In vitro activity of a histone H3 methyltransferase inhibitor BIX-01294 against Echinococcus granulosus

doi:10.12140/j.issn.1000-7423.2026.03.002

Abstract

Abstract:

Objective To investigate the effects of BIX-01294, a histone H3 methyltransferase inhibitor, on the viability of protoscoleces and metacestode vesicles of Echinococcus granulosus. Methods Liver samples were collected from E. granulosus-infected sheep in local slaughterhouses. E. granulosus protoscoleces were isolated under sterile conditions and exposed to BIX-01294 solutions (dissolved in DMSO) at concentrations of 1, 5, 10, 25, and 50 µmol/L, while a solvent control group (DMSO group) served as controls. Protoscoleces were collected at 3, 6, 9, 12, and 15 days post-exposure and stained with methylene blue to observe viability. The survival rate of protoscoleces was calculated, and a survival curve was plotted to estimate the area under the survival curve (AUC) was determined using the trapezoidal rule. Protoscoleces were collected following exposure to 25 µmol/L BIX-01294 for 12 days and stained with hematoxylin and eosin (HE) to observe post-exposure changes. Protoscoleces exposed to 1 and 25 µmol/L BIX-01294 for 1, 6, 9, and 12 days were collected and stained with Caspase-3/7 dyes to assess the apoptosis of protoscoleces. The expression of H3K9me2 was determined using Western blotting in protoscoleces exposed to 25 µmol/L BIX-01294 for 12 days. The morphology of vesicles was observed under a microscope following exposure to 25 µmol/L BIX-01294 in vitro for 1, 3, 6, 9, and 12 days, and ultrastructural changes in vesicles were examined using scanning electron microscopy following exposure to BIX-01294 for 9 days. Results The viability of protoscoleces was (36.45 ± 7.14)% on day 6 post-treatment with 25 µmol/L BIX-01294, which significantly reduced as compared with that [(90.21 ± 3.72)%] on day 3 post-treatment (t = 15.86, P < 0.01), and the viability of protoscoleces was (71.91 ± 7.92)% on day 3 post-treatment with 50 µmol/L BIX-01294, which significantly reduced as compared with that (100%) on day 0 post-treatment (t = 6.25, P < 0.01), while the viability was (5.64 ± 1.48)% on day 6 post-treatment with 50 µmol/L BIX-01294, which continued to reduce as compared with that on day 3 post-treatment (t = 15.89, P < 0.01). The AUC values of BIX-01294 decreased in a dose-dependent manner with increasing concentrations, with the lowest AUC seen in the 50 µmol/L BIX-01294 treatment group (395.25%·d). Microscopy displayed shrinkage of protoscoleces, shedding of rostellum and hooklets, reduction of calcium granules and decreased motility following treatment with 50 µmol/L BIX-01294 for 6 days, and HE staining revealed loose, cavitary and irregular protoscoleces with a reduced volume, and thinned and incomplete body wall following treatment with 25 µmol/L BIX-01294 for 12 days. The highest Caspase-3/7 fluorescence intensity was recorded in protoscoleces exposed to 25 µmol/L BIX-01294 for 12 days. Western blotting assay determined lower H3K9me2 expression in protoscoleces treated with BIX-01294 for 12 days than in the DMSO group [(0.53 ± 0.08) vs. (1.00 ± 0.17); t = 4.226, P < 0.05]. Collapse or shrinkage of vesicles was found since day 6 following treatment with 25 µmol/L BIX-01294. Scanning electron microscopy displayed remarkable ultrastructural alterations, separation of germinal layer and cuticle and disorganization of the germinal layer structure in vesicles 9 days post-treatment with BIX-01294. Conclusion BIX-01294 exhibits a remarkable in vitro activity against E. granulosus protoscoleces and vesicles in a time- and concentration-dependent manner, accompanied by reduced H3K9me2 expression and upregulation of apoptosis-related signals, indicating that BIX-01294 has a potential value against echinococcosis.

Key words: Echinococcus granulosus, Histone H3 methyltransferase, BIX-01294

CLC Number:

R383.33

GE Conghui, ABIDAN Ainiwaer, XIAO Wenying, TANG Na, SUN Sheng, WANG Mengying, GAO Yi, AYINAER Jiensi, HU Qiu, LI Jing, WANG Hui, ZHANG Chuanshan. In vitro activity of a histone H3 methyltransferase inhibitor BIX-01294 against Echinococcus granulosus[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2026, 44(3): 320-327.

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References 26

[1]	Wen H, Vuitton L, Tuxun T, et al. Echinococcosis: advances in the 21st century[J]. Clin Microbiol Rev, 2019, 32(2): e00075-e00018.
[2]	WHO. WHO guidelines for the treatment of patients with cystic echinococcosis. Web Annex B. Evidence-to-decision framework[M]. World Health Organization, 2025.
[3]	Wen LM, Lv GD, Zhao J, et al. In vitro and in vivo effects of artesunate on Echinococcus granulosus protoscoleces and metacestodes[J]. Drug Des Dev Ther, 2020, 14: 4685-4694.
[4]	Tawfeek GM, Fahmy HM, Faried ES, et al. Genotoxic and protoscolicidal effects of Gallic acid and its nanoformulation against Echinococcus granulosus protoscoleces: in vitro and ex vivo insights from Egypt[J]. Vet Parasitol, 2026, 343: 110731.
[5]	Kaethner M, Preza M, Kaempfer T, et al. Establishment and application of unbiased in vitro drug screening assays for the identification of compounds against Echinococcus granulosus sensu stricto[J]. PLoS Negl Trop Dis, 2023, 17(8): e0011343.
[6]	Saha N, Muntean AG. Insight into the multi-faceted role of the SUV family of H3K9 methyltransferases in carcinogenesis and cancer progression[J]. Biochim Biophys Acta BBA Rev Cancer, 2021, 1875(1): 188498.
[7]	Ni YC, Shi MC, Liu LL, et al. G9a in cancer: mechanisms, therapeutic advancements, and clinical implications[J]. Cancers, 2024, 16(12): 2175.
[8]	Padeken J, Methot SP, Gasser SM. Establishment of H3K9-methylated heterochromatin and its functions in tissue differentiation and maintenance[J]. Nat Rev Mol Cell Biol, 2022, 23(9): 623-640.
[9]	Haebe JR, Bergin CJ, Sandouka T, et al. Emerging role of G9a in cancer stemness and promises as a therapeutic target[J]. Oncogenesis, 2021, 10(11): 76.
[10]	Jiang LB, López-Barragán MJ, Jiang HY, et al. Epigenetic control of the variable expression of a Plasmodium falciparum receptor protein for erythrocyte invasion[J]. Proc Natl Acad Sci U S A, 2010, 107(5): 2224-2229.
[11]	Sundriyal S, Malmquist NA, Caron J, et al. Development of diaminoquinazoline histone lysine methyltransferase inhibitors as potent blood-stage antimalarial compounds[J]. Chem MedChem, 2014, 9(10): 2360-2373.
[12]	Xu LY, Gao X, Sang W, et al. EHMT2 inhibitor BIX-01294 induces endoplasmic reticulum stress mediated apoptosis and autophagy in diffuse large B cell lymphoma cells[J]. Blood, 2019, 134: 2787.
[13]	Zhang CS, Li J, Aji T, et al. Identification of functional MKK3/6 and MEK1/2 homologs from Echinococcus granulosus and investigation of protoscolecidal activity of mitogen-activated protein kinase signaling pathway Inhibitors in vitro and in vivo[J]. Antimicrob Agents Chemother, 2019, 63: e01043-e01018.
[14]	Musabyimana JP, Musa S, Manti J, et al. The Plasmodium falciparum histone methyltransferase SET10 participates in a chromatin modulation network crucial for intraerythrocytic development[J]. mSphere, 2024, 9(11): e00495-e00424.
[15]	Gissot M, Kelly KA, Ajioka JW, et al. Epigenomic modifications predict active promoters and gene structure in Toxoplasma gondii[J]. PLoS Pathog, 2007, 3(6): e77.
[16]	Appetecchia F, Fabbrizi E, Fiorentino F, et al. Transmission-blocking strategies for malaria eradication: recent advances in small-molecule drug development[J]. Pharmaceuticals, 2024, 17(7): 962.
[17]	Ngwa CJ, Kiesow MJ, Orchard LM, et al. The G9a histone methyltransferase inhibitor BIX-01294 modulates gene expression during Plasmodium falciparum gametocyte development and transmission[J]. Int J Mol Sci, 2019, 20(20): 5087.
[18]	Malmquist NA, Sundriyal S, Caron J, et al. Histone methyltransferase inhibitors are orally bioavailable, fast-acting molecules with activity against different species causing malaria in humans[J]. Antimicrob Agents Chemother, 2015, 59(2): 950-959.
[19]	Yason JA, Koh KARP, Tan KSW. Viability screen of LOPAC¹²⁸⁰ Reveals phosphorylation inhibitor auranofin as a potent inhibitor of Blastocystis subtype 1, 4, and 7 isolates[J]. Antimicrob Agents Chemother, 2018, 62(8): e00208-e00218.
[20]	Liu SK, Jiang YH, Yang H, et al. BIX-01294 enhances the effect of chemotherapy on colorectal cancer by inhibiting the expression of stemness genes[J]. Biochem Biophys Res Commun, 2022, 590: 169-176.
[21]	Smith SA, Richards KS. Ultrastructure and microanalyses of the calcareous corpuscles of the protoscoleces of Echinococcus granulosus[J]. Parasitol Res, 1993, 79(3): 245-250.
[22]	Loos JA, Caparros PA, Nicolao MC, et al. Identification and pharmacological induction of autophagy in the larval stages of Echinococcus granulosus: An active catabolic process in calcareous corpuscles[J]. Int J Parasitol, 2014, 44(7): 415-427.
[23]	Antoniou M, Tselentis Y. Studies on Echinococcus granulosus using the scanning electron microscope[J]. Parasitol Res, 1993, 79(7): 537-542.
[24]	Asadi M, Taghizadeh S, Kaviani E, et al. Caspase-3: structure, function, and biotechnological aspects[J]. Biotech And App Biochem, 2022, 69(4): 1633-1645.
[25]	Yuan ZQ, Miao ZL, Gong XN, et al. Changes on lipid peroxidation, enzymatic activities and gene expression in planarian (Dugesia japonica) following exposure to perfluorooctanoic acid[J]. Ecotoxicol Environ Saf, 2017, 145: 564-568.
[26]	de Paula Aguiar D, Brunetto Moreira Moscardini M, Rezende Morais E, et al. Curcumin generates oxidative stress and induces apoptosis in adult Schistosoma mansoni worms[J]. PLoS One, 2016, 11(11): e0167135.

组别 Group	第3天 Day 3	第6天 Day 6	第9天 Day 9	第12天 Day 12	第15天 Day 15
CON	95.41 ± 2.49	96.42 ± 2.23	99.29 ± 1.23	96.41 ± 2.26	97.08 ± 0.79
DMSO	96.97 ± 0.60	96.13 ± 0.95	98.46 ± 1.42	99.38 ± 0.74	97.94 ± 0.62
1 µmol/L BIX-01294	96.22 ± 1.82	96.48 ± 0.75	97.76 ± 0.53	98.32 ± 0.24	97.51 ± 0.38
5 µmol/L BIX-01294	96.36 ± 1.70	95.87 ± 1.06	97.11 ± 1.52	96.78 ± 2.79	94.37 ± 3.90
10 µmol/L BIX-01294	96.94 ± 0.61	94.37 ± 1.11	97.12 ± 0.35	96.00 ± 0.81	94.75 ± 1.60
25 µmol/L BIX-01294	90.21 ± 3.72	36.45 ± 7.14^b	5.50 ± 3.54^b	2.59 ± 0.85^b	0.25 ± 0.28^b
50 µmol/L BIX-01294	71.91 ± 7.92^a	5.64 ± 1.48^b	1.61 ± 1.73^b	0.00 ± 0.00^b	0.00 ± 0.00^b