Study Of Disulfiram/cytarabine Eradicating Acute Myeloid Leukemia Stem Cell-like Cells With High Aldehyde Dehydrogenase Expression

ObjectiveAcute myeloid leukemia?AML?is a malignant tumor with a highly heterogeneous hematopoietic system^[1,2].In the past two decades,with the continuous improvement of treatment methods,the vast majority of patients with AML achieved complete remission after standardized induction chemotherapy,but some patients subsequently relapsed and died of the disease,so the study affects the recurrence and survival of AML patients after chemotherapy factors can help judge high-risk patients and give corresponding treatment measures to reduce the risk of recurrence and improve prognosis and survival.The reason for the relapse of leukemia patients was the presence of a group of leukemia stem cells?LSCs?in the body,similar to normal hematopoietic stem cells,in a relatively resting state,with the potential for self-renewal,infinite proliferation and multi-directional differentiation^[3-5].At present,the standardized induction therapy of AML has a high killing ability on leukemia cells,but LSCs are not sensitive to this chemotherapy^[6-8].Therefore,how to identify and eliminate LSCs is the key to complete treatment of AML.Many studies have found that aldehyde dehydrogenase?ALDH?activity can be used as a marker for cancer stem cells in different tissues^[9-1].ALDH can oxidize apoptotic aldehydes to non-apoptotic carboxylic acids,protecting cancer cells from these aldehydes apoptosis^[12].AML patients with high expression of ALDH were resistant to standardized treatments,and AML cells with high ALDH activity were highly enriched in LSCs^[13-15].In this study,the clinical characteristics and prognosis of primary AML patients?high expression of ALDH and low expression of ALDH?were compared and analyzed to explore whether ALDH can be used as a marker of leukemia stem cell load.and then the use of high expression ALDH cell model and NOD/SCID mouse leukemia model to study the combined use of DSF and Ara-C Chemosensitivity of chemotherapeutic drugs,while more effectively reducing leukemia burden in mice,and exploring possible molecular mechanisms to provide new ideas for further improving the remission rate and survival rate of AML.Method1.Case collectionWe collected 69 patients with primary AML who were admitted to the Department of Hematology of the Second Hospital of Shanxi Medical University from January 2015 to December 2017,including 29 males and 40 females,and bone marrow samples from 10normal people?suspected leukemia patients who were rejected after testing?.All specimens were the remaining samples for clinical testing.We collected laboratory-related data of patients?including basic information,treatment remission and recurrence,prognostic survival,ALDH expression,gene mutations and karyotypes,etc.?.2.To determine the cutoff value and analyze the prognosis of ALDH expression according to the ROC curveThe ROC curve was drawn according to the expression level of ALDH and the cutoff value was determined.The patients were divided into two groups:ALDH^high and ALDH^low.The relationship between ALDH expression level and cytogenetics and the relationship between ALDH expression and patient prognosis were analyzed.3.Construction of ALDH Lentiviral Expression VectorRT-PCR method was used to obtain ALDH cDNA by reverse transcription.After using specific primers to amplify the ALDH gene coding sequence?CDS?,double-enzyme digestion?Not I and EcoR I?PCR products and lentiviral expression vector pCDH-MCS-T2A-copGFP-MSCV,the digested product is recovered,ligated,transformed into DH5?,amplified,and then the plasmid is extracted.The target gene sequence was identified by one-generation sequencing.No mismatches,deletions and inserted base clones were selected and were amplified,then the plasmids were extracted on a large scale.4.To establish UT7 and THP1 cell models that stably express ALDH and MSCVLentiviral expression vectors ALDH and MSCV and packaging plasmid were co-transfected into 293T cells at a certain ratio by liposome method.After 48 hours,the virus suspension was collected and infected with UT7 and THP1 cells.After 72 hours,the infection efficiency was observed and tested.Cells with an infection efficiency of less than 90%were sorted to obtain UT7 and THP1 cell models that stably express ALDH and MSCV.5.After UT7 and THP1 series cells were treated with DSF or/and Ara-C,cell viability was detected using CCK-8 methodIn this study,UT7 series?UT7,UT7-ALDH,and UT7-MSCV?cells and THP1 series?THP1,THP1-ALDH,and THP1-MSCV?cells were treated separately and jointly in DSF and Ara-C at 0h,24h,48h,72h,then,CCK-8 kit was used to detect and analyze cell viability in each group.6.After DSF or/and Ara-C treatment of UT7 and THP1 series cells,apoptosis was detected using Annexin V methodAfter DSF and Ara-C were used alone and in combination to treat UT7 series cells and THP1series cells,apoptosis was detected and analyzed by Annexin V-PE.7.After treatment of UT7-ALDH and THP1-ALDH cells by DSF or/and Ara-C for 48hours,P65 was detected by Western blot methodP65 is a core member of the NF-?B signaling pathway.After UT7-ALDH and THP1-ALDH cells were treated by DSF/Ara-C separately and simultaneously for 48 hours,cells were lysed,protein was extracted,and the expression level of P65 in the treatment group cells was detected and analyzed using Westen blot.8.After treating UT7-ALDH and THP1-ALDH cells with DSF or/and Ara-C for 48hours,intracellular DNA was detected and analyzed by?-H₂AX?pS139?After UT7-ALDH and THP1-ALDH cells were treated with DSF and Ara-C alone or/and in combination for 48 h,the formation of intracellular?-H₂AX?pS139?lesions was detected and analyzed using?-H₂AX?pS139?immunofluorescent antibodies.9.To study and analysis of leukemia burden in NOD/SCID leukemia mice after DSF or/and Ara-C treatmentNOD/SCID mice were subjected to sub-lethal dose of radiation to clear the bone marrow,and then implanted with THP1-ALDH cells to successfully construct a mouse model of leukemia.After continuous treatment with DSF or/and Ara-C for 5 days,the observation was continued until 14 days after stopping the drug,and the leukemia load in the mice was analyzed.Result1.Determine the cutoff value by ROC curve and analyze the prognosis of ALDH expressionAccording to the ROC curve,the cut-off value for expressing ALDH was 1.85%,ALDH in the CD34+cells of normal human bone marrow is at a low level?0.5±0.17%?,the expression of ALDH in AML is very different?0-79%?,and some of them?50.7%?have higher expression of ALDH??1.85%?,Called ALDH^high patients,ALDH^low patients with lower ALDH expression??1.85%?.In the correlation analysis between ALDH expression and cytogenetics,it was found that the expression of ALDH was higher in patients with high risk of cytogenetics?15.36±4.46%?,and the expression of ALDH was lower in patients with low risk of cytogenetics?1.57±0.74%?.According to ALDH expression,patients were divided into:ALDH^high patients and ALDH^low patients.The overall survival?OS?of ALDH^highigh patients was significantly lower than that of ALDH^low patients?P<0.01?.Since the expression of ALDH in patients in the intermediate risk group of cytogenetics was significantly different?0.1-44.4%?,They were divided into two groups according to ALDH expression and the OS was analyzed.The overall survival of ALDH^high patients in the intermediate risk group of cytogenetics was also found to be significantly lower For ALDH^low patients?P<0.01?.2.Construction of ALDH Lentiviral Expression VectorALDH was successfully ligated into the lentiviral expression vector pCDH-MCS-T2A-copGFP-MSCV.The target gene ALDH sequence was identified by sequencing,and no deletions,insertions,and mismatched bases were found.The sequence was accurate after Blast alignment,and a viral expression vector was successfully constructed.3.UT7 and THP1 cells were transduced by lentivirus to establish a cell model that stably expressed the target geneAfter the lentivirus expression vector was successfully constructed,293 T cells were co-transfected with the liposome method to prepare ALDH and MSCV lentiviral suspensions and infect UT7 and THP1 cells.After 72h of cell culture,the positive rate of cell infection was detected by flow cytometry.The cell infection efficiency?GFP?of each group was greater than 90%,indicating that the UT7 and THP1 cell models that stably express the target gene were successfully constructed.4.Changes in cell viability of UT7 and THP1 series cells treated with DSF or/and Ara-CThe IC50 values of DSF and Ara-C on both cells were measured.After UT7 and THP1 series cells were treated with Ara-C at the specified concentration for 48h,the vitalities of THP1-ALDH and THP1-ALDH cells was not significantly inhibited,indicating that they were resistant to Ara-C.DSF inhibited the viability of THP1 and UT7 series cells.THP1-ALDH and UT7-ALDH cells were treated with DSF and Ara-c for 48 hours,and the cell viabilities were significantly inhibited,indicating that they reversed Ara-C resistance,however,the viability of cord blood stem cells was not affected?P>0.05?.5.Changes in apoptosis of UT7 and THP1 series cells treated with DSF or/and Ara-C After UT7 and THP1 series cells were treated with Ara-c or/and DSF for 48 hours,the percentage of apoptotic cells was analyzed by flow cytometry,indicating that treatment with Ara-c or DSF alone could induce apoptosis in UT7 and THP1 series cells?P<0.05?,however,the combined treatment of DSF and Ara-c could significantly enhance the apoptosis effect?P<0.01?.But no corresponding apoptosis was found in CD34+CD38-umbilical cord blood stem cells?P>0.05?.6.DSF or/and Ara-C inhibited p65 expression in UT7-ALDH and THP1-ALDH cells and increased intracellular?-H₂AXTreatment of THP1-ALDH and UT7-ALDH cells with DSF or Ara-c alone for 48 hours could reduce the expression level of p65 protein,a core member of the NF-?B signaling pathway?P<0.05?,at the same time,?-H₂AX foci were formed in the cells.However,after these cells were treated with DSF and Ara-c,the expression levels of p65 protein were significantly reduced?P<0.001?and the formations of?-H₂AX foci were significantly increased?P<0.001?,but no similar results were found in umbilical cord blood stem cells?P>0.05?.7.Study on leukemia load in NOD/SCID mice treated with DSF or/and Ara-CAfter treatment with DSF or/and Ara-C for consecutive 5 days,mice were sacrificed at 14days,and the leukemia burdens of peripheral blood,bone marrow and spleen in the mice were reduced to varying degrees.However,the combined use of DSF and Ara-C reduced the leukemia burden in mice more significantly than the drug alone?P<0.05?.However,compared with the drug alone,the combined use of DSF and Ara-C treatment significantly reduced the leukemia load in mice?P<0.05?.Conclusion1.The expression level of ALDH was lower in the CD34+cells of normal human bone marrow cells,however?0.5±0.17%?,the expression level of ALDH was higher in some AML patients??1.85%?.AML patients with high risk of cytogenetics had higher levels of ALDH?15.36±4.46%?,whereas patients with low risk of cytogenetics had lower levels of ALDH?1.57±0.74%?.The overall survival?OS?of ALDH^high patients was significantly lower than that of ALDH^low patients?P<0.01?.Since the expression of ALDH in patients in the intermediate risk group of cytogenetics was significantly different?0.1-44.4%.?,They were divided into two groups according to ALDH expression and the OS was analyzed.The overall survival of ALDH^high patients in the intermediate risk group of cytogenetics was also found to be significantly lower For ALDH^low patients?P<0.01?.2.Leukemia stem cell-like cells expressing ALDH at high levels were successfully constructed,and it was confirmed that cells expressing ALDH were relatively resistant to the chemotherapeutic drug Ara-C at the cellular level.DSF alone could inhibit the viability of leukemia stem-like cells,induce apoptosis,inhibit the expression of p65 protein,a core member of the NF-?B signaling pathway,and promote intracellular DNA fragmentation,however,the combined use of DSF and Ara-C significantly increased these effects.3.In the NOD/SCID leukemia mouse model,DSF combined with Ara-C could reduce the leukemia load in mice more than alone.