Korean J Physiol Pharmacol.  2014 Apr;18(2):109-120. 10.4196/kjpp.2014.18.2.109.

Multiple Effects of a Novel Epothilone Analog on Cellular Processes and Signaling Pathways Regulated by Rac1 GTPase in the Human Breast Cancer Cells

• Zhang H
• An F
• Tang L
• Qiu R

• KMID: 2285499
• DOI: http://doi.org/10.4196/kjpp.2014.18.2.109

Abstract

The epothilones are a class of microtubule inhibitors that exhibit a strong antitumor activity. UTD2 is a novel epothilone analog generated by genetic manipulation of the polyketide biosynthetic gene cluster. This study investigated the effects of UTD2 on the actin cytoskeleton and its critical regulators, and the signaling pathways which are essential for cell motility, growth and survival in MCF-7 breast cancer cells. Results showed that UTD2 inhibited the cellular functions of actin cytoskeleton, such as wound-closure, migration and invasion, as well as adhesion. Our study further demonstrated that UTD2 suppressed Rac1 GTPase activation and reduced the activity of PAK1, which is a downstream effector of Rac1, while the activity of Cdc42 was not affected. Additionally, the phosphorylation of p38 and ERK were significantly inhibited, but the phosphorylation of JNK remained the same after UTD2 treatment. Moreover, UTD2 inhibited the activity and mRNA expression of MMP-2, which plays a key role in cell motility. UTD2 also reduced the phosphorylation of Akt, which is an important signaling kinase regulating the cell survival through Rac1. Furthermore, UTD2 interrupted the synergy between Rac1 and Raf in focus formation assays. Taken together, these results indicated that UTD2 exerted multiple effects on the actin cytoskeleton and signaling pathways associated with Rac1. This study provided novel insights into the molecular mechanism of the antineoplastic and antimetastatic activities of epothilones. Our findings also suggest that the signaling pathways regulated by Rac1 may be evaluated as biomarkers for the response to therapy in clinical trials of epothilones.

Keyword

Actin cytoskeleton; Epothilone; Rac1 GTPase signaling; UTD2

MeSH Terms

Actin Cytoskeleton
Biomarkers
Breast Neoplasms*
Breast*
Cell Movement
Cell Survival
Epothilones*
GTP Phosphohydrolases*
Humans
Microtubules
Multigene Family
Phosphorylation
Phosphotransferases
RNA, Messenger
Epothilones
GTP Phosphohydrolases
Phosphotransferases
RNA, Messenger

Figure

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  Fig. 1 Effect of UTD2 on the cytoskeleton in MCF-7 cells. β-tubulin (green) and nuclear (blue) staining of cells after they were treated with or without UTD2 for 24 hours. Images were acquired by confocal microscopy. The images are representative of four independent experiments. (A) Untreated MCF-7 cells. (B) MCF-7 cells treated with IC10 dose of UTD2. (C) MCF-7 treated with IC50 dose of UTD2. Bar=50µm.

• 

  Fig. 2 Effects of UTD2 on the cell motility of MCF-7 cells. The cells were preincubated with or without the indicated concentrations of UTD2. The cell motility was measured as described in materials and methods. (A) Wound-closure of MCF-7 cells treated with IC50 dose of UTD2 for 24 hours. (B) Wound closure of cells treated with IC10 and IC50 doses of UTD2 for 24 hours as a ratio relative to control cells. (C) Migration of MCF-7 cells treated with IC10 and IC50 concentrations of UTD2 for 16 hours. (D) Migration of the drug treated MCF-7 cells presented as a ratio relative to control cells. (E) Invasion of MCF-7 cells incubated with IC10 and IC50 doses of UTD2 for 16 hours. (F) Invasion of the drug treated MCF-7 cells presented as a ratio relative to control cells. (G) The adhesion of MCF-7 cells incubated with IC10 and IC50 doses of UTD2 for 2 hours relative to control cell adhesion. The data are presented as mean±SE of these independent experiments (n=4), **p<0.01.

• 

  Fig. 3 Effects of UTD2 on the activity of Rho GTPases in MCF-7 cells. MCF-7 cells were treated with the indicated concentrations of UTD2, and the amount of activated Rac1/Cdc42 was analyzed using the Rac1/Cdc42 activation assay as described in materials and methods. (A) The activated of Rac1 in MCF-7 cells treated with UTD2 for 4 hours (B) The activated of Cdc42 in MCF-7 cells treated with UTD2 for 4 hours. The data are presented as mean±SE of these independent experiments (n=4), **p<0.01.

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  Fig. 4 Effect of UTD2 on the migration of stable cell lines expressing Rac1 mutants. (A) Expression of correct myc-tagged Rac1 mutants in MCF-7 subclones. Constitutively active Rac1-(mRac1V12) and dominant negative Rac1-(mRac1N17) was determined by Western blot using a myc tag antibody. Vector represents vector control. (B) The migration of Rac1 mutant subclones was evaluated. The migration was measured as described in migration assay and cells were preincubated with or without 20 nM of UTD2. The data are presented as mean±SE of these independent experiments (n=4), **p<0.01.

• 

  Fig. 5 Effects of UTD2 on the phosphorylation of PAK1 and Akt in MCF-7 cells. Cells were treated with the indicated concentrations of UTD2 for 24 hours. The cell lysates were prepared to analyze the levels of phosphorylated PAK1 (p-PAK1) and phosphorylated Akt (p-Akt) as described in materials and methods. (A) p-PAK1 assay in MCF-7 cells treated with UTD2. (B) p-Akt assay in MCF-7 cells treated with UTD2. The data are presented as mean±SE of these independent experiments (n=4), *p<0.05 and **p<0.01.

• 

  Fig. 6 Effects of UTD2 on activation MAPKs. MCF-7 cells were treated with the indicated concentrations of UTD2 for 24 hours, and the cell lysates were assayed for phosphorylation of ERK1/2, p38 and JNK, as described in materials and methods. (A) The phosphorylation of ERK1/2, p38 and JNK in MCF-7 cells treated with UTD2 for 24 hours. (B) Optical density of phosphorylated ERK/total ERK. (C) Optical density of phosphorylated p38/total p38. (D) Optical density of phosphorylated JNK/total JNK. The data are presented as mean±SE of these independent experiments (n=4), *p<0.05 and **p<0.01.

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  Fig. 7 Effects of UTD2 on the activity and expression of MMP-2 in MDA-MB-231 cells. (A) MMP2 in MDA-MB-231 cells was determined by zymography; the cells and cell lysates were treated as described in materials and methods. (B) RT-PCR analysis of the mRNA levels of MMP-2 in MDA-MB-231 cells. (C) Actin was used as loading control for the RT-PCR analysis. The data are presented as mean±SE. of these independent experiments (n=4).

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  Fig. 8 A proposed model for signaling pathways affected by UTD2 in breast cancer cells. By targeting tubulin and promoting tubulin polymerization, UTD2 inhibits the Rac1/PAK1/p38/MMP-2 signaling pathway, which is critical for the UTD2-induced actin cytoskeleton dysfunction. Meanwhile, the Rac1/PI3K/Akt/MMP-2 pathway may be also involved in UTD2-induced actin cytoskeleton dysfunction. In addition, represented by a thinner arrow in this model, UTD2 can moderately inhibit the Raf-MEK-ERK pathway. Together with the effect on survival factor Akt, these account for at least part of UTD2's antitumor activities.

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