| T-cell acute lymphoblastic leukemia（T-ALL）is a heterogeneous aggressive hematopoietic malignancy that accounts for 30%of adult and 10%-15%of pediatric with BCR-ABL–negative ALL cases.Despite considerable advances in the treatment of ALL,there are still 10%-40%of BCR-ABL–negative ALL cases failing to reach complete remission（CR）.A number of ALL cases relapse occur during therapy or soon after completion of therapy and eventually experience refractory leukemia associated with a poor likelihood of cure.Long-term prognostic of T-ALL cases remains unsatisfactory,with a 5-year overall survival（OS）rate of about 50%.Chemo-resistance,which is the continuous multiple of leukemia cells insensitive or resistant to anti-neoplastic agents,is considered to be the principal cause of ineffective treatment.Many factors,such as genetic alteration,the overexpression of membrane ATP-binding cassette（ABC）transporters in leukemia cells,bone marrow microenvironment changes and Micro RNA regulation are responsible for the development of chemo-resistance.Human Nucleolin（NCL,C23）,is a multifunctional protein that has a diversified cellular activities due to three discrete structural domains,which binding to DNA,RNA and protein that enable the nucleolin protein to perform its various functions.Known as a shuttling protein,nucleolin distributes over different cellular aspects,including cell surface,cytoplasm and the nucleus,playing a role on ribosomal biogenesis,mRNA translation,signal transduction,cell proliferation and apoptosis.Moreover,the NCL expression level is elevated in highly proliferating cells especially in cancerous cells.Recent studies have shown that overexpression of nucleolin in gastric carcinoma cells,leukemia cells,cervical carcinoma cells and lung cancer cells.Our previous studies have elucidated that NCL was highly expressed on drug- resistant leukemia cell lines and PBMC of relapse/refractory acute leukemia patients.Difference gel electrophoresis（DIGE）technology analysis revealed that NCL together with Nucleophosmin（NPM）,another multifunctional nuclear protein,were extremely distinct up-regulated in drug resistant acute myeloid leukemia cell line HL-60/ADR in comparison with its parent cell line HL-60.Further research showed that NCL and NPM level were raised in relapse/refractory acute leukemia patients and positively correlated with poor prognosis.Moreover,the expressions of the two proteins were continued to be verified on 9 hematopoietic malignant cell lines,including drug-resistant cell line HL-60/ADR,KG/01,and K562/ADR.Based on above findings,we hypothesize that NCL probably is relevant to the performance of chemo-resistance in leukemia cells.Nonetheless few studies have analyzed the overexpression of NCL on leukemia cells in relation to the chemo-resistance.To investigate the effect of NCL on drug sensitivity in leukemia cells,we modulated NCL level with over-expression or inhibition in acute T lymphoblastic leukemia cell lines.Methods1.NCL protein expression was decteced in bone marrow and/or peripheral bloodclinical samples of de novo ALL patients.The effect of NCL level on complete remission（CR）rate,relapse rate,overall survival（OS）and relapse free survival were calculated.COX analysis were performanced to exporle prognostic value of NCL in ALL.2.NCL expression vector and vector encoding short hairpin RNA（shRNA）targeted NCL mRNA were designed and constructed.Lentiviral vectors were packaged.Jurkat cells were transfected with LV-NCL-OE and LV-NCL-RNAi respectively.Molt-4 cells were transfected with LV-NCL-OE.Transfection rates and overexpression or interference efficiency were examined.3.Cells viability after transfection were measured using cells growth curve tests,colony formation analysis,cells cycle and cells apoptosis detection.4.Cells drug sensitivity after transfection were evaluated by IC50 detection,drug accumulation assay and drug efflux test.5.Variation of cells apoptosis and drug resistance associated molecules at mRNA and protein level were evaluated by qRT-PCR and western blot test.6.Related signaling pathways were examined using western blot test.7.Rescue tests were performed to exclude off-targeted effect of NCL shRNA,including construction of NCL reverse mutation expression Lentiviral and detection recovery efficiency of cell phenotype.8.Protein co-immunoprecipitations were performed to expound the NCL protein-other protein interactions.Possible mechanisms of NCL regulatory were investigated by inhibition of ERK signaling specifically.Results1.Compared with NCL negative expression patients,positive expression patiens presented lower CR rates,higher relapse rate,poor OS and RFS.NCL expression is associated withpoor OS as an independent prognostic factor.2.LV-NCL-OE were successfully constructed with a titer of 2.50E+08 TU/ml and transfected Jurkat and Molt-4 cells with a 3.40±0.08 and 2.48±0.13-fold increased expression level of NCL.LV-NCL-OE were constructed with a titer of 3.00E+08 TU/ml and transfected Jurkat cells with a more than 70%inhibition rates of RNAi.3.Overexpression of NCL in Jurkat and Molt-4 cells led to a significant increase of cell proliferation and colony formation ability in both Jurkat and Molt-4 cells.Knockdown of NCL in Jurkat cells damaged cells proliferation and colony formation ability,induced cells apoptosis and suppressed cells cycle transition from G2M phase.4.An evidence drug insensitive in NCL-OE lines were observed.IC50 of ADM on Jurkat and Molt-4 OE lines were 1.362±0.271μg/ml and 2.821±0.733μg/ml,which multiplied8.42-fold and 6.60-fold relative to negative control lines.Both Jurkat and Molt-4 OE lines exhibited notably decreased accumulation of ADM and elevated efflux of rhodamine-123.While in KD lines,IC50 of ADM was 2.32 times lower than Scr group.KD lines showed remarkable increased accumulation of ADM and suppressed effluxion of rhodamine-123.5.Some apoptosis and drug resistance associated molecules such as Bcl-2,c-Myc,BCRP,LRP,MRP1 and ERK signaling were up-regulated of in OE cells while down-regulated in KD cells.6.Functional rescue experiments in Jurkat/KD cells showed partly recovered expression level of NCL,BCRP.7.Interaction of NCL protein with BCRP,MRP1,Ras,ERK protein respectively were confirmed.Inhibition of Raf,MEK1/2,ERK blocked the function and expression of BCRP and MRP1,without influence expression of NCL.Conclusions1.NCL expression level is related to reduced survival of ALL and is an independent marker of poor prognostic.2.Overexpression of NCL could promote cells proliferation and drug resistance,up-regulate drug resistance molecules such as Bcl-2,c-Myc,BCRP,LRP,MRP1,active ERK signal.While knockdown of NCL led to inhibition of cells proliferation and drug resistance,down-regulation of apoptosis inhibition and drug resistance molecules,suppression of ERK signal.3.Interaction of NCL protein with BCRP,MRP1,Ras,ERK proteins respectively were confirmed.4.Inhibition of ERK signal could weaken function and level of BCRP and MRP1 but didn’t affect expression of NCL.Concluding the above findings,we speculated that NCL may affect leukemia cells drug resistance in the following two ways:acceleration leukemia cells proliferation,interaction with BCRP,MRP1 and increased its capabilities,regulation expression of Bcl-2,c-Myc,BCRP,LRP,MRP1 via ERK signal pathway. |
