All cell lines were obtained from Cell Resource Center for Biomedical Research Institute of Development, Aging and Cancer, Tohoku University. Cells were cultured in Roswell Park Memorial Institute-1640 (RPMI-1640; Gibco, 05918, Gaithersburg, MD, USA) medium supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco, 26140-079) and 21 mM of L-glutamine, at 37°C, in a humidified 5% CO₂-95% air mixture. GEM-resistant cells derived from KLM1 (KLM1-R) was established as previous descriptions (19,20). KLM1 cells were tretaed with 10 µg/ml of GEM for 2 weeks, after which, cells were washed and resuspended in fresh medium. This was followed by a 2-week culture period which served as a recovery procedure. This procedure was repeated for another 3 cycles.

LY294002 (9901) and wortmannin (9951S) were purchased from Cell Signaling Technology (Boston, MA, USA). AKTi-1/2 (ab142088) was purchased from Abcam Biochemicals (Cambridge, MA, USA). Epithelial growth factor (E9644) was purchased from Sigma (St. Louis, MO, USA). The antibodies specific for p-ERK^T204/202 (sc-7383), ERK (sc-94200), Hsp27 (sc-13132) and actin (sc-1616) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The antibodies specific for Src (Src Antibody Sampler kit, 9935), p-Hsp27^S78 (2405), p-Hsp27^S82 (2401), PTEN (9552S), AKT (9272) and p-AKT^S473 (4058) were purchased from Cell Signaling Technology.

Total protein was extracted from the cells using lysis buffer (1% NP-40, 1 mM sodium vanadate, 11 mM PMSF, 501 mM Tris, 101 mM NaF, 101 mM EDTA, 1651 mM NaCl, 10 µg/ml leupeptin, and 10 µg/ml aprotinin) (21). Equal amounts of protein (20 µg) were resolved by 5–20% SDS-polyacrylamide gel and then transferred onto PVDF membrane (Immobilon-P; Millipore, Bedford, MA, USA). The membrane was incubated with a primary antibody at 4°C overnight and a horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at room temperature. The immunoblots were visualized with a chemiluminescent reagent (Immunostar, Wako, Osaka, Japan) and detected by using an Image Reader LAS-1000 Pro (Fujifilm Corp., Tokyo, Japan).

Cells were cultured on coverslips in 12-well plates at a density of 1×10⁵ cells per well. Cells were fixed using fresh 3.7% paraformaldehyde in phosphate-buffered saline (PBS) and permeabilized with 0.1% Triton X-100. After washing with PBS they were incubated in blocking solution (1% goat serum or 1% donkey serum in PBS with 0.1% Tween-20) for 1 h at room temperature (22). Cells were treated with a primary antibody in blocking solution overnight at 4°C and a secondary antibody for 1 h at room temperature. Cell nuclei were counter-stained with 1.43 µM DAPI (4,6′-diamidino-2-phenylindole). Confocal images were obtained by using Laser Scan Confocal Microscope (LSM 510 META; Carl Zeiss, Mobicity, Australia).

Cells were cultured in 96-well plates. After treatment, 20 µl of the 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethonyphenol)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) dye (Promega, Madison, WI, USA) was added to each well of the plate and incubated for 2 h at 37°C, in a humidified 5% CO₂-95% air mixture. Optical density (OD) was read directly at 492 nm using the iMark™ microplate absorbance reader (Bio-Rad, Hercules, CA, USA). Each experiment was repeated three times.

The effect of the PI3K inhibitor, LY294002, on AKT phosphorylation at serine 473 in PC PK59 cells was detected by western blot analysis. Unexpectedly, p-AKT was significantly increased rather than decreased by treatment with LY294002 for 24 h in a dose-dependent manner and over a time course (Fig. 1A and C). LY294002 was also shown to enhance epithelial growth factor (EGF)-triggered AKT phosphorylation (Fig. 1B). However, the other PI3K inhibitor, wortmannin, showed the expected performance in inhibiting the PI3K/AKT pathway (Fig. 1B and C). Interestingly, the effect of wortmannin on inhibiting AKT phosphorylation was weakened after 6 h of treatment and completely disappeared after 24 h (Fig. 1C). In addition, the phosphorylation of tyrosine-protein kinase CSK (Src), extracellular regulated protein kinases (ERK) and heat shock protein 27 (Hsp27), which have been shown to be involved in AKT activation (23–26), were not altered by treatment with LY294002 (Fig. 1D). These results indicate that LY294002, but not wortmannin, displays the opposite function to that expected in regulating the PI3K/AKT signaling pathway in PK59 cells.

LY294002, as well as wortmannin, have shown their ability to inhibit PI3K/AKT activity in HeLa and HEK293 cells (Fig. 2A and B). LY294002 could also inhibit AKT phosphorylation in various types of PC cancer cell lines, such as in Panc-1, MIA-PaCa-2, AsPC-1 and BxPC-3 cells (27–29). We previously reported that PK59 is much less sensitive to GEM (IC₅₀, 294.72 µg/ml) than Panc-1 (IC₅₀, 8.07 µg/ml), MIA-PaCa-2 (IC₅₀, 6.81 µg/ml), AsPC-1 (IC₅₀, 1.05 µg/ml) or BxPC-3 (IC₅₀, 6.67 µg/ml) cells (30). We thus tested whether LY294002-induced AKT phosphorylation specifically occurs in GEM-resistant PC cells. A GEM-resistant PC cell line KLM1-R has been established by culturing the gemcitabine-sensitive KLM1 cells with 10 µg/ml of GEM (19,20), as shown (Fig. 2C). As hypothesized, upregulation of p-AKT instead of downregulation was observed by treatment with LY294002 in GEM-resistant KLM1-R compared to KLM1 cells in a dose-dependent manner (Fig. 2D). These results suggest that the inhibiting ability of LY294002 on PI3K could be altered in PC, once cells acquire GEM-resistance. PI3K activity is required for the LY294002-induced AKT phosphorylation and associated intracellular membrane translocation.

Using immunofluorescent analysis, p-AKT could only be observed in the nuclei of untreated PK59 cells (Fig. 3). However, AKT phosphorylation was initiated in the cytoplasm of LY294002-treated cells and the p-AKT eventually translocated onto the intracellular membrane after treatment for 24 h (Fig. 3), indicating that the LY294002-induced p-AKT may have functions associated with this specific localization. This kind of phosphorylation and intracellular membrane translocation of AKT could be abolished by combined treatment with an AKT specific inhibitor, AKTi-1/2 (Fig. 4A). We next examined whether LY294002-induced AKT phosphorylation is mediated by PI3K. Following treatment with LY294002 alone, p-AKT was significantly upregulated, but following combined treatment with LY294002 and either AKTi-1/2 or wortmannin, p-AKT was completely inhibited in both PK59 and KLM1-R cells (Fig. 4B and C). These data reveal that LY294002-induced AKT phosphorylation and intracellular membrane translocation are still dependent on PI3K.

We next assessed the role of LY294002-induced p-AKT in cell proliferation. Treatment with wortmannin was shown to increase or have no effect on cell proliferation in PK59 and KLM1-R cells, respectively (Fig. 5A and B), indicating that GEM-resistant PC cells may also be resistant to the inhibitor of PI3K. Treatment with LY294002 alone significantly inhibited cell proliferation in PK59 and KLM1-R cells and this inhibition could be further enhanced by combined treatment with either AKTi-1/2 or wortmannin (Fig. 5A and B). These results indicate that LY294002-induced p-AKT plays a negative role in LY294002-triggered cytotoxicity in GEM-resistant PC cells. In addition, LY294002-induced p-AKT did not contribute to reducing the sensitivity of PK59 or KLM1-R cells to GEM (Fig. 5C and D).