α2β1 integrin promotes chemoresistance against doxorubicin in cancer cells through extracellular signal-regulated kinase (ERK)

Abstract

The role and the mechanisms by which β1 integrins regulate the survival and chemoresistance of T cell acute lymphoblastic leukemia (T-ALL) still are poorly addressed. In this study, we demonstrate in T-ALL cell lines and primary blasts, that engagement of α2β1 integrin with its ligand collagen I (ColI), reduces doxorubicin-induced apoptosis, whereas fibronectin (Fn) had no effect. ColI but not Fn inhibited doxorubicin-induced mitochondrial depolarization, cytochrome c release, and activation of caspase-9 and -3. ColI but not Fn also prevented doxorubicin from down-regulating the levels of the prosurvival Bcl-2 protein family member Mcl-1. The effect of ColI on Mcl-1 occurred through the inhibition of doxorubicin-induced activation of c-Jun N-terminal kinase (JNK). Mcl-1 knockdown experiments showed that the maintenance of Mcl-1 levels is essential for ColI-mediated T-ALL cell survival. Furthermore, activation of MAPK/ERK, but not PI3K/AKT, is required for ColI-mediated inhibition of doxorubicin-induced JNK activation and apoptosis and for ColI-mediated maintenance of Mcl-1 levels. Thus, our study identifies α2β1 integrin as an important survival pathway in drug-induced apoptosis of T-ALL cells and suggests that its activation can contribute to the generation of drug resistance.

FIGURE 1.

ColI inhibits doxorubicin-induced apoptosis. The T cell lines Jurkat (a) and HSB-2 (b) were preactivated or not for 4 h with ColI (Coll I) or with Fn (Fbn), after which they were treated or not for 16 h with doxorubicin. At the end of cell culture, cell apoptosis was determined by annexin V staining and flow cytometry analysis (Jurkat) or by DNA fragmentation ELISA assay (HSB-2) as described under “Experimental Procedures.” Similar results were obtained when apoptosis was measured after 24 h of doxorubicin treatment (not shown). c, Jurkat cells were preactivated for 4 h with ColI, Fn, or with ColI+Fn. The cells were then treated or not for 16 h with doxorubicin, and apoptosis was determined by annexin V staining and flow cytometry analysis. d, Jurkat cells were preincubated or not for 1 h with 10 μg/ml of control (IgG), anti-α2, or anti-β1 integrin blocking antibodies before being activated with ColI for 4 h. The cells were then treated or not with doxorubicin for 16 h, and apoptosis was determined by DNA fragmentation ELISA assay. The results in the four panels are presented as mean values from three independent experiments with S.D. as indicated. *, p < 0.05 between doxorubicin+ColI and doxorubicin samples. **, p < 0.05 between doxorubicin+ColI and doxorubicin samples, and between doxorubicin+ColI and doxorubicin+ColI+anti-α2- or doxorubicin+ColI+anti-β1 samples. e, MSCs inhibit doxorubicin-induced apoptosis of Jurkat cells via α2β1 integrin. Jurkat cells were cultured in suspension or with a monolayer of MSCs as described under “Experimental Procedures.” Twenty four hours after doxorubicin treatment, Jurkat cells were separated from MSCs by pipetting with ice-cold PBS. As indicated, Jurkat cells were preincubated for 1 h with 10 μg/ml of anti-α2 or anti-β1 integrin blocking antibodies or control isotypic antibody (IgG), washed with PBS, and then co-cultured with MSCs. Apoptosis was determined by annexin V staining and FACS analysis. The results are presented as mean values from two independent experiments. *, p < 0.05 between samples of Jurkat cells co-cultured with MSCs treated with doxorubicin and samples of Jurkat cells cultured in suspension treated with doxorubicin, or with samples of Jurkat cells co-cultured with MSCs treated with doxorubicin in the presence of anti-α2- and anti-β1 antibodies. f, clonogenic survival of the T-ALL cell lines Jurkat (left panel) and HSB-2 (right panel) was determined as described under “Experimental Procedures.” The results represent mean values of two independent experiments with S.D. as indicated. *, p < 0.05 between doxorubicin+ColI samples and doxorubicin+Fn or doxorubicin samples.

FIGURE 2.

ColI inhibits doxorubicin-induced mitochondrial apoptosis by maintaining the levels of Mcl-1. a, ColI (Coll I) inhibits doxorubicin-induced loss of mitochondrial membrane potential. Jurkat cells were preactivated for 4 h with ColI or Fn (Fbn), after which they were treated for 16 h with doxorubicin (Dox). The loss of the mitochondrial membrane potential (low ΔΨ_m) was measured by DioC₆(3) staining and flow cytometry analysis. The results represent mean values of three independent experiments with S.D. as indicated. *, p < 0.05 between doxorubicin+ColI and doxorubicin samples. b, ColI inhibits doxorubicin-induced cytochrome c release. After activation, the cytosolic fractions were prepared and analyzed by immunoblotting using anti-cytochrome c (cyt c) Ab. The blot was stripped and reprobed with anti-β-actin Ab to confirm equal loadings. c, ColI inhibits doxorubicin-induced caspase activation. The cells were activated as described in a, and after cell lysis, activation of caspase-9 and -3 was determined by immunoblot analysis using specific anti-caspase-9 and anti-caspase-3 antibodies that recognize the native forms and the active fragments of caspases. The membrane was stripped and reprobed with anti-β-actin antibody to confirm equal loading. The results in b and c are representative of three independent experiments. d, ColI maintains Mcl-1 levels in doxorubicin-treated cells. The cells were preactivated with ColI or Fn, after which they were treated for 12 h with doxorubicin (Dox). After treatment, the cells were lysed, and the expression of Mcl-1, Bcl-2, and Bcl-x_L was determined by immunoblot analysis using specific antibodies. The blots were stripped and reprobed with anti-β-actin antibody to ensure equal loading. The results are representative of three independent experiments. e, Mcl-1 siRNA abolishes the protective effect of ColI. Jurkat cells were transfected with control siRNA or with Mcl-1 siRNA. After transfection, the cells were activated with ColI and treated or not with doxorubicin (Dox). Expression of Mcl-1 protein in control and in Mcl-1 siRNA-transfected cells was assessed by immunoblot analysis, and β-actin levels were determined as control to ensure equal loading (left panel). Apoptosis of cells transfected with control and Mcl-1 siRNA was determined after 16 h of treatment with doxorubicin by measuring the loss of the mitochondrial membrane potential (right panel). The results represent mean values of three independent experiments with S.D. as indicated. *, p < 0.05 between doxorubicin+ColI and doxorubicin samples in control siRNA-transfected cells.

FIGURE 3.

ColI inhibits doxorubicin-induced JNK activation. a, doxorubicin (Dox) activates JNK in Jurkat cells. The cells were treated with doxorubicin for the indicated periods of time, and JNK activation was determined by immunoblot analysis using a specific antibody recognizing the phosphorylated forms of JNK1/2. The blot was stripped and reprobed with anti-JNK-2 antibody to ensure equal loading. b, activation of JNK is necessary for doxorubicin-induced Mcl-1 down-regulation. The cells were treated for 12 h with doxorubicin (Dox) in the absence or in the presence of 10 μm of the JNK inhibitor SP600125. The cells were lysed, and expression of Mcl-1 protein was determined by immunoblot analysis. The blot was stripped and reprobed with anti-β-actin antibody to ensure equal loading. c, ColI (Coll I) but not Fn (Fbn) blocks the ability of doxorubicin to activate JNK. The cells were preactivated or not with ColI or Fn and then treated for 12 h with doxorubicin. JNK activation was determined by immunoblot analysis as described above. The results shown in the three panels are representative of three independent experiments. Nonspecific bands are indicated as NS (a and c).

FIGURE 4.

The protective effect of ColI is dependent on MAPK/ERK but not on PI3K/AKT pathway. Jurkat cells were activated for 4 h with ColI (Coll I) in the presence or absence of doxorubicin (Dox) and activation of ERK (a) and AKT (b) was determined by immunoblot analysis using specific antibodies recognizing the phosphorylated forms of ERK1/2 and AKT. The blots were stripped and reprobed with anti-ERK2 and anti-AKT antibodies to ensure equal loadings. Similar results were found with HSB-2 cells (data not shown). c, HSB-2 cells were pretreated or not with 10 μm of the MEK-1 inhibitor (U0126) or with 10 μm of the PI3K/AKT inhibitor (LY294002) for 1 h before being activated with ColI for 4 h. The cells were then treated or not with doxorubicin, and cell apoptosis was evaluated by DNA fragmentation ELISA assay. The results represent mean values from three independent experiments with S.D. as indicated. *, p < 0.05 between doxorubicin+ColI or doxorubicin+ColI+LY294002 and doxorubicin samples. Similar results were found with Jurkat cells (not shown). d, Jurkat cells were transfected with control plasmid or with plasmids encoding dominant-negative forms of MEK-1 (DN-MEK-1) and AKT (DN-AKT). After transfection, the cells were activated with ColI for 4 h and then treated with doxorubicin (Dox). Apoptosis was then determined by annexin V staining and flow cytometry analysis. The results represent mean values from three independent experiments with S.D. as indicated. *, p < 0.05 between doxorubicin+ColI and doxorubicin samples in control and in DN-AKT-transfected cells. e, Jurkat cells were pretreated with U0126 or with LY294002 inhibitors prior to their activation with ColI. The cells were then treated for 12 h with doxorubicin (Dox), and the expression of Mcl-1 was determined by immunoblot analysis. The blot was stripped and reprobed with anti-β-actin antibody to ensure equal loading. The results are representative of three independent experiments. f, the cells were treated and activated as described in e, and activation of JNK was determined by immunoblot analysis as described above. The results are representative of three independent experiments. Nonspecific bands are indicated as NS.

FIGURE 5.

ColI/α2β1 integrin inhibits doxorubicin-induced apoptosis in primary T-ALL blasts. a, expression of α2 and β1 integrin chains on primary T-ALL blasts was determined by immunostaining and flow cytometry analysis. The number of positive cells (%) and the mean fluorescence intensity values are indicated. b, ColI (Coll I) reduces doxorubicin-induced apoptosis of primary T-ALL blasts. The cells were preactivated or not for 4 h with ColI or Fn (Fbn), after which they were treated for 24 h with doxorubicin. Apoptosis was then determined by annexin V staining and flow cytometry analysis. The results represent mean values of triplicates with S.D. as indicated. *, p < 0.05 between doxorubicin+ColI and doxorubicin samples. c, ColI-mediated inhibition of doxorubicin-induced apoptosis in primary T-ALL blasts (patient 2) is MAPK/ERK-dependent. The cells were pretreated or not with 10 μm of the MEK-1 inhibitor (U0126) or with 10 μm of the PI3K/AKT inhibitor (LY294002) for 1 h before being activated with ColI for 4 h. The cells were then treated or not for 24 h with doxorubicin, and cell apoptosis was evaluated by DNA fragmentation ELISA assay. The results represent mean values of triplicates with S.D. as indicated. *, p < 0.05 between doxorubicin+ColI and doxorubicin samples.

FIGURE 6.

Proposed model by which α2β1 integrin mediates doxorubicin resistance in T-ALL cells. Doxorubicin (Dox) activates JNK, which induces the downmodulation of Mcl-1 levels, thus allowing activation of the mitochondrial death pathway. Ligation of α2β1 integrin with ColI activates the MAPK/ERK survival pathway, which inhibits the ability of doxorubicin to activate JNK and subsequently to downregulate the levels of prosurvival Mcl-1. Maintenance of Mcl-1 levels leads to the protection of mitochondria and to the inhibition of cytochrome c release and caspase activation, and subsequently to the decrease in doxorubicin-induced apoptosis.