两种菌种来源的左旋门冬酰胺酶抗白血病作用与机制比较

Comparison of the Anti-Leukemia Effect and Mechanism of L-Asparaginase between Two Different Strains

比较大肠杆菌来源的左旋门冬酰胺酶(E.coli-L-Asp)与欧文菌来源的左旋门冬酰胺酶(Erwinia-L-Asp)体外抗白血病作用,并探讨其作用机制。以两种来源的L-Asp分别处理急性淋巴细胞白血病(ALL)细胞株(REH、Jurkat),采用CCK-8试剂盒检测细胞增殖变化(IC50),Annexin V/PI双染流式细胞术检测细胞凋亡,高压液相色谱(HPLC)检测用药后培养液中4种氨基酸[门冬酰胺(Asn)、门冬氨酸(Aspa)、谷氨酰胺(Gln)、谷氨酸(Glu)]浓度的变化。结果表明,REH、Jurkat细胞株对两种L-Asp的抑制作用均敏感,且在一定时间内存在明显剂量效应关系,即在较低的药物浓度下,细胞增殖抑制率处于较低水平,而药物浓度增加后,细胞的增殖抑制率明显升高,其中作用后24 h,Erwinia-L-Asp组细胞抑制率、细胞凋亡率均明显高于E.coli-L-Asp组(P<0.05),但至48 h两组比较差异无显著性(P>0.05)。两种药物在低浓度时均能耗竭培养液中的Asn,且作用相当(P>0.05),高浓度时耗竭Gln作用才凸显,而Erwinia-L-Asp的作用明显强于E.coli-L-Asp(P<0.05)。结论:两种菌种来源的L-Asp抗白血病作用与剂量存在量效关系,除耗竭外环境中的Asn和Gln外,诱导细胞凋亡也是其抗白血病作用之一。ErwiniaL-Asp具有起效快、Gln耗竭作用较强的特点,可作为临床儿童ALL治疗的一线药物选择。

This study was purposed to compare the anti-leukemic effects of E. coli-L-Asp and Erwinia-L-Asp in vitro, and to investigate their mechanism. The cell apoptosis and proliferation inhibition rate were measured by CCK-8 kit, and IC50 of two drugs was calculated by using SPSS software. Pro-apoptosis effect of E. coli-L-Asp and Erwinia-L- Asp on REH and Jurkat cell lines was also determined by flow cytometry with Annexin V/PI double staining. Concentration changes of 4 amino acids （Asn, Aspa, Gin, and Glu） before and after medication were detected by using high pressure liquid chromatography （HPLC） assay. The results showed that both REH and Jurkat cell lines were sensitive to L-Asp drugs from two different strains, and E. coli-L-Asp and Erwinia-L-Asp displayed the inhibition effect on the proliferation of Jurkat and REH cell lines in dose-dependent and time-dependent manners. The inhibition cell of proliferation and cell apoptosis in Erwinia-L-Asp group were higher than those in E. coli-L-Asp group after 24 hours （ P 〈 0.05）, However, after treatment of REN and Jurkat cells with 2 kind of L-Asp for 48 hours, the inhibition of cell proliferation and apoptosis rates were not significantly different between the 2 L-Asp drugs （P 〉 0.05 ）. The Asn in medium could be depleted by two different L-Asp drugs with low concentration. Both the two L-Asp drugs had the same capability to deplete the Asn surrounding leukemia cells （ P 〉 0.05 ）. The Gin in medium could be depleted by two L- Asp drugs with high concentration. The hydrolysis effect of Erwinia-L-Asp on Gin was stronger than that of E. coli-L- Asp （ P 〈 0.05 ）. It is concluded that in a certain range of concentrations, E. coli-L-Asp and Erwinia-L-Asp exert anti- leukemia effect in dose-dependent manner. Depletion of Gin and Asn in surrounding environment and induction of cell apoptosis are two potential mechanisms, by which leukemia cells can be killed. Erwinia-L-Asp may be choseen as the first-line drug to treat childhood ALL for its fast action and stronger hydrolysis effect on Gin.