Cas IIgly induces apoptosis in glioma C6 cells in vitro and in vivo through caspase-dependent and caspase-independent mechanisms

Abstract

In this work, we investigated the effects of Casiopeina II-gly (Cas IIgly)--a new copper compound exhibiting antineoplastic activity--on glioma C6 cells under both in vitro and in vivo conditions, as an approach to identify potential therapeutic agents against malignant glioma. The exposure of C6 cells to Cas IIgly significantly inhibited cell proliferation, increased reactive oxygen species (ROS) formation, and induced apoptosis in a dose-dependent manner. In cultured C6 cells, Cas IIgly caused mitochondrio-nuclear translocation of apoptosis induction factor (AIF) and endonuclease G at all concentrations tested; in contrast, fragmentation of nucleosomal DNA, cytochrome c release, and caspase-3 activation were observed at high concentrations. Administration of N-acetyl-L-cystein, an antioxidant, resulted in significant inhibition of AIF translocation, nucleosomal DNA fragmentation, and caspase-3 activation induced by Cas IIgly. These results suggest that caspase-dependent and caspase-independent pathways both participate in apoptotic events elicited by Cas IIgly. ROS formation induced by Cas IIgly might also be involved in the mitochondrio-nuclear translocation of AIF and apoptosis. In addition, treatment of glioma C6-positive rats with Cas IIgly reduced tumor volume and mitotic and cell proliferation indexes, and increased apoptotic index. Our findings support the use of Cas IIgly for the treatment of malignant gliomas.

Figure 1

Cas IIgly inhibits cell proliferation and induces morphologic changes in tumor cells. (A) Morphology of untreated glioma C6 cells (left panel) and glioma C6 cells treated with 1 µg/ml Cas IIgly for 24 hours (right panel). Cell morphology was visualized with a light microscope at x20 using H&E staining. (B) Dose-dependent effect of Cas IIgly on cell viability in C6 glioma cells. Cell viability was measured by the MTT assay; data represent the mean ± SD (*P ≤ .05, **P ≤ .001, and ***P ≤ .0001) from six independent experiments. (C) Dose-dependent effect in the expression of PCNA in cell lysates of control and Cas IIgly-treated glioma C6 cells (left panel). The bar graphs indicate the relative units of PCNA normalized with actin (right panel). Each bar represents the mean ± SD (*P ≤ .05, **P ≤ .001, and *** P ≤ .0001) of three independent experiments.

Figure 2

Treatment with Cas IIgly induces apoptosis in tumor cells. (A) Cell death of control and 24-hour Cas IIgly-treated glioma C6 cells was measured by flow cytometry after propidium iodine staining. The data represent the mean ± SD (*P ≤ .05, **P ≤ .001, and ***P ≤ .0001) of three independent determinations. (B) Dose-dependent effect of Cas IIgly on apoptosis. Apoptosis of control and 24-hour Cas IIgly-treated glioma C6 cells was determined by TUNEL assay. The results are the mean ± SD (*P ≤ .05 and ***P ≤ .0001) of three independent determinations. Quantitative estimation of TUNEL-positive nuclei at different doses was determined by counting five fields at x10 for each determination. (C) Effect of Cas IIgly on DNA. The marker is a 100-bp DNA ladder (left lane). DNA was isolated from control and 24-hour Cas IIgly-treated glioma C6 cells.

Figure 3

Cas IIgly induced the loss of mitochondrial membrane potential and the release of apoptogenic factors. (A) Mitochondrial membrane potential from control and 24-hour Cas IIgly-treated glioma C6 cells was measured by accumulated R123 fluorescence determined by flow cytometry. The data represent the mean ± SD (*P ≤ .05, **P ≤ .001, and ***P ≤ .0001) of three independent determinations. (B) Western blot analysis for cyt c, the cytosolic fraction obtained from control and 24-hour Cas IIgly-treated C6 cells (left panel). The bar graphs indicate the relative amounts of cyt c normalized to the respective actin level (right panel). Each bar represents the mean of three independent experiments. (C) Caspase-3 activity is presented as fold increases over control. Each bar represents a mean of three independent experiments. (D) Western blot analysis for AIF and Endo G in the nuclear fraction obtained from control and 24 h Cas IIgly-treated C6 cells (left panel). The bar graphs indicate the relative amounts of AIF and Endo G normalized to the respective laminin B level (right panel). Each bar represents the mean of three independent experiments.

Figure 4

Treatment with Cas IIgly induces ROS and lipid peroxidation. (A) ROS generation was determined in lysed cells obtained from control and 24-hour Cas IIgly-treated C6 cells as described in Materials and Methods section. Each bar represents the mean ± SD (**P ≤ .001 and ***P ≤ .0001) of three independent experiments. (B) Levels of lipid peroxidation were determined in lysed cells obtained from control and 24-hour Cas IIgly-treated C6 cells, as described in Materials and Methods section. Each bar represents the mean ± SD (**P ≤ .001 and ***P ≤ .0001) of three independent experiments.

Figure 5

Increase in cellular ROS is associated with apoptosis induced by Cas IIgly. (A) Effects of NAC on DNA of 24-hour Cas IIgly-treated cells in the presence of 20 mM NAC. DNA extraction was performed as described in Materials and Methods section. The marker is a 1-kb extension ladder. (B) Immunocytochemistry of AIF in control C6 cells, treated with Cas IIgly and Cas IIgly +20 mM NAC for 24 hours (left panel). The figures shown are representative of at least three different experiments for each experimental condition. Original magnification, x20. The bar graphs indicate the mean of three independent determinations (left panel). Quantitative estimation of AIF-positive nuclei at different doses was determined by counting five fields of x20 for each determination. (C) Caspase-3 activity in control C6 cells, treated with Cas IIgly and Cas IIgly +20 mM NAC for 24 hours. Caspase-3 activity was determined as described in Materials and Methods section. Caspase-3 activities were presented as fold increases over control. Each bar represents the mean ± SD (**P ≤ .001 and ***P ≤ .0001) of three independent experiments.

Figure 6

In vivo antitumoral effect of Cas IIgly. (A) Comparison of tumoral volume determined through water displacement. (B) Mitotic index determined by microscopic analysis. (C) Cellular proliferation index determined by inmunohistochemistry for PCNA. (D) Apoptotic index determined by TUNEL assay for rats with glioma C6 treated with Cas IIgly at doses of 0.4 and 0.8 mg/kg per day for 21 days.