Combination Therapy with PIK3R3-siRNA and EGFR-TKI Erlotinib Synergistically Suppresses Glioblastoma Cell Growth In Vitro

Abstract

Background: Up-regulation of PIK3R3 (Phosphoinositide-3-Kinase Regulatory Subunit 3), the regulatory subunit of PI3K is correlated with the drug resistance of the glioblastoma cells. In the present study, the effect of PIK3R3 siRNA on erlotinib sensitivity of the U373-MG glioblastoma cells was explored.

Methods: After PIK3R3 siRNA transfection, the expression of PIK3R3 mRNA was measured using RT-qPCR. Trypan blue exclusion assay was used to explore the effect of PIK3R3 siRNA on cell proliferation. The effects of PIK3R3 siRNA and erlotinib, alone and in combination, on cell survival and apoptosis were measured using MTT assay and ELISA cell death assay, respectively.

Results: Our data showed that PIK3R3 siRNA markedly suppressed the expression of PIK3R3 in a time dependent way, inhibited the proliferation of the U373-MG cells and triggered apoptosis (p <0.05, relative to blank control). Pretreatment with PIK3R3 siRNA synergistically decreased the cell survival rate and lowered the IC50 of erlotinib. Moreover, PIK3R3 siRNA markedly enhanced the apoptotic effect of erlotinib.

Conclusions: Our data propose that suppression of PIK3R3 can effectively triggers apoptosis and enhances the sensitivity of the glioblastoma cells to EGFR-TKI erlotinib. Thus, PIK3R3 can be a potential therapeutic target in glioblastoma patients.<br />.

Keywords: Glioblastoma; PI3K; PIK3R3; erlotinib; siRNA.

Figure 1

Gene Expression Analysis in U-373 Cells Treated with PIK3R3 siRNA and NC siRNA . The cells were transfected with PIK3R3 siRNA and NC siRNA for 24, 48 and 72 h. Relative PIK3R3 mRNA expression was calculated using RT-qPCR and 2 ^{- (∆∆Ct)} method (A). Figure 1B and 1C show the melting curves, and Figure 1D and 1E show the proliferation curves of β-actin and PIK3R3 genes, respectively. The results are showed as mean ± SD of the results of three experiments. *p<0.05 versus corresponding blank control or NC siRNA transfected cells

Figure 2

The Effect of PIK3R3 siRNA on Sensitivity of the U-373 Cells to Erlotinib. Cells were treated with PIK3R3 siRNA and different concentrations of erlotinib for 24 h (A and C) and 48 h (B and D). Next, the cell survival was measured using MTT assay. Cell survival curves (A and B) were plotted using Prism software. The results are presented as mean ± SD (n=3). Data from three independent experiments were used to plot the CI versus (Fa) (C and D) by the Chou and Talalay method and CalcuSyn software. Dashed lines represent CI=1

Figure 3

Proliferation Curve of U-373 Cells Treated with PIK3R3 siRNA. Cell proliferation was determined using trypan blue staining over a period of 5 days. Data are expressed as mean ± SD (n=3). *p<0.05 versus blank control or NC siRNA

Figure 4

The Effect of Treatments on Apoptosis of Tumor Cells. The U-737 cells were treated with PIK3R3 siRNA (50 nM), negative control (NC) siRNA (50 nM) and erlotinib (IC₅₀ doses of 24 and 48 h), alone and in combination. The apoptosis was quantified using ELISA cell death assay after 24 (A) and 48 h (B). The data are presented mean ± SD (n=3) of three independent experiments. *p<0.05 compared with control