The Effect of Ras Homolog C/Rho-Associated Coiled-Protein Kinase (Rho/ROCK) Signaling Pathways on Proliferation and Apoptosis of Human Myeloma Cells

Abstract

BACKGROUND The aim of this study was to explore the impact of Ras homolog C/Rho-associated coiled-protein kinase (Rho/ROCK) signaling pathways intervention on biological characteristics of the human multiple myeloma cell lines RPMI-8226 and U266 cells, and to investigate the expression of RhoC, ROCK1, and ROCK2 in RPMI-8226 and U266 cells. MATERIAL AND METHODS RPMI8226 and U266 cell lines were treated by 5-aza-2-deoxycytidine (5-Aza-Dc), trichostatin A (TSA), RhoA inhibitor CCG-1423, Rac1 inhibitor NSC23766, and ROCK inhibitor fasudil. Cell proliferation was examined by Cell Counting Kit-8 (CCK-8) assay and clone formation. Cell apoptosis was examined by flow cytometry and TUNEL assay. The mRNA and protein expressions of RhoC, ROCK1, and ROCK2 were detected by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and western blot, respectively. RESULTS CCG-1423, NSC23766, and fasudil could significantly inhibit the proliferation of RPMI8226 and U266 cells. The inhibitory effect was dose- and time-dependent within a certain concentration range (P<0.05). After treatment with CCG-1423, NSC23766, and fasudil for 24 hours, the apoptosis rates of RPMI8226 and U266 cells were significantly higher than those of the control group, which were dose-dependent (P<0.05). Compared with the control group, the mRNA and protein expressions of RhoC, ROCK1, and ROCK2 in RPMI8226 and U266 cells were significantly decreased with single 5-Aza-Dc or TSA treatment. However, the effects were obviously stronger after combined treatment of 5-Aza-CdR and TSA (P<0.05). CONCLUSIONS We found that 5-Aza-Dc and TSA can effectively decrease the mRNA and protein expressions of RhoC, ROCK1, and ROCK2. Furthermore, Rho and ROCK inhibitors significantly inhibit cell growth and induce cell apoptosis in the human multiple myeloma cell lines RPMI-8226 and U266.

Figure 1

RhoA, Rac1 and ROCK inhibitors could inhibit the proliferation of RPMI-8226 cells in a dose- and time-dependent manner. (A) After treatment of RhoA inhibitor CCG-1423 for 12, 24, 48 hours, cell viability was determined by CCK-8. (B) Rac1 inhibitors NSC23766 could inhibit proliferation of RPMI-8226 cells. (C) Compared with CCG-1423 and NSC23766 groups, the viability of cells was higher in cells treated with ROCK inhibitor fasudil for 48 hours. (D) Plate cloning experiments demonstrated that RhoA, Rac1 and ROCK inhibitors could significantly inhibited proliferation of RPMI-8226 cells.

Figure 2

Rac1 and ROCK inhibitors could inhibit the proliferation of U266 cells in a dose- and time-dependent manner. (A) After CCG-1423 treatment for 12, 24, 48 hours, cell viability rates were significantly decreased. (B) Rac1 inhibitors NSC23766 could inhibit proliferation of U266 cells. (C) Similarly, fasudil could obviously inhibit cell proliferation. (D) Plate cloning experiments demonstrated that RhoA, Rac1, and ROCK inhibitors could significantly inhibited proliferation of U266 cells.

Figure 3

Effect of CCG-1423, NSC23766, and fasudil on rates of apoptosis in RPMI-8226 cells. (A) TUNEL assay indicated that CCG-1423, NSC23766, and fasudil promoted apoptosis of RPMI8226 cells. (B) After treatment with CCG-1423, NSC23766, and fasudil for 24 hours, the apoptosis rates of RPMI8226 cells were significantly higher than those of the control group, which were dose-dependent (P<0.05).

Figure 4

Effect of CCG-1423, NSC23766, and fasudil on rates of apoptosis in U266 cells. (A) TUNEL assay indicated that CCG-1423, NSC23766, and fasudil promoted apoptosis of RPMI8226 cells. (B) After treatment with CCG-1423, NSC23766, and fasudil for 24 hours, the apoptosis rates of U266 cells were significantly higher than those of the control group, which were dose-dependent (P<0.05).

Figure 5

The mRNA expression of RhoC, ROCK1, and ROCK2 in RPMI8226 cells after treated with 5-Aza-Dc, TSA, and combined 5-Aza-Dc and TSA. (A) The mRNA expression of RhoC, ROCK1, and ROCK in RPMI8226 cells with different concentrations of 5-Aza-Dc. 5-Aza-Dc could effectively reduce the expression of RhoC, ROCK1, and ROCK (P<0.05). (B) The mRNA expression of RhoC, ROCK1, and ROCK with different concentrations of TSA. Slimily, TSA could reduce the expression of RhoC, ROCK1, and ROCK (P<0.05). (C) The mRNA expression of RhoC, ROCK1, and ROCK with 5 μmol/L 5-Aza-Dc combined TSA group. When the 2 drugs were combined, the mRNA expression of RhoC, ROCK1, and ROCK were significantly decreased. 5-Aza-Dc – 5-aza-2-deoxycytidine; TSA – trichostatin A.

Figure 6

The mRNA expression of RhoC, ROCK1, and ROCK2 in U266 cells after treated with 5-Aza-Dc, TSA, and combined 5-Aza-Dc and TSA. (A) 5-Aza-Dc could effectively reduce the expression of RhoC, ROCK1, and ROCK in U266 cells (P<0.05). (B) Slimily, TSA could reduce the expression of RhoC, ROCK1, and ROCK in U266 cells (P<0.05). (C) When the 2 drugs were combined, the mRNA expression of RhoC, ROCK1, and ROCK were significantly decreased in U266 cells (P<0.01). 5-Aza-Dc – 5-aza-2-deoxycytidine; TSA – trichostatin A.

Figure 7

The protein expression of RhoC, ROCK1, and ROCK2 in RPMI8226 cells after treated with 5-Aza-Dc, TSA, and combined 5-Aza-Dc and TSA. (A–C) The protein expression of RhoC in control and experimental groups. (D–F) The protein expression of ROCK1 in control and experimental groups. (G–I) The protein expression of ROCK2 in control and experimental groups. The results showed that the protein expressions of RhoC, ROCK1, and ROCK2 in RPMI8226 cells were gradually decreased with different concentrations of 5-Aza-Dc and/or TSA treatment (P<0.05). 5-Aza-Dc – 5-aza-2-deoxycytidine; TSA – trichostatin A.

Figure 8

The protein expression of RhoC, ROCK1, and ROCK2 in U266 cells after treated with 5-Aza-Dc, TSA, and combined 5-Aza-Dc and TSA. (A–C) The protein expression of RhoC in control and experimental groups. (D–F) The protein expression of ROCK1 in control and experimental groups. (G–I) The protein expression of ROCK2 in control and experimental groups. The results showed that the protein expressions of RhoC, ROCK1, and ROCK2 in U266 cells were gradually decreased with different concentrations of 5-Aza-Dc and/or TSA treatment (P<0.05). 5-Aza-Dc – 5-aza-2-deoxycytidine; TSA – trichostatin A.