{"title":"库欣病的高通量筛选:硫司群通通过细胞周期调节的治疗潜力。","authors":"Takuro Hakata, Ichiro Yamauchi, Daisuke Kosugi, Taku Sugawa, Haruka Fujita, Kentaro Okamoto, Yohei Ueda, Toshihito Fujii, Daisuke Taura, Nobuya Inagaki","doi":"10.1210/endocr/bqae089","DOIUrl":null,"url":null,"abstract":"<p><p>Cushing disease is a life-threatening disorder caused by autonomous secretion of ACTH from pituitary neuroendocrine tumors (PitNETs). Few drugs are indicated for inoperative Cushing disease, in particular that due to aggressive PitNETs. To explore agents that regulate ACTH-secreting PitNETs, we conducted high-throughput screening (HTS) using AtT-20, a murine pituitary tumor cell line characterized by ACTH secretion. For the HTS, we constructed a live cell-based ACTH reporter assay for high-throughput evaluation of ACTH changes. This assay was based on HEK293T cells overexpressing components of the ACTH receptor and a fluorescent cAMP biosensor, with high-throughput acquisition of fluorescence images. We treated AtT-20 cells with compounds and assessed ACTH concentrations in the conditioned media using the reporter assay. Of 2480 screened bioactive compounds, over 50% inhibition of ACTH secreted from AtT-20 cells was seen with 84 compounds at 10 μM and 20 compounds at 1 μM. Among these hit compounds, we focused on thiostrepton (TS) and determined its antitumor effects in both in vitro and in vivo xenograft models of Cushing disease. Transcriptome and flow cytometry analyses revealed that TS administration induced AtT-20 cell cycle arrest at the G2/M phase, which was mediated by FOXM1-independent mechanisms including downregulation of cyclins. Simultaneous TS administration with a cyclin-dependent kinase 4/6 inhibitor that affected the cell cycle at the G0/1 phase showed cooperative antitumor effects. Thus, TS is a promising therapeutic agent for Cushing disease. Our list of hit compounds and new mechanistic insights into TS effects serve as a valuable foundation for future research.</p>","PeriodicalId":11819,"journal":{"name":"Endocrinology","volume":null,"pages":null},"PeriodicalIF":3.8000,"publicationDate":"2024-07-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"High-throughput Screening for Cushing Disease: Therapeutic Potential of Thiostrepton via Cell Cycle Regulation.\",\"authors\":\"Takuro Hakata, Ichiro Yamauchi, Daisuke Kosugi, Taku Sugawa, Haruka Fujita, Kentaro Okamoto, Yohei Ueda, Toshihito Fujii, Daisuke Taura, Nobuya Inagaki\",\"doi\":\"10.1210/endocr/bqae089\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>Cushing disease is a life-threatening disorder caused by autonomous secretion of ACTH from pituitary neuroendocrine tumors (PitNETs). Few drugs are indicated for inoperative Cushing disease, in particular that due to aggressive PitNETs. To explore agents that regulate ACTH-secreting PitNETs, we conducted high-throughput screening (HTS) using AtT-20, a murine pituitary tumor cell line characterized by ACTH secretion. For the HTS, we constructed a live cell-based ACTH reporter assay for high-throughput evaluation of ACTH changes. This assay was based on HEK293T cells overexpressing components of the ACTH receptor and a fluorescent cAMP biosensor, with high-throughput acquisition of fluorescence images. We treated AtT-20 cells with compounds and assessed ACTH concentrations in the conditioned media using the reporter assay. Of 2480 screened bioactive compounds, over 50% inhibition of ACTH secreted from AtT-20 cells was seen with 84 compounds at 10 μM and 20 compounds at 1 μM. Among these hit compounds, we focused on thiostrepton (TS) and determined its antitumor effects in both in vitro and in vivo xenograft models of Cushing disease. Transcriptome and flow cytometry analyses revealed that TS administration induced AtT-20 cell cycle arrest at the G2/M phase, which was mediated by FOXM1-independent mechanisms including downregulation of cyclins. Simultaneous TS administration with a cyclin-dependent kinase 4/6 inhibitor that affected the cell cycle at the G0/1 phase showed cooperative antitumor effects. Thus, TS is a promising therapeutic agent for Cushing disease. Our list of hit compounds and new mechanistic insights into TS effects serve as a valuable foundation for future research.</p>\",\"PeriodicalId\":11819,\"journal\":{\"name\":\"Endocrinology\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":3.8000,\"publicationDate\":\"2024-07-26\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Endocrinology\",\"FirstCategoryId\":\"3\",\"ListUrlMain\":\"https://doi.org/10.1210/endocr/bqae089\",\"RegionNum\":3,\"RegionCategory\":\"医学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q2\",\"JCRName\":\"ENDOCRINOLOGY & METABOLISM\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Endocrinology","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.1210/endocr/bqae089","RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"ENDOCRINOLOGY & METABOLISM","Score":null,"Total":0}
High-throughput Screening for Cushing Disease: Therapeutic Potential of Thiostrepton via Cell Cycle Regulation.
Cushing disease is a life-threatening disorder caused by autonomous secretion of ACTH from pituitary neuroendocrine tumors (PitNETs). Few drugs are indicated for inoperative Cushing disease, in particular that due to aggressive PitNETs. To explore agents that regulate ACTH-secreting PitNETs, we conducted high-throughput screening (HTS) using AtT-20, a murine pituitary tumor cell line characterized by ACTH secretion. For the HTS, we constructed a live cell-based ACTH reporter assay for high-throughput evaluation of ACTH changes. This assay was based on HEK293T cells overexpressing components of the ACTH receptor and a fluorescent cAMP biosensor, with high-throughput acquisition of fluorescence images. We treated AtT-20 cells with compounds and assessed ACTH concentrations in the conditioned media using the reporter assay. Of 2480 screened bioactive compounds, over 50% inhibition of ACTH secreted from AtT-20 cells was seen with 84 compounds at 10 μM and 20 compounds at 1 μM. Among these hit compounds, we focused on thiostrepton (TS) and determined its antitumor effects in both in vitro and in vivo xenograft models of Cushing disease. Transcriptome and flow cytometry analyses revealed that TS administration induced AtT-20 cell cycle arrest at the G2/M phase, which was mediated by FOXM1-independent mechanisms including downregulation of cyclins. Simultaneous TS administration with a cyclin-dependent kinase 4/6 inhibitor that affected the cell cycle at the G0/1 phase showed cooperative antitumor effects. Thus, TS is a promising therapeutic agent for Cushing disease. Our list of hit compounds and new mechanistic insights into TS effects serve as a valuable foundation for future research.
期刊介绍:
The mission of Endocrinology is to be the authoritative source of emerging hormone science and to disseminate that new knowledge to scientists, clinicians, and the public in a way that will enable "hormone science to health." Endocrinology welcomes the submission of original research investigating endocrine systems and diseases at all levels of biological organization, incorporating molecular mechanistic studies, such as hormone-receptor interactions, in all areas of endocrinology, as well as cross-disciplinary and integrative studies. The editors of Endocrinology encourage the submission of research in emerging areas not traditionally recognized as endocrinology or metabolism in addition to the following traditionally recognized fields: Adrenal; Bone Health and Osteoporosis; Cardiovascular Endocrinology; Diabetes; Endocrine-Disrupting Chemicals; Endocrine Neoplasia and Cancer; Growth; Neuroendocrinology; Nuclear Receptors and Their Ligands; Obesity; Reproductive Endocrinology; Signaling Pathways; and Thyroid.