Anisomycin induces glioma cell death via down-regulation of PP2A catalytic subunit in vitro

Abstract

Aim: To examine the effects of anisomycin on glioma cells and the related mechanisms in vitro.

Methods: The U251 and U87 human glioblastoma cell lines were tested. The growth of the cells was analyzed using a CCK-8 cell viability assay. Apoptosis was detected using a flow cytometry assay. The expression of proteins and phosphorylated kinases was detected using Western blotting.

Results: Treatment of U251 and U87 cells with anisomycin (0.01-8 μmol/L) inhibited the cell growth in time- and concentration-dependent manners (the IC(50) values at 48 h were 0.233±0.021 and 0.192±0.018 μmol/L, respectively). Anisomycin (4 μmol/L) caused 21.5%±2.2% and 25.3%±3.1% of apoptosis proportion, respectively, in U251 and U87 cells. In the two cell lines, anisomycin (4 μmol/L) activated p38 MAPK and JNK, and inactivated ERK1/2. However, neither the p38 MAPK inhibitor SB203580 (10 μmol/L) nor the JNK inhibitor SP600125 (10 μmol/L) prevented anisomycin-induced cell death. On the other hand, anisomycin (4 μmol/L) reduced the level of PP2A/C subunit (catalytic subunit) in a time-dependent manner in the two cell lines. Treatment of the two cell lines with the PP2A inhibitor okadaic acid (100 nmol/L) caused marked cell death.

Conclusion: Anisomycin induces glioma cell death via down-regulation of PP2A catalytic subunit. The regulation of PP2A/C exression by anisomycin provides a clue to further study on its role in glioma therapy.

Figure 1

Anisomycin inhibits U251 and U87 cell growth and induces cell apoptosis. (A) U251 and U87 cells were separately treated with anisomycin at concentrations of 0, 0.004, 0.01, 0.04, 0.1, 0.4, 1, 2, 4, or 8 μmol/L for 48 h. Cell viability was analyzed using CCK-8 kit. Anisomycin started to suppress U251 and U87 cell growth at concentration of 0.01 μmol/L (cell viability is 75.3%±6.1% for U251 and 74.4%±4.3% for U87). At 8 μmol/L, the cell viability is only 18.4%±2.1% for U251 and 15.6%±1.3% for U87. Anisomycin inhibits U251 and U87 cell growth in a concentration-dependent manner. (B) U251 or U87 cells were treated with 0 or 4 μmol/L anisomycin for 72 h, and double stained with annexin V-FITC conjugates and propidium iodide followed by analysing in a flow cytometer. The Q2 plus Q4 proportions indicated apoptosis cells, 4 μmol/L anisomycin caused 21.5%±2.2% of apoptosis proportion in U251 cells and 25.3%±3.1% in U87 cells.

Figure 2

Anisomycin activates p38MAPK and JNK but inactivates ERK1/2 in U251 and U87 cells. Cells were non-treated (–) or treated with 4 μmo/L anisomycin for 30 min, or pre-treated with SB203580 or SP600125 for 1 h and then treated with 4 μmol/L anisomycin for 30 min. The phosphorylated and unphosphorylated p38MAPK, JNK, and ERK1/2 were detected by Western blotting using corresponding antibodies. The activations (phosphorylations) of the downstream substrates of p38 and JNK, Hsp27, and c-JUN were also detected. The unphosphorylated MAPKs served as loading control. A: Anisomycin; SB: SB203580; SP: SP600125.

Figure 3

Neither SB203580 nor SP600125 prevents anisomycin-induced U251 or U87 cell death. U251 or U87 cells were divided into four groups and separately pre-treated with DMSO vehicle, 10 μmol/L SB203580, 10 μmol/L SP600125, or 10 μmol/L SB203580 plus 10 μmol/L SP600125. After 1 h, the pre-treated cells were treated with 0 or 4 μmol/L anisomycin for 48 h. Then, cell viability was detected using CCK-8 kit. In groups that pre-treated with 10 μmol/L SB203580, 10 μmol/L SP600125, or 10 μmol/L SB203580 plus 10 μmol/L SP600125, anisomycin-induced cell death was not prevented (P>0.05).

Figure 4

Anisomycin down-regulates PP2A C subunit level in U251 and U87 cells. (A) U251 and U87 cells were non-treated (–) or treated with 4 μmol/L anisomycin for 6, 12, 24, or 48 h. PP2A C subunit level was detected by Western blotting. GAPDH served as loading control. (B) U251 and U87 cells were treated with 0 or 100 nmol/L okadaic acid (OA) for 48 h, then the cell viability were detected by using CCK-8 kit. In OA treated cells, the cell viability was 15.3%±4.2% for U251 and 12.8%±3.5% for U87.