Many studies investigated the possibility of repurposing antidepressants for GBM treatment. Levkovitz et al. [91
] studied the effect of several different antidepressants on apoptotic markers in both glioma C6 and neuroblastoma SH-SY5Y cell lines. They reported that paroxetine and fluoxetine, two serotonin selective reuptake inhibitors (SSRIs), and clomipramine, a TCA, caused apoptosis in both cell lines. Interestingly, the toxic effect of clomipramine on C6 cells developed in almost all-or-nothing manner. At 12 μM there was hardly any toxicity while at 25 μM the effect was already maximal. Similarly, with fluoxetine there was little toxicity at 25 μM but 50 μM had a strong negative effect on viability. This is consistent with the results from a much earlier study in the C6 cell line, showing that fluoxetine caused DNA fragmentation, which is a major known step in apoptosis [92
]. Similarly, Liu et al. [93
] reported that fluoxetine suppressed the growth of GMB cell lines. The effective concentration of fluoxetine in that study was 25–30 μM in vitro. The authors explained this effect by activation of the intrinsic apoptotic pathway (see below). In vivo, fluoxetine strongly suppressed growth of tumors from U87 implants in the brains of Nu/Nu mice when administered daily at 10 mg/kg orally. Its effect was comparable to that of TMZ at 5 mg/kg intraperitonially. This study illustrates a stark contrast between the available models of GBM and clinic, where fluoxetine has never shown such potency against GMB. Fluvoxamine, another SSRI, at 40 μM was able to suppress migration and invasion of human GBM cell lines (A172, U87-MG, and U251-MG) [94
]. This effect was accompanied by inhibition of FAK/Akt mTOR pathway activity. Regarding the feasibility of the concentrations of fluoxetine and other TCA used in anti-GBM studies, human data suggest that they do accumulate in the brain, reaching remarkably high concentrations, up to 10 μg/ml, which converts to approximately 20–30 μM [95
]. Bielecka-Wajdman et al. [97
] examined the influence of six different antidepressants on the phenotypic signature and viability of GSCs isolated from a human GBM cell line. In that study only imipramine and amitriptyline significantly altered cell viability. Imipramine and amitriptyline were most effective in reducing quantity and expression of various stem cell markers, thus silencing the GSC profile. Jeon et al. [98
] also used two different GBM cell lines (U87 and C6) and reported that 40 and 60 μM of imipramine-induced cell death in GBM models but, remarkably, not normal primary rat astrocytes. The authors explain the effects of imipramine by activation of autophagy and implicate protein Beclin-1 in this process, because short hairpin RNA (sh-RNA) mediated knock-down of this protein conferred resistance to imipramine-induced cell death. Again, limitations of this study are the use of very old and hypermutated cell lines, and the use of very high concentrations of the antidepressant [98
]. In yet another study, Shchors et al. [99
] reported that imipramine treatment prolonged the overall survival of glioma-bearing mice by 18 days compared to that of a control cohort. These authors also concluded that TCAs induce autophagic cell death. Their explanation for this effect, however, was different to the previous two studies. The authors proposed that TCAs activate the G-protein αs subunit which, in turn, activates adenylyl cyclase resulting in an elevation of cellular cyclic adenosine monophosphate (cAMP). This was thought to induce autophagy associated cell death in glioma cells via the EPAC branch of the cAMP signaling cascade. This hypothesis was supported by an additional finding that inhibition of the purinergic receptor P2Y12
, activation of which inhibits adenylyl cyclase, potentiated the effects of imipramine, making the combination of drugs particularly effective [99
]. The problem with this explanation is that it relies on the monoamine theory for the mechanism of action of TCA which is the canonical explanation of the antidepressant effect of TCA. It poses that TCA act by inhibiting reuptake of noradrenaline and serotonin into the monoaminergic terminals from which they are released in the brain. Meanwhile in the in vitro experiments on GBM cultures there are neither monoamines, nor the terminals which could release and then reuptake them and therefore the very substrate for the “classic” monoamine-dependent action is lacking. In addition, TCAs block re-uptake of monoamines in nanomolar concentrations, which is orders of magnitude lower than what is commonly used in GBM experiments. Therefore, the effects reported in that paper require a different explanation.
The studies listed above illustrate the issues common to the literature on anti-GBM effects of TCAs (and, in fact, other repurposed drugs). These issues include: (a) use of the GBM cell lines such as C6, which have been in vitro for decades and accumulated mutations and acquired qualities which make them very different to the real tumors in human brain, (b) the use of unrealistically high concentrations of antidepressants, and (c) lack of coherency in terms of the proposed molecular targets for these drugs between different studies. Another major general limitation is the lack of an adequate model for studying toxic effects of these drugs on healthy human cells. Typically, researchers use either primary rodent astrocytes or human embryonic astrocytes. Neither of these are a close replica of mature human astrocytes or a good match for the GBM cells found in the human brain in the second half of life. Therefore, we do not know whether high concentrations of antidepressants used in GMB studies can be tolerated by healthy adult human brain cells or we are dealing with some un-specific cellular toxicity.