Only recently have functional NOX (NOX1–5) activities, similar to the well-documented NOX2 in phagocytes, been identified in many non-phagocytic cell types (microglia, astrocytes, neurons) as a source of ROS production in response to stimuli such as TNFα.4
Importantly, several lipids including arachidonic acid and anionic phospholipids are critical in the activation mechanism of NOX enzymes.4,31
A recent study also demonstrated that ceramide synthesized de novo from the saturated fatty acid palmitate, induced the activation of NOX in retinal pericytes.5
Many chemotherapeutics, as well as endogenous stimuli such as TNFα, stimulate ceramide accumulation and induce apoptosis in tumor cells.2,32,33
Mechanisms of chemoresistance have therefore developed to evade the toxic effects of ceramide, by neutralization, degradation or conversion to pro-mitogenic and pro-survival metabolites.2
Moreover, oxidative stress resistance of tumor cells has also recently been described in the form of increased catalase activity in rat glioblastoma cells.15
Therefore, it became important to evaluate the role of chemoresistant pathways that metabolize ceramide, such as GCS, in the regulation of NOX activity. Intriguingly, glucosylceramide was shown to interfere with NOX in crude cell-free systems.18,34
In this respect, the phenotype of Gaucher disease (type I), a cerebrosidase deficiency, is strikingly similar to that of the NOX-deficiency chronic granulomatous disease.4,18
We therefore hypothesized that glucosylceramide could interfere with NOX activity and that this was critical to the chemotherapeutic-resistance mechanism of GCS.
In this study, we observed that various chemotherapeutics stimulated an increase in intracellular ROS production, which was completely blocked by an inhibitor of NOX enzymes (). Additionally, liposomal ceramide stimulated NOX-dependent intracellular ROS accumulation as opposed to a liposomal formulation containing no ceramide. More so, addition of the GCS inhibitor, PDMP, to the liposomal ceramide formulation dramatically augmented its ability to stimulate NOX-dependent intracellular ROS accumulation. Altogether, these results strongly indicated that ceramide and generators of ceramide stimulated intracellular production of ROS in SH-SY5Y neuroblastoma cells, which was attributed to NOX enzymes. We next observed that U-87 MG and LN-18 glioblastoma cells had elevated GCS, catalase and superoxide dismutase activities, consistent with potential mechanisms of multidrug-resistance (). Not surprisingly, these glioblastoma cell lines had a diminished capacity to generate intracellular ROS in response to doxorubicin (). Further investigation showed that pharmacological inhibitors of catalase or GCS or siRNA directed against GCS, restored or augmented the ability of the glioblastoma and neuroblastoma cell lines to produce intracellular ROS in response to doxorubicin (). More importantly, this restoration in ROS generation was accompanied by a concomitant decrease in the viability of these cancerous cell lines. In contrast, the overexpression of GCS in the more sensitive neuroblastoma cells rendered them resistant to doxorubicin or TNFα ( and B).
These findings demonstrating that targeting of GCS can improve the efficacy of chemotherapy, or that overexpression of GCS can induce resistance, were consistent with the defined multidrug-resistance mechanism of ceramide neutralization by GCS. However, we also observed that addition of exogenous glucosylceramide could block the responsiveness of SH-SY5Y cells to stimuli mediating intracellular ROS production (). Moreover, exogenous glucosylceramide directly interfered with p67phox translocation to the plasma membrane, a critical step in functional NOX assembly (). These findings were corroborated with additional molecular approaches manipulating endogenous glucosylceramide levels such as utilizing siRNA directed against GCS or overexpression of GCS. Depleting GCS activity with siRNA augmented p67phox translocation to the plasma membrane, whereas overexpression of GCS blocked this same translocation (). The ability of exogenous and endogenous glucosylceramide to block NOX assembly was further studied at a biophysical level utilizing model membranes in the form of liposomes. Exogenous glucosylceramide incorporates into the outer leaflet of the plasma membrane, with no lateral diffusion across this membrane. In comparison, endogenous glucosylceramide, which is generated on the cytosolic leaflet of the Golgi, is ultimately relocated to the inner leaflet of the Golgi where it is metabolized into other glycosphingolipids or transported to the outer leaflet of the plasma membrane. It was therefore important to understand how a lipid added to the outer leaflet of the plasma membrane, exogenously or endogenously, could interfere with NOX enzymes localized on the cytosolic leaflet. Using model membrane liposomes, we demonstrated that glucosylceramide induced positive curvature, evidenced by a significant decrease in the size of the liposomes (). We therefore speculated that positive curvature of a membrane may interfere with the assembly of NOX enzymes (). Altogether, our findings demonstrated a novel mechanism whereby glucosylceramide, the product of GCS activity, could interfere with NOX activity by preventing proper assembly of the enzyme.
These findings argue that GCS's role in chemoresistance is more profound than previously thought. While ceramide neutralization by GCS may also result in NOX inactivity, as ceramide stimulates NOX, the ability of exogenous glucosylceramide to block NOX activity clearly demonstrates a distinct inhibitory mechanism. Another important aspect of this study is the link of NOX enzymes to the efficacy of chemotherapy. In contrast, antioxidant therapy is a well recognized preventative and therapeutic approach to cancer. In addition, a recent study specifically defined NOX4 as a possible oncoprotein.10
Importantly, NOX4's localization to the mitochondria was evaluated and suggested to be important to the transformation of cells. In reconciling the differences with this NOX4 study, it is important to note that NOX4 is considered to be a constitutively active enzyme with no requirement for cytosolic cofactors to assemble with membrane-bound subunits.4,8
In contrast to the NOX4 study, we have showed in previous studies,19
as well as the current study, that agonist-dependent stimulation of NOX-dependent activity involves cytosolic cofactors translocating to the plasma membrane. More so, in the current study, interference of agonist-stimulated NOX assembly by exogenously-delivered glucosylceramide critically depended on the presence of the enzyme at the plasma membrane. Overall, our findings are specific to a chemotherapy-responsive NOX enzyme that is distinct from the potential cancer-promoting NOX4.
In summary, ceramide neutralization through the formation of glucosylceramide not only decreases the proapoptotic stimulus ceramide but also abolishes NOX assembly and activity () and in conjunction with increased antioxidant enzyme activities, greatly alleviates cell death-inducing oxidative stress associated with chemotherapy or physiological agonists. Consequently, pharmacological inhibition of antioxidant enzymes, such as catalase or pharmacological or molecular inhibition of GCS restored doxorubicin-toxicity, offering promise as a useful therapeutic avenue for the treatment of advanced cancers of the CNS. While treatment of patients with siRNA is currently not possible due to toxicity associated with delivery systems, our non-toxic cationic nanoliposomal formulation is designed specifically to overcome this hurdle. Additionally, our nanoliposomal formulation combining C6-ceramide and PDMP overcomes the current hurdle to clinical delivery of the GCS inhibitor PDMP associated with its low solubility. Nanoscale delivery systems further offer considerable clinical potential in the delivery of lower, yet more concentrated, therapeutic doses.26,35
Many advanced CNS tumors, such as glioblastomas, respond poorly to chemotherapeutic and radiation therapies, while in comparison, resistance in neuroblastomas is less common.14,36
Complicating matters and driving the demand for new and better therapeutics, is the fact that glioblastoma multiform, the most common primary brain tumor, has one of the worst survival rates among all cancers.14
This research offers an important advance to the understanding of mechanisms of multidrug-resistance in glioblastoma and the design of better therapeutics. On the other hand, it is worth noting that degenerative pathologies, including acute and chronic CNS disorders as well as psychiatric disorders, exhibit oxidative stress as a major component.37,38
This is important because in contrast to the goals of cancer therapy, the goals in the treatment of degenerative disorders are to improve the survival and function of normal cells. While ceramide neutralization and degradation are hallmarks of chemoresistance, ceramide accumulation is characteristic of degenerative disorders.6,32,33,38
Altogether, nano-encapsulated pharmacological or molecular agents targeting GCS offer exceptional promise in the treatment of chemoresistant CNS cancers, based on our findings that downregulating GCS activity not only can override ceramide neutralization by tumor cells, but also reengage stimuli-dependent NOX activity, restoring an ability to produce ROS and ultimately inducing tumor cell death.