Tie2-mediated multidrug resistance in malignant gliomas is associated with upregulation of ABC transporters

Abstract

Resistance and relapse are still primary causes that result in poor effectiveness of chemotherapy in malignant gliomas. Therefore, development of new therapeutic strategies requires the identification of key molecular pathways regulating chemoresistance. We previously found that abnormal high expression of the Tie2 receptor in gliomas was associated with tumor malignancy. Here, we studied the role of Tie2 activation in drug resistance by testing the cytotoxicity of several chemotherapeutic drugs in a panel of human glioma cell lines and brain tumor stem cells and found that Tie2 activation was significantly related to chemoresistance. The essential role of Tie2 in this phenotype was illustrated by silencing Tie2 using specific siRNA, and the subsequent abrogation of the angiopoietin 1 (Ang1)-mediated chemoresistance. Using quantitative real-time PCR and functional drug efflux studies, we observed that Tie2 activation resulted in increased expression of ATP-binding cassette (ABC) transporters. Consistent with these results, downmodulation of ABCG2 or ABCC2 resulted in the inability of Tie2 activation to induce a chemoresistant phenotype. Our results indicate that Tie2 activation may be important in modifying the evolution of gliomas during conventional chemotherapy regimens, and open new avenues for the search of more effective therapies to avoid the inevitable brain tumor recurrence.

Figure 1

Tie2 activation resulted in an increased IC₅₀ of the different chemotherapeutic drugs in the human glioma cells. (a) Tie2 expression in glioma cells (A172, U373 MG) and brain tumor stem cells (NSC20). Cellular membrane fractions (M) were obtained as previously described (Lee et al., 2006), and subjected to western blotting analysis, using antibody against Tie2 (1:500, C-20; Santa Cruz Biotechnology, Santa Cruz, CA, USA). Tie2 expression in isogenic cells (Lee et al., 2006), U251.vector and U251.Tie2 cells, was used as controls (C = cytosol fraction). (b) IC₅₀ for the panel of chemotherapeutics in cells expressing endogenous Tie2. Cells (5000 cells per well in 96-well plates) were treated with angiopoietin 1 (Ang1, 250 ng/ml; R&D Systems, Minneapolis, MN, USA) or vehicle (0.1% bovine serum albumin, BSA) overnight, and then treated with the different drugs for 48 h (doses of 1–100 µm for cisplatin and 10 nm–5 µm for SN38, mitoxantrone (MTX), or doxorubicin (Dox)). Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and then we analysed the dose–response curves using CalcuSyn software version 2.1 (Biosoft, Cambridge, UK) and calculated the IC₅₀ values (dose causing 50% cell survival) for each drug and treatment, as previously described (Alonso et al., 2007). Data are represented as the fold of increase in the IC₅₀ relative to control (which was set equal to 1). Figure numbers represent mean ± s.d. from three independent experiments performed in triplicate. (c) U251.vector or U251.Tie2 cells overexpressing constitutively active Tie2 were treated with the indicated drugs for 48 h. Cell viability was assessed by MTT assay, and the IC₅₀ values were calculated and represented as in Figure 1b. (d) Tie2⁻ U373 MG cells were exposed to Ang1 for 18 h and then treated with the indicated panel of chemotherapeutics. Cell viability was assessed by MTT assay 48 h later, and the IC₅₀ values were calculated and represented as in Figure 1b. *P < 0.01, compared to mock-treated cells (A172, NSC20, in b) or to U251.vector cells (in c) (t-test).

Figure 2

Role of Tie2 in chemoresistance of glioma cells. (a) Left, reverse transcriptase (RT)–PCR and western blotting analyses showed downregulation of Tie2 mRNA and protein levels in A172 cells 48 h after transfection with Tie2 siRNA (40 nm; Santa Cruz Biotechnology), as compared to the controls (vehicle or control (Co) siRNA, 40 nm; Santa Cruz Biotechnology). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and epidermal growth factor receptor (EGFR) levels are shown as loading controls (NT = no DNA template). Methodology was reported previously (Lee et al., 2006). Right, A172 human glioma cells were transfected with siRNA against Tie2 or control siRNA or were mock treated. After 48 h, cultures were incubated overnight with angiopoietin 1 (Ang1, 250 ng/ml) or vehicle (veh) and then exposed to cisplatin (100 µm). After 2 days, cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay. Cell death is expressed relative to the cisplatin-induced cell death in untreated A172 cells (= 100%). Data are represented as the mean ± s.d. from three independent experiments performed in triplicate. (b) Left, RT–PCR and western blotting analyses showed downregulation of Tie2 mRNA and protein in U251.Tie2 cells 48 h after transfection with Tie2 siRNA (40 nm), as compared to the controls (veh or control (Co) siRNA). Right, U251.Tie2 and U251.vector glioma cells were transfected with siRNA against Tie2 or control siRNA or were mock treated. After 48 h, cultures were exposed to cisplatin (25 µm). After 2 days, cell viability was measured by MTT reduction assay. Cell death is expressed relative to cisplatin-induced cell death in untreated U251.vector cultures (= 100%). Data are represented as median ± s.d. from three independent experiments performed in triplicate. *P < 0.01 in either vehicle-treated A172 cells (as in a) or U251.vector cells (as in b) (one-way analysis of variance (ANOVA) followed by a Student—Newman–Keuls multiple range test).

Figure 3

Tie2 activation resulted in upregulation of ATP-binding cassette (ABC) transporters expression levels in human glioma cells and brain tumor stem cells. (a) ABC transporter RNA expression (ABCG2, ABCC2, MDR1, MRP1) in human glioma cells as assessed by qPCR. Glioma cells (A172 and U373 MG cells) and brain tumor stem cells (NSC20) were treated with either angiopoietin 1 (Ang1) or vehicle. Total RNA was extracted 18 h later and qPCR was performed. Relative expression levels of ABCG2, ABCC2, MDR1 and MRP1 are represented as the fold increase compared to basal levels (= 1). The mean ± s.d. are shown from three independent experiments performed in triplicate. *P < 0.01 Ang1- vs vehicle-treated cells (A172 and NS20) and U251.Tie2 vs U251.vector cells (t-test). (b) Active Tie2 is related to increased drug efflux. U251.vector (Tie2⁻) and U251.Tie2 (Tie2⁺) cells were treated with mitoxantrone (MTX, 1 µm) or vehicle for 30 min, at 37 °C in a 5% CO₂ atmosphere. Cells were then incubated for 1 h at 37 °C in mitoxantrone-free medium, and then analysed with a FACS Calibur flow cytometer (BD Biosciences, San Jose, CA, USA). Shown are representative results from one of the three independent experiments. (c) Examination of ABCG2 and ABCC2 RNA levels of glioma cells after Tie2 downmodulation. Cells were transfected with siRNA against Tie2 (siTie2; 40 nm) or control siRNA (siCo; 40 nm) or were mock treated. After 48 h, A172 cells were incubated overnight with Ang1 (250 ng/ml) or vehicle. Total RNA was extracted, and the ABCG2 and ABCC2 RNA levels were measured by qPCR. Relative expression levels of ABCG2 and ABCC2 are represented as the x-fold increase compared to the basal expression levels in U251.vector or vehicle-treated A172 cells (= 1, dotted lines). Shown are the mean ± s.d. from three independent experiments performed in triplicate. *P < 0.01 vs mock-treated cells (one-way analysis of variance (ANOVA) followed by a Student–Newman–Keuls multiple range test).

Figure 4

Tie2-induced chemoresistance is mediated by upregulation of ATP-binding cassette (ABC) transporters. (a) Reverse transcriptase (RT)–PCR showing a decrease in the ABCG2 and ABCC2 RNA levels in the A172 and U251 MG cells 48 h after transfection of siRNA against ABCG2 (siABCG2), siRNA against ABCC2 (siABCC2), control siRNA (siCo) (50 nm; Ambion Inc., Applied Biosystems, Foster City, CA, USA), or mock-treated cells. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) amplification was used as the internal control. NT, no DNA template. (b) Drug-induced cell death in A172 human glioma cells after incubation with ABCG2 siRNA or ABCC2 siRNA. Cells were then treated overnight with angiopoietin 1 (Ang1, 250 ng/ml) or vehicle, and mitoxantrone (500 nm) or cisplatin (100 µm) were added to the cultures. After 48 h, cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay. The relative drug-induced cell death is represented as the mean ± s.d. of three independent experiments performed in triplicate (cell death induced by drug in untreated A172 was set equal to 100%). (c) Drug-induced cell death in the U251 MG cells treated with mitoxantrone (500 nm) after incubation with ABCG2 siRNA, or cisplatin (10 µm) after incubation with ABCC2 siRNA. After 48 h, cell viability was measured by MTT reduction assay. Relative drug-induced cell death is represented as the mean ± s.d. of three independent experiments performed in triplicate (cell death induced by drug in mock-treated U251.vector was set equal to 100%). *P < 0.01 vs either vehicle- or mock-treated (A172) or U251.vector cells (one-way analysis of variance (ANOVA) followed by a Student–Newman–Keuls multiple range test). (d) Mitoxantrone incorporation functional study in Ang1-stimulated A172 and U251.Tie2 cells after downmodulating ABCG2 expression. Cells were transfected with siRNA against ABCG2 or control siRNA (both 50 nm), and 48 h later, they were treated with mitoxantrone (MTX; 1 µm) or the vehicle for 30 min. Cells were then washed, allowed to incubate for 1 h in mitoxantrone-free media and then fluorescence was determined as explained in Figure 3. Shown are representative results from one of the three independently performed experiments.