Rapamycin suppresses small bowel adenocarcinoma HUTU 80 cells proliferation by inhibiting hypoxia-inducible factor-1α mediated metabolic reprogramming

doi:10.4251/wjgo.v17.i9.109378

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World Journal of Gastrointestinal Oncology

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1948-5204

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Baishideng Publishing Group Inc, 7041 Koll Center Parkway, Suite 160, Pleasanton, CA 94566, USA

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World J Gastrointest Oncol. Sep 15, 2025; 17(9): 109378
Published online Sep 15, 2025. doi: 10.4251/wjgo.v17.i9.109378

Rapamycin suppresses small bowel adenocarcinoma HUTU 80 cells proliferation by inhibiting hypoxia-inducible factor-1α mediated metabolic reprogramming

Bao-Peng Pu, Peng-Hui Wang, Kai-Kai Guo, Chun Liu, Si-Run Chen, Xiao-Meng Li, Shi-Min Chen, Xiang-Zhou Zeng, Chang Gao

Bao-Peng Pu, Peng-Hui Wang, Kai-Kai Guo, Xiao-Meng Li, Shi-Min Chen, Xiang-Zhou Zeng, Chang Gao, Key Laboratory of Tropical Translational Medicine of Ministry of Education, Hainan Provincial Key Laboratory of Carcinogenesis and Intervention, School of Basic Medicine and Life Sciences, Hainan Medical University, Haikou 571199, Hainan Province, China

Peng-Hui Wang, Department of Pathology, Nanjing Hospital of Chinese Medicine, Nanjing 210022, Jiangsu Province, China

Kai-Kai Guo, Dazhou Vocational College of Chinese Medicine, Dazhou 635000, Sichuan Province, China

Chun Liu, Hainan Institute for Drug Control, Haikou 570216, Hainan Province, China

Si-Run Chen, Hainan Medical University Press, Haikou 571199, Hainan Province, China

Co-first authors: Bao-Peng Pu and Peng-Hui Wang.

Co-corresponding authors: Xiang-Zhou Zeng and Chang Gao.

Author contributions: Pu BP performed experiments, analyzed data, provided all figures and wrote the article; Wang PH and Guo KK performed research; Liu C, Chen SR and Li XM designed the experiments and interpreted the results; Gao C, Zeng XZ, and Chen SM contributed to the central idea, designed the research, provided reagents and supported funds, and confirm the authenticity of all the raw data; Pu BP and Wang PH are designated as co-first authors based on their nearly equal contributions to multiple aspects of the project. Both played pivotal roles in study design, data collection and analysis, and manuscript writing. Their dedication and collaborative efforts at these critical stages underscore their strong professional competence, which significantly contributed to the smooth progress and high quality of the research. Given their comparable workload and impact, the co-first authorship designation fairly reflects their substantial and balanced contributions; The designation of Gao C and Zeng XZ as co-corresponding authors is justified for three key reasons. First, both researchers made equally significant contributions to the research, investing comparable effort in experimental design and manuscript preparation, warranting fair recognition. Second, each contributed distinct expertise and assumed complementary responsibilities in the study. Third, dual corresponding authorship enhances accessibility, providing readers with broader communication channels and facilitating engagement with the research team, thereby amplifying the work’s dissemination and impact; All authors discussed the results and revised the manuscript, and approved the final version to be published.

Supported by the National Natural Science Foundation of China, No. 81660270; Hainan Provincial Natural Science Foundation of China, No. 823RC497; Project of Nanhai Series of Talent Cultivation Program, No. 20192031; Key Discipline Project of Pathophysiology at Hainan Medical University, No. 05; and The Open Project Fund for Provincial Key Disciplines of Basic Medicine at Hainan Medical University, Hainan Medical College Talent Research Start up Fund, No. RZ300006194.

Institutional review board statement: This study does not involve human subjects, and therefore does not require approval from the Institutional Review Board Approval Form or Document.

Institutional animal care and use committee statement: The animal experiments were approved by the Ethics Committee of Hainan Medical College (2024 Animal Ethics Preliminary Examination No. 568). All protocols were in accordance with the approved guidelines and regulations.

Conflict-of-interest statement: The authors declare that they have no conflict of interest.

ARRIVE guidelines statement: The authors have read the ARRIVE guidelines, and the manuscript was prepared and revised according to the ARRIVE guidelines.

Data sharing statement: The sequencing data have been deposited to National Center for Biotechnology Information (NCBI) under the BioProject number PRJNA1228170. The metabolome data generated in the present study may be found in the metabolights database under accession number MTBLS12268. The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Open Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: https://creativecommons.org/Licenses/by-nc/4.0/

Corresponding author: Chang Gao, PhD, Associate Professor, Key Laboratory of Tropical Translational Medicine of Ministry of Education, Hainan Provincial Key Laboratory of Carcinogenesis and Intervention, School of Basic Medicine and Life Sciences, Hainan Medical University, No. 3 Academy Road, Haikou 571199, Hainan Province, China. gaochang@muhn.edu.cn

Received: May 9, 2025
Revised: June 20, 2025
Accepted: August 18, 2025
Published online: September 15, 2025
Processing time: 129 Days and 15.2 Hours

Abstract

BACKGROUND

Small bowel adenocarcinoma (SBA) is a rare malignant tumor of gastrointestinal tract. Currently, there is no standard treatment approach for late-stage SBA, which lead to poor outcome and prognosis. Rapamycin is an immunosuppressive agent that has been reported to inhibit the proliferation of tumor cells. However, whether rapamycin inhibit the growth of SBA remains to be investigated.

AIM

To observe the inhibitory effect of rapamycin on small intestinal adenocarcinoma cells.

METHODS

Methylthiazolyldiphenyl-tetrazolium bromide assay, colony formation assay, cell cycle analysis, and glycolysis assay were used to observe the phenotypic changes of rapamycin-treated HUTU 80 cells. RNA sequencing and untargeted ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) metabolomics were also used to find the potential targets of action of rapamycin in inhibiting HUTU 80 cells proliferation, and validate potential targets by quantitative polymerase chain reaction and western blotting. The construction of a subcutaneous HUTU 80 xenograft in BALB/c nude mice was used to explore the tumor suppression effect of rapamycin.

RESULTS

Rapamycin inhibited HUTU 80 cell proliferation in vitro and in vivo. Rapamycin inhibited the migration, invasion, and glycolysis of HUTU 80 cells, and induced cell cycle arrest. RNA sequencing and untargeted UHPLC-MS/MS metabolomic analysis indicated that the mechanism of rapamycin action was linked to the hypoxia-inducible factor (HIF)-1α signaling pathway and the related gluconeogenesis/glycolysis pathways. Subsequent experiments found that rapamycin downregulated the messenger RNA expression of HIF-1α and its downstream target genes, LDHA, PDK1 and VEGF. Additionally, rapamycin inhibited expression of phosphorylated mammalian target of rapamycin (mTOR), phosphorylated-70 kDa ribosomal protein S6 kinase (p70S6k), phosphorylated eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) and HIF-1α proteins in vitro and in vivo.

CONCLUSION

Downregulation of mTOR/p70S6k/4E-BP1/HIF-1α signaling pathway activation, leading to decreased glycolysis and cell cycle arrest, may be the pivotal mechanism by which rapamycin inhibits SBA.

Keywords: Rapamycin; Small bowel adenocarcinoma; Mammalian target of rapamycin/hypoxia-inducible facto-1α; Metabolic reprogramming; Warburg effect; Cell cycle arrest; Xenograft

Core Tip: Rapamycin is an immunosuppressive agent that inhibits the proliferation of tumor cells. HUTU 80 cells were used as a representative model for small bowel adenocarcinoma (SBA). Rapamycin inhibited the proliferation, migration, and invasion of HUTU 80 cells and downregulated glycolysis levels and cell cycle arrest. Transcriptomic and metabolomic analyses were used to find the potential targets of action of rapamycin in inhibiting HUTU 80 cell proliferation. The key mechanism of rapamycin against SBA is related to the mammalian target of rapamycin/70 kDa ribosomal protein S6 kinase/4E-binding protein 1/hypoxia-inducible factor-1α signaling pathway.