West Asia Organization for Cancer Prevention (WAOCP), APOCP's West Asia Chapter. Asian Pacific Journal of Cancer Prevention 1513-7368 23 7 2022 07 01 Anticancer Properties of N,N-dibenzylasparagine as an Asparagine (Asp) analog, Using Colon Cancer Caco-2 Cell Line 2531 2540 90206 10.31557/APJCP.2022.23.7.2531 EN El-Shimaa A. Mohamed Department of Molecular Biology, Genetic Engineering and Biotechnology Research Institute, University of Sadat City, 32897 Sadat City, Egypt. Khalil Bassiouny Department of Molecular Biology, Genetic Engineering and Biotechnology Research Institute, University of Sadat City, 32897 Sadat City, Egypt. Abeer A. Alshambky 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. Hany Khalil Department of Molecular Biology, Genetic Engineering and Biotechnology Research Institute, University of Sadat City, 32897 Sadat City, Egypt. Journal Article 2022 04 11 Objectives: 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. Methods: 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. Results: 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. Conclusion: These data provide evidence for the effectiveness of NNDAsp in cancer treatment via selective degradation of ASNS protein expression in colon cancer cells. https://journal.waocp.org/article_90206_1b9cb33b22613263e5bebbafe81c822c.pdf