LY294002 and sorafenib as inhibitors of intracellular survival pathways in the elimination of human glioma cells by programmed cell death

Abstract

Gliomas are aggressive brain tumors with very high resistance to chemotherapy throughout the overexpression of multiple intracellular survival pathways. Therefore, the aim of the present study was to investigate for the first time the anticancer activity of LY294002, phosphatidylinositol 3-kinase (PI3K) inhibitor and sorafenib, and rapidly accelerated fibrosarcoma kinase (Raf) inhibitor in the elimination of human glioma cells by programmed cell death. MOGGCCM (anaplastic astrocytoma, III) and T98G (glioblastoma multiforme, IV) cell lines incubated with LY294002 and/or sorafenib were used in the experiments. Simultaneous treatment with both drugs was more effective in the elimination of cancer cells on the way of apoptosis with no significant necrotic effect than single application. It was correlated with decreasing the mitochondrial membrane potential and activation of caspase 3 and 9. The expression of Raf and PI3K was also inhibited. Blocking of those kinases expression by specific siRNA revealed significant apoptosis induction, exceeding the level observed after LY294002 and sorafenib treatment in non-transfected lines but only in MOGGCCM cells. Our results indicated that combination of LY294002 and sorafenib was very efficient in apoptosis induction in glioma cells. Anaplastic astrocytoma cells turned out to be more sensitive for apoptosis induction than glioblastoma multiforme after blocking PI3K and Raf expression with siRNA.

Keywords: Apoptosis; Autophagy; Gliomas; LY294002; Sorafenib.

Fig. 1

Effect of 24-h-long incubation of MOGGCCM (a) and T98G (b) cells with different (0–30 µm) concentration of LY294002. *P < 0.01

Fig. 2

Evaluation of the level of apoptosis, necrosis and autophagy in MOGGCCM (a) and T98G (b, c, d) cells (scale bar—white line—50 µm) after 24 h sorafenib (1 µl) and LY294002 (10 µl) treatment. L LY294002, S sorafenib, LS LY294002 + sorafenib, *P < 0.01

Fig. 3

Analysis of mitochondrial membrane potential after LY294002 and sorafenib incubated MOGGCCM (a) and T98G (b) cells. L LY294002, S sorafenib, LS LY294002 + sorafenib, *P < 0.01

Fig. 4

Evaluation of programmed cell death marker proteins expression: densitograms (a, b, c, d) and immunoblotting membranes (a′, b′, c′, d′) of beclin-1 (a, b) and caspase 3 (c, d) and activity: caspases 3, 8, and 9 (e, f) in MOGGCCM (a, c, e) and T98G (b, d, f) cells. L LY294002, S sorafenib, LS LY294002 + sorafenib, *P < 0.01

Fig. 5

Estimation of PI3K (a, b) and Raf (c, d) kinases expression [densitograms (a, b, c, d) and immunoblotting membranes (a′, b′, c′, d′)] and AKT (e) and ERK (f) activity in MOGGCCM (a, c) and T98G (b, d) cells after studied drugs application. L LY294002, S sorafenib, LS LY294002 + sorafenib, *P < 0.01

Fig. 6

The effect of blocking PI3K (a, b) and Raf (c, d) expression [densitograms (a, b, c, d) and immunoblotting membranes (a′, b′, c′, d′)] with specific siRNA in MOGGCCM (a, c) and T98G (b, d) cells. TR transfection reagent, L LY294002, S sorafenib, *P < 0.01

Fig. 7

Examination of programmed cell death level in MOGGCCM (a, c) and T98G (b, d) after blocking expression with specific siRNAs, TR transfection reagent, L LY294002, S sorafenib, *P < 0.01