Knocking Down Low Molecular Weight Protein Tyrosine Phosphatase (LMW-PTP) Reverts Chemoresistance through Inactivation of Src and Bcr-Abl Proteins

The development of multidrug resistance (MDR) limits the efficacy of continuous chemotherapeutic treatment in chronic myelogenous leukemia (CML). Low molecular weight protein tyrosine phosphatase (LMW-PTP) is up-regulated in several cancers and has been associated to poor prognosis. This prompted us to investigate the involvement of LMW-PTP in MDR. In this study, we investigated the role of LMW-PTP in a chemoresistant CML cell line, Lucena-1. Our results showed that LMW-PTP is highly expressed and 7-fold more active in Lucena-1 cells compared to K562 cells, the non-resistant cell line. Knocking down LMW-PTP in Lucena-1 cells reverted chemoresistance to vincristine and imatinib mesylate, followed by a decrease of Src and Bcr-Abl phosphorylation at the activating sites, inactivating both kinases. On the other hand, overexpression of LMW-PTP in K562 cells led to chemoresistance to vincristine. Our findings describe, for the first time, that LMW-PTP cooperates with MDR phenotype, at least in part, through maintaining Src and Bcr-Abl kinases in more active statuses. These findings suggest that inhibition of LMW-PTP may be a useful strategy for the development of therapies for multidrug resistant CML

Identificação antropológica: superposição de imagens pelos ossos nasais.

Os aspectos periciais a respeito da identificação humana por meio do segmento cefálico utilizando os princípios atuais de Antropologia Forense, considerando o uso dos métodos diretos e indiretos, bem como, em especial os arcos dentais e as variações anatômicas morfológicas dos ossos nasais, como particularidades, foram úteis numa investigação forense de superposição de imagens.

Knocking down <i>LMW-PTP</i> decreased phosphorylation of Src and ABL.

<p>(<b>A</b>) Phosphorylation status of Src kinase and ABL in leukemia cells with silenced <i>LMW-PTP</i>. (<b>B</b>) The ratio between p-Brc-Abl(210 kDa)/GAPDH and p-c-Abl (135 kDa)/GAPDH, from K562 and Lucena-1 cells with silenced <i>LMW-PTP</i> (<i>LMW-PTP</i> siRNA) or not (scramble siRNA) were quantified by densitometry and plot in the graph. *** p<0.001- significant differences relative to the respective scramble siRNA.</p

Evaluation of P-glycoprotein activity.

<p>To measure P-gp activity the substrate Rhodamine 123 was used as substrate. <i>LMW-PTP</i> was silenced and after 24 h cells were further incubated for 1 h in the presence of 200 ng/mL Rhodamine 123 with or without a P-gp inhibitor (5 µM verapamil). Afterwards, cells were washed with PBS and Rhodamine 123 mean fluorescence intensity was assessed by flow cytometry analysis within the live-gate, using a FACScalibur (Becton and Dickinson, USA). Decreasing of mean fluorescence intensity indicates higher P-gp activity.</p

Expression of LMW-PTP and modulation of c-Src and FAK in leukemia cells treated with vincristine.

<p>Cells were treated with VCR for 24 h and LMW-PTP expression, phosphorylation status of c-Src and FAK were analyzed. (A) K562 and Lucena-1 cells were treated with VCR for 24 h and the protein levels were analyzed by Western blotting. (B) The ratio between LMW-PTP/GAPDH, Src/GAPDH, FAK/GAPDH, p-Src/Src, p-FAK/FAK from VCR treatment samples were quantified by densitometry and plot in the graph.</p

P-glycoprotein and LMW-PTP on both leukemia cell lines.

<p>Cells (100.000/ mL) were plated and lysed after 24 h. (<b>A</b>) The expression of P-gp and LMW-PTP were evaluated by immunoblotting. Soluble lysates were matched for protein content and analyzed by Western blot and β-actin was used as an internal control. (<b>B</b>) LMW-PTP from K562 and Lucena-1 cells was immunoprecipitated and afterwards had its activity determined by using p-nitrophenyl phosphate as a substrate.</p

K562 cells overexpressing LMW-PTP are less sensitive towards vincristine.

<p>(A) LMW-PTP was overexpressed in K562 and its expression was examined in the total lysate of leukemia cells. (<b>B</b>) K562 cells overexpression LMW-PTP (K562^LMW-PTP) or not (K562^WT) were treated with VCR 125 nM for 24 h and the viable cells were counted. Each value represents the mean ± S.D. of three independent experiments (n = 3). ***p<0.001, *p<0.5 - significant differences relative to control (no VCR) or as indicated in the graph.</p

<i>LMW-PTP</i> silencing increases the cytotoxicity effect of chemotherapeutics on Lucena-1 cells.

<p>(<b>A</b>) LWM-PTP was knocked down in Lucena-1 cells and its expression was checked by Western (<b>B</b>) After 48 h of <i>LMW-PTP</i> silencing Lucena-1 cells (100.000 cells/ml) were treated with VCR 500 nM or imatinib 500 nM for 24 h, subsequently, the viable cells were assessed by trypan blue exclusion and (<b>C</b>) apoptosis induction by caspase 3 activity. Each value represents the mean ± S.E.M. of three independent experiments (n = 3) and has been normalized to scramble siRNA sample. *** p<0.001 - significant differences relative to its respective scramble siRNA sample.</p