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IPA-3 inhibits the growth of liver cancer cells by suppressing PAK1 and NF-κB activation.

Wong LL, Lam IP, Wong TY, Lai WL, Liu HF, Yeung LL, Ching YP - PLoS ONE (2013)

Bottom Line: 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 in vivo 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China.

ABSTRACT
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 in vivo 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.

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The inhibitory effect of IPA-3 on NF-κB nuclear translocation.(A) Effect of IPA-3 on subcellular localization of NF-κB was evaluated by immunofluorescence staining. After overnight serum starvation, H2M (left panel) and MIHA (right panel) cells were treated with either DMSO or IPA-3 (20 µM, 15 minutes) followed by an addition of TNF-α (20 ng/ml, 15 minutes). NF-κB was detected with a specific antibody (Green) and nucleus was stained with DAPI (Blue). (B) Western blotting analysis of P-PAK1 (T423) and total PAK1 were detected in the H2M cells stimulated by TNF-α (20 ng/ml) with or without IPA-3 pretreatment (20 µM, 15 minutes). TNF-α was included in the culture medium for 0, 0.5, 1, 2 or 4 hours. (C) Expression of quantitative real-time PCR was performed to analyze the mRNA level of MMP-9 (left panel) and COX-2 (right panel). Serum-starved H2M cells were treated with or without IPA-3 pretreatment (10 or 20 µM, 15 minutes) followed by TNF-α (10 or 20 ng/ml, 24 hours). Quantitative results of MMP-9 and COX-2 mRNA levels were normalized to β-actin. The values represented the mean ± SD of three independent experiments. *P<0.001 (ANOVA), ***P<0.05 (ANOVA) compared with the TNF-α control.
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pone-0068843-g005: The inhibitory effect of IPA-3 on NF-κB nuclear translocation.(A) Effect of IPA-3 on subcellular localization of NF-κB was evaluated by immunofluorescence staining. After overnight serum starvation, H2M (left panel) and MIHA (right panel) cells were treated with either DMSO or IPA-3 (20 µM, 15 minutes) followed by an addition of TNF-α (20 ng/ml, 15 minutes). NF-κB was detected with a specific antibody (Green) and nucleus was stained with DAPI (Blue). (B) Western blotting analysis of P-PAK1 (T423) and total PAK1 were detected in the H2M cells stimulated by TNF-α (20 ng/ml) with or without IPA-3 pretreatment (20 µM, 15 minutes). TNF-α was included in the culture medium for 0, 0.5, 1, 2 or 4 hours. (C) Expression of quantitative real-time PCR was performed to analyze the mRNA level of MMP-9 (left panel) and COX-2 (right panel). Serum-starved H2M cells were treated with or without IPA-3 pretreatment (10 or 20 µM, 15 minutes) followed by TNF-α (10 or 20 ng/ml, 24 hours). Quantitative results of MMP-9 and COX-2 mRNA levels were normalized to β-actin. The values represented the mean ± SD of three independent experiments. *P<0.001 (ANOVA), ***P<0.05 (ANOVA) compared with the TNF-α control.

Mentions: Previous reports demonstrated that PAK1 stimulates the activity and subcellular translocation of nuclear factor light-chain enhancer of activated B cells (NF-κB), and promotes cell survival [17], [18]. Thus, we examined whether IPA-3 is able to inhibit the activity of NF-κB. H2M with a high endogenous expression level of PAK1 and immortalized hepatocytes, MIHA cells were serum-starved and then separately treated with IPA-3 followed by tumor necrosis factor-alpha (TNF-α). As showed in Fig. 5A, NF-κB positive staining was predominantly detected in the cytoplasm of the DMSO control, whereas NF-κB staining was accumulated in the nucleus after TNF-α induction. Interestingly, H2M cells pretreated with IPA-3 resulted in a cytoplasmic staining of NF-κB, indicating that IPA-3 suppressed the TNF-α-induced nuclear targeting of NF-κB. Unlike H2M cells, IPA-3 did not suppress TNF-α-induced NF-κB activation in MIHA cells, which lack the endogenous PAK1. This result suggested that the inhibition of NF-κB activation by IPA-3 is PAK1-dependent. To further investigate whether PAK1 inactivation is involved in the IPA-3-induced suppression of NF-κB translocation, phosphorylation of PAK1 was determined (Fig. 5B). Consistent with an earlier study which demonstrated that PAK1 was promptly activated by TNF-α in various cell lines [19], the phospho-PAK1 level was elevated in cells stimulated with TNF-α (20 ng/ml). The phosphorylation level was highest at 0.5-hour and then gradually reduced afterwards. On the other hand, the phosphorylation of PAK1 was completely suppressed in IPA-3 pretreated cells. This result suggested that IPA-3 is able to abolish the PAK1 activation induced by TNF-α, which correlated well with the IPA-3 inhibition on TNF-α-induced NF-κB translocation. The induction of metalloproteinase (MMP)-9 by TNF-α was shown to be mediated by PAK1 [19]. To elucidate the effect of IPA-3 on the TNF-α-induced activity of MMP-9 and COX-2, which are downstream targets of NF-κB [20], [21], we performed qRT-PCR to quantify the mRNA production of MMP-9 and COX-2. After normalization with β-actin, H2M cells responded to TNF-α with an increasing mRNA production of MMP-9 and COX-2 (Fig. 5C). However, the results showed that treatment of IPA-3 significantly suppressed the expression of MMP-9 and COX-2 transcripts in a dose dependent manner.


IPA-3 inhibits the growth of liver cancer cells by suppressing PAK1 and NF-κB activation.

Wong LL, Lam IP, Wong TY, Lai WL, Liu HF, Yeung LL, Ching YP - PLoS ONE (2013)

The inhibitory effect of IPA-3 on NF-κB nuclear translocation.(A) Effect of IPA-3 on subcellular localization of NF-κB was evaluated by immunofluorescence staining. After overnight serum starvation, H2M (left panel) and MIHA (right panel) cells were treated with either DMSO or IPA-3 (20 µM, 15 minutes) followed by an addition of TNF-α (20 ng/ml, 15 minutes). NF-κB was detected with a specific antibody (Green) and nucleus was stained with DAPI (Blue). (B) Western blotting analysis of P-PAK1 (T423) and total PAK1 were detected in the H2M cells stimulated by TNF-α (20 ng/ml) with or without IPA-3 pretreatment (20 µM, 15 minutes). TNF-α was included in the culture medium for 0, 0.5, 1, 2 or 4 hours. (C) Expression of quantitative real-time PCR was performed to analyze the mRNA level of MMP-9 (left panel) and COX-2 (right panel). Serum-starved H2M cells were treated with or without IPA-3 pretreatment (10 or 20 µM, 15 minutes) followed by TNF-α (10 or 20 ng/ml, 24 hours). Quantitative results of MMP-9 and COX-2 mRNA levels were normalized to β-actin. The values represented the mean ± SD of three independent experiments. *P<0.001 (ANOVA), ***P<0.05 (ANOVA) compared with the TNF-α control.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3716906&req=5

pone-0068843-g005: The inhibitory effect of IPA-3 on NF-κB nuclear translocation.(A) Effect of IPA-3 on subcellular localization of NF-κB was evaluated by immunofluorescence staining. After overnight serum starvation, H2M (left panel) and MIHA (right panel) cells were treated with either DMSO or IPA-3 (20 µM, 15 minutes) followed by an addition of TNF-α (20 ng/ml, 15 minutes). NF-κB was detected with a specific antibody (Green) and nucleus was stained with DAPI (Blue). (B) Western blotting analysis of P-PAK1 (T423) and total PAK1 were detected in the H2M cells stimulated by TNF-α (20 ng/ml) with or without IPA-3 pretreatment (20 µM, 15 minutes). TNF-α was included in the culture medium for 0, 0.5, 1, 2 or 4 hours. (C) Expression of quantitative real-time PCR was performed to analyze the mRNA level of MMP-9 (left panel) and COX-2 (right panel). Serum-starved H2M cells were treated with or without IPA-3 pretreatment (10 or 20 µM, 15 minutes) followed by TNF-α (10 or 20 ng/ml, 24 hours). Quantitative results of MMP-9 and COX-2 mRNA levels were normalized to β-actin. The values represented the mean ± SD of three independent experiments. *P<0.001 (ANOVA), ***P<0.05 (ANOVA) compared with the TNF-α control.
Mentions: Previous reports demonstrated that PAK1 stimulates the activity and subcellular translocation of nuclear factor light-chain enhancer of activated B cells (NF-κB), and promotes cell survival [17], [18]. Thus, we examined whether IPA-3 is able to inhibit the activity of NF-κB. H2M with a high endogenous expression level of PAK1 and immortalized hepatocytes, MIHA cells were serum-starved and then separately treated with IPA-3 followed by tumor necrosis factor-alpha (TNF-α). As showed in Fig. 5A, NF-κB positive staining was predominantly detected in the cytoplasm of the DMSO control, whereas NF-κB staining was accumulated in the nucleus after TNF-α induction. Interestingly, H2M cells pretreated with IPA-3 resulted in a cytoplasmic staining of NF-κB, indicating that IPA-3 suppressed the TNF-α-induced nuclear targeting of NF-κB. Unlike H2M cells, IPA-3 did not suppress TNF-α-induced NF-κB activation in MIHA cells, which lack the endogenous PAK1. This result suggested that the inhibition of NF-κB activation by IPA-3 is PAK1-dependent. To further investigate whether PAK1 inactivation is involved in the IPA-3-induced suppression of NF-κB translocation, phosphorylation of PAK1 was determined (Fig. 5B). Consistent with an earlier study which demonstrated that PAK1 was promptly activated by TNF-α in various cell lines [19], the phospho-PAK1 level was elevated in cells stimulated with TNF-α (20 ng/ml). The phosphorylation level was highest at 0.5-hour and then gradually reduced afterwards. On the other hand, the phosphorylation of PAK1 was completely suppressed in IPA-3 pretreated cells. This result suggested that IPA-3 is able to abolish the PAK1 activation induced by TNF-α, which correlated well with the IPA-3 inhibition on TNF-α-induced NF-κB translocation. The induction of metalloproteinase (MMP)-9 by TNF-α was shown to be mediated by PAK1 [19]. To elucidate the effect of IPA-3 on the TNF-α-induced activity of MMP-9 and COX-2, which are downstream targets of NF-κB [20], [21], we performed qRT-PCR to quantify the mRNA production of MMP-9 and COX-2. After normalization with β-actin, H2M cells responded to TNF-α with an increasing mRNA production of MMP-9 and COX-2 (Fig. 5C). However, the results showed that treatment of IPA-3 significantly suppressed the expression of MMP-9 and COX-2 transcripts in a dose dependent manner.

Bottom Line: 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 in vivo 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China.

ABSTRACT
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 in vivo 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.

Show MeSH
Related in: MedlinePlus