Walterinnesia aegyptia venom combined with silica nanoparticles enhances the functioning of normal lymphocytes through PI3K/AKT, NFκB and ERK signaling
© Badr et al; licensee BioMed Central Ltd. 2012
Received: 9 January 2012
Accepted: 15 February 2012
Published: 15 February 2012
The toxicity of snake venom varies over time in some species. The venom of newborn and small juvenile snakes appears to be more potent than adults of the same species, and a bite from a snake that has not fed recently, such as one that has just emerged from hibernation, is more dangerous than one that has recently fed due to the larger volume of venom injected. Therefore, the potency of a snake's venom is typically determined using the LD50 or IC50 tests. In the present study, we evaluated the anti-tumor potential of snake venom from Walterinnesia aegyptia (WEV) on the human breast carcinoma cell line MDA-MB-231, as well as its effect on the normal mice peripheral blood mononuclear cells (PBMCs).
This venom was used alone (WEV) or in combination with silica nanoparticles (WEV+NP). The IC50 values of WEV alone and WEV+NP in the MDA-MB-231 cells were determined to be 50 ng/ml and 20 ng/ml, respectively. Interestingly, at these concentrations, the venom did not affect the viability of normal human PBMCs. To investigate the in vivo effects of this venom further, three groups of mice were used (15 mice in each group): Group I was the control, Group II was subcutaneously injected with WEV, and Group III was injected with WEV+NP. Using flow cytometry and western blot analysis, we found that the blood lymphocytes of WEV-injected mice exhibited a significant increase in actin polymerization and cytoskeletal rearrangement in response to CXCL12 through the activation of AKT, NF-κB and ERK. These lymphocytes also showed a significant increase in their proliferative capacity in response to mitogen stimulation compared with those isolated from the control mice (P < 0.05). More importantly, in the WEV+NP-treated mice, the biological functions of normal lymphocytes were significantly (P < 0.05) enhanced in comparison with those of WEV-treated mice.
Our data reveal the unique biological effects of WEV, and we demonstrated that its combination with nanoparticles strongly enhanced these biological effects.
KeywordsCytoskeleton Growth arrest Lymphocytes Nanoparticles Proliferation Cell signaling Snake venom
Cell proliferation is a basic biological process that occurs continuously in higher organisms in response to changes in their external and internal environments. In the immune system, lymphocyte proliferation is an important parameter indicating the status of the body's defenses . Recent evidence from both animal and human studies further supports the concept that lymphopenia can drive homeostatic proliferation and the development of autoimmune disease . The uncontrolled proliferation of cells leads to cancer metastasis; therefore, several studies have focused on inhibiting the proliferative capacity of cancer cells using various drugs. Directional cell migration is an integral component of cancer cell invasion during metastasis and involves changes in the cytoskeleton and cell adhesion . The migration of cells through an extracellular matrix is a multistep process that begins with the extension of lamellipodia, cell-surface protrusions comprised of actin filaments, which are anchored to the underlying substratum by small, integrin-dependent focal adhesions. In both normal and cancer cells, the polymerization of actin pushes against the plasma membrane and provides the force for forward movement. Within the cell body, actin stress fibers generate contractile forces by pulling against focal adhesions to induce retraction at the rear of the cell membrane. The bundling of actin filaments into stress fibers clusters and activates integrins, leading to the formation of new focal adhesions . In normal cells, several transcription factors are activated following cell stimulation, leading to cytoskeletal rearrangement and proliferation. The extracellular-signal-regulated kinase (ERK), NF-κB and AKT signaling pathways are major determinants in the control of diverse cellular processes in normal lymphocytes such as proliferation, survival, differentiation and motility. Mechanisms for the inhibition of these pathways thus present targets for cancer therapy . Additionally, previous studies have reported that the constitutive activation of NF-κB in human melanoma cells is linked to the activation of AKT kinase, suggesting that the activation of AKT may be an early marker of tumor progression in melanoma  and that inhibitors of NF-κB activation can block the neoplastic transformation response [7, 8].
Natural products are well recognized as sources for drugs in several human ailments, including cancers. Despite the discovery of many drugs of natural origin, the search for new anticancer agents remains necessary to increase the number of available options and to identify less toxic and more effective drugs . Snake venom is a complex mixture of many substances with a wide spectrum of biological activities including toxins, enzymes, growth factors, activators and inhibitors. Natural toxins, especially sub lethal doses of snake venom, have shown the potential to reduce the size of solid tumors and block angiogenesis . Nanoparticles carrying chemical therapeutics have shown great promise in treating cancer patients. When loaded with anticancer agents, nanoparticles can successfully increase drug concentrations in cancer tissues and act at the cellular level, enhancing antitumor efficacy. The nanoparticles can be endocytosed and/or phagocytosed by cells, with resulting cell internalization of the encapsulated drug .
Few studies have investigated the effects of snake venom in combination with nanoparticles on normal and cancer cells. Therefore, in the present study, we investigated the effects of Walterinnesia aegyptia venom (WEV), alone and in combination with silica nanoparticles (WEV+NP), on the survival of a human breast carcinoma cell line (MDA-MB-231) and human peripheral blood mononuclear cells (PBMCs) in vitro and the in vivo effects of WEV and WEV+NP on mouse lymphocytes.
Materials and methods
Preparation of Walterinnesia aegyptia venom
Walterinnesia aegyptia snakes were collected from the central region of Saudi Arabia. The snakes were kept in a serpentarium in the Zoology Department, College of Science, King Saud University. The snakes were warmed daily using a 100-watt lamp for nine hours, and water was always available. The snakes were fed purpose-bred mice every 10 to 14 days. The venom was milked from adult snakes, lyophilized and reconstituted in 1X phosphate-buffered saline (PBS) prior to use.
Combination of snake venom with silica nanoparticles
A total of 25 mg of mesoporous silica nanoparticles was added to a solution of 50 mg/ml venom in water. The suspension was stirred for 2 hour; the evaporation of water was prevented. The mesoporous silica nanoparticles loaded with venom were recovered using high-speed centrifugation and dried in a vacuum oven at 60°C.
Cell culture and reagents
Human MDA-MB-231 breast cancer cells were obtained from Dr. Douaa Sayed at Assiut University, Egypt and maintained in a culture medium consisting of MEM supplemented with 10% heat-inactivated fetal bovine serum (FBS, EuroClone, Life Science Division, Milan, Italy). The anti-proliferative effect of WEV and WEV+NP on MDA-MB-231 cells was determined using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) uptake method. The cells were plated at 1 × 106 cells/ml in 2 ml of culture medium in six-well Costar plates (Corning, Corning, NY). The cells were treated with different concentrations of WEV or WEV+NP for 1, 2, 6, 12, 24 or 48 h, and cytotoxicity was expressed as a relative percentage of the OD values measured in the control and WEV- and WEV+NP-treated cells. The experiments were repeated using human peripheral blood mononuclear cells (PBMCs). Morphological changes were observed after exposure to WEV and WEV+NP using a phase-contrast inverse microscope (Olympus, Japan).
Animals and lymphocyte isolation
Forty-five sexually mature 12-week-old male Swiss Webster (SW) mice weighing 25-30 g each were obtained from the Central Animal House of the Faculty of Pharmacy at King Saud University. All animal procedures were performed in accordance with the standards set forth in the Guidelines for the Care and Use of Experimental Animals by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) and the National Institutes of Health (NIH). The study protocol was approved by the Animal Ethics Committee of the Zoology Department, College of Science, King Saud University. All animals were allowed to acclimatize in metal cages inside a well-ventilated room for 2 weeks prior to the experiment. The animals were maintained under standard laboratory conditions (a temperature of 23°C, a relative humidity of 60-70% and a 12-hour light/dark cycle) and were fed a diet of standard commercial pellets and given water ad libitum. The animals were divided into 3 experimental groups (n = 15/group): group I was a control group that was subcutaneously injected with PBS, group II was subcutaneously injected with WEV (50 ng/ml for 12 hrs) and group III was subcutaneously injected with WEV+NP (20 ng/ml for 12 hrs). Lymphocytes were isolated from the animals' blood using Ficoll-Paque density gradients. The remaining red blood cells were osmotically lysed using ACK buffer. The cells were washed with phosphate-buffered saline (PBS), counted using the Trypan blue exclusion test, and cultured in R-10 culture medium (complete RPMI 1640 medium supplemented with 10% FCS, 2 mM L-glutamine, 100 IU/ml penicillin, 100 μg/ml streptomycin, 1 mM sodium pyruvate, and 50 μM 2-mercaptoethanol).
F-actin polymerization assay
The isolated blood lymphocytes were cultured for two hour in culture medium before the F-actin polymerization test. Intracellular F-actin polymerization was assessed as previously described . Briefly, cells were harvested and resuspended (4 × 106/ml) in HEPES-buffered RPMI 1640 at 37°C with or without CXCL12 (250 ng/ml). At the indicated times, cell suspensions (100 μl) were added to 400 μl of assay buffer containing 4 × 10-7 M FITC-labeled phalloidin, 0.5 mg/ml L-α-lysophosphatidylcholine (both from Sigma-Aldrich) and 4.5% formaldehyde in PBS. The fixed cells were analyzed using flow cytometry, and the mean fluorescence intensity (MFI) was determined for each sample. The percentage change in MFI was calculated for each sample at each time point using the following formula: (1-(MFI before the addition of CXCL12/MFI after the addition of CXCL12) × 100.
Whole-cell lysates were prepared from lymphocytes that were isolated from the control and WEV- and WEV+NP-treated mice in RIPA buffer (20 mM Tris-HCl, pH 7.5, 120 mM NaCl, 1.0% Triton X100, 0.1% SDS, 1% sodium deoxycholate, 10% glycerol, 1 mM EDTA and 1% protease inhibitor cocktail, Roche). Following centrifugation at 16,000 × g at 4°C for 15 min, the protein concentrations in the supernatants were determined using a protein assay kit (Bio-Rad, Hercules, CA). Equal amounts of whole-cell protein (50 μg) were mixed with reducing sample buffer (0.92 M Tris-HCl, pH 8.8, 1.5% SDS, 4% glycerol, and 280 mM 2-ME) and separated using discontinuous SDS-PAGE. Proteins were transferred onto nitrocellulose membranes using a Bio-Rad Trans-Blot electrophoretic transfer device, and the membranes were blocked for 1 h at room temperature with 1% BSA or 5% skim milk dissolved in TBS (20 mM Tris-HCl, pH 7.4, and 150 mM NaCl) supplemented with 0.1% Tween 20. The membranes were then incubated in the same blocking buffer with anti-phospho-ERK, anti-phospho-AKT, anti-phospho-IκBα, anti-phospho-p38MAPK or anti-β-actin antibodies (1:1,000; Cell Signaling Technology, Beverly, MA). The blots were thoroughly rinsed and then incubated with an HRP-labeled species-matched secondary antibody for another 1 h. Protein bands were detected using enhanced chemiluminescence reagents (ECL, SuperSignal West Pico Chemiluminescent Substrate, Perbio, Bezons, France), and the ECL signals were recorded on Hyperfilm ECL. To quantify band intensities, the films were scanned, saved as TIFF files and analyzed using NIH ImageJ software.
CFSE proliferation assay
Isolated blood lymphocytes were harvested, washed twice in PBS and stained with 0.63 mM carboxyfluorescein diacetate succinimidyl ester (CFSE) (Molecular Probes, Eugene, OR) for 8 min at room temperature. Residual CFSE was removed by washing three times in PBS, and CFSE-labeled cells were seeded in 6-well plates, treated with or without a mitogen cocktail and grown for 4 days in cell culture medium. The CFSE fluorescence intensity was measured using FACS analysis.
Data are expressed as the mean ± standard error of the mean (SEM). Significant differences among groups were analyzed using a one-way analysis of variance (for more than two groups) followed by Tukey's post-test using SPSS software, version 17. Differences were considered statistically significant at P < 0.05. *P < 0.05, WEV-treated vs control; #P < 0.05, WEV+NP-treated vs. control; +P < 0.05, WEV+NP-treated vs. WEV-treated groups.
WEV affects the cell viability of breast cancer cells but not normal cells
WEV combined with NP enhances CXCL12-mediated actin polymerization
WEV combined with NP increases CXCL12-mediated signaling through AKT, NF-κB and ERK but not p38MAPK
WEV combined with NP enhances mitogen-induced cell proliferation
Although many studies have investigated the anti-tumor and cytotoxic effects of snake venom on numerous types of cancer cells [13, 14], little is known regarding its effects on breast cancer. Here, we investigated the effects of snake venom on the MDA-MB-231 cell line. Proliferation and survival are critically important for tumor growth and metastatic spreading; therefore, proliferation and survival constitute attractive targets for tumor therapy. First, we assessed the ability of WEV to arrest the growth of the MDA-MB-231 cell line, and we found that WEV affected the cell viability of breast cancer cells without inhibiting the viability of normal cells. The combination of WEV with NP (WEV+NP) enhanced the effect of WEV on the cancer cells. Our results agree with the results of Park et al. (2009) , who attributed this growth inhibition to apoptosis and cell cycle arrest. Interestingly, at the same concentrations, the venom neither alone nor in combination with nanoparticles induced the in vitro growth arrest of normal PBMCs. We next investigated how this venom might alter the biology of normal lymphocytes in vivo. Actin cytoskeletal reorganization is the primary mechanism of cell motility and is essential for lymphocyte migration to the secondary lymphoid organs, which is regulated by chemokines [12, 15]. Therefore, we monitored actin polymerization in response to CXCL12 stimulation and found that WEV combined with NP enhanced CXCL12-mediated actin polymerization. Similarly, Oliva et al. (2007)  suggested that RGD-disintegrins isolated from snake venom were potent anti-metastatic agents that contributed to the inhibition of melanoma cell invasion through the involvement of the actin cytoskeleton. The control of microfilament actin remodeling thus represents a potential target for the development of anticancer drugs . It has been reported that chemokines such as CCL20, CCL21 and CXCL12 induce actin polymerization and chemotaxis in B lymphocytes through the activation of PI3K/AKT, NF-κB, PLC, ERK and P38MAPK signaling [18, 19]. Our data revealed that WEV combined with NP increased CXCL12-mediated signaling through AKT, NF-κB and ERK but not through p38MAPK. A previous study found that tungsten carbide-cobalt nanoparticles at 5 μg/cm2 induced the production of reactive oxygen species (ROS) that activated AKT and ERK signal pathways in murine epidermal cells . Additionally, in this study, WEV combined with NP enhanced mitogen-induced lymphocyte proliferation. Similar observations have been reported in the induction of endothelial cell proliferation, migration, and angiogenesis by the interaction of aggretin, a component of snake venom, with integrin α2β1, leading to the activation of PI3K and AKT . Taken together, our data demonstrate a new effect of snake venom in combination with nanoparticles on the biological functioning of normal lymphocytes. However, the growth arrest of the breast cancer cell line by WEV and WEV+NP is more interesting. Therefore, in an ongoing work, we are studying the effects of WEV and WEV+NP on the growth arrest of cancer cell lines in an attempt to elucidate the molecular mechanism(s) by which this venom affects cancer cells.
Carboxyfluorescein diacetate succinimidyl ester
CXC chemokine ligand 12
Extracellular signal-regulated kinase
Inhibitor of nuclear factor kappa B alpha
Nuclear factor kappa B
- PKB or AKT:
Protein kinase B
Walterinnesia aegyptia venom
Walterinnesia aegyptia venom combined with nanoparticles.
This work was supported by the National Plan for Science and Technology (NPST) funded by King Abdulaziz City for Science and Technology (KACST) through project number 10-BIO969-02. The authors declare that they have no conflicts of interest.
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