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Evaluation of the anti-cervical cancer effect of a prodrug ：CBZ-AAN-DOX with hypoxic cell culture and tumor-bearing zebrafish models.
Experimental Cell Research ( IF 3.3 ) Pub Date : 2020-03-27 , DOI: 10.1016/j.yexcr.2020.111980 Hong-Ce Chen 1 , Wen Rui 2 , Si-Yuan You 1 , Xia-Wan Liu 1 , Jun Huang 3 , Hong-Yuan Chen 4
Experimental Cell Research ( IF 3.3 ) Pub Date : 2020-03-27 , DOI: 10.1016/j.yexcr.2020.111980 Hong-Ce Chen 1 , Wen Rui 2 , Si-Yuan You 1 , Xia-Wan Liu 1 , Jun Huang 3 , Hong-Yuan Chen 4
Affiliation
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BACKGROUND
Prodrugs are medications or compounds that, after administration, can be converted into pharmacologically active drugs through metabolism. Unlike conventional drugs, prodrugs have reduced adverse or unintended effects, which could become critical limitations in treatments such as chemotherapy. Previously through computer-aided drug design and chemical synthesis, we have obtained and examined a prodrug N-benzyloxycarbonyl-Ala-Asn-Doxorubicin (CBZ-AAN-DOX). CBZ-AAN-DOX is essentially Doxorubicin that is chemically-modified with tripeptides to target Legumain, a highly expressed protein in cancer cells and is involved in tumor metastasis and tumor microvessel formation. The difficulty to test the safety and efficacy of the prodrug (including the pharmacodynamic parameters of CBZ-AAN-DOX on metastasis and invasion of tumors, as well as cardiac and vascular toxicity) primarily comes from the lack of appropriate experimental models.
METHODS
Human cervical cancer cell lines CaSki under hypoxic conditions were used to evaluate the cell viability by CCK-8 assay after the prodrug treatment. Western blotting method was performed for Legumain protein determination in the cell culture. Wound healing and transwell invasion assays were performed to determine the effects of the prodrug on tumor metastasis and invasion, respectively. Zebrafish models were constructed for toxicity and angiogenesis visual analysis after in vivo treatment with the prodrug.
RESULTS
The CCK-8 results showed that CBZ-AAN-DOX exhibits an IC50 of 28.7 μM in 48 h on CaSki cells that had a lower cell inhibition rate than DOX 80.3 μM for 24 h. Legumain expression was significantly increased in a time-dependent manner in 48 h under hypoxia conditions. The results also showed that 13.9 μM of the prodrug significantly inhibited the migration and invasion of cells and the effects were significantly stronger than that of 41.8 μM of DOX under hypoxia conditions after 48 h. The effects of 160 μM of the prodrug on the survival rate of zebrafish after 72 h and heart-toxicity showed no obvious abnormalities. Cell metastasis and angiogenesis were also inhibited in tumor-bearing zebrafish model.
CONCLUSION
The findings in this study demonstrated that CBZ-AAN-DOX is a promising chemotherapy candidate with low toxicity and high efficiency for cervical cancer. Remarkably, the hypoxic culture model together with the zebrafish model serve as a good system for the evaluation of the toxicity, targeting and impact of the prodrug on tumor invasion and metastasis.
中文翻译：
低氧细胞培养和荷瘤斑马鱼模型评估前药：CBZ-AAN-DOX的抗宫颈癌作用。
背景技术前药是在给药后可以通过代谢转化为药理活性药物的药物或化合物。与常规药物不同，前药具有减少的不良或意外影响，这可能成为化学疗法等治疗中的关键限制。以前，通过计算机辅助药物设计和化学合成，我们已经获得并检查了前药N-苄氧羰基-Ala-Asn-阿霉素（CBZ-AAN-DOX）。CBZ-AAN-DOX本质上是阿霉素，被三肽化学修饰以靶向Legumain（一种在癌细胞中高度表达的蛋白质），并参与肿瘤转移和肿瘤微血管形成。测试前药安全性和有效性的困难（包括CBZ-AAN-DOX对肿瘤转移和侵袭的药效学参数，以及心脏和血管毒性）主要来自缺乏适当的实验模型。方法采用缺氧条件下的人宫颈癌细胞株CaSki，经前药处理后，通过CCK-8检测细胞的生存能力。蛋白质印迹法用于细胞培养物中豆科动物蛋白的测定。进行伤口愈合和transwell侵袭测定，以确定前药分别对肿瘤转移和侵袭的影响。在用前药进行体内治疗后，构建了用于毒性和血管生成视觉分析的斑马鱼模型。结果CCK-8结果显示，在48小时内，CBZ-AAN-DOX在CaSki细胞上的IC50为28.7μM，在24小时内的细胞抑制率低于DOX 80.3μM。在缺氧条件下48小时内，Legumain表达以时间依赖性方式显着增加。结果还表明，在缺氧条件下48小时后，有13.9μM的前药显着抑制了细胞的迁移和侵袭，其作用明显强于41.8μM的DOX。160μM的前药对斑马鱼72小时后的存活率和心脏毒性的影响未见明显异常。在荷瘤斑马鱼模型中，细胞转移和血管生成也受到抑制。结论本研究的结果表明，CBZ-AAN-DOX是一种有前途的化学疗法，对宫颈癌具有低毒性和高效率。值得注意的是，低氧培养模型与斑马鱼模型一起成为评估毒性的良好系统，
更新日期：2020-03-27
中文翻译：
低氧细胞培养和荷瘤斑马鱼模型评估前药：CBZ-AAN-DOX的抗宫颈癌作用。
背景技术前药是在给药后可以通过代谢转化为药理活性药物的药物或化合物。与常规药物不同，前药具有减少的不良或意外影响，这可能成为化学疗法等治疗中的关键限制。以前，通过计算机辅助药物设计和化学合成，我们已经获得并检查了前药N-苄氧羰基-Ala-Asn-阿霉素（CBZ-AAN-DOX）。CBZ-AAN-DOX本质上是阿霉素，被三肽化学修饰以靶向Legumain（一种在癌细胞中高度表达的蛋白质），并参与肿瘤转移和肿瘤微血管形成。测试前药安全性和有效性的困难（包括CBZ-AAN-DOX对肿瘤转移和侵袭的药效学参数，以及心脏和血管毒性）主要来自缺乏适当的实验模型。方法采用缺氧条件下的人宫颈癌细胞株CaSki，经前药处理后，通过CCK-8检测细胞的生存能力。蛋白质印迹法用于细胞培养物中豆科动物蛋白的测定。进行伤口愈合和transwell侵袭测定，以确定前药分别对肿瘤转移和侵袭的影响。在用前药进行体内治疗后，构建了用于毒性和血管生成视觉分析的斑马鱼模型。结果CCK-8结果显示，在48小时内，CBZ-AAN-DOX在CaSki细胞上的IC50为28.7μM，在24小时内的细胞抑制率低于DOX 80.3μM。在缺氧条件下48小时内，Legumain表达以时间依赖性方式显着增加。结果还表明，在缺氧条件下48小时后，有13.9μM的前药显着抑制了细胞的迁移和侵袭，其作用明显强于41.8μM的DOX。160μM的前药对斑马鱼72小时后的存活率和心脏毒性的影响未见明显异常。在荷瘤斑马鱼模型中，细胞转移和血管生成也受到抑制。结论本研究的结果表明，CBZ-AAN-DOX是一种有前途的化学疗法，对宫颈癌具有低毒性和高效率。值得注意的是，低氧培养模型与斑马鱼模型一起成为评估毒性的良好系统，
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