclusions:Shikonin was able to induce human medullary thyroid carcinoma TT cells autophagy. We could inspect autophagosome by electron microscope. The autophagy marker protein LC3-Ⅱ could be inspected by fluorescence microscope. Shikonin could induce TT cells autophagy, and it may be related with the decrease expression of autophagy suppressor gene Beclin1.