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. 2014 Jan;139(1):117-24.

Validity of SW982 synovial cell line for studying the drugs against rheumatoid arthritis in fluvastatin-induced apoptosis signaling model

Jae-Ho Chang, Kyu-Jae Lee, Soo-Ki Kim  1 , Dae-Hyun Yoo, Tae-Young Kang  2
Affiliations

Validity of SW982 synovial cell line for studying the drugs against rheumatoid arthritis in fluvastatin-induced apoptosis signaling model

Jae-Ho Chang et al. Indian J Med Res. 2014 Jan.

Abstract

Background & objectives: To study effects of drugs against rheumatoid arthritis (RA) synoviocytes or fibroblast like synoviocytes (FLS) are used. To overcome the drawbacks of using FLS, this study was conducted to show the validity of SW982 synovial cell line in RA study.

Methods: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, Annexin V propidium iodide (PI) staining, mitochondrial membrane potential assay, Triton X-114 Phase partitioning, and immunolot for apoptosis signaling in SW982 human synovial cell line were performed.

Results: Fluvastatin induced apoptosis in a dose- and time-dependent manner in TNFα -stimulated SW982 human synovial cells. A geranylgeranylpyrophosphate (GGPP) inhibitor, but not a farnesylpyrophosphate (FPP) inhibitor, induced apoptosis, and fluvastatin-induced apoptosis was associated with the translocation of isoprenylated RhoA and Rac1 proteins from the cell membrane to the cytosol. Fluvastatin-induced downstream apoptotic signals were associated with inhibition of the phosphoinositide 3-kinase (PI3K)/Akt pathway. Accordingly, 89 kDa apoptotic cleavage fragment of poly (ADP-ribose) polymerase (PARP) was detected.

Interpretation & conclusions: Collectively, our data indicate that fluvastatin induces apoptotic cell death in TNFα-stimulated SW982 human synovial cells through the inactivation of the geranylgerenylated membrane fraction of RhoA and Rac1 proteins and the subsequent inhibition of the PI3K/Akt signaling pathway. This finding shows the validity of SW982 cell line for RA study.

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Figures

Fig. 1
Fig. 1
Reduction of cell viability by fluvastatin. TNFα-stimulated SW982 synovial cells were incubated with or without 0-50 μM fluvastatin for 24 h. Cell viability was determined by MTT assay. Data were obtained from duplicate experiments using three different samples. *P<0.05, **P<0.01 compared to control (medium only).
Fig. 2
Fig. 2
Influence of fluvastatin on the apoptosis of TNFα-stimulated SW982 synovial cells. Apoptosis was measured by flow cytometry after staining with annexin V. (A) The escalated fluvastatin concentrations (0-50 μM) resulted in a linear increase in the apoptotic response. (B) Cells were cultured for various durations (0-48 h) in the presence of 10 μM fluvastatin. **P<0.01compared to control (medium only).
Fig. 3
Fig. 3
Effects of RhoA kinase inhibitors on apoptosis of TNF-α-stimulated SW982 cells. Cells were incubated for 48 h with medium alone, 10 μM fluvastatin, or 20 μM Y-27632. Apoptosis was measured by flow cytometer after staining with annexin V. *P<0.05, **P<0.01 compared to control (medium only).
Fig. 4
Fig. 4
Effects of the geranylgeranyl transferase inhibitor GGTI-298 and the farnesyl transferase inhibitor FTI-277 on cell viability and apoptosis. TNFα-stimulated SW982 cells were incubated with 10 μM GGTI-298 or 10 μM FTI-277. The geranylgeranyl transferase inhibitor, but not the farnesyltransferase inhibitor, induced apoptosis. Data were obtained from duplicate experiments using three different samples. (A) Cell viability was determined by MTT assays. **P<0.01 compared between CGTI and FTI. (B) Apoptosis was measured by flow cytometry after staining with annexin V. **P<0.01 compared to control (medium only).
Fig. 5
Fig. 5
Effects of fluvastatin on the phosphorylations of ERK1/2, p38 MAPK, Akt, and Bad. After treatment with fluvastatin, phospho-Akt expression was suppressed whereas phosphorylations of ERK1/2 and p38 MAPK were sustained. Furthermore, phospho-Bad expression was significantly reduced. (A) TNFα-stimulated SW982 cells were treated with fluvastatin for 24 h. Cell lysates were subjected to SDS-PAGE and immunoblotted with anti-phospho-ERK1/2, anti-phospho-p38 MAPK, anti-phospho-Akt, or anti-phospho-Bad antibodies. Equal amounts of protein were loaded in each lane, and equal loading was confirmed by immunoblotting with an anti-β-actin antibody. (B) TNFα-stimulated SW982 cells were incubated for 24 h in the presence of 10 μM fluvastatin, 50 μM PD98059, 10 μM SB203580, or 15 μM LY294002. Apoptosis was measured by flow cytometry after staining with annexin V. The results are representative of two independents experiments. *P<0.05 compared to control (medium only).
Fig. 6
Fig. 6
Effects of fluvastatin on mitochondrial transmembrane potentials (MMP) and PARP cleavage activity. TNFα-stimulated SW982 cells were cultured for 24 h in the presence of 10 μM fluvastatin. (A) Cells were stained with JC-1. Data are representative of three independent experiments. MMP, mitochondrial membrane potential. (B) The expression of cleaved PARP was detected by immunoblotting. The results are representative of two independent experiments. **P<0.01 compared to control (medium only).
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