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Inhibition of Rho-associated coiled-coil containing protein kinase enhances the activation of epidermal growth factor receptor in pancreatic cancer cells.

Nakashima M, Adachi S, Yasuda I, Yamauchi T, Kawaguchi J, Hanamatsu T, Yoshioka T, Okano Y, Hirose Y, Kozawa O, Moriwaki H - Mol. Cancer (2011)

Bottom Line: As a result, the precise role of ROCK remains controversial.We also obtained similar results using transforming growth factor-α.Taken together, these data indicate that ROCK functions to switch off EGFR signaling by promoting the internalization of the EGFR.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Gastroenterology, Gifu University Graduate School of Medicine, Gifu 501-1194, Japan.

ABSTRACT

Background: Rho-associated coiled-coil containing protein kinase (Rho-kinase/ROCK) is involved in various cellular functions including cell proliferation, and is generally considered to be oncogenic, while some studies show that ROCK functions as a negative regulator of cancer progression. As a result, the precise role of ROCK remains controversial. We have previously reported that Rho-kinase/ROCK negatively regulates epidermal growth factor (EGF)-induced cell proliferation in SW480 colon cancer cells. In the present study, we investigated the role of ROCK in EGF receptor (EGFR) signaling in the pancreatic cancer cell lines, Panc1, KP3 and AsPc1.

Results: In these cells, Y27632, a specific ROCK inhibitor, enhanced EGF-induced BrdU incorporation. The blockade of EGF stimulation utilizing anti-EGFR-neutralizing antibodies suppressed Panc1 cell proliferation. EGF induced RhoA activity, as well as the phosphorylation of cofilin and myosin light chain (MLC), both targets of ROCK signaling, and Y27632 suppressed both of these processes, indicating that the phosphorylation of cofilin and MLC by EGF occurs through ROCK in Panc1 cells. EGF-induced phosphorylation of EGFR at tyrosine residues was augmented when the cells were pretreated with Y27632 or were subjected to gene silencing using ROCK-siRNA. We also obtained similar results using transforming growth factor-α. In addition, EGF-induced phosphorylation of p44/p42 mitogen-activated protein kinase and Akt were also enhanced by Y27632 or ROCK-siRNA. Moreover, an immunofluorescence microscope study revealed that pretreatment with Y27632 delayed EGF-induced internalization of EGFR. Taken together, these data indicate that ROCK functions to switch off EGFR signaling by promoting the internalization of the EGFR.

Conclusions: While EGF first stimulates the activation of the EGFR and subsequently increases cancer cell proliferation, EGF concurrently induces the activation of ROCK, which then turns off the activated EGFR pathway via a negative feedback system.

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The effects of Y27632 and EGF on pancreatic cancer cell proliferation. (A) Panc1, KP3 and AsPc1 cells were pretreated with 3 μM Y27632 or vehicle in RPMI containing 0.3% FCS for 1 h, and then stimulated with 30 ng/ml of EGF or vehicle for 24 h. The BrdU incorporation as the % of the control (lane 1) is shown. (B) Panc1 cells were treated with the indicated doses of Y27632 or vehicle in RPMI containing 3% FCS for 48 h, and the cell viability assay was performed using the MTT cell proliferation kit I. (C) The attached cells were treated with 0.5 μg/ml of normal mouse-IgG (open circle) or anti-EGFR-neutralizing antibodies (closed circle) for the indicated periods in medium containing 3% FCS, and the surviving cells were counted using the MTT cell proliferation kit I. The results are expressed as the absorbance (OD 560 nm-OD 750 nm). All assays were done in triplicate. (*) indicates a significant difference (p < 0.05) compared with lane 1, (#) indicates a significant difference (p < 0.05), compared with lane 2.
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Figure 1: The effects of Y27632 and EGF on pancreatic cancer cell proliferation. (A) Panc1, KP3 and AsPc1 cells were pretreated with 3 μM Y27632 or vehicle in RPMI containing 0.3% FCS for 1 h, and then stimulated with 30 ng/ml of EGF or vehicle for 24 h. The BrdU incorporation as the % of the control (lane 1) is shown. (B) Panc1 cells were treated with the indicated doses of Y27632 or vehicle in RPMI containing 3% FCS for 48 h, and the cell viability assay was performed using the MTT cell proliferation kit I. (C) The attached cells were treated with 0.5 μg/ml of normal mouse-IgG (open circle) or anti-EGFR-neutralizing antibodies (closed circle) for the indicated periods in medium containing 3% FCS, and the surviving cells were counted using the MTT cell proliferation kit I. The results are expressed as the absorbance (OD 560 nm-OD 750 nm). All assays were done in triplicate. (*) indicates a significant difference (p < 0.05) compared with lane 1, (#) indicates a significant difference (p < 0.05), compared with lane 2.

Mentions: In order to examine whether or not EGF and ROCK are involved in pancreatic cancer cell proliferation, we first evaluated BrdU incorporation in Panc1, KP3 and AsPc1 cells utilizing Y27632 as a specific ROCK inhibitor. When these cells were treated with EGF (Figure 1A, lane 2, respectively), the BrdU incorporation was increased. Interestingly, BrdU incorporation was also increased when these cells were treated with Y27632 alone (Figure 1A, lane 3 compared to lane 1, respectively). In addition, the BrdU incorporation induced by EGF was further enhanced when these cells were pretreated with Y27632 (Figure 1A, lane 4 compared to lane 2, respectively). To verify these results, we also performed another experiment using the MTT assay. The growth of Panc1 cells was significantly enhanced when the cells were pretreated with Y27632 at a dose over 1 μM (Figure 1B). Taken together, these results indicate that ROCK plays a suppressive role in pancreatic cancer cell proliferation.


Inhibition of Rho-associated coiled-coil containing protein kinase enhances the activation of epidermal growth factor receptor in pancreatic cancer cells.

Nakashima M, Adachi S, Yasuda I, Yamauchi T, Kawaguchi J, Hanamatsu T, Yoshioka T, Okano Y, Hirose Y, Kozawa O, Moriwaki H - Mol. Cancer (2011)

The effects of Y27632 and EGF on pancreatic cancer cell proliferation. (A) Panc1, KP3 and AsPc1 cells were pretreated with 3 μM Y27632 or vehicle in RPMI containing 0.3% FCS for 1 h, and then stimulated with 30 ng/ml of EGF or vehicle for 24 h. The BrdU incorporation as the % of the control (lane 1) is shown. (B) Panc1 cells were treated with the indicated doses of Y27632 or vehicle in RPMI containing 3% FCS for 48 h, and the cell viability assay was performed using the MTT cell proliferation kit I. (C) The attached cells were treated with 0.5 μg/ml of normal mouse-IgG (open circle) or anti-EGFR-neutralizing antibodies (closed circle) for the indicated periods in medium containing 3% FCS, and the surviving cells were counted using the MTT cell proliferation kit I. The results are expressed as the absorbance (OD 560 nm-OD 750 nm). All assays were done in triplicate. (*) indicates a significant difference (p < 0.05) compared with lane 1, (#) indicates a significant difference (p < 0.05), compared with lane 2.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3141756&req=5

Figure 1: The effects of Y27632 and EGF on pancreatic cancer cell proliferation. (A) Panc1, KP3 and AsPc1 cells were pretreated with 3 μM Y27632 or vehicle in RPMI containing 0.3% FCS for 1 h, and then stimulated with 30 ng/ml of EGF or vehicle for 24 h. The BrdU incorporation as the % of the control (lane 1) is shown. (B) Panc1 cells were treated with the indicated doses of Y27632 or vehicle in RPMI containing 3% FCS for 48 h, and the cell viability assay was performed using the MTT cell proliferation kit I. (C) The attached cells were treated with 0.5 μg/ml of normal mouse-IgG (open circle) or anti-EGFR-neutralizing antibodies (closed circle) for the indicated periods in medium containing 3% FCS, and the surviving cells were counted using the MTT cell proliferation kit I. The results are expressed as the absorbance (OD 560 nm-OD 750 nm). All assays were done in triplicate. (*) indicates a significant difference (p < 0.05) compared with lane 1, (#) indicates a significant difference (p < 0.05), compared with lane 2.
Mentions: In order to examine whether or not EGF and ROCK are involved in pancreatic cancer cell proliferation, we first evaluated BrdU incorporation in Panc1, KP3 and AsPc1 cells utilizing Y27632 as a specific ROCK inhibitor. When these cells were treated with EGF (Figure 1A, lane 2, respectively), the BrdU incorporation was increased. Interestingly, BrdU incorporation was also increased when these cells were treated with Y27632 alone (Figure 1A, lane 3 compared to lane 1, respectively). In addition, the BrdU incorporation induced by EGF was further enhanced when these cells were pretreated with Y27632 (Figure 1A, lane 4 compared to lane 2, respectively). To verify these results, we also performed another experiment using the MTT assay. The growth of Panc1 cells was significantly enhanced when the cells were pretreated with Y27632 at a dose over 1 μM (Figure 1B). Taken together, these results indicate that ROCK plays a suppressive role in pancreatic cancer cell proliferation.

Bottom Line: As a result, the precise role of ROCK remains controversial.We also obtained similar results using transforming growth factor-α.Taken together, these data indicate that ROCK functions to switch off EGFR signaling by promoting the internalization of the EGFR.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Gastroenterology, Gifu University Graduate School of Medicine, Gifu 501-1194, Japan.

ABSTRACT

Background: Rho-associated coiled-coil containing protein kinase (Rho-kinase/ROCK) is involved in various cellular functions including cell proliferation, and is generally considered to be oncogenic, while some studies show that ROCK functions as a negative regulator of cancer progression. As a result, the precise role of ROCK remains controversial. We have previously reported that Rho-kinase/ROCK negatively regulates epidermal growth factor (EGF)-induced cell proliferation in SW480 colon cancer cells. In the present study, we investigated the role of ROCK in EGF receptor (EGFR) signaling in the pancreatic cancer cell lines, Panc1, KP3 and AsPc1.

Results: In these cells, Y27632, a specific ROCK inhibitor, enhanced EGF-induced BrdU incorporation. The blockade of EGF stimulation utilizing anti-EGFR-neutralizing antibodies suppressed Panc1 cell proliferation. EGF induced RhoA activity, as well as the phosphorylation of cofilin and myosin light chain (MLC), both targets of ROCK signaling, and Y27632 suppressed both of these processes, indicating that the phosphorylation of cofilin and MLC by EGF occurs through ROCK in Panc1 cells. EGF-induced phosphorylation of EGFR at tyrosine residues was augmented when the cells were pretreated with Y27632 or were subjected to gene silencing using ROCK-siRNA. We also obtained similar results using transforming growth factor-α. In addition, EGF-induced phosphorylation of p44/p42 mitogen-activated protein kinase and Akt were also enhanced by Y27632 or ROCK-siRNA. Moreover, an immunofluorescence microscope study revealed that pretreatment with Y27632 delayed EGF-induced internalization of EGFR. Taken together, these data indicate that ROCK functions to switch off EGFR signaling by promoting the internalization of the EGFR.

Conclusions: While EGF first stimulates the activation of the EGFR and subsequently increases cancer cell proliferation, EGF concurrently induces the activation of ROCK, which then turns off the activated EGFR pathway via a negative feedback system.

Show MeSH
Related in: MedlinePlus