可利霉素通过抑制AXL/c-Met/c-Myc信号轴调控肝内胆管细胞癌细胞恶性行为的机制

doi:10.3969/j.issn.1006-5725.2025.15.003

摘要/Abstract

摘要：

目的通过体外实验探究可利霉素（carrimycin， CAM）对肝内胆管细胞癌HuCCT1细胞生物学功能的影响，并初步探讨其作用机制。方法选择肝内胆管细胞癌HuCCT1细胞系，使用CCK-8试剂盒检测CAM对细胞活力的影响，并计算IC₅₀浓度；通过划痕实验验证CAM对细胞迁移能力的影响；使用平板克隆实验检测CAM对细胞克隆形成能力的影响；Transwell侵袭实验检测CAM对细胞侵袭能力的影响；流式细胞术分析CAM对细胞周期进程的影响；WB实验检测CAM对上皮-间充质转化、细胞周期等关键蛋白的表达情况的影响；通过Western blot实验检测CAM对AXL/c-Myc/c-Met信号轴表达情况的影响。结果与对照组相比，CAM能显著抑制HuCCT1细胞的增殖能力，并且呈浓度依赖性（P < 0.05）；平板克隆结果显示，CAM能显著抑制HuCCT1细胞克隆形成能力（P < 0.05）；划痕实验证实经CAM处理后，HuCCT1细胞迁移速度减缓，并且迁移面积变小（P < 0.05）。经CAM处理的HuCCT1细胞G₀/G₁期占比上调，S期下调（P < 0.05），进一步通过Western blot实验证实，G₁/S期转化关键蛋白CCND1和CDK4表达下调，p21表达上调（P < 0.05）。Transwell侵袭实验结果提示，CAM能抑制HuCCT1细胞侵袭能力；Western blot实验结果提示E-Cadherin表达上调（P＜0.05），N-Cadherin以及Vimentin表达下调（P < 0.05）。通过Western blot实验验证经AXL重组蛋白处理后的HuCCT1细胞AXL、c-Met和c-Myc的表达上调（P < 0.05），而经CAM联合AXL重组蛋白共同处理后HuCCT1细胞的上述蛋白表达均下调（P < 0.05）。结论 CAM可以抑制肝内胆管细胞癌HuCCT1细胞的增殖、迁移以及侵袭等过程从而发挥抗肿瘤特性，这种作用可能是通过AXL /c-Met/c-Myc介导的。

关键词: 肝内胆管细胞癌, 细胞增殖, 细胞周期, 上皮-间充质转化

Abstract:

Objective The effects of carrimycin （CAM） on the biological functions of intrahepatic cholangiocarcinoma HuCCT1 cells were examined through in vitro experiments， and a preliminary investigation was conducted into its mechanism of action. Methods The intrahepatic cholangiocarcinoma cell line HuCCT1 was selected for the study. The effect of CAM on cell viability was assessed using the CCK-8 assay， and the IC₅₀ concentration was determined accordingly. The impact of CAM on cell migration was evaluated through a scratch wound healing assay. In addition， the effect of CAM on clonogenic ability was examined using a colony formation assay. Cell invasion capacity was assessed using a Transwell invasion assay. Flow cytometry was employed to analyze the effect of CAM on cell cycle progression. Furthermore， Western blotting was conducted to evaluate the expression levels of key proteins associated with epithelial-mesenchymal transition and the cell cycle. Finally， the influence of CAM on the AXL/c-Myc/c-Met signaling axis was also investigated. Results Compared with the control group， CAM significantly inhibited the proliferation of HuCCT1 cells in a concentration-dependent manner （P < 0.05）. Plate cloning assays demonstrated that CAM markedly suppressed the colony-forming ability of HuCCT1 cells （P < 0.05）. Scratch wound healing assays confirmed that CAM treatment significantly reduced the migration speed and narrowed the migration area of HuCCT1 cells （P < 0.05）. Flow cytometry analysis revealed that CAM treatment led to a significant increase in the proportion of cells in the G₀/G₁ phase and a decrease in the S phase （P < 0.05）. Western blot analysis further confirmed that the expression levels of key regulatory proteins CCND1 and CDK4， which are involved in the G_1/S phase transition， were down-regulated， while the expression of p21 was up-regulated （P < 0.05）. Transwell invasion assays indicated that CAM inhibited the invasive capacity of HuCCT1 cells. Consistently， Western blot results showed that E-Cadherin expression was increased （P < 0.05）， whereas the expression levels of N-Cadherin and Vimentin were decreased （P < 0.05）. Moreover， Western blot analysis verified that the expression of AXL， c-Met， and c-Myc was up-regulated in HuCCT1 cells treated with AXL recombinant protein （P < 0.05）. However， co-treatment with CAM and AXL recombinant protein significantly attenuated the expression of these proteins （P < 0.05）. Conclusions CAM inhibits the proliferation， migration， and invasion of intrahepatic cholangiocarcinoma HuCCT1 cells， thereby demonstrating antitumor effects， which may be associated with the AXL/c-Met/c-Myc signaling pathway.

Key words: intrahepatic cholangiocarcinoma, cell proliferation, cell cycle, epithelial-mesenchymal transition

中图分类号:

R730.5

王童舒,周敏,刘慧敏,朴红心. 可利霉素通过抑制AXL/c-Met/c-Myc信号轴调控肝内胆管细胞癌细胞恶性行为的机制[J]. 实用医学杂志, 2025, 41(15): 2304-2310.

Tongshu WANG,Min ZHOU,Huimin LIU,Hongxin PIAO. Carrimycin modulates malignant behavior of intrahepatic cholangiocarcinoma cells through inhibition of the AXL/c⁃Met/c⁃Myc signaling axis[J]. The Journal of Practical Medicine, 2025, 41(15): 2304-2310.

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项目	对照组	4 μg/mL CAM
24 h迁移率	27.68 ± 1.32	12.43 ± 1.38^*
48 h迁移率	69.39 ± 1.21	44.58 ± 1.25^*
72 h迁移率	82.53 ± 0.84	54.04 ± 1.03^*

组别	CCND1	CDK4	p21
对照组	0.19 ± 0.01	0.23 ± 0.01	0.13 ± 0.01
4 μg/mL CAM	0.14 ± 0.02^*	0.15 ± 0.02^*	0.20 ± 0.01^*

组别	E-Cadherin	N-Cadherin	Vimentin
对照组	0.26 ± 0.02	0.35 ± 0.01	0.49 ± 0.02
4 μg/mL CAM	0.32 ± 0.01^?	0.27 ± 0.03^?	0.35 ± 0.01^?

组别	AXL	c-Met	c-Myc
对照组	0.25 ± 0.01	0.16 ± 0.01	0.41 ± 0.01
rAXL	0.32 ± 0.01^*	0.26 ± 0.01^*	0.92 ± 0.01^*
CAM+rAXL	0.27 ± 0.02^#	0.19 ± 0.01^#	0.67 ± 0.02^#