α-Conotoxins and α-Cobratoxin Promote, while Lipoxygenase and Cyclooxygenase Inhibitors Suppress the Proliferation of Glioma C6 Cells

Abstract

Among the brain tumors, glioma is the most common. In general, different biochemical mechanisms, involving nicotinic acetylcholine receptors (nAChRs) and the arachidonic acid cascade are involved in oncogenesis. Although the engagement of the latter in survival and proliferation of rat C6 glioma has been shown, there are practically no data about the presence and the role of nAChRs in C6 cells. In this work we studied the effects of nAChR antagonists, marine snail α-conotoxins and snake α-cobratoxin, on the survival and proliferation of C6 glioma cells. The effects of the lipoxygenase and cyclooxygenase inhibitors either alone or together with α-conotoxins and α-cobratoxin were studied in parallel. It was found that α-conotoxins and α-cobratoxin promoted the proliferation of C6 glioma cells, while nicotine had practically no effect at concentrations below 1 µL/mL. Nordihydroguaiaretic acid, a nonspecific lipoxygenase inhibitor, and baicalein, a 12-lipoxygenase inhibitor, exerted antiproliferative and cytotoxic effects on C6 cells. nAChR inhibitors weaken this effect after 24 h cultivation but produced no effects at longer times. Quantitative real-time polymerase chain reaction showed that mRNA for α4, α7, β2 and β4 subunits of nAChR were expressed in C6 glioma cells. This is the first indication for involvement of nAChRs in mechanisms of glioma cell proliferation.

Keywords: cyclooxygenase inhibitor; glioma C6; lipoxygenase inhibitor; proliferation; real-time polymerase chain reaction; viability; α-cobratoxin; α-conotoxin.

Figure 1

The effect of α-conotoxins MII; PnIA; RgIA; [V11L,V16D]ArIB ([L,D]ArIB) and α-cobratoxin (CTX) at concentration of 1 nM on the viability (a) and on the proliferative activity (b) of C6 glioma cells after 24 h (n = 20), 48 h (n = 20), and 72 h (n = 10–12) of incubation. The cell viability and concentration were determined by flow cytometry. * p < 0.05 according to Student’s t-test when compared to control.

Figure 2

Effect of CTX on the survival of rat C6 glioma cells. The cells were incubated in the presence of various CTX concentrations (3 pM–3 μM) for 72 h. Cytotoxin I (Cyt I) from Naja oxiana venom was used for a positive control. The cell viability was determined by MTT test. Data are shown as % of control (untreated cells). The results are presented as the mean ± SEM in three (for concentrations of 3 nM and 300 pM—four) independent experiments with 12 replicates in each. * p < 0.05, Student’s two-sample t-test compared to control.

Figure 3

Glioma C6 cells treated with nicotine at a concentration of 1 μL/mL (6.1 mM).

Figure 4

Effect of α-conotoxins MII; PnIA; RgIA; [V11L,V16D]ArIB ([L,D]ArIB) and α-cobratoxin (CTX) at concentration of 1 nM and COX inhibitors (IM, indomethacin, 10 µМ; NS-560, 5 µM; SC-398, 0.1 µM) applied separately or in the indicated combinations on viability (a) and on the proliferative activity (b) of C6 glioma cells after 24 h (n = 10), 48 h (n = 15) and 72 h (n = 10) of incubation. The cell viability and concentration were determined by flow cytometry. * p < 0.05 according to Student’s t-test when compared to control; ** p < 0.05 according to Student’s t-test when compared to the corresponding COX inhibitor.

Figure 5

Effect of α-conotoxins MII; PnIA; RgIA; [V11L,V16D]ArIB ([L,D]ArIB) and α-cobratoxin (CTX) at concentration of 1 nM and LOX inhibitors (NDGA, nordihydroguaiaretic acid, 30 µM; Bai, baicalein, 50 µM; Zil, zileuton, 50 µM) on the viability (a) and on the proliferative activity (b) of glioma C6 cells after 24 h (n = 15), 48 h (n = 14) and 72 h (n = 15) of incubation. (c) Effect of the combined use of NDGA and COX inhibitors (IM, indomethacin, 10 µМ; NS-560, 5 µM; SC-398, 0.1 µM) with the α7 nAChR blockers on the proliferation of C6 glioma cells after 24 h of incubation (n = 5). The cell viability and concentration were determined by flow cytometry. * p < 0.05 according to Student’s t-test when compared to control; ** p < 0.05 according to Student’s t-test when compared with the corresponding LOX/COX inhibitor.

Figure 6

Real-time PCR detection of mRNA for nAChR subunits. Only α4, α7, β2 and β4 subunits were detected. ΔCT values normalized to the α7 ΔCT value are shown. Results are presented as mean ± SE of two independent experiments. Not detected, n.d.

Figure 7

Binding of radioactive ¹²⁵I-labeled α-bungarotoxin (¹²⁵I-αBgt) to glioma C6 cells. Nonspecific ¹²⁵I-αBgt binding was determined in the presence of 200-fold excess of CTX. Results are presented as mean ± SE of two independent experiments (n = 5 in each experiment).