Potentiation of paclitaxel cytotoxicity in lung and esophageal cancer cells by pharmacologic inhibition of the phosphoinositide 3-kinase/protein kinase B (Akt)–mediated signaling pathway

Downstream signaling of activated Akt. Binding of growth factors to their cognate receptors results in receptor phosphorylation, recruitment of multiple adaptor proteins (SOS and Grb2), activation of PI3K, and generation of PIP₃ in the plasma membrane. PIP₃ provides docking sites for proteins containing the Pleckstrin homology domain (eg, Akt and PDK1) to accumulate at the sites of PI3K activation. Proximity of Akt and PDK results in phosphorylation and activation of Akt. Activated Akt in turn phosphorylates and modulates functions of a myriad of cellular proteins, creating an antiapoptotic milieu and enhanced cell-survival capacity. NF, Nuclear factor; PIP₃, phosphatidylinositol-3,4,5-triphosphate.

A, Western blot analysis of LY-mediated dose-dependent suppression of Akt phosphorylation in NSCLC and esophageal cancer cell lines in vitro. B, Dose-dependent inhibition of cell proliferation by LY in NSCLC and esophageal cancer cells. Cells were treated with increasing concentrations of LY for 60 hours, and viable cells were quantitated by MTT assay. Data are presented as mean ± SD of 3 independent experiments.

Dose- and time-dependent LY-mediated cell-cycle arrest at G0/G1 checkpoint in H460 NSCLC and TE2 esophageal cancer cells in vitro. Cell-cycle analysis was performed using propidium iodide staining and flow cytometry.

A, LY-mediated dose-dependent reduction of phosphorylated Akt levels in H322 and H460 NSCLC cells and SKGT5 and TE2 esophageal cancer cells was paralleled with increase of IκB levels and reduced expression of NF-κB–dependent antiapoptotic proteins cIAP1, cIAP2, and BclXL. B, LY-mediated dose-dependent suppression of intrinsic NF-κB activity in cultured NSCLC and esophageal cancer cells as determined by the NF-κB-Luciferase reporter system. Luciferase activity in LY-treated cells was expressed as percentages of activity of untreated control cells. Data are expressed as mean ± SD of 3 independent experiments (*P < .001-P = .004 vs controls).

Supra-additive enhancement of cytotoxicity mediated by the Taxol + LY combinations. Although LY induced mild reduction of cell proliferation, exposure of Taxol-treated cells to LY resulted in further reduction of cell viability that exceeded the growth-inhibitory effect mediated by LY. Cultured cancer cells were treated with varying doses of Taxol for 90 minutes, followed by exposure to LY (10, 20, or 40 μmol/L) 12 hours later. Viable cells were quantitated by MTT assay 72 hours after the onset of Taxol treatment. Data are expressed as mean ± SD of 4 independent experiments.

Activation of Akt (increased phosphorylated Akt levels) (A) and significant elevation of NF-κB transcriptional activity (as determined by NF-κB Luciferase reporter system) (B) in H322 NSCLC and TE2 esophageal cancer cells treated with increasing concentrations of Taxol (50, 100, or 500 nmol/L) was totally suppressed by PI3K inhibitor LY (40 μmol/L). Mean ± SD; 3 independent experiments (*P = .012 vs controls, #P < .001 vs Taxol-treated cells). The combinations of Taxol and LY synergistically induced apoptosis in NSCLC and esophageal cancer cells (C). Cells were sequentially treated with Taxol and LY for 60 hours before harvest and assayed for DNA fragmentation of apoptosis using the TUNEL-based ApoBrdU technique. Data are expressed as mean ± SD of 3 independent experiments (*P < .001-P = .007 of Taxol + LY vs Taxol alone or LY alone). TUNEL, Terminal deoxynucleotide transferase-mediated dUTP nick-end labeling.

A, BAY, through direct inhibition of IκK, also suppressed intrinsic NF-κB activity in NSCLC and esophageal cancer cells. Data are expressed as mean ± SD of 4 independent experiments (*P < .001-P = .013 vs controls). B, This was paralleled with an enhancement of the magnitude of apoptosis in cells treated with the Taxol + BAY combination. Representative data of 3 independent experiments that yielded similar results are shown here.