| BackgroundAlpha-momorcharin(α-MMC) is a type I ribosome-inactivating protein extracted and purified from the seeds of bitter melon(Momordica charantia), which has been shown to exert strong anti-tumor activity and may serve as a potential anti-tumor agent.However, α-MMC has severe hepatotoxicity, reducing of cytotoxic lymphocytes and other cytotoxicity when applied in vivo. The cytotoxicity of α-MMC was an obstacle for its clinical application. It is a requirement that excluding the toxicity mechanism and reducing cytotoxicity for assuring clinical safety.ObjectiveTo verify the LRP1 receptor medicated of α-MMC’s hepertoxicity mechanism, using L02 normal human embryonic liver cells.Materials and MethodsBeing used CCK-8 cytotoxical detecting reagents, the cell proliferation rate ofα-MMC induced in liver cell L02, normal mammary cells MCF-10 A, normal kidney cells AD293 and normal lung cells BEAS-2B were detected, and their half inhibitory rate IC50 were to calculate subsequently. Then the α-MMC-induced apoptosis was detected by flow cytometry, and after that, the endocytosis of α-MMC in L02 cells,BEAS-2B cells and AD293 cells were observed with laser scanning confocal microscopy. Afterwards, the binding affinity and receptor density of α-MMC’s cell receptor was calculated using radioligand binding assay. Finally, we used gene knockdown(siRNA) technique to silence the LPR1 protein in L02 cells, then theα-MMC’s cytotoxic effect and its apoptosis signaling protein were detected accordingly.Results(1) The IC50 of α-MMC to L02 cells, BEAS-2B cells、MCF-7 cells、MCF-10 A cells and AD293 cells were 5.077±1.03μg/ml, 15.03±2.26μg/ml, 30.19±2.45μg/ml,77.43±2.54 μg/ml and 104.9± 4.73 μg/ml respectively. α-MMC can induced cell apoptosis.(2) There were obvious differences in the FITC-α-MMC internalization in various cell lines, that is, the levels in L02 cells were the greatest, followed by BEAS-2B, andthe AD293 cells had the lowest levels.(3) The receptor affinity constant Kd values were 47.1 ± 7.54 nM/L. L02 cell receptor density is 14260.7±1422.8 sites/cell, while the BEAS-2B and AD293 receptor density were 7823±447.5 sites/cell and 872±91 sites/cell respectively.(4) After LRP1 silenced, the IC50 of α-MMC to L02 cells rose to 173.6±3.79μg/ml from 5.077±1.03 μg/ml. The JNK, Bad, P53 and Caspase9 was activated by α-MMC while it was treated with siRNA, this effect was inhibited obviously.ConclusionThe experimental results showed(1) that α-MMC was toxic to the human embryonic liver cell line L02 and the toxic effect on liver cells was exerted through inducing apoptosis;(2) α-MMC entered cells by binding to a specific LRP1 receptor on cell membranes; therefore, the higher the receptor density was, the more α-MMC entered cells and the greater its toxicity to the cells;(3) that α-MMC could also induce apoptosis through the LRP1-JNK signal pathway. We can conclude that the hepatotoxicity mechanism of α-MMC is to induce liver cell apoptosis, leading to hepatotoxicity, through the following two routes: LRP1 receptor-mediated endocytosis and LRP1 receptor-mediated JNK signalling. |
PDF Full Text Request