14,15-EET Promote Colony Forming Via Activating PI3K-AKT Pathway In Chronic Myelogenous Leukemia

Background: Epoxyeicosatrienoic acid(EET)is one of the metabolic products of arachidonic acid(AA).In recent years,its role in the survival,proliferation,apoptosis and migration of various tumor cells has been extensively studied.The current bottleneck in the treatment of chronic myelogenous leukemia(CML)is mainly due to the inability to clear TKI-resistant leukemia stem cells(LSC).This research is in order to explore whether EET plays a role in the progression of CML and can it help to further eliminate chronic myelogenous leukemia stem cells.Methods: CCK8 method was used to screen out appropriate concentrations and treatment durations of 11,12-EET,14,15-EET and 17-ODYA;mononuclear cells were separated from bone marrow of chronic phase(whose time of taking TKI is no longer than one year)and newly diagnosed patients by Ficoll method;the CD34+ cells were sorted by using a CD34 Micro Bead Kit(Miltenyi Biotec)according to the manufacturer’s instructions and treated with different concentrations of 11,12-EET and 14,15-EET(with or without 17-ODYA)for 1-7 days,then cultured in methylcellulose medium for about two weeks,after that the colony formation number was counted under microscope;K562 cells were treated with agarose gel for cloning,and photographed after 14 days;the colony number differentially significant groups were sent to sequencing to screen out differential genes,then the differential genes were verified by q-PCR method;WB method was used to verify the expression of differential genes’ related pathway proteins(PI3K-AKT,MAPK-ERK);the CML review specimens and bone marrow mononuclear cells of healthy controls were collected,and the expression levels of CYP2C8 and CYP2J2 m RNA in the cells were detected by PCR;PI stain for detection of K562 cell cycle changes in different concentration 11,12-EET or 14,15-EET treatment groups by flow cytometry;Annexin V/PI double stained to detect the apoptosis levels of K562 cells after exposed in different concentrations of 11,12-EET,14,15-EET,17-ODYA and imatinib treatment groups by flow cytometry.Results: 1.14,15-EET promotes colony formation in CML CD34+ cells and K562 cells in low concentration by activating PI3K-AKT pathway.2.The m RNA of CYP2C8 enzyme was found highly expressed in bone marrow mononuclear cells of CML patients compared with control group.3.17-ODYA promotes apoptosis of K562 cells via a concentration gradient dependent method,and this pro-apoptotic effect can be reversed by 14,15-EET.4.17-ODYA can increase the sensitivity of K562 cells to imatinib.Conclusion: Our results indicate that 14,15-EET can promote the K562 cells colony forming and promote CML stem cells to form more mature cell colonies in a low concentration range,and this effect is related to the activation of the PI3K-AKT pathway,in addition,the combination of 17-ODYA and TKI can improve the killing effect of TKI to K562 cells,which can bring new hope for improving the cure rate of CML.