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Abstract
Purpose
This study demonstrates that pharmacologic induction of heme oygenase-1 (HO-1) along with catalytic activation significantly modulated apoptosis of Jurkat cells induced by mycophenolic acid (MPA).
Methods
Cells were cultured with the presence or absence of MPA. Flow cytometric analysis was performed after propidium iodide staining. Western blotting of HO-1, Bcl, and Bax was also performed. Cells were stained 4'-6-Diamidino-2-phenylindole (DAPI) and measured by flow cytometry in the absence or presence of CoPPIX.
Results
Treatment of MPA decreased cell viability in a dose- and time-dependent manner. MPA-induced cell death was confirmed as apoptosis characterized by sub G0/G1 phase arrest. Expression of HO-1 assumes a pattern of decline after rising at the initial phase. CoPPIX, HO-1 inducer, significantly inhibited the cisplatin-induced apoptosis. Treatment of MPA resulted in reactive oxygen species (ROS) generation in Jurkat cells. CoPPIX attenuated ROS production in MPA-treated cells.
Figures and Tables
Fig. 1
MPA decreased the viability of Jurkat cells in a dose- and time-dependent manner. (A) Cells were treated with various concentrations of MPA for 24 hr and cell viability measured by MTT assay. (B) Cell viability was measured by MTT assay after 5µM MPA treatment for various times. *P<0.05 by student t-test, compared to control group.
Fig. 2
MPA increased the sub-G0/G1 fraction in Jurkat cells. (A) Cells were treated with 5µM MPA for various periods. After PI staining, sub-G0/G1 fraction (%) was measured by flow cytometry. (B) Apoptosis (%) in (A) was measured and plotted against time. Data represent the mean±S.D. of quadruplicates. *P<0.05 by Student's t-test, compared to control group.
Fig. 3
Production of H2O2 in MPA treated Jurkat cells. Cells were treated with 5µM MPA for various periods. Then, cells were incubated with the dye 2', 7'-dichlorofluorescin diacetate (5µM) and the fluorescence intensity of more than 10,000 cells was analyzed using flow cytometry.
Fig. 4
Effects of HO-1 expression on MPA treated-Jurkat cells in a time-dependent manner. Cells were treated with 5µM MPA for various periods. Cell lysates were separated on 15% SDS-PAGE and immunoblotted for anti-HO-1 and β-actin. The immunoreactive signals were visualized by ECL detection kit.
Fig. 5
Effect of CoPPIX on expression of HO-1 proteins in MPA treated Jurkat cells. Cells were treated with 5µM MPA in the absence and presence of 20µM MPA for 36 hr. Equal amounts of protein from cell lysate were subjected on 15% SDS-PAGE, transferred onto nitrocellulose membrane and immunoblotted with anti-HO-1 and anti-β-actin antibodies. The immunoreactive signals were visualized by ECL detection kit.
Fig. 6
Effect of CoPPIX, HO-1 inducer, in MPA induced apoptosis on Jurkat cells. (A) Cells were treated with 5µM MPA in the absence and presence of 20µM MPA for 48 hr. After PI staining, the fluorescence intensity of more than 10,000 cells was analyzed using a flow cytometry. (B) Apoptosis (%) in A was measured and plotted against dose. Data represent the mean±standard deviation (S.D.) of quadruplicates. (C) Cells were stained with 10µg/ml DAPI and visualized under fluorescence microscope.
Fig. 7
Effects of CoPPIX on expression of Bcl-2 and Bax proteins in MPA treated Jurkat cells. Cells were treated with MPA for 36 hr in the absence or presence of CoPPIX. Equal amounts of protein from cell lysate were subjected on 15% SDS-PAGE, transferred onto nitrocellulose membrane and immunoblotted with anti-Bcl-2, anti-Bax and anti-β-actin antibodies. The immunoreactive signals were visualized by ECL detection kit.
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