MicroRNA-21 inhibitor sensitizes human glioblastoma cells U251 (PTEN-mutant) and LN229 (PTEN-wild type) to taxol

Abstract

Background: Substantial data indicate that the oncogene microRNA 21 (miR-21) is significantly elevated in glioblastoma multiforme (GBM) and regulates multiple genes associated with cancer cell proliferation, apoptosis, and invasiveness. Thus, miR-21 can theoretically become a target to enhance the chemotherapeutic effect in cancer therapy. So far, the effect of downregulating miR-21 to enhance the chemotherapeutic effect to taxol has not been studied in human GBM.

Methods: Human glioblastoma U251 (PTEN-mutant) and LN229 (PTEN wild-type) cells were treated with taxol and the miR-21 inhibitor (in a poly (amidoamine) (PAMAM) dendrimer), alone or in combination. The 50% inhibitory concentration and cell viability were determined by the MTT assay. The mechanism between the miR-21 inhibitor and the anticancer drug taxol was analyzed using the Zheng-Jun Jin method. Annexin V/PI staining was performed, and apoptosis and the cell cycle were evaluated by flow cytometry analysis. Expression of miR-21 was investigated by RT-PCR, and western blotting was performed to evaluate malignancy related protein alteration.

Results: IC(50) values were dramatically decreased in cells treated with miR-21 inhibitor combine with taxol, to a greater extent than those treated with taxol alone. Furthermore, the miR-21 inhibitor significantly enhanced apoptosis in both U251 cells and LN229 cells, and cell invasiveness was obviously weakened. Interestingly, the above data suggested that in both the PTEN mutant and the wild-type GBM cells, miR-21 blockage increased the chemosensitivity to taxol. It is worth noting that the miR-21 inhibitor additively interacted with taxol on U251cells and synergistically on LN229 cells. Thus, the miR-21 inhibitor might interrupt the activity of EGFR pathways, independently of PTEN status. Meanwhile, the expression of STAT3 and p-STAT3 decreased to relatively low levels after miR-21 inhibitor and taxol treatment. The data strongly suggested that a regulatory loop between miR-21 and STAT3 might provide an insight into the mechanism of modulating EGFR/STAT3 signaling.

Conclusions: Taken together, the miR-21 inhibitor could enhance the chemo-sensitivity of human glioblastoma cells to taxol. A combination of miR-21 inhibitor and taxol could be an effective therapeutic strategy for controlling the growth of GBM by inhibiting STAT3 expression and phosphorylation.

Figure 1

Real time PCR analysis of miR-21 expression in glioblastoma cells treated with taxol and miR-21 inhibitor, alone or combination. RT-PCR results showing significant knockdown of miR-21 expression in U251 and LN229 cells.

Figure 2

Effect of the miR-21 inhibitor on the chemo-sensitivity of U251 and LN229 cells to taxol treatment. The growth of U251 (Figure 2a) and LN229 (Figure 2b) cells were inhibited by the miR-21 inhibitor, taxol only, and the indicated combinations. The cells were treated with the miR-21 inhibitor complexed to PAMAM for 6 h at 37°C. The medium was then replaced with media containing various concentrations of taxol. After 72 h of incubation, an MTT assay was performed. Absorbance at 570 nm was normalized to the control (untreated cells) to determine cell viability. Each value represents the mean ±SD from triplicate determinations. An aqueous solution of taxol (circles) and miR-21 inhibitor-loaded PAMAM (triangle) was incubated with human glioblastoma U251 (Figure 2c) and LN229 (Figure 2d) cells for six days. Drug-induced decrease in cell numbers was measured using the MTT assay. Values represent the mean ± SD (n = 6 replicates).

Figure 3

Activity of the AKT pathway in human glioblastoma LN229 and U251 cell lines. Representative Western blots showing the levels of p-AKT expression and the results of densitometric determinations. Significant differences from the control value are indicated by **p < 0.01.

Figure 4

Evaluation of the expression of PTEN, EGFR, STAT3, and p-STAT3 in human glioblastoma LN229 and U251 cell lines. Western blot of protein extracts from cells treated with the miR-21 inhibitor or taxol, alone or combination. The expression of β-actin was examined to ensure uniform protein loading in all lanes.

Figure 5

The miR-21 inhibitor enhanced taxol induced apoptosis. Flow cytometry analyses of propidium iodide-stained cells were performed in triplicate (Fig. 5A). Percentages of apoptotic cells are shown in the histogram (Fig. 5B). The effectiveness of the miR-21 inhibitor combined with taxol on BcL-2 and caspase-3 was analyzed by western blotting, with mock cells as controls (Fig. 5C). Antibodies against β-actin were used as a loading control to ensure uniform protein loading in all lanes.

Figure 6

miR-21 inhibitor and taxol induce G1 and S phase arrest on cell cycle distribution. U251 and LN229 cells were treated with the miR-21 inhibitor and taxol alone or in combination, and cell cycle distributions were detected by Flow cytometry 48 h later (Fig. 6A). Percentages of cells in different phases of the cell cycle are shown in the histogram (Fig. 6B). Western blotting showing cyclin D1 activity in U251 and LN229 cells. (Fig. 6C) Representative Western blots showing altered levels of active cyclin D1 expression. The expression of β-actin was examined to ensure uniform protein loading in all lanes.

Figure 7

Effects of miR-21 inhibitor and taxol on cell invasion ability in U251 and LN229 cell lines. Cell invasion ability was assessed by a transwell assay after 48 h treatment with the miR-21 inhibitor and taxol, alone or in combination (Fig. 7A). The numbers of cells that could invade via the membrane are shown as a histogram (Fig. 7B). Western blotting validation of proteins (MMP-2 and MMP-9) downregulated by miR-21 inhibitor and taxol combination therapy in both U251 and LN229 cell lines (Fig. 7C). The expression of β-actin was examined to ensure uniform protein loading in all lanes.