{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,23]],"date-time":"2026-06-23T10:25:58Z","timestamp":1782210358434,"version":"3.54.5"},"reference-count":39,"publisher":"MDPI AG","issue":"4","license":[{"start":{"date-parts":[[2018,4,6]],"date-time":"2018-04-06T00:00:00Z","timestamp":1522972800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Colorectal cancer is one of the most common causes of cancer death worldwide. In patients with metastatic colorectal cancer, combination treatment with several anti-cancer drugs is employed and improves overall survival in some patients. Nevertheless, most patients with metastatic disease are not cured owing to the drug resistance. Cancer stem cells are known to regulate resistance to chemotherapy. In the previous study, we established a novel three-dimensional organoid culture model from tumor colorectal tissues of human patients using an air\u2013liquid interface (ALI) method, which contained numerous cancer stem cells and showed resistance to 5-fluorouracil (5-FU) and Irinotecan. Here, we investigate which inhibitor for stem cell-related signal improves the sensitivity for anti-cancer drug treatment in tumor ALI organoids. Treatment with Hedgehog signal inhibitors (AY9944, GANT61) decreases the cell viability of organoids compared with Notch (YO-01027, DAPT) and Wnt (WAV939, Wnt-C59) signal inhibitors. Combination treatment of AY9944 or GANT61 with 5-FU, Irinotecan or Oxaliplatin decreases the cell viability of tumor organoids compared with each anti-cancer drug alone treatment. Treatment with AY9944 or GANT61 inhibits expression of stem cell markers c-Myc, CD44 and Nanog, likely through the decrease of their transcription factor, GLI-1 expression. Combination treatment of AY9944 or GANT61 with 5-FU or Irinotecan also prevents colony formation of colorectal cancer cell lines HCT116 and SW480. These findings suggest that Hedgehog signals mediate anti-cancer drug resistance in colorectal tumor patient-derived ALI organoids and that the inhibitors are useful as a combinational therapeutic strategy against colorectal cancer.<\/jats:p>","DOI":"10.3390\/ijms19041098","type":"journal-article","created":{"date-parts":[[2018,4,10]],"date-time":"2018-04-10T13:06:08Z","timestamp":1523365568000},"page":"1098","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":90,"title":["Hedgehog Signals Mediate Anti-Cancer Drug Resistance in Three-Dimensional Primary Colorectal Cancer Organoid Culture"],"prefix":"10.3390","volume":"19","author":[{"given":"Tatsuya","family":"Usui","sequence":"first","affiliation":[{"name":"Laboratory of Veterinary Pharmacology, Department of Veterinary Medicine, Faculty of Agriculture, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo 183-8509, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Masashi","family":"Sakurai","sequence":"additional","affiliation":[{"name":"Laboratory of Veterinary Pathology, Joint Faculty of Veterinary Medicine, Yamaguchi University, 1677-1 Yoshida, Yamaguchi 753-8515, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Koji","family":"Umata","sequence":"additional","affiliation":[{"name":"Laboratory of Veterinary Pharmacology, Joint Faculty of Veterinary Medicine, Yamaguchi University, 1677-1 Yoshida, Yamaguchi 753-8515, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9368-1535","authenticated-orcid":false,"given":"Mohamed","family":"Elbadawy","sequence":"additional","affiliation":[{"name":"Laboratory of Veterinary Pharmacology, Department of Veterinary Medicine, Faculty of Agriculture, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo 183-8509, Japan"},{"name":"Department of Pharmacology, Faculty of Veterinary Medicine, Benha University, Moshtohor, Toukh, Elqaliobiya 13736, Egypt"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Takashi","family":"Ohama","sequence":"additional","affiliation":[{"name":"Laboratory of Veterinary Pharmacology, Joint Faculty of Veterinary Medicine, Yamaguchi University, 1677-1 Yoshida, Yamaguchi 753-8515, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hideyuki","family":"Yamawaki","sequence":"additional","affiliation":[{"name":"Laboratory of Veterinary Pharmacology, School of Veterinary Medicine, Kitasato University, Higashi 23 bancho 35-1, Towada City, Aomori 034-8628, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shoichi","family":"Hazama","sequence":"additional","affiliation":[{"name":"Department of Translational Research and Developmental Therapeutics against Cancer, School of Medicine, Yamaguchi University, 1-1-1 Ogushi, Ube, Yamaguchi 755-8505, 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School of Medicine, Yamaguchi University, 1-1-1 Ogushi, Ube, Yamaguchi 755-8505, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nobuaki","family":"Suzuki","sequence":"additional","affiliation":[{"name":"Department of Gastroenterological, Breast and Endocrine Surgery, Graduate School of Medicine, Yamaguchi University, 1-1-1 Ogushi, Ube, Yamaguchi 755-8505, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hiroaki","family":"Nagano","sequence":"additional","affiliation":[{"name":"Department of Gastroenterological, Breast and Endocrine Surgery, Graduate School of Medicine, Yamaguchi University, 1-1-1 Ogushi, Ube, Yamaguchi 755-8505, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5265-0775","authenticated-orcid":false,"given":"Koichi","family":"Sato","sequence":"additional","affiliation":[{"name":"Laboratory of Veterinary Pharmacology, Joint Faculty of Veterinary Medicine, Yamaguchi University, 1677-1 Yoshida, Yamaguchi 753-8515, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0660-7156","authenticated-orcid":false,"given":"Masahiro","family":"Kaneda","sequence":"additional","affiliation":[{"name":"Laboratory of Veterinary Anatomy, Department of Veterinary Medicine, Faculty of Agriculture, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo 183-8509, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kazuaki","family":"Sasaki","sequence":"additional","affiliation":[{"name":"Laboratory of Veterinary Pharmacology, Department of Veterinary Medicine, Faculty of Agriculture, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo 183-8509, 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J. Cancer"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"87","DOI":"10.3322\/caac.21262","article-title":"Global cancer statistics, 2012","volume":"65","author":"Torre","year":"2015","journal-title":"CA Cancer J. Clin."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"1442","DOI":"10.1200\/JCO.2002.20.6.1442","article-title":"Liver metastases from colorectal cancer: The turning point","volume":"20","author":"Nordlinger","year":"2002","journal-title":"J. Clin. Oncol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"S3","DOI":"10.1016\/S0305-7372(08)70002-1","article-title":"First-line combination treatment of colorectal cancer with hepatic metastases: Choosing a targeted agent","volume":"34","author":"Lenz","year":"2008","journal-title":"Cancer Treat. Rev."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"374","DOI":"10.1111\/j.1582-4934.2007.00211.x","article-title":"Cancer stem cells: Implications for the progression and treatment of metastatic disease","volume":"12","author":"Croker","year":"2008","journal-title":"J. Cell. Mol. Med."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"503","DOI":"10.1016\/j.molmed.2008.09.005","article-title":"Colon cancer stem cells: Implications for prevention and therapy","volume":"14","author":"Huang","year":"2008","journal-title":"Trends Mol. Med."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"2151","DOI":"10.1053\/j.gastro.2009.12.063","article-title":"Colon cancer stem cells: Promise of targeted therapy","volume":"138","author":"Todaro","year":"2010","journal-title":"Gastroenterology"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"91","DOI":"10.1016\/S1535-6108(03)00334-9","article-title":"Small-molecule antagonists of the oncogenic Tcf\/\u03b2-catenin protein complex","volume":"5","author":"Lepourcelet","year":"2004","journal-title":"Cancer Cell"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"168","DOI":"10.1016\/j.stem.2009.05.019","article-title":"Dll4 blockade inhibits tumor growth and reduces tumor-initiating cell frequency","volume":"5","author":"Hoey","year":"2009","journal-title":"Cell Stem Cell"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"7053872","DOI":"10.1155\/2016\/7053872","article-title":"Establishment of a novel model for anticancer drug resistance in three-dimensional primary culture of tumor microenvironment","volume":"2016","author":"Usui","year":"2016","journal-title":"Stem Cells Int."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"3059","DOI":"10.1101\/gad.938601","article-title":"Hedgehog signaling in animal development: Paradigms and principles","volume":"15","author":"Ingham","year":"2001","journal-title":"Genes Dev."},{"key":"ref_12","first-page":"6339","article-title":"Cyclopamine decreased the expression of sonic hedgehog and its downstream genes in colon cancer stem cells","volume":"34","author":"Batsaikhan","year":"2014","journal-title":"Anticancer Res."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"37","DOI":"10.2174\/1574888X11666160607211436","article-title":"Cancer Stem Cells\u2014Are Surface Markers Alone Sufficient?","volume":"12","author":"Nagare","year":"2017","journal-title":"Curr. Stem Cell Res. Ther."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"2330","DOI":"10.3390\/cancers7040894","article-title":"Aberrant gli1 activation in DNA damage response, carcinogenesis and chemoresistance","volume":"7","author":"Palle","year":"2015","journal-title":"Cancers"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"243","DOI":"10.1007\/s00432-016-2273-6","article-title":"Gli1, a potential regulator of esophageal cancer stem cell, is identified as an independent adverse prognostic factor in esophageal squamous cell carcinoma","volume":"143","author":"Yang","year":"2017","journal-title":"J. Cancer Res. Clin. Oncol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"15239","DOI":"10.1038\/srep15239","article-title":"Pterostilbine, an active component of blueberries, sensitizes colon cancer cells to 5-fluorouracil cytotoxicity","volume":"5","author":"Tolba","year":"2015","journal-title":"Sci. Rep."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"17","DOI":"10.1186\/s13578-017-0145-7","article-title":"The role of gli1 for 5-fu resistance in colorectal cancer","volume":"7","author":"Zhang","year":"2017","journal-title":"Cell Biosci."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"1898","DOI":"10.3748\/wjg.v20.i8.1898","article-title":"Intestinal stem cells and the colorectal cancer microenvironment","volume":"20","author":"Ong","year":"2014","journal-title":"World J. Gastroenterol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1147","DOI":"10.1158\/1541-7786.MCR-12-0022","article-title":"Inhibition of the hedgehog pathway targets the tumor-associated stroma in pancreatic cancer","volume":"10","author":"Hwang","year":"2012","journal-title":"Mol. Cancer Res."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"4254","DOI":"10.1073\/pnas.0813203106","article-title":"Hedgehog signaling is restricted to the stromal compartment during pancreatic carcinogenesis","volume":"106","author":"Tian","year":"2009","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1457","DOI":"10.1126\/science.1171362","article-title":"Inhibition of hedgehog signaling enhances delivery of chemotherapy in a mouse model of pancreatic cancer","volume":"324","author":"Olive","year":"2009","journal-title":"Science"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"170","DOI":"10.1053\/j.gastro.2014.10.006","article-title":"Stromal indian hedgehog signaling is required for intestinal adenoma formation in mice","volume":"148","author":"Buller","year":"2015","journal-title":"Gastroenterology"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"14228","DOI":"10.1073\/pnas.0400067101","article-title":"A distinct \u201cside population\u201d of cells with high drug efflux capacity in human tumor cells","volume":"101","author":"Foster","year":"2004","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"8980","DOI":"10.1158\/0008-5472.CAN-07-0895","article-title":"Cancer stem cells in radiation resistance","volume":"67","author":"Rich","year":"2007","journal-title":"Cancer Res."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"151","DOI":"10.1053\/ctrv.1999.0161","article-title":"Systematic reviews of chemotherapy and endocrine therapy in metastatic breast cancer","volume":"26","author":"Stockler","year":"2000","journal-title":"Cancer Treat. Rev."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"9","DOI":"10.3892\/ijmm.2016.2815","article-title":"Self-renewal molecular mechanisms of colorectal cancer stem cells","volume":"39","author":"Pan","year":"2017","journal-title":"Int. J. Mol. Med."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"1102","DOI":"10.1053\/j.gastro.2009.05.053","article-title":"Combined targeted treatment to eliminate tumorigenic cancer stem cells in human pancreatic cancer","volume":"137","author":"Mueller","year":"2009","journal-title":"Gastroenterology"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"1679","DOI":"10.1007\/s00432-011-1038-5","article-title":"Identification of CD44+CD24+ gastric cancer stem cells","volume":"137","author":"Zhang","year":"2011","journal-title":"J. Cancer Res. Clin. Oncol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"338","DOI":"10.1002\/emmm.200900039","article-title":"Human colon cancer epithelial cells harbour active hedgehog-gli signalling that is essential for tumour growth, recurrence, metastasis and stem cell survival and expansion","volume":"1","author":"Varnat","year":"2009","journal-title":"EMBO Mol. Med."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"1751","DOI":"10.3892\/ijo.2012.1599","article-title":"Response to inhibition of smoothened in diverse epithelial cancer cells that lack smoothened or patched 1 mutations","volume":"41","author":"Galimberti","year":"2012","journal-title":"Int. J. Oncol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"5321","DOI":"10.1200\/JCO.2010.27.9943","article-title":"Clinical experience with hedgehog pathway inhibitors","volume":"28","author":"Low","year":"2010","journal-title":"J. Clin. Oncol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"4883","DOI":"10.1158\/1078-0432.CCR-11-2509","article-title":"Molecular pathways: The hedgehog signaling pathway in cancer","volume":"18","author":"McMillan","year":"2012","journal-title":"Clin. Cancer Res."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"572","DOI":"10.1126\/science.1179386","article-title":"Smoothened mutation confers resistance to a hedgehog pathway inhibitor in medulloblastoma","volume":"326","author":"Yauch","year":"2009","journal-title":"Science"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"854","DOI":"10.18632\/oncotarget.586","article-title":"Regulation of DNA damage following termination of hedgehog (hh) survival signaling at the level of the gli genes in human colon cancer","volume":"3","author":"Agyeman","year":"2012","journal-title":"Oncotarget"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"769","DOI":"10.1038\/nm.3585","article-title":"Oncogenic transformation of diverse gastrointestinal tissues in primary organoid culture","volume":"20","author":"Li","year":"2014","journal-title":"Nat. Med."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"701","DOI":"10.1038\/nm.1951","article-title":"Sustained in vitro intestinal epithelial culture within a wnt-dependent stem cell niche","volume":"15","author":"Ootani","year":"2009","journal-title":"Nat. Med."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"1100","DOI":"10.3892\/or.2017.5359","article-title":"Death-associated protein kinase 3 controls the tumor progression of a549 cells through erk mapk\/c-myc signaling","volume":"37","author":"Kake","year":"2017","journal-title":"Oncol. Rep."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"10858","DOI":"10.1074\/jbc.M115.704908","article-title":"Regulation of beclin 1 protein phosphorylation and autophagy by protein phosphatase 2a (pp2a) and death-associated protein kinase 3 (dapk3)","volume":"291","author":"Fujiwara","year":"2016","journal-title":"J. Biol. Chem."},{"key":"ref_39","doi-asserted-by":"crossref","unstructured":"Yabe, R., Miura, A., Usui, T., Mudrak, I., Ogris, E., Ohama, T., and Sato, K. (2015). Protein phosphatase methyl-esterase pme-1 protects protein phosphatase 2a from ubiquitin\/proteasome degradation. 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