Activation of c-Jun N-terminal kinase 1 and caspase 3 in the tamoxifen-induced apoptosis of rat glioma cells

Abstract

Purpose: The mechanisms of the antitumor effects of tamoxifen upon gliomas are still unclear. In this study, we investigated the role of c-Jun N-terminal kinase-1 (JNK1) and caspase 3 in the tamoxifen-induced apoptosis of rat glioma cells.

Methods: Glioma cells were treated with tamoxifen, followed by a cytotoxicity assay to study its effects on the cells, and then a flow-activated cell sorter (FACS) analysis was performed to analyze the cellular apoptosis of the glioma cells. The expression of JNK1 and phospho-specific JNK1 in glioma cells treated with tamoxifen was investigated by Western blot analysis. The activity of caspase 3 in glioma cells was analyzed by caspase activity assay.

Results: Tamoxifen was demonstrated to exert cytotoxic effects upon and induced apoptosis of the glioma cells in a concentration- and time-dependent manner (P<0.05). Western blot analysis demonstrated that tamoxifen increased the expression of phospho-specific JNK1 in glioma cells, and an increasing concentration of tamoxifen induced an increasing expression of phospho-specific JNK1. Four-hour 50-microM tamoxifen treatment increased the expression of phospho-specific JNK1 to 3.2 times that of the control level in glioma cells. Tamoxifen also increased the activity of caspase 3 in glioma cells. Pretreatment of glioma cells with the antisense oligonucleotide (OGN) of JNK1 immediately prior to tamoxifen treatment suppressed the expression of phospho-specific JNK1 and the activity of caspase 3. The apoptosis fraction of glioma cells induced by 4-h treatment with 50 microM tamoxifen was decreased from 51% to 28% by pretreatment with the antisense OGN of JNK1 (P<0.003), and to 20% by pretreatment with caspase 3 inhibitor (DEVD-CHO) (P<0.0008).

Conclusions: The results suggest that the tamoxifen-induced apoptosis of rat glioma cells is related to the activation of the JNK1/caspase 3 signaling pathway; however, the confirmation of the occurrence of such activation in vivo needs further investigation.

Fig. 1

Cytotoxicity effects of tamoxifen on the RT-2 glioma cells. 5×10³ RT-2 cells were seeded in triplicate wells in flat-bottomed 24-well microtiter plates. Subsequently, the cells were exposed to various concentrations (0 μM, 0.1 μM, 1 μM, 5 μM, 10 μM, 15 μM, 20 μM, 30 μM, 40 μM, and 50 μM) of tamoxifen for various time intervals including 4 h, 12 h, or 24 h. Following the removal of the drug, the cells were incubated for a total of 5 days following cell seeding. The cell proliferation and viability were then determined by an MTT (3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide)-based colorimetric assay. Percent survival is defined as the optical density at a given drug concentration divided by the optical density for controls treated with dimethyl sulfoxide (DMSO) alone multiplied by 100. Each point is the average of three independent trials (nine determinations for each concentration) and presented as mean±standard deviation. One-way analysis of variance (ANOVA) by Scheffe’s multiple comparison was used for statistical analyses of the extent of cytotoxicity of the glioma cells induced by various kinds of regimens. The significance was accepted as P<0.05. The concentration at which 50% of the cells were killed was designated as the IC₅₀. The IC50 was 7.4 μM following a 4-h-tamoxifen treatment, 5.4 μM post 12 h of such treatment, and 0.97 μM subsequent to 24 h treatment

Fig. 2

Apoptosis of the RT-2 glioma cells subsequent to tamoxifen treatment. After treatment with various concentration (0 μM, 0.1 μM, 1 μM, 5 μM, 10 μM, 15 μM, 20 μM, 30 μM, 40 μM, and 50 μM) of tamoxifen for 4 h, 12 h, or 24 h exposure periods, the apoptosis fraction of the RT-2 glioma cells was analyzed by way of flow-activated cell sorter (FACS) flow cytometry. Each point is the average of three independent trials (nine determinations for each concentration) and presented as mean±standard deviation. One-way analysis of variance (ANOVA) by Scheffe’s multiple comparison was used for statistical analyses of the cellular apoptosis of the glioma cells induced by various kinds of regimens. The significance was accepted as P<0.05

Fig. 3

Increased expression of phospho-specific JNK1 in the RT-2 glioma cells treated with tamoxifen. Western blot analysis of the JNK1 and phospho-specific JNK1 in the glioma cells treated with various concentrations (0 μM, 1 μM, 10 μM, 20 μM, 30 μM, 35 μM, 40 μM, 45 μM, and 50 μM) of tamoxifen for a period of 4 h. The upper panel represents the expression of the JNK1, the middle panel represents the phospho-specific JNK1, and the lower panel represents the α-tubulin (control)

Fig. 4A, B

Effects of the antisense oligonucleotide of JNK1 upon the expression of JNK1 and phospho-specific JNK1 in the RT-2 glioma cells. A Western blot analysis of the JNK1 and phospho-specific JNK1 in the glioma cells treated with antisense and sense oligonucleotides (OGNs) of JNK1, or random OGN of the same length and containing the same bases as the antisense OGN of JNK1. The upper panel represents the expression of the JNK1, the middle panel represents the phospho-specific JNK1, and the lower panel represents the α-tubulin (control). The expression of the JNK1 and the phospho-specific JNK1 was suppressed by the antisense OGN, but was changed insignificantly by the sense and random OGNs, as compared with the control; B Western blot analysis of the JNK1 and phospho-specific JNK1 in the glioma cells treated with antisense oligonucleotide of JNK1 or antisense oligonucleotide of JNK1 followed by 50 μM of tamoxifen treatment. The upper panel represents the expression of the JNK1, the middle panel represents the phospho-specific JNK1, and the lower panel represents the α-tubulin (control). The expression of the JNK1 and the phospho-specific JNK1 was markedly reduced by the antisense OGN, even after 50 μM of tamoxifen treatment, as compared with the control

Fig. 5A, B

Effects of the antisense oligonucleotide of JNK1 and caspase 3 inhibitor upon the tamoxifen-induced apoptosis of RT-2 glioma cells. Following glioma cell treatment with various kinds of treatment regimens, the apoptosis fraction of the glioma cells was measured by FACScan analysis. The treatment regimens included dimethyl sulfoxide for 4 h (a, tamoxifen 0 μM), 50 μM tamoxifen for 4 h (b, tamoxifen 50 μM), 25 μM of antisense oligonucleotide (OGN) of c-Jun N-terminal kinase-1 (JNK1) for 16 h (c, antisense OGN), 25 μM of antisense OGN of JNK1 for 16 h followed by 50 μM of tamoxifen for 4 h (d, tamoxifen 50 μM +Antisense OGN), 100 μM of caspase 3 inhibitor (DEVD-CHO) for 2 h (e, DEVD-CHO), and 100 μM of DEVD-CHO for 2 h followed by 50 μM of tamoxifen for 4 h (f, tamoxifen 50 μM + DEVD-CHO). A The apoptotic fraction of the glioma cells treated with various regimens. Data points indicate mean percentages of apoptosis fraction of glioma cells from three independent experiments; bars standard deviation; B FACScan analysis of the glioma cells treated with various kinds of regimens. One-way analysis of variance (ANOVA) by Scheffe’s multiple comparison was used for statistical analyses of the cellular apoptosis of the glioma cells induced by various kinds of regimens. The apoptosis fraction of glioma cells from three independent experiments was shown as mean±standard deviation. Significance was accepted as P<0.05 (P<0.0002 for tamoxifen 0 μM vs tamoxifen 50 μM; P=0.45 for tamoxifen 0 μM vs antisense OGN; P<0.003 for tamoxifen 0 μM vs DEVD-CHO; P<0.003 for tamoxifen 50 μM vs tamoxifen 50 μM + antisense OGN; P<0.0008 for tamoxifen 50 μM vs tamoxifen 50 μM + DEVD-CHO). The extent of tamoxifen-induced apoptosis was reduced by the antisense oligonucleotide of JNK1 and caspase 3 inhibitor

Fig. 6

Possible signaling pathway during tamoxifen-induced apoptosis of the glioma cells. Tamoxifen triggered c-Jun N-terminal kinase (JNK1) activation to result in the phosphorylation of JNK1 (phosphorylated JNK1), subsequently activated the downstream caspase 3, and, ultimately, apoptosis. The antisense oligonucleotide (OGN) of JNK1 suppressed the expression of JNK1 and the activation of the downstream signaling pathway. (-) indicates inhibition of the expression of JNK1 by antisense OGN of JNK1