The Cytotoxic Action of Cytochrome C/Cardiolipin Nanocomplex (Cyt-CL) on Cancer Cells in Culture
The effect of existing anti-cancer therapies is based mainly on the stimulation of apoptosis in cancer cells. Here, we have demonstrated the ability of a catalytically-reactive nanoparticle-based complex of cytochrome c with cardiolipin (Cyt-CL) to induce the apoptosis and killing of cancer cells in a monolayer cell culture.
Cyt-CL nanoparticles were prepared by complexing CytC with different molar excesses of CL. Following characterization, cytotoxicity and apoptosis inducing effects of nanoparticles were investigated. In an attempt to identify the anticancer activity mechanism of Cyt-CL, pseudo-lipoxygenase and lipoperoxidase reaction kinetics were measured by chemiluminescence.
Using chemiluminescence, we have demonstrated that the Cyt-CL complex produces lipoperoxide radicals in two reactions: by decomposition of lipid hydroperoxides, and by lipid peroxidation under the action of H2O2. Antioxidants inhibited the formation of lipid radicals. Cyt-CL nanoparticles, but not the CytC alone, dramatically enhanced the level of apoptosis and cell death in two cell lines: drug-sensitive (A2780) and doxorubicin-resistant (A2780-Adr). The proposed mechanism of the cytotoxic action of Cyt-CL involves either penetration through the cytoplasm and outer mitochondrial membrane and catalysis of lipid peroxidation reactions at the inner mitochondrial membrane, or/and activation of lipid peroxidation within the cytoplasmic membrane.
Here we propose a new type of anticancer nano-formulation, with an action based on the catalytic action of Cyt-CL nanoparticles on the cell membrane and and/or mitochondrial membranes that results in lipid peroxidation reactions, which give rise to activation of apoptosis in cancer cells, including multidrug resistant cells.
Key Wordsapoptosis cytotoxicity cytochrome c-cardiolipin complex lipid peroxidation lipid peroxyl radicals
Bovine heart cardiolipin
Complex of cytochrome с with cardiolipin
- IMM and OMM
Outer and inner mitochondrial membranes
10 mM NaH2PO4-Na2HPO4 (pH = 7.4)
Cell death mechanism called apoptosis (1) is the major way to maintain the homeostasis in the vertebrates (2). Decoding the principles of apoptosis revealed a tightly controlled genetic process that usually proceeds via the intrinsic, so-called mitochondrial pathway (3). Upon the activation of the mitochondrial apoptosis pathway by cell stress and DNA damage-inducing reagents and/or conditions, several key proteins are released from the mitochondria into the cytosol including cytochrome c (CytC) and the apoptosis-promoting factor Apaf-1. The release of CytC to the cytosol is controlled by the outer mitochondrial membrane permeabilization. Paradoxically, the permeability of the outer mitochondrial membrane is caused by events in the inner mitochondrial membrane, primarily the complex formation of CytC with specific mitochondrial highly anionic phospholipid, cardiolipin (CL), and lipid peroxidation in the inner membrane catalyzed by the complex. In the inner mitochondrial membrane, CL accounts for 25% of all phospholipids it consists of (4, 5, 6). When CytC assumes its electron shuttle role, the functional positions in its heme iron are occupied. However, when CytC is bound to CL, its confirmation changes and it becomes partially unfolded (7). This change allows an opening for hydrogen peroxide to bind with the heme and imparts CytC a peroxidase activity (8). This change in activity is followed by CL oxidation, mitochondrial inner membrane permeability transition, outer membrane permeabilization, and CytC release into the cytosol (9).
One of the major characteristics of the cancer cells is their ability to evade or down-regulate the apoptosis. The oncogenic mutations that disrupt apoptosis cause tumor initiation, progression, and metastasis. That is why many agents used for cancer chemotherapy kill tumor cells in vitro and in vivo through launching/restoring the mechanisms of apoptosis (10). Anticancer agents directed at this target are listed in the review (11), where they are classified as those acting on Apoptotic Proteins in the Extrinsic, Intrinsic, and Common Pathways (see also (12)). In their turn, cancer cells develop resistance to drugs, based on the elimination of the drugs from the cell or overexpression of cellular antiapoptotic proteins. The majority of these are the mitochondrial membrane proteins called B-cell/lymphoma 2 (Bcl-2) family, which prevent permeabilization of outer mitochondrial membrane, and thus the release of CytC (13). Bcl-2 and Bcl-xL are the most commonly overexpressed proteins in cancer cells that act as anti-apoptotic factors and prevent CytC release into the cytoplasm (14). The overexpression of Bcl-2 is also associated with multidrug resistance (MDR) phenotype of the cancer cells, which results in the decreased sensitivity to chemically unrelated anticancer agents and diminishes the clinical success of chemotherapy.
Thus, one can assume that the intracellular delivery of CytC from outside of the cell could initiate the apoptosis in the cancer cells by bypassing the factors that prevent the “own” CytC release from the cell mitochondria. However, most proteins, including CytC, are cellular membrane impermeable. To overcome this obstacle, several different nano-sized CytC delivery systems including mesoporous silica nanoparticles (15), polymer-based nanoparticles (16) or lipoprotein-based nanoparticles (17) have been recently studied. Most of the mentioned systems have used CytC carriers formulated with synthetic compounds, in other words, have tried to mimic a naturally occurring event using non-naturally occurring compounds.
In our study, in the light of the current information, we have developed a biomaterial-based nanoparticle formulation containing CytC and a natural lipid (cardiolipin). Due to the great affinity of CytC for cardiolipins compared to other lipids, such as phosphatidylcholine or phosphatidylserine, we have used tetraoleoyl cardiolipin (TOCL), which is not susceptible to lipid peroxidation, inevitable in the system of “CytC + oxidizable lipid”. Cyt-CL complexes that contain TOCL have been studied extensively by different methods such as NMR, mass spectrometry and X-ray structural analysis (see review (18)). According to the small-angle X-Ray scattering data, the Cyt-CL complexes represent closely packed nanospheres of 11.1 ± 1 nm in diameter (19). Along with the spectrophotometric titration (19) and protein fluorescence measurements, the data show that each nanosphere represents a melted globe of CytC with the diameter of about 5.5 nm (18), surrounded by cardiolipin monolayer, with hydrophobic tails turned to outside. Such hydrophobic nanoparticles may penetrate lipid bilayer (20, 21, 22), (see also discussion in (23)) and hence – cell and mitochondrial membranes.
It should be stressed that Cyt-CL nanosphere is not merely the CytC carrier across the cell membrane, but mostly important the enzymatic complex performing lipoperoxidase action inside the cell. The complex of CytC with anionic lipids, including CL in the inner and outer membrane of mitochondria (9,24, 25, 26, 27, 28, 29) and phosphatidylserine in the cytoplasmic membrane (30, 31, 32, 33), catalyzes the lipid peroxidation that results in permeability transition in mitochondria, the efflux of CytC, and apoptosis initiation on one hand, and provokes phosphatidyl serine externalization on the other hand to send out ‘eat-me signal’ for phagocytes.
Based on all the above considerations, we have investigated the cytotoxic and pro-apoptotic action of the Cyt-CL complex in cancer cells, both drug-sensitive and -resistant, and studied the mechanism of reactions of free radical formation by the Cyt-CL complex, since these reactions could mediate the cytotoxic and pro-apoptotic effects observed. Our results allow us to propose a new type of anticancer agent, the action of which is based on introducing Cyt-CL into the cancer cell, catalysis of lipid peroxidation reactions in mitochondrial and cytoplasmic membranes, and activation of apoptosis in cancer cells, including multidrug resistant cells.
1,1′,2,2′-Tetraoleoyl cardiolipin (TOCL, CAS 115404–77-8) in chloroform (10 mg/ml) was purchased from Avanti Polar Lipids (Alabaster, AL, USA). Cytochrome c from bovine heart (CytC, CAS 9007–43-6) was purchased from Sigma-Aldrich (St. Louise, MO, USA). The phosphate buffer pH 7.4 (10 mM NaH2PO4-Na2HPO4) was prepared in ultrapure water. Streptomycin (25 μg/ml)/Penicillin (10,000 U/ml) solution, RPMI-1640 media and Trypsin/EDTA were purchased from Corning/Mediatech (Manassas, VA). Fetal bovine serum (FBS) was from Atlanta Biologicals (Flowery Branch, GA). A2780 human ovarian carcinoma cell line and its doxorubicin-resistant derivative (A2780-Adr) were from ECACC and purchased from Sigma-Aldrich (USA). All media were supplemented with 10% (v/v) FBS and 1% (v/v) streptomycin/penicillin, unless otherwise indicated. Cells were all grown at 37°C with 5% CO2. CellTiter-Blue® cell viability assay was purchased from Promega Corporation (Fitchburg, WI). Alexa Fluor® 488 Annexin V/Dead Cell Apoptosis Kit with Alexa Fluor 488 Annexin V and PI was from Molecular Probes (Eugene, OR, USA).
Cyt-CL Complex Preparation
To prepare the nanospheres of CytC and CL (Cyt-CL), first CytC was dissolved in the phosphate buffer. Different molar excesses (25, 35, and 40) of TOCL over CytC were calculated and required volumes were taken from the TOCL chloroform stock solution into separate tubes. Chloroform was evaporated under nitrogen to form a thin film and the tubes were further freeze-dried to remove the trace solvent. Resulted TOCL film was dissolved in methanol. Concentrated TOCL solution was then added to the CytC solution dropwise while mixing. The methanol concentration was kept at not more than 5% (v/v). Then the dispersion was subjected to probe sonication on ice for 20 s at 10 W for 6 times to obtain a clear nanoparticle complex solution. The empty CL particles consisted of only TOCL were prepared in the same way but without CytC in the buffer.
Particle Size and Polydispersity Index Measurements
Following the preparation of the Cyt-CL nanoparticles, the particle sizes of the formulations were measured using Malvern Zetasizer ZS90 (Malvern Instruments, UK). The formulations were diluted in ultrapure water, and particle size distribution of all samples was measured in triplicate at 90-degree scattering angle. Same instrument was used to determine the zeta potential of the formulations.
Chemiluminescence Kinetic Measurements
The chemiluminescence kinetics was measured using Lum-100 luminometer (DiSoft, Russia). The operation of the device, the analysis of the results of photon counting, and plotting were performed using the computer program Power Graph (34). The method described in this paper, involves the sequential measurements of lipoxygenase activity and lipoperoxidase activity in one experiment.
The measurements began with the recording of background luminescence: 100 μl of 100 μM CytC in PBS, 25 μl of 1mМ coumarin C-334 in methanol and 775 μl of PBS were placed in the cuvette of luminometer, and the registration was started, which lasted for 25 s.
To start the record of lipoxygenase reaction kinetics, the luminometer lid was opened, 100 μl of 6 mM BCL methanol solution were rapidly injected into the cuvette, the lid was closed, and the recording of the kinetics of lipoxygenase reaction was immediately started, so that the non-registered interval between reagent mixing and measurement was as small as 5–7 s. In the absence of coumarin C-334, the chemiluminescence curve was about five times as low as with C-334, due to the sensitizing effect of the dye, typical of quinolizin (9a,9,l-gh)-substituted coumarins (35). Because the luminescence was oxygen-dependent (data not shown), but observed in the absence of H2O2, it could be accounted for quasi-lipoxygenase activity of CytC (36), in particular in complex with cardiolipin (9). After adding BCL methanol solution, the chemiluminescence intensity was measured for 3.5 min, and then the cuvette was removed from a chemiluminometer, covered with filter paper, and allowed to stand.
To record the kinetics of lipoperoxidase reaction, hydrogen peroxide was added to the mixture of reagents in the first cuvette. The lid of the luminometer was opened and second cuvette was placed there, which contained 100 μl of 1.5 mM H2O2 solution, then the luminometer lid was closed, and the record of the chemiluminescence was continued for 10 s. Then, the lid was opened, and 900 μL of the first cuvette content were added in the second cuvette, the lid closed, and the recording continued for 6 min. By adding a larger volume into a smaller, we provided a good stirring of the sample.
The influence of the hydrogen peroxide concentration on the lipoperoxidase function of Cyt-CL was studied by varying the concentration of hydrogen peroxide added before the registration of the lipoperoxidase reaction kinetics.
The effect of antioxidants on the lipoxygenase and lipoperoxidase functions of Cyt-CL was studied by adding 50 μL of an antioxidant solution of a certain concentration before the background luminescence registration started.
A2780 cells were cultured in RPMI-1640 complete medium in cell culture flasks and passaged two times a week for routine culture. A2780-Adr cells were cultured in RPMI-1640 complete medium containing 100 nM doxorubicin HCl. For cytotoxicity experiments, 3000 A2780 and A2780-Adr cells were seeded in each well of 96-well flat-bottom cell culture treated plates and allowed to grow 24 h prior to the treatments. The cells were treated with empty CL particles, Cyt-CL particles, or free CytC in phosphate buffer in serum-containing complete medium for 24 h and 48 h continuously. 5% (v/v) methanol-containing buffer was used as the control and cell viability was measured by the CellTiter-Blue® assay according to the manufacturer’s protocol. All treatments were carried out in triplicate.
Apoptosis Enhancement by Cyt-CL
The externalization of phosphatidylserine at the cell surface as an indicator of apoptosis was investigated by flow cytometry using annexin V and propidium iodide (PI). A2780 and A2780-Adr cells were seeded into the wells of 12-well plate at a density of 80,000 cells/well. The cells were allowed to attach and grow for 24 h and then treated with empty CL particles, Cyt-CL particles, free CytC in phosphate buffer, and buffer control for 48 h in serum-containing complete medium at the CytC concentration of 62.5 μg/ml. Following the treatments, the cells were harvested, washed with the PBS three times and resuspended in the annexin-binding buffer. The cells were analyzed by flow cytometer (FACSCalibur, Beckton Dickinson, Franklin Lakes, NJ) using Alexa Fluor® 488 annexin V/Dead Cell Apoptosis Kit according to the manufacturer’s protocol. The fluorescent signal from the cells was measured using 488 nm blue laser for excitation and FL1 (for Alexa Fluor® 488, 530/30 bandpass filter) and FL3 (for PI, 670 longpass filter) channels for recording. A total of 10,000 live cells were acquired after gating the population.
Cyt-CL Complex Preparation and Characterization
Summary of particle size, polydispersity index and zeta potential values of the complexes
Time after preparation (hours)
Mean particle size (nm)
Zeta Potential (mV)
t = 0
217.1 ± 77
−54.62 ± 2.16
225.1 ± 79
t = 0
248.6 ± 86
−64.77 ± 1.94
252.5 ± 90
t = 0
218.7 ± 73
−63.94 ± 1.64
218.0 ± 75
After probe sonication
93.8 ± 1.35
−61.11 ± 1.21
After probe sonication
154.3 ± 3.73
-58.66 ± 6.21
Cyt-CL Nanoparticles Demonstrate Efficient Cancer Cell Killing
Cyt-CL Nanoparticle Treatment Enhances the Apoptosis
To investigate the factors that are resulted in the effective cell killing of sensitive and resistant ovarian cancer cells, we have analyzed the apoptosis enhancement following the formulation treatment. The results are given in Fig. 4. Annexin V was used as the early apoptosis marker of the cells that preserve the membrane integrity. The translocation of the phosphatidylserine from the inner cell membrane to the outer surface allows annexin V to recognize the apoptotic cells and stain them. PI was used as the second dye to distinguish the cells with damaged membranes, i.e. necrotic and dead cells. It should be noted that using this double stain method does not allow to distinguish the necrotic cells from the late apoptotic cells due to the diminished membrane integrity.
To better investigate the apoptosis enhancement properties of the formulations, instead of their cell killing action, we have treated the cells at 62.5 μg/ml CytC concentration. As can be seen from the Fig. 4, even at this low concentration of CytC, the Cyt-CL complexes caused significantly increased apoptotic cell populations in both sensitive and MDR cell lines. The viable cell population was somewhat higher in the A2780-Adr cells, which supports the cytotoxicity results. At this low concentration, empty CL formulations did not cause any significant cell death or apoptosis when compared to free CytC or the control. The same concentration of CytC in nanoparticle complexes resulted in more apoptosis enhancement in the A2780-Adr cells compared to A2780 cells, in which they show mainly cell killing effect. The results suggest that the Cyt-CL complexes successfully enhance the apoptotic mechanisms in the both studied sensitive and resistant cell lines, which in turn causes significant cytotoxic efficacy against them.
Lipoperoxyl Radical Formation in Pseudo-Lipoxygenase and Lipoperoxidase Reactions Catalyzed by the Complex Cyt-CL Detected by C-334 Enhanced Chemiluminescence
In an attempt to identify the mechanisms underlying the anticancer activity of Cyt-CL nanocomplexes, we have investigated some aspects of the enzymatic activity of these complexes. Lipid peroxidation is a chain reaction mediated by the reactions of lipid free radicals L• and LOO•, forming and disappearing at each coin of the chain. The interaction of two LOO• radicals is accompanied by the “ultra-weak” luminescence (41,42), which can be increased by several orders of magnitude with special CL enhancers, quinolizin-(9a,9,l-gh)-substituted coumarins, such as C-525, C-338, etc. (35). They typically do not influence the kinetics of the chemical reaction (35) and the biological status of cells (43), but strongly increase the quantum yield of the chemiluminescence due to the electronic energy transfer from the CL-reaction product to the enhancer (35). In this paper, we have used the C-334 as the enhancer in order to determine the steady state concentration of LOO• in reactions, catalyzed by Cyt-CL in poly-unsaturated fatty acid (PUFA)-containing systems, hoping to model the reactions occurring in biological membranes in the presence of Cyt-CL complex.
The curves shown on the left panel in the Fig. 5 represent the pseudo-lipoxygenase process induced by Cyt-CL and show also the comparison of the chemiluminescence kinetics in presence of Cyt-CL with the kinetics provided by the stand-alone components of Cyt-CL system. The addition of the free CytC in PBS did not show any chemiluminescence (the beginning of the CL-curves in both diagrams in Fig. 5). In contrast, the bovine heart cardiolipin mediated the C-334-dependent chemiluminescence even in the absence of the CytC (Left diagram, curve BCL). Whatever the mechanism is, the chemiluminescence shows the current concentration of lipid peroxyl radical (LOO•) appearing in the system as a result of the autoxidation of PUFA residues, spontaneous or catalyzed by the transient metal contaminations in the system (35,45).
This “background” luminescence should be subtracted from that associated with the formation of LOO• radicals during the decomposition of the hydroperoxides catalyzed by Cyt-CL. Subtracting the BCL curve from the CytC-BCL curve, we obtain the kinetics of the lipoxygenase processes catalyzed by the Cyt-CL complex, which, as expected, has an exponential form (calculation data not shown).
Since the formation of lipid peroxyl radicals occurs in the absence of H2O2, but in the presence of oxygen, the reaction may be called pseudo-oxygenase.
The mechanism of this reaction is similar to the mechanism of the luminol radical formation when luminol is oxidized by the horseradish peroxidase or Cyt-CL, and therefore such a reaction should be called a lipoperoxidase reaction.
We hypothesize that the quasi-lipoxygenase and lipoperoxidase reactions occur in mitochondria under the conditions of the oxidative stress and lead to the apoptosis, which is consistent with the literature data (7,9,27).
The Effect of Antioxidants on Cyt-CL Lipoperoxidase Function
The luminescence kinetics found in the study on the effect of antioxidants on the formation of radicals in the reaction catalyzed by Cyt-CL complex in the presence of hydrogen peroxide, confirms the hypothesis that in presence of hydrogen peroxide, the Cyt-CL complex acquires the properties of the peroxidase.
Taxifolin (Fig. 6, right diagram) in given concentrations insignificantly reduced the intensity of the luminescence in the lipoxygenase reaction. In the same concentrations, taxifolin depressed free radical formation in lipoperoxidase reaction showing the significant decrease in the light intensity over the entire time interval. The fact that two different antioxidants demonstrated the same effect on the kinetics of the formation of radicals in the “classic” peroxidase reaction (horseradish peroxidase with luminol as a substrate) and on the kinetics of the lipid radical production by the Cyt-CL complex in presence of H2O2, one can consider as an evidence that the mechanism of the lipoperoxidase reaction catalyzed by the Cyt-CL is similar to that in typical peroxidase reactions.
The apoptosis is the main physiological process that cleans and safely eliminates the damaged cells. Mitochondria serve as the arena of the cascade of events that lead the cell to its death. CytC release from the mitochondria into the cytoplasm due to mitochondrial membrane permeabilization and subsequent activation of caspases is considered as the starting point of this pathway (47). In cancers, this pathway of programmed cell death is inhibited via many different mechanisms (48) to allow cancer cells to proliferate, metastasize and differentiate. When CytC is released into the cytosol, it directly activates Apaf-1, which leads to the cascade resulting in the activation of the effector caspase-3. Bcl-2 or Bcl-xL expression inhibits the CytC release from the mitochondria in cells as well as from the isolated mitochondria (49,50). These genes are significantly overexpressed in many MDR cancers.
In the current study, our aim was to introduce the apoptosis in cancer cells by adding Cyt-CL nanoparticle complexes from outside. While the formation of the reverse-micelle like structure of the Cyt-CL complex formation has been explained earlier, briefly, the addition of the concentrated methanolic CL to the CytC solutions results in the coating of the CytC surface by the CL molecules, and the resulting nanospheres form microcrystals having low solubility in water (19,21). We have used TOCL as the lipid of choice, which is not susceptible to the lipid peroxidation by itself and by CytC (51).
When the particle size data of the formulations were evaluated (Table 1), it can be seen that the size of the particles does not change with the increasing ratios of TOCL over CytC, which supports the previously published hypothesis that there is no intermediate phase of the complex formation. The positive charge of the CytC was shielded by coverage the protein surface by the TOCL molecules and the surface area of the CytC was completely covered at and above 35-fold mol excess of the highly negatively charged lipid. When the equilibrium of complete surface coating of the CytC is reached, the negative zeta potential of the particles increases. Probe sonication of the complexes with 40x mol excess of TOCL yielded a clear, red-pink colored solution without any visible precipitate. The micelle-looking dispersion gave smaller particle size but same zeta potential as its non-sonicated counterpart. Further centrifugation at 13000 rpm for 30 min did not result any precipitation due to small particle size of the microcrystals. Short sonication pulse time (20 s) and long delays between the pulses prevented both the heat generation which can cause significant degradation of the protein and also the formation of excessive air cavities during the process. In the light of the mentioned results, we believe that the sonication energy did not destruct the Cyt-CL nanoparticles but rather dispersed the dense aggregates of the microcrystals.
Most commonly used anticancer drugs and their IC50 values for A2780 human ovarian cancer cell line
The third possible way of cell death, consistent with our experiments, is shown in Figs. 7, 9. According to this hypothesis, Cyt-CL incorporates directly into the cell membrane and catalyzes the lipid peroxidation via quasi-lipoxygenase and lipoperoxidase mechanisms described earlier in the results. This is followed by the lipid peroxidation and phosphatidyl serine (PS) appearance on the cell surface. The PS is a “eat me” signal for macrophages, which recognize the damaged cell and remove it. The mechanisms of the lipid peroxidation in the cell membrane, catalyzed by CytC and followed by scramblase inactivation, PS oxidation, and its externalization, are now well documented (60,61). In our experiments, these phenomena may explain the data obtained using the annexin V to recognize the apoptotic cells (Fig. 4). The more durable treatment of cells with Cyt-CL could destroy the membrane lipid layer integrity and increase the membrane permeability for PI (late apoptosis/necrosis in Fig. 4). As a result, the cells became not viable (that is they lose the ability to convert a redox dye (resazurin) into the fluorescent end product, Figs. 1, 2 and 3). Note that the mechanism based on the assumption that the target for Cyt-CL peroxidase activity is the cell membrane (1, 9 in Fig. 7), explains the equal response of sensitive A2780 cells and doxorubicin-resistant A2780-Adr cells (as well as the mechanism based on the assumption that the inner mitochondrial membrane is the target, Figs. 7, 1–3, 4–8). To differentiate between these two possibilities, further investigations will be performed. It should be noted that our study outlines the proof of principle of an alternative approach and reports that the Cyt-CL nanoparticles acts as a complete system with all of its components. Thus, in the future studies cancer cell targeting would be required, as for any other neoplastic agents, not only to minimize the toxicity against non-cancerous cells in vivo but also to overcome the off-site empty CL cytotoxicity.
Whichever the precise mechanism of the Cyt-CL cytotoxic and pro-apoptotic action, our results indicate that the extra/intracellularly delivered Cyt-CL nanoparticle complexes could successfully start the apoptosis cascade in both the sensitive and drug resistant cancer cells (ovarian cancer cells in this particular case). Delivering these complexes into the cells can push the cells to enter the apoptosis even when they overexpress the factors that normally prevent them from doing so. The completely biomaterial-based Cyt-CL formulation is able to act like a nano-sized machinery in different parts of the cancer cells to start different cascades that cumulatively result in effective anticancer activity. Our finding could be of interest for developing novel approaches to cancer therapy.
Acknowledgments and Disclosures
This study was supported by the Russian Foundation for Basic Research [grant number 14–04-01361]. Yury A. Vladimirov and Can Sarisozen contributed equally as first co-authors.
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