Limits...
Targeting Cdc42 with the small molecule drug AZA197 suppresses primary colon cancer growth and prolongs survival in a preclinical mouse xenograft model by downregulation of PAK1 activity.

Zins K, Gunawardhana S, Lucas T, Abraham D, Aharinejad S - J Transl Med (2013)

Bottom Line: Rho GTPases play important roles in cytoskeleton organization, cell cycle progression and are key regulators of tumor progression.Strategies to modulate increased Rho GTPase activities during cancer progression could have therapeutic potential.These data indicate the therapeutic potential of the small-molecule inhibitor AZA197 based on targeting Cdc42 GTPase activity to modulate colorectal cancer growth.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory for Molecular Cellular Biology, Center for Anatomy and Cell Biology, Medical University of Vienna, Waehringerstrasse 13, A-1090 Vienna, Austria. dietmar.abraham@meduniwien.ac.at.

ABSTRACT

Background: Rho GTPases play important roles in cytoskeleton organization, cell cycle progression and are key regulators of tumor progression. Strategies to modulate increased Rho GTPase activities during cancer progression could have therapeutic potential.

Methods: We report here the characterization of a Cdc42-selective small-molecule inhibitor AZA197 for the treatment of colon cancer that was developed based on structural information known from previously developed compounds affecting Rho GTPase activation. We investigated the effects of AZA197 treatment on RhoA, Rac1 and Cdc42 activities and associated molecular mechanisms in colon cancer cells in vitro. Therapeutic effects of AZA197 were examined in vivo using a xenograft mouse model of SW620 human colon cancer cells. After treatment, tumors were excised and processed for Ki-67 staining, TUNEL assays and Western blotting to evaluate proliferative and apoptotic effects induced by AZA197.

Results: In SW620 and HT-29 human colon cancer cells, AZA197 demonstrated selectivity for Cdc42 without inhibition of Rac1 or RhoA GTPases from the same family. AZA197 suppressed colon cancer cell proliferation, cell migration and invasion and increased apoptosis associated with down-regulation of the PAK1 and ERK signaling pathways in vitro. Furthermore, systemic AZA197 treatment reduced tumor growth in vivo and significantly increased mouse survival in SW620 tumor xenografts. Ki-67 staining and tissue TUNEL assays showed that both inhibition of cell proliferation and induction of apoptosis associated with reduced PAK/ERK activation contributed to the AZA197-induced therapeutic effects in vivo.

Conclusions: These data indicate the therapeutic potential of the small-molecule inhibitor AZA197 based on targeting Cdc42 GTPase activity to modulate colorectal cancer growth.

Show MeSH

Related in: MedlinePlus

Cdc42 blockade by AZA197 suppresses tumor growth and prolongs animal survival. A Left panel: representative images of human SW620 tumor xenografts on day 22 from mice treated with solvent (Control) or 100 μg/day AZA197 (AZA197 d22) (bar = 10 mm). Right panel: AZA197 significantly suppresses tumor weight of human colon cancer xenografts in mice. Data are shown as mean tumor weights on day 22. *, significantly different from control on day 22 (P = 0.006). B Effect of AZA197 on proliferation in SW620 xenografts. Left panel: representative immunohistochemistry images of tumor tissue sections from mice treated with solvent (Control) or 100 μg/day AZA197 on day 22 stained with Ki-67 antibody (bar = 200 μm). Right panel: Quantitative histomorphometric analysis of Ki-67-positive, proliferating tumor cells. *, significantly different from control (P = 0.046). C Effect of AZA197 on apoptosis in SW620 xenografts. TUNEL labeling of cells undergoing DNA fragmentation: Left panel: representative images of tumor tissue sections from mice treated with solvent (Control) or 100 μg/day AZA197 on day 22 (bar = 50 μm). Right panel: Quantitative histomorphometric analysis of TUNEL-positive, apoptotic tumor cells. *, significantly different from control (P = 0.035). D Analysis of total Cdc42 protein expression and levels of PAK1 and ERK phosphorylation in SW620 colon cancer tissue on day 22 from mice treated with solvent (Control) or 100 μg/day AZA197 (AZA197 d22). Representative Western blot images and quantification of immunoblots stained with Cdc42, PAK1 and phospho-PAK1/2 (pPAK), ERK and phospho-ERK antibodies. Cdc42 blockade by AZA197 reduces PAK1/2 and ERK phosphorylation in SW620 tumor tissue. *, significantly different from control. E Effect of AZA197 treatment on animal survival in SW620 colon cancer bearing mice. Kaplan-Meier survival curves for controls and AZA197 treated mice. AZA197 significantly prolongs animal survival.*, significantly different to control (P = 0.042).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4222769&req=5

Figure 6: Cdc42 blockade by AZA197 suppresses tumor growth and prolongs animal survival. A Left panel: representative images of human SW620 tumor xenografts on day 22 from mice treated with solvent (Control) or 100 μg/day AZA197 (AZA197 d22) (bar = 10 mm). Right panel: AZA197 significantly suppresses tumor weight of human colon cancer xenografts in mice. Data are shown as mean tumor weights on day 22. *, significantly different from control on day 22 (P = 0.006). B Effect of AZA197 on proliferation in SW620 xenografts. Left panel: representative immunohistochemistry images of tumor tissue sections from mice treated with solvent (Control) or 100 μg/day AZA197 on day 22 stained with Ki-67 antibody (bar = 200 μm). Right panel: Quantitative histomorphometric analysis of Ki-67-positive, proliferating tumor cells. *, significantly different from control (P = 0.046). C Effect of AZA197 on apoptosis in SW620 xenografts. TUNEL labeling of cells undergoing DNA fragmentation: Left panel: representative images of tumor tissue sections from mice treated with solvent (Control) or 100 μg/day AZA197 on day 22 (bar = 50 μm). Right panel: Quantitative histomorphometric analysis of TUNEL-positive, apoptotic tumor cells. *, significantly different from control (P = 0.035). D Analysis of total Cdc42 protein expression and levels of PAK1 and ERK phosphorylation in SW620 colon cancer tissue on day 22 from mice treated with solvent (Control) or 100 μg/day AZA197 (AZA197 d22). Representative Western blot images and quantification of immunoblots stained with Cdc42, PAK1 and phospho-PAK1/2 (pPAK), ERK and phospho-ERK antibodies. Cdc42 blockade by AZA197 reduces PAK1/2 and ERK phosphorylation in SW620 tumor tissue. *, significantly different from control. E Effect of AZA197 treatment on animal survival in SW620 colon cancer bearing mice. Kaplan-Meier survival curves for controls and AZA197 treated mice. AZA197 significantly prolongs animal survival.*, significantly different to control (P = 0.042).

Mentions: To analyze whether treatment with AZA197 can modulate tumor growth in vivo, we treated mice bearing human SW620 colon cancer xenografts with AZA197 or vehicle as controls. To assess treatment modalities in vivo, we initially assessed AZA197 stability in vitro (data not shown) and cycled treatment daily for two weeks to guarantee continuous delivery of the compound. At the beginning of treatment on day 8, mice developed tumor xenografts of comparable size. On day 22, the mean tumor weight was significantly (P = 0.006) reduced in mice treated with AZA197 (676.7 ± 106 mg) compared to control mice (968 ± 208 mg) and treatment was well tolerated (Figure 6A). To compare the proliferation and apoptotic rate of untreated tumors and tumors treated with AZA197, tumor sections were stained for expression of Ki-67 and DNA fragmentation by TUNEL assays, respectively. In accordance with the tumor weight reduction findings, treatment with AZA197 decreased the number of Ki-67-positive cells in tumors based on counting 20 randomly selected microscopic fields by 27.4 ± 14.2% (P = 0.046) in AZA197-treated tumors, suggesting an anti-proliferative effect for AZA197 (Figure 6B). Moreover, AZA197-treated tumors showed increased numbers of apoptotic cells as assessed by positive staining for TUNEL compared with untreated controls. Based on the counting of randomly selected microscopic fields, the number of apoptotic cells was increased by 80.6 ± 58.3% (P = 0.035) from controls to AZA197-treated tumors (Figure 6C). Western blotting of isolated tumor tissue indicated that AZA197 treatment does not change Cdc42 and total PAK and ERK expression. Phospho-PAK1 expression in tumors treated with AZA197 was significantly reduced by 48.5 ± 11.4% (P = 0.027) compared to untreated controls (Figure 6D). Similarly, in tumors treated with AZA197, phospho-ERK levels decreased significantly by 59.2 ± 17.1% (P = 0.003) compared to untreated controls (Figure 6D). These data show that the PAK-ERK signaling pathway is a downstream target of the small molecule inhibitor AZA197 in SW620 colon cancer tissue confirming our findings in vitro.


Targeting Cdc42 with the small molecule drug AZA197 suppresses primary colon cancer growth and prolongs survival in a preclinical mouse xenograft model by downregulation of PAK1 activity.

Zins K, Gunawardhana S, Lucas T, Abraham D, Aharinejad S - J Transl Med (2013)

Cdc42 blockade by AZA197 suppresses tumor growth and prolongs animal survival. A Left panel: representative images of human SW620 tumor xenografts on day 22 from mice treated with solvent (Control) or 100 μg/day AZA197 (AZA197 d22) (bar = 10 mm). Right panel: AZA197 significantly suppresses tumor weight of human colon cancer xenografts in mice. Data are shown as mean tumor weights on day 22. *, significantly different from control on day 22 (P = 0.006). B Effect of AZA197 on proliferation in SW620 xenografts. Left panel: representative immunohistochemistry images of tumor tissue sections from mice treated with solvent (Control) or 100 μg/day AZA197 on day 22 stained with Ki-67 antibody (bar = 200 μm). Right panel: Quantitative histomorphometric analysis of Ki-67-positive, proliferating tumor cells. *, significantly different from control (P = 0.046). C Effect of AZA197 on apoptosis in SW620 xenografts. TUNEL labeling of cells undergoing DNA fragmentation: Left panel: representative images of tumor tissue sections from mice treated with solvent (Control) or 100 μg/day AZA197 on day 22 (bar = 50 μm). Right panel: Quantitative histomorphometric analysis of TUNEL-positive, apoptotic tumor cells. *, significantly different from control (P = 0.035). D Analysis of total Cdc42 protein expression and levels of PAK1 and ERK phosphorylation in SW620 colon cancer tissue on day 22 from mice treated with solvent (Control) or 100 μg/day AZA197 (AZA197 d22). Representative Western blot images and quantification of immunoblots stained with Cdc42, PAK1 and phospho-PAK1/2 (pPAK), ERK and phospho-ERK antibodies. Cdc42 blockade by AZA197 reduces PAK1/2 and ERK phosphorylation in SW620 tumor tissue. *, significantly different from control. E Effect of AZA197 treatment on animal survival in SW620 colon cancer bearing mice. Kaplan-Meier survival curves for controls and AZA197 treated mice. AZA197 significantly prolongs animal survival.*, significantly different to control (P = 0.042).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4222769&req=5

Figure 6: Cdc42 blockade by AZA197 suppresses tumor growth and prolongs animal survival. A Left panel: representative images of human SW620 tumor xenografts on day 22 from mice treated with solvent (Control) or 100 μg/day AZA197 (AZA197 d22) (bar = 10 mm). Right panel: AZA197 significantly suppresses tumor weight of human colon cancer xenografts in mice. Data are shown as mean tumor weights on day 22. *, significantly different from control on day 22 (P = 0.006). B Effect of AZA197 on proliferation in SW620 xenografts. Left panel: representative immunohistochemistry images of tumor tissue sections from mice treated with solvent (Control) or 100 μg/day AZA197 on day 22 stained with Ki-67 antibody (bar = 200 μm). Right panel: Quantitative histomorphometric analysis of Ki-67-positive, proliferating tumor cells. *, significantly different from control (P = 0.046). C Effect of AZA197 on apoptosis in SW620 xenografts. TUNEL labeling of cells undergoing DNA fragmentation: Left panel: representative images of tumor tissue sections from mice treated with solvent (Control) or 100 μg/day AZA197 on day 22 (bar = 50 μm). Right panel: Quantitative histomorphometric analysis of TUNEL-positive, apoptotic tumor cells. *, significantly different from control (P = 0.035). D Analysis of total Cdc42 protein expression and levels of PAK1 and ERK phosphorylation in SW620 colon cancer tissue on day 22 from mice treated with solvent (Control) or 100 μg/day AZA197 (AZA197 d22). Representative Western blot images and quantification of immunoblots stained with Cdc42, PAK1 and phospho-PAK1/2 (pPAK), ERK and phospho-ERK antibodies. Cdc42 blockade by AZA197 reduces PAK1/2 and ERK phosphorylation in SW620 tumor tissue. *, significantly different from control. E Effect of AZA197 treatment on animal survival in SW620 colon cancer bearing mice. Kaplan-Meier survival curves for controls and AZA197 treated mice. AZA197 significantly prolongs animal survival.*, significantly different to control (P = 0.042).
Mentions: To analyze whether treatment with AZA197 can modulate tumor growth in vivo, we treated mice bearing human SW620 colon cancer xenografts with AZA197 or vehicle as controls. To assess treatment modalities in vivo, we initially assessed AZA197 stability in vitro (data not shown) and cycled treatment daily for two weeks to guarantee continuous delivery of the compound. At the beginning of treatment on day 8, mice developed tumor xenografts of comparable size. On day 22, the mean tumor weight was significantly (P = 0.006) reduced in mice treated with AZA197 (676.7 ± 106 mg) compared to control mice (968 ± 208 mg) and treatment was well tolerated (Figure 6A). To compare the proliferation and apoptotic rate of untreated tumors and tumors treated with AZA197, tumor sections were stained for expression of Ki-67 and DNA fragmentation by TUNEL assays, respectively. In accordance with the tumor weight reduction findings, treatment with AZA197 decreased the number of Ki-67-positive cells in tumors based on counting 20 randomly selected microscopic fields by 27.4 ± 14.2% (P = 0.046) in AZA197-treated tumors, suggesting an anti-proliferative effect for AZA197 (Figure 6B). Moreover, AZA197-treated tumors showed increased numbers of apoptotic cells as assessed by positive staining for TUNEL compared with untreated controls. Based on the counting of randomly selected microscopic fields, the number of apoptotic cells was increased by 80.6 ± 58.3% (P = 0.035) from controls to AZA197-treated tumors (Figure 6C). Western blotting of isolated tumor tissue indicated that AZA197 treatment does not change Cdc42 and total PAK and ERK expression. Phospho-PAK1 expression in tumors treated with AZA197 was significantly reduced by 48.5 ± 11.4% (P = 0.027) compared to untreated controls (Figure 6D). Similarly, in tumors treated with AZA197, phospho-ERK levels decreased significantly by 59.2 ± 17.1% (P = 0.003) compared to untreated controls (Figure 6D). These data show that the PAK-ERK signaling pathway is a downstream target of the small molecule inhibitor AZA197 in SW620 colon cancer tissue confirming our findings in vitro.

Bottom Line: Rho GTPases play important roles in cytoskeleton organization, cell cycle progression and are key regulators of tumor progression.Strategies to modulate increased Rho GTPase activities during cancer progression could have therapeutic potential.These data indicate the therapeutic potential of the small-molecule inhibitor AZA197 based on targeting Cdc42 GTPase activity to modulate colorectal cancer growth.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory for Molecular Cellular Biology, Center for Anatomy and Cell Biology, Medical University of Vienna, Waehringerstrasse 13, A-1090 Vienna, Austria. dietmar.abraham@meduniwien.ac.at.

ABSTRACT

Background: Rho GTPases play important roles in cytoskeleton organization, cell cycle progression and are key regulators of tumor progression. Strategies to modulate increased Rho GTPase activities during cancer progression could have therapeutic potential.

Methods: We report here the characterization of a Cdc42-selective small-molecule inhibitor AZA197 for the treatment of colon cancer that was developed based on structural information known from previously developed compounds affecting Rho GTPase activation. We investigated the effects of AZA197 treatment on RhoA, Rac1 and Cdc42 activities and associated molecular mechanisms in colon cancer cells in vitro. Therapeutic effects of AZA197 were examined in vivo using a xenograft mouse model of SW620 human colon cancer cells. After treatment, tumors were excised and processed for Ki-67 staining, TUNEL assays and Western blotting to evaluate proliferative and apoptotic effects induced by AZA197.

Results: In SW620 and HT-29 human colon cancer cells, AZA197 demonstrated selectivity for Cdc42 without inhibition of Rac1 or RhoA GTPases from the same family. AZA197 suppressed colon cancer cell proliferation, cell migration and invasion and increased apoptosis associated with down-regulation of the PAK1 and ERK signaling pathways in vitro. Furthermore, systemic AZA197 treatment reduced tumor growth in vivo and significantly increased mouse survival in SW620 tumor xenografts. Ki-67 staining and tissue TUNEL assays showed that both inhibition of cell proliferation and induction of apoptosis associated with reduced PAK/ERK activation contributed to the AZA197-induced therapeutic effects in vivo.

Conclusions: These data indicate the therapeutic potential of the small-molecule inhibitor AZA197 based on targeting Cdc42 GTPase activity to modulate colorectal cancer growth.

Show MeSH
Related in: MedlinePlus