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Gamma Linolenic Acid (GLA) as a Therapeutic tool in the Management of Glioblastoma

Word Cloud By Danielle Smolyar

Eric Fine* (1), Mike Briggs* (1,2), Raphael Nir^# (1,2,3)

Sefacor, LLC (1); Woodland Pharmaceuticals, LLC (2); SBH Sciences, Inc (3). 

* These authors contributed equally; # Corresponding author (rnir@sbhsciences.com).

 

I. Introduction

Glioblastoma multiform is a fast-growing, invasive central nervous system tumor that forms from glial (supportive) tissue of the brain and spinal cord. Glioblastoma multiform also called glioblastoma or glioma along with grade III/IV astrocytoma and abbreviated herein and elsewhere as GBM. It usually occurs in adults and affects the brain more often than the spinal cord.  Brain tumor patients with GBM have a severely major unmet medical need. Current treatment for stage IV glioblastoma provides only 16-month median survival from time of diagnosis.

There has been and continues to be a tremendous amount of research with the goal of finding a cure for brain tumors, yet there are only 3 FDA approved drugs for this indication, BCNU in the form of Gliadel® wafers, temozolomide (Temodar®), since 2005 and most recently, 2009 bevacizumab (Avastin®; 10 mg/Kg intra venous) for recurrent GBM. Patients with grade IV glioma undergoing surgical resection of the tumor combined with radiation therapy (RT) to prevent any remaining cancer cells from regrowing have shown historical median survival of 11.5 to 12 months. The first FDA approved glioma treatment was the Gliadel wafer that is placed in the brain tumor bed after surgery, where it degrades, releasing the drug carmustine. This treatment that included surgery and radiation has been shown to extend the median survival of these patients to about 14 months approximately 2 months longer than the group that received placebo wafers (Westphal M, 2003, 2006), (Attenello FJ, 2008). However, the rate of complications, including an increase in cerebrospinal fluid leaks and intracranial hypertension, has limited their use (Nagpal S., 2012).  The current ‘gold standard’ treatment to which all new experimental treatments are compared is temozolomide. Patients with high grade glioma receiving surgery, temozolomide and radiation therapy have a mean survival of 14.5 to 16 months (Stupp R, 2005), (Grossman SA, 2010). Avastin (bevacizumab), is a humanized monoclonal antibody that inhibits vascular endothelial growth factor A (VEGF-A) administered by intravenous infusion and has been approved for treating the recurrence of glioma only after the cancer has become refractory to temozolomide (Cohen MH, 2009), (Chamberlain MC, 2010). Still, GBM remains one of the two worst-case scenarios in the spectrum of cancer, sharing with pancreatic cancer a less than 5% five-year survival rate.

Due to the current success of polyunsaturated fatty acid (PUFA) based therapeutics including Lovasa (GlaxoSmithKline/ Reliant Pharmaceuticals) and Vascepa (Amarin) for high triglycerides with mixed dyslipidemia, there seems to be a renewed interest in PUFA’s therapeutic effects in different disease indications, especially cancer.

The scientific literature reports various results for the many different PUFA forms and their affects in a wide variety of cancer cell line tests.  The use of PUFA in the clinical setting has shown a slight enhancement of tamoxifen treatment in breast cancer patients when taken as an oral supplement (Kenny FS, 2000). But the lack of clear clinical improvement predominates in most trials such as those for bladder cancer (Harris NM, 2002) and pancreatic cancer (Johnson CD, 2001). Intravenous infusion of the polyunsaturated fatty acid gamma linolenic acid (GLA) for pancreatic cancer patients had met with little success in extending these patients’ lives (Johnson CD, 2001).

We hypothesize that the systemic administration of PUFAs has had limited success in cancer treatment mainly due to their being highly protein bound in the blood upon infusion and the need for an apparently high local concentration in the vicinity of the cancer tissue. In the face of the confounding data for the utility of PUFAs in cancer treatment, our hypothesis has been supported by the promising results found in a small, but uncontrolled pilot clinical trial using a protocol entailing local application of GLA directly into the resected tumor bed of High Grade GBM patients (Das UN, 1995).

 

 

II.  Polyunsaturated fatty acids in Glioblastoma

 

Fatty acids are key nutrients that affect early growth and development, as well as chronic and other diseases. A fatty acid containing more than one carbon double bond is termed polyunsaturated fatty acid (PUFA). PUFA affect the prevalence and severity of cardiovascular disease, diabetes, inflammation, cancer, and age-related functional decline. PUFA are components of the structural phospholipids in cell membranes; they modulate cellular signaling, cellular interaction, and membrane fluidity. The two most important groups of PUFA are the Omega 3 and Omega 6 fatty acids. Alpha-linolenic acid (ALA or 18 : 3n-3) is the parent of Omega 3 fatty acids, and linoleic acid (LA or 18 : 2n-6), the parent of the n-6 PUFA family. The human body is unable to readily synthesize ALA, and LA, classifying them both as essential fatty acids that one must ingest in the diet.    LA and ALA are converted to their respective n-6 and n-3 PUFA families by a series of independent reactions of which both pathways require the same enzymes, Δ6 Desaturase and Δ5 Desaturase, for desaturation and elongation (Sprecher H, 2002).

Common polyunsaturated fatty acid forms tested for their anti-tumor effect include gamma linolenic acid (GLA), arachidonic acid (AA) from the n-6 series and eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) from the n-3 series. One of the most promising PUFA in the development of cancer therapeutics is the GLA.  GLA is a carboxylic acid with an 18-carbon chain and three cis double bonds. Although the cytotoxicity of GLA, AA, EA and DHA is very high in cancer cell-lines, GLA shows the greatest specificity of destroying only cancerous cells and leaving non-cancerous cells intact (Bégin ME, 1986) (Das UN, 1991). For this reason we will narrow the focus of this review to GLA.

In-Vitro analysis of GLA on various cancer cell lines

GLA has shown cytotoxicity to a number of cancer cell lines including breast (ZR-75-11), lung (A-549), prostate (PC-3) (Begin ME, 1986), pancreas (Ravichandran D, 2000), liver (Itoh S, 2010).   GLA was the most effective in selectively killing the tumor cells. In a co-culture experiment wherein normal human skin fibroblasts (CCD-41-SK) and human breast cancer cells (ZR-75-1) were grown together in a Petri dish and supplemented with GLA, only human breast cancer cells were eliminated without any effect on normal skin fibroblasts  (Bégin ME, 1986).

The studies outlined below focus on GBM:

Bell et al, (1999) examined the invasion and growth of cell spheroids of human GBM cell lines U87, U373 and MOG-G-CCM.  The spheroids were grown on collagen with up to 1 mM GLA for 5 days. Measurements showed that low concentrations of GLA (< 100uM) increased both apoptosis and proliferation while higher concentrations (>250 uM) significantly impaired spheroid growth. All spheroid preparations showed 100% growth inhibition after 5 days of culture with 500–1000 uM GLA. Similar experiments by Leaver HA et al, [2002a] found that the Lithium  (Li+) salt of GLA was more potent than GLA, most likely due to its increased solubility. Li⁺GLA showed statistically significant pro-apoptotic and anti-proliferative effects in C6 rat glioma cell line culture at 40 uM PUFA as observed using the MTT assay compared to nontreated controls.  Meglumine gammalinolenate (MeGLA) was also developed for enhancing the water solubility of the PUFA and it showed greater activity than Li⁺GLA (Ilc K, 1999). Work reported by Scheim (Scheim DE, 2009) on human cell cultures derived from human GBM biopsy treated with 500 uM GLA showed complete cytotoxicity to the cancerous cells, while maintaining complete viability in noncancerous cell organ cultures from human biopsy.

III. Mechanism of Action for GLA against cancer cells

The mechanisms by which PUFA act on normal and cancerous cells are complex and not well understood. In tumor cells, addition of PUFAs results in the generation of free radicals, enhancement of lipid peroxidation and the suppression of cell rescue proteins and pathways thereby leading to cell apoptosis.  However, in normal cells, supplementation of PUFAs produce adequate amounts of lipoxins, resolvins and protectins that protect the cells from free radicals and reactive oxygen species, suppress inflammation and prevent actions of mutagens and carcinogens (Das UN and Madhavi N, 2011).

1. A.    Free radical generation:

In vitro experiments testing the cytotoxic effects of  PUFA has shown that GLA application induced lipid peroxidation products may have a high affinity to Bcl-2, an integral membrane oncoprotein that is unique in its ability to suppress apoptosis. This interaction prevents Bcl-2 from suppressing apoptosis even in cancer cells. Haldar et al (1995) concluded that Bcl-2 is deactivated upon phosphorylation and Bodur et al (2012), have shown that the exposure to 4-hydroxynonenal (HNE) the main aldehydic product of plasma LDL peroxidation induces Bcl-2 phosphorylation (Haldar S, 1995), (Bodur C, 2012).

To decipher the mechanism of the cytotoxic action of GLA and other fatty acids, cyclo-oxygenase, lipoxygenase inhibitors, and anti-oxidants and free radical quenchers have been added to cancer cell line cultures.  The GLA may induce different cell death pathways in different cell lines. In HeLa cells, indomethacin, a cyclo-oxygenase and inhibitor, and NDGA, a lipoxygenase inhibitor, that were added to cell cultures were ineffective in blocking the cytotoxic action of GLA and DHA (Das UN and Madhavi N, 2011).  However, SOD and Vitamin E, both free radical scavengers blocked the tumoricidal action of GLA on human cervical carcinoma, (HeLa) cells, human leukemia, HL-60 cells, breast cancer, ZR-75-1, cells (Das UN, 1991, 2007), (Sagar PS, 1995).   The increased production of free radicals by GLA treated cancer cells may be one of the reasons for enhanced cytotoxicity of glioma tumors seen in the pilot human clinical trials.

1. B.    GLA influence on Angiogenesis:

Inclusion of GLA in a 3D matrix culture system of the rat aortic ring assay, significantly inhibited angiogenesis in a concentration-dependent manner and a significant reduction of vascular endothelial cell motility was observed (Cai J, 1999).  Localized administration of GLA to orthotopically implanted C6 glioma cell line in the rat brain decreased the tumor cell’s protein expression of the pro-angiogenic factor vascular endothelial growth factor (VEGF) by 71% (± 16%) and the VEGF receptor Flt1 by 57% (± 5.8%) (Miyake JA, 2009). The GLA treatment reduced the micro vessel density of the tumors by 41% compared to control tumors.  In addition, the GLA treatment caused a significant decrease in ERK1 and ERK2 protein expression of (27 ± 7.7%) and (31±8.7%), respectively. More recently, Miyake et al report that neoangiogenesis is regulated through the ERK1/2 pathway (Miyake M, 2013).

1. C.    GLA influence on cancer related genes:

Miyake et al, [2009] examined the changes in cancer related gene expression in C6 glioma cells growing in rat brains when treated with local GLA brain infusion as compared to vehicle controls. The GLA treatment shows evidence for the upregulation of proteins that would inhibit cell cycle growth and division and induce apoptosis. The expression of p53 was increased (44 ±16%) by GLA as compared to control.

The tumor suppressor protein p53 has many mechanisms of anticancer function, playing a role in apoptosis, genomic stability, and inhibition of angiogenesis. The mechanisms by which p53 works include: activating DNA repair proteins when DNA has sustained damage; arresting growth by holding the cell cycle at the G1/S regulation point if DNA damage is recognized allowing for repair or it can initiate apoptosis, or it can initiate programmed cell death, if DNA damage proves to be irreparable (Liang Y, 2013).  Similarly, the expression of p27 (another tumor suppressor protein) was also increased (27 ± 7.3%) in GLA treated animals (Miyake JA, 2009).

 

1. D.    Caspase:

Apoptosis is induced by caspase signaling pathways in many cells (Kim R, 2002) (Philchenkov A, 2004). One of the mechanisms of apoptosis involves a mitochondrial signaling pathway, which entails the efflux of cytochrome c from mitochondria to the cytosol (Ge H, 2009). Cytosolic cytochrome c together with Apaf-1 activates caspase-9, which then activates caspase-3 (Cain K, 2002), (Wang X, 2001). Caspase-3 play an important role in apoptosis and degrades proteins such as PARP, which is a nuclear enzyme implicated in many cellular process including apoptosis and DNA repair. Studies by Ge et al, (2009) suggest that GLA treatment induces a dose-dependent increase in cytochrome c and activation of caspase-3 that correlates with the apoptosis of human chronic myelogenous leukemia K562 cells (Kong X, 2006). Further, the apoptosis could be inhibited by a pan-caspase inhibitor (z-VAD-fmk) (Ge H, 2009).

1. E.    Ku Proteins:

The heterodimeric Ku70/Ku80 protein complex is important for DNA repair and plays an important role in double strand breaks especially in gamma irradiation resistant tumor cells where high levels of these proteins are related to hyper proliferation and carcinogenesis (Gullo, 2006). Ku proteins have shown that loss or reduction in their expression causes increased DNA damage and micronucleus formation in the presence of radiation (Yang QS, 2008). GLA treatment of C6 rat glioma cells was accompanied by a 71% reduction in Ku80 protein expression and a 39% increase in the number of micronuclei detected by Hoechst fluorescence, as well as a 49% reduction of cells in S-phase even at concentrations that do not produce significant increases in apoptosis when measured within only a 24 hour exposure (Benadiba M, 2009).

1. IV.  In Vivo effect of GLA

As previously discussed, GLA has been reported to have effects in many cancers in vivo with treatments ranging from direct anti-tumor activity in clinical studies with injected GLA to dietary supplementation as an adjuvant to more traditional chemotherapy (Fetrow CW, 1999) (Kleijnen J, 1994). There are a number of anecdotal reports of increased response and duration, but none of these studies have shown convincing evidence to support the continued use of GLA against any specific cancer subtype. In one small clinical pancreatic cancer study using an injectable form of GLA there was some apparent benefit (Fearon KC, 1996), which failed to be reproduced in a larger study (Johnson CD, 2001). Other tumor types for which there have been reports regarding use of GLA in cancer include breast cancer (Kenny FS, 2000, 2001), (Menendez JA, 2004, 2005) bladder cancer (Harris NM, 2002) and even leukemia (Kong X, 2009). In even earlier studies, PUFAs including GLA were shown to have some efficacy against both chemically induced skin carcinogenesis in mice (Ramesh G, 1998) and hepatocarcinoma models in rats (Ramesh G, 1995) although again, these studies were not definitive.  A recurring theme seems to be that for utility, the GLA needs to be present at reasonably high doses in the vicinity of the tumor, indicating the some form of local delivery must be considered, or perhaps some kind of targeted therapy.

A. GLA tumorcidal effect on rat glioma:

The Leaver group (Leaver HA, 2002 b) continued their work examining the effects of GLA treatment.  Rats with orthotopically placed C6 glioma tumor in their brains were locally infused with PBS vehicle or GLA solution from 200 uM to 2 mM. The most active was 2 mM, infused at 1 ul/hr over 7 days. In contrast 1mM total dose had no significant difference from the controls.  In the positive response group, tumor regression, increased apoptosis and decreased proliferation were observed. Minimal effects on normal neuronal tissue was detected, with the caveat that their methods were not comprehensive (see discussion on safety, section IV.B. and Conclusion discussion, section VI). Tumor volume was less than 50% of controls in the 2 mM infused rats. However, histology and TUNEL reactivity of the remaining tumor indicated that this may be an under-estimate of residual viable tumor as substantial areas of treated tumors showed characteristics of necrotic tissue and apoptotic cell death. Supporting this hypothesis, tumor tissue sections evaluated by IHC with the proliferative marker Ki67 in the 2mM GLA treated animals showed < 20% of PBS control expression. Note: in these experiments there was no initial debulking surgery of the tumor mass.

Further studies by Miyake JA et al, (2009) showed that increasing the concentration of GLA delivered to the implanted C6 cell glioma in rat brains by treating them with 5 mM GLA/d in cerebrospinal fluid (CSF) caused an even greater decrease in C6 tumor growth in vivo. The average tumor area was reduced by 75 ± 8.8% in comparison with CSF alone.  VEGF protein expression was reduced 77 ± 16%. GLA had an inhibitory effect on vessel number causing a 44 ± 5.4% reduction in tumor micro vessel density.

While the in vivo data have a mixed response when looking at different tumor types and delivery methods, it appears that there may be some utility in GBM, particularly when the drug is delivered locally.  Further exploration of delivery methods for GBM and other tumor types need to be explored including the use of more targeted therapies such as targeted nano-particle delivery and even antibody-drug conjugates (ADC).  The research models also need to reinforce and support if possible the clinical observation of efficacy seen with direct intratumoral (or resected cavity) delivery noted in previous studies carried out in India.

B. Safety Studies in the Canine Model:

A safety study in 3 healthy dogs showed that daily injection of 0.25 mg in 1ml of saline for six days into the brain parenchyma under aseptic conditions was found to be safe (Das U N, 1995). CT scan and gross examination of the meninges and subarachnoid space as well as histopathological exams showed no abnormality and no difference between injected side and non-injected side. None of the animals developed any side effects or complications due to the procedure or GLA injection. Note that humans were given 1 mg GLA per day (see next section).  These are at best preliminary findings and further evaluation of safety in normal brain tissues and CSF need to be considered.

 

1. V.            Clinical application of GLA for Glioma Patients

The most compelling argument for the usefulness of GLA in the treatment of glioblastoma comes from a series of open label, non-randomized trials that were run in India by Drs. Das and Reddy nearly 2 decades ago.  In these studies, summarized below, they found that direct administration of the GLA to the tumor site via infusion over several days provided no observable toxicities or side effects although there were not complete cognitive or behavioral studies done on the patients.  It remains to be shown that there are no significant liabilities to the administration of GLA to brain cancer patients to provide both an extension of life (overall survival benefit) as well as not impinging on the quality of life for the patient.

1. A.    Recurrent glioma patients:

The initial study treating patients with local administration of GLA was performed on patients with recurrent GBM. GLA was injected directly into the tumor and/or an Ommaya reservoir was used to deliver the GLA to the tumor bed after surgical tumor resection followed by standard RT (see Naidu MR , 1992).  This procedure not only showed substantial efficacy but also there were no drug related side effects. Although only a small group of 6 patients, 3 of the 6 were alive at their last follow-up check-in 2 yrs 4 months to 2 yrs 8 months. These patients with recurrent glioma when administered the GLA therapy were in critical condition with life expectancy of 9 months or less. A 50 % survival at ~ 2.5 yrs is much better than historic average of 27% survival at 2 years in primary glioma patients with what is now the “gold standard” treatment of radiation and temozolomide and thus warranted further study.

 

1. B.    GLA treatment of primary tumor patients:

The next study performed was on patients with grade III Astrocytoma and Grade IV glioblastoma receiving their first intervention. Patients underwent neurosurgery to remove as much of tumor as possible. Before closure of the dura, 1 mg GLA was instilled into the tumor bed and cerebral catheter and reservoir were positioned for subsequent injections. On day 7 post operation, a baseline CT brain scan was taken. One mg daily of GLA in 2-3 ml of sterile saline was instilled for 10 days before a repeat CT scan was taken for comparison  This procedure not only showed substantial efficacy but also there were no drug related side effects. Surgery plus RT supplemented with GLA treatment extended patient survival for 80% of treated patients (12/15) to 34 months with very limited drug-related side effects (Das U N, 1995).

 

1. VI.           Conclusion

As some of the patients (Trial B, above) were alive and apparently well more than 2 years after receiving treatment, it is rather incredible that this treatment has not been more widely tested in the west in the last 18 years.  It is likely due to the fact that no robust and reproducible preclinical studies have come forward and that more standard GLP toxicology studies were not done.  Safety needs to be the first concern and whether in rats, dogs or monkeys, if direct delivery of GLA to the brain cavity is the best treatment, then it is imperative to have these studies carried out with a full analysis of both histopathological findings as well as the more indirect cognitive and behavioral studies that will be very important in human therapy.  As direct delivery to the brain is not a typical therapeutic approach, it remains to be seen what the regulatory agencies will demand for this kind of novel treatment.  The most pressing need is to have a thorough assessment of normal brain tissue exposure at the doses that are likely to be administered to a human and to include some surgical intervention (slicing through the brain) to mimic the surgical resection of the glioma.  Thus just delivering to the cerebrospinal fluid, while an intermediate assessment tool, may not have full predictive value for the adjuvant application of GLA in the treatment of glioblastoma.  For true safety studies, multiples of the minimum efficacious dose would ideally be done to ensure that there is a safety margin for dose administration errors.  These studies are enabled by Alzet mini-pump technologies as well as direct cannulation and a sterile port for the daily administration of drugs to the test subject.

As systemic exposures will be minimized from direct brain delivery of small amounts such as the 1-2 mg per day in the referenced trials, there would be almost no way to evaluate for typical toxicology organ effects, coupled with the fact that GLA is an endogenous component of fatty acid metabolism.  With drugs such as Gliadel® having been used, with its poor safety profile (Based on Pharmacy Codes: The oral LD₅₀ in rat and mouse are 20 mg/kg and 45 mg/kg, respectively. Side effects include leukopenia, thrombocytopenia, and nausea.) Toxic effects include pulmonary fibrosis and bone marrow toxicity). Moreover, recent studies showing combining carmustine with temozolomide reduces survival time compared to temozolomide alone (Prados MD, 2004). The safety hurdle is fairly low for this devastating and fast growing tumor, however, that is not an excuse to forgo the safety studies that apparently were casually done previously and have kept this potential therapy out of the mainstream medicine for the past 18 years.

Taken together, these reports from the intriguing conundrum provided by the various outcomes of the animal efficacy studies to the patient feeding studies and the various delivery routes tested suggest that there is some rationale for utility of GLA in the treatment of cancer. Disciplined and well-controlled studies need to be undertaken with GLA / GLA salt or derivative forms of GLA that may have better pharmaceutical properties coupled with optimal delivery of the agent to the tumor with or without another therapy (chemotherapy or electrical field therapy ).

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