In the present study, we sought to screen a library of FDA-approved compounds to rapidly identify new, non-GBM drugs that could be readily introduced into GBM clinical trials. Using a platform that employed a wide range of human GBM lines, including clinically relevant patient-derived primary GBM lines, our screening uncovered 22 compounds from different classes with anti-neoplastic activity in GBM. Among others, the cardiovascular drugs statins showed high efficacy in reducing tumor growth both in vitro and in vivo, drawing our attention to these relatively non-toxic cholesterol lowering drugs. The present study demonstrates the potency of pitavastatin relative to other statins. Importantly, our results demonstrated that co-administration of pitavastatin with low-dose chemotherapy, greatly increased the potency of the latter, lowering the IC50 values for irinotecan by 40- to 70-fold, with few adverse effects. Experimentally, we found that statins independently induced autophagy in GBM and that statins may potentiate chemotherapeutic agents by inhibiting MDR-1 function. This was consistent with in silico screening results using our virtual tumor cell technology, which suggested that pitavastatin affects cell viability by inducing autophagy.
Cholesterol has a key role in cell membranes, cell metabolism, cell signaling and has been implicated in tumor development and progression. Therefore, as cholesterol-lowering agents, questions about the anti-tumor effects of statins have been already posed
[42, 43]. Statins decrease cholesterol levels by inhibiting the enzyme HMG-CoA reductase in the liver. In addition, mevalonate, and isoprenoid intermediates such as geranylgeranylpyrophosphate (GGPP) and farnesylpyrophosphate (FPP) in the cholesterol synthesis pathway are also depleted after statin treatment
. Another intermediate, dolichol, an essential substrate for protein N-glycosylation, is also blocked by statins
[45, 46]. Considering that GBMs are highly proliferative taking up large quantities of cholesterol, potentially they may be vulnerable to statin treatment
[47, 48]. However, the mechanism of sensitivity of GBM to statins has not been elucidated. Recent studies have shown that statins may have an anti-GBM effect in xenograft mouse models, by targeting the low-density lipoprotein receptor (LDLR), inducing apoptosis via ERK/AKT pathway
[20, 47]. Other data hypothesize that statins may inhibit tumor growth by inducing autophagy via the NF-κB pathway in human colon cancer cell line
. Our data obtained in both stable cell lines and primary patient samples clearly demonstrated that pitavastatin induced macro-autophagy in GBM cells
[20, 21]. Further experiments are now ongoing to investigate the signaling pathway(s) involved in this effect.
Importantly, we have shown that pitavastatin potentiated the anti-tumor effects of low-dose irinotecan, a topoisomerase inhibitor. Pitavastatin is know to be a substrate of the multi-drug resistance protein, MDR-1, which is overexpressed in GBM upon drug treatment and is partly responsible for the resistance of GBM to chemotherapy. Our data indicate that, in combination with irinotecan, pitavastatin suppressed glycosylation of MDR-1, thereby inhibiting its function and allowing irinotecan to accumulate intracellularly
[37, 39, 40]. Accumulation of irinotecan is likely responsible for the increased apoptosis in the presence of pitavastatin. The MDR-1 expression in cancer cells can be a significant obstacle to the success of chemotherapy. Many MDR-1 inhibitors have been extensively tested in clinical trials but the results have been inconclusive. According to TCGA data, down-regulated ABCB1 (MDR-1) predicted better survival of GBM patients. Combining a statin with a chemotherapeutic agent represents a powerful, potential strategy for circumventing resistance and significantly enhancing efficacy. Here we have confirmed that pitavastatin may improve the therapeutic response to TOPO-1 inhibitors, by inhibiting MDR-1 function, and may be beneficial for GBM patients. It remains to be determined whether other statins exert a similar or a different anti-neoplastic mechanism as compared to pitavastatin, and whether different subtypes of GBM have different sensitivity to pitavastatin or display other mechanisms for statin actions. GBM is a complex and heterogeneous disease that likely accounts for the different results obtained across various studies.
Irinotecan is broadly used in solid cancer therapy, especially in combination with other drugs
[50, 51]. In clinical use, the toxicity of irinotecan is generally manageable and reversible
[52, 53]. However, in some patients it may lead to severe side effects, such as diarrhea and neutropenia that can be life threatening. In our animal model, co-administration of pitavastatin allowed for a reduced dosage of irinotecan and avoided drug toxicity at higher dosage. These data indicate a new strategy to develop better irinotecan-based drug combination.
Based on the promising results of our present study, we are now undertaking additional preclinical studies of GBM to optimize dosing and characterize efficacy, thus providing a solid basis for a clinical trial with pitavastatin and irinotecan for the treatment of glioblastoma patients.