Cancer Res Treat.  2005 Aug;37(4):251-256.

Characterization of RhoA-mediated Chemoresistance in Gastric Cancer Cells

• Kang WK
• Lee I
• Park C

Abstract

PURPOSE
RhoA is a critical transducer of extracellular signals, which leads to organization of actin cytoskeleton, motility, adhesion and gene regulation. The present study aimed to explore whether RhoA influences the susceptibility of gastric cancer cells to chemotherapeutic drugs. MATERIALS AND METHODS: SNU638 cells were transfected with a mock vector (pcDNA3.1), RhoA (pcDNA/RhoA), or constitutively active RhoA (pcDNA/caRhoA). MTT assay and Western blot analysis were performed to study the growth response to several chemotherapeutic drugs in the gastric cancer cell line, SNU638, with different RhoA levels. RESULTS: RhoA significantly enhanced the resistance to lovastatin, 5-FU, taxol and vincristine, but did not affect the sensitivity to cisplatin or etoposide in SNU638. In the Western blot analysis, RhoA decreased the PARP cleavage, which was accompanied by a concurrent reduction in cell death. The gene expression profile after a cDNA microarray analysis demonstrated that RhoA was associated with the differential expression of 19 genes, including those involved in anti-oxidant defense, glucose metabolism, anti-apoptosis and protein turnover. CONCLUSION: Gastric cancer cells with a high expression of RhoA could be resistant to chemotherapeutic drugs, such as taxol or vincristine, implying that treatment strategies aimed at inactivation of RhoA might be promising for improving the efficacy of these chemotherapeutic drugs.

Keyword

Rho; Resistance; Microarray analysis

MeSH Terms

Actin Cytoskeleton
Blotting, Western
Cell Death
Cell Line
Cisplatin
Etoposide
Fluorouracil
Glucose
Lovastatin
Metabolism
Microarray Analysis
Oligonucleotide Array Sequence Analysis
Paclitaxel
Stomach Neoplasms*
Transcriptome
Transducers
Vincristine
Cisplatin
Etoposide
Fluorouracil
Glucose
Lovastatin
Paclitaxel
Vincristine

Figure

• 

  Fig. 1 Ectopic expression of RhoA induces resistance to lovastatininduced cell death. (A) Western blot analysis demonstrating ectopic expressions of myc-tagged recombinant RhoA and caRhoA in SNU638 cells. (B) Dose response curves detailing the effect of stable expressions of RhoA and caRhoA on the cytotoxicity of lovastatin in SNU638 cells. Cells (5×103/well) were plated in 96-well plates and treated with lovastatin for 72 h on the following day. A MTT assay was performed to detect live cells. IC50 values were calculated from the sigmoidal dose response curves.

• 

  Fig. 2 RhoA is a selective modulator of chemotherapeutic drug-induced apoptosis. PARP cleavage assay demonstrates the absence of the cleaved PARP fragment in RhoA (or caRhoA)-transfected cells compared with the parental (SNU638) or mock-transfected cells (pcDNA3.1), after treatments with either 2 µg/ml cisplatin, 5 µg/ml etoposide, 1 µg/ml 5-FU, 2 ng/ml taxol or 10 ng/ml vincristine. Identical blots were reprobed with β-actin as a control for loading.

• 

  Fig. 3 Western blot analyses of chemoresistance-related proteins in total cell lysates from parental (SNU638), mock-transfected (pcDNA3.1), RhoA overexpressing (RhoA) and caRhoA expressing (caRhoA) cells. Identical blots were reprobed with β-actin as a control for loading.

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