Induction of cell cycle arrest by increasing GTP‑RhoA levels via Taxol‑induced microtubule polymerization in renal cell carcinoma

Abstract

Renal cell carcinoma (RCC) is the most common neoplasm of the kidney in adults, accounting for ~3% of adult malignancies. Understanding the underlying mechanism of RCC tumorigenesis is necessary to improve patient survival. The present study revealed that Taxol‑induced microtubule (MT) polymerization causes cell cycle arrest and an increase in guanosine triphosphate‑Ras homology gene family, member A (GTP‑RhoA) protein expression. Disruption of Taxol‑induced MT polymerization reversed GTP‑RhoA expression and cell cycle arrest. The localization and redistribution of MTs and RhoA were consistent in cells with MT bundles and those without. Decreased GTP‑RhoA had no marked effect on Taxol‑induced MT bundling, however, it reduced the proportion of cells in G2/M phase. Taken together, Taxol‑induced MT polymerization regulated the protein expression levels of GTP‑RhoA and cell cycle arrest. However, the alteration in GTP‑RhoA expression did not influence MT arrangement, suggesting that GTP‑RhoA serves a pivotal role in Taxol‑induced MT polymerization and cell cycle arrest in RCC.

Figure 1.

Taxol-induced MT polymerization increases GTP-RhoA protein expression and cell cycle arrest. MT polymerization was elevated following Taxol treatment, as determined by immunofluorescent staining and confocal microscopy. (A) Confocal images of MFs stained with rhodamine-phalloidin (red), MTs stained with Alexa 488-labeled β-tubulin antibody (green) and nuclei stained with DAPI (blue) are presented. Scale bar=10 µm. (B) Western blot analysis and (C) Flow cytometry assessed GTP-RhoA protein expression levels following Taxol treatment. Expression of GTP-RhoA was significantly upregulated in a time-dependent manner. (D) Taxol treatment caused a significant increase in the proportion of cells in G2/M phase, as determined by flow cytometry. Data are presented as the mean ± standard deviation of three independent experiments. *P<0.05 and **P<0.01 vs. control. MT, microtubule; GTP, guanosine triphosphate; RhoA, Ras homology gene family, member A; MF, actin/myosin microfilaments.

Figure 2.

Taxol treatment of cells induces MT and GTP-RhoA co-localization, as determined by immunofluorescent staining. Confocal images demonstrate cell staining of RhoA (red), MTs (green) and nuclei (blue). Scale bar=10 µm. MT, microtubule; GTP, guanosine triphosphate; RhoA, Ras homology gene family, member A.

Figure 3.

Disruption of Taxol-induced MT polymerization decreases GTP-RhoA protein expression levels and reverses cell cycle arrest. Cells pre-treated with the MT depolymerizing agent, Col, (A) exhibited reduced MT bundling and (B) GTP-RhoA protein expression levels, which was determined by western blotting and (C) flow cytometry. (D) Taxol-induced cell cycle arrest was reversed following col treatment. Data are presented as the mean ± standard deviation of three independent experiments. *P<0.05 and **P<0.01 vs. control; ^#P<0.05 vs. Taxol. Scale bar=10 µm. MT, microtubule; GTP, guanosine triphosphate; RhoA, Ras homology gene family, member A; Col, colchicine; MF, actin/myosin microfilaments.

Figure 4.

GTP-RhoA inhibition has no effect on Taxol-induced MT bundling, but partially inhibits the Taxol-induced increase in G2/M cell number. (A) Western blotting demonstrated that transfection with the RhoAT19N mutant or using RhoA inhibitor (C3 transferase) significantly decreased GTP-RhoA expression. (B and C) The combination of Taxol with RhoAT19N plasmid or C3 transferase decreased the Taxol-induced increase in the proportion of cells in the G2/M phase however, (D) it had no marked effects on Taxol-induced MT bundling. Data are presented as the mean ± standard deviation of three independent experiments. *P<0.05 vs. control; ^#P<0.05 vs. Taxol. Scale bar=10 µm. GTP, guanosine triphosphate; RhoA, Ras homology gene family, member A; MT, microtubule; MF, actin/myosin microfilaments.