Abstract
Glioblastoma is the most aggressive and common type of malignant brain tumor in humans, with a median survival of 15 months. There is a great need for more therapies for the treatment of glioblastoma. Naturally occurring phytochemicals have received much scientific attention because many exhibit potent tumor killing action. Thymoquinone (TQ) is the bioactive compound of the Nigella sativa seed oil. TQ has anti-oxidant, anti-inflammatory and anti-neoplastic actions with selective cytotoxicity for human cancer cells compared to normal cells. Here, we show that TQ selectively inhibits the clonogenicity of glioblastoma cells as compared to normal human astrocytes. Also, glioblastoma cell proliferation could be impaired by chloroquine, an autophagy inhibitor, suggesting that glioblastoma cells may be dependent on the autophagic pathway for survival. Exposure to TQ caused an increase in the recruitment and accumulation of the microtubule-associated protein light chain 3-II (LC3-II). TQ also caused an accumulation of the LC3-associated protein p62, confirming the inhibition of autophagy. Furthermore, the levels of Beclin-1 protein expression were unchanged, indicating that TQ interferes with a later stage of autophagy. Finally, treatment with TQ induces lysosome membrane permeabilization, as determined by a specific loss of red acridine orange staining. Lysosome membrane permeabilization resulted in a leakage of cathepsin B into the cytosol, which mediates caspase-independent cell death that can be prevented by pre-treatment with a cathepsin B inhibitor. TQ induced apoptosis, as determined by an increase in PI and Annexin V positive cells. However, apoptosis appears to be caspase-independent due to failure of the caspase inhibitor z-VAD-FMK to prevent cell death and absence of the typical apoptosis related signature DNA fragmentation. Inhibition of autophagy is an exciting and emerging strategy in cancer therapy. In this vein, our results describe a novel mechanism of action for TQ as an autophagy inhibitor selectively targeting glioblastoma cells.
Highlights
Glioblastoma is a grade IV glioma and remains the most aggressive and devastating cancer of the central nervous system [1]
To test the effect of TQ on glioblastoma cell proliferation, we subjected a heterogeneous set of glioblastoma cells to a clonogenicity based cell survival assay, which accurately assesses the inhibition of cell proliferation in response to a cytotoxic or cytostatic agent
The sensitivity of each cell line does not appear to correlate with p53 status, which is in agreement with earlier studies showing that TQ is cytotoxic to cancer cells independent of p53 status [22,32]
Summary
Glioblastoma is a grade IV glioma and remains the most aggressive and devastating cancer of the central nervous system [1] It is the most common brain tumor diagnosed in adults, with about 9,000 new diagnoses annually in the United States alone. The standard of care for newly diagnosed glioblastoma is surgical resection of the tumor, followed by radiation therapy with concomitant and adjuvant chemotherapy with the alkylating agent temozolomide (TMZ). Anti-apoptotic Bcl-2 and Bcl-XL proteins of the Bcl-2 family are up-regulated, but the pro-apoptotic Bax and Bak proteins are down-regulated This suggests that glioblastomas might naturally be under a selection pressure to develop resistance to apoptosis [2]
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