RHOA and mDia1 Promotes Apoptosis of Breast Cancer Cells Via a High Dose of Doxorubicin Treatment

Abstract

Background: Transforming RhoA proteins (RHOA) and their downstream Diaphanous homolog 1 proteins (DIAPH1) or mDia1 participate in the regulation of actin cytoskeleton which plays critical role in cells, i.e., morphologic changes and apoptosis.

Methodology: To determine the cell viability the real time cell analysis (RTCA) and flow cytometry were used. To perform proteomic analysis, the label-free quantitative method and post-translation modification by the nano-HPLC and ESI-MS ion trap mass analyser were used.

Results: The results of the cell viability showed an increase of dead cells (around 30 %) in MCF-7/DOX-1 (i.e., 1μM of doxorubicin was added to MCF-7/WT breast cancer cell line) compared to MCF-7/WT (control) after 24 h doxorubicin (DOX) treatment. The signalling pathway of the Regulation of actin cytoskeleton (p<0.0026) was determined, where RHOA and mDia1 proteins were up-regulated. Also, post-translational modification analysis of these proteins in MCF-7/DOX-1 cells revealed dysregulation of the actin cytoskeleton, specifically the collapse of actin stress fibbers due to phosphorylation of RHOA at serine 188 and mDia1 at serine 22, resulting in their deactivation and cell apoptosis.

Conclusion: These results pointed to an assumed role of DOX to dysregulation of actin cytoskeleton and cell death.

Keywords: Diaphanous homolog 1 protein; Transforming RhoA protein; actin cytoskeleton; stress fibre.

Figure 1

The real-time monitoring of DOX cytotoxic effect in MCF-7 cells. (A) Normalized cell index values of MCF-7 cell line after addition of DOX in the range of 0,001-1 μM. (B) The time dependent IC₅₀ of DOX (time range: 21:35:30 ~ 48:07:13) (C) The Dose-Response Curve (CI at a time point vs conc.) of the highest cytotoxicity of DOX (IC₅₀ = 3.53) after 24 h treatment.

Figure 2

Determination of the number of MCF-7 cells and cell viability using Muse™ Cell Analyzer. Each sample was subjected to run in duplicate. Error bars represent standard deviation. Significant differences relative to the control are marked with an * (p < 0.05).

Figure 3

Signalling pathways analysis on the all 1772 identified proteins (log2-fold change >0.6, p ≤ 0.05) via iPathwayGuide analysis tool.

Figure 4

The regulation of actin cytoskeleton obtained from the KEGG pathways database (p < 0.0026). The 8 proteins like: RAC1, CYFP1, GNA13, IQGAP1, ITGB1, RHOA, GSN and DIAPH1 with different regulation were identified.

Figure 5

Quantification of post-translational modifications (PTMs) of RHOA and mDia1 in MCF-7/WT and MCF-7/DOX-1 cells. (A, C) Modified serine residues of RHOA, mDia1 and number of detected peptides with corresponding modification(s) observed in MCF-7/WT and MCF-7/DOX-1. (B, D) Sequence and mass spectrum of the peptide with phosphorylated residues (Ser-188 and Ser-22) located in RHOA and mDia1 proteins.

Figure 6

(A) Crystal structure of phosphorylated RHOA-RhoGDIα complex using PyTMs plugin in PyMOL software [PDB: 1CC0] (B) A detailed structure of phosphorylated serine 188.

Figure 7

(A) Plasma membrane bound active RHOA-mDia1 in MCF-7/WT cells (B) Inactivated RHOA and auto-inhibited mDia1 in MCF-7/DOX-1 cells.