Journal of Kidney Cancer and VHL 2015; 2(1): 15-24. Doi: http://dx.doi.org/10.15586/jkcvhl.2015.21
Review
Article
Gramicidin A: A New Mission for an Old Antibiotic
Justin M. David1, Ayyappan K. Rajasekaran1,2
Abstract
Gramicidin
A (GA) is a channel-forming ionophore that renders biological membranes
permeable to specific cations which disrupts cellular ionic homeostasis. It is a well-known antibiotic, however it’s
potential as a therapeutic agent for cancer has not been widely evaluated. In two recently published studies, we showed
that GA treatment is toxic to cell lines and tumor xenografts derived from
renal cell carcinoma (RCC), a devastating disease that is highly resistant to
conventional therapy. GA was found to
possess the qualities of both a cytotoxic drug and a targeted angiogenesis
inhibitor, and this combination significantly compromised RCC growth in vitro
and in vivo. In this review, we
summarize our recent research on GA, discuss the possible mechanisms whereby it
exerts its anti-tumor effects, and share our perspectives on the future
opportunities and challenges to the use of GA as a new anticancer agent. Copyright: The
Authors.
How
to cite: David
JM, Rajasekaran AK. Gramicidin A: A New Mission for an Old Antibiotic. Journal of Kidney
Cancer and VHL 2015;2(1):15-24. Doi: http://dx.doi.org/10.15586/jkcvhl.2015.21
Introduction
Ionophores have traditionally found utility as
antibiotics in veterinary medicine and as growth-promoting feed additives for
agriculture (1, 2), but research over the past decade has now recognized that
they also possess extraordinary anticancer properties. The vast majority of this work has focused on
the mobile-carriers monensin and salinomycin.
These agents have been shown to induce antiproliferative and cytotoxic
effects, overcome therapy resistance, target cancer stem-like cells, and
disrupt specific oncogenic signaling pathways in a diverse array of cancer
types (reviewed in (2-5)). Furthermore,
salinomycin has been used in a small "first-in-man" pilot study with
two patients. It was reported to induce
tumor/metastasis regression, partial clinical response, and decreased levels of
circulating tumor markers without any of the severe and long-term side effects
that are commonly observed with conventional chemotherapeutics (4). Continued clinical development of salinomycin
is ongoing, and in 2012, the pharmaceutical companies Eisai and Verastem joined
together to develop a “proprietary analog of salinomycin” to use as a Wnt
inhibitor and anti-cancer stem cell drug for breast cancer.
In contrast to the mobile-carriers, the potential anticancer properties of channel-formers have been largely overlooked. Gramicidin A (GA) is the simplest and best-characterized channel-forming ionophore. It was the very first antibiotic to be isolated and used in a clinical setting, and its initial success paved the way for the clinical development of penicillin and the dawn of the antibiotic era (6). Structurally, GA is a short linear peptide of 15 alternating L- and D- amino acids with a formyl group at the N-terminus and ethanolamine at the C-terminus. GA is extremely hydrophobic, and within biological membranes two GA monomers dimerize end-to-end to form an unusual β-helix nanopore that spans the membrane (7) (Figure 1A). Water and inorganic monovalent cations can freely diffuse through the channel formed by GA dimers, and in biological systems this results in Na+ influx/K+ efflux, membrane depolarization, osmotic swelling, and cell lysis (7, 8) (Figure 1B). GA is well known to display potent broad-spectrum antibiotic activity (9-12), and we can now confirm that it also exhibits compelling anticancer properties that are both similar to, and distinct from, the mobile-carrier ionophores.
Figure 1. Mechanism of action of gramicidin A. (A) Gramicidin monomers form a β-helix
conformation within membranes. Dynamic
dimerization of two monomers forms the functional channel, which consequently
induces local membrane deformation. (B)
Cells maintain a low concentration of intracellular Na+ and a high
concentration of intracellular K+ relative to the extracellular
environment. Formation of the gramicidin
channel (green cylinder) upsets this balance by permitting the passive
diffusion of these cations along their respective concentration gradients
(arrows) resulting in Na+ influx and K+ efflux.
Gramicidin A is Cytotoxic
In our initial study (13), we evaluated the
cytotoxicity of GA using a panel of human cancer cell lines derived from renal
cell carcinoma (RCC). RCC is a
relatively rare but deadly disease that is histologically heterogeneous and
highly resistant to both chemotherapy and radiation. The 5-year disease-specific survival rate for
invasive RCC is only 10% (14, 15). We
found that treatment with GA decreased the viability of all six of the RCC cell
lines tested at submicromolar concentrations (all IC50 < 1.0μM). GA was uniformly toxic regardless of
histological subtype or the expression of various molecular markers of relevance
to RCC pathophysiology. This finding
indicates that GA may be effective in multiple RCC subtypes, which is important
because there are as yet no established therapies for the more rare subtypes of
RCC (papillary, chromophobe, collecting duct carcinoma, etc.). When we compared GA to the ionophore
monensin, a mobile-carrier with similar cation selectivity, we found that GA
reduced cell viability equal to or even greater than monensin depending on the
cell line tested. However, further
examination revealed that whereas monensin provoked apoptotic responses in
treated cells, GA induced cell death through a necrotic mechanism that was
associated with profound ATP depletion elicited by a blockade of both the
oxidative phosphorylation and glycolytic metabolic pathways. GA was also found to effectively suppress
tumor growth in vivo.
Collectively, this work demonstrated that perturbation of Na+ and K+ homeostasis by GA impairs cellular metabolism and starves cancer cells of energy. Precisely how this occurs remains to be fully determined, however our evidence supports a model in which oxidative stress is a potential link between GA and energy depletion (Figure 2). Oxidative stress appears to be a common feature of ionophores as both monensin and salinomycin were reported to increase the production of reactive oxygen species (ROS) (16-19). Cells respond to oxidative stress by upregulating ROS detoxifying pathways, and nicotinamide adenine dinucleotide phosphate (NADPH) is a crucial coenzyme that is required for the regeneration of reduced glutathione that is used to detoxify ROS (20). AMP-activated protein kinase (AMPK) was recently shown to increase NADPH production via enhancing glycolytic flux (21), and we observed both increased AMPK activation and a transient initial increase in glycolysis in GA-treated cells. If GA does in fact induce oxidative stress, then it is possible that AMPK responds by upregulating glycolysis to enhance NADPH production in order to alleviate this stress.
Figure 2: Proposed model of GA cytotoxicity. GA may induce oxidative stress, which can
activate AMPK to increase glycolytic flux.
This in turn can increase NADPH production via the pentose phosphate
pathway, and NADPH regenerates glutathione to detoxify ROS. Oxidative stress also damages DNA leading to
the activation of PARP. Overactive PARP
depletes NAD+, which inhibits glycolysis leading to ATP depletion and
subsequent necrotic cell death.
In addition, oxidative stress by ionophores damages
DNA (16-19). Cells use the enzyme poly
(ADP-ribose) polymerase (PARP) to signal damaged DNA by catalyzing the addition
of ADP-ribose moieties to nuclear proteins at the site of damage in a reaction
that consumes NAD+ (22). In the case of
extensive DNA damage, PARP can become overstimulated and deplete cellular NAD+
(22). Glycolysis depends upon the
reduction of NAD+ to NADH, and loss of NAD+ blocks glycolysis (22). We did not observe PARP cleavage
(inactivation) in GA-treated cells, but we did observe a marked decrease in
cellular redox activity and eventual loss of glycolytic activity, suggesting
that NAD+ may have been depleted by treatment with GA. Loss of glycolysis would impair NADPH
production and rapidly deplete ATP, ultimately leading to necrotic cell
death. This mechanism of bioenergetic
catastrophe leading to necrosis has been reported for DNA-damaging alkylating
agents (e.g. nitrogen mustards) (23), suggesting that GA shares important
characteristics with conventional chemotherapeutics. Experimental validation of this proposed
model (Figure 2) would provide key insights into the mechanism of cytoxicity by
gramicidin A.
Gramicidin A Inhibits Angiogenesis
Hypoxia and RCC
Oxygen deprivation is a common feature of solid
tumors as the tumor microenvironment is characterized by a steep oxygen
concentration gradient that regularly experiences temporal fluctuations in
oxygenation. Accordingly, tumors exhibit
many molecular and biochemical features associated with the cellular response
to low oxygen (hypoxia), which is controlled by the transcription factor
hypoxia-inducible factor (HIF). Numerous
functional investigations have revealed that HIF promotes tumor growth,
vascularization, and metastatic spread, and a large body of clinical evidence
has linked HIF activation with cancer progression, and reduced patient survival
(24-26). RCC tumors in particular
display features associated with chronic hypoxia responses (27), and it is now
recognized that constitutive activation of HIF is a key etiologic feature of
RCC.
HIF exists as a heterodimer that consists of an
oxygen-sensitive α-subunit and a constitutively expressed β-subunit (HIF-β)
(28). Regulation of HIF activity is
mediated by strict control of the protein levels of the α-subunits. When oxygen levels are adequate (normoxia),
HIF-α is rapidly hydroxylated and bound by the von Hippel-Lindau tumor
suppressor protein (VHL) which promotes the ubiquitylation and subsequent
degradation of HIF-α (27). Conversely,
HIF-α stabilizes in hypoxic conditions as O2 deprivation inhibits protein
hydroxylation. Constitutive activation
of HIF occurs in RCC through the loss of VHL expression/activity in the clear
cell subtype (ccRCC), and through additional VHL-independent means in other
subtypes (14). Anti-angiogenesis
therapies that antagonize HIF function (e.g. sunitinib, sorafenib, bevacizumab,
etc.) have succeeded in increasing progression-free survival and quality of
life for ccRCC patients, however durable and complete remissions remain rare
(29).
Gramicidin A Inhibits HIF
Studies conducted over the past 10-15 years have
demonstrated that a diverse array of chemotherapeutic agents, including
topoisomerase inhibitors, microtubule-targeting drugs, and anthracyclines, can
inhibit HIF transcriptional activity (24).
Furthermore, low-dose cyclophosphamide given at more frequent intervals
has been shown to block tumor angiogenesis (30). Given the aforementioned cytotoxic
similarities between GA and chemotherapy drugs, we sought to examine the
effects of GA upon hypoxia responses in RCC cells. We discovered that treatment of cells with GA
reduced the expression of the HIF-1α and HIF-2α isoforms in both normoxic and
hypoxic conditions. This in turn
suppressed HIF-dependent hypoxia responses and occurred even at doses lower
than those used in our prior cytotoxicity studies. Comparison of GA with the mobile-carriers
monensin, valinomycin, and calcimycin showed that only GA elicited a dramatic
and persistent decrease in HIF-1α and HIF-2α expression. These effects only occurred in VHL-positive
but not VHL-negative cell lines, and mechanistic examination revealed that GA
specifically upregulates the VHL tumor suppressor to accelerate the
O2-dependent destabilization of HIF.
These effects were confirmed in vivo, as treatment with GA reduced the
growth and angiogenesis in VHL-positive RCC tumors.
VHL upregulation by Gramicidin A
The anti-angiogenic effects of GA raise several
provocative questions and possibilities.
First, precisely how perturbing the intracellular ionic milieu affects
VHL expression is not fully understood.
GA exhibits similar sensitivity for Na+ and K+ (31) and induces the
simultaneous influx of Na+ and efflux of K+ in living cells. When we compared GA with three mobile-carrier
ionophores, only valinomycin provoked a partial decrease in HIF
expression. Since valinomycin is highly
selective for K+ over Na+ (32), this result suggests that increased VHL
expression is due primarily to the loss of intracellular K+, assuming the
mechanism of HIF downregulation is identical for both drugs. Further experiments will be necessary to
confirm this supposition. Second, our
results showed that only VHL protein increased in GA-treated cells implying
that either the translation of VHL transcripts or the stability of VHL protein
was increased. Factor(s) that regulate
VHL mRNA translation have yet to be identified, but several factors are known
to influence VHL protein stability. VHL
is stabilized when bound to its associated ubiquitin ligase components
(elongins B and C, RBX1, cullin 2) (33), and GA may promote this binding. Alternatively, several proteins are known to
specifically target and destabilize VHL: 1) E2-EPF ubiquitin carrier protein is
another ubiquitin ligase component that directly targets VHL for proteasomal
degradation and is expressed in primary and metastatic tumors (34); 2) casein
kinase 2 destabilizes VHL through phosphorylation of serines 33, 38, and 43 and
is upregulated in most human cancers (35); 3) transglutaminase 2 is a
crosslinking enzyme that causes VHL degradation by polymerization and is also
overexpressed in many cancers (36).
Whether GA inhibits any of these cancer-associated proteins to stabilize
VHL expression remains to be determined.
Third, our findings indicate that upregulation of VHL by GA blocks tumor
angiogenesis and growth, yet we found no relationship between VHL expression
and in vitro viability in response to GA (13).
This finding was actually not surprising as studies have reported that
VHL overexpression in naturally VHL-deficient cell lines caused dramatic
suppression of in vivo tumor formation and growth without concomitant
inhibition of in vitro cell growth (37, 38).
However, exactly how much of the reduction in tumor growth by GA is due
to direct cytotoxicity (VHL-independent) as opposed to the blockade of tumor
angiogenesis (VHL-dependent) is not yet known.
Lastly, it has become increasingly apparent in
recent years that VHL suppresses tumorigenesis not only through the
downregulation of HIF, but also through a myriad of HIF-independent
mechanisms. VHL has been shown to
directly bind both fibronectin and collagen IV alpha 2 and promote the proper
assembly of the extracellular matrix, and loss of VHL disrupts the normal
tissue and extracellular matrix architecture in a way that better facilitates
tumor growth, invasion, and blood vessel infiltration (39). VHL also downregulates integrins which
prevent cell motility and invasion by preserving the cell-cell adhesions of
both the tight and adherens junctions (39).
Furthermore, VHL stabilizes microtubules at the cell periphery, which
positively regulates the biogenesis and function of the primary cilium. The primary cilium is a microtuble-based
organelle found in all cells that functions as a chemo-, osmo-, and
mechano-sensor of the extracellular environment, and its loss in VHL-deficient
kidney cells leads to inappropriate proliferation and the formation of
preneoplastic renal cysts (i.e. polycystic kidney disease) (39, 40). Finally, VHL stabilizes the fellow tumors
suppressor proteins p53 and Jade-1 (gene for apoptosis and differentiation in
epithelia), which preserves DNA damage responses and inhibits oncogenic
Wnt/β-catenin signaling, respectively (40, 41).
An exciting proposition to consider is whether upregulation of VHL by GA
promotes these additional HIF-independent mechanisms to block tumor growth and
development. Validation of this notion
would broaden the therapeutic appeal of GA as a treatment for VHL-positive
cancers of the kidney and other tissues alike.
Future Development of GA
Generalized toxicity is a significant challenge to
the development of ionophores as therapies for human cancer. GA causes hemolysis and is toxic to the
liver, kidney, meninges, and olfactory apparatus (7, 42), and polyether
mobile-carrier ionophores are also toxic and elicit neurological side effects (1,
43). However, a variety of normal and
nonmalignant cells were reported to be less sensitive to mobile-carrier
ionophores (16-18, 44, 45) and murine xenograft experiments from ours and other
investigators have demonstrated the in vivo efficacy of ionophores without
significant side effects (46-50).
Furthermore, salinomycin was shown to be effective in two human cancer
patients without eliciting any severe toxicities (4). Nevertheless, a comprehensive understanding
of effects of ionophore drugs upon cancer cells vs. normal tissues is currently
lacking and will be necessary before clinical development can progress to a
larger scale.
The generalized toxicity of GA can be alleviated by
intratumoral injection. This method of
administration improves the therapeutic index of drugs by concentrating the
drug at the tumor site only to spare the rest of the body. We found intratumoral injection of GA to be
both safe and effective in our murine xenograft studies. Through the use of X-ray computed tomography,
intratumoral injection in the clinic is now possible for metastatic and/or
inoperable tumors, and we suggest that wider use of the technique will allow
agents such as GA to advance into clinical use more rapidly.
Chemical modification or mutation of the GA peptide
has proven effective at increasing microbial targeting and decreasing
non-specific toxicity (7, 8, 51, 52). Such mutagenesis approach could be utilized
to identify a non-toxic but efficacious form of GA that could be used systemic
delivery for treating tumors in in vivo.
Alternatively, encapsulation of GA in nanoparticles targeted to the
tumor could be used to safely deliver GA for treatment purposes. A recent report by Wijesinghe et al. used a
novel pH-sensitive liposomal approach to deliver encapsulated GA into the
membranes of cancer cells, resulting in cancer cell death (53). Such an approach could be used to target
cancer cells within the acidic tumor microenvironment only, thereby reducing
non-specific toxicity by sparing normal tissues.
Conclusion
GA, the channel-forming ionophore, has cytotoxic
and antiangiogenic activities in RCC tumors.
The cytotoxic activity is due to ATP depletion, and the anti-angiogenic
effect is due to the inhibition of HIF via the induction of endogenously
expressed VHL. Our in vitro and in vivo
studies strongly suggest that GA has the potential to be developed into a
therapeutic agent for RCC and possibly other cancers.
Conflicts of Interest
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