ABSTRACT
OBJECTIVE@#To establish HL-60 cells and adriamycin resistant HL-60 cells (H-60/ADR) in which the expression of homologous box gene 1 (SIX1) was inhibited, and investigate the effect of inhibiting the expression of SIX1 on the drug resistance.@*METHODS@#Lentivirus was used to transfect HL-60 and HL-60/ADR cells, and the cell lines stably inhibiting the expression of SIX1 were screened by puromycin. CCK-8 assay was used to detect the proliferation ability of cells in each group, apoptosis kit was used to detect the cell apoptosis, and real-time quantitative PCR was used to detect the expression level of drug-resistant related genes.@*RESULTS@#HL-60 and HL-60/ADR stably transfected cell lines with down-regulation of SIX1 expression were successfully constructed. Compared with control group, the inhibition of SIX1 expression significantly inhibited the proliferation of HL-60 and HL-60/ADR cells (P <0.05), increased the apoptosis rate (P <0.05), and the sensitivity of cells to adriamycin increased after inhibition of SIX1 expression.@*CONCLUSION@#Inhibition of SIX1 expression can improve cell sensitivity to adriamycin, and its role in reversing drug resistance may be related to the promotion of apoptosis gene expression.
Subject(s)
Humans , HL-60 Cells , Drug Resistance, Neoplasm/genetics , Leukemia, Myeloid, Acute , Doxorubicin/pharmacology , Apoptosis , Cell Proliferation , Homeodomain Proteins/geneticsABSTRACT
Objective To construct a colon cancer chemotherapy-resistant cell line COLO,and study its characteristics and its relationship with tumor stem cells.Methods We constructed two 5-fluorouraci (5-FU)-resistant colon cancer cell line COLO/5-FU-1 and COLO/5-FU-2, which were resistant to 0.1 0 μmol/ml and 0.20 μmol/ml 5-FU respectively through gradiently increased drug concentration.The cha-racteristics of 5-FU-resistant cell lines were compared with parental colon cancer cell line COLO related to proli-feration,colony forming ability,migration and invasion,sphere forming ability,expression of stemness genes and cross drug-resistance.Results In the cell viability assay,4 days after regular training,the absorbancy of colon cancer 5-FU-resistant cell lines COLO/5-FU-2,COLO/5-FU-1 and parental colon cancer cell line COLO were 0.61 ±0.1 3,0.54 ±0.07 and 0.41 ±0.09 respectively,with significant difference (F =63.43,P =0.033).With the increased concentration of 5-FU,5-FU-resistant cell lines presented increasing clonality. The cloning efficiency of COLO/5-FU-2,COLO/5-FU-1 and parental colon cancer cell line COLO were (87.6 ±1 2.7)%,(65.3 ±9.7)% and (38.5 ±7.6)% respectively,with significant difference (F =33.64, P =0.01 7).In each high power field of vision,the cell numbers of migration through the basement membrane of COLO/5-FU-2,COLO/5-FU-1 and parental colon cancer cell line COLO were 482 ±39,434 ±45 and 373 ±38 respectively;and the cell numbers of invasion through the basement membrane were 1 74 ±42,1 1 2 ± 31 and 87 ±29 respectively,with significant differences (F =1 09.61 ,P =0.009;F =67.31 ,P =0.032). Compared with parental colon cancer cell line COLO,5-FU-resistant cell lines had higher expression of stem-ness genes (F =47.31 ,P =0.042).5-FU-resistant cell lines were cross-resistant to other chemotherapeutic drugs such as mitoxantrone.For example,after incubation for 96 hours,inhibition rate of mitoxantrone to parent colon cancer cell line COLO was higher significantly than COLO/5-FU-1 and COLO/5-FU-2 (0.749 ± 0.042,0.423 ±0.024,0.342 ±0.01 8),with significant difference (F =1 2.61 ,P =0.028).The micro-sphere forming rates of COLO/5-FU-2,COLO/5-FU-1 and parental colon cancer cell line COLO were (8.90 ± 0.97)%,(6.20 ±0.75)% and (3.90 ±0.32)% respectively,with significant difference (F =1 64.32,P =0.006).Conclusion Colon cancer drug-resistant cell line COLO possess tumor stem cell-like characteristics, which are enriched in cancer stem cells.
ABSTRACT
Objective: To analyze the expression of HLA class Ⅰ molecules and MHC classⅠ related chain A and B (MICA/MICB) in human nasopharyngeal carcinoma cell line (CNE2) and multi-drug resistant nasopharyngeal carcinoma cell line (CNE2/ DDP), and to assess their influence on NK cell-mediated lysis.Methods: Expression of HLA classⅠ molecules and MICA/MICB on the surface of CNE2 and CNE2/DDP cell lines was analyzed by flow cytometry. Cytotoxicity of NK cells (isolated from 3 healthy persons) against CNE2 and CNE2/DDP cells were detected by LDH releasing assay at different effect-to-target cell ratios (E∶T). In blocking experiments, anti-MHC class Ⅰ monoclonal antibody (mAb) (W6/32, a pan anti-HLA class Ⅰ antibody) and anti-MHC class I chain related molecules mAb (BAMO-1, specificly against MICA and MICB) were added to the target cells at a E∶T ratio of 10∶1. Results:It was found that the expression of HLA class Ⅰ molecules and MICA/MICB on CNE2 was higher than that on CNE2/DDP(P
ABSTRACT
Objective To establish a bladder cancer MDR cell line by gene transfection and to lay a foundation for the research of occurrence and reversion mechanisms of MDR. Methods The invasive bladder cancer T24 cells were transfected with mdr1 cDNA by cationic liposome (DOTAP) introduced gene transfection.The adriamycin (ADM) resistant cells were screened as MDR cells,named TADM. The MDR phenotype of TADM was identified by MTT, immunohistochemistry,immunofluorescence,flow cytometry,PCR and RT PCR. Results The relative resistant index of TADM was 41.6,mdr1 cDNA being integrated into the genome of T24.As a result,the expression of P gp and mdr1 mRNA in TADM increased. Conclusions The integration of mdr1 cDNA into the T24 cells greatly increases the expression of P gp.TADM cells manifests excellent MDR phenotype.The establishment of MDR cell lines by gene tranfection has the benefits of less time consuming,stronger drug resistivity and more stable.