Curcumin decreases malignant characteristics of glioblastoma stem cells via induction of reactive oxygen species

A growing body of evidence indicates that GSCs are responsible for tumor formation, progression and recurrence and that targeting these cells may be paramount in the eradication of GBM [63, 64]. Studying GSCs from patient derived GBM samples is the best model of disease in humans, as it has been shown that established, indefinitely passaged GBM cell lines do not predict clinical drug efficacy and are not representative of patient tumors [65]. Here we demonstrate the anti-neoplastic effects of curcumin, a blood brain barrier permeable compound shown to be non-toxic to normal astrocytes and neurons, on patient derived GSCs [66, 67].

In this study we demonstrate through a neurosphere growth pattern (with the exception of the adherent Gio9), chemoresistance and the expression of all tested stem cell markers (with the exception of SOX2 in Glio9) in all cell lines that our samples are indeed GSCs (Fig. 1). Due to its adherent nature and lack of SOX2 expression, we hypothesize that Glio9 is of the mesenchymal GBM subtype [46, 68]. We show that curcumin decreases viability of GSCs in a dose dependent manner (Fig. 2) and that low doses of curcumin inhibit proliferation, sphere formation and colony formation of GSCs (Fig. 3). Experiments at doses this low are lacking from the GBM literature. We have shown that treatment with curcumin induces ROS activity (Fig. 4) and that pretreatment with the antioxidant n-acetylcysteine reverses curcumin’s effects on viability and molecular pathways (Fig. 6). It has been shown that the ERK pathway is inducible through ROS [58, 59] and that activated ERK can cause repression of STAT3 and downregulation of its downstream targets though an inhibition of its tyrosine 705 phosphorylation and activation of its serine 727 phosphorylation [60]. Although more work needs to be done, our data suggests that curcumin may exert its effects through this mechanism via induction of ROS.

The role of ROS in cancer is dichotomist in nature. Low levels of ROS have been shown to promote cancer through stimulation of cell proliferation, increased cell survival and amplified angiogenesis through activation of several pathways including NF-?B [69–71]. High levels of ROS have been shown to have anti-cancer effects by inducing cell cycle arrest and apoptosis via several mechanisms including Rac-1/NADPH oxidase pathway induction [72, 73]. CSCs have been shown to have lower intracellular ROS content due to increased expression of free radical scavenging systems [74]. Although this may indicate CSC ROS resistance, several studies have demonstrated ROS-induced targeting of CSCs. Induction of ROS through niclosamide treatment in AML, parthenolide treatment in AML and CML, and arsenic trioxide treatment in PML (promyelocytic leukemia) target CSCs [75–77]. In this study we demonstrate that curcumin-induced ROS targets glioblastoma stem cells.

Curcumin has been shown to be an effective CSC targeting molecule in glioma as well as other tumor types [9–12] while maintaining a minimal side effect profile even at high doses of 12 g/day [78]. The main hurdle facing curcumin as a potential chemotherapeutic agent is its bioavailability [14]. When dosed orally, unformulated curcumin has been shown to reach peak plasma levels of 2 ?M in humans [79]. In order to overcome this limitation, researchers have formulated several bioavailable forms of curcumin. Nano-emulsion curcumin, thermacurcumin (curcumin within colloidal nanoparticles), and curcumin within N-trimethyl chitosan coated solid lipid nanoparticles have been shown to reach peak plasma levels of 12.6 ?M, 4.6 ?M, and 3.28 ?M respectively in rodent models [80–82]. In this study we demonstrate that 2.5 ?M of curcumin inhibits the self-renewal properties of GSCs. In order to target GSC viability at curcumin levels of 25 ?M (Fig. 6) and above, alternative routes of administration must be considered. Polymeric drug and convection-enhanced delivery systems have been shown to deliver high local concentrations of active agents while decreasing systemic toxicities in GBM and may serve to circumvent the bioavailability issues facing curcumin [83]. Currently curcumin is being evaluated clinically for neurological diseases including bi-polar disorder and Alzheimer’s disease as well as for multiple cancers, however clinical trials are needed to determine the potential of curcumin alone and in combination with radiotherapy and or chemotherapy for GBM patients.