Patent Grant
9289456
The invention relates to a system and method for statin modulation of oncolytic virus and upregulation of nuclear pore complex proteins and antiviral factors, and in particular, modulation and upregulation using simvastatin.
Cancer is the second leading cause of death in the United States and worldwide. Current anticancer treatments have low specificity, affecting cancerous as well as normal cells. New therapeutic approaches, such as the use of viruses with cancer-selective killing properties (oncolytic viruses), are being explored.
In the United States, the number of cancer deaths among people younger than 85, surpasses the number of deaths due to heart disease (1) (Numbers in parentheses refer to the Bibliography which follows this section). Currently, there are many anti-cancer treatments, however, cure or long term remission and clinical success have come with significant toxic side effects, acute and chronic morbidity. The low efficacy attributed to anti-cancer treatment creates an urgent need for innovative therapeutic strategies.
One such strategy known as virotherapy, utilizes live viruses that have selective cancer-killing properties. However, oncolytic virotherapy is in its infancy and must overcome several obstacles before rendered as safe and effective for the management of cancer patients.
The art described in this section is not intended to constitute an admission that any patent, publication or other information referred to herein is “prior art” with respect to this invention, unless specifically designated as such. In addition, this section should not be construed to mean that a search has been made or that no other pertinent information as defined in 37 CFR §1.56(a) exists.
In accordance with the disclosure, a method of treating cancer in a patient comprises administering to the patient having cancer an effective amount of an oncolytic virus and modulating virus replication with a statin. In various embodiments, the statin modulates virus replication by upregulating nuclear pore complex antiviral factors; the nuclear pore complex antiviral factors are Rae1 and Nup98; the oncolytic virus is Vesicular Stomatitis Virus; and the statin is simvastatin.
In another embodiment, a combination cancer treatment therapy comprises an effective amount of Vesicular Stomatitis Virus (VSV), and an effective amount of simvastatin (Sim), wherein the effective amount of VSV and the effective amount of Sim synergistically enhance cancer-cell apoptosis. In further embodiment, the Sim modulates the virus by upregulating nuclear pore complex antiviral factors; the nuclear pore complex antiviral factors are Rae1 and Nup98; and the patient is first pretreated with said statin and then administered an effective amount of an oncolytic virus.
In a yet further embodiment, a cancer treatment therapy comprises administering a statin at a first dosage level to cancer cells of the patient; infecting cancer cells of the patient with Vesicular Stomatitis Virus (VSV) between 0 and 24 hours after administering the statin, growth of VSV and associated oncolysis thereby enhanced by the statin. Embodiments include said statin is selected from the group consisting of a lipophilic statin, simvastatin; said statin is simvastatin, and is administered at a concentration of between about 0.01 and about 0.175 μM; said statin is simvastatin, and is administered at a concentration of about 0.125 μM; infection is with VSV at a Multiplicity of Infection of about 0.01 to 2.0; infection is with VSV at a Multiplicity of Infection of about 1.0.
The therapy, in another embodiment, further includes the step of administering a statin at a second, higher dosage level, to cells of the patient, thereby causing the statin to exhibit an antiviral effect on the VSV. Other embodiments include the higher dosage level being about 0.25 μM or higher; further including the step, a substantial time after administering the statin, of administering to the patient a different anticancer therapeutic substance; the different anticancer therapeutic substance is an inhibitor of the RAS/Raf/ERK pathway; the different anticancer therapeutic substance is administered at least about 4 hours after the statin; and, the statin is simvastatin and the different anticancer therapeutic substance is PLX4032.
A more complete understanding of the present invention, and the attendant advantages and features thereof, will be more readily understood by reference to the following detailed description when considered in conjunction with the accompanying drawings, described as follows.
Detection of viral proteins are shown by immunoblot assays performed with anti-VSV-GFP antibodies.
FIGS. 13 and 14A-14B illustrate a profile of lipid raft protein Flotillin-1.
As required, detailed embodiments are disclosed herein; however, it is to be understood that the disclosed embodiments are merely examples and that the systems and methods described below can be embodied in various forms. Therefore, specific structural and functional details disclosed herein are not to be interpreted as limiting, but merely as a basis for the claims and as a representative basis for teaching one skilled in the art to variously employ the present subject matter in virtually any appropriately detailed structure and function. Further, the terms and phrases used herein are not intended to be limiting, but rather, to provide an understandable description of the concepts.
The terms “a” or “an”, as used herein, are defined as one or more than one. The term plurality, as used herein, is defined as two or more than two. The term another, as used herein, is defined as at least a second or more. The terms “including” and “having,” as used herein, are defined as comprising (i.e., open language). The term “coupled,” as used herein, is defined as “connected,” although not necessarily directly, and not necessarily mechanically.
Vesicular Stomatitis Virus (VSV) is relatively non-pathogenic to humans, and preferentially replicates in cancer cells, due to these cells' defects on interferon (IFN) and MAPK cellular innate immunity (RAS/Raf/ERK and MxA) signaling pathway. In accordance with the disclosure, normal cells tend to recover quickly from VSV infections, however, there are risks associated with uncontrolled infections and neurotoxicity.
In accordance with the disclosure, the mechanism by which VSV causes cytopathic effects is mainly by interacting with the Nuclear Pore Complex (NPC) mRNA export factors Rae1 and Nup98, blocking mRNA nuclear export, and by arresting cell division. The instant disclosure uncovers a class of drugs capable of controlling VSV replication/infection and simultaneously protecting healthy cells, thereby enhancing VSV as an oncolytic therapy. More particularly, statins, the extensively used cholesterol-lowering drugs, exert other actions, known as pleiotropic effects. Among them, statins are shown to (a) induce cytoprotection on normal cells; (b) inhibit the replication of viruses, such as HIV, HCV, polio and influenza; and (c) to increase the replication of oncolytic adenovirus.
At the cell surface, statins alter the composition of lipid rafts, which exert important functions in viral replication/budding. Intracellularly, statins modulate the expression of interferon, as well as downstream signaling events, such as MAPK (RAS/Raf/ERK) pathway. Here, we have investigated the actions of statins (simvastatin, lovastatin and pravastatin), as well as Methyl-β-cyclodextrin (MβCD, a cholesterol-sequestering drug) on VSV infection, lipid raft integrity, and protein levels of Rae1, Nup98, ERK1/2, and MxA.
Overview
Methods:
HeLa cells were exposed to different concentrations of statins or MβCD. Cells were infected with VSV-GFP. Protein expression was analyzed by immunoblots. Viral titers were obtained from culture supernatants. In situ hybridization of mRNA was performed and imaging analysis was done using confocal microscopy. Cell viability was determined by MTT assays.
Results:
Low concentration (0.125 μM) of simvastatin or MβCD treatment enhanced VSV replication. Higher doses of simvastatin (0.25-4 μM) caused a concentration-dependent antiviral effect. Simvastatin increased the protein levels of Rae1 and Nup98, and reversed the VSV-induced mRNA export block in a dose-dependent manner. Protein levels of lipid raft marker Flotillin-1, as well as levels of ERK1/2, and MxA were also altered by simvastatin treatment. In accordance with the disclosure, low concentrations are between 0.01 and less than 0.25 μM, and higher concentrations between about 0.5 and about 4.0 μM.
Conclusions:
Simvastatin appears to modulate VSV infection at the level of viral replication. Both lipid-lowering and pleiotropic cellular effects of simvastatin may be important for such modulation. Simvastatin appears to modulate the expression of Rae1 and Nup98, therefore altering cellular mRNA distribution and reverting VSV's mRNA export block. Furthermore, simvastatin caused redistribution of Flotillin-1, a lipid raft marker, which may be important for VSV replication/budding. It is disclosed that simvastatin may advantageously be used as a neoadjuvant for the selective modulatory control of live VSV oncolytic therapy.
Two of the major barriers of viral oncolysis are: (a) the innate immune system, which can rapidly destroy oncolytic viruses, and (b) the risk of uncontrollable systemic viral infections, which can cause neurotoxicity (2, 3). Overcoming there barriers requires the use of drugs capable of minimizing infectivity to normal cells and/or enhancing the viral oncolytic potency.
Viral infections in general, persistently threaten human health, and are difficult to control. A classical approach towards the development of antiviral drugs is the discovery of agents that directly bind/inhibit viral particles. These drugs tend to have small and specific ranges of drug action, and are further limited by the fact that many viruses mutate frequently, quickly falling out of interacting range. An approach disclosed herein to antiviral drug development, is to target strategic antiviral host factors, and not viral particles directly. When drugs are directed towards cellular components, they can be effective against several unrelated viruses, since those need common host cellular factors for proper replication and budding (4). This approach enables the development of broad range antiviral agents, which may be useful not only towards effective viral oncolysis, but also as mainstream antiviral therapy.
The disclosure details an investigation of the oncolytic properties of Vesicular Stomatitis Virus (VSV), as well as drugs capable of controlling VSV infection. VSV is oncolytic in nature, meaning that it preferentially kills cancer cells (5). Importantly, VSV's preferential replication in cancer cells is due to these cells' flaws in interferon (IFN) antiviral immune pathways (5-7). Such defects involve flawed induction of RAS/Raf/ERK signaling, which in turn upregulate antiviral MxA protein. It has been suggested that a cellular compensation for such flaws involves constitutive activation of RAS/Raf/ERK signaling pathway. The later promotes enhanced VSV replication, as well as the promotion of cell division and survival (8). Approximately 70% of all tumors, and in particular types that are persistently resistant to conventional therapy, have constitutive upregulation of RAS/Raf/ERK pathway proteins. Moreover, it is known that this same defective pathway favors robust replication of VSV in particular, therefore allowing for increased viral oncolysis (8).
VSVis advantageously used in clinical trials as a cancer-selective, replication-competent therapeutic vector (9, 10). It exclusively replicates in the cytoplasm, and not in the nucleus of infected cells, therefore rendering it advantageous over other oncolytic viruses. VSV's genetic material does not become integrated into the host's chromosomes, avoiding controversial risks of oncogenic nature. VSV is relatively non-pathogenic to humans; nevertheless, it is capable of finding and destroying localized inaccessible tumors, such as brain cancers, as well as dispersed metastatic cells, considered difficult to detect/treat with available therapeutic means.
With reference to
VSV induces a variety of cytopathic effects in the cells it infects, including morphological changes, cell rounding and apoptosis (11, 12). Most of VSV's cytopathic effects are caused by M protein, which is responsible for blocking mRNA nuclear export, inhibiting host protein synthesis, which favors VSV's own cytoplasmic replication and viability (12, 13).
Viruses in general need to use the host cellular machinery in order to replicate, therefore they must take-over primary cell functions. The most striking characteristic of eukaryotic cells is the presence of nuclei that compartmentalize the processes of transcription and translation. With reference to
In accordance with the disclosure, VSV takes over cellular function as follows: VSV M protein causes the blockage of mRNA export by interacting with the mRNA Export factor 1 (Rae1, also known as mrnp41) (13). Rae1 is a nucleoporin (Nup) responsible for the export of hnRNPs through the central channel of the NPC via interactions with Nup98 (16) 212. Upregulation of Rae1, or of Nup98, can revert the VSV mRNA export block, and therefore restore normal mRNA export, which leads to a fast cellular innate immune response and recovery from infection (13, 17). As is the case for other oncolytic viruses, the use of VSV as a mainstream systemic oncolytic agent presents the risks of unwanted infections of healthy cells. This disclosure presents a class of drugs which protect healthy cells, and allow control of VSV infection/replication on cancerous cells.
The viruses Adenovirus, measles, dengue, CMV, HIV and RSV replicate in the nucleus, and exit their genomes into the cytoplasm via the CRM1/Nup98 pathway, which is used by the host cell mainly as means for protein export. VSV 100 replicates in the cytoplasm and directly blocks Rae1/Nup98 cellular mRNA export (11-13, 17), although the disclosure is not bound to a particular modality of operation. Most viruses known to take over NPC transport machinery target, either directly or indirectly, components of Nup98 transport. Nup98 212 is essential for NPC 200 function, and is important for transport of proteins as well as for export of mRNAs (27). Nup98 plays roles in several important biological events such as gene expression, mitotic control and pathogenesis (reviewed in Iwamoto, 2010). Nup98 chromosomal rearrangements have been implicated in many forms of cancer (28). Furthermore, Nup98 is emerging as a new subtype of protein that utilizes autoprocessing to control biogenesis pathways and intracellular translocation. Autoprocessing is a rare biological event, important for protein/antigen processing, G protein-coupled receptor in cell signaling, hypercholesterolemia, and maturation of viral proteins (29).
The class of drugs known as statins, or 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors have an ability to effectively lower circulating levels of LDL-cholesterol (LDL-C) (30). Statins affect many metabolic pathways and organ systems, and they may impact several disease states, independently of LDL-C reduction. Such protective non-lipid-lowering effects of statins are referred to as pleiotropic effects. One advantageous pleiotropic effect of statins is disclosed to be an interference with the infectivity and proliferation of viruses. Examples of viruses that are modulated by statins include the human immunodeficiency virus (HIV) (31, 32), influenza (33), poliovirus (34), hepatitis C virus (HCV) (35, 36), hepatitis B virus (HBV) (37), cytomegalovirus (CMV) (38), respiratory syncytial virus (RSV) (39), Dengue (40), measles (41), and adenovirus (42). Statins possess antiviral actions against unrelated viruses, possibly via shared cellular antiviral pathways. Most of the viruses affected by statins may also take-over NPC functions.
Antiviral mechanisms for statins may include: (a) disruption of viral entry due to cholesterol depletion of lipid rafts (34); (b) formation of intracellular vesicles that do not support viral replication (34); (c) inhibition of cellular protein prenylation by lipophilic statins, leading to reduction of viral RNA as well as viral protein synthesis and suppression of virion release (34, 43); and (d) targeting of Rho GTPases, which in turn alter actin cytoskeleton re-arrangements needed for viral entry and budding (31, 32, 44).
The disclosure clarifies that statins can interfere with VSV infection, and can modulate the expression of key nucleoporins, such as Rae1 and Nup98.
Other underlying cellular factors involved in the delicate balance between infected host vs. viral control of cellular functions, are the proteins of antiviral interferon response RAS/Raf/ERK pathway and the human Myxovirus Resistance 1 (MxA). MxA is a powerful antiviral agent against VSV. It is a member of the large dynamin-like Mx protein family, and a large guanosine-5′-triphosphatase (GTPase). MxA can shuttle between the nucleus and the cytoplasm, where it interacts with lipids and forms vesicle-like structures. When overexpressed, it accumulates in distinguishable speck-like nuclear structures known as Nuclear Bodies. Mx proteins are also involved in fundamental cellular processes, such as membrane-associated intracellular vesicle transport (45).
The disclosure advantageously takes an entire-cell approach, and focuses on features that are important for viral infection, replication and cellular defense. A factor needed for viral replication/budding is the cellular membrane, and in particular the integrity of membrane microdomains, or lipid rafts. VSV depends on lipid raft-like structures that interact with viral M protein and promote envelopment of viral particles. Statins also disrupt lipid rafts, therefore, the disclosure exploits the effects of statins and VSV infection on a lipid raft protein marker, Flotillin-1. This marker aggregates on microdomains, and when overexpressed, forms vesicle-like structures in the cytoplasm, which most likely will coincide with sites of high viral activity (46).
The disclosure identifies common features of oncolytic viral infection, replication and cellular defense, and advantageously reveals insights into the mechanisms of statins. The effect of statins upon VSV replication, viral protein expression, and viable progeny was tested, including statins' effects on: (a) levels of nucleoporins Rae1 and Nup98; (b) cellular distribution of mRNA; (c) distribution and levels of lipid raft marker Flotillin-1; (d) levels of signaling proteins ERK1/2 and antiviral MxA. In accordance with the disclosure, it appears that simvastatin exerts effects via the following mechanisms: (1) by modulating the expression and function of key antiviral components of NPC transport, Rae1 and Nup98, simvastatin may counteract the cytopathic effects of VSV; (2) by directly lowering cholesterol on the membrane, and disrupting microdomains, simvastatin may render the cellular membrane more permeable to VSV infection, which in turn, facilitates viral replication and/or budding; and (3) by modulating cellular innate immune proteins, ERK1/2 and MxA, simvastatin may shift the balance of virus vs. host antiviral processes, possibly enabling fast elimination of VSV-infected cells (i.e. increased levels of apoptosis).
Materials and Methods
Cell Culture
HeLa (human cervical adenocarcinoma) cells and BHK-21 cells (Baby Hamster Kidney) cells were obtained from the American Tissue Culture Collection (ATCC Cat. N. CCL-2 and C-13). Cells are grown in DMEM (Cellgro, Fisher Scientific, PA) containing 10% fetal bovine serum (FBS, Fisher Scientific HyClone, PA), supplemented with 1% antibiotic/antimycotic solution (FBS, Fisher Scientific HyClone, PA) in atmosphere of 5% CO2, at 37° C.
Cholesterol Inhibition and Infections
For cholesterol synthesis inhibition studies, HeLa cells were treated with either simvastatin or pravastatin (EMD/Merck, NJ), for the indicated concentrations and time points. HeLa infections with VSV-GFP virus (gift from Dr. Glen Barber, University of Miami Sylvester Cancer Center), were performed as previously described (13). Briefly, HeLa cells were plated in either 100 mm2 plates, or on glass cover slips placed on 6-well plates. At 100% confluency, cells were washed with PBS, and infected with VSV-GFP in low volume DMEM without FBS or antibiotics. Infection proceeded for 1 hr with rocking every 10 minutes. Consequently, the infecting media was removed, and complete media was added. Cells were further incubated for 16 hrs (O/N).
For Cholesterol-sequestering from cellular membranes, we used Methyl-β-Cyclodextrin (MβCD). MβCD, like lipophilic statins, lowered levels of membrane cholesterol and also disrupts microdomains called lipid rafts. HeLa cells were treated with indicated concentrations of MβCD (Sigma-Aldrich, MO) for 45 min. prior to VSV-GFP infection as described above.
Immunoblots
Cells were lysed with the following buffer: 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 15 mM MgCl2, 0.5% NP40, 0.1 mM Na3V04, 0.1 M NaF, and protease inhibitors cocktail (Sigma-Aldrich, MO). Lysates were kept on ice for 20 min, sonicated, and then centrifuged at 14,000×g for 30 min. Protein concentration was determined by the Lowry Protein Assay Method. Reducing agent and loading buffer with bromophenol blue were added to the samples, and immunoblot analysis was performed. Proteins were separated by 12% SDS-P AGE, and transferred into PVDF membranes (GE healthcare, NJ). Non-specific sites were blocked with 5% milk in PBS/0.05% NP-40 (Sigma-Aldrich, MO). The membranes were incubated with primary antibodies: mouse polyclonal anti-VSV-GFP (gift from Dr. G. Barber, University of Miami Sylvester Cancer Center, FL); rabbit polyclonal anti-Nup98 (gift from Dr. B. Fontoura, University of Texas, Dallas, Tex.); goat polyclonal anti-Rae1 (Abcam, MA); MxA (Santa Cruz Biotechnology, CA); ERK1/2 (Cell Signaling Technology, MA). The membranes were washed with PBS/0.05% NP-40 and incubated with a secondary antibody conjugated to HRP (American Qualex, CA). The VSV protein bands were detected by chemiluminescence using West-Phemto Supersignal Substrate (Thermo-Fisher Scientific, PA). Quantitation of bands was performed using Image-J software program (NIH).
Viral Titer Assay
HeLa cells were treated as described above. After 16 hrs of VSV-GFP infection, a sample of the supernatant was collected and frozen at −80° C. BHK-21 cells were plated into six-well plates and cultured to 80% confluence. Culture medium was removed from each well and replaced with 300 nl of serially diluted viral samples. The inoculated monolayers were left at room temperature for 1 h to allow virus adsorption. The plates were rocked gently every 10 minutes. At the end of the adsorption period, the inoculum was removed from each BHK monolayer and then replaced with 1.5 ml of DMEM 1% Low-Melting-Point agarose (LMPA) (Sigma-Aldrich, MO) in order to immobilize viral plaques. The plates were left at room temperature for an additional 10 minutes in order to solidify the plaque isolation media and then returned to the incubator for 24 h. The LMPA was melted by microwave (10 sec, maximum power) and removed. The BHK-21 monolayers were fixed and stained by the addition of 1 ml of Gentian violet diluted in methanol (0.01% (w/v), Sigma-Aldrich, MO). Particles of Infectitious Units (pfu) were calculated per ml. of supernatant (pfu/ml) of duplicate samples. Data were analyzed using One-way ANOVA (GraphPad Prism5). Statistical significance was determined by p values less than 0.05.
Cell Viability MTT Assays
Cells were seeded at a density of 1×10^4 in 96-well plates. Each of 4 experiments were performed using eight wells per treatment condition. Cells were pretreated with the respective concentrations of cholesterol-lowering drugs, followed by O/N infection with VSV-GFP, as described in the immunoblot protocol. After infection, cells were treated with 5 μg/ml MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Sigma-Aldrich, MO) and incubated for 4 hrs at 37° C., 5% CO2. MTT is a yellow tetrazole that is reduced to purple formazan by enzymes in living cells. After incubation, the media was aspirated, and the converted dye was solubilized using 200 μl Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, MO) per well. Endpoint absorbance was measured with a plate-reader at wavelength of 570 nm.
Oligo-dT In Situ Hybridization and Confocal Microscopy
Cells were grown on glass coverslips, pretreated with different concentrations of simvastatin for 8 hrs, and then infected with VSV-GFP at MOI of 1.0. After 16 hrs of VSV-GFP infection, in situ hybridization of poly (A) RNA using a biotinylated oligo-dT 45mer was performed, as previously described (13). Briefly, cells were fixed with 4% formaldehyde for 8 min, extensively washed with PBS (3 washes, 15 min each), permeabilized with 0.5% Triton X-100 (Sigma-Aldrich, MO) for 5 min, washed again in PBS (2 washes, 15 min each), then equilibrated in pre-hybridization mix (without oligo-dT) for 20 min at 42° C. in a humidifying chamber placed in a hybridization oven followed by overnight hybridization with biotinylated oligo-dT probes. Hybridization mix was composed of 2×SSC with 1 mg/ml tRNA, 10% dextran sulfate, 25% formamide, and 50 μg/mlbiotinilated-oligo-d(T). After hybridization, samples were washed twice for 15 min with 2×SSC at 42° C. and once for 15 min with 0.5×SSC at 42° C. Cells were fixed with formaldehyde, and washed in PBS as described above. Cells were then incubated with CY5-streptavidin (Jackson Immunoresearch, PA) for 30 min at room temperature. Cells were washed 3× with PBS and stained with Hoescht 33258 (Invitrogen, CA) for 5 min in a humidifying chamber at room temperature. Cells were washed 3× with PBS, then mounted on glass slides using the ProLongGold Antifade reagent (Invitrogen, CA). Samples were examined on a Zeiss LSM510 confocal microscope. Fields of cells representing generalized effects were depicted.
Results
Effects of Simvastatin and Pravastatin on VSV Protein Expression
The effects of statins on VSV infection were studied in HeLa cells exposed to the virus. Cells were pretreated with simvastatin, pravastatin or the statin-vehicle (DMSO) for different times and several drug concentrations. At the end of the statin or statin-vehicle pretreatment, HeLa cells were infected with VSV-GFP for 16 additional hours. Subsequently, the cells were harvested and the supernatants and cell lysates analyzed.
VSV proteins were detected by immunoblot employing anti-VSV-GFP antibodies. Supernatants and cell lysates from VSV-infected cells showed three major bands compatible with G, N/P, and M viral proteins (
Time of exposure of HeLa cells to simvastatin prior to the VSV infection (pretreatment time) was important in determining the magnitude of the drug effects (
Pretreatment (2-24 hours) with pravastatin, a non-lipophilic statin with poor cell penetration, failed to alter VSV protein expression at any of the concentrations tested (0.1 to 2.0 μM) (data not shown).
Effects of Methyl-β-Cyclodextrin on VSV Protein Expression
MβCD sequesters cholesterol from the membrane by forming water-soluble complexes. It is also efficient in disrupting membrane microdomains known as lipid rafts. HeLa cells were pretreated with increasing concentrations of MβCD (from 0.125 mM to 5.0 mM) for 45 minutes prior to O/N infection with VSV-GFP at MOI of 1.0.
but M, again was not altered to a great extent (103%). 5.0 mM MβCD increased levels of: G to 182%; N/P to 275%; and M was increased to 125% (
Without being bound to a particular theory, these results suggest that the cholesterol-lowering property of simvastatin may be connected to the stimulation of VSV replication, and not to antiviral effects.
Effects of VSV Infection on Cellular Distribution of Lipid Raft Protein Flotilin-1.
Flotillin-1 is an integral protein that localizes on lipid rafts. When overexpressed, it generates de novo membranes microdomains with lipid raft properties, and induces the formation of dynamic membrane invaginations as well as intracellular vesicles (46). Since simvastatin disrupts lipid rafts, which are important for viral replication, and simultaneously causes vesicle-like formations (
Indeed, FIGS. 13 and 14A-14B show that VSV infection re-distributed endogenous Flotillin-1 from cytoplasmic/membranous sites (control) to vesicle-like structures near the membrane (VSV-GFP). These vesicle-like structures co-localized to sites of intense viral replication (
Effects of Simvastatin and Pravastatin on Viral Titers.
The disclosure details an investigation of whether the effects of simvastatin and MβCD on VSV protein expression correlated with differences in the production of viral titers in supernatants of HeLa cells. In order to measure attenuated production of VSV infectious particles (pfu), viral titer assays were performed of supernatants obtained from either simvastatin, pravastatin, or MβCD pretreated cells that had been infected with VSV-GFP at MOI of 1.0.
The average titer value for VSV control infections was of 8.75×106 (+/−1.05×106) pfu/ml. Concentrations of simvastatin pretreatment ranging from 0.25 μM to 4.0 μM efficiently decreased VSV titers in a dose-dependent response. The simvastatin concentration needed for reduction of pfu values to half of control (IC50) was estimated to 0.278 μM. Pfu values obtained using 4 μM simvastatin pretreatment [1.80×104 (+/−3×103) pfu/ml] were 660 fold lower than control pfu counts obtained from non-treated, control VSV-infected cells (
Viability of HeLa Cells after Statins or MβCD Treatment and Viral Infection.
The disclosure further details an investigation of whether the antiviral effects were true drug effects on viral replication, or were artifacts due to cell toxicity and decreased viability. Overall, the experimental conditions were designed to maximize drug effects on the virus, and minimize cell killing. Cells were reasonably tolerant of the combined treatment of simvastatin, as well as pravastatin, and viral infection.
Treatment of cells with pravastatin also caused decreased cell survival as a function of drug concentration, with cell death rates of −1.78% cells/μM Prava (
Lovastatin, as well as MβCD were highly toxic to HeLa cells, showing cell survival at ˜20% for 5 mM MβCD and ˜30% for 3 μM lovastatin (
Pre-Treatment of Simvastatin Reverts VSV-Mediated Inhibition of mRNA Export.
In accordance with the disclosure, VSV causes blockage of mRNA nuclear export. In order to visualize the mRNA distribution in situ, a hybridization of mRNAs poly-A tails using oligo-dT 40-mer probes was performed.
In order to observe the effects of simvastatin on mRNA distribution, non-infected cells (mock infections) were treated with either 0.125 μM or 2.0 μM of the drug. Simvastatin treated cells showed a more pronounced mRNA cytoplasmic distribution than mock untreated cells (
Compared to control VSV infections, Hela pretreatment of 0.125 μM simvastatin partially reversed the viral mRNA export block (
Pretreatment with 2 μM simvastatin induced recovery from viral mRNA export block, which was indicated by the normal distribution of mRNA between the nucleus and the cytoplasm of infected cells (
These results coincided with immunoblot data shown in
Simvastatin Upregulates NPC Antiviral Proteins Rae1 and Nup98.
It has been shown that Rae1 is the binding target of VSV M protein (13). Upregulation of Nup98 and/or Rae1 either by interferon treatment or by ectopic expression reverts the mRNA export block mediated by VSV M protein (13, 17). This effect indicates that both Rae1 and Nup98/Nup96 genes are involved in antiviral response. These findings are further supported by the fact that both Rae1 and Nup98 are upregulated by interferon (13, 17). Low concentrations of simvastatin (0.01-1 μM) are known to upregulate interferon-γ (47), and since simvastatin pretreatment presented a distinguished anti-VSV effect, the disclosure tests whether simvastatin could alter the expression levels of Rae1 and/or Nup98. As shown in
Simvastatin Modulates the Expression of Key Proteins Involved in VSV Replication: MxA and ERK1/2.
It is known that in approximately 30% of cancers, RAS/Raf/ERK pathway is upregulated (48). Importantly, these cancers do not respond to conventional therapeutics (49). Cancer cells that have RAS/Raf/ERK pathway upregulated, do not induce MxA protein, which is a powerful antiviral agent, and therefore favor the replication of viruses, such as VSV. Here we have shown that, in accordance with studies by He et al. (50), simvastatin exerted a biphasic action upon ERK protein regulation: at low simvastatin concentrations, ERK protein was upregulated. At higher concentrations, ERK levels fell. Further investigating the levels of MxA, protein levels were increased to ˜400%, 650%, and 150% when pretreated with simvastatin at 0.125, 0.5, and 2.0 μM, respectively (
Discussion
The disclosure describes four exemplary novel pleiotropic properties of simvastatin:
(1) Simvastatin caused a biphasic, concentration-dependent effect towards modulation of VSV infection and replication. At low concentration (0.125 μM), simvastatin promoted high levels of viral protein expression and replication, which may be due to cholesterol depletion, since MβCD produced similar results. At subsequent higher concentrations (from 0.25 to 2.0 μM), simvastatin inhibited VSV proteins expression and viral replication. This antiviral activity may have been mediated by effects of simvastatin on host cell gene expression levels. Supporting this deduction is the fact that cells needed to be incubated with simvastatin for at least 4 hrs prior to VSV infection in order for changes in viral content to be observed.
(2) Simvastatin modulated endogenous mRNA distribution by upregulating mRNAexport factors Rae1 and Nup98, which also possess antiviral properties.
(3) Simvastatin upregulated antiviral protein MxA via modulation of IFN-inducible RAS/Raf/ERK pathway.
(4) Simvastatin redistributed lipid raft marker protein flotillin-1, which co-localized with high replication sites of VSV.
The results disclosed for the biphasic effects of simvastatin differed from experiments by Delang et al, who had tested the pleiotropic properties of simvastatin against several viruses, including VSV. Delang's group had simultaneously conducted simvastatin treatment and viral infections on cells, therefore not observing the drug's effects towards the tested viruses (35). Another observation that supports simvastatin's pretreatment time requirement is the fact that VSV blocks host gene expression within 2 hrs of infection by inhibiting Rae1/Nup98 cellular mRNA export. Therefore, if cells are simultaneously treated with statins and infected, one should expect not to observe innate antiviral effects in vitro, since cellular responses via transcriptional/translational upregulation is blocked by the viral matrix protein.
The disclosed data suggest that simvastatin, at low concentration, favors the VSV take-over of cellular translational mechanisms, and simultaneously enhance the budding ability of newly formed progeny. These results are most likely derived from an immediate decrease in intracellular as well as membranous cholesterol levels, as supported by experiments using MβCD. Significantly, expression levels of N and P proteins are known to control viral phase-switch from transcription/translation to genome replication. The increases in N/P protein levels observed with 0.125 mM simvastatin and with MβCD treatment, indicated that VSV was able to replicate more efficiently under those conditions. Thus, it is feasible to infer that cholesterol depletion favors the initial phase of VSV's life cycle, causing a delay of phase-switch from transcription/translation to genome replication. This delay allows for a greater degree of VSV protein expression, as observed.
A non-lipophilic statin drug, pravastatin had previously been shown not to possess antiviral activity against several viruses, including Hepatitis C(HCV) (51), Human Immunodeficiency Virus (HIV) (52), and oncolytic Adenovirus (AdV) (42, 51, 52).
It is becoming increasingly aparent that most viruses favor their own replication by hijacking basic nuclear membrane functions. Since host gene expression is compartmentalized, and all mRNA need to be exported to the cytoplasm in order to be expressed into protein, the gateways to the nucleus (NPCs) become vulnerable targets to viral attack. Recal that the antiviral Nup Rae1 is the target of VSV M protein, and a major factor that mediates mRNA export from the nucleus via Nup98. In the instant disclosure, for the first time, it is shown that statins interfere with the fundamental cellular function of mRNA export by upregulating nucleoporins Rae1 and Nup98, which are in turn, capable of exerting antiviral activity.
The ultimate goal of anti-viral therapy is to completely restore all functions to the infected cells. This goal is currently unattainable, since irreversible cellular damage occurs very early in most viral infections. A more realistic goal for anti-viral therapy, and for the prevention of healthy cells, is to inhibit virus replication and/or the spread of viruses to healthy cells. In order to achieve this level of protection, it is important to take in consideration the cellular first line of defense against viral infections: interferon. IFN signaling promotes the activation of innate immune response by synthesizing antiviral proteins, such as nucleoporins Nup98 and Rae1, and proteins of RAS/Raf/ERK signaling pathway, ERK1/2 and MxA.
As mentioned, VSV preferentially replicates in the cytoplasm of cancer cells due to these cells' flaws in IFN innate immunity/antiviral response. The mechanism responsible for the cancer-cell enhanced replication involves constitutive activation of the RAS/Raf/ERK pathway (8). Previously, a study on statins pleiotropic effects have also shown a biphasic dose-related action of the drug on RAS/Raf/ERK signaling pathway (53). Medina et al demonstrated that low concentrations of simvastatin (0.01-0.1 μM) significantly promoted cell proliferation via upregulation of RAS/Raf/ERK pathway, while higher concentrations (>1.0 μM) halted proliferation and induced cell death via downregulation of the same pathway. In accordance with the disclosure, observations of simvastatin biphasic response, and the knowledge that modulation of antiviral MxA expression is also dependent on the RAS/Raf/ERK pathway, the profiles of both ERK1/2 and MxA proteins after simvastatin treatment are studied. The disclosure shows that low concentration of simvastatin pretreatment correlate to an increase in the levels of VSV detected. Furthermore, cancer cells that present aberrant cell division due to RAS/Raf/ERK constitutive upregulation, can be synergistically suppressed by strategic drug combinations of specific inhibitors of the RAS/Raf/ERK pathway, as for example the experimental drug PLX4032 (54), with simvastatin pretreatment followed by oncolytic VSV infection.
Another mechanism of simvastatin anti-VSV action might be related to disruptions of membrane microdomains. We have shown that when cells are infected with VSV, endogenous lipid raft protein Flotillin-1 is re-distributed from a uniform distribution to concentrated vesicle-like structures near the cellular membrane. These vesicle-like structures co-localized with sites of high VSV replication. The observed shift in flotillin-1 distribution pattern, with subsequent accumulation of this lipid raft protein on cell lysates, indicates that simvastatin caused cytoplasmic trapping and a dysfunction of viral budding.
The disclosure applies principles of viral-host interactions in order to investigate the effects of simvastatin on overall VSV replication and on possible antiviral cytoprotection. A multi-dimensional approach has been applied in order to uncover converging cellular factors that are affected by statin treatment. In accordance with the disclosure, it may be advantageous to use simvastatin as a non-specific, or broad-range antiviral agent capable of regulating host cell antiviral proteins, rather than directly interacting with viral components. Furthermore, since simvastatin can potentially either enhance or eliminate VSV infection in a dose-dependent manner, studies in accordance with the disclosure suggest that the drug may advantageously be used as a priming therapeutic neoadjuvant for oncolysis via replication-competent VSV. The disclosure provides insight into novel strategies for the use of statin drugs as both broad-range antiviral agents and oncolytic adjuvants, thereby broadening perspectives towards novel antiviral and anticancer therapies.
It will be appreciated by persons skilled in the art that the present invention is not limited to what has been particularly shown and described herein above. In addition, unless mention was made above to the contrary, it should be noted that all of the accompanying drawings are not to scale. A variety of modifications and variations are possible in light of the above teachings without departing from the scope and spirit of the invention.
All references cited herein are expressly incorporated by reference in their entirety. In addition, unless mention was made above to the contrary, it should be noted that all of the accompanying drawings are not to scale. There are many different features to the present invention and it is contemplated that these features may be used together or separately. Thus, the invention should not be limited to any particular combination of features or to a particular application of the invention. Further, it should be understood that variations and modifications within the spirit and scope of the invention might occur to those skilled in the art to which the invention pertains. Accordingly, all expedient modifications readily attainable by one versed in the art from the disclosure set forth herein that are within the scope and spirit of the present invention are to be included as further embodiments of the present invention.
This application claims the benefit of related U.S. Provisional Patent Application No. 61/416,080, filed Nov. 22, 2010, the contents of which are incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US2011/061920 | 11/22/2011 | WO | 00 | 7/29/2013 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2012/071453 | 5/31/2012 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 20090081161 | Roberts et al. | Mar 2009 | A1 |
| 20100172877 | van den Pol | Jul 2010 | A1 |
| 20100303839 | Bose | Dec 2010 | A1 |
| 20110044952 | Bell | Feb 2011 | A1 |
| 20130302284 | Faria-Waziry | Nov 2013 | A1 |
| Number | Date | Country |
|---|---|---|
| 2818701 | Nov 2011 | CA |
| 1932518 | Jun 2008 | EP |
| 11843911.6 | Nov 2011 | EP |
| 2009067808 | Jun 2009 | WO |
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| Number | Date | Country | |
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| 20130302284 A1 | Nov 2013 | US |
| Number | Date | Country | |
|---|---|---|---|
| 61252303 | Nov 2010 | US |
