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

Abstract

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

CLC Number:

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.

Figures/Tables 12

Table 1

Fig. 1

Fig. 2

Fig. 3

Fig. 4

Fig. 5

Fig. 6

Fig. 7

Fig. 8

Fig. 9

Fig. 10

Fig. 11

References 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]	ZHENG Yilei, SHEN Fengting, XIN Liang, YUAN Xiwang, LIN Lin, CAO Di, WU Jiusheng, SHI Fushan, WANG Huanan. Pathological and Immunohistochemical Analysis of A Case of Feline Nasal Malignant Neuroendocrine Neoplasm [J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(6): 2948-2956.
[2]	LI Xueyuan, YANG Lifeng, ZHAO Deming. Pathologic Diagnosis and Analysis of Ovarian Tumors in Dogs [J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(5): 2393-2402.
[3]	Yi ZHANG, Jie XU, Xiaoyuan SONG, Shiwei ZHOU, Yumeng TENG, Xiaoli LIU, Guofu CHENG, Changqin GU, Wanpo ZHANG, Xueying HU. Clinico-histopathological Traits of Canine Mammary Tumors in Wuhan Area and Their Correlation with Benign and Malignant Tumors [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(9): 4121-4130.
[4]	Xiying XU, Yiheng WANG, Qianting OU, Linyuan HONG, Xujing LIU, Xianying LU, Kun JIA. Effects of Silencing PREX1 Expression on Proliferation and Invasiveness of CHMp [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(8): 3706-3713.
[5]	Yingxin TU, Xiangmei ZHOU, Deming ZHAO, Lifeng YANG. Canine Testicular Tumors: a Retrospective Pathological Study of 119 Cases [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(7): 3177-3184.
[6]	SHI Zefeng, LI Lingxu, GUO Yiwen, LIAO Yun, SUN Zhaoyu, WANG Lairong, YANG Deji, YAO Dawei. Detection of Microsatellite Instability in Marek's Disease Tumor based on High-resolution Melting [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(2): 759-769.
[7]	DAI Yuexin, YANG Lifeng, ZHAO Deming, ZHOU Xiangmei. A Retrospective Pathological Study of Odontogenic Tumors in Dogs and Cats [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(10): 4372-4378.
[8]	ZHANG Yan, LIU Jiayue, WU Meijin, ZHOU Jiahao, DIAO Hongxiu. Research Progress of Non-coding RNA as A Potential Biomarker for Canine Tumors [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(6): 2264-2271.
[9]	REN Xiaoli, FAN Yuying, HUANGFU Heping, LIU Yun, SHI Dongmei. Effect of GSK126 on Epithelial-mesenchymal Transition of Canine Mammary Tumor Cells [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(4): 1721-1729.
[10]	XU Huihao, LIU Jiangyu, LI Qijuan, ZHENG Xiaobo, LIN Jiahao, JIN Yipeng, LIN Degui. Expression and Prognosis of Hexokinase 2 in Canine Mammary Tumors [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(3): 1310-1324.
[11]	WU Juye, LU Baochun, ZHU Yufan, JIA Kun. The Effects of Thrombospondin 1 on the Biological Characteristics of Canine Mammary Tumor Cells in vitro [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(12): 4470-4481.
[12]	LI Yan, LAN Tingying, PANG Bo, YANG Xiaonong, HUANG Jian. Expression of NLRP3 Inflammasome Signal Associated Genes in Canine Mammary Tumors and Clinical Significance [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(4): 1252-1258.
[13]	DENG Zhaoyang, WANG Zheng, ZHANG Jiaren, HE Xijun, ZHENG Jiasan. Pathological and Immunohistochemical Analysis of Splenic Metastasis of Adrenal Cortical Carcinoma in a Cat [J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(12): 3669-3674.
[14]	PAN Qidong, ZENG Xiancheng, YANG Bincai, LIU Qinghua, XU Quanming, CHEN Jilong. Analysis of Complete Genomic Sequences of Enzootic Nasal Tumor Virus 2 and Role of Its gag Gene in Tumorigenesis [J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(11): 3165-3174.
[15]	CHEN Mengyue, ZHANG Yuchen, LOU Jiangcheng, LUO Chuanzhen, ZHAO Hongli, LIU Xiaoli, HU Xueying. Histopathological Diagnosis and Immunohistochemical Analysis of 26 Cases of Canine Hair Follicle Tumors [J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(5): 1407-1413.

Mechanistic Studies on the Inhibition of Malignant Biological Behavior of Canine Osteosarcoma Cells by Gambogenic Acid Regulated Glycogen Metabolism Pathway

RichHTML

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

Figures/Tables 12

References 36

Related Articles 15

Recommended Articles

Metrics

Comments