School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong, China.
Toxicol In Vitro. 2010 Jun;24(4):1176-82. doi: 10.1016/j.tiv.2010.02.014. Epub 2010 Feb 17.
This study was aimed at investigating and comparing the cytotoxicities of two structurally similar type I RIPs, namely trichosanthin (TCS) and free ricin A chain (RTA). A type II RIP, namely Ricinus communis agglutinin (RCA), was also included for comparison. The three RIPs were added separately to cultures of NIH 3T3 cells. The effective doses and time courses were analyzed using cell counts. Polyclonal antibodies against TCS and RTA were produced in rabbits and purified by a protein A-Sepharose CL-4B column. The mechanisms of cell death were determined by TUNEL, immunohistochemical staining, flow cytometry, and Western blotting. The effective doses for TCS, RTA and RCA were found to be 800, 50, and 50 nM, respectively. All three RIPs induced apoptosis. In all cases, activation of caspase-3 and caspase-8, but not caspase-9, was detected. Additionally, RTA caused in vivo tissue necrosis in rabbits after intradermal administration. Hence the mechanism of cell death due to RTA intoxication may vary depending on the experimental conditions, being necrosis in vivo and apoptosis in vitro. The present findings may shed light on the apoptotic pathway induced by RIPs. RTA may be useful for studying the shift in cell death.
本研究旨在探讨和比较两种结构相似的 I 型 RIP，即天花粉蛋白（TCS）和游离蓖麻毒素 A 链（RTA）的细胞毒性。还包括一种 II 型 RIP，即蓖麻凝集素（RCA）进行比较。将这三种 RIP 分别加入 NIH 3T3 细胞培养物中。通过细胞计数分析有效剂量和时间进程。针对 TCS 和 RTA 的多克隆抗体在兔子中产生，并通过蛋白 A-Sepharose CL-4B 柱进行纯化。通过 TUNEL、免疫组织化学染色、流式细胞术和 Western blot 确定细胞死亡的机制。TCS、RTA 和 RCA 的有效剂量分别为 800、50 和 50 nM。这三种 RIP 均诱导细胞凋亡。在所有情况下，均检测到 caspase-3 和 caspase-8 的激活，但 caspase-9 没有被激活。此外，RTA 在皮内给药后会导致兔子体内组织坏死。因此，RTA 引起的细胞毒性的细胞死亡机制可能因实验条件而异，在体内为坏死，在体外为凋亡。本研究结果可能为 RIP 诱导的细胞凋亡途径提供了新的认识。RTA 可能有助于研究细胞死亡的转变。
