RESUMEN
The aim of this study was to clarify whether bortezomib might induce apoptosis in Burkitt's lymphoma Raji cell line and its mechanism. Different concentrations of bortezomib were used to treat Raji cells and its effects of time and dose were observed. Cell morphology was observed under light microscope; flow cytometry was used to analyze cell apoptosis; RT-PCR was used to detect the expressions of NF-kappaB and p53 gene mRNAs. The results showed that the bortezomib could inhibit Raji cell growth within a certain range of treating time and dose. Apoptosis were induced in relation to time and dose. The expression of NF-kappaB mRNA and p53 mRNA decreased after treatment with bortezomib. It is concluded that the bortezomib can induce Raji cell apoptosis, which provides a theoretical basis for clinical treatment. NF-kappaB and p53 gene are supposed to participate in the bortezomib induced apoptosis of Raji cells.
Asunto(s)
Humanos , Apoptosis , Ácidos Borónicos , Farmacología , Bortezomib , Linfoma de Burkitt , Metabolismo , Patología , Línea Celular Tumoral , Proliferación Celular , Citometría de Flujo , FN-kappa B , Metabolismo , Pirazinas , Farmacología , Proteína p53 Supresora de Tumor , MetabolismoRESUMEN
The objective of study was to investigate the effect of ribosomal protein L6 (RPL6) gene expression on the drug resistance of leukemia cells and its possible mechanism. RPL6 cDNA was obtained by RT-PCR, both sense and antisense cDNA recombinants of RPL6-encoding gene were constructed with pcDNA3. 1 (+) expression vector. Subsequently, the sense RPL6 cDNA recombinant was transfected into K562 cells while the antisense one into K562/A02 cells by liposomal reagent. The chemosensitivity, apoptosis and caspase-3 activity of K562 and K562/A02 cells were evaluated by MTT assay, flow cytometer and fluorometer respectively. The results indicated that expression of RPL6 in K562/A02 was higher than that in K562; resistance of sense-transfected K562 cells to doxorubicin was 325% of control cells, apoptosis and caspase-3 activity decreased (P<0.005); whereas resistance of antisense-transfected K562/A02 cells to adriamycin was 38% of control cells, apoptosis and caspase-3 activity significantly increased (P<0.005). It is concluded that RPL6 gene plays an important role in the development of drug resistance in K562/A02 cells by changing drug-induced apoptosis.
Asunto(s)
Humanos , Antibióticos Antineoplásicos , Farmacología , Apoptosis , Genética , Doxorrubicina , Farmacología , Resistencia a Antineoplásicos , Genética , Regulación Leucémica de la Expresión Génica , Células K562 , Proteínas Ribosómicas , GenéticaRESUMEN
To construct a plasmid expressing MDR1 short hairpin RNA (shRNA) mediated by pEGFP-C1/U6 vector, two coding sequences of 19 nucleotides were selected from MDR. Two pairs of oligonucleotides were designed for these two fragments. After annealing the formed double-stranded DNAs were ligated with plasmid pEGFP-C1/U6 (pEGFP-C1 vector with U6 promoter). The plasmids producing MDR1 shRNA were constructed from the inverted motif containing 9 spacers and four Ts. The results showed that the constructed plasmids were named pEGFP-C1/U6/A and pEGFP-C1/U6/B, and the constructs were identified by restriction and sequence analysis, no any base mutation was observed. It is concluded that plasmids of pEGFP-C1/U6/A and pEGFP-C1/U6/B expressing MDR1 shRNA were successfully constructed, providing a highly effective method for reversing the multidrug resistance in clinic.
Asunto(s)
Humanos , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP , Genética , Proteínas de Unión al ADN , Resistencia a Múltiples Medicamentos , Genética , Resistencia a Antineoplásicos , Genética , Vectores Genéticos , Genética , Proteínas Fluorescentes Verdes , Genética , Plásmidos , Genética , ARN Mensajero , Genética , ARN Interferente Pequeño , Genética , ARN Nuclear Pequeño , GenéticaRESUMEN
<p><b>OBJECTIVE</b>To construct a short hairpin RNA (shRNA) eukaryotic expression vector specific to MDR1 gene in multidrug resistance (MDR) human leukemia cell line K562/A02 to observe its silencing effect on MDR1 and P-glycprotein (P-gp) expression.</p><p><b>METHODS</b>The shRNA expression vector was constructed by gene recombination, then transfected into the cultured K562/A02 cells. The transcription of MDR1 gene was detected by semi-quantitative RT-PCR and the expression level of P-gp was determined by Western blot. 50% inhibition concentration (IC50) of ADM in K562/A02 cells was determined by MTT method. The intracellular doxorubicin (ADM) concentration was determined by HPLC.</p><p><b>RESULTS</b>The introduction of pEGFP-C1/U6/MDR1-A or pEGFP-C1/U6/MDR1-B expression vector was shown to efficiently and specifically inhibit the expression of P-gp according to results of Western blot, with an inhibitory rate of 50.67%. Semi-quantitative RT-PCR showed that mRNA transcription of MDR1 gene was reduced by (48.2 +/- 2.5)%. On the contrast, the control plasmid did not exhibit inhibitory effect on the protein expression and mRNA transcription of MDR1. The relative efficiency of K562/A02 to ADM was 40.8% or 62.4%, respectively, and the intracellular accumulation of ADM increased after shRNA treatment.</p><p><b>CONCLUSION</b>The shRNA expression vector targeting MDR1 gene showed dramatic inhibition on RNA transcription and protein expression. It could effectively restore the sensitivity of K562/A02 cells to conventional chemotherapeutic agents.</p>
Asunto(s)
Humanos , Subfamilia B de Transportador de Casetes de Unión a ATP , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP , Genética , Metabolismo , Antibióticos Antineoplásicos , Metabolismo , Farmacología , Western Blotting , Supervivencia Celular , Relación Dosis-Respuesta a Droga , Doxorrubicina , Metabolismo , Farmacología , Resistencia a Múltiples Medicamentos , Genética , Resistencia a Antineoplásicos , Genética , Silenciador del Gen , Vectores Genéticos , Genética , Células K562 , ARN Mensajero , Genética , ARN Interferente Pequeño , Genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , TransfecciónRESUMEN
OBJECTIVE@#To explore the effect of neferine on the chemotherapic sensitivity of STI 571 to K562/A02 cells and to reveal its mechanism.@*METHODS@#MTT method was used to observe the alteration of the proliferation of K562/A02 cell line treated with STI571 alone or combined with neferine. The transcription of mdrl gene was detected by semi-quantitative RT-PCR and the P-gp expression was determined by Western blot after STI 571 alone or combined with neferine treatment.@*RESULTS@#The cytotoxic effect of STI 571 (1 micromol/L) combined with neferine (IC50 = 3.02 micromol/L) on K562/A02 cell line was significantly higher than that of STI 571 alone (IC50 = 0.689 micromol/L). After treating with STI571 combined with neferine, the synergistic interaction on K562/A02 cells increased 4.38 folds (P < 0.05); the mdrl mRNA expression by semi-quantitative RT-PCR was significantly reduced by (45.4 +/- 2.5) % (P < 0.01); and the P-gp expression by Western blot was deregulated by 40.58% (P < 0.05).@*CONCLUSION@#Neferine significantly enhances the antineoplastic effect of STI 571 on K562/A02 cells by reducing mdrl mRNA transcription and blocking P-gp expression.
Asunto(s)
Humanos , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP , Genética , Antineoplásicos Fitogénicos , Farmacología , Benzamidas , Bencilisoquinolinas , Farmacología , Proliferación Celular , Resistencia a Múltiples Medicamentos , Resistencia a Antineoplásicos , Sinergismo Farmacológico , Mesilato de Imatinib , Células K562 , Piperazinas , Farmacología , Proteínas Tirosina Quinasas , Pirimidinas , FarmacologíaRESUMEN
OBJECTIVE@#To explore the relationship among intracellular glutathione S-transferase activity (GST), the expression of lung resistance-related proteins (LRP) in acute leukemia, and its clinical effects.@*METHODS@#The GST activity of bone marrow mononuclear cells and LRP expression in 57 acute leukemia patients were detected by the spectrophotometry assay and immuno-cytochemistry (SABC), respectively.@*RESULTS@#The GST activity of bone marrow mononuclear cells in the acute leukemia group was significantly higher than that of the control group (P < 0.01). The GST activity of mononuclear cells in acute leukemia was positively correlated with the percentage of blast in the bone marrow (r = 0.30, P < 0.05). The GST activity of mononuclear cells in the untreated acute leukemia group was obviously higher than that of the complete remission group (P <0.01). The GST activity in the refractory or relapsed acute leukemia group was significantly higher than that of the complete remission group and untreated leukemia group (P <0.05). In post-chemotherapy 13 of 17 the LRP-positive patients were the non-remission, 12 of the 20 LRP-negative patients were the complete remission. The curative rate of the LRP-positive group was the significantly lower than the LRP-negative group (P < 0.05). The GST activities of non-remission patients in the LRP-positive and LRP-negative group obviously increased.@*CONCLUSION@#The increase of GST activity in the bone marrow mononuclear cells is related to the clinical curative effects and the proliferation of blast in acute leukemia. Detection of LRP and GST activities in acute leukemia may have a reference value in judging the leukemia with drug resistance and estimating the prognosis.