Network pharmacology-based investigation of potential mechanism of Triptolide against Thyroid Cancer

Network pharmacology-based investigation of potential mechanism of Triptolide against Thyroid Cancer | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Network pharmacology-based investigation of potential mechanism of Triptolide against Thyroid Cancer Bing Chen, Qian Shen, Jie Shen, Hongyang Xie, Yiping Wang, Hongkan Lou This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4779748/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Objective This study aimed to explore the mechanism of triptolide (TPL) in the treatment of thyroid cancer (TC). Methods The targets of TPL in TC were collected from databases. A protein-protein interaction network was constructed using the common targets of TPL and TC. Enrichment analysis was performed using the DAVID database. The effects of TPL on cell activity, apoptosis, and cell cycle were assessed using CCK-8 assay and flow cytometry. Western blot analysis was conducted to measure the levels of caspase 3, caspase 7, PCNA, ki67, cleaved caspase 7, cleaved caspase 3, and p-P53. RT-PCR was used to measure TP53 mRNA levels. Results The protein-protein interaction network revealed 8 potential targets for TPL in TC treatment. Enrichment analysis indicated that TPL mainly involved in cell apoptosis, proliferation, and inflammation response. In vitro studies showed that TPL inhibited K1 cell activity, down-regulated PCNA and ki67 levels, and up-regulated caspase 3, caspase 7, cleaved caspase 3, cleaved caspase 7, p-P53 protein expressions, and TP53 mRNA levels. TPL also promoted K1 cell apoptosis and arrested cell cycle at the G2/M and S phase. Conclusion TPL exhibits antitumor effects on K1 cells by inhibiting cell proliferation, inducing cell apoptosis, and disrupting cell cycle. Triptolide Thyroid cancer Cell apoptosis Cell proliferation TP53 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1 Introduction Thyroid cancer (TC), a kind of endocrine malignancy and ranked ninth among the newly diagnosed cancers cases in 2020, could be induced by sex, a diet low in iodine, smoking, and exposure to radiation (Myung et al., 2017 ; Niciporuka et al., 2021 ; Silva, 2021 ). According to the report, China (11016 in 1990 and 41511 in 2017), America (10833 in 1990 and 25896 in 2017) and India (7369 in 1990 and 25 675 in 2017) were the top three countries which have the highest incident cases of TC, and South Asia (3630 in 1990 and 8930 in 2017) had the largest numbers of TC deaths (Deng et al., 2020 ). Based on TC morphological variations, it could be classified as four types and papillary thyroid carcinoma (PTC) and follicular thyroid carcinoma (FTC) represent 90–95% (Sarkar and Li, 2009 ). Although surgical resection is the common therapy for the most TC patients and the new therapies including thyroid hormone therapy, targeted drug therapy, etc are constantly emerging, but effective treatments are still lacking (Haugen et al., 2016 ; Laha et al., 2020 ). Therefore, it is necessary to find novel therapeutic method for TC. Triptolide (TPL), a diterpenoid epoxide, is extracted from Tripterygium wilfordii , with the biological activity of anti-inflammatory, immunosuppressive and anti-cancer (Zhao et al., 2020a ). Especially, TPL is known as the new star for treating human malignancies, such as lung cancer, pancreatic cancer, and thyroid cancer (Yan and Sun, 2018 ). Its anti- neoplastic properties have been proposed for the anti-proliferative and pro-apoptotic activities (Zhu et al., 2010 ). TPL suppresses invasion of TC have been demonstrated, but it’s mechanism of TC is reminded to be explored. In this study, we used network pharmacology to find the potential target and pathway about TPL for TC treatment and further verified the results via experiments, which aimed to find a new direction for TC treatment. 2 Methods 2.1 The collection of TPL and TC targets Firstly, the targets of TPL (MOL003187) were collected from TCMSP ( https://tcmsp-e.com/tcmsp.php ). The targets of TC were obtained from Gene Cards database ( https://www.genecards.org/ ), OMIM database ( https://omim.org/ ) and TTD ( http://db.idrblab.net/ ) database, and removed the duplicate targets. Using Venn map to present the common targets of TPL and TC. 2.2 Protein-protein (PPI) interaction network analysis The targets mentioned above were uploaded to STRING database ( https://string-db.org ) with the limited conditions (Species: Homo sapiens; medium confidence:0.4000). Then the PPI network was visualized using Cytoscape software 3.6.0. 2.3 Go and KEGG enrichment performance The common targets were uploaded to DAVIDA database ( https://davidd.ncifcrf.gov ) by setting the conditions (Species: Homo sapiens; Identifier: Official Gene Symbol; List type: Gene List;). Gene Ontology (GO) including molecular function (MF), cellular component (CC), biological process (BP) and Kyoto Encyclopedia of Genes and Genomes (KEGG) information were collected. Th top 10 items of them were showed as the histogram and bubble chart, respectively. 2.4 Cell and regents K1 human thyroid cancer cells (K1) were obtained from Shanghai Zyyan Biotechnology Co., LTD. Triptolide was bought from Yuanye (B20709, Shanghai, China). Dulbecco’s modified Eagle’s medium (DMEM) was obtained from Corning (10-013-CVRC, Shanghai, China). CCK-8 was bought from APExBIO (K1018, Houston, US). The antibodies of caspase 3 (bsm-33284M), caspase 7 (bsm-60304R), PCNA (bs-2006R), ki67 (bs-23102R) were obtained from Bioss (Beijing, China). The antibodies of cleaved caspase 7 (Ab256469), cleaved caspase 3 (ab2302) were bought from abcam (Shanghai, China). HRP-labeled Goat Anti-Rabbit IgG(H + L) (A0208) and Annexin V-FITC Apoptosis Detection Kit (C1062M) and Cell Cycle and Apoptosis Analysis Kit (C1052) were got from Beyotime (Shanghai, China). 2×Hieff Robust PCR Master Mix, HifairⅢ 1st Strand cDNA Synthesis SuperMix for qRCR, Hieff qPCR SYBR Green Master Mix were bought from Yi Sheng Biotechnology Co., LTD. 2.5 Cell culture K1 cells were cultured with DEME medium (containing 5% fetal bovine serum, 1% Penicillin - Streptomycin - amphotericin B- solution) under a 5% CO 2 atmosphere at 37°C. K1 cells were resuspended in DEME medium (containing 5% fetal bovine serum) and seeded in 24 well plates at the density of 4×10 5 cells/well. 2.6 Cell treatment and CCK-8 assay K1 cells were seed in 96 well plates at the density of 1×10 4 cells/well under a 5% CO 2 atmosphere at 37°C until the cell density reached 70%-80% confluence. The cells were treated with TPL with different concentrations (0, 5, 10, 25, 100, 500 nM) for 24 hours. Next, the cells were added into 10 µL CCK-8 and incubated for another 2 hours. Finally, the OD value was detected under the wavelength of 450 nm by microplate reader (Infinite E Plex, Tecan). 2.7 Western blot The protein of cells was extracted by adding radio immunoprecipitation assay (RIPA) lysis buffer and its concentrations were detected by BCA protein assay kit. The proteins were separated by 10% sodium dodecyl sulfate (SDS) polyacrylamide gels and then transferred into polyvinylidene fluoride (PVDF) membranes. Next, the membranes were blocked by 5% skim milk for 2 hours and incubated with primary antibodies of caspase 3 (1:1000), caspase 7 (1:500), PCNA (1:1000), ki67 (1:1000), cleaved caspase 7 (1:500) and cleaved caspase 3 (1:500) at 4℃overnight. Next day, the membranes were washed by TBST for 3 times and incubated with HRP-labeled Goat Anti-Rabbit IgG(H + L) for 2 hours. Finally, the blots were visualized using the ECL chemiluminescent substrate reagent by chemiluminescence imager (ChemiDoc XRS+, Bio-Rad). The bands were qualified by Image J software. 2.8 Quantitative Real-time PCR Total mRNA was extracted by TRIzol® reagent and reversed complementary DNA (cDNA) synthesis with the Reverse Transcription system. The cDNA samples were diluted to 100 µl with RNase-free water and 1 µl cDNA mixture was used for qPCR in a total volume of 20 µL with SYBR Green Reagent with the following condition: Predenaturation at 95℃ for 5 min, denaturation at 95℃ for 10 s, annealing/extension at 60℃ for 30sec, and the number of cycles was 40. GAPDH acted as an endogenous control of TP53 . using the 2 −ΔΔCt method and normalized to the level of TP53 . Primer sequences (TP53: F 5’-FAGGCCTTGGAACTCAAGGAT-3’, R: 5’-CCCTTTTTGGACTTCAGGTG-3’; GPADH: F: 5’-GGAGCGAGATCCCTCCAAAAT-3’; R: 5’-GGCTGTTGTCATACTTCTCATGG-3’). 2.9 Flow cytometry The cells were obtained with trypsin enzyme digesting technique (without Ethylene Diamine Tetraacetic Acid) and resuspended by PBS. The resuspended cells (number: 10×10 4 cells) were centrifuged (1000 g) for 5 min and obtained the pellet which was added into 195 µL Annexin V-FITC binding buffer to resuspend cells. Then, 5 µL Annexin V-FITC, 10 µL propidium iodide staining solution added to the tube, in order, blending gently. The sample were incubated at room temperature for 20 min and then placed in the ice bath. Finally, the cell apoptosis was detected on flow cytometer (Flowsight, Merck). The cells were obtained with trypsin enzyme digesting technique (without Ethylene Diamine Tetraacetic Acid) and resuspended by PBS. Then, 1 mL 70% ethanol at 4℃ for 30 min to fix cells. Next, centrifuging (1000 g) for 5 min to obtain pellet. The sample were added to 0.5 mL propidium iodide staining solution to resuspended the cells, which were incubated at 37℃ for 30 min. Finally, the cell cycle was detected on flow cytometer (Flowsight, Merck) at the excitation wavelength of 488 nm. 2.10 Statistical analysis All data were repeated in triplicate and expressed as mean ± standard deviation (SD). All statistical analyses were performed using GraphPad Prism software (Version 8.0, LA Jolla, CA, USA). One-way analysis of variances (ANOVAs) was performed for multiple group comparisons, and two group comparisons were conducted with Student's t test. P < 0.05 was considered to be statistically significant. 3 Results 3.1 The potential targets of TPL for TC treatment In order to find the potential targets of TPL in the treatment of TC, we utilized Venn map to present the results which demonstrated that there were 2166 and 34 targets of TC and TPL, respectively, and the common targets of them were 25 (Fig. 1 ). It suggested that TPL have enormous potentialities for TC treatment. 3.2 PPI network analysis According to the results of Venn map, there were 25 potential targets of TPL for TC treatment. We further analyzed the interaction among these targets and found that the degree values of TP53, Caspase 3, TNF, CD40, JUN, VEGFA, PTGS2, STAT3 were all more than 20 (Fig. 2 ). The results hinted that TPL for TC treatment were possible via these targets. 3.3 Go enrichment analysis Through Go enrichment analysis, there have 261, 19, 36 terms of BP, CC and MF, respectively, and the top 10 of them were selected to present. As is shown in Fig. 3 A-C, BP mainly included positive regulation of transcription from RNA polymerase II promoter, negative regulation of apoptotic process, apoptotic process, etc.; CC involved with cytoplasm, nucleus, nucleoplasm, etc., MF were related to protein binding, identical protein binding, etc. 3.4 KEGG pathway enrichment analysis We performed KEGG pathway enrichment analysis on the above potential targets. It showed that TPL for TC treatment were associated with Pathway in cancer, apoptosis, MAPK signaling way and so on (Fig. 4 ). It could infer that TPL regulated cell apoptosis, cell proliferation, inflammation response etc., to inhibit TC development. 3.5 The inhibiting effect of TPL on K1 cell activity Firstly, we investigated the effect of different concentrations TPL (0, 5, 10, 25, 100, 500 nM) on cell activity. The results showed that TPL (5, 10) have no inhibiting effect on K1 cells ( P > 0.05). However, when the dose at 25, 100, 500 nM, the inhibition ratios were achieved 32.75%, 43.93, 61.70%, respectively (Fig. 5 B). The images of cells under bright field were also presented the similarly (Fig. 5 A). The results suggested that TPL could inhibit K1 cells viability. 3.6 The expressions of apoptosis and proliferation-related protein of TPL treated on K1 cells According to the analysis of PPI network, GO and KEGG enrichment, TPL in the treatment of TC were associated with cell apoptosis and proliferation. Therefore, we detected the apoptosis and proliferation-related proteins levels by Western blot (Fig. 6 A). As is shown in Fig. 6 B-G, TPL at the dose of 10 and 25 nM obviously down-regulated PNCA and ki67 levels and regulated with caspase 3, caspase 7, cleaved caspase 3, cleaved caspase 7 ( P < 0.05, 0.01). The results hint that TPL could promote cancer cell apoptosis and reduce cell proliferation in order to prevent TC development. 3.7 The effect of TPL on K1 cell apoptosis We used flow cytometry to assess the effect of TPL on K1 cell apoptosis (Fig. 7 A). It indicated that TPL at the dose of 10, 25 nM significantly increased the cell apoptosis in both early and later period compared with control group (TPL: 0 nM, P < 0.01, Fig. 7 B-C). These data emphasized that TPL could increase cancer cell apoptosis. 3.8 TPL arrests K1 cells cycle at the G2/M and S phase To determine the effect of TPL on K1 cell cycle, we checked G0/G1, G2/M and S period of cell (Fig. 8 A). K1 cells at the state of reversible cell cycle (G0 phase)/G1 phase of the cells synthesizes RNA and ribosome in TPL (10, 25 nM) group were higher than in control group (TPL: 0 nM, P < 0.05, 0.01, Fig. 8 B). K1 cells at G2 phase (protein synthesis)/ mitosis (M phase) in TPL (10, 25 nM) group were lower than in control group (TPL: 0 nM, P < 0.05, Fig. 8 C). TPL at the dose of 10, 25 nM significantly reduce the amount of K1 cells during S phase which takes place DNA replication compared to control group (Fig. 8 D, P < 0.05, 0.01). This suggests that K1 cell cycle blocked at G2/M and S phase. 3.9 The effect of TPL on the expression of TP53 PPI analysis showed that TP53 was the potential target of TPL for TC treatment. Thus, we further detected the expressions of p-P53 protein and TP53 mRNA by WB and RT-PCR. The results demonstrated that p-P53 protein and TP53 mRNA expressions were obviously increased after TPL (5, 10, 25 nM) treatment, compared with the control group ( P < 0.05 or 0.01, Fig. 9 A-C). It proved that TPL could up-regulated the TP53 level to inhibit K1 cell apoptosis. 4 Discussion TC is a the most prevalent form of endocrine malignancy and its incidence has increased worldwide over the past four decades (Nabhan et al., 2021 ). TPL has the biological activity of anti-cancer, such as liver cancer, head and neck cancer, breast cancer etc., via inducing cell apoptosis, resisting drug resistance and arresting tumor cell cycle (Li et al., 2021a ; Yang et al., 2022 ). In this study, we utilized network to analyze the putative targets and mechanism of TPC for TC treatment. The results showed TPL mainly involved with cell apoptosis and proliferation related pathways. Therefore, the further experiments verified the effect of TPL for TC treatment by regulating related the proteins in order to determine the clearly the mechanism. In this study, the data firstly proved that TPL significant decreased K1 cells activity when the concentration achieved 25, 100, 500 nM. Zhu et al also demonstrated that TPL obviously reduced TC cells viability with dose-dependent effects (Zhu et al., 2009 ). Additionally, on the basis of network pharmacology, caspases were participated in the process of TC in the treatment of TC. Caspase-3 plays an important in regulating the growth and homeostatic maintenance of malignant cells and tissues in multicellular organisms (Eskandari and Eaves, 2022 ). Wang et al demonstrated that the in malignant tumor cells, the increased expression of caspase-3 promoted cell apoptosis (Wang et al., 2019 ). Caspase-7, as the same with caspase-3, is also, belong to effector caspases, which of them are responsible for initiating the cascade reaction of protein hydrolysis (Dubash et al., 2018 ). The result in this study, caspase-3 and caspase-7 were both decreased after the treatment of TPL in K1 cells. Furthermore, in order to explore the effect of TPL on TC cell apoptosis, flow cytometry was performed and the data also showed TPL induced cell apoptosis at later and early period. In addition, KEGG enrichment pathway analysis also showed that TPL for TC treatment were involved with MAPK signaling way which not only regulate cell apoptosis but also the cell proliferation (Su et al., 2019 ). Thus, ki-67, the cell proliferation markers, and proliferating cell nuclear antigen (PCNA) were detected by WB (Zhao et al., 2020b ). PCNA and ki67 expression. PCNA serves an important role in the priming of cell proliferation and ki67 is also expressed at high levels only in proliferating cells (Sobecki et al., 2016 ; Wang et al., 2017 ). Li et al demonstrated that inhibiting the expression levels of ki-67 and PCNA obviously blocked cancer cell proliferation (Li et al., 2021b ). Our data revealed that TPL significantly inhibited ki-67 and PCNA expressions. Furthermore, cell cycle is one of the most successfully drugged targets in cancers (Icard et al., 2019 ). In eukaryotic cells, it mainly produced proteins for M phase, at G2 phase. G2 and M phase processes appear to overlap temporally due to the relatively short lived of M phase, the two phase are taken together as G2/M refers to the cell is not actively dividing (Jain et al., 2015 ). Cells always duplicate their chromosomes and synthesize histones at S phase (Wang et al., 2018 ). Our data indicated that TPL compromises the mitosis of K1 cells leading to G2/M and S phase arrest. Thus, TPL promotes cells gets into the program of the apoptosis, suppresses cells proliferation and disturbs cell cycle to prevent TC development. TP53 protein is a key tumor suppressor whose activation may result in a variety of cellular responses, including apoptosis, cell senescence, cell-cycle arrest and et al (Aubrey et al., 2016 ). PPI network analysis presented TP53 is the potential target of TPL for TC treatment. In vitro experiment, the expressions of TP53 protein and TP53 mRNA were indeed up-regulated after TPL treatment. Huang et al indicated that down-regulated p-p53 was the critical reason for low sensitivity to oridonin-induced apoptosis (Huang et al., 2005 ). It was inferred that TPL might target in TP53 to induce cancer K1 cell apoptosis and prevent cell proliferation. In summary, the data demonstrated TPL exerts antitumor effectors on K1 cells through inhibition of cell proliferation, induction of cell apoptosis and disruption cell cycle by targeting in TP53. However, further animal experiment and methods for this study is needed to reveal the deep mechanism of TPL for TC treatment. Declarations Conflicts of Interest/Competing Interests Hongkan Lou, Bing Chen, Qian Shen, Jie Shen, Hongyang Xie and Yiping Wang have no conficts of interest that are directly relevant to the content of this article. Ethics Approval Not applicable. Consent to Participate Not applicable. Consent for Publication Not applicable. Funding This research was funded by Natural Science Foundation of Ningbo City (NO. 202003N4252). Author Contribution B. C. and H.K. L. conceived and designed the experiments. Q. S. and J. S. contributed significantly to analysis and manuscript preparation. H.Y. X. and Y.P. W. helped perform the analysis with constructive discussions. Acknowledgements Not applicable. Availability of data and materials The data used to support the findings of this study are included within the article. Code Availability Not applicable. References Aubrey, B.J., Strasser, A., and Kelly, G.L. (2016). Tumor-Suppressor Functions of the TP53 Pathway. Cold Spring Harb Perspect Med 6 . Deng, Y., Li, H., Wang, M., Li, N., Tian, T., Wu, Y., Xu, P., Yang, S., Zhai, Z., Zhou, L. , et al. (2020). Global Burden of Thyroid Cancer From 1990 to 2017. JAMA Netw Open 3 , e208759. Dubash, S.R., Merchant, S., Heinzmann, K., Mauri, F., Lavdas, I., Inglese, M., Kozlowski, K., Rama, N., Masrour, N., Steel, J.F. , et al. (2018). Clinical translation of [(18)F]ICMT-11 for measuring chemotherapy-induced caspase 3/7 activation in breast and lung cancer. Eur J Nucl Med Mol Imaging 45 , 2285-2299. Eskandari, E., and Eaves, C.J. (2022). Paradoxical roles of caspase-3 in regulating cell survival, proliferation, and tumorigenesis. J Cell Biol 221 . Haugen, B.R., Alexander, E.K., Bible, K.C., Doherty, G.M., Mandel, S.J., Nikiforov, Y.E., Pacini, F., Randolph, G.W., Sawka, A.M., Schlumberger, M. , et al. (2016). 2015 American Thyroid Association Management Guidelines for Adult Patients with Thyroid Nodules and Differentiated Thyroid Cancer: The American Thyroid Association Guidelines Task Force on Thyroid Nodules and Differentiated Thyroid Cancer. Thyroid 26 , 1-133. Huang, J., Wu, L., Tashiro, S., Onodera, S., and Ikejima, T. (2005). Bcl-2 up-regulation and P-p53 down-regulation account for the low sensitivity of murine L929 fibrosarcoma cells to oridonin-induced apoptosis. Biol Pharm Bull 28 , 2068-2074. Icard, P., Fournel, L., Wu, Z., Alifano, M., and Lincet, H. (2019). Interconnection between Metabolism and Cell Cycle in Cancer. Trends Biochem Sci 44 , 490-501. Jain, R.K., Hong, D.S., Naing, A., Wheler, J., Helgason, T., Shi, N.Y., Gad, Y., and Kurzrock, R. (2015). Novel phase I study combining G1 phase, S phase, and G2/M phase cell cycle inhibitors in patients with advanced malignancies. Cell Cycle 14 , 3434-3440. Laha, D., Nilubol, N., and Boufraqech, M. (2020). New Therapies for Advanced Thyroid Cancer. Front Endocrinol (Lausanne) 11 , 82. Li, J.X., Shi, J.F., Wu, Y.H., Xu, H.T., Fu, C.M., and Zhang, J.M. (2021a). [Mechanisms and application of triptolide against breast cancer]. Zhongguo Zhong Yao Za Zhi 46 , 3249-3256. Li, Y., Lv, M., Wang, J., Gao, C., and Wu, Y. (2021b). LINC00641 inhibits the proliferation and invasion of ovarian cancer cells by targeting miR-320a. Transl Cancer Res 10 , 4894-4904. Myung, S.K., Lee, C.W., Lee, J., Kim, J., and Kim, H.S. (2017). Risk Factors for Thyroid Cancer: A Hospital-Based Case-Control Study in Korean Adults. Cancer Res Treat 49 , 70-78. Nabhan, F., Dedhia, P.H., and Ringel, M.D. (2021). Thyroid cancer, recent advances in diagnosis and therapy. Int J Cancer 149 , 984-992. Niciporuka, R., Nazarovs, J., Ozolins, A., Narbuts, Z., Miklasevics, E., and Gardovskis, J. (2021). Can We Predict Differentiated Thyroid Cancer Behavior? Role of Genetic and Molecular Markers. Medicina (Kaunas) 57 . Sarkar, F.H., and Li, Y. (2009). Harnessing the fruits of nature for the development of multi-targeted cancer therapeutics. Cancer Treat Rev 35 , 597-607. Silva, S.N. (2021). Special Issue: Genetic Perspectives in Thyroid Cancer. Genes (Basel) 12 . Sobecki, M., Mrouj, K., Camasses, A., Parisis, N., Nicolas, E., Lleres, D., Gerbe, F., Prieto, S., Krasinska, L., David, A. , et al. (2016). The cell proliferation antigen Ki-67 organises heterochromatin. Elife 5 , e13722. Su, X., Shen, Z., Yang, Q., Sui, F., Pu, J., Ma, J., Ma, S., Yao, D., Ji, M., and Hou, P. (2019). Vitamin C kills thyroid cancer cells through ROS-dependent inhibition of MAPK/ERK and PI3K/AKT pathways via distinct mechanisms. Theranostics 9 , 4461-4473. Wang, J.L., Quan, Q., Ji, R., Guo, X.Y., Zhang, J.M., Li, X., and Liu, Y.G. (2018). Isorhamnetin suppresses PANC-1 pancreatic cancer cell proliferation through S phase arrest. Biomed Pharmacother 108 , 925-933. Wang, M., Qiu, S., and Qin, J. (2019). Baicalein induced apoptosis and autophagy of undifferentiated thyroid cancer cells by the ERK/PI3K/Akt pathway. Am J Transl Res 11 , 3341-3352. Wang, X.H., Chen, Z.G., Xu, R.L., Lv, C.Q., Liu, J., and Du, B. (2017). TGF-beta1 signaling pathway serves a role in HepG2 cell regulation by affecting the protein expression of PCNA, gankyrin, p115, XIAP and survivin. Oncol Lett 13 , 3239-3246. Yan, P., and Sun, X. (2018). Triptolide: A new star for treating human malignancies. J Cancer Res Ther 14 , S271-S275. Yang, J., Guo, W., and Lu, M. (2022). Confusion about the article: Natural product triptolide induces GSDME-mediated pyroptosis in head and neck cancer through suppressing mitochondrial hexokinase-IotaIota. Cancer Lett 534 , 115568. Zhao, J., Xie, C., Wang, K., Takahashi, S., Krausz, K.W., Lu, D., Wang, Q., Luo, Y., Gong, X., Mu, X. , et al. (2020a). Comprehensive analysis of transcriptomics and metabolomics to understand triptolide-induced liver injury in mice. Toxicol Lett 333 , 290-302. Zhao, J., Xie, C., Wang, K., Takahashi, S., Krausz, K.W., Lu, D., Wang, Q., Luo, Y., Gong, X., Mu, X. , et al. (2020b). Comprehensive analysis of transcriptomics and metabolomics to understand triptolide-induced liver injury in mice. Toxicology Letters 333 , 290-302. Zhu, W., He, S., Li, Y., Qiu, P., Shu, M., Ou, Y., Zhou, Y., Leng, T., Xie, J., Zheng, X. , et al. (2010). Anti-angiogenic activity of triptolide in anaplastic thyroid carcinoma is mediated by targeting vascular endothelial and tumor cells. Vascul Pharmacol 52 , 46-54. Zhu, W., Hu, H., Qiu, P., and Yan, G. (2009). Triptolide induces apoptosis in human anaplastic thyroid carcinoma cells by a p53-independent but NF-kappaB-related mechanism. Oncol Rep 22 , 1397-1401. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4779748","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":330326561,"identity":"dcf71882-9db2-41d9-9849-bfb4d31bfb03","order_by":0,"name":"Bing Chen","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Bing","middleName":"","lastName":"Chen","suffix":""},{"id":330326562,"identity":"64fd5a1e-7902-4ef7-8c16-a77360744797","order_by":1,"name":"Qian Shen","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Qian","middleName":"","lastName":"Shen","suffix":""},{"id":330326563,"identity":"3ed8aecc-ca49-42a3-9856-1233b32aef6e","order_by":2,"name":"Jie Shen","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Jie","middleName":"","lastName":"Shen","suffix":""},{"id":330326564,"identity":"354a2255-daa7-4955-a05a-5e196d8fc5ca","order_by":3,"name":"Hongyang Xie","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Hongyang","middleName":"","lastName":"Xie","suffix":""},{"id":330326565,"identity":"2536ffc6-d8ae-492f-a033-d1851e6c1c23","order_by":4,"name":"Yiping Wang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Yiping","middleName":"","lastName":"Wang","suffix":""},{"id":330326566,"identity":"24ab0173-922a-4a12-97ff-da1216965dc8","order_by":5,"name":"Hongkan Lou","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5klEQVRIiWNgGAWjYBACPgYGNhBtwMDAfuDghwoJOXlCWtgQWngSH0ucsTA2bCBeC4OxAW9bRSLDAUJa2HvMHnzcUWvMP7shTUJynkQCYwPzw0c38GnhOWNuOPPMcTOJOwePSRRuk8hjZ2AzNs7Bp0Uix0yat+2YDcONBKAt2ySKGRt42KSJ0iJ/I8FMgneORGLDAeK01JgZ3EgAer+BGC08x8okZ7YdMDa8kQMM5GMSxobNBPzCz968TeJjW53hvBvpwKisqZOTZ29++BifFig4jMRmJqwcBOqIUzYKRsEoGAUjEwAA2hhHEaC9j4YAAAAASUVORK5CYII=","orcid":"","institution":"","correspondingAuthor":true,"prefix":"","firstName":"Hongkan","middleName":"","lastName":"Lou","suffix":""}],"badges":[],"createdAt":"2024-07-22 06:57:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4779748/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4779748/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":62596555,"identity":"feb51847-5523-4574-8a59-6e2ff4a2ef34","added_by":"auto","created_at":"2024-08-16 09:03:33","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":14732,"visible":true,"origin":"","legend":"\u003cp\u003eVenn map of targets in TC and TPL\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4779748/v1/0b5ecf6cbf35d052e0d3afa2.png"},{"id":62597267,"identity":"1c087cea-2be2-444b-9db9-dcf1431d32cd","added_by":"auto","created_at":"2024-08-16 09:11:33","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":115654,"visible":true,"origin":"","legend":"\u003cp\u003ePPI network analysis for the common targets of TC and TPL\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4779748/v1/529f9ff71edaf987a5a921d8.png"},{"id":62597947,"identity":"9517017d-0cc8-4fd1-92aa-a8f2b88230de","added_by":"auto","created_at":"2024-08-16 09:19:34","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":310093,"visible":true,"origin":"","legend":"\u003cp\u003eGO enrichment analysis of genes for TPL in the treatment. GO analysis included BP (A), CC (B) and MF (C).\u003c/p\u003e","description":"","filename":"floatimage3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4779748/v1/4b84e81825fe45336eb7ef57.jpg"},{"id":62596557,"identity":"2ca7c71f-6d90-4ad7-bf8d-e15c7e1ad219","added_by":"auto","created_at":"2024-08-16 09:03:33","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":73004,"visible":true,"origin":"","legend":"\u003cp\u003eKEGG pathway enrichment analysis\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4779748/v1/51d4b5bf4803508cfe5d6292.png"},{"id":62596561,"identity":"6a89b33e-d3ed-46b4-937f-1b7006d908ca","added_by":"auto","created_at":"2024-08-16 09:03:34","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":210979,"visible":true,"origin":"","legend":"\u003cp\u003eTPL inhibited K1 cell activity. (A) The images of K1 cells treated with different concentrations TPL under bright field. (B) OD value (fold to control group) of K1 cells treated with different concentrations. The experiments were repeated for 3 times.\u003c/p\u003e","description":"","filename":"floatimage5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4779748/v1/a0e9d908277c09b0839bfb4f.jpg"},{"id":62596560,"identity":"448683fa-f464-4b86-89f6-dbb18c3a535f","added_by":"auto","created_at":"2024-08-16 09:03:34","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":164894,"visible":true,"origin":"","legend":"\u003cp\u003eTPL regulated the expression of apoptosis and proliferation- related proteins. (A) Western blot was used to detect the expression of (B) caspase 3, (C) caspase 7, (D) cleaved caspase 3, (E) cleaved caspase 7, (F) ki67, (G) PNCA which were qualified by Image J software. The experiments were repeated for 3 times.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-4779748/v1/7c74b113746fb82df90ea3c7.png"},{"id":62597268,"identity":"b7e6f706-4136-4593-aad0-11f6044f25e2","added_by":"auto","created_at":"2024-08-16 09:11:34","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":234055,"visible":true,"origin":"","legend":"\u003cp\u003eTPL increased K1 cells apoptosis. (A) Flow cytometry was used to detect the cell apoptosis. (B) The ratio of cell apoptosis at the later period (C) The ratio of cell apoptosis at the early period. The experiments were repeated for 3 times.\u003c/p\u003e","description":"","filename":"floatimage7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4779748/v1/1343bef3a3a9d7139b8ddcc6.jpg"},{"id":62597271,"identity":"5ea03caf-850d-478d-b48e-58e967801955","added_by":"auto","created_at":"2024-08-16 09:11:34","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":184276,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of TPL on K1 cells cycle. (A) Flow cytometry was used to detect the cell cycle. (B) KO K1 cells cycle at G0/G1 phase (C) K1 cells cycle at G2/M phase. (C) K1 cells cycle at S phase. The experiments were repeated for 3 times.\u003c/p\u003e","description":"","filename":"floatimage8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4779748/v1/af504d342c819e21de031555.jpg"},{"id":62596562,"identity":"e07f1ba3-7f42-402f-a539-29248e980dbe","added_by":"auto","created_at":"2024-08-16 09:03:34","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":95318,"visible":true,"origin":"","legend":"\u003cp\u003eTPL regulated the expression of TP53. (A) Western blot was used to detect the expression of ((B) P53 protein which were qualified by Image J software. (C) RT-PCR detected the relative expression of \u003cem\u003eTP53\u003c/em\u003e mRNA. The experiments were repeated for 3 times.\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-4779748/v1/f2b4107f6473674676fa308e.png"},{"id":71573020,"identity":"0f032939-0ec9-4513-b850-27c9eb3cb73a","added_by":"auto","created_at":"2024-12-16 20:16:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1949364,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4779748/v1/7b409120-e925-4c6e-beb6-9fb889f4c917.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Network pharmacology-based investigation of potential mechanism of Triptolide against Thyroid Cancer","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eThyroid cancer (TC), a kind of endocrine malignancy and ranked ninth among the newly diagnosed cancers cases in 2020, could be induced by sex, a diet low in iodine, smoking, and exposure to radiation (Myung et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Niciporuka et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Silva, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). According to the report, China (11016 in 1990 and 41511 in 2017), America (10833 in 1990 and 25896 in 2017) and India (7369 in 1990 and 25 675 in 2017) were the top three countries which have the highest incident cases of TC, and South Asia (3630 in 1990 and 8930 in 2017) had the largest numbers of TC deaths (Deng et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Based on TC morphological variations, it could be classified as four types and papillary thyroid carcinoma (PTC) and follicular thyroid carcinoma (FTC) represent 90\u0026ndash;95% (Sarkar and Li, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Although surgical resection is the common therapy for the most TC patients and the new therapies including thyroid hormone therapy, targeted drug therapy, etc are constantly emerging, but effective treatments are still lacking (Haugen et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Laha et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Therefore, it is necessary to find novel therapeutic method for TC.\u003c/p\u003e \u003cp\u003eTriptolide (TPL), a diterpenoid epoxide, is extracted from \u003cem\u003eTripterygium wilfordii\u003c/em\u003e, with the biological activity of anti-inflammatory, immunosuppressive and anti-cancer (Zhao et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020a\u003c/span\u003e). Especially, TPL is known as the new star for treating human malignancies, such as lung cancer, pancreatic cancer, and thyroid cancer (Yan and Sun, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Its anti- neoplastic properties have been proposed for the anti-proliferative and pro-apoptotic activities (Zhu et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). TPL suppresses invasion of TC have been demonstrated, but it\u0026rsquo;s mechanism of TC is reminded to be explored. In this study, we used network pharmacology to find the potential target and pathway about TPL for TC treatment and further verified the results via experiments, which aimed to find a new direction for TC treatment.\u003c/p\u003e"},{"header":"2 Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 The collection of TPL and TC targets\u003c/h2\u003e \u003cp\u003eFirstly, the targets of TPL (MOL003187) were collected from TCMSP (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://tcmsp-e.com/tcmsp.php\u003c/span\u003e\u003cspan address=\"https://tcmsp-e.com/tcmsp.php\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The targets of TC were obtained from Gene Cards database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.genecards.org/\u003c/span\u003e\u003cspan address=\"https://www.genecards.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), OMIM database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://omim.org/\u003c/span\u003e\u003cspan address=\"https://omim.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and TTD (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://db.idrblab.net/\u003c/span\u003e\u003cspan address=\"http://db.idrblab.net/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) database, and removed the duplicate targets. Using Venn map to present the common targets of TPL and TC.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Protein-protein (PPI) interaction network analysis\u003c/h2\u003e \u003cp\u003eThe targets mentioned above were uploaded to STRING database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://string-db.org\u003c/span\u003e\u003cspan address=\"https://string-db.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) with the limited conditions (Species: Homo sapiens; medium confidence:0.4000). Then the PPI network was visualized using Cytoscape software 3.6.0.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Go and KEGG enrichment performance\u003c/h2\u003e \u003cp\u003eThe common targets were uploaded to DAVIDA database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://davidd.ncifcrf.gov\u003c/span\u003e\u003cspan address=\"https://davidd.ncifcrf.gov\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) by setting the conditions (Species: Homo sapiens; Identifier: Official Gene Symbol; List type: Gene List;). Gene Ontology (GO) including molecular function (MF), cellular component (CC), biological process (BP) and Kyoto Encyclopedia of Genes and Genomes (KEGG) information were collected. Th top 10 items of them were showed as the histogram and bubble chart, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Cell and regents\u003c/h2\u003e \u003cp\u003eK1 human thyroid cancer cells (K1) were obtained from Shanghai Zyyan Biotechnology Co., LTD. Triptolide was bought from Yuanye (B20709, Shanghai, China). Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM) was obtained from Corning (10-013-CVRC, Shanghai, China). CCK-8 was bought from APExBIO (K1018, Houston, US). The antibodies of caspase 3 (bsm-33284M), caspase 7 (bsm-60304R), PCNA (bs-2006R), ki67 (bs-23102R) were obtained from Bioss (Beijing, China). The antibodies of cleaved caspase 7 (Ab256469), cleaved caspase 3 (ab2302) were bought from abcam (Shanghai, China). HRP-labeled Goat Anti-Rabbit IgG(H\u0026thinsp;+\u0026thinsp;L) (A0208) and Annexin V-FITC Apoptosis Detection Kit (C1062M) and Cell Cycle and Apoptosis Analysis Kit (C1052) were got from Beyotime (Shanghai, China). 2\u0026times;Hieff Robust PCR Master Mix, HifairⅢ 1st Strand cDNA Synthesis SuperMix for qRCR, Hieff qPCR SYBR Green Master Mix were bought from Yi Sheng Biotechnology Co., LTD.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Cell culture\u003c/h2\u003e \u003cp\u003eK1 cells were cultured with DEME medium (containing 5% fetal bovine serum, 1% Penicillin - Streptomycin - amphotericin B- solution) under a 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere at 37\u0026deg;C. K1 cells were resuspended in DEME medium (containing 5% fetal bovine serum) and seeded in 24 well plates at the density of 4\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/well.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Cell treatment and CCK-8 assay\u003c/h2\u003e \u003cp\u003eK1 cells were seed in 96 well plates at the density of 1\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells/well under a 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere at 37\u0026deg;C until the cell density reached 70%-80% confluence. The cells were treated with TPL with different concentrations (0, 5, 10, 25, 100, 500 nM) for 24 hours. Next, the cells were added into 10 \u0026micro;L CCK-8 and incubated for another 2 hours. Finally, the OD value was detected under the wavelength of 450 nm by microplate reader (Infinite E Plex, Tecan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Western blot\u003c/h2\u003e \u003cp\u003eThe protein of cells was extracted by adding radio immunoprecipitation assay (RIPA) lysis buffer and its concentrations were detected by BCA protein assay kit. The proteins were separated by 10% sodium dodecyl sulfate (SDS) polyacrylamide gels and then transferred into polyvinylidene fluoride (PVDF) membranes. Next, the membranes were blocked by 5% skim milk for 2 hours and incubated with primary antibodies of caspase 3 (1:1000), caspase 7 (1:500), PCNA (1:1000), ki67 (1:1000), cleaved caspase 7 (1:500) and cleaved caspase 3 (1:500) at 4℃overnight. Next day, the membranes were washed by TBST for 3 times and incubated with HRP-labeled Goat Anti-Rabbit IgG(H\u0026thinsp;+\u0026thinsp;L) for 2 hours. Finally, the blots were visualized using the ECL chemiluminescent substrate reagent by chemiluminescence imager (ChemiDoc XRS+, Bio-Rad). The bands were qualified by Image J software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Quantitative Real-time PCR\u003c/h2\u003e \u003cp\u003eTotal mRNA was extracted by TRIzol\u0026reg; reagent and reversed complementary DNA (cDNA) synthesis with the Reverse Transcription system. The cDNA samples were diluted to 100 \u0026micro;l with RNase-free water and 1 \u0026micro;l cDNA mixture was used for qPCR in a total volume of 20 \u0026micro;L with SYBR Green Reagent with the following condition: Predenaturation at 95℃ for 5 min, denaturation at 95℃ for 10 s, annealing/extension at 60℃ for 30sec, and the number of cycles was 40. GAPDH acted as an endogenous control of \u003cem\u003eTP53\u003c/em\u003e. using the 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method and normalized to the level of \u003cem\u003eTP53\u003c/em\u003e. Primer sequences (TP53: F 5\u0026rsquo;-FAGGCCTTGGAACTCAAGGAT-3\u0026rsquo;, R: 5\u0026rsquo;-CCCTTTTTGGACTTCAGGTG-3\u0026rsquo;; GPADH: F: 5\u0026rsquo;-GGAGCGAGATCCCTCCAAAAT-3\u0026rsquo;; R: 5\u0026rsquo;-GGCTGTTGTCATACTTCTCATGG-3\u0026rsquo;).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Flow cytometry\u003c/h2\u003e \u003cp\u003eThe cells were obtained with trypsin enzyme digesting technique (without Ethylene Diamine Tetraacetic Acid) and resuspended by PBS. The resuspended cells (number: 10\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells) were centrifuged (1000 g) for 5 min and obtained the pellet which was added into 195 \u0026micro;L Annexin V-FITC binding buffer to resuspend cells. Then, 5 \u0026micro;L Annexin V-FITC, 10 \u0026micro;L propidium iodide staining solution added to the tube, in order, blending gently. The sample were incubated at room temperature for 20 min and then placed in the ice bath. Finally, the cell apoptosis was detected on flow cytometer (Flowsight, Merck).\u003c/p\u003e \u003cp\u003eThe cells were obtained with trypsin enzyme digesting technique (without Ethylene Diamine Tetraacetic Acid) and resuspended by PBS. Then, 1 mL 70% ethanol at 4℃ for 30 min to fix cells. Next, centrifuging (1000 g) for 5 min to obtain pellet. The sample were added to 0.5 mL propidium iodide staining solution to resuspended the cells, which were incubated at 37℃ for 30 min. Finally, the cell cycle was detected on flow cytometer (Flowsight, Merck) at the excitation wavelength of 488 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Statistical analysis\u003c/h2\u003e \u003cp\u003eAll data were repeated in triplicate and expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). All statistical analyses were performed using GraphPad Prism software (Version 8.0, LA Jolla, CA, USA). One-way analysis of variances (ANOVAs) was performed for multiple group comparisons, and two group comparisons were conducted with Student's t test. \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered to be statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.1 The potential targets of TPL for TC treatment\u003c/h2\u003e \u003cp\u003eIn order to find the potential targets of TPL in the treatment of TC, we utilized Venn map to present the results which demonstrated that there were 2166 and 34 targets of TC and TPL, respectively, and the common targets of them were 25 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). It suggested that TPL have enormous potentialities for TC treatment.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.2 PPI network analysis\u003c/h2\u003e \u003cp\u003eAccording to the results of Venn map, there were 25 potential targets of TPL for TC treatment. We further analyzed the interaction among these targets and found that the degree values of TP53, Caspase 3, TNF, CD40, JUN, VEGFA, PTGS2, STAT3 were all more than 20 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The results hinted that TPL for TC treatment were possible via these targets.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Go enrichment analysis\u003c/h2\u003e \u003cp\u003eThrough Go enrichment analysis, there have 261, 19, 36 terms of BP, CC and MF, respectively, and the top 10 of them were selected to present. As is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-C, BP mainly included positive regulation of transcription from RNA polymerase II promoter, negative regulation of apoptotic process, apoptotic process, etc.; CC involved with cytoplasm, nucleus, nucleoplasm, etc., MF were related to protein binding, identical protein binding, etc.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.4 KEGG pathway enrichment analysis\u003c/h2\u003e \u003cp\u003eWe performed KEGG pathway enrichment analysis on the above potential targets. It showed that TPL for TC treatment were associated with Pathway in cancer, apoptosis, MAPK signaling way and so on (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). It could infer that TPL regulated cell apoptosis, cell proliferation, inflammation response etc., to inhibit TC development.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.5 The inhibiting effect of TPL on K1 cell activity\u003c/h2\u003e \u003cp\u003eFirstly, we investigated the effect of different concentrations TPL (0, 5, 10, 25, 100, 500 nM) on cell activity. The results showed that TPL (5, 10) have no inhibiting effect on K1 cells (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). However, when the dose at 25, 100, 500 nM, the inhibition ratios were achieved 32.75%, 43.93, 61.70%, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). The images of cells under bright field were also presented the similarly (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). The results suggested that TPL could inhibit K1 cells viability.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.6 The expressions of apoptosis and proliferation-related protein of TPL treated on K1 cells\u003c/h2\u003e \u003cp\u003eAccording to the analysis of PPI network, GO and KEGG enrichment, TPL in the treatment of TC were associated with cell apoptosis and proliferation. Therefore, we detected the apoptosis and proliferation-related proteins levels by Western blot (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). As is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB-G, TPL at the dose of 10 and 25 nM obviously down-regulated PNCA and ki67 levels and regulated with caspase 3, caspase 7, cleaved caspase 3, cleaved caspase 7 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, 0.01). The results hint that TPL could promote cancer cell apoptosis and reduce cell proliferation in order to prevent TC development.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.7 The effect of TPL on K1 cell apoptosis\u003c/h2\u003e \u003cp\u003eWe used flow cytometry to assess the effect of TPL on K1 cell apoptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). It indicated that TPL at the dose of 10, 25 nM significantly increased the cell apoptosis in both early and later period compared with control group (TPL: 0 nM, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB-C). These data emphasized that TPL could increase cancer cell apoptosis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.8 TPL arrests K1 cells cycle at the G2/M and S phase\u003c/h2\u003e \u003cp\u003eTo determine the effect of TPL on K1 cell cycle, we checked G0/G1, G2/M and S period of cell (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). K1 cells at the state of reversible cell cycle (G0 phase)/G1 phase of the cells synthesizes RNA and ribosome in TPL (10, 25 nM) group were higher than in control group (TPL: 0 nM, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, 0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB). K1 cells at G2 phase (protein synthesis)/ mitosis (M phase) in TPL (10, 25 nM) group were lower than in control group (TPL: 0 nM, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC). TPL at the dose of 10, 25 nM significantly reduce the amount of K1 cells during S phase which takes place DNA replication compared to control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, 0.01). This suggests that K1 cell cycle blocked at G2/M and S phase.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.9 The effect of TPL on the expression of TP53\u003c/h2\u003e \u003cp\u003ePPI analysis showed that TP53 was the potential target of TPL for TC treatment. Thus, we further detected the expressions of p-P53 protein and \u003cem\u003eTP53\u003c/em\u003e mRNA by WB and RT-PCR. The results demonstrated that p-P53 protein and \u003cem\u003eTP53\u003c/em\u003e mRNA expressions were obviously increased after TPL (5, 10, 25 nM) treatment, compared with the control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 or 0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA-C). It proved that TPL could up-regulated the TP53 level to inhibit K1 cell apoptosis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eTC is a the most prevalent form of endocrine malignancy and its incidence has increased worldwide over the past four decades (Nabhan et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). TPL has the biological activity of anti-cancer, such as liver cancer, head and neck cancer, breast cancer etc., via inducing cell apoptosis, resisting drug resistance and arresting tumor cell cycle (Li et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021a\u003c/span\u003e; Yang et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In this study, we utilized network to analyze the putative targets and mechanism of TPC for TC treatment. The results showed TPL mainly involved with cell apoptosis and proliferation related pathways. Therefore, the further experiments verified the effect of TPL for TC treatment by regulating related the proteins in order to determine the clearly the mechanism.\u003c/p\u003e \u003cp\u003eIn this study, the data firstly proved that TPL significant decreased K1 cells activity when the concentration achieved 25, 100, 500 nM. Zhu et al also demonstrated that TPL obviously reduced TC cells viability with dose-dependent effects (Zhu et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Additionally, on the basis of network pharmacology, caspases were participated in the process of TC in the treatment of TC. Caspase-3 plays an important in regulating the growth and homeostatic maintenance of malignant cells and tissues in multicellular organisms (Eskandari and Eaves, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Wang et al demonstrated that the in malignant tumor cells, the increased expression of caspase-3 promoted cell apoptosis (Wang et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Caspase-7, as the same with caspase-3, is also, belong to effector caspases, which of them are responsible for initiating the cascade reaction of protein hydrolysis (Dubash et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The result in this study, caspase-3 and caspase-7 were both decreased after the treatment of TPL in K1 cells. Furthermore, in order to explore the effect of TPL on TC cell apoptosis, flow cytometry was performed and the data also showed TPL induced cell apoptosis at later and early period. In addition, KEGG enrichment pathway analysis also showed that TPL for TC treatment were involved with MAPK signaling way which not only regulate cell apoptosis but also the cell proliferation (Su et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Thus, ki-67, the cell proliferation markers, and proliferating cell nuclear antigen (PCNA) were detected by WB (Zhao et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2020b\u003c/span\u003e). PCNA and ki67 expression. PCNA serves an important role in the priming of cell proliferation and ki67 is also expressed at high levels only in proliferating cells (Sobecki et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Li et al demonstrated that inhibiting the expression levels of ki-67 and PCNA obviously blocked cancer cell proliferation (Li et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021b\u003c/span\u003e). Our data revealed that TPL significantly inhibited ki-67 and PCNA expressions. Furthermore, cell cycle is one of the most successfully drugged targets in cancers (Icard et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In eukaryotic cells, it mainly produced proteins for M phase, at G2 phase. G2 and M phase processes appear to overlap temporally due to the relatively short lived of M phase, the two phase are taken together as G2/M refers to the cell is not actively dividing (Jain et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Cells always duplicate their chromosomes and synthesize histones at S phase (Wang et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Our data indicated that TPL compromises the mitosis of K1 cells leading to G2/M and S phase arrest. Thus, TPL promotes cells gets into the program of the apoptosis, suppresses cells proliferation and disturbs cell cycle to prevent TC development.\u003c/p\u003e \u003cp\u003eTP53 protein is a key tumor suppressor whose activation may result in a variety of cellular responses, including apoptosis, cell senescence, cell-cycle arrest and et al (Aubrey et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). PPI network analysis presented TP53 is the potential target of TPL for TC treatment. In vitro experiment, the expressions of TP53 protein and \u003cem\u003eTP53\u003c/em\u003e mRNA were indeed up-regulated after TPL treatment. Huang et al indicated that down-regulated p-p53 was the critical reason for low sensitivity to oridonin-induced apoptosis (Huang et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). It was inferred that TPL might target in TP53 to induce cancer K1 cell apoptosis and prevent cell proliferation.\u003c/p\u003e \u003cp\u003eIn summary, the data demonstrated TPL exerts antitumor effectors on K1 cells through inhibition of cell proliferation, induction of cell apoptosis and disruption cell cycle by targeting in TP53. However, further animal experiment and methods for this study is needed to reveal the deep mechanism of TPL for TC treatment.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflicts of Interest/Competing Interests\u003c/h2\u003e \u003cp\u003eHongkan Lou, Bing Chen, Qian Shen, Jie Shen, Hongyang Xie and Yiping Wang have no conficts of interest that are directly relevant to the content of this article.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eEthics Approval\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent to Participate\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for Publication\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis research was funded by Natural Science Foundation of Ningbo City (NO. 202003N4252).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eB. C. and H.K. L. conceived and designed the experiments. Q. S. and J. S. contributed significantly to analysis and manuscript preparation. H.Y. X. and Y.P. W. helped perform the analysis with constructive discussions.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eNot applicable.\u003c/p\u003e\u003ch2\u003eAvailability of data and materials\u003c/h2\u003e \u003cp\u003eThe data used to support the findings of this study are included within the article.\u003c/p\u003e\u003ch2\u003eCode Availability\u003c/h2\u003e \u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAubrey, B.J., Strasser, A., and Kelly, G.L. (2016). Tumor-Suppressor Functions of the TP53 Pathway. Cold Spring Harb Perspect Med\u003cem\u003e 6\u003c/em\u003e.\u003c/li\u003e\n\u003cli\u003eDeng, Y., Li, H., Wang, M., Li, N., Tian, T., Wu, Y., Xu, P., Yang, S., Zhai, Z., Zhou, L.\u003cem\u003e, et al.\u003c/em\u003e (2020). Global Burden of Thyroid Cancer From 1990 to 2017. JAMA Netw Open\u003cem\u003e 3\u003c/em\u003e, e208759.\u003c/li\u003e\n\u003cli\u003eDubash, S.R., Merchant, S., Heinzmann, K., Mauri, F., Lavdas, I., Inglese, M., Kozlowski, K., Rama, N., Masrour, N., Steel, J.F.\u003cem\u003e, et al.\u003c/em\u003e (2018). Clinical translation of [(18)F]ICMT-11 for measuring chemotherapy-induced caspase 3/7 activation in breast and lung cancer. Eur J Nucl Med Mol Imaging\u003cem\u003e 45\u003c/em\u003e, 2285-2299.\u003c/li\u003e\n\u003cli\u003eEskandari, E., and Eaves, C.J. (2022). Paradoxical roles of caspase-3 in regulating cell survival, proliferation, and tumorigenesis. J Cell Biol\u003cem\u003e 221\u003c/em\u003e.\u003c/li\u003e\n\u003cli\u003eHaugen, B.R., Alexander, E.K., Bible, K.C., Doherty, G.M., Mandel, S.J., Nikiforov, Y.E., Pacini, F., Randolph, G.W., Sawka, A.M., Schlumberger, M.\u003cem\u003e, et al.\u003c/em\u003e (2016). 2015 American Thyroid Association Management Guidelines for Adult Patients with Thyroid Nodules and Differentiated Thyroid Cancer: The American Thyroid Association Guidelines Task Force on Thyroid Nodules and Differentiated Thyroid Cancer. Thyroid\u003cem\u003e 26\u003c/em\u003e, 1-133.\u003c/li\u003e\n\u003cli\u003eHuang, J., Wu, L., Tashiro, S., Onodera, S., and Ikejima, T. (2005). Bcl-2 up-regulation and P-p53 down-regulation account for the low sensitivity of murine L929 fibrosarcoma cells to oridonin-induced apoptosis. Biol Pharm Bull\u003cem\u003e 28\u003c/em\u003e, 2068-2074.\u003c/li\u003e\n\u003cli\u003eIcard, P., Fournel, L., Wu, Z., Alifano, M., and Lincet, H. (2019). Interconnection between Metabolism and Cell Cycle in Cancer. Trends Biochem Sci\u003cem\u003e 44\u003c/em\u003e, 490-501.\u003c/li\u003e\n\u003cli\u003eJain, R.K., Hong, D.S., Naing, A., Wheler, J., Helgason, T., Shi, N.Y., Gad, Y., and Kurzrock, R. (2015). Novel phase I study combining G1 phase, S phase, and G2/M phase cell cycle inhibitors in patients with advanced malignancies. Cell Cycle\u003cem\u003e 14\u003c/em\u003e, 3434-3440.\u003c/li\u003e\n\u003cli\u003eLaha, D., Nilubol, N., and Boufraqech, M. (2020). New Therapies for Advanced Thyroid Cancer. Front Endocrinol (Lausanne)\u003cem\u003e 11\u003c/em\u003e, 82.\u003c/li\u003e\n\u003cli\u003eLi, J.X., Shi, J.F., Wu, Y.H., Xu, H.T., Fu, C.M., and Zhang, J.M. (2021a). [Mechanisms and application of triptolide against breast cancer]. Zhongguo Zhong Yao Za Zhi\u003cem\u003e 46\u003c/em\u003e, 3249-3256.\u003c/li\u003e\n\u003cli\u003eLi, Y., Lv, M., Wang, J., Gao, C., and Wu, Y. (2021b). LINC00641 inhibits the proliferation and invasion of ovarian cancer cells by targeting miR-320a. Transl Cancer Res\u003cem\u003e 10\u003c/em\u003e, 4894-4904.\u003c/li\u003e\n\u003cli\u003eMyung, S.K., Lee, C.W., Lee, J., Kim, J., and Kim, H.S. (2017). Risk Factors for Thyroid Cancer: A Hospital-Based Case-Control Study in Korean Adults. Cancer Res Treat\u003cem\u003e 49\u003c/em\u003e, 70-78.\u003c/li\u003e\n\u003cli\u003eNabhan, F., Dedhia, P.H., and Ringel, M.D. (2021). Thyroid cancer, recent advances in diagnosis and therapy. Int J Cancer\u003cem\u003e 149\u003c/em\u003e, 984-992.\u003c/li\u003e\n\u003cli\u003eNiciporuka, R., Nazarovs, J., Ozolins, A., Narbuts, Z., Miklasevics, E., and Gardovskis, J. (2021). Can We Predict Differentiated Thyroid Cancer Behavior? Role of Genetic and Molecular Markers. Medicina (Kaunas)\u003cem\u003e 57\u003c/em\u003e.\u003c/li\u003e\n\u003cli\u003eSarkar, F.H., and Li, Y. (2009). Harnessing the fruits of nature for the development of multi-targeted cancer therapeutics. Cancer Treat Rev\u003cem\u003e 35\u003c/em\u003e, 597-607.\u003c/li\u003e\n\u003cli\u003eSilva, S.N. (2021). Special Issue: Genetic Perspectives in Thyroid Cancer. Genes (Basel)\u003cem\u003e 12\u003c/em\u003e.\u003c/li\u003e\n\u003cli\u003eSobecki, M., Mrouj, K., Camasses, A., Parisis, N., Nicolas, E., Lleres, D., Gerbe, F., Prieto, S., Krasinska, L., David, A.\u003cem\u003e, et al.\u003c/em\u003e (2016). The cell proliferation antigen Ki-67 organises heterochromatin. Elife\u003cem\u003e 5\u003c/em\u003e, e13722.\u003c/li\u003e\n\u003cli\u003eSu, X., Shen, Z., Yang, Q., Sui, F., Pu, J., Ma, J., Ma, S., Yao, D., Ji, M., and Hou, P. (2019). Vitamin C kills thyroid cancer cells through ROS-dependent inhibition of MAPK/ERK and PI3K/AKT pathways via distinct mechanisms. Theranostics\u003cem\u003e 9\u003c/em\u003e, 4461-4473.\u003c/li\u003e\n\u003cli\u003eWang, J.L., Quan, Q., Ji, R., Guo, X.Y., Zhang, J.M., Li, X., and Liu, Y.G. (2018). Isorhamnetin suppresses PANC-1 pancreatic cancer cell proliferation through S phase arrest. Biomed Pharmacother\u003cem\u003e 108\u003c/em\u003e, 925-933.\u003c/li\u003e\n\u003cli\u003eWang, M., Qiu, S., and Qin, J. (2019). Baicalein induced apoptosis and autophagy of undifferentiated thyroid cancer cells by the ERK/PI3K/Akt pathway. Am J Transl Res\u003cem\u003e 11\u003c/em\u003e, 3341-3352.\u003c/li\u003e\n\u003cli\u003eWang, X.H., Chen, Z.G., Xu, R.L., Lv, C.Q., Liu, J., and Du, B. (2017). TGF-beta1 signaling pathway serves a role in HepG2 cell regulation by affecting the protein expression of PCNA, gankyrin, p115, XIAP and survivin. Oncol Lett\u003cem\u003e 13\u003c/em\u003e, 3239-3246.\u003c/li\u003e\n\u003cli\u003eYan, P., and Sun, X. (2018). Triptolide: A new star for treating human malignancies. J Cancer Res Ther\u003cem\u003e 14\u003c/em\u003e, S271-S275.\u003c/li\u003e\n\u003cli\u003eYang, J., Guo, W., and Lu, M. (2022). Confusion about the article: Natural product triptolide induces GSDME-mediated pyroptosis in head and neck cancer through suppressing mitochondrial hexokinase-IotaIota. Cancer Lett\u003cem\u003e 534\u003c/em\u003e, 115568.\u003c/li\u003e\n\u003cli\u003eZhao, J., Xie, C., Wang, K., Takahashi, S., Krausz, K.W., Lu, D., Wang, Q., Luo, Y., Gong, X., Mu, X.\u003cem\u003e, et al.\u003c/em\u003e (2020a). Comprehensive analysis of transcriptomics and metabolomics to understand triptolide-induced liver injury in mice. Toxicol Lett\u003cem\u003e 333\u003c/em\u003e, 290-302.\u003c/li\u003e\n\u003cli\u003eZhao, J., Xie, C., Wang, K., Takahashi, S., Krausz, K.W., Lu, D., Wang, Q., Luo, Y., Gong, X., Mu, X.\u003cem\u003e, et al.\u003c/em\u003e (2020b). Comprehensive analysis of transcriptomics and metabolomics to understand triptolide-induced liver injury in mice. Toxicology Letters\u003cem\u003e 333\u003c/em\u003e, 290-302.\u003c/li\u003e\n\u003cli\u003eZhu, W., He, S., Li, Y., Qiu, P., Shu, M., Ou, Y., Zhou, Y., Leng, T., Xie, J., Zheng, X.\u003cem\u003e, et al.\u003c/em\u003e (2010). Anti-angiogenic activity of triptolide in anaplastic thyroid carcinoma is mediated by targeting vascular endothelial and tumor cells. Vascul Pharmacol\u003cem\u003e 52\u003c/em\u003e, 46-54.\u003c/li\u003e\n\u003cli\u003eZhu, W., Hu, H., Qiu, P., and Yan, G. (2009). Triptolide induces apoptosis in human anaplastic thyroid carcinoma cells by a p53-independent but NF-kappaB-related mechanism. Oncol Rep\u003cem\u003e 22\u003c/em\u003e, 1397-1401.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Triptolide, Thyroid cancer, Cell apoptosis, Cell proliferation, TP53","lastPublishedDoi":"10.21203/rs.3.rs-4779748/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4779748/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cb\u003eObjective\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThis study aimed to explore the mechanism of triptolide (TPL) in the treatment of thyroid cancer (TC).\u003c/p\u003e\u003cp\u003e\u003cb\u003eMethods\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe targets of TPL in TC were collected from databases. A protein-protein interaction network was constructed using the common targets of TPL and TC. Enrichment analysis was performed using the DAVID database. The effects of TPL on cell activity, apoptosis, and cell cycle were assessed using CCK-8 assay and flow cytometry. Western blot analysis was conducted to measure the levels of caspase 3, caspase 7, PCNA, ki67, cleaved caspase 7, cleaved caspase 3, and p-P53. RT-PCR was used to measure TP53 mRNA levels.\u003c/p\u003e\u003cp\u003e\u003cb\u003eResults\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe protein-protein interaction network revealed 8 potential targets for TPL in TC treatment. Enrichment analysis indicated that TPL mainly involved in cell apoptosis, proliferation, and inflammation response. In vitro studies showed that TPL inhibited K1 cell activity, down-regulated PCNA and ki67 levels, and up-regulated caspase 3, caspase 7, cleaved caspase 3, cleaved caspase 7, p-P53 protein expressions, and TP53 mRNA levels. TPL also promoted K1 cell apoptosis and arrested cell cycle at the G2/M and S phase.\u003c/p\u003e\u003cp\u003e\u003cb\u003eConclusion\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTPL exhibits antitumor effects on K1 cells by inhibiting cell proliferation, inducing cell apoptosis, and disrupting cell cycle.\u003c/p\u003e","manuscriptTitle":"Network pharmacology-based investigation of potential mechanism of Triptolide against Thyroid Cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-16 09:03:29","doi":"10.21203/rs.3.rs-4779748/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"66c90987-717c-40d8-a731-11cb72b77f4a","owner":[],"postedDate":"August 16th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-12-16T20:08:31+00:00","versionOfRecord":[],"versionCreatedAt":"2024-08-16 09:03:29","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4779748","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4779748","identity":"rs-4779748","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}