Figure 1.
GAK is overexpressed in the nuclei of cancer cells.
Western blot analysis of GAK, lamin A/C, and α-tubulin (control) in cytoplasmic (Cyt) and nuclear (Nuc) extracts from prostate cancer (PC-3 and LNCaP), breast cancer (MDA-MB231 and MCF-7) and cervical cancer (HeLaS3) cell lines. Lamin A/C was used as a nuclear marker. Two different anti-GAK antibodies were used. The arrows and arrowheads indicate the GAK bands identified primarily in the nuclear and cytoplasmic fractions, respectively.
Figure 2.
GAK is overexpressed in radical prostatectomy specimens from prostate cancer patients.
A–D, hematoxylin and eosin (H-E) staining (A, B) and anti-GAK immunostaining (C, D) of representative radical prostatectomy sections of cancerous (A, C) and normal (B, D) tissues from prostate cancer patients (n = 42). Scale bar = 50 µm. E, higher power magnification of the boxed area shown in C. Scale bar = 50 µm.
Table 1.
Statistical analysis of the relationship between the Gleason score and GAK immunostaining in the prostatectomy specimens.
Figure 3.
Loss of GAK activity leads to constitutive hyper-phosphorylation of the EGFR.
A, western blot analysis of EGFR expression in WT (GAK-kd+/+) and mutant (GAK-kd−/−) MEFs. B, the effects of a Tyr-phosphatase inhibitor (50 mM Na3VO4) and Ser/Thr phosphatase inhibitors (50 mM NaF and 50 mM β-glycerophosphate, or 2.5 µM okadaic acid) on the inhibition of EGFR phosphorylation by λ-phosphatase (λPPase; 200 U). A, B, the arrows indicate the phosphorylated EGFR protein. Alpha-tubulin (α-Tub) was used as a loading control. (C, D) Western blot analysis of expression levels of the EGFR and ERK1/2 in WT (+/+) and GAK-kd (−/−) MEFs following EGF stimulation for the indicated times. Cycloheximide (50 µg/ml) was added to the culture medium 1 h prior to EGF (10 µg/ml) to inhibit novel protein synthesis. The tilted and horizontal arrows indicate the phosphorylated and hyper-phosphorylated EGFR bands, respectively. GAPDH was used as a loading control. C, the arrowheads indicate differential expression of phosphorylated ERK1/2 in WT and GAK-kd MEFs. D, NT, non-treated. E, immunostaining of the EGFR protein in WT (+/+) and mutant (−/−) MEFs treated with or without the proteasome inhibitor MG132 (50 µg/ml). Notable panels are encircled by turquoise and green lines. F, the numbers of EGFR-positive cells in WT and GAK-kd (Homo) cells in the presence or absence of MG132. The data are represented as the mean ± SEM of n = 3 independent experiments at each time point.
Figure 4.
Luteolin and erlotinib inhibit GAK activity similarly to gefitinib and SB203580.
A–C, representative SDS-PAGE analysis of proteins subjected to in vitro kinase assays using 32P-γATP, GAK (∼100 µg/ml) as the enzyme, PP2A B′γ (∼10 µg/ml) as the substrate, and the indicated concentrations of gefitinib, erlotinib, and SB203580 as inhibitors. The signals incorporated were detected by autoradiography and the amounts of proteins loaded were assessed by Coomassie Brilliant Blue (CBB) staining. The graphs show the intensity ratios of phosphorylated PP2A B′γ and GAK relative to that of non-treated samples. The data are represented as the mean ± SEM of n = 3 independent experiments at each concentration.
Figure 5.
Luteolin and gefitinib induce death of PC-3 cells.
A, the numbers of viable PC-3 cells 0, 24, 48, and 72 h after the addition of solvent alone (DMSO), 60 µM luteolin, 60 µM gefitinib, or 60 µM luteolin plus 60 µM gefitinib to the culture medium. The data are represented as the mean ± SEM of n = 3 independent experiments at each concentration. B, immunoblot analyses of GAK, AR, caspase-3, and α-tubulin (control) protein levels in PC-3 cells exposed to solvent alone (DMSO), 60 µM luteolin, 60 µM gefitinib, or 60 µM luteolin plus 60 µM gefitinib for 0–72 h. The arrowheads indicate reduced expression levels of GAK and AR in cells exposed to the drugs for 72 h. The arrows indicate the emergence of activated caspase-3, which suggested that gefitinib and luteolin induced apoptosis. C, FACS analysis of PC-3 cells PC-3 cells exposed to solvent alone (DMSO), 60 µM luteolin, 60 µM gefitinib, or 60 µM luteolin plus 60 µM gefitinib for 72 h. The arrowheads indicate the subG1 populations. D, the percentages of PC-3 cells in the indicated phases of the cell cycle after exposure to the same treatments for 72 h.
Figure 6.
Exogenous expression of miR-630 inhibits the growth of PC-3 cells.
A, confirmation by qRT-PCR of the successful overexpression of exogenous miR-630 from the pEP-hsa-miR-630 expression vector in PC-3 cells. The data are represented as the mean ± SEM of n = 3 independent experiments. B, The growth rate of PC-3 cells transfected with the pEP-hsa-miR-630 expression vector. Cells were also transfected with GFP to monitor the successful expression of miR-630. The data are represented as the mean ± SEM of n = 3 independent experiments for each condition. P<0.01 by a Student's t-test.
Figure 7.
Working model showing the molecular mechanisms involved in the induction of growth arrest and apoptosis of PC-3 cells by luteolin and gefitinib.
The arrows and T-shaped lines signify positive and negative actions, respectively. The thickness of each line is proportional to the strength of the denoted action. The dashed green line and arrow indicate that these actions were verified by another group [10]. The red and blue arrows indicate up-regulation and down-regulation, respectively. Down-regulation of GAK leads to hyper-phosphorylation of tyrosine residues (pY) in EGFR.