Chloroquine activates the p53 pathway and induces apoptosis in human glioma cells

Abstract

Glioblastoma is the most common malignant brain tumor in adults. The currently available treatments offer only a palliative survival advantage and the need for effective treatments remains an urgent priority. Activation of the p53 growth suppression/apoptotic pathway is one of the promising strategies in targeting glioma cells. We show that the quinoline derivative chloroquine activates the p53 pathway and suppresses growth of glioma cells in vitro and in vivo in an orthotopic (U87MG) human glioblastoma mouse model. Induction of apoptosis is one of the mechanisms underlying the effects of chloroquine on suppressing glioma cell growth and viability. siRNA-mediated downregulation of p53 in wild-type but not mutant p53 glioblastoma cells substantially impaired chloroquine-induced apoptosis. In addition to its p53-activating effects, chloroquine may also inhibit glioma cell growth via p53-independent mechanisms. Our results clarify the mechanistic basis underlying the antineoplastic effect of chloroquine and reveal its therapeutic potential as an adjunct to glioma chemotherapy.

Fig. 1.

Chloroquine inhibits glioma cell growth and viability in culture. (A) Assessment of cell growth rates in glioma cells with known functional status of p53.³¹ Cells were treated with a range of chloroquine concentrations indicated in the legends. (B) Assessment of cell death rates in glioma cells lines with wtp53 (left panel) or deficient p53 function (middle and right panels). Values represent the mean of 6 replicates.

Fig. 2.

Chloroquine induces apoptosis in cultured glioma cells. (A and B) Time-dependent activation of caspase-3 by chloroquine in U87MG cells. Untreated or chloroquine-treated cells were stained for the cleaved form of caspase-3 and counterstained by DAPI. The inset in (A) shows the characteristic nuclear morphology of chloroquine-treated cells. (C) Summary of the caspase-3 activation assessment in glioma cells lines with different status of p53. The percentage of cells positive for cleaved caspase-3 was determined by counting a minimum of 500 cells in 5–10 microscopic fields in replicates of 3 for each condition. (D) Assessment of apoptosis in U87MG cells by TUNEL. A propidium iodide (PI) counterstain was used. (E) The effects of chloroquine on the mitochondrial membrane potential integrity assessed by measurements of the mitochondrial accumulation of fluorescent JC-1 in glioma cells with wtp53 (U87MG) or mutp53 (G112). The ratio of red/green JC-1 fluorescence was determined in cells untreated or treated with chloroquine for 24 or 48 hours.

Fig. 2.

Chloroquine induces apoptosis in cultured glioma cells. (A and B) Time-dependent activation of caspase-3 by chloroquine in U87MG cells. Untreated or chloroquine-treated cells were stained for the cleaved form of caspase-3 and counterstained by DAPI. The inset in (A) shows the characteristic nuclear morphology of chloroquine-treated cells. (C) Summary of the caspase-3 activation assessment in glioma cells lines with different status of p53. The percentage of cells positive for cleaved caspase-3 was determined by counting a minimum of 500 cells in 5–10 microscopic fields in replicates of 3 for each condition. (D) Assessment of apoptosis in U87MG cells by TUNEL. A propidium iodide (PI) counterstain was used. (E) The effects of chloroquine on the mitochondrial membrane potential integrity assessed by measurements of the mitochondrial accumulation of fluorescent JC-1 in glioma cells with wtp53 (U87MG) or mutp53 (G112). The ratio of red/green JC-1 fluorescence was determined in cells untreated or treated with chloroquine for 24 or 48 hours.

Fig. 3.

The p53 pathway is responsive to chloroquine. p53 protein and products of the known p53 targets p21, mdm2, bax1, or pig3 were assessed in glioma cell lines with different p53 status (A–C) and in the human colon carcinoma cell line HCT116 expressing wtp53 (B) by Western blot. Cells were treated with varying concentrations of chloroquine for 24 hours (A) or with a constant dose of chloroquine (30 µg/mL) for the indicated time periods (B and C). (D) Assessment of the p53 phosphorylation status at a serine residue Ser15 in U87MG (upper panel) and HCT116 (bottom panel) cells treated with chloroquine or ionizing radiation. The phosphorylated form of p53 (p53-Ser15^P) was detected using a phosphorylation-sensitive antibody 16G8, which recognizes only the phosphorylated p53-Ser15^P isoform. Total p53 detection was by antibody DO-7. The ubiquitously expressed cytoskeleton component α-tubulin or the basal transcription factor TBP was assessed to assure equal protein loading.

Fig. 4.

Inhibition of p53 diminishes apoptotic response to chloroquine in glioma cell lines with wtp53. (A) Assessment of the efficacy of endogenous p53 inhibition by transfection with unspecific scr-siRNA or p53-siRNA. U251 cells, which express high levels of endogenous mutp53 were transfected with scr-siRNA or p53-siRNA and stained with antibody DO-7 to ascertain the effect of siRNAs. (B) p53 inhibition by p53-siRNA diminished significantly (P < .02) the activation of caspase-3 in glioma cell lines with wtp53 but not in mutp53 lines. The percentage of cells with activated caspase-3 was determined by counting at least 500 cells in 5–10 microscopic fields from three replicate cover slips. The results shown represent the mean of two experiments.

Fig. 5.

Treatment of experimental glioma with chloroquine in vivo. Tumor volumes and mitotic and apoptotic indexes were determined in chloroquine- or PBS-treated U87MG tumors as described in the Materials and Methods section. (A) Chloroquine-treated tumors show a significant decrease in the average tumor volume and a lower mitotic index, but significantly elevated rates of apoptotic cells. The data analysis was performed using a one-way analysis of variance. (B) Representative hematoxylin/eosin-stained histological sections. The arrowheads indicate mitotic cells. (C) Representative TUNEL-stained histological sections. The arrowheads indicate TUNEL-positive cells.