Molecular alterations in apoptotic pathways after PKB/Akt-mediated chemoresistance in NCI H460 cells

Abstract

Protein kinase B/Akt has been described as a central mediator of antiapoptotic signals in cancer cells. Furthermore, Akt has been shown to affect cell cycle progression and proliferative pathways and to possess a potential function in tumorigenesis and chemoresistance. In this study, we show that the ectopic expression of a constitutively active form of Akt1 (CA-Akt1) results in enhanced chemoresistance of NCI H460 human NSCLC cells towards a panel of chemotherapeutic agents. To understand the molecular alterations leading to impaired chemosensitivity mediated by activated Akt, we analysed various apoptotic pathways, including the activation of p53, caspases 3, 7, 8, and 9, release of cytochrome c from mitochondria, and the expression levels of pro- and antiapoptotic proteins such as Bcl-2, Bcl-x(L), Bcl-x(s), Bax, or Bfl-1. We observed that expression of CA-Akt did not interfere with single defined apoptotic switches, but modulated the apoptotic threshold of several apoptotic pathways towards increasing the threshold of onset. In particular, we found that CA-Akt-expressing cells displayed increased expression of the antiapoptotic Bcl-2 family member protein Bcl-x(l), and a delayed onset of the p53 pathway after treatment with cisplatin or Mitoxantrone. Thus, our data suggest that Akt mediates chemoresistance in NHI H460 cells by interfering with and delaying the onset of various apoptotic pathways. A complete inactivation of apoptotic pathways was observed in none of the molecular alterations investigated. Our data strengthen the role of Akt as a central mediator of cell survival signals and/or chemoresistance and as an attractive target for cancer cell chemosensitisation.

Figure 1

(A) Expression and activation of ectopically expressed farnesylated Akt1 in NCI H460 cells. Control transfected NCI H460 cells or cells expressing farnesylated Akt1 were serum starved overnight (-) and then stimulated for 15 min with medium containing 10% FCS (S) or 10% FCS plus a cocktail of 10 ng ml⁻¹ each EGF, IGF-1, and PDGF (GF). Cell lysates were analysed by immunoblotting with antibodies specific for Akt1, FLAG-epitope, phospho-Akt (Thr 308) or phospho-Akt (Ser 473) as indicated. The ectopically expressed farnesylated Akt1 devoid of the PH domain migrates at an MW of approximately 50 kDa. (B) Akt kinase activity in NCI H460-Akt1 cells and in control cells. Cells were serum starved for 16 h and then stimulated as described above. Endogenous Akt1 from control transfected cells and endogenous Akt1 plus farnesylated Akt1 from NCI H460-Akt1 cells were immunoprecipitated with an Akt1 specific antibody. Immunoprecipitates were used in kinase assay reactions with GSK-3-fusion protein as substrate. Immunoblots of the reactions were assayed with antibodies specific for phospho-GSK (Ser 21/9) (lower panel) and Akt1 (upper panel). (C) Proliferation rates of NCI H460 control transfectants and NCI H460-Akt1 cells. 3 × 10⁴ cells (as indicated) per well were seeded into six wells into a medium containing 0.5% FCS. At the indicated time points, the cells were harvested by trypsinisation and cell numbers were determined using a Coulter counter. Each sample was determined in triplicates. Two independent NCI H460-Akt1 cell clones were analysed. (D) Expression analysis of p21^Waf1 and p27^Kip1 in NCI H460-Akt1 cells vs control cells. Cell lysates were analysed by immunoblotting with antibodies specific for p21^Waf1, p27^Kip1, and β-actin as a loading control.

Figure 2

Decreased chemosensitivity of NCI H460-Akt1 cells towards doxorubicin, cisplatin, and Mitoxantrone. Control transfected NCI H460 cells (filled squares) or NCI H460-Akt1 cells (open triangles) were seeded in 96-well plates in medium containing 0.5% serum. After 24 h cells were treated with different concentrations of doxorubicin, cisplatin, or Mitoxantrone as indicated and incubated for another 72 h at 37°C. Cell viability was then determined with a standard XTT assay as described in ‘Materials and methods’. Values are the mean of at least three independent experiments.

Figure 3

(A) Activation of initiator caspases after treatment of NCI H460 cells with cisplatin. Control transfected NCI H460 cells or NCI H460-Akt1 cells were grown in medium containing 0.5% serum overnight and afterwards treated with cisplatin (20 μM) for 36 h. Cell lysates were analysed by immunoblotting with antibodies specific for caspase 8 or caspase 9 as indicated. (B) Activation of effector caspases and PARP after treatment of NCI H460 cells with cisplatin. Lysates from cells treated as described above were prepared and analysed by immunoblotting with antibodies specific for caspase 3, caspase 7, and PARP as indicated. (C) Enzymatic assays of caspase activity. Cells were treated as described above. Additionally, samples from cells treated with 400 nM Mitoxantrone for 24 h were included. Caspase 3, 8, and 9 activities from lysates were determined by a colorimetric assay as described in ‘Materials and methods’.

Figure 4

Expression analysis of Bcl-2 family proteins in NCI H460 cells. Control transfected NCI H460 cells or NCI H460-Akt1 cells were grown in medium containing 0.5% serum overnight (-) and afterwards treated with 150 nM Mitoxantrone (Mito) for 24 h or 20 μM cisplatin (CP) for 36 h, respectively. Equal amounts of cell lysates were analysed by immunoblotting with antibodies specific for Bcl-2, A1/Bfl-1, Bax, and Bcl-x_L as indicated.

Figure 5

Cytochrome c release from mitochondria after treatment of NCI H460 cells with Mitoxantrone or cisplatin. Control transfected NCI H460 cells or NCI H460-Akt1 cells were grown in medium containing 0.5% serum overnight (-) and treated with 400 nM Mitoxantrone (Mito) or 20 μM cisplatin (CP) for 8 h, respectively. (A) Cell lysates were fractionally centrifugated and analysed with a cytochrome c ELISA as described in ‘Materials and methods’. Each value was determined as triplicate. (B) Aliquots of cell lysates were analysed by Western blotting with an antibody specific for caspase 8 as a control for cellular fractionation.

Figure 6

Induction of p53 in Akt1-transfected NCI H460 cells. Control transfected NCI H460 cells or NCI H460-Akt1 cells as indicated were grown in medium containing 0.5% serum and treated with cisplatin (20 μM) for 2, 4, 8, 16, and 36 h, respectively. At each time point, cell lysates were generated and analysed by immunoblotting with antibodies specific for p53, MDM2, and β-actin.