| Myeloid leukemia is the most common type of adult leukemia,including acute myeloid leukemia(AML)and chronic myeloid leukemia(CML).AML is a heterogeneous disease with high frequency of mutation of oncogenes or abnormality of chromosomes.Treatment to AML contains two phrases,the first is the induction therapy in which cytosine arabinoside and anthracycline are most adopted.The second step is after consolidation therapy in which combined chemotherapy or hematopoietic stem cell transplantation are adopted.In the past thirty years,the survival of AML patients younger than 65 has been improved mainly due to progress of supportive therapy making combined chemotherapy and stem cell transplantation possible intolerant so that lead to long-term survival for AML patients.However,more than one third patients relapse.The survival of old AML patients hasn't improved with five-year OS less than 10%because intolerance of intensive chemotherapy and stem cell transplantation.Treatment of refractory,relapsed AMLand AML of old patients has been the difficult point of AML treatment and need exploration of new treatment.Targeted therapy has been the hot spot during the recent years in the field of cancer study due to its specificity and low side effect.Inhibitor of mutated FLT3 kinase,ABT-199,inhibitor of IDH and et al have been investigated in clinical trials showing their antileukemia advantages.Despite modest progress in targeted therapy in AML,no drug could has a dramatic improvement on all the AML patients due to heterogenicty,thus more targets need to be explored to provide customed therapy to AML patients.As for CML targeted drug imatinib and second,third tyrosine kinase inhibitors have made great success.However,about 20-51%CML patients develop resistance to TKIs.The causes of resistance have many,including point mutation in Abl kinase domain,amplification and overexpression of BCR-ABL,elevated expression of ATP drug transportors,abnormal activation of kinase of Src kinases,stem cell mediated resistance,bone marrow microenviroment mediated resistance,epigenetic mediated resistance and et al.TKI resistance remains the key point to CML treatment and need alternative treatments.Epidermal growth factor substrate 8(EPS8)is a adaptor protein in the cytoplasm.Eps8 is the substrate of receptor and non-receptor tyrosine kinase like EGFR.Its structure from N terminal to C terminal contains phosphotyrosine binding protein(PTB),SH3,SAM-PNT,with the ability to form a complex with other proteins to transmit signaling.Studies have shown Eps8 can combine with Abi1,RN-tre,Shc,Shb,Dvl-1,IRSp53 and other proteins to form complexes to transduct signals and is involved in physical processes including remodeling of cell cytoskeleton,proliferation,apoptosis,adhesion,migration,internalization of receptor.It has been shown that Eps8 is overexpressed and involved in proliferation and invasive migration in squamous cancers,cervical cancers,ovarium cancers,lung cancers,neuroglimas and other solid malignant tumors and is related to invasion and prognosis and regarded as an oncogene in solid tumors.Role of eps8 on hematological malignant tumors has been little addressed and it's not clear that whether it's involved in malignant process of leukemia.To address this problem we studied the role of eps8 on process of myeloid leukemia cells and its mechanism.The study was divided into three parts:expression of eps8 in Bcr-abl positive cell lines ad CML patients;the effect of overexpression of eps8 on myeloid leukemia and its mechanism;the effect of knockdown of eps8 on myeloid leukemia and its mechanisms.The results showed that the expression of eps8 was elevated in newly diagnosed CML compared with normal bone marrow and was reduced after treatment remission.Overexpression of eps8 led to increased proliferation,reduced apoptosis,elevated adhesion and migration of HL60 cells and reduced sensitivities to chemotherapy drugs possibly through activation of PI3K/AKT,RAS/ERK,JAK2/STAT5 signaling pathways,knockdown of eps8 led to the opposite results.The present study confirms that eps8 is involved in process of myeloid leukemia cells suggesting EPS8 may be a promising target for treatment of myeloid leukemia.Part One Study of expression of EPS8 in Bcr-abl positive cell lines and CML patientsObjective:To study the expression of eps8 in CML patients and Bcr-abl+cell linesMethods:Q-RT-PCR was adopted to measure the expression of EPS8 in bone marrow in 98 cases of CML and 11 cases of normal contrast from Hematological Department of Zhujiang Hospital,Southern medical university and GuangZhou KingMed Diagnostics during 2013 to 2015.CML of 98 cases included 58 chronic phase(CP),11 accelerated phase(AP),21 blast crisis(BC)and 8 remission cases.2-??Ct was used to represent relative value of eps8 to internal reference.Westeren Blot was adopted to detect eps8 protein expression in CML cell lines of KBM5,MEG01,K562 and murine temperature sensitive abl positive B cell lines S4C2-1,S9,murine cell lines of 32D-p210-widetype and 32D-p210-T315I.Results1.The median of EPS 8 expression was 4.12(0.76-13.2)in CP,3.25(1.77-9.01)in AP,4.12(2.11-9.41)in BC,1.46(0.39-1.79)in remission and 1.00(0.90-1.24)in normal contrast,respectively.Non-parametric Rank test was adopted to statistical analysis.The mean rank of CP,AP,BC,remission,normal contrast was 58,16?64.14?63.48?32.62?29.32,respectively(P=0.006).Eps8 expression in CP was higher than that in remission arm and normal contrast(P=0.047 and 0.029),eps8 expression in AP was higher than that in remission arm and normal contrast(P=0.010 and 0.001),Eps8 expression in BC was higher than that in remission arm and normal contrast(P=0.005 and 0.000).2.Expression of eps8 was higher in KBM5 and K562 compared with MEG01.Eps8 was elevated in malignant temperature-sensitive murine virus abl transducted B cell line S4C2-1 compared with its parental B cell line S9.Eps8 was elevated in TKI resistant cell line 32D-p210-T315I compared with 32D-p210-widetype.Conclusion:Eps8 mRNA is higher in newly diagnosed CML compared to normal contrast and reduced in remission patients.Eps8 protein is elevated in malignant B cell line S4 than its parental non-malignant cell line SP9.Eps8 protein is elevated in TKI resistant cell line 32D-p210-T315I compared with 32D-p210-widetype.Part Two Effect of overexpression of eps8 on biological process of myeloid leukemia cells and mechanismObjective:Study the effect of overexpression of eps8 on biological process of myeloid leukemia cells and its mechanismMetholds:Vectors of pLVX-hEPS8-Puro and pLVX-AcGFP1-N1 were constructed and transducted into 293T cells.HL60 cells were infected by lentivirus carrying eps8 or GFP and selected by puromycin to construct stably eps8-overexpressed cell line HL60-EPS8 and its contrast cell line HL60-GFP.Proliferation was detected by CCK8,apoptosis was measured by Annexin-APC/7-AAD staining.Cell cycles were analyzed by cytometric flow after PI staining.Adhesion was detected after cultured in fibronectin-coated plate for 1.5 hours by observation under microscope and measure of OD value of adhesive cells remained on plate by CCK8.Migration was analyzed by Transwell.F-actin was stained by fluorescence-staining phalloidin to measure motility ability.Cells were treated by doxorubicin and cytosine arabinoside at different concentrations for different time,CCK8 was adopted to measure viability.P--AKT?p-ERK?p-STAT5?p-PP2Ac were detected by western blot to measure the activation of PI3K/AKT?RAS/ERK?JAK2/STAT5 signaling pathways.Results:1.Stably eps8-overexpressed HL60-EPS8 cell line was constructed,mRNA level of HL60-EPS8 was 52.09 times that of HL60-GFP,protein level was 10.62 fold ofHL60-GFP.2.OD value of HL60,HL60-GFP,HL60-EPS8 at 24 hour was 0.44±0,0.44±0.01,0.47±0(F=31.864,P=0.001).Difference between HL60-EPS8 and HL60,HL60-GFP was significant(P<0.01).OD value of HL60,HL60-GFP,HL60-EPS8 was 0.62±0.01,0.62±0.01,0.67±0.01,respectively(F=18.827,P=0.003).The difference between HL60-EPS8 and HL60,HL60-GFP was significant(P<0.01).OD value of HL60,HL60-GFP,HL60-EPS8 was 0.86±0.01,0.87±0.01,1.01±0.03,respectively(welch=28.388,P=0.008).The difference between HL60-EPS8 and HL60,HL60-GFP was significant(P<0.05).3.Cells were cultured in fibronetin-coated plate for 1.5 hours,observation under microscope showed more adhesion cells of HL60-EPS8 than those of HL60,HL60-GFP.OD values of adhesion cells were 0.43±0.05?0.51±0.06?0.69±0.07 for HL60,HL60-GFP,HL60-EPS8,respectively(F=28.432,P=0.000).The difference between HL60-EPS8 and HL60,HL60-GFP was significant(P<0.001).4.Apoptosis was detected by annexin V/7-AAD staining,early apoptosis of HL60,HL60-GFP,HL60-EPS8 was 3.31±0.21%.3.7±0.2%?2.43±0.06%(F=43.712,P=0.000).The difference between HL60-EPS8 and HL60,HL60-GFP was significant(P<0.01).Late apoptosis of HL60,HL60-GFP,HL60-EPS8 was 1.13±0.09%,1.04±0.13%,1.01±0.07%,respectively(F=1.098,P=0.392).Total apoptosis of HL60,HL60-GFP and HL60-EPS8 was 4.44±0.28%,4.74±0.26%,3.44±0.03%(Welch=44.730,P=0.008).The difference between HL60-EPS8 and HL60,HL60-GFP was significant(P<0.05).5.Cell cycles were detected by cytometric flow after PI staining,proporation of S phase of HL60,HL60-GFP,HL60-EPS8 cells was 48.39±0.87%?55.44±0.25%?60.57±0.87%(F=213.519,P=0.000).The difference beween HL60-EPS8 and HL60,HL60-GFP was significant,respectively(P<0.05).6.Migration of cells were detected by transwell methold,observation under microscope showed HL60-EPS8 cells in lower body of transwell were more than HL60 and HL60-GFP.OD value of lower body of transwell of HL60?HL60-GFP?HL60-EPS8 was 0.99±0.01,0.96±0.02,1.23±0.02(F=544.573,P=0.000).The OD value of HL60-EPS8 was significantly more than that of HL60(P<0.001)and HL60-GFP(P<0.001).7.F-actin was stained by fluorescence-staining phalloidin,observation under confocal microscopy showed no dramatic difference among HL60,HL60-GFP,HL60-EPS8 cells.8.HL60-GFP and HL60-EPS8 cells were treated by doxorubicin at different concentrations for 24 hours,48 hours and 72 hours.Viability of HL60-EPS8 at 15nM,30nM,60nM,120nM for 24 hours was higher than that of HL60-GFP(t=-7.120,P=0.002;t=-6.664,P=0.003;t=-34.689,P=0.000,t=-19.724,P=0.000).Viability of HL60-EPS8 at 15nM,30nM,60nM,120nM for 48 hours was higher than that of HL60-GFP(t=-25.216,P=0.000;t=-69.148,P=0.000;t=-158.408,P=0.000,t=-16.612,P=0.000).Viability of HL60-EPS8 at 15nM,30nM,60nM for 72 hours was higher than that of HL60-GFP(t=-50.289,P=0.000;t=-38.240,P=0.000;t=8.316,P=0.001).9.Total proteins of HL60,HL60-GFP,HL60-EPS8 were abstracted and several signaling proteins were detected by western blot.The levels of p-ERK?p-AKT?p-STAT5 were elevated in HL60-EPS8 than those in HL60 and HL60-GFP.The levels of p-PP2Ac were lowered in HL60-EPS8 than those in HL60 and HL60-GFP.Conclusion Overexpression of EPS8 leads increased proliferation,reduced apoptosis,elevated adhesion and migration and reduced sensitivity to chemotherapy by activating p-ERK,p-AKT,P-STAT5.Part Three Effect of knockdown of EPS8 on biological process of myeloid leukemia and mechanismObjection:Study the effect of knockdown of EPS8 on biological process of myeloid leukemia and its mechanismMethods:Four vectors of shRNA-EPS8 were constructed and transducted into 293T cells to pack up lentivirus interfering expression of eps8.K562 cells were infected by shRNA-EPS8 carrying lentivirus and selected by puromycin to construct stably eps8-attenuated cell line K562-shRNA-EPS8 and its contrast cell line K562-GFP.Proliferation of K562-shRNA-EPS8 and parental cells was detected by MTT.Apoptosis was measured by cytometric flow by annexin V-APC/7-AAD staining.Cell cycles were analyzed by cytometric flow by PI staining.Viability of cells treated with different imatinib at different time points by MTT.Levels of p-AKT?p-ERK?p-PP2Ac?p-STAT5?PTEN were detected by western blot.Results1.Four sequences were designed to interfering EPS8 and connected to vectors.Sequencing analysis confirmed proper sequences connected.2.K562 cells were infected by lentivirus,infection rate was more than 70%after three days observed under fluorescence microscope.After amplification and selection by puromycin inhibition rate of eps8 was detected by Q-RT-PCR.The interfering rate of eps8 of four lentivirus was 58.03%?14.6%?13.67%?74.93%,respectively.Interfering rate of the fourth sequence was the most dramatic confirmed by western blot.3.Proliferation was detected by MTT,OD values of K562,K562-GFP,K562-shRNA-EPS8 cells at 24h were 0.61±0.01,0.58±0.02,0.56±0.02,respectively(F=5.328,P=0.030).There was no significant difference between OD value of K562-shRNA-EPS8 and that of K562-GFP(P>0.05).OD values of K562,K562-GFP,K562-shRNA-EPS8 cells at 48h were 0.90±0.02,0.87±0.03,0.79±0.02,respectively(F=23.073,P=0.000).There was no significant difference between OD value of K562-shRNA-EPS8 and that of K562-GFP(P<0.05).OD values of K562,K562-GFP,K562-shRNA-EPS8 cells at 72h were 1.19±0.08,1.13±0.07,0.99±0.11,respectively(F=5.377,P=0.029).There was no significant difference between OD value of K562-shRNA-EPS8 and that of K562-GFP(P<0.05).4.Apoptosis was detected by cytometric flow after annexin V-APC/7-AAD staining.The early apoptosis of K562?K562-GFP?K562-shRNA-EPS8 was 2.66±0.14%,6.25±0.10%,14.27±0.06%(F=9506,P=0.000).The differences between K562-shRNA-EPS8 cells and K562,K562-GFP cells were significant,respectively(P<0.001).Late apoptosis of K562?K562-GFP?K562-shRNA-EPS8 was 1.19±0.06%,2.18±0.17%,6.45±0.08%.The differences between K562-shRNA-EPS8 cells and K562,K562-GFP cells were significant,respectively(P<0.001).Total apoptosis of K562?K562-GFP?K562-shRNA-EPS8 was 3.85±0.20%,8.43±0.11%,20.72±0.13%.The differences between K562-shRNA-EPS8 cells and K562,K562-GFP cells were significant,respectively(P<0.001).5.Cycle phases were analyzed by cytometric flow after PI staining.Proportion of G1 phase of K562?K562-GFP?K562-SHRNA-EPS8 was 47.78±0.48%48.04±0.66%,54.85±0.45%(F=163.847,P=0.000).The differences between K562-shRNA-EPS8 cells and K562,K562-GFP cells were significant,respectively(P<0.001).6.Cells were cultured in fibronetin-coated plate for 1.5 hours,observation under microscope showed less adhesion cells of K562-shRNA-EPS8 than those of K562,K562-GFP.OD values of adhesion cells were 0.48±0.05?0.46±0.29?0.35±0.06 for K562,K562-GFP,K562-shRNA-EPS8,respectively(F=12.060,P=0.001).The difference between K562-shRNA-EPS8 and K562,K562-GFPwas significant(P<0.01).7.Migration of cells were detected by transwell methold,observation under microscope showed K562-shRNA-EPS8 cells in lower body of transwell were less than K562,K562-GFP.OD value of lower body of transwell of K562,K562-GFP,K562-shRNA-EPS8 was 0.14±0.00,0.14±0.00,0.10±0.00(F=208.245,P=0.000).The OD value of K562-shRNA-EPS8 was significantly less than that of K562(P<0.001)and K562-GFP(P<0.001).8.K562-GFP and K562-shRNA-EPS8 cells were treated with imatinib at different concentrations and different periods.It was found that viability of K562-shRNA-EPS8 was significantly lower than that of K562-GFP at 1.2?M,1.6?M 2.0?M for 48 hours(t=3.441,P=0.03;t=12.093,P=0.000;t=11.078,P=0.000).Viability of K562-shRNA-EPS8 was significantly lower than that of K562-GFP at 0.8?M,1.2?M,1.6?M,2.0?M for 72 hours(t=17.079,P=0.000;t=5.204,P=0.029;t=34.692,P=0.000;t=23.603,P=0.000).K562-GFP and K562-shRNA-EPS 8 cells were treated with doxorubicin at different concentrations and different periods.Viability of K562-shRNA-EPS8 was significantly lower than that of K562-GFP at 0.05?M,0.1?M,0.15?M for 48 hours(t=17.802,P=0.000;t=15.563,P=0.000;t=5.665,P=0.001).Viability of K562-shRNA-EPS8 was significantly lower than that of K562-GFP at0.025?M,0.05?M,0.075?M,0.1?M for 72 hours(t=6.736,P=0.001;t=7.857,P=0.000;t=8.617,P=0.000;t=4.638,P=0.004).9.The levels of p-ERK?p-AKT?p-STAT5?PTEN were measured by western blot and knockdown of EPS8 led to reduced levels of p-ERK?p-AKT?p-STAT5 and increased level of PTEN.The level of p-BCR/ABL was reduced and levels of p-PP2Ac were increased after attenuation of eps8.Conclusion:Stable eps8-attenuated K562-shRNA-EPS8 cell line and contrast cell line K562-GFP were constructed.Knockdown of eps8 leads to reduced proliferation,increased apoptosis of K562.Cells are blocked at G1 phase possibly through inactivation of RAS/ERK,PI3K/AKT,JAK2/STAT5.Eps8 may be a promising target and provide new strategy to overcome TKI resistance. |
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