Glioblastoma (GBM) is a highly aggressive primary brain cancer that is difficult to treat. The current standard of care consists of surgery, followed by radiation and chemotherapy with the alkylating agent temozolomide (TMZ) (1). However, the therapeutic benefit of TMZ is restricted to the patients presenting with epigenetic silencing of the O6-methylguanine-DNA methyltransferase (MGMT) DNA repair gene in the tumor (2). Otherwise, high expression levels of MGMT allow tumor cells to repair O6-guanine subsequent to methylation by TMZ, thereby preventing drug-induced DNA double-strand breaks and tumor cell death. As a consequence, median overall survival time differs depending on MGMT promoter methylation status, with 15 months for patients with an unmethylated promoter and 22 months for those with the methylated promoter status (3). In either case, however, (89–97%) of patients succumb to the disease within 5 years, as even for patients initially responding favorably to TMZ, the effectiveness wanes over time as tumors develop drug resistance (4).

A newly-approved vaccine-based therapy, rindopepimut (CDX-110), has demonstrated further extension of median survival time; however, this regimen is only applicable to the 30% of GBM patients who are positive for the epidermal growth factor receptor variant EGFRvIII (5). An approved treatment for the recurrent setting is the angiogenesis inhibitor bevacizumab (6). Although it improves the outcome for the majority of GBM patients, the duration of benefit is limited and the survival rate from the time of initial bevacizumab progression is poor (7,8). The latest therapy to be approved is the alternating electric fields generator-based NovoTTF therapy, (9) which has been demonstrated to extend median overall survival time by ~5 months (10). Regardless of the treatment regimen, however, patients with GBM continue to exhibit a dismal prognosis and novel methods of treatment are urgently required.

In one novel approach, the intranasal delivery of perillyl alcohol (POH), a naturally occurring monoterpene related to limonene (11), was investigated in two clinical studies with grade III and grade IV glioma patients (12–14). POH was administered via a lightweight nebulizer and nose mask up to four times daily on a continuous basis. The results were highly promising, indicating therapeutic activity along with good tolerance and no long-term central nervous system or systemic severe adverse events, even following years of daily administration (12–14). POH activity was also noted in recurrent patients, i.e., patients who had become unresponsive to standard of care, and 19% of all patients remained in clinical remission following 4 years of continuous, exclusive treatment with intranasal POH (13,14). As of April 2017, a clinical trial is currently underway in the United States that aims to investigate the safety and efficacy of intranasal NEO100, a highly purified version of perillyl alcohol, in patients with recurrent grade IV glioma (NCT02704858).

Another newly emerging principle being investigated for the treatment of GBM is the ketogenic diet (KD). Besides being a lifestyle choice or a method for weight loss, in medicine the KD is prescribed primarily as a treatment for refractory epilepsy in children (15). Based on its high-fat/low-carbohydrate content, the diet forces the body to metabolize fats and ketone bodies rather than glucose; the state of ketosis is hypothesized to reduce the frequency of epileptic seizures (16).

In the context of brain tumor treatment, the KD is expected to provide two main benefits. First, as epilepsy commonly develops among GBM patients (17), it may aid a reduction in the severity and number of epileptic episodes and alleviate the requirement for anti-epileptic drugs, which are associated with detrimental neuropsychological effects and often interfere with the therapeutic regimen (18,19). Second, KD may increase the efficacy of cancer therapy. Tumor development is frequently associated with a metabolic alteration, in which cells switch from aerobic respiration towards increased glycolysis and lactate production, despite the availability of oxygen (20). This so-called Warburg effect (aerobic glycolysis) leads to the increased consumption of glucose and the inability of the affected cells to utilize ketone bodies as an energy source (21). In response to KD, it is hypothesized that tumor cells will starve, or revert to normal mitochondrial activity and respiration, altogether resulting in decreased proliferation and metastatic ability, along with a heightened sensitivity to chemotherapy (22–24).

Previous preclinical studies demonstrated notable benefits of the KD in a variety of animal tumor models, including in malignant brain tumors (25) and in the meta-analysis by Klement et al (26). For example, among groups of mice with luciferase-labeled GL261 glioma cells implanted into their brains, median survival time was significantly extended in mice on a KD compared with mice on a standard diet; in addition, when such mice received two fractions of radiation (2×4 Gy of whole brain radiation), the bioluminescent signals from the intracranial tumors was diminished below the detectable limit in 82% of the mice, and no signs of tumor recurrence were observed for >200 days (27).

Encouraging results from the use of a KD were also reported in patients, and a small number of case studies have indicated a benefit of the KD in patients with malignant glioma, as reviewed by Maroon et al (28). It was consistently reported that patients undergoing the KD presented with reduced blood glucose levels and beneficial therapeutic responses, including the transient disappearance of the brain tumor tissue (29–31). Thus, these case reports, along with numerous anecdotal observations, presented encouraging signs of metabolic reprogramming by the KD, along with the increased efficacy of standard of care treatment for malignant glioma (23,29). This preliminary evidence of the benefit of KD for cancer therapy is currently being validated in ongoing, controlled clinical trials, which include patients with malignant glioma (25).

At the molecular level, reduced glucose availability may cause endoplasmic reticulum (ER) stress, which triggers the unfolded protein response (UPR) cellular process, consisting of an interplay of antagonistic mechanisms; low to moderate activity is cell protective and supports chemoresistance, but more severe conditions aggravate these mechanisms to the point where protective efforts are abandoned and the cell death program is induced instead (32). As tumor cells frequently experience chronic stress conditions (due to hypoxia, hypoglycemia, acidification, oxidative stress, etc.), the protective components of their ER stress response are continuously engaged and thus, less able to neutralize additional insults that tax the ER stress response (33,34). Of note, the ER stress/UPR process has been described as a potential therapeutic target in GBM (35,36) and this cellular mechanism has been demonstrated to be targeted by POH in GBM cells in vitro (37).

It is thus conceivable that the concerted effect of KD-induced hypoglycemia together with POH-induced responses may trigger severely aggravated ER stress, resulting in tumor cell apoptosis (38,39). Therefore, the present study combined a KD with the intranasal delivery of POH, and investigated the effects of this novel combination in a cohort of 32 patients who had previously relapsed from standard of care treatment.

The lack of effective treatments for patients with malignant glioma requires the design of novel therapeutic strategies that take advantage of common glioma phenotypes, including the altered metabolism of glioma cells (23). It has been postulated that a ketogenic diet, i.e., a diet consisting of high fat, low carbohydrates and adequate protein, may be useful in the treatment of brain tumors (25–31). The present study was conducted to assess the therapeutic efficacy and safety of a KD concomitant with intranasal perillyl alcohol as a novel therapeutic strategy for recurrent GBM. Following three months of adherence to the treatment, the following were observed: i) The PR rate was 77.8% (7/9 patients), including the reduction of the MRI tumor image and peritumoral edema; ii) reduced serum lipid levels; and v) decreased LDL-C levels.

Kambach et al (40) demonstrated that glioma cells are sensitive to cholesterol synthesis inhibition downstream of the mevalonate pathway, suggesting that specifically targeting cholesterol synthesis could be an adjuvant treatment for GBM. Another study (41) proposed a potential molecular mechanism by which cholesterol reduction may be effective for preventing and treating the progression of malignant tumors; it suggested that upregulation of the cholesterol pathway may be associated with a poor prognosis in patients with GBM. Consistent with this model, the data of the present study indicated that there is a statistically significant difference in total cholesterol levels prior to subsequent to treatment with a KD with concomitant intranasal POH, with lower values at the end of treatment (Fig. 3). However, no such difference was observed in the standard diet group. Furthermore, there was a statistically significant difference in LDL-C and triglyceride levels prior to subsequent to treatment in the KD group, with higher values at the onset (Table II).

In our previous study, it was demonstrated that although the consistent use of intranasal POH in recurrent GBM patients induced clinical responses, MRI data presented no evidence for the regression in the extent of peritumoral edema (42). In the present study, the extent of peritumoral edema was notably reduced, along with a decrease of the tumor area (Figs. 1 and 2). These results are consistent with another study (43) that investigated the effects of the KD on tumor growth and progression in animal models. The results revealed that peritumoral edema was significantly reduced in animals fed the KD, and protein analyses showed altered expression of zona occludens-1 and aquaporin-4. The authors concluded that the KD directly or indirectly altered the expression of several proteins involved in malignant progression and may be a useful tool for the treatment of gliomas. In addition to the MRI-based observation of decreased cerebral edema and tumor volume, a significant decrease in cholesterol and cholesterol esters in particles of LDL was identified in the present study. This observation is in line with an earlier study that demonstrated that proliferating tumor cells utilize serum-derived cholesterol esters, which are presumably carried by LDL particles (44). The decrease of cholesterol in the present study can be explained as there was attention to the quality of the lipids offered (monounsaturated fatty acid > polyunsaturated > saturated fatty acid) and the amount of cholesterol was limited to 200 mg/day. These results are not consistent with other studies (44–46), which concluded that KD increased the serum levels of total lipids and LDL-C. However, Sharma et al (47) did not observe significant alterations to the lipid profile of children with refractory epilepsy treated with KD, which remained within normal limits throughout the study.

In the present study, a moderate decrease in fasting glucose levels was documented, although without statistical significance (Table IV). The reason for this discrepancy is unclear and further investigation of this aspect will be required. It is important to note that the results of the present study were obtained with a 90-day period of dietary intervention. It is conceivable that a longer duration of the concomitant regimen may provide additional insight into the benefits of this regimen. However, shorter interventions may prove beneficial as well; for example, using a type 2 diabetic mouse model, Zhang et al (45) demonstrated that just 8 weeks of KD was sufficient to improve glucose and insulin tolerance, although hepatic lipid accumulation and hepatic steatosis were observed. Future studies investigating the KD as an adjuvant strategy to combat malignant gliomas should explore the optimal duration of this dietary intervention to support superior therapeutic outcomes.

In summary, the results of the present study are encouraging and suggest that KD associated with intranasal POH may represent a viable option as an adjunct therapy for recurrent GBM. Currently established therapeutic approaches for this patient cohort are lacking comprehensive efficacy, and the vast majority of patients succumb to the disease within a few years. Clearly, more effective treatments are urgently required. This small study will hopefully fuel further discussion and stimulate additional investigations to explore the potential benefit of adding a KD to existing regimens for patients with GBM.