The affect of metformin on autophagy and apoptosis of Echinococcus multilocularis cysts and protoscoleces

doi:10.12140/j.issn.1000-7423.2022.01.006

Abstract

Abstract:

Objective To investigate the affect of different concentrations of metformin on autophagy and apoptosis of Echinococcus multilocularis cysts and protoscoleces cultured in vitro. Methods The E. multilocularis cysts and protoscoleces were co-cultured in vitro with 1, 5, and 10 mmol/L metformin for 24 and 48 h, respectively, while PBS was used in culture as control. The cysts were observed a microscopically for their viability, size and transparency, and the protoscoleces were stained with hematoxylin and eosin, and observed for viability to estimate survival rate. After co-cultured with metformin at 1, 5, and 10 mmol/L for 48 h, the cell apoptosis-related enzymatic activity of caspase3 and caspase9 in protoscoleces were determined using assay kit. The germinal layer cells in the cysts were collected for determination the apoptosis using flow cytometry. Proteins from the cysts and protoscoleces were extracted to analyze the expression of apoptosis related cleaved-caspase3, casapase9, and B-cell lymphoma/leukemia-2 using Western blotting. After the cysts and protoscoleces were co-cultured with 1, 5, 10 mmol/L metformin for 48 h, the changes in expression of adenosine 5′-monophosphate (AMP)-dependent protein kinase (P-AMPK), mammalian rapamycin target protein (mTOR), autophagy-related genes, and autophagy-related protein LC3-Ⅱ, were analyzed using Western blotting. The difference between the two groups was compared by t test, and the difference between multiple groups was compared by analysis of variance. Results After co-culturing of the cysts with metformin at concentration of 1, 5, and 10 mmol/L, the cyst viability gradually decreased with time, the internal structure was disordered, and the turbidity increased. Apoptotic body-like structures were observed in the cysts after 48 h co-culturing with 10 mm/L metformin. After 24 h of co-cultivation of protoscoleces with different concentrations of metformin, the survival rate in the 1, 5, and 10 mmol/L groups was 0.91 ± 0.10, 0.80 ± 0.12, 0.57 ± 0.11, respectively, which are lower than the control group (0.99 ± 0.02) (F = 95.829, P < 0.05). Afrer 48 h, the survival rate of the protoscoleces in the 1, 5, and 10 mmol/L groups was 0.68 ± 0.18, 0.46 ± 0.15, 0.04 ± 0.01, respectively, which are lower than the control group (0.80 ± 0.22) (F = 287.524, P < 0.05). After co-cultivation with 10 mmol/L metformin for 48 h, all the protoscoleces showed red stained. The relative expression levels of caspase3 in the 1, 5, and 10 mmol/L groups were 33.32 ± 2.94, 53.89 ± 1.01, 124.88 ± 26.44, respectively, which were higher than the control group (16.21 ± 6.20) (F = 36.628, P < 0.05). While the relative expression level of caspase9 were 47.48 ± 9.56, 54.50 ± 1.29, 165.09 ± 16.85, respectively, which was higher than that of the control group (25.29 ± 53.560) (F = 120.612, P < 0.05). The apoptosis rates of 1, 5, and 10 mmol/L group were (14.94 ± 1.35)%, (23.85 ± 2.97)%, (33.87 ± 4.93)%, respectively, which were higher than the control group (8.92 ± 1.95)% (F = 293.452, P < 0.05). The relative expression levels of cleaved-caspase3 and caspase9 in the 1, 5, and 10 mmol/L groups were higher than the control group (F = 144.574, 45.675, P < 0.05); the relative expression levels of Bcl-2 were all lower than the control group (F = 39.487, P < 0.05). After co-culturing the cysts with metformin at different concentrations for 48 h, the relative expressions of P-AMPK and mTOR in the 1, 5, and 10 mmol/L groups were higher than the control group (F = 33.342, 60.617, P < 0.05), and the relative expressions of Atg14 and LC3-Ⅱ was lower than the control group (F = 41.688, 41.375, P < 0.05). Following co-cultured of protoscoleces with metformin for 48 h, the relative expressions of P-AMPK, mTOR, Atg14, and LC3-Ⅱ were higher than the control group (F =25.853, 10.695, 12.528, 17.613, P < 0.05). Conclusion Metformin may suppress the viability of E. multilocularis cysts and protoscoleces and accelerate their apoptosis in vitro, showing concentration- and time-dependent pattern. It was demonstrated that AMPK-mTOR signaling pathway may contribute to initiate autophagy of the parasite.

Key words: Alveolar echinococcosis, Metformin, Apoptosis, Autophagy

CLC Number:

532.32

SHAO Han, LI Si-yuan, LI Jun. The affect of metformin on autophagy and apoptosis of Echinococcus multilocularis cysts and protoscoleces[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2022, 40(1): 43-49.

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References 29

[1]	Wen H, Vuitton L, Tuxun T, et al. Echinococcosis: advances in the 21st century[J]. Clin Microbiol Rev, 2019, 32(2): 1-2.
[2]	Siles-Lucas M, Casulli A, Cirilli R, et al. Progress in the pharmacological treatment of human cystic and alveolar echinococcosis: compounds and therapeutic targets[J]. PLoS Negl Trop Dis, 2018, 12(4): e0006422.
[3]	Stojkovic M, Mickan C, Weber TF, et al. Pitfalls in diagnosis and treatment of alveolar echinococcosis: a sentinel case series[J]. BMJ Open Gastroenterol, 2015, 2(1): e000036.
[4]	Spiliotis M, Lechner S, Tappe D, et al. Transient transfection of Echinococcus multilocularis primary cells and complete in vitro regeneration of metacestode vesicles[J]. Int J Parasitol, 2008, 38(8/9): 1025-1039.
[5]	Spiliotis M, Brehm K. Axenic in vitro cultivation of Echinococcus multilocularis metacestode vesicles and the generation of primary cell cultures[J]. Methods Mol Biol, 2009, 470(1): 245-262.
[6]	Flory J, Lipska K. Metformin in 2019[J]. JAMA, 2019, 321(19): 1926-1927.
[7]	Harati R, Vandamme M, Blanchet B, et al. Drug-drug interaction between metformin and sorafenib alters antitumor effect in hepatocellular carcinoma cells[J]. Mol Pharmacol, 2021, 100(1): 32-45.
[8]	Ashrafizadeh M, Mirzaei S, Hushmandi K, et al. Therapeutic potential of AMPK signaling targeting in lung cancer: advances, challenges and future prospects[J]. Life Sci, 2021, 278: 119649.
[9]	Koroglu-Aydn P, Bayrak BB, Bugan I, et al. Histological and biochemical investigation of the renoprotective effects of metformin in diabetic and prostate cancer model[J]. Toxicol Mech Methods, 2021, 31(7): 489-500.
[10]	Alhourani AH, Tidwell TR, Bokil AA, et al. Metformin treatment response is dependent on glucose growth conditions and metabolic phenotype in colorectal cancer cells[J]. Sci Rep, 2021, 11(1): 1-10.
[11]	Wang Z, Guo JJ, Han XQ, et al. Metformin represses the pathophysiology of AAA by suppressing the activation of PI3K/AKT/mTOR/autophagy pathway in ApoE(-/-) mice[J]. Cell Biosci, 2019, 9(1): 1-15.
[12]	Guo LM, Cui J, Wang HR, et al. Metformin enhances anti-cancer effects of cisplatin in meningioma through AMPK-mTOR signaling pathways[J]. Mol Ther Oncolytics, 2021, 20: 119-131.
[13]	Ouyang JY, Parakhia RA, Ochs RS. Metformin activates AMP kinase through inhibition of AMP deaminase[J]. J Biol Chem, 2011, 286(1): 1-11.
[14]	Zheng LY, Yang W, Wu FQ, et al. Prognostic significance of AMPK activation and therapeutic effects of metformin in hepatocellular carcinoma[J]. Clin Cancer Res, 2013, 19(19): 5372-5380.
[15]	Loos JA, Dávila VA, Brehm K, et al. Metformin suppresses development of the Echinococcus multilocularis larval stage by targeting the TOR pathway[J]. Antimicrob Agents Chemother, 2020, 64(9): e01808-e01827.
[16]	Brehm K, Wolf M, Beland H, et al. Analysis of differential gene expression in Echinococcus multilocularis larval stages by means of spliced leader differential display[J]. Int J Parasitol, 2003, 33(11): 1145-1159.
[17]	Hemer S, Brehm K. In vitro efficacy of the anticancer drug imatinib on Echinococcus multilocularis larvae[J]. Int J Antimicrob Agents, 2012, 40(5): 458-462.
[18]	Stadelmann B, Aeschbacher D, Huber C, et al. Profound activity of the anti-cancer drug bortezomib against Echinococcus multilocularis metacestodes identifies the proteasome as a novel drug target for cestodes[J]. PLoS Negl Trop Dis, 2014, 8(12): e3352.
[19]	Cheng Z, Xu Z, Tian H, et al. In vitro and efficacies of inhibitors of the EGFR/MEK/ERK signaling in the treatment of alveolar echinococcosis[J]. Antimicro Agents Chemo, 2020, 64(8): 234-236.
[20]	Saraei P, Asadi I, Kakar MA, et al. The beneficial effects of metformin on cancer prevention and therapy: a comprehensive review of recent advances[J]. Cancer Manag Res, 2019, 11: 3295-3313.
[21]	Sanli T, Steinberg GR, Singh G, et al. AMP-activated protein kinase (AMPK) beyond metabolism[J]. Cancer Biol Ther, 2014, 15(2): 156-169.
[22]	Jia JY, Bissa B, Brecht L, et al. AMPK, a regulator of metabolism and autophagy, is activated by lysosomal damage via a novel galectin-directed ubiquitin signal transduction system[J]. Mol Cell, 2020, 77(5): 951-969.
[23]	Zaidi S, Gandhi J, Joshi G, et al. The anticancer potential of metformin on prostate cancer[J]. Prostate Cancer Prostatic Dis, 2019, 22(3): 351-361.
[24]	Fan H, Yu X, Zou Z, et al. Metformin suppresses the esophageal carcinogenesis in rats treated with NMBzA through inhibiting AMPK/mTOR signaling pathway[J]. Carcinogenesis, 2019, 40(5): 669-679.
[25]	Lee J, Hong EM, Kim JH, et al. Metformin induces apoptosis and inhibits proliferation through the AMP-activated protein kinase and insulin-like growth factor 1 receptor pathways in the bile duct cancer cells[J]. J Cancer, 2019, 10(7): 1734-1744.
[26]	Guillermo M, Mireia NS, Eric HB, et al. Self-consumption: the interplay of autophagy and apoptosis[J]. Nat Rev Mol Cell Biol, 2014, 15(2): 81-90.
[27]	Wang Y, Zhang YF, Feng XM, et al. Metformin inhibits mTOR and c-Myc by decreasing YAP protein expression in OSCC cells[J]. Oncol Rep, 2021, 45(3): 1249-1260.
[28]	Yang HC, Zhang HW, Shi KJ, et al. Autocrine osteopontin promotes the growth and metastasis of Echinococcus multilocu-laris via the EGFR signaling pathway[J]. Chin J Parasitol Parasit Dis, 2021, 39(2): 226-232. (in Chinese)
[28]	(杨海成, 张宏伟, 史康杰, 等. 自分泌骨桥蛋白通过EGFR信号通路促进多房棘球蚴生长和转移的研究[J]. 中国寄生虫学与寄生虫病杂志, 2021, 39(2): 226-232.)
[29]	Tan XW, Yu XF, Jiang HQ, et al. Inhibitory effect of xanthohumol on the growth of Echinococcus multilocularis[J]. Chin J Parasitol Parasit Dis, 2021, 39(3): 304-310. (in Chinese)
[29]	(谭小武, 俞晓凡, 姜慧娇, 等. 黄腐酚对小鼠肝多房棘球蚴生长的抑制作用[J]. 中国寄生虫学与寄生虫病杂志, 2021, 39(3): 304-310.)

蛋白Protein	相对表达量 Relative expression
蛋白Protein	1 mmol/L二甲双胍 1 mmol/L Metformin	5 mmol/L二甲双胍 5 mmol/L Metformin	10 mmol/L二甲双胍 10 mmol/L Metformin	对照组 Control group
囊泡 Vesicles B细胞淋巴瘤/白血病-2 Bcl-2	0.82 ± 0.05	0.89 ± 0.02	0.93 ± 0.04	1.09 ± 0.07
剪切的半胱氨酸肽酶3 Cleaved-caspase3	1.00 ± 0.09	1.34 ± 0.06	1.61 ± 0.05	0.67 ± 0.01
半胱氨酸肽酶9 Caspase9	0.94 ± 0.02	1.03 ± 0.11	0.78 ± 0.03	0.46 ± 0.05
原头节Protoscoleces B细胞淋巴瘤/白血病-2 Bcl-2	1.05 ± 0.12	0.60 ± 0.06	0.84 ± 0.12	1.55 ± 0.12
剪切的半胱氨酸肽酶3 Cleaved-caspase3	0.66 ± 0.09	0.80 ± 0.02	1.01 ± 0.05	0.46 ± 0.07
半胱氨酸肽酶9 Caspase9	0.72 ± 0.02	0.75 ± 0.05	0.97 ± 0.10	0.54 ± 0.07

蛋白 Protein	相对表达量 Relative expression
蛋白 Protein	1 mmol/L二甲双胍 1 mmol/L Metformin	5 mmol/L二甲双胍 5 mmol/L Metformin	10 mmol/L二甲双胍 10 mmol/L Metformin	对照组 Control group
囊泡 Vesicles 蛋白轻链3Ⅱ LC3-Ⅱ	0.66 ± 0.06	0.86 ± 0.13	1.07 ± 0.06	0.75 ± 0.10
自噬基因14 Atg14	1.28 ± 0.21	1.33 ± 0.23	1.44 ± 0.23	0.52 ± 0.14
雷帕霉素机械靶蛋白 mTOR	1.47 ± 0.27	1.42 ± 0.25	1.58 ± 0.31	0.63 ± 0.11
磷酸化的一磷酸腺苷依赖的蛋白激酶 P-AMPK	0.77 ± 0.11	0.83 ± 0.12	0.69 ± 0.11	0.53 ± 0.09
原头节 Protoscoleces 蛋白轻链3Ⅱ LC3-Ⅱ	0.84 ± 0.14	1.45 ± 0.23	1.68 ± 0.24	0.33 ± 0.07
自噬基因14 Atg14	0.89 ± 0.03	1.16 ± 0.10	1.33 ± 0.11	0.55 ± 0.09
雷帕霉素机械靶蛋白 mTOR	1.10 ± 0.02	1.49 ± 0.11	1.25 ± 0.03	0.59 ± 0.06
磷酸化的一磷酸腺苷依赖的蛋白激酶 P-AMPK	1.18 ± 0.09	1.52 ± 0.09	1.27 ± 0.19	0.20 ± 0.01