华蟾素通过增强 FOXO1 介导的 FCGBP 在骨肉瘤中的转录来抑制肿瘤进展和降低多柔比星耐药性。

Cinobufagin inhibits tumor progression and reduces doxorubicin resistance by enhancing FOXO1-mediated transcription of FCGBP in osteosarcoma.

机构信息

Department of Bone and Soft Tissue Oncology, Gansu Provincial People's Hospital, Lanzhou, 730001, Gansu, PR China.

Department of Spinal Surgery, General Hospital of Ningxia Medical University, Yinchuan, 750004, Ningxia, PR China.

出版信息

J Ethnopharmacol. 2022 Oct 5;296:115433. doi: 10.1016/j.jep.2022.115433. Epub 2022 Jun 8.

DOI:10.1016/j.jep.2022.115433

PMID:35690338

Abstract

ETHNOPHARMACOLOGICAL RELEVANCE

Cinobufagin (Huachansu), an aqueous extract from the dried skin of the toad Bufo bufo gargarizans Cantor (frog skin), is a biologically active ingredient of a traditional Chinese medicine cinobufacini that can treat multiple bone pathological conditions such as bone pain, bone tumors, and osteosarcoma.

AIM OF THE STUDY

The study aimed to explore the roles and molecular mechanisms of cinobufagin underlying osteosarcoma development and doxorubicin (ADR) resistance.

MATERIALS AND METHODS

Cell viability, migration, and invasion were examined by CCK-8, wound healing, and Transwell invasion assays, respectively. RNA sequencing analysis was performed in MNNG/HOS cells treated with or without cinobufagin. The relationships of cinobufagin, forkhead box O1 (FOXO1), and Fc fragment of IgG binding protein (FCGBP) were examined by luciferase reporter, immunofluorescence (IF), RT-qPCR, and chromatin immunoprecipitation (ChIP) assays together with weighted gene co-expression network analysis (WGCNA) analysis. Epithelial-mesenchymal transition (EMT) marker levels were examined through the Western blot assay. The function and molecular basis of cinobufagin in osteosarcoma were further investigated by mouse xenograft experiments.

RESULTS

Cinobufagin reduced cell viability, weakened ADR resistance, and inhibited cell migration/invasion/EMT in osteosarcoma cells. Cinobufagin enhanced FOXO1-mediated transcription of downstream genes including FCGBP. FCGBP knockdown partly abrogated the effect of cinobufagin on osteosarcoma cell development. Cinobufagin inhibited the growth of mouse osteosarcoma xenografts in vivo. Cinobufagin reduced the expression of Ki-67 and MMP9 and facilitated caspase-3 expression in osteosarcoma xenografts.

CONCLUSION

Cinobufagin suppressed tumor progression and reduced ADR resistance by potentiating FOXO1-mediated transcription of FCGBP in osteosarcoma.

摘要

民族药理学相关性

华蟾酥毒基（蟾酥），是中华大蟾蜍 Bufo bufo gargarizans Cantor （蟾皮）干燥皮的水提物，是一种生物活性成分，用于治疗多种骨骼病理状况，如骨痛、骨肿瘤和骨肉瘤。

研究目的

本研究旨在探讨华蟾酥毒基在骨肉瘤发生和多柔比星（阿霉素）（ADR）耐药性中的作用和分子机制。

材料和方法

通过 CCK-8、划痕愈合和 Transwell 侵袭实验分别检测细胞活力、迁移和侵袭。用华蟾酥毒基处理 MNNG/HOS 细胞进行 RNA 测序分析。通过荧光素酶报告、免疫荧光（IF）、RT-qPCR 和染色质免疫沉淀（ChIP）实验以及加权基因共表达网络分析（WGCNA）分析，研究华蟾酥毒基、叉头框 O1（FOXO1）和免疫球蛋白 G 结合蛋白 Fc 段（FCGBP）之间的关系。通过 Western blot 实验检测上皮-间充质转化（EMT）标志物水平。通过小鼠异种移植实验进一步研究华蟾酥毒基在骨肉瘤中的作用和分子基础。

结果

华蟾酥毒基降低细胞活力，减弱 ADR 耐药性，并抑制骨肉瘤细胞的迁移/侵袭/EMT。华蟾酥毒基增强了 FOXO1 介导的下游基因包括 FCGBP 的转录。FCGBP 敲低部分削弱了华蟾酥毒基对骨肉瘤细胞发育的影响。华蟾酥毒基抑制体内骨肉瘤异种移植的生长。华蟾酥毒基降低骨肉瘤异种移植中的 Ki-67 和 MMP9 表达，并促进 caspase-3 表达。