新藤黄酸调控糖原代谢途径抑制犬骨肉瘤细胞恶性生物学行为的机制研究

doi:10.11843/j.issn.0366-6964.2025.06.042

摘要/Abstract

摘要：

本研究基于肿瘤细胞糖原代谢途径探究新藤黄酸(gambogenic acid，GNA)抑制犬骨肉瘤(osteosarcoma，OS)细胞恶性生物学行为的作用及机制。选用Mckinely犬OS细胞系，通过CCK-8试验确定GNA的给药剂量；通过平板克隆试验探究GNA对犬OS细胞增殖的抑制作用；通过划痕愈合试验、Transwell试验及Western blot试验探究GNA对犬OS细胞迁移及侵袭的抑制作用；通过糖原代谢物试剂盒、酶活力试剂盒、RT-PCR及Western blot试验探究GNA对犬OS糖原代谢途径的影响。通过CCK-8试验确定了后续细胞试验所需剂量，低剂量(0.24 μmol·L^-1)、中剂量(0.28 μmol·L^-1)及高剂量(0.32 μmol·L^-1)；细胞恶性生物学行为相关试验结果表明，GNA呈剂量依赖性地显著抑制犬OS的增殖、迁移及侵袭行为(P < 0.05)；Western blot试验结果表明，GNA呈剂量依赖性地显著抑制Vimentin、MMP-9及Snail蛋白表达(P < 0.05)；糖原代谢检测结果表明，GNA能显著促进犬OS细胞葡萄糖的摄取及细胞内糖原含量的增加，并显著抑制GPa的酶活力；RT-PCR结果显示，GNA显著促进犬OS细胞GLUT1表达(P < 0.05)，但对犬OS细胞HK2及PYGL的表达均无显著影响(P>0.05)；Western blot试验结果与RT-PCR结果基本一致；细胞免疫荧光检测结果表明，GNA能抑制PYGL入核，且PYGL的平均荧光强度随GNA浓度升高而逐渐降低(P < 0.05)。GNA能抑制Mckinely犬OS细胞的增殖、迁移与侵袭且与PYGL所介导的糖原分解途径被抑制相关。

关键词: 犬骨肉瘤, 新藤黄酸, 糖原, 恶性生物学行为

Abstract:

This study investigated the role and mechanism of Gambogenic acid (GNA) in inhibiting the malignant biological behavior of canine osteosarcoma (OS) cells based on tumor cell glycogen metabolism pathway. We used the Mckinely canine OS cell line to determine the dosage of GNA by CCK-8 assay; to investigate the inhibitory effect of GNA on the proliferation of canine OS cells by plate cloning assay; to investigate the inhibitory effect of GNA on the migration and invasion of canine OS cells by scratch healing assay, Transwell assay and Western blot assay; to investigate the effect of GNA on the pathway of glycogen metabolism of canine OS by Glycogen Metabolite Kit, Enzyme activity kit, RT-PCR and Western blot assays to investigate the effect of GNA on the glycogen metabolism pathway of canine OS. The CCK-8 assay was used to determine the doses required for subsequent cell experiments, with low dose of 0.24 μmol·L^-1, medium dose of 0.28 μmol·L^-1 and high dose of 0.32 μmol·L^-1; the results of the experiments related to the malignant biological behaviors of the cells showed that GNA significantly inhibited the proliferation, migration and invasive behaviors of the canine OS in a dose-dependent manner (P < 0.05); the results of the Western blot assay showed that GNA significantly inhibited Vimentin, MMP-9 and Snail protein expression in a dose-dependent manner (P < 0.05); Glycogen metabolism assay showed that GNA significantly promoted glucose uptake and increased intracellular glycogen content in canine OS cells, and significantly inhibited GPa enzyme activity; RT-PCR results showed that GNA significantly promoted the expression of GLUT1 in canine OS cells (P < 0.05); and the results of Western blot assay showed that GNA significantly inhibited GLUT1 expression in canine OS cells (P < 0.05), but had no significant effect on the expression of HK2 and PYGL in canine OS cells (P>0.05); the results of Western blot assay were basically the same as those of RT-PCR; the results of cellular immunofluorescence assay showed that GNA inhibited the entry of PYGL into the nucleus, and the average fluorescence intensity of PYGL was gradually decreased with the increase of the concentration of GNA (P < 0.05). GNA inhibited proliferation, migration and invasion of Mckinely canine OS cells and was associated with inhibition of the PYGL-mediated glycogenolysis pathway.

Key words: canine osteosarcoma, gambogenic acid, glycogen, malignant biological behavior

中图分类号:

康慧杰, 沙季辰, 杨天元, 张云彤, 侯晓昱, 李思瑶, 王维千, 侯庆典, 张帅, 杨昊天, 赵元, 范宏刚. 新藤黄酸调控糖原代谢途径抑制犬骨肉瘤细胞恶性生物学行为的机制研究[J]. 畜牧兽医学报, 2025, 56(6): 3002-3013.

KANG Huijie, SHA Jichen, YANG Tianyuan, ZHANG Yuntong, HOU Xiaoyu, LI Siyao, WANG Weiqian, HOU Qingdian, ZHANG Shuai, YANG Haotian, ZHAO Yuan, FAN Honggang. Mechanistic Studies on the Inhibition of Malignant Biological Behavior of Canine Osteosarcoma Cells by Gambogenic Acid Regulated Glycogen Metabolism Pathway[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(6): 3002-3013.

图/表 12

表 1

图 1

图 2

图 3

图 4

图 5

图 6

图 7

图 8

图 9

图 10

图 11

参考文献 36

1	RAFALKO J M , KRUGLYAK K M , MCCLEARY-WHEELER A L , et al. Age at cancer diagnosis by breed, weight, sex, and cancer type in a cohort of more than 3, 000 dogs: Determining the optimal age to initiate cancer screening in canine patients[J]. PLoS One, 2023, 18 (2): e0280795. doi: 10.1371/journal.pone.0280795
2	ROMANUCCI M , DE MARIA R , MORELLO E M , et al. Editorial: Canine osteosarcoma as a model in comparative oncology: Advances and perspective[J]. Front Vet Sci, 2023, 10, 1141666. doi: 10.3389/fvets.2023.1141666
3	BECK J , REN L , HUANG S , et al. Canine and murine models of osteosarcoma[J]. Vet Pathol, 2022, 59 (3): 399- 414. doi: 10.1177/03009858221083038
4	WILK S S , ZABIELSKA-KOCZYW S K A . Molecular mechanisms of canine osteosarcoma metastasis[J]. Int J Mol Sci, 2021, 22 (7): 3639. doi: 10.3390/ijms22073639
5	SAMUELS S K , COOK M R , GREEN E , et al. Case report: Metastatic parosteal osteosarcoma in a dog[J]. Front Vet Sci, 2021, 8, 715908. doi: 10.3389/fvets.2021.715908
6	BREHM A , WILSON-ROBLES H , MILLER T , et al. Feasibility and safety of whole lung irradiation in the treatment of canine appendicular osteosarcoma[J]. Vet Comp Oncol, 2022, 20 (1): 20- 28. doi: 10.1111/vco.12702
7	WEINMAN M A , RAMSEY S A , LEEPER H J , et al. Exosomal proteomic signatures correlate with drug resistance and carboplatin treatment outcome in a spontaneous model of canine osteosarcoma[J]. Cancer Cell Int, 2021, 21 (1): 245. doi: 10.1186/s12935-021-01943-7
8	JIA Z , ZHU X , ZHOU Y , et al. Polypeptides from traditional Chinese medicine: Comprehensive review of perspective towards cancer management[J]. Int J Biol Macromol, 2024, 260 (Pt 1): 129423.
9	YANG C , LI D , KO C N , et al. Active ingredients of traditional Chinese medicine for enhancing the effect of tumor immunotherapy[J]. Front Immunol, 2023, 14, 1133050. doi: 10.3389/fimmu.2023.1133050
10	WANG Y , ZHANG X , WANG Y , et al. Application of immune checkpoint targets in the anti-tumor novel drugs and traditional Chinese medicine development[J]. Acta Pharm Sin B, 2021, 11 (10): 2957- 2972. doi: 10.1016/j.apsb.2021.03.004
11	高慧敏, 彭代银, 王雷, 等. 藤黄化学成分和药理作用的研究进展及其质量标志物(Q-Marker)预测分析[J]. 中草药, 2023, 54 (8): 2668- 2685.
	GAO H M , PENG D Y , WANG L , et al. Research progress on chemical composition and pharmacological effects of gamboge and predictive analysis on quality marker[J]. Chinese Traditional and Herbal Drugs, 2023, 54 (8): 2668- 2685.
12	MI L , XING Z , ZHANG Y , et al. Unveiling gambogenic acid as a promising antitumor compound: A review[J]. Planta Med, 2024, 90 (5): 353- 367. doi: 10.1055/a-2258-6663
13	LIU C , XU J , GUO C , et al. Gambogenic acid induces endoplasmic reticulum stress in colorectal cancer via the aurora A pathway[J]. Front Cell Dev Biol, 2021, 9, 736350. doi: 10.3389/fcell.2021.736350
14	WANG M , TU Y , LIU C , et al. Gambogenic acid inhibits invasion and metastasis of melanoma through regulation of lncRNA MEG3[J]. Biol Pharm Bull, 2023, 46 (10): 1385- 1393. doi: 10.1248/bpb.b23-00156
15	AN F , CHANG W , SONG J , et al. Reprogramming of glucose metabolism: Metabolic alterations in the progression of osteosarcoma[J]. J Bone Oncol, 2024, 44, 100521. doi: 10.1016/j.jbo.2024.100521
16	FU J Y , HUANG S J , WANG B L , et al. Lysine acetyltransferase 6A maintains CD4(+) T cell response via epigenetic reprogramming of glucose metabolism in autoimmunity[J]. Cell Metab, 2024, 36 (3): 557- 574. doi: 10.1016/j.cmet.2023.12.016
17	LIU J , CAO X . Glucose metabolism of TAMs in tumor chemoresistance and metastasis[J]. Trends Cell Biol, 2023, 33 (11): 967- 978. doi: 10.1016/j.tcb.2023.03.008
18	DAUER P , LENGYEL E . New Roles for Glycogen in Tumor Progression[J]. Trends Cancer, 2019, 5 (7): 396- 399. doi: 10.1016/j.trecan.2019.05.003
19	FAVARO E , BENSAAD K , CHONG M G , et al. Glucose utilization via glycogen phosphorylase sustains proliferation and prevents premature senescence in cancer cells[J]. Cell Metab, 2012, 16 (6): 751- 764. doi: 10.1016/j.cmet.2012.10.017
20	LIU Q , LI J , ZHANG W , et al. Glycogen accumulation and phase separation drives liver tumor initiation[J]. Cell, 2021, 184 (22): 5559- 5576. doi: 10.1016/j.cell.2021.10.001
21	YANG Y T , ENGLEBERG A I , YUZBASIYAN-GURKAN V . Establishment and Characterization of cell lines from canine metastatic osteosarcoma[J]. Cells, 2023, 13 (1): 25. doi: 10.3390/cells13010025
22	CHEN X , ZHANG X , CAI H , et al. Targeting USP9x/SOX2 axis contributes to the anti-osteosarcoma effect of neogambogic acid[J]. Cancer Lett, 2020, 469, 277- 286. doi: 10.1016/j.canlet.2019.10.015
23	WEISS M C , EULO V , VAN TINE B A . Truly man's best friend: Canine cancers drive drug repurposing in osteosarcoma[J]. Clin Cancer Res, 2022, 28 (4): 571- 572. doi: 10.1158/1078-0432.CCR-21-3471
24	GIRI S , ALLEN K J H , PRABAHARAN C B , et al. Initial insights into the interaction of antibodies radiolabeled with lutetium-177 and actinium-225 with tumor microenvironment in experimental human and canine osteosarcoma[J]. Nucl Med Biol, 2024, 134-135, 108917. doi: 10.1016/j.nucmedbio.2024.108917
25	MASON N J . Comparative immunology and immunotherapy of canine osteosarcoma[J]. Adv Exp Med Biol, 2020, 1258, 199- 221.
26	胥辉豪, 刘江渝, 李启卷, 等. 己糖激酶2在犬乳腺肿瘤中的表达及预后研究[J]. 畜牧兽医学报, 2023, 54 (3): 1310- 1324. doi: 10.11843/j.issn.0366-6964.2023.03.041
	XU H H , LIU J Y , LI Q J , et al. Expression and prognosis of Hexokinase 2 in canine mammary tumors[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54 (3): 1310- 1324. doi: 10.11843/j.issn.0366-6964.2023.03.041
27	CHEN B , HONG Y , GUI R , et al. N6-methyladenosine modification of circ_0003215 suppresses the pentose phosphate pathway and malignancy of colorectal cancer through the miR-663b/DLG4/G6PD axis[J]. Cell Death Dis, 2022, 13 (9): 804. doi: 10.1038/s41419-022-05245-2
28	YANG E , WANG X , GONG Z , et al. Exosome-mediated metabolic reprogramming: the emerging role in tumor microenvironment remodeling and its influence on cancer progression[J]. Signal Transduct Target Ther, 2020, 5 (1): 242. doi: 10.1038/s41392-020-00359-5
29	YOUNG L E A , CONROY L R , CLARKE H A , et al. In situ mass spectrometry imaging reveals heterogeneous glycogen stores in human normal and cancerous tissues[J]. EMBO Mol Med, 2022, 14 (11): e16029. doi: 10.15252/emmm.202216029
30	REN L K , LU R S , FEI X B , et al. Unveiling the role of PYGB in pancreatic cancer: a novel diagnostic biomarker and gene therapy target[J]. J Cancer Res Clin Oncol, 2024, 150 (3): 127. doi: 10.1007/s00432-024-05644-2
31	JIANG L , LIU S , DENG T , et al. Analysis of the expression, function and signaling of glycogen phosphorylase isoforms in hepatocellular carcinoma[J]. Oncol Lett, 2022, 24 (2): 244. doi: 10.3892/ol.2022.13364
32	ZOIS C E , HENDRIKS A M , HAIDER S , et al. Liver glycogen phosphorylase is upregulated in glioblastoma and provides a metabolic vulnerability to high dose radiation[J]. Cell Death Dis, 2022, 13 (6): 573. doi: 10.1038/s41419-022-05005-2
33	JI Q , LI H , CAI Z , et al. PYGL-mediated glucose metabolism reprogramming promotes EMT phenotype and metastasis of pancreatic cancer[J]. Int J Biol Sci, 2023, 19 (6): 1894- 1909. doi: 10.7150/ijbs.76756
34	CHEN Y F , ZHU J J , LI J , et al. O-GlcNAcylation increases PYGL activity by promoting phosphorylation[J]. Glycobiology, 2022, 32 (2): 101- 109. doi: 10.1093/glycob/cwab114
35	ZHAO X , WANG C , ZHAO L , et al. HBV DNA polymerase regulates tumor cell glycogen to enhance the malignancy of HCC cells[J]. Hepatol Commun, 2024, 8 (3): e0387.
36	XU J , LIU X , LIU X , et al. Long noncoding RNA KCNMB2-AS1 promotes the development of esophageal cancer by modulating the miR-3194-3p/PYGL axis[J]. Bioengineered, 2021, 12 (1): 6687- 6702. doi: 10.1080/21655979.2021.1973775

基因 Gene	序列(5′→3′) Sequence
基因 Gene	上游引物Upstream primer	下游引物Downstream primer
ACTB(β-actin)	GGAACCACCATGTACCCTGG	TGCTGTCACCTTCTCCGTTC
SLC2A1(GLUT1)	AAGGAGGAGAGTCGGCAGAT	GACGTACGGACCACACAGTT
HK2	GTAACAGACAACGGGCTCCA	GTCATCGAGTATGCCGTCGT
PYGL	ATCGCCAATGTGAAGCAGGA	TCCTCTGCCATTTCCACGTT

[1]	郑艺蕾, 沈冯婷, 辛良, 袁希望, 林琳, 曹迪, 吴旧生, 师福山, 王华南. 一例猫鼻神经内分泌恶性肿瘤的病理和免疫组化分析[J]. 畜牧兽医学报, 2025, 56(6): 2948-2956.
[2]	李雪源, 杨利峰, 赵德明. 犬卵巢肿瘤病理诊断及分析[J]. 畜牧兽医学报, 2025, 56(5): 2393-2402.
[3]	张诣, 徐婕, 宋晓愿, 周世伟, 滕雨萌, 刘晓丽, 程国富, 谷长勤, 张万坡, 胡薛英. 武汉地区犬乳腺肿瘤的临床病理特征与良恶性的相关性分析[J]. 畜牧兽医学报, 2024, 55(9): 4121-4130.
[4]	徐禧莹, 王毅恒, 区倩婷, 洪琳媛, 刘栩靖, 卢羡盈, 贾坤. 沉默PREX1表达对CHMp细胞增殖及侵袭性的影响[J]. 畜牧兽医学报, 2024, 55(8): 3706-3713.
[5]	涂颖欣, 周向梅, 赵德明, 杨利峰. 119例犬原发性睾丸肿瘤的回顾性病理学分析[J]. 畜牧兽医学报, 2024, 55(7): 3177-3184.
[6]	史泽风, 李翎旭, 郭译文, 廖云, 孙昭宇, 王来荣, 杨德吉, 姚大伟. 基于高分辨率熔解曲线检测MD肿瘤微卫星不稳定性[J]. 畜牧兽医学报, 2024, 55(2): 759-769.
[7]	戴悦欣, 杨利峰, 赵德明, 周向梅. 犬猫牙源性肿瘤的回顾性病理学分析[J]. 畜牧兽医学报, 2023, 54(10): 4372-4378.
[8]	张琰, 刘佳悦, 吴梅金, 周家豪, 刁洪秀. 非编码RNA作为犬肿瘤潜在生物标志物的研究进展[J]. 畜牧兽医学报, 2023, 54(6): 2264-2271.
[9]	任晓丽, 范玉营, 皇甫和平, 刘云, 石冬梅. GSK126对犬乳腺肿瘤细胞上皮间质转化的影响[J]. 畜牧兽医学报, 2023, 54(4): 1721-1729.
[10]	胥辉豪, 刘江渝, 李启卷, 郑小波, 林珈好, 金艺鹏, 林德贵. 己糖激酶2在犬乳腺肿瘤中的表达及预后研究[J]. 畜牧兽医学报, 2023, 54(3): 1310-1324.
[11]	吴居业, 卢宝春, 祝宇凡, 贾坤. 血小板凝血酶蛋白1对犬乳腺肿瘤细胞体外生物学特性的影响分析[J]. 畜牧兽医学报, 2022, 53(12): 4470-4481.
[12]	李妍, 蓝婷英, 庞博, 杨晓农, 黄坚. NLRP3炎症小体及其下游炎症因子在犬乳腺肿瘤组织中的表达[J]. 畜牧兽医学报, 2022, 53(4): 1252-1258.
[13]	邓朝阳, 王峥, 张佳韧, 何希君, 郑家三. 1例猫肾上腺皮质癌脾转移的病理及免疫组化分析[J]. 畜牧兽医学报, 2021, 52(12): 3669-3674.
[14]	潘启东, 曾显成, 杨彬偲, 刘庆华, 徐泉明, 陈吉龙. 山羊地方性鼻内肿瘤病毒全基因组序列分析及其gag基因促肿瘤发生的机制研究[J]. 畜牧兽医学报, 2021, 52(11): 3165-3174.
[15]	陈梦月, 张羽晨, 娄江城, 罗传真, 赵红利, 刘晓丽, 胡薛英. 26例犬毛囊肿瘤的组织病理学诊断和免疫组织化学分析[J]. 畜牧兽医学报, 2021, 52(5): 1407-1413.