关键词: 氯普噻吨, 蛋白激酶B/哺乳动物雷帕霉素靶蛋白通路, 人急性髓系白血病细胞, 自噬, 凋亡

Abstract:

Objective To investigate the impacts of chlorprothixene on autophagy and apoptosis in human acute myeloid leukemia （AML） cells by regulating the protein kinase B （Akt）/mammalian target of rapamycin （mTOR） pathway. Methods Two cell lines， SKNO-1 and MOLM-13， were used as the research objects， and they were treated with （0， 20， 40， 60） μmol/L chlorprothixene for （6， 12， 24） h， respectively， CCK-8 was performed to detect cell proliferation.Experiments were divided into control group （normal culture）， SC79 group （5 mg/L SC79 treated cells for 2 h），chlorprothixene group （40 μmol/L chloroprotixol treated cells for 12 h）， chlorprothixene + SC79 group （40 μmol/L chlorprothixene was added to the cells for 12 h on the basis of the group），monodansylcadaverine （MDC） staining was performed to measure the occurrence of autophagy；flow cytometry was performed to measure cell apoptosis rate；Western blot was performed to measure autophagy-related protein SQSTM1/p62， microtubule-related protein 1 light chain 3B （LC3B）， apoptosis-related protein poly （ADP-ribosyltransferase） （PARP）， aspartate-specific cysteine protease 3 （caspase3）， activated caspase3 （cleaved caspase3） and Akt/mTOR signaling pathway related proteins Akt， phosphorylated （p）-Akt， mTOR and p-mTOR. Results In SKNO-1 and MOLM-13 cells， at （6， 12， 24） h， compared with 0 μmol/L chlorprothixene， （20， 40， 60） μmol/L chlorprothixene treatment increased the inhibition rate of cell proliferation （P < 0.05）；at the same time point， as the concentration of chlorprothixene increased， the inhibition rate of cell proliferation increased， and 40 μmol/L chlorprothixene treatment for 12 h was used for the next study.In SKNO-1 and MOLM-13 cells， compared with the control group， the apoptosis rate， autophagosome positive rate， LC3B， PARP， cleaved caspase3/caspase3 protein expression levels in SC79 group decreased （P < 0.05）， the SQSTM1/p62， p-Akt/Akt and p-mTOR/mTOR protein expression levels increased （P < 0.05）；the chlorprothixene group showed the opposite trends （P < 0.05）；after adding chlorprothixene on the basis of SC79 group， the apoptosis rate， autophagosome positive rate， LC3B， PARP， cleaved caspase3/caspase3 protein expression levels decreased than chlorprothixene group （P < 0.05）， the protein expression levels of SQSTM1/p62， p-Akt/Akt and p-mTOR/mTOR increased （P < 0.05）. Conclusion Chlorprothixene can inhibit the Akt/mTOR pathway and promote autophagy and apoptosis in human AML cells.

Key words: chlorprothixene, protein kinase B/mammalian target of rapamycin pathway, human acute myeloid leukemia cells, autophagy, apoptosis