Blockage of PI3K/PKB/P27kip1 signaling pathway can antagonize 17β-estradiol-induced Ishikawa proliferation and cell cycle progression

It is well-known that risk for endometrial adenocar-cinoma increases in patients with high level of estrogen that is unopposed by progestin. And activation of extracellular signal-regulated kinase (ERK)1 and phosphatidylinositol 3 kinase/protein kinase B (PI3K/PKB) pathway are responsible for hormone-dependent cell growth in endometrial carcinoma.2–4 PI3K produces phosphatidylinositol-3-phosphates by phosphory- lating the D3 hydroxyl of phosphoinositides, leading to membrane translocation of PKB, on which PKB is phosphorylated and activated. Then the activated PKB can stimulate antiapoptotic cellular responses and block apoptotic functions of the cells, thus regulate the apoptosis and proliferation of cell, cell cycle progression, glucose usage, and angiogenesis. In general, cell cycle transitions are controlled by cyclin-dependent kinases (CDKs). Activation of cyclin/CDK activities is required for cell cycle progression and G1/S transition in response to growth factor stimulation. In cancer cells, CDKs, cyclins, and CDK inhibitors are frequently deregulated. P27kip1 and P21WAF-1, as CDK inhibitors, can mediate cell cycle arrest following growth inhibitory stimuli.5 In this study, we focused on the inhibitory effect of LY294002, a specific PI3K inhibitor, on cell proliferation and cell cycle progression induced by 17β-estadiol in endometrial cancer cell line (Ishikawa). We analysed the expression of cell cycle associated proteins, CDK inhibitor, P27kip1, and proapoptotic protein Bad in the cells during PI3K mediated cell cycle progression induced by estrogen. METHODS Materials Water-soluble 17β-estradiol (E2), serum replace- ment (SR2) containing no steroids, no growth factors and monotetrazolium (MTT) were purchased from Sigma Co. (USA). Annexin V-FITC (fluorescein-isothiocyanate) kit for apoptosis detection was from BaoSai Biological Co. (China). The antibodies against cyclin E and β-actin were from Santa Cruz Biotechnology (USA) and the antibody against P27kip1 was from MaiXin Co. (China). The antibodies against phospho-PKB at Ser473 (p-PKB), phospho-Bad at Ser136 (p-Bad), and LY294002 were from Cell Signaling Technology (USA). Cell culture A well differentiated adenocarcinoma cell line (Ishikawa) bearing estrogen receptor (ER)6 was maintained in phenol red-free RPMI1640 medium supple-mented with 10% Fetal Calf Serum (FCS),100 U/ml penicillin and 100 μg/ml streptomycin, and cultured at 37°C in a 5% CO2 incubator. Cell proliferation assay Cells were seeded in parallel 96-well microtiter plates in Phenol red-free RPMI1640 containing 5% steroid-stripped FCS (DCC-FCS, using dextran-coated charcoal). After 24 hours, the cells were incubated in RPMI1640 containing 0.1% (v/v) SR2 (0.1% SR2-RPMI1640) for 24 hours, then were stimulated by DMSO plus 1 μmol/L E2 combined with different doses of LY294002. In MTT assay, cells were placed in medium with 1 mg/ml of MTT, then incubated at 37°C in a 5% CO2 incubator for 3 hours. The formazan crystals were dissolved in 200 μl DMSO. A microplate reader (Bio-Rad, Germany) was used to determine the absorbance at 570 nm. The viability was calculated with the formula: Viability (%) = 100% × (Absorbance of treated ample)/(Absorbance of cells treated with DMSO) Analysis of cell cycle and apoptosis Cells were plated in 60-mm dishes in phenol red-free RPMI1640 containing 5% DCC-FCS. After 24 hours, the cells were incubated in 0.1% SR2-RPMI1640 medium for another 24 hours, and then were exposed to 0.1% SR2-RPMI1640 medium with 1 μmol/L E2 combined with 0, 10, or 50 μmol/L LY29002 for 24 or 48 hours. For cell cycle analysis, the cell pellets were fixed in 80% ethanol overnight and then resuspended in 50 μl PBS containing 1 mg/ml RNase and kept in a thermostatic cabinet at 37°C for 30 minutes. DNA was stained with 0.5 μg/ml propidium iodide (PI). After analysis of PI-stained cells by fluorescence-activated cell sorting (FACS), cell cycle histograms were generated and analyzed to determine the percentage of cells in each phase (G0-G1, S, G2/M). The number of apoptotic cells was estimated by binding annexin V to externalized phosphatidyl-serine. After being incubated in 0.1% SR2-RPMI1640 medium for 24 hours, the cells were treated with 1 μmol/L E2 combined with 0, 10, or 50 μmol/L LY29002, and harvested at 48 hours. Early apoptotic cells would be stained with annexin V-FITC, viable cells would show no staining, and necrotic cells would be stained by both PI and annexin V-FITC. Western blotting Cells were plated in 100-mm plates in phenol red-free RPMI1640 containing 5% DCC-FCS. After subconfluence, cells were incubated in 0.1% SR2-RPMI1640 medium for 24 hours, then exposed to DMSO plus 1 μmol/L E2 combined with 0 or 50 μmol/L LY29002 for 24 or 48 hours. After being lysed, the total protein extracts from the cells were separated by SDS-PAGE and were probed with the antibodies against p-PKB, p-Bad, cyclin E, P27kip1, or β-actin. The proteins were detected by using an enhanced chemiluminescence system. The levels of the proteins were normalized with respect to β-actin and were quantified after measuring the optical density of the protein bands. Statistical analysis Data were expressed as means ± standard deviation (SD) and analyzed using balanced ANOVA and Fisher's least significant difference post hoc test. Statistical significance was defined as P<0.05. Expression level of protein was expressed as the ratio of protein/β-actin. RESULTS Inhibitory effect of LY294002 On exposure to 1 μmol/L E2, Ishikawa cells proliferated in a time-dependent manner with a viability of 150.23% at 72 hours, which decreased with the increase of LY294002 doses, and was significantly higher than that of cells stimulated by DMSO (P<0.01). Cell proliferation, either induced by or not by E2, was abrogated by LY294002, and was almost completely suppressed with 50 μmol/L of LY294002. Cell cycle progression arrested in G1 phase The percentage of Ishikawa cells were increased significantly by LY294002 at G0-G1 phase and were decreased at S phase (Table).Table: Distribution of cell cycles in Ishikawa stimulated by 1 μmol/L E2 with or without LY294002 (mean±SD)LY294002-induced apoptosis At 48 hours, the rate of apoptosis and necrosis in the Ishikawa cells stimulated by 1 μmol/L E2 plus 10 μmol/L LY294002 were 26.6% and 12.71% respectively, while those in the cells treated by1 μmol/L E2 plus 50 μmol/L LY294002 were 63.33% and 26.28%. And the rate of viable cells decreased to 10.38% from 81.82% without stimulation by LY294002. Phosphorylation of PKB regulated by PI3K By treating with E2, PKB phosphorylation and the expression level of cyclin E in Ishikawa were increased, but P27kip1 expression level was decreased. All of them were inhibited by LY294002. The level of P-Bad was unchanged during the whole experiment, and so did the level of β-actin relatively. The effects of LY294002 were more prominent at 48 hours than that at 24 hours. (Fig.)Fig.: The Ishikawa cells were exposed to DMSO, 1 μmol/L E2, or 1 μmol/L E2 plus 50 μmol/L LY294002 for 24 or 48 hours. Total cellular protein extracts were prepared and subjected to immunoblot analysis using specific antibodies against p-PKB, p-Bad, Cyclin E, P27kip1, and β-actin. Lanes 1–3: 24 hours; lanes 4–6: 48 hours. E2: 17 β-estradiol; LY: LY294002.DISUSSION In the last 5 years, it has been shown that PI3K/PKB signaling is critical for cell survival triggered by mitogenic factors, and that alterations to this pathway are frequent in human cancers, playing pivotal roles in proliferation, survival, apoptosis, and cycle progression of cancer cells by phosphorylating and dephosphorylating some substrates.4,7,8 Thus it might be valid to treat human malignancies by inhibiting the activation of PI3K/PKB signaling specifically. It was reported that estrogen can activate PI3K/PKB signaling in normal human endometrial cells.9 The results of this study indicate that, in the endometrial cancer cells stimulated by estrogen, LY294002-inhibited PI3K can decrease cell proliferation, induce arrested cell growth at G1 phase, and trigger cell apoptosis. The mechanism involved in the change of cell proliferation and apoptosis by PI3K may be related with direct interaction of PI3K with some signal proteins, in which the protein kinase activity of PI3K plays a critical role. It is well known that cell cycle progression is governed by CDKs. P27kip1, a CDKs inhibitor, can inhibit their activity by binding to cyclin E-CDK2 complex, and thus block entry into S phase. Our results indicate that PI3K was required for the interaction between cyclin E or cyclin E/CDK2 and P27kip1, which causes cell cycle arrest in G1 phase. And PTEN, a PI3K negative regulator, plays a role as tumor suppressor gene by upregulating P27kip1 and downregulating cyclin E.10,11 PKB is the most important downstream target of PI3K. It mediates cell events by phosphorylating cell cycle-related proteins (such as P27kip1), cyclin D, and proapoptotic protein.4 The result that LY294002 markedly inhibited PKB phosphorylation suggests that LY294002 is involved in regulation of PI3K-dependent cell cycle as a specific inhibitor of PI3K and thus an inhibitor of PKB activation. PKB can directly phosphorylate some key regulators of apoptotic cascade, such as Bad, a member of the Bcl-2 family. In some types of cells, PKB-dependent phosphorylation of Bad is related to the promotion of cell survival via PI3K/PKB signaling,12 which is different from our results. In our study, blockage of PI3K/PKB pathway by LY294002 had no influence on the levels of p-Bad. In summary, this study demonstrates that LY204002, an inhibitor of PI3K, inhibits E2-induced proliferation of endometrial cancer cells by inducing apoptosis and cell cycle arrest at G1 phase. Inhibition of PI3K restrains PKB and cyclin E form phosphorylation, but increases expression level of P27kip1. In endometrial cancer cells stimulated by E2, PI3K signaling regulates proliferation of the cells, and the progression cell cycle at G1 phase through the increased expression of cyclins. Inhibited PI3K activity can induce the expression of P27kip1, leading to decreased expression of cyclin E in endometrial cancer cells. The results suggest that PI3K/PKB/P27kip1 signaling can be a novel target for therapeutic intervention in endometrial cancers.