Department of Molecular Biology, Genetic Engineering and Biotechnology Research Institute, University of Sadat City, 32897 Sadat City, Egypt.
Biochemistry Department, Animal Health Research Institute, 33374856 Cairo, Egypt.
Asian Pac J Cancer Prev. 2022 Jul 1;23(7):2531-2540. doi: 10.31557/APJCP.2022.23.7.2531.
This study was conducted to investigate the potential anticancer properties of N, N-dibenzyl asparagine (NNDAsp), as an Asparagine (Asp) analog, using colon cancer Caco-2 cell and the normal NCM-460 cell line.
Cell viability rate and levels of produced lactate dehydrogenase (LDH) were achieved upon treatment with NNDAsp compared to Asp treatment using MTT assay and LDH production kit. The protein expression profile of asparagine synthetase (ASNS) was achieved by using ELISA and flow cytometry assay. The levels of released inflammatory cytokines, including interleukin-1 alpha (IL-1α) and IL-1 beta (IL-β), were monitored using an ELISA assay.
Our findings showed significant inhibition of colon cancer cell proliferation accompanied by a high level of produced LDH in a dose-dependent of an NNDAsp treatment without detectable toxic effect in normal cells. Interestingly, NNDAsp showed competitive inhibition of ASNS protein expression, in almost 3% of stained cancer cells, compared to 18% and 35% of untreated cells and cells pre-treated with Asp, respectively. Likewise, the concentration of ASNS protein was dramatically depleted in a dose and time-dependent of NNDAsp treatment in comparison with Asp treatment indicated by ELISA assay. Furthermore, as an apoptotic indicator, the expression of P53 and Caspase 3 (Caps3) was significantly increased in Caco-2 cells treated with NNDAsp at both RNA and protein levels. In contrast, their expression was markedly depleted in Asp-treated cells. In addition, the expression of both IL-1α and IL-1 β was markedly increased in Caco-2 cells in a dose and time-dependent of NNDAsp exogenous treatment. Moreover, targeting of ASNS by the Asp analog, NNDAsp, was further confirmed by the docking analysis of inhibitors ligands and crystal structure of ASNS protein.
These data provide evidence for the effectiveness of NNDAsp in cancer treatment via selective degradation of ASNS protein expression in colon cancer cells.
本研究旨在探讨 N,N-二苄基天冬酰胺（NNDAsp）作为天冬酰胺（Asp）类似物的潜在抗癌特性，使用结肠癌 Caco-2 细胞和正常 NCM-460 细胞系进行研究。
通过 MTT 检测和 LDH 产生试剂盒检测 NNDAsp 处理后细胞活力和产生的乳酸脱氢酶（LDH）水平，与 Asp 处理相比。使用 ELISA 和流式细胞术检测 asparagine synthetase（ASNS）的蛋白表达谱。通过 ELISA 测定释放的炎症细胞因子，包括白细胞介素-1α（IL-1α）和白细胞介素-1β（IL-β）的水平。
我们的研究结果表明，NNDAsp 以剂量依赖性方式显著抑制结肠癌细胞增殖，并伴有高水平的 LDH 产生，而对正常细胞无明显毒性作用。有趣的是，与未经处理的细胞和用 Asp 预处理的细胞相比，NNDAsp 对 ASNS 蛋白表达的抑制作用呈竞争型，在大约 3%的染色癌细胞中，而分别为 18%和 35%。同样，ELISA 检测表明，NNDAsp 处理以剂量和时间依赖性方式显著耗尽 ASNS 蛋白浓度，与 Asp 处理相比。此外，作为凋亡指标，P53 和 Caspase 3（Caps3）在 Caco-2 细胞中的表达在 RNA 和蛋白质水平上均显著增加，而在 Asp 处理的细胞中则明显减少。此外，在 Caco-2 细胞中，IL-1α和 IL-1β的表达均随着 NNDAsp 外源性处理的剂量和时间的增加而显著增加。此外，通过 Asp 类似物 NNDAsp 对 ASNS 的靶向作用进一步通过抑制剂配体和 ASNS 蛋白晶体结构的对接分析得到证实。
这些数据为 NNDAsp 通过选择性降解结肠癌细胞中 ASNS 蛋白表达来治疗癌症的有效性提供了证据。
