Nuclear localization of CDK5RAP3 was important for the suppression of p14ARF promoter activity.

<p>(<b>a</b>) <i>S</i>chematic diagram of CDK5RAP3 mutants (<b>b</b>) Western blotting showing the expression levels of CDK5RAP3 mutants overexpressed in HepG2. Protein lysates from reporter assay were used for Western blotting probed with anti-Myc antibody. (<b>c</b>) <i>D</i>ual luciferase reporter was performed by co-transfection of CDK5RAP3 mutants with p14^ARF-luc reporter in HepG2. Results were mean of three independent experiments, with promoter activity of vector control set as 100%. *, <i>P</i><0.05 and **, <i>P</i><0.005 compared with vector control, Student’s <i>t</i>-test. (<b>d</b>) Confocal images of wild type (WT) and the indicated deletion mutants of Myc-CDK5RAP3.</p

Suppression of endogenous expression of p14^ARF by CDK5RAP3.

<p>(<b>a</b>) The <i>p14^ARF</i> and <i>CDK5RAP3</i> mRNA expression in stable CDK5RAP3 knockdown SMMC-7721 stable clones was determined by Quantitative real-time PCR (qPCR). Data was analyzed by comparative Ct method. Band intensity was analyzed using AlphaEasePC software and normalized with <i>β-actin</i>. Results were mean of three independent experiments. *, <i>P</i><0.005, Student’s <i>t</i>-test. (b) Similar to (a), the CDK5RAP3 stable expressing HepG2 clones (CDK5RAP3#1 and #2), vector control and parental cells were used for qPCR assay. Results were mean of three independent experiments. *<i>P</i><0.04 and **<i>P</i><0.02 compared with vector control, Student’s <i>t</i>-test. (c) The CDK5RAP3 expression construct and p14^ARF luciferase reporter, pGL3-p14^ARF were co-transfected into SMMC-7721 cells for dual-luciferase reporter assay. Results represent mean ±SD for triplicate wells. *, <i>P</i><0.05 compared with vector control, Student’s <i>t</i>-test. (d) Similar to (c), luciferase reporters carrying truncation mutants of the p14^ARF promoter, CDK5RAP3 expression construct (0.3 µg) and vector (0.3 µg) were used for dual-luciferase reporter assay. Results represent mean ±SD for triplicate wells. *, <i>P</i><0.02 compared with vector control, Student’s <i>t</i>-test. (e) CDK5RAP3 bound <i>p14^ARF</i> promoter by performing chromatin immunoprecipitation (ChIP) analysis on CDK5RAP3 stable overexpression clone #2 HepG2 cells (1×10⁷). Input (IN) and no antibody control (No Ab) were included. Error bars: mean ±SD.</p

Knockdown of p14^ARF reversed the suppression of cell migration and invasiveness in CDK5RAP3 knockdown HCC cells.

<p>(<b>a</b>) The CDK5RAP3 stable knockdown SMMC-7721 cells were transfected with p14^ARF or control siRNA. <i>Top,</i> Western blotting showing p14^ARF knockdown in cells; <i>bottom,</i> The bar chart showed the quantitation of migrated cells in three independent experiment (*, <i>P</i> = 0.005, Student’s <i>t</i>-test). Representative photomicrographs were shown. (<b>b</b>) Similar to <b>a</b>), but invasion assay were performed. The bar chart showed the quantitation of the invaded cells in three independent experiment (*, <i>P</i> = 0.05, Student’s <i>t</i>-test).</p

Regulation of p14^ARF localization and protein expression by CDK5RAP3.

<p>(<b>a</b>) The p14^ARF and CDK5RAP3 protein levels in stable CDK5RAP3 knockdown SMMC-7721 clones (shCDK5RAP3#1 and #2), vector control and parental cells <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042210#pone.0042210-Mak1" target="_blank">[5]</a> were compared by Western blotting using indicated antibodies, respectively. (<b>b</b>) The stable CDK5RAP3 knockdown SMMC-7721 (shCDK5RAP3#2) and vector control cells were treated with 100 µg/ml cycloheximide or DMSO (vehicle), and harvested at the indicated time points. p14^ARF and <i>β</i>-actin protein levels were determined by Western blotting. V: DMSO treatment. <i>Top</i>: Western blotting; <i>bottom</i>: quantification of p14^ARF protein level.</p

CDK5RAP3 transcriptionally regulated p14^ARF in a E2F1 independent manner.

<p>(<b>a</b>) CDK5RAP3 was co-transfected with p53-responsive element reporter for luciferase assay in HepG2 cells. Results represent mean ±SD for triplicate wells. *, <i>P</i><0.05, **, <i>P</i><0.005 compared with vector control, Student’s <i>t-</i>test. (<b>b</b>) Expression constructs of CDK5RAP3 was co-transfected with HA-E2F1 and p14^ARF-luc reporter for luciferase assay in HepG2 cells. Results represent mean ±SD for triplicate wells. *, <i>P</i><0.005 compared with vector control, Student’s <i>t</i>-test.</p

IPA-3 suppressed cell proliferation.

<p>(A) The effect of IPA-3 on the cell proliferation rates of MIHA (upper, left panel), HepG2 (upper, middle panel) H2P (upper, right panel), H2M (lower, left panel) and MHCC97L (lower, right panel) cells. Statistical analysis was performed by comparing with the value of DMSO control. *<i>P</i><0.05, **<i>P</i><0.01 (ANOVA). (<b>B</b>) BrdU labeling assays of MIHA (upper, left panel), HepG2 (upper, middle panel), H2P (upper, right panel), H2M (lower, left panel) and MHCC97L (lower, right panel) cells. *<i>P</i><0.05 (ANOVA) compared with the DMSO control. Error bars, mean ± SD of triplicate samples. (<b>C</b>) Representative plates of colony formation assay of MIHA (left panel), HepG2 (middle panel) and H2M cells (right panel). Bar chart of colony formation assay (lower panel). *<i>P</i><0.001, **<i>P</i><0.01 (ANOVA) compared with the DMSO control. Error bars, mean ± SD of triplicate samples.</p

IPA-3 Inhibits the Growth of Liver Cancer Cells By Suppressing PAK1 and NF-κB Activation

<div><p>Hepatocellular carcinoma (HCC) is one of the major malignancies worldwide and is associated with poor prognosis due to the high incidences of metastasis and tumor recurrence. Our previous study showed that overexpression of p21-activated protein kinase 1 (PAK1) is frequently observed in HCC and is associated with a more aggressive tumor behavior, suggesting that PAK1 is a potential therapeutic target in HCC. In the current study, an allosteric small molecule PAK1 inhibitor, IPA-3, was evaluated for the potential in suppressing hepatocarcinogenesis. Consistent with other reports, inhibition of PAK1 activity was observed in several human HCC cell lines treated with various dosages of IPA-3. Using cell proliferation, colony formation and BrdU incorporation assays, we demonstrated that IPA-3 treatment significantly inhibited the growth of HCC cells. The mechanisms through which IPA-3 treatment suppresses HCC cell growth are enhancement of apoptosis and blockage of activation of NF-κB. Furthermore, our data suggested that IPA-3 not only inhibits the HCC cell growth, but also suppresses the metastatic potential of HCC cells. Nude mouse xenograft assay demonstrated that IPA-3 treatment significantly reduced the tumor growth rate and decreased tumor volume, indicating that IPA-3 can suppress the <i>in vivo</i> tumor growth of HCC cells. Taken together, our demonstration of the potential preclinical efficacy of IPA-3 in HCC provides the rationale for cancer therapy.</p></div

Inhibitory effect of IPA-3 on PAK1.

<p>(<b>A</b>) Relative protein levels of PAK1 in various cell lines. The signal intensities of the bands were quantified and normalized by taking that level of MIHA as 1. (<b>B</b>) MTT assay on Day 1 and 2. H2M cells (4×10³) were seeded onto 96-well plates and treated with different dosages of IPA-3. The cells were harvested after incubation and the MTT assay was performed as mentioned in Material and Method. The graph showed the percentage of viable cells plotted against the dosage of IPA-3 (<b>C</b>) Western blotting analysis of the P-PAK1, total PAK1, P-JNK and total JNK. Serum-starved H2M cells were treated with either DMSO or IPA-3 at the indicated concentration for 15 minutes and followed by FBS replenishment and overnight culture. (<b>D</b>) H2M cells were serum-starved and treated with IPA-3 (10, 20 or 40 µM) in complete medium and allowed to grow overnight. The cell morphology images were captured at a magnification of 40X. Scale bar, 0.4 mm.</p

The suppressive effect of IPA-3 in nude mouse xenograft model.

<p>(<b>A</b>) MHCC97L cells were used for the xenograft model. Mice were treated three times weekly either with DMSO or IPA-3 (2 mg/kg or 4 mg/kg, i.p.). *<i>P</i><0.001 (ANOVA) compared with the DMSO control group. (<b>B</b>) Tumor weights were measured at the end of study. *<i>P</i><0.001, **<i>P</i><0.01, (ANOVA) compared with the DMSO control group. (<b>C</b>) Representative results of Western blotting analysis. P-PAK1 (T423), total PAK1, P-JNK and total JNK were detected. ***<i>P</i><0.05 (ANOVA) compared with the DMSO control. Error bars, mean ± SD of 5 animals per group.</p

The suppressive effect of IPA-3 on migration of H2M cells.

<p>(<b>A</b>) Paxillin protein expression was detected by immunofluorescence analysis under IPA-3 treatment. H2M and H2P (upper panel) cells were serum-starved overnight and treated with either DMSO control or IPA-3 (20 µM) for 15 minutes, followed by FBS replenishment for 10 minutes. Immunofluoresence signals of phalloidin (Red), paxillin (Green) and DAPI (Blue) represent stress fiber, focal adhesion and nucleus, respectively (magnification 40X). The number of focal adhesion (paxillin) were counted in H2M (lower, left panel) and H2P (lower, right panel), and represented in the bar chart. Error bars, mean ± SD of triplicate samples. *<i>P</i><0.01 (<i>t</i>-test) compared with DMSO control. (<b>B</b>) Western blotting analysis on the phosphorylation levels of PAK1 and paxillin. Serum-starved cells were treated with various concentrations of IPA-3 as indicated for 15 minutes, followed by FBS replenishment for 10 minutes. (<b>C</b>) Representative images of Transwell migration assay of H2M cells. Cells were treated with either DMSO or 10 µM IPA-3, and were allowed to migrate for 24 hours. Images show the cells having migrated to the lower chamber (upper panel). The number of migrated cells were counted and represented in the bar chart (lower panel). Error bars, mean ± SD of triplicate samples. *<i>P</i><0.01 (<i>t</i>-test) compared with DMSO control.</p