Toxicity of doxorubicin on Echinococcus granulosus protoscoleces in vitro

doi:10.12140/j.issn.1000-7423.2019.05.011

Abstract

Abstract:

Objective To evaluate the toxicity of doxorubicin (Dox) on Echinococcus granulosus protoscoleces in vitro. Methods Total 200 E. granulosus protoscoleces in each group were incubated with different concentration of doxorubicin (25, 50, 100, 200, 400, 600 μmol/L) in triplicate for 24 hours in vitro. Other two groups were incubated with medium(blank) or 1% DMSO controls. The viability of protoscoleces was determined by eosin staining and survival rate was calculated. The structural damage of treated protoscoleces was observed under scanning electron microscope (SEM). Single cell gel electrophoresis was used to detect DNA damage in treated protoscoleces. Real-time quantitative PCR (qRT-PCR) was used to detect the mRNA expression levels of ataxia telangiectasia mutated gene (ATM), p53 and DNA topoisomerase 2-alpha (Topo2a) in treated protoscoleces. Results Based on the eosin dye staining, the survival rates of treated protoscoleces were (96.0 ± 1.4)%, (80.3 ± 4.8)%, (75.6 ± 6.2)%, (53.2 ± 3.0)%, (26.4 ± 8.1)% and 0 when the concentrations of Dox were 25, 50, 100, 200, 400, 600 μmol/L, respectively, compared to that of protoscoleces incubated with blank control [(99.0 ± 0.5)%] and 1% DMSO (98.6 ± 0.3)%], with significant difference when incubated concentration is over 25 μmol/L (P < 0.01). The Dox IC₅₀ on protoscoleces was (267.92 ± 7.14) μmol/L. SEM results showed that E. granulosus protoscoleces in the blank control group and 1% DMSO group had smooth surface with a spherical or elliptic shaped scolex and complete apical structure. There were clearly visible microtricles on the surface. When incubated with 400 μmol/L Dox, the protoscoleces scolex was distorted and damaged and whole body collapsed. The apical structure slightly distorted. The microtriches on the surface were lost obviously. The body surface was shrinked. The single cell gel electrophoresis showed trailed cell when the concentration of Dox reached to 100 μmol/L, indicating the cell damage. The olive tail moment (OTM) was 16.6 ± 1.7 for cells treated with 100 μmol/L of Dox, which was significantly longer compared with the blank control group(0.1 ± 0.0) (P < 0.01). The qRT-PCR results showed that the relative expressions of ATM, p53 and Topo2a mRNA in 1% DMSO group were 1.0 ± 0.1, 1.1 ± 0.1 and 1.3 ± 0.9, respectively, with no statistical significance compared with the blank control group (1.0 ± 0.1, 1.0 ± 0.4, 1.0 ± 0.1, respectively) (P > 0.05). However, the relative expression levels of ATM, p53 and Topo2a mRNA in Dox group were 38.6 ± 3.5, 10.0 ± 2.5 and 54.0 ± 0.8, respectively, which were significantly increased compared with the blank control group (P < 0.05). Conclusion Dox has toxicity on the protoscoleces of E. granuloides in vitro, leading to cell and DNA damage and rpossibly related to the mRNA expression of of ATM, p53 and Topo2a.

Key words: Doxorubicin, Echinococcus granulosus protoscoleces, DNA damage, Ataxia telangiectasia mutated, p53, Topo2a

CLC Number:

R383.33

Chun-yan TIAN, Bei CHEN, Shuai LU, Li-mei WEN, Jun ZHAO, Xuan ZHENG, Kuerbannisha·AMAHONG, Yi GAO, Jian-hua WANG. Toxicity of doxorubicin on Echinococcus granulosus protoscoleces in vitro[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2019, 37(5): 571-576.

Figures/Tables 5

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基因 Gene	引物序列（5′→3′） Primer sequences（5′→3′）	退火温度/℃ Tm/℃	长度/bp Length/bp
ATM	F：GTTCCTACAGTCCATCCTAAT	73.3	146
	R：CTCCATCAAGCCAGCATT
p53	F：AACCACCGAACTCACAAC	78.2	216
	R：AACCGACACAACTCATCAA
Topo2a	F：ACCTTCCTCGCCAACTAT	82.4	256
	R：GTCTGTCTCCTCCATCAAC
β-actin	F：GCGATGTATGTAGCTATCCAGGC- AGTGCTCTCGCT	81.5	431
	R：CAATCCAGACAGAGTATTTGCGT- TCCGGAGGA