The effects of ursolic acid on cytokine production via the MAPK pathways in leukemic T-cells

Abstract

Ursolic acid (UA) is a pentacyclic triterpenoid carboxylic acid that is found in plants and herbal products. It is one of the chemopreventive agents, which can suppress cancer cell proliferation and induce apoptosis. UA possesses various biological activities including anticancer, anti-inflammatory, anti-oxidative and hepatoprotective activity. We investigated the effect of UA on cytokine production via the MAPK pathways in Jurkat leukemic T-cells, showing that UA inhibited cell growth and proliferation of Jurkat cells, as well as suppressing PMA/PHA induced IL-2 and TNF-α production in a concentration and time dependent manner. The inhibition of IL-2 and TNF-α production by UA involved the c-Jun N-terminal kinase (JNK) pathway, but not the extracellular-signal regulated protein kinase (ERK) pathway. Future utilization of UA as a chemopreventive or therapeutic agent may provide an alternative option for leukemia treatments.

Keywords: ERK; IL-2; JNK; Jurkat cells; TNF-alpha; ursolic acid.

Figure 1. Inhibition of leukemic T-cell growth by ursolic acid. Jurkat cells were treated with UA (5 to 100 M) or DMSO for 12 hours. Cell viability was determined using the XTT assay. Results are expressed as mean SEM from three independent experiments. Statistical analysis was carried out with one-way ANOVA followed by Tukey's multiple comparison test compared with DMSO (** P < 0.01, *** P < 0.001).

Figure 2. Effect of PMA/PHA on IL-2 and TNF-α production in Jurkat cells. Cells were treated with PMA/PHA (12.5 ng ml^-1 / 0.25 µg ml^-1) for 1 to 24 hours. IL-2 and TNF-α released to the media were determined using ELISA. Results are expressed as mean SEM from three independent experiments. Statistical analysis was carried out with one-way ANOVA followed by Tukey's multiple comparison test compared with DMSO control (** P < 0.01, *** P < 0.001).

Figure 3. Effect of UA and MAPK inhibitors on inhibition of PMA/PHA-induced IL-2 and TNF-α production. Cells were treated with DMSO (control), UA, PD98059 or SP600125 for 0 to 12 hours and then stimulated with PMA/PHA for a further 12 hours. The IL-2 (a), (c) and TNF-α (b), (d) were determined using ELISA. Results are expressed as mean SEM from three independent experiments. Statistical analyses were carried out with one-way ANOVA followed by Tukey's multiple comparison test compared with positive controls (PMA/PHA) (* P < 0.05, ** P < 0.01, *** P < 0.001).

Figure 4. Effect of PMA/PHA on the activity of ERK1/2 and JNK in Jurkat leukemic T-cells; Western blot analysis (representative figure of three independent experiments). Cells were incubated with PMA/PHA for 1 to 24 hours, or only DMSO (C) for 24 hours. Protein expression of (a) p-ERK1/2 (42, 44 kDa), ERK1/2 (42, 44 kDa) and β-actin (45kDa) was detected by Western blot and normalized with β-actin. (b), (c), (d) Relative p-ERK, ERK and p-ERK/ERK protein level was calculated. Protein expression of (e) p-JNK (46, 54 kDa), JNK (46, 54 kDa) and β-actin (45kDa) was detected by Western blot and normalized with β-actin. (f), (g), (h) Relative p-JNK, JNK and p-JNK/JNK protein level was calculated. Statistical analysis was carried out with one-way ANOVA followed by Tukey's multiple comparison test compared with positive controls (PMA/PHA) (* P < 0.05, ** P < 0.01)

Figure 5. Effect of UA on the activity of ERK1/2 and JNK in Jurkat leukemic T-cell; Western blot analysis (representative figure of three independent experiments). Cells were incubated with 20 µM UA for 1 to 24 hours, or only DMSO (C) for 24 hours. Protein expression of (a) p-ERK1/2 (42, 44 kDa), ERK1/2 (42, 44 kDa) and β-actin (45kDa) was detected by Western blot and normalized with β-actin. (b), (c), (d) Relative p-ERK, ERK and p-ERK/ERK protein levels were calculated. Protein expression of (e) p-JNK (46, 54 kDa), JNK (46, 54 kDa) and β-actin (45kDa) were detected by Western blot and normalized with β-actin. (f), (g), (h) Relative p-JNK, JNK and p-JNK/JNK protein levels were calculated. Statistical analysis was carried out with one-way ANOVA followed by Tukey's multiple comparison test compared with positive controls (PMA/PHA) (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).

Figure 6. Inhibition of PMA/PHA induced-IL-2 and TNF-α production by ursolic acid via the JNK pathway; Western blot analysis (representative figure of three independent experiments). Cell were pre-incubated with UA for 0 to 12 hours, PD98059 or SP600125 for 12 hours prior to addition of PMA/PHA for a further 12 hours, or only DMSO (C) for 24 hours. For positive control (P) cells were left without treatment for 12 hours prior to addition of PMA/PHA for a further 12 hours. Protein expression of (a) p-ERK1/2 (42, 44 kDa), ERK1/2 (42, 44 kDa) and β-actin (45kDa) was detected by Western blot and normalized with β-actin. (b), (c), (d) Relative p-ERK, ERK and p-ERK/ERK protein level was calculated. Protein expression of (e) p-JNK (46, 54 kDa), JNK (46, 54 kDa) and β-actin (45kDa) was detected by Western blot and normalized with β-actin. (f), (g), (h) Relative p-JNK, JNK and p-JNK/JNK protein level was calculated. Statistical analysis was carried out with one-way ANOVA followed by Tukey's multiple comparison test compared with positive controls (PMA/PHA) (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).