Study On The Mechanism Of PHI Inducing Apoptosis In Acute Myeloid Leukemia M2b Cells

Objective: To study the effect of 6-phenylhexyl isothiocyanate?PHI?inhibiting cell proliferation and inducing apoptosis in acute myeloid leukemia M2 b Cell lines.We investigated the signal pathway and other possible mechanisms which PHI involved in protein,mRNA and long non encoding RNA?lncRNA?in Kasumi-1.Methods: We evaluated the effect of PHI on inhibiting cell proliferation in several hematologic malignancies cells by CCK8 in vitro.Apoptosis and cell cycle arrest of Kasumi-1 and SKNO-1 after exposure to PHI were detected by flow cytometry?FCM?.Colony forming ability of Kasumi-1 cells evaluated using methyl cellulose semi-solid medium.Establishment of xenograft tumor nude mice model of Kasumi-1 cells to study anti-tumor effects of PHI in vivo.Apoptosis was detected by TUNEL.Apoptosis-related protein after exposure Kasumi-1 to PHI was measured by western blotting.Microarraywas used to analyze alteration expression of lncRNA and mRNA after treated Kasumi-1 to PHI.Differential expression of lncRNA and mRNA were analyzed by using KEGG,GO analysis and lncRNA target gene prediction.Reverse transcription real-time quantitative PCR?RT-qPCR?was used to verify the expression of RNA.Results: 1.PHI could inhibit cell growth in several hematologic malignancies cell lines.Acute myeloid leukemia cell lines?Kasumi-1 and SKNO-1?were more sensitive.2.PHI inhibited cell clone formation of Kasumi-1 cell.Further it induced apoptosis and cell cycle arrest at G0/G1 phase in Kasumi-1 and SKNO-1 cells in time and concentration dependent manner in vitro.PHI could inhibit cell growth and cause cell apoptosisin xenograft tumor in nude mice in vivo.3.PHI induced apoptosis in Kasumi-1 cells through the mitochondrial and Fas death receptor pathway.It activated p21,and consequening cell cycle arrest in the G0/G1 phase.We found that activation of P53 pathway was the key point in the mechanism of PHI inducing apoptosis and cell cycle blocking in Kasumi-1.4.it was demonstrated that TNFR death receptor pathway might be involved in PHI inducing apoptosis,and E2F1,CDKN 2A,MDM2 might be involved in regulation of P53 signaling pathway by the study of lncRNA chip analysis and RT-qPCR verification.5,Alteration expression of lncRNA were found after treated by PHI in Kasumi-1,such as GAPLINC downregulation,lnc-HDAC9-1:2 upregulation,some p53 downstream lncRNAs like NEAT1 and LncPINT upregulation..Meanwhile,PHI could regulate ENST00-000590797 and NR₀24330 targeting miRNA-125 a and miRNA122 respectively.It was showed that PHI could regulate apoptosis by the mechanism of ceRNA?competing endogenous RNA?.Conclusion: 1.PHI inhibits cell proliferation in several hematologic malignancies cells,especially in M2 b cells.PHI inhibits cells proliferation by inducing tumor cell apoptosis and arresting cell cycle in G0/G1 phase.2.P53 pathway play a key role in PHI leading cells apoptosis and cell cycle arrest in Kasumi-1.3.TNFR death receptor pathway may involve in PHI inducing apoptosis,and E2F1,CDKN 2A,MDM2,etc.might also involve in the regulation of P53 signaling pathway.4.PHI may regulate the expression of lncRNA,like some p53 downstream lncRNAs and induce apoptosis by the mechanism of ceRNA.