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AKT and ERK signaling
Cellular Immunology 273 (2012) 10–16
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Human breast carcinoma cells are induced to apoptosis by samsum ant venom through an IGF-1-dependant pathway, PI3K/AKT and ERK signaling Gamal Badr a,d,⇑, Olivier Garraud b,e, Maha Daghestani c, Mohammed Saleh Al-Khalifa c, Yolande Richard f a
Deanship of Scientific Research, King Saud University, Riyadh, Saudi Arabia Graduate Studies and Research-Visiting Professor Program, King Saud University, Saudi Arabia c Zoology Department, College of Science, King Saud University, Riyadh, Saudi Arabia d Zoology Department, Faculty of Science, Assiut University, Assiut, Egypt e EA3064—GIMAP, Université de Lyon, F-42023 Saint-Etienne (Cedex 2), France f INSERM U1016, CNRS UMR 8104, Département d’Immunologie, Institut Cochin, F-75014 Paris, France b
a r t i c l e
i n f o
Article history: Received 8 July 2011 Accepted 5 December 2011 Available online 17 December 2011 Keywords: Ant Apoptosis Breast cancer Cell signaling Venom
a b s t r a c t In the present study we evaluated the anti-tumor potential of samsum ant venom (SAV) from Pachycondyla sennaarensis on the human breast carcinoma cell line MCF-7. We found that SAV induced growth arrest of MCF-7 cells without affecting the viability of MCF-10 (non-tumorigenic normal breast epithelial cells) and normal PBMCs. We then analyzed its impact on IGF-1-mediated MCF-7 cell proliferation and its effect on the underlying IGF-1 signaling pathways. Using flow cytometry analysis, we showed that the percentage of apoptotic cells was fourfold higher in SAV-treated cells as compared to untreated cells. More importantly, treatment with SAV induced a marked reduction in actin polymerization and a subsequent marked reduction in IGF-1-mediated cell proliferation. In addition to growth-inhibitory and proapoptotic effects, significant reductions were also observed in the phosphorylation of AKT and ERK, but not p38MAPK, in SAV-treated cells as compared to untreated cells. Our data reveal unique anti-tumor effects of samsum ant venom. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction Pachycondyla sennaarensis, commonly known as the samsum ant, was first identified in Saudi Arabia in 1985 [1]. Since then, there have been reports on the species’ distribution, which is concentrated in the central area of the country [2]. The stings caused by the Hymenoptera order can cause reactions ranging from mild local reactions with painful erythromatous swelling to severe life-threatening anaphylaxis [3]. The anaphylactic effect of P. sennaarensis in Saudi Arabia was confirmed by Al-Shahwan and colleagues [2]. However, some reports have demonstrated that ant venom has many beneficial pharmacological effects, such as reduction of inflammation, pain relief, inhibition of tumor growth, improvement of immunological and hepatic function, and protection of the liver [4–6]. It has been confirmed that the venom of
Abbreviations: CFSE, carboxyfluorescein diacetate succinimidyl ester; ERK, extracellular signal-regulated kinase; IGF-1, insulin-like growth factor-1; PI3K, phosphatidylinositol-3 kinase; PKB or AKT, protein kinase B; SAV, samsum ant venom. ⇑ Corresponding author at: Deanship of Scientific Research, King Saud University, P.O. Box 2454, Riyadh 11451, Saudi Arabia. Fax: +966 14679781. E-mail address: [email protected] (G. Badr). 0008-8749/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.cellimm.2011.12.003
the ant species Polyrhachis lamellidens exerts potent analgesic and anti-inflammatory effects in mouse models [5]. Breast cancer remains the second most common lethal malignancy in women worldwide. It is generally accepted that the balance between proliferation and apoptosis influences the response of tumors to treatments such as chemotherapy, radiotherapy, and hormonal therapy. Apoptosis is an essential and highly conserved mode of cell death that is important for normal development, host defense, and the suppression of oncogenesis. Apoptosis helps discarding cancerous and virally infected cells, and the aberrant regulation of apoptosis is the major cause of tumor development and progression [7]. Cell adhesion and motility are fundamental functions of both normal cells and metastatic tumor cells that involve both transmembrane adhesion receptors and intracellular signaling molecules [8]. These signaling molecules modulate a wide range of intracellular events, including the regulation of actin polymerization, which is necessary for cells to change shape, form lamellipodia, and migrate [9]. The insulin-like growth factor-1 (IGF-1) pathway plays a major role in cancer cell proliferation, survival and resistance to anti-cancer therapies in many human malignancies, including breast cancer. Targeting the IGF signaling pathway represents a promising strategy for the development of novel
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anti-cancer therapeutics. As a drug target, the IGF system has a number of appealing key features. The expression of IGF-1R, which is the major signal-transducing receptor of the pathway, appears to be necessary for malignant transformation in preclinical models. IGF-1-deficient mice have a greatly reduced capacity to support tumor growth and metastasis [10]. The therapeutic potential of targeting the IGF signaling pathway is derived from the roles it plays in the promotion of cell growth and the inhibition of apoptosis. These properties are mediated by crosstalk between two IGF -R-activated pathways, namely, the MEK/ERK/MAPK and PI3K/ AKT pathways [11]. The IGF system has been implicated in chemotherapy resistance. For instance, HBL100 human breast cancer cells become resistant to 5-fluorouracil, methotrexate, and camptothecin when stimulated with IGF-1 [12]. Similarly, IGF-1 administration rescues MCF-7 cells from doxorubicin and paclitaxel treatment [13]. In both studies, IGF-1 signaling provided a growth advantage by either promoting cell proliferation or inhibiting apoptosis. Based on the above information, we investigated the anti-tumor effects of samsum ant venom (SAV) on the growth arrest and induction of apoptosis in MCF-7 cells as well as its ability to alter IGF-1-mediated proliferation and signaling. 2. Materials and methods 2.1. Preparation of samsum ant venom Samsum ants were collected from southern Riyadh, Saudi Arabia. Ants were dissected in distilled water using a binuclear microscope (Olympus SZX10, Seoul, Korea). The sting apparatus was removed with a pair of forceps by grabbing the last segment of the abdomen and detaching it with the sting apparatus. The venom gland was pinched out and placed in a small tube [14]. The glands were homogenized and then centrifuged at 1000 rpm for 2 min. The supernatant (i.e., venom) was collected. Total protein levels in the venom were measured by spectrophotometry according to the Bradford assay. 2.2. Cell culture and reagents Human MCF-7 breast cancer cells and non-tumorigenic normal breast epithelial cells (MCF-10) were obtained from Dr. Maha Daghestani at King Saud University and maintained in culture medium: MEM supplemented with 10% heat-inactivated fetal bovine serum (FBS, EuroClone, Life Science Division, Milan, Italy). The anti-proliferative effect of SAV on MCF-7 and MCF-10 cells was determined using 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 SAV for 6, 12, 24 and 48 h, and cytotoxicity was expressed as a relative percentage of the OD values measured in the control and SAV-treated cells. Morphological changes after exposure to SAV were observed by a phase-contrast inverse microscope (Olympus, Japan). 2.3. Detection of apoptotic cells MCF-7 cells were treated with various concentrations of SAV, and the percentage of apoptotic cells was determined using flow cytometry, as described previously [15]. For further discrimination between viable and apoptotic cells, untreated and SAV-treated MCF-7 cells were washed and incubated in PBS containing 30% heat-inactivated human AB serum at 4 °C for 30 min prior to staining with Annexin V-FITC and PI for 15 min at 25 °C using a com-
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mercial kit according to manufacturer’s instructions (Abcam, Canada). The cells were washed twice with PBS and re-suspended in buffer solution (1 106 cells/ml). Stained cells were analyzed using a flow cytometer (BD FACSCalibur, San Jose, CA) within 1 h of staining, following manufacturer’s protocol. The percentage of cells undergoing apoptosis was determined by calculating (PIneg)/ (Annexin Vpositive); dead cells were double positive, and viable cells were double negative. 2.4. F-actin polymerization assay MCF-7 cells were cultured for 24 h in culture medium with or without SAV. Intracellular F-actin polymerization was assessed as previously described [16]. Briefly, cells were harvested and re-suspended (4 106 ml 1) in HEPES-buffered RPMI 1640 at 37 °C with or without IGF-1 (100 ng/ml). At the indicated times, cells (100 ll) were added to 400 ll of assay buffer containing 4 10 7 M FITClabeled phalloidin, 0.5 mg/ml L-a-lysophosphatidylcholine (both from Sigma–Aldrich) and 4.5% formaldehyde in PBS. Fixed cells were analyzed by 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 with the formula (1-(MFI before addition of IGF-1/MFI and after addition of IGF-1) 100. 2.5. CFSE proliferation assays MCF-7 cells were harvested and washed twice in PBS and then 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 and treated with or without SAV in the presence or absence of IGF-1 (100 ng/ml, from Intergen, France) and grown for 5 days in cell culture medium. The CFSE fluorescence intensity was measured by FACS analysis. 2.6. Intracellular phospho-specific flow cytometry MCF-7 cells were stimulated with IGF-1 (100 ng/ml at 37 °C) or left unstimulated and then fixed for 10 min in pre-warmed cytofix buffer (BD Cytofix # 554655). The cells were permeabilized for 30 min on ice in PERM-III buffer (BD PERM-III buffer # 558050). Permeabilized cells were washed twice, re-suspended in staining buffer (PBS plus 0.5% bovine serum albumin), and stained in a final volume of 100 lL for 30 min at room temperature. Direct conjugated antibodies from Cell Signaling Technology included human phospho-ERK (Thr202/Tyr204), phospho-AKT (S473), phosphop38MAPK (T180/Y182) and control IgG. Cells were fixed and directly analyzed using flow cytometry to determine the percentage of phospho-ERK, phospho-AKT and phospho-p38MAPK positive cells in the gate of viable cells (FSC/SSC). 2.7. Immunoblotting Whole-cell lysates were prepared from SAV-treated and untreated MCF-7 cells 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 16,000g at 4 °C for 15 min, the protein concentrations in the supernatants were determined with a protein assay kit (Bio-Rad, Hercules, CA). Equal amounts of protein (50 lg) in whole cell lysates were mixed with reducing sample buffer (0.92 M Tris–HCl pH 8.8, 1.5% SDS, 4% glycerol, and 280 mM 2ME) and separated in discontinuous SDS–PAGE products. Proteins were transferred with a Bio-Rad Trans-Blot electrophoretic transfer
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device onto nitrocellulose membranes, which was followed by blocking of membranes for 1 h at room temperature with 1% BSA or 5% skim milk dissolved in TBS (20 mM Tris–HCl pH 7.4, 150 mM NaCl) supplemented with 0.1% Tween 20. The membranes were then incubated in the same blocking buffer with anti-phospho-ERK, pan-ERK, phospho-Akt, pan-Akt, phospho-p38MAPK or pan-p38MAPK antibodies (1:1000; Cell Signaling Technology, Beverly, MA). The blots were extensively 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 ECL signals were recorded on hyperfilm ECL. To quantify band intensities, films were scanned, saved as TIFF files and then analyzed using NIH ImageJ software. 2.8. Statistical analyses The data were analyzed using SPSS software version 16 and expressed as means ± SEM. The differences between groups were assessed using analysis of variance (ANOVA). Data were considered significant if the calculated p values were less than 0.05. 3. Results 3.1. Inhibition of cell viability by samsum ant venom The effects of SAV on the viability of MCF-7 cells were assessed using MTT uptake method. SAV inhibited the growth of MCF-7 cells in a concentration- and time-dependent manner (Fig. 1A) without affecting the survival of MCF-10 cells and peripheral blood mononuclear cells (PBMC) (Fig. 1B and C). SAV induced the death of MCF-7 cells, with minimal effects 6 h after treatment. The maximal inhibitory effect (59%) on cell viability was observed at 24 h after treatment with 10 ng/ml of SAV. At this point, MCF-7 cells exhibited a lower survival rate (41%) than that of the untreated cells (91%). The experiment was performed in triplicate, and the results are expressed as the mean percentage of viable cells ± SEM. Trypan blue exclusion test was also simultaneously used for detecting cell viability with cell number count for all the experiments. The percentage of viable cell number was significantly (p < 0.05) reduced from 100% in untreated to 39% in SAV (10 ng/ml)-treated MCF-7 cells for 24 incubation hour (Fig. 1D). Nevertheless, treatment with SAV for 24 h did not significantly affect the viability of control MCF-10 cells and PBMCs. The experiment was performed in triplicate, and the results are expressed as the mean percentage of viable cells ± SEM. 3.2. Samsum ant venom induces apoptosis of MCF-7 cells SAV treatment at a concentration of 10 ng/ml potently induced apoptosis in MCF-7 cells 24 h after treatment. Using an Annexin V-FITC binding assay and flow cytometric analysis, we found that the percentage of apoptotic cells exhibited a significant fourfold elevation in the presence of SAV (48%) as compared to untreated MCF-7 cells (12%; p < 0.5; Fig. 2A). Annexin V-conjugated FITC and propidium iodide (PI) double stain was used to discriminate between viable, apoptotic and necrotic cells in the absence or presence of SAV. Early apoptotic cells were identified by positive Annexin V-FITC and negative PI staining, whereas cells that were in late apoptosis or necrotic cells were positive for both Annexin V-FITC and PI. Viable cells were negative for both Annexin V-FITC and PI. The total percentage of apoptotic and necrotic MCF-7 cells increased to 49% and 6%, respectively, in SAV-treated MCF-7 cells as compared to 6% and 3%, respectively, in control untreated MCF-7 cells (Fig. 2B). The percentage of apoptotic MCF-7 cells in-
creased from 7 ± 1.1% in untreated control cells to 43 ± 3.9% when the cells were treated with SAV for 24 h. Similarly, the percentage of necrotic MCF-7 cells increased from 3 ± 0.25% in untreated control cells to 6 ± 0.7% when the cells were treated with SAV (n = 6; Fig. 2C). Nevertheless, The percentage of apoptotic cells was not significantly changed from untreated control MCF-10 cells (6 ± 0.5%) to SAV-treated (8 ± 0.4%) MCF-10 cells. Similarly, the percentage of necrotic cells was not significantly changed from untreated control MCF-10 cells (3 ± 0.29%) to SAV-treated (5 ± 0.45%) MCF-10 cells. 3.3. Samsum ant venom inhibits IGF-1-induced proliferation of MCF-7 cells At a concentration of 10 ng/ml, SAV was found to induce apoptosis more potently in MCF-7 cells 24 h after treatment. IGF-1 plays a major role in the development and maintenance of breast cancer cells. Therefore, we monitored the effects of SAV on IGF-1-mediated MCF-7 cell proliferation using CFSE proliferation and flow cytometric analysis. As shown in Fig. 3A, the percentage of spontaneously proliferating MCF-7 cells was markedly reduced from 25% in the untreated control cells to 11% in the SAV-treated cells. When the cells were stimulated with IGF-1, the percentage of proliferation was 56% in untreated control cells versus 30% in SAV-treated cells (Fig. 3B). Our data revealed that the proliferative capacity of MCF-7 cells was significantly reduced from 20 ± 2.1% in the untreated control cells to 13 ± 1.3% in the SAV-treated cells (p < 0.05; Fig. 3C; n = 20). Similarly, treatment with SAV significantly reduced IGF-1-mediated cell proliferation from 64 ± 4.5% in the untreated control cells to 24 ± 2.3% in the SAV-treated cells (p < 0.05; Fig. 3C; n = 20). 3.4. Samsum ant venom decreases IGF-1-mediated actin polymerization Actin and microtubules provide a dynamic cellular framework to orchestrate and ultimately control cellular activation and cancer metastasis. Therefore, we monitored actin polymerization upon IGF-1 stimulation in MCF-7 cells in the absence or presence of SAV. The cells were stimulated every 15 s with IGF-1 (100 ng/ ml), and the degree of F-actin polymerization was determined by flow cytometry. In untreated control cells, the percentage of F-actin polymerization was 45 ± 4.8, 83 ± 6, 70 ± 5.4 and 22 ± 3.1 at 15, 30, 45 and 60 s, respectively (open symbols) (Fig. 4). The percentage of F-actin polymerization was significantly reduced to 26 ± 2.8, 41 ± 4.4, 32 ± 3.1 and 5 ± 2 at 15, 30, 45 and 60 s, respectively, in SAV-treated cells (black symbols) (p < 0.05; n = 6). 3.5. Samsum ant venom blocks the action of IGF-1 through AKT and ERK but not p38MAPK Because SAV impairs IGF-1 dependent MCF-7 cell proliferation (Fig. 3) and PI3K/AKT and ERK are important effectors downstream IGF-R, we investigated whether IGF-1 signaling, which is important for breast cancer cell maintenance and survival, was impaired by the treatment with SAV. Using Phosflow assays and flow cytometric analysis, we found that IGF-1-enhanced phosphorylation of ERK and AKT was clearly reduced (p < 0.01) in SAV-treated cells (MFI = 12 and 87, respectively) as compared to the control cells (MFI = 98 and 428, respectively). Nevertheless, SAV did not modulate IGF-1-mediated phosphorylation of p38MAPK (MFI = 282 in control versus 277 in SAV-treated cells; Fig. 5A). Similarly, data from 6 independent experiments revealed that the IGF-mediated phosphorylation of ERK and AKT was significantly (p < 0.03) reduced from 100 ± 6.8 and 435 ± 9.3, respectively, in untreated control cells to 13 ± 2 and 85 ± 5.9, respectively, in SAV-treated cells.
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Fig. 1. Time and dose responses of cell death after SAV treatment. MCF-7 (A), MCF-10 (B) and PBMC cells (C) were treated for different incubation times (6, 12, 24 and 48 h) with SAV at concentrations of 1, 2, 5, 10, 20 and 40 ng/mL. The experiment was performed in triplicate, and the results are expressed as the mean percentage of viable cells ± SEM. Untreated (open bars) and SAV (10 ng/ml)-treated cells for 24 h were subjected to Trypan blue exclusion test. The experiment was performed in triplicate, and the results are expressed as the mean percentage of viable cell number ± SEM.
However, IGF-1-mediated phosphorylation of p38MAPK was similar in control untreated cells (285 ± 7.4) and SAV-treated cells (274 ± 7.8; Fig. 5B). Because antibodies directed against phospho-proteins vary in their sensitivity depending on the technique used, we confirmed our results using antibodies against pERK, pAKT and p38MAPK with western blot analysis. The levels of phosphorylated ERK, AKT and p38MAPK were normalized to the amounts of total ERK, AKT and p38MAPK, respectively. We observed that IGF-1-mediated phosphorylation of ERK and AKT was significantly diminished from 610 ± 12 and 751 ± 13, respectively, in untreated control cells to 60 ± 6.7 and 62 ± 7, respectively, in SAV-treated cells (p < 0.01; n = 6; Fig. 5C). In SAV-treated cells, IGF-1-induced p38MAPK phosphorylation was similar in untreated control cells (125 ± 7.8) and SAV-treated cells (120 ± 6.9).
4. Discussion Several studies have shown that different types of ant venom possess many pharmacological effects, such as the reduction of inflammation, pain relief, the inhibition of tumor growth, improving immunological function, liver protection and hepatitis treatment [4–6,14]. Because of their active substances, such as citral, ATP, histamine, growth hormone, testosterone and superoxide dismutase, ant venoms have been used as medicinal therapies [5]. The venoms of ants share some common proteins, but each species ap-
pears to have a number of unique components. Recent study has revealed the structure and importance of ant venoms [17]. Phenol-2, 4-bis[1,1-dimethylethyl] and trimethyl pyrazine are the main volatile components in the venom gland of samsum ants [18]. In this study, the anti-tumor activities of SAV on the human breast carcinoma cell line MCF-7 was investigated for the first time with promising results. SAV significantly affected cell viability of MCF-7 in a dose- and time-dependent manner, without affecting the viability of non-tumorigenic normal breast epithelial cells and normal PBMCs. Moreover, the percentages of apoptotic and, to a lesser extent of necrotic cells were significantly increased in SAV-treated MCF-7 cells. Our results are consistent with those of Goa and colleagues [19], who found that the venom of a spider (Macrothele raven) affected MCF-7 cell viability in a dose- and time-dependent manner. They also demonstrated by flow cytometry that the spider venom induced the apoptosis and necrosis of MCF-7 cells. Interestingly, SAV did not significantly induce neither apoptosis nor necrosis in both normal controls: an epithelial cell line, MCF-10 cells and PBMCs. The specificity of SAV mediating apoptosis of cancer cells without affecting their normal counterpart need further investigations for clarifying the underlying mechanisms. The insulin-like growth factor pathway has been shown to play a major role in cancer cell proliferation, survival and resistance to anti-cancer therapies in many human malignancies, including breast cancer. As a key signaling component of the IGF system, the IGF-1 receptor is the target of several investigational agents in clinical and preclinical development [20]. Targeting the IGF sig-
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Fig. 2. SAV-induced apoptosis of MCF-7 cells. MCF-7 cells were treated for 24 h with or without 10 ng/ml SAV. (A) MCF-7 cells were stained with Annexin V-FITC alone for early apoptosis detection and then analyzed by FACS. The percentage of early apoptotic cells is represented by Annexin V positive histograms. One representative experiment of three is shown. (B) After incubation of MCF-7 cells for 24 h with or without SAV, apoptotic and necrotic MCF-7 cells were distinguished using the Annexin V/PI kit according to manufacturer’s instructions, and the cells were analyzed by flow cytometry. The data presented in dot blots are as follows. UR indicates the percentage of necrotic cells (Annexin V and PI positive cells), and LR indicates the percentage of early apoptotic cells (Annexin V positive cells). The percentage of cells in each quadrant is shown. One representative experiment of three is shown. (C) Data from six independent experiments are expressed as the mean percentage of apoptotic and necrotic cells ± SEM in untreated control MCF-7 cells (open bars), SAV-treated MCF-7 cells (closed black bars), control MCF-10 cell (closed gray bars) and SAV-treated MCF-10 cells (hatched bars). ⁄p < 0.05 and ⁄⁄ p < 0.01.
naling pathway represents a promising strategy for the development of novel anti-cancer therapeutics. As a drug target, the IGF system has a number of appealing key features. The expression of IGF-1R, which is the major signal-transducing receptor of the pathway, appears to be necessary for malignant transformation in preclinical models of breast cancer [21]. Indeed, forced overexpression of IGF-1R not only increases the likelihood of tumor development but also accelerates the process in animal models [22,23]. Additionally, others have shown that IGF-1-deficient mice have a greatly reduced capacity to support tumor growth and metastasis [10]. Therefore, we attempted to investigate the effects of SAV on IGF-1 signaling; for example, we tested whether it impairs the organization of the cytoskeleton and the proliferation and maintenance of breast cancer cells. IGF-1 has been shown to promote cellular proliferation through multiple signaling pathways. Through their antiproliferative effects, inhibitors of the IGF-1R system may provide a number of clinically important benefits. For instance, maintenance therapy, which is aimed at suppressing the growth of residual subclinical disease, could have a
Fig. 3. SAV abrogates IGF-1-induced proliferation of MCF-7 cells. The ability of MCF-7 cells to proliferate spontaneously or in response to IGF-1 stimulation was evaluated after treatment with SAV using CFSE assays and flow cytometry. MCF-7 cells were treated for 24 h with or without 10 ng/ml SAV. CFSE-labeled cells were then left unstimulated (A) or stimulated with IGF-1 (B). Analysis of CFSE staining was performed after gating on viable cells. The percentage of proliferating cells (CFSE-lo) is indicated for each panel. One representative experiment of six is shown. (C) Data from 20 different experiments are expressed as the mean percentage of proliferating cells ± SEM, either without stimulation or in response to IGF-1 stimulation for untreated control cells (open bars) or SAV-treated cells (black bars). ⁄p < 0.05.
Fig. 4. Effects of SAV on IGF-1-mediated F-actin polymerization. MCF-7 cells were untreated (open circles) or treated with SAV for 24 h (black triangles). F-actin polymerization in response to IGF-1 was measured. The results are expressed as the percentage change in MFI (n = 6) ± SEM, as described in Section 2.
major impact if IGF signaling proves to be a critical factor, as suggested by prognostic data in patients with breast and ovarian can-
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Fig. 5. IGF-1-mediated PI3K/AKT and ERK signaling in MCF-7 cells impaired by treatment with SAV. Phosphorylation of ERK, AKT and p38MAPK, which are the main signaling pathways downstream of IGF-1, in MCF-7 cells was monitored during 24-h incubation in the presence or absence of SAV using Phosflow mAbs and flow cytometric analysis. (A) Phospho-ERK (pERK), pAKT and p38MAPK levels are displayed in untreated control cells (gray-filled histograms) and SAV-treated cells (open bold line histograms), while the isotype controls are represented by open dotted line histograms. One representative experiment of six in shown. (B) The results from six different independent experiments are expressed as the mean ± SEM of MFI values from untreated control cells (open bars) or SAV-treated cells (black bars). (C) To further confirm the impairment of IGF-1 signaling by SAV, after 24 h of incubation in the presence or absence of SAV, MCF-7 cells were stimulated with or without IGF-1, lysed and analyzed using Western blot analysis. Protein bands from one representative experiment out of the six performed are shown. The phosphorylation of ERK, AKT and p38MAPK was normalized to the total relevant protein on stripped blots. The results are expressed as the mean ± SEM of normalized phosphorylation values. ⁄⁄p < 0.01.
cer [24,25]. Our data demonstrate that SAV treatment which strongly decreases survival of MCF-7 cells also abrogates IGF-1mediated MCF-7 proliferation and actin polymerization. Actin polymerization is a critical event that determines the invasiveness of breast cancer cells, including MCF-7 cells [26]. One mechanism by which IGF-1-treated breast cancer cells have been shown to evade tamoxifen-induced apoptosis is through IGF-mediated activation of AKT. These results have revealed the importance of PI3 K/AKT signaling in the metastasis of breast cancer cells [27]. Therefore, defects in PI3 K/AKT signaling increase cellular susceptibility to apoptosis. Our results demonstrate that SAV abrogates the IGF-1-mediated phosphorylation of ERK and AKT, but not p38MAPK. Recent data have shown that ERK plays an important role in the resistance of MCF-7 cells to cell death, which demonstrates the importance of ERK signaling in the maintenance of breast cancer cells [28]. More importantly, a recent study has demonstrated that the suppression of IGF-1 receptor expression in MCF-7 cells causes a decrease in the growth rate and an increase in the rate of apoptosis by triggering the rapid phosphorylation of p38MAPK, which highlights the essential role of its signaling in the induction of apoptosis [29]. Insect-derived products have been widely used in folk medicine in many parts of the world since ancient times, and promising
treatments have been preliminarily studied experimentally. Combining insects with conventional treatments may provide further benefits [30]. Our data reveal a unique anti-tumor effect caused by the venom derived from the samsum ant. Therefore, SAV can be considered a new approach that enhances immunogenicity, reduces cell proliferation and increases apoptosis in cancer cells through the disruption of IGF-1 signaling. Although significant reductions in the phosphorylation of AKT and ERK, but not p38MAPK, were shown in this study, further research detailing the regulatory mechanisms underlying these events is needed. Further investigations should determine the mechanism by which SAV disrupts the action of IGF-1. For example, does SAV interfere with the binding of IGF-1 to its receptor IGF-1R or decrease IGF-1R membrane expression? In addition, other mechanisms must be taken into consideration. Studies focusing on the mechanism of SAV may be key to achieving effective anti-tumor immune responses that can be translated to clinical settings.
Competing interests None.
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Acknowledgments This work was supported by King Saud University, Saudi Arabia, through the Nobel Laureate Collaboration Project (No. NLCP-1/ 2009). Therefore, we thank Nobel Laureate Professor Gunter Blobel (Laboratory of Cell Biology, Howard Hughes Medical Institute, Rockefeller University, New York), the consultant on this project. References [1] C.A. Collingwood, Hymenoptera: Fam. Formicidae of Saudi Arabia, Fauna Saudi Arabia 7 (1985) 230–302. [2] M. Al-Shahwan, S. Al-Khenaizan, M. Al-Khalifa, Black [Samsum] ant induced anaphylaxis in Saudi Arabia, Saudi Med. J. 27 (2006) 1761–1763. [3] A. Potier, C. Lavigne, D. Chappard, J.L. Verret, A. Chevailler, B. Nicolie, et al., Cutaneous manifestations in Hymenoptera and Diptera anaphylaxis: relationship with basal serum tryptase, Clin. Exp. Allergy 39 (2009) 717–725. [4] R.D. Altman, D.R. Schultz, B. Collins-Yudiskas, J. Aldrich, P.I. Arnold, H.E. Brown, The effect of a partially purified fraction of ant venom in rheumatoid arthritis, Arthritis Rheum. 27 (1984) 277–285. [5] J. Kou, Y. Ni, N. Li, J. Wang, L. Liu, Z.H. Jiang, Analgesic and anti-inflammatory activities of total extract and individual fractions of Chinese medicinal ants Polyrhachis lamellidens, Biol. Pharm. Bull. 28 (2005) 176–180. [6] C.P. Wang, Y.L. Wu, Study on mechanism underlying the treatment of rheumatoid arthritis by Keshiling, Zhongguo Zhong Yao Za Zhi 31 (2006) 155–158. [7] D.K. Han, C.C. Haudenschild, M.K. Hong, B.T. Tinkle, M.B. Leon, G. Liau, Evidence for apoptosis in human atherogenesis and in a rat vascular injury model, Am. J. Pathol. 147 (1995) 267–277. [8] D. Kedrin, J. van Rheenen, L. Hernandez, J. Condeelis, J.E. Segall, Cell motility and cytoskeletal regulation in invasion and metastasis, J. Mammary Gland Biol. Neoplasia 12 (2007) 143–152. [9] T.D. Pollard, The cytoskeleton cellular motility and the reductionist agenda, Nature 17 (422) (2003) 741–745. [10] S. Yakar, D. Leroith, P. Brodt, The role of the growth hormone/insulin-like growth factor axis in tumor growth and progression: lessons from animal models, Cytokine Growth Factor Rev. 16 (2005) 407–420. [11] J.N. Lavoie, G. L’Allemain, A. Brunet, R. Müller, J. Pouysségur, Cyclin D1 expression is regulated positively by the p42/p44MAPK and negatively by the p38MAPK/HOGMAPK pathway, J. Biol. Chem. 23 (271) (1996) 20608–20616. [12] S.E. Dunn, R.A. Hardman, F.W. Kari, J.C. Barrett, Insulin-like growth factor 1 [IGF-1] alters drug sensitivity of HBL100 human breast cancer cells by inhibition of apoptosis induced by diverse anticancer drugs, Cancer Res. 157 (1997) 2687–2693. [13] J.L. Gooch, C.L. Van Den Berg, D. Yee, Insulin-like growth factor [IGF]-I rescues breast cancer cells from chemotherapy-induced cell death – proliferative and anti-apoptotic effects, Breast Cancer Res. Treat. 56 (1999) 1–10. [14] M. Dkhil, A. Abdel-Baki, S. Al-Quraishi, M. Al-Khalifa, Anti-inflammatory activity of the venom from samsumants Pachycondyla sennaarensis, Afr. J. Pharm. Pharmacol. 4 (2010) 115–118.
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Cellular Immunology journal homepage: www.elsevier.com/locate/ycimm
Human breast carcinoma cells are induced to apoptosis by samsum ant venom through an IGF-1-dependant pathway, PI3K/AKT and ERK signaling Gamal Badr a,d,⇑, Olivier Garraud b,e, Maha Daghestani c, Mohammed Saleh Al-Khalifa c, Yolande Richard f a
Deanship of Scientific Research, King Saud University, Riyadh, Saudi Arabia Graduate Studies and Research-Visiting Professor Program, King Saud University, Saudi Arabia c Zoology Department, College of Science, King Saud University, Riyadh, Saudi Arabia d Zoology Department, Faculty of Science, Assiut University, Assiut, Egypt e EA3064—GIMAP, Université de Lyon, F-42023 Saint-Etienne (Cedex 2), France f INSERM U1016, CNRS UMR 8104, Département d’Immunologie, Institut Cochin, F-75014 Paris, France b
a r t i c l e
i n f o
Article history: Received 8 July 2011 Accepted 5 December 2011 Available online 17 December 2011 Keywords: Ant Apoptosis Breast cancer Cell signaling Venom
a b s t r a c t In the present study we evaluated the anti-tumor potential of samsum ant venom (SAV) from Pachycondyla sennaarensis on the human breast carcinoma cell line MCF-7. We found that SAV induced growth arrest of MCF-7 cells without affecting the viability of MCF-10 (non-tumorigenic normal breast epithelial cells) and normal PBMCs. We then analyzed its impact on IGF-1-mediated MCF-7 cell proliferation and its effect on the underlying IGF-1 signaling pathways. Using flow cytometry analysis, we showed that the percentage of apoptotic cells was fourfold higher in SAV-treated cells as compared to untreated cells. More importantly, treatment with SAV induced a marked reduction in actin polymerization and a subsequent marked reduction in IGF-1-mediated cell proliferation. In addition to growth-inhibitory and proapoptotic effects, significant reductions were also observed in the phosphorylation of AKT and ERK, but not p38MAPK, in SAV-treated cells as compared to untreated cells. Our data reveal unique anti-tumor effects of samsum ant venom. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction Pachycondyla sennaarensis, commonly known as the samsum ant, was first identified in Saudi Arabia in 1985 [1]. Since then, there have been reports on the species’ distribution, which is concentrated in the central area of the country [2]. The stings caused by the Hymenoptera order can cause reactions ranging from mild local reactions with painful erythromatous swelling to severe life-threatening anaphylaxis [3]. The anaphylactic effect of P. sennaarensis in Saudi Arabia was confirmed by Al-Shahwan and colleagues [2]. However, some reports have demonstrated that ant venom has many beneficial pharmacological effects, such as reduction of inflammation, pain relief, inhibition of tumor growth, improvement of immunological and hepatic function, and protection of the liver [4–6]. It has been confirmed that the venom of
Abbreviations: CFSE, carboxyfluorescein diacetate succinimidyl ester; ERK, extracellular signal-regulated kinase; IGF-1, insulin-like growth factor-1; PI3K, phosphatidylinositol-3 kinase; PKB or AKT, protein kinase B; SAV, samsum ant venom. ⇑ Corresponding author at: Deanship of Scientific Research, King Saud University, P.O. Box 2454, Riyadh 11451, Saudi Arabia. Fax: +966 14679781. E-mail address: [email protected] (G. Badr). 0008-8749/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.cellimm.2011.12.003
the ant species Polyrhachis lamellidens exerts potent analgesic and anti-inflammatory effects in mouse models [5]. Breast cancer remains the second most common lethal malignancy in women worldwide. It is generally accepted that the balance between proliferation and apoptosis influences the response of tumors to treatments such as chemotherapy, radiotherapy, and hormonal therapy. Apoptosis is an essential and highly conserved mode of cell death that is important for normal development, host defense, and the suppression of oncogenesis. Apoptosis helps discarding cancerous and virally infected cells, and the aberrant regulation of apoptosis is the major cause of tumor development and progression [7]. Cell adhesion and motility are fundamental functions of both normal cells and metastatic tumor cells that involve both transmembrane adhesion receptors and intracellular signaling molecules [8]. These signaling molecules modulate a wide range of intracellular events, including the regulation of actin polymerization, which is necessary for cells to change shape, form lamellipodia, and migrate [9]. The insulin-like growth factor-1 (IGF-1) pathway plays a major role in cancer cell proliferation, survival and resistance to anti-cancer therapies in many human malignancies, including breast cancer. Targeting the IGF signaling pathway represents a promising strategy for the development of novel
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anti-cancer therapeutics. As a drug target, the IGF system has a number of appealing key features. The expression of IGF-1R, which is the major signal-transducing receptor of the pathway, appears to be necessary for malignant transformation in preclinical models. IGF-1-deficient mice have a greatly reduced capacity to support tumor growth and metastasis [10]. The therapeutic potential of targeting the IGF signaling pathway is derived from the roles it plays in the promotion of cell growth and the inhibition of apoptosis. These properties are mediated by crosstalk between two IGF -R-activated pathways, namely, the MEK/ERK/MAPK and PI3K/ AKT pathways [11]. The IGF system has been implicated in chemotherapy resistance. For instance, HBL100 human breast cancer cells become resistant to 5-fluorouracil, methotrexate, and camptothecin when stimulated with IGF-1 [12]. Similarly, IGF-1 administration rescues MCF-7 cells from doxorubicin and paclitaxel treatment [13]. In both studies, IGF-1 signaling provided a growth advantage by either promoting cell proliferation or inhibiting apoptosis. Based on the above information, we investigated the anti-tumor effects of samsum ant venom (SAV) on the growth arrest and induction of apoptosis in MCF-7 cells as well as its ability to alter IGF-1-mediated proliferation and signaling. 2. Materials and methods 2.1. Preparation of samsum ant venom Samsum ants were collected from southern Riyadh, Saudi Arabia. Ants were dissected in distilled water using a binuclear microscope (Olympus SZX10, Seoul, Korea). The sting apparatus was removed with a pair of forceps by grabbing the last segment of the abdomen and detaching it with the sting apparatus. The venom gland was pinched out and placed in a small tube [14]. The glands were homogenized and then centrifuged at 1000 rpm for 2 min. The supernatant (i.e., venom) was collected. Total protein levels in the venom were measured by spectrophotometry according to the Bradford assay. 2.2. Cell culture and reagents Human MCF-7 breast cancer cells and non-tumorigenic normal breast epithelial cells (MCF-10) were obtained from Dr. Maha Daghestani at King Saud University and maintained in culture medium: MEM supplemented with 10% heat-inactivated fetal bovine serum (FBS, EuroClone, Life Science Division, Milan, Italy). The anti-proliferative effect of SAV on MCF-7 and MCF-10 cells was determined using 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 SAV for 6, 12, 24 and 48 h, and cytotoxicity was expressed as a relative percentage of the OD values measured in the control and SAV-treated cells. Morphological changes after exposure to SAV were observed by a phase-contrast inverse microscope (Olympus, Japan). 2.3. Detection of apoptotic cells MCF-7 cells were treated with various concentrations of SAV, and the percentage of apoptotic cells was determined using flow cytometry, as described previously [15]. For further discrimination between viable and apoptotic cells, untreated and SAV-treated MCF-7 cells were washed and incubated in PBS containing 30% heat-inactivated human AB serum at 4 °C for 30 min prior to staining with Annexin V-FITC and PI for 15 min at 25 °C using a com-
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mercial kit according to manufacturer’s instructions (Abcam, Canada). The cells were washed twice with PBS and re-suspended in buffer solution (1 106 cells/ml). Stained cells were analyzed using a flow cytometer (BD FACSCalibur, San Jose, CA) within 1 h of staining, following manufacturer’s protocol. The percentage of cells undergoing apoptosis was determined by calculating (PIneg)/ (Annexin Vpositive); dead cells were double positive, and viable cells were double negative. 2.4. F-actin polymerization assay MCF-7 cells were cultured for 24 h in culture medium with or without SAV. Intracellular F-actin polymerization was assessed as previously described [16]. Briefly, cells were harvested and re-suspended (4 106 ml 1) in HEPES-buffered RPMI 1640 at 37 °C with or without IGF-1 (100 ng/ml). At the indicated times, cells (100 ll) were added to 400 ll of assay buffer containing 4 10 7 M FITClabeled phalloidin, 0.5 mg/ml L-a-lysophosphatidylcholine (both from Sigma–Aldrich) and 4.5% formaldehyde in PBS. Fixed cells were analyzed by 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 with the formula (1-(MFI before addition of IGF-1/MFI and after addition of IGF-1) 100. 2.5. CFSE proliferation assays MCF-7 cells were harvested and washed twice in PBS and then 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 and treated with or without SAV in the presence or absence of IGF-1 (100 ng/ml, from Intergen, France) and grown for 5 days in cell culture medium. The CFSE fluorescence intensity was measured by FACS analysis. 2.6. Intracellular phospho-specific flow cytometry MCF-7 cells were stimulated with IGF-1 (100 ng/ml at 37 °C) or left unstimulated and then fixed for 10 min in pre-warmed cytofix buffer (BD Cytofix # 554655). The cells were permeabilized for 30 min on ice in PERM-III buffer (BD PERM-III buffer # 558050). Permeabilized cells were washed twice, re-suspended in staining buffer (PBS plus 0.5% bovine serum albumin), and stained in a final volume of 100 lL for 30 min at room temperature. Direct conjugated antibodies from Cell Signaling Technology included human phospho-ERK (Thr202/Tyr204), phospho-AKT (S473), phosphop38MAPK (T180/Y182) and control IgG. Cells were fixed and directly analyzed using flow cytometry to determine the percentage of phospho-ERK, phospho-AKT and phospho-p38MAPK positive cells in the gate of viable cells (FSC/SSC). 2.7. Immunoblotting Whole-cell lysates were prepared from SAV-treated and untreated MCF-7 cells 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 16,000g at 4 °C for 15 min, the protein concentrations in the supernatants were determined with a protein assay kit (Bio-Rad, Hercules, CA). Equal amounts of protein (50 lg) in whole cell lysates were mixed with reducing sample buffer (0.92 M Tris–HCl pH 8.8, 1.5% SDS, 4% glycerol, and 280 mM 2ME) and separated in discontinuous SDS–PAGE products. Proteins were transferred with a Bio-Rad Trans-Blot electrophoretic transfer
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device onto nitrocellulose membranes, which was followed by blocking of membranes for 1 h at room temperature with 1% BSA or 5% skim milk dissolved in TBS (20 mM Tris–HCl pH 7.4, 150 mM NaCl) supplemented with 0.1% Tween 20. The membranes were then incubated in the same blocking buffer with anti-phospho-ERK, pan-ERK, phospho-Akt, pan-Akt, phospho-p38MAPK or pan-p38MAPK antibodies (1:1000; Cell Signaling Technology, Beverly, MA). The blots were extensively 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 ECL signals were recorded on hyperfilm ECL. To quantify band intensities, films were scanned, saved as TIFF files and then analyzed using NIH ImageJ software. 2.8. Statistical analyses The data were analyzed using SPSS software version 16 and expressed as means ± SEM. The differences between groups were assessed using analysis of variance (ANOVA). Data were considered significant if the calculated p values were less than 0.05. 3. Results 3.1. Inhibition of cell viability by samsum ant venom The effects of SAV on the viability of MCF-7 cells were assessed using MTT uptake method. SAV inhibited the growth of MCF-7 cells in a concentration- and time-dependent manner (Fig. 1A) without affecting the survival of MCF-10 cells and peripheral blood mononuclear cells (PBMC) (Fig. 1B and C). SAV induced the death of MCF-7 cells, with minimal effects 6 h after treatment. The maximal inhibitory effect (59%) on cell viability was observed at 24 h after treatment with 10 ng/ml of SAV. At this point, MCF-7 cells exhibited a lower survival rate (41%) than that of the untreated cells (91%). The experiment was performed in triplicate, and the results are expressed as the mean percentage of viable cells ± SEM. Trypan blue exclusion test was also simultaneously used for detecting cell viability with cell number count for all the experiments. The percentage of viable cell number was significantly (p < 0.05) reduced from 100% in untreated to 39% in SAV (10 ng/ml)-treated MCF-7 cells for 24 incubation hour (Fig. 1D). Nevertheless, treatment with SAV for 24 h did not significantly affect the viability of control MCF-10 cells and PBMCs. The experiment was performed in triplicate, and the results are expressed as the mean percentage of viable cells ± SEM. 3.2. Samsum ant venom induces apoptosis of MCF-7 cells SAV treatment at a concentration of 10 ng/ml potently induced apoptosis in MCF-7 cells 24 h after treatment. Using an Annexin V-FITC binding assay and flow cytometric analysis, we found that the percentage of apoptotic cells exhibited a significant fourfold elevation in the presence of SAV (48%) as compared to untreated MCF-7 cells (12%; p < 0.5; Fig. 2A). Annexin V-conjugated FITC and propidium iodide (PI) double stain was used to discriminate between viable, apoptotic and necrotic cells in the absence or presence of SAV. Early apoptotic cells were identified by positive Annexin V-FITC and negative PI staining, whereas cells that were in late apoptosis or necrotic cells were positive for both Annexin V-FITC and PI. Viable cells were negative for both Annexin V-FITC and PI. The total percentage of apoptotic and necrotic MCF-7 cells increased to 49% and 6%, respectively, in SAV-treated MCF-7 cells as compared to 6% and 3%, respectively, in control untreated MCF-7 cells (Fig. 2B). The percentage of apoptotic MCF-7 cells in-
creased from 7 ± 1.1% in untreated control cells to 43 ± 3.9% when the cells were treated with SAV for 24 h. Similarly, the percentage of necrotic MCF-7 cells increased from 3 ± 0.25% in untreated control cells to 6 ± 0.7% when the cells were treated with SAV (n = 6; Fig. 2C). Nevertheless, The percentage of apoptotic cells was not significantly changed from untreated control MCF-10 cells (6 ± 0.5%) to SAV-treated (8 ± 0.4%) MCF-10 cells. Similarly, the percentage of necrotic cells was not significantly changed from untreated control MCF-10 cells (3 ± 0.29%) to SAV-treated (5 ± 0.45%) MCF-10 cells. 3.3. Samsum ant venom inhibits IGF-1-induced proliferation of MCF-7 cells At a concentration of 10 ng/ml, SAV was found to induce apoptosis more potently in MCF-7 cells 24 h after treatment. IGF-1 plays a major role in the development and maintenance of breast cancer cells. Therefore, we monitored the effects of SAV on IGF-1-mediated MCF-7 cell proliferation using CFSE proliferation and flow cytometric analysis. As shown in Fig. 3A, the percentage of spontaneously proliferating MCF-7 cells was markedly reduced from 25% in the untreated control cells to 11% in the SAV-treated cells. When the cells were stimulated with IGF-1, the percentage of proliferation was 56% in untreated control cells versus 30% in SAV-treated cells (Fig. 3B). Our data revealed that the proliferative capacity of MCF-7 cells was significantly reduced from 20 ± 2.1% in the untreated control cells to 13 ± 1.3% in the SAV-treated cells (p < 0.05; Fig. 3C; n = 20). Similarly, treatment with SAV significantly reduced IGF-1-mediated cell proliferation from 64 ± 4.5% in the untreated control cells to 24 ± 2.3% in the SAV-treated cells (p < 0.05; Fig. 3C; n = 20). 3.4. Samsum ant venom decreases IGF-1-mediated actin polymerization Actin and microtubules provide a dynamic cellular framework to orchestrate and ultimately control cellular activation and cancer metastasis. Therefore, we monitored actin polymerization upon IGF-1 stimulation in MCF-7 cells in the absence or presence of SAV. The cells were stimulated every 15 s with IGF-1 (100 ng/ ml), and the degree of F-actin polymerization was determined by flow cytometry. In untreated control cells, the percentage of F-actin polymerization was 45 ± 4.8, 83 ± 6, 70 ± 5.4 and 22 ± 3.1 at 15, 30, 45 and 60 s, respectively (open symbols) (Fig. 4). The percentage of F-actin polymerization was significantly reduced to 26 ± 2.8, 41 ± 4.4, 32 ± 3.1 and 5 ± 2 at 15, 30, 45 and 60 s, respectively, in SAV-treated cells (black symbols) (p < 0.05; n = 6). 3.5. Samsum ant venom blocks the action of IGF-1 through AKT and ERK but not p38MAPK Because SAV impairs IGF-1 dependent MCF-7 cell proliferation (Fig. 3) and PI3K/AKT and ERK are important effectors downstream IGF-R, we investigated whether IGF-1 signaling, which is important for breast cancer cell maintenance and survival, was impaired by the treatment with SAV. Using Phosflow assays and flow cytometric analysis, we found that IGF-1-enhanced phosphorylation of ERK and AKT was clearly reduced (p < 0.01) in SAV-treated cells (MFI = 12 and 87, respectively) as compared to the control cells (MFI = 98 and 428, respectively). Nevertheless, SAV did not modulate IGF-1-mediated phosphorylation of p38MAPK (MFI = 282 in control versus 277 in SAV-treated cells; Fig. 5A). Similarly, data from 6 independent experiments revealed that the IGF-mediated phosphorylation of ERK and AKT was significantly (p < 0.03) reduced from 100 ± 6.8 and 435 ± 9.3, respectively, in untreated control cells to 13 ± 2 and 85 ± 5.9, respectively, in SAV-treated cells.
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Fig. 1. Time and dose responses of cell death after SAV treatment. MCF-7 (A), MCF-10 (B) and PBMC cells (C) were treated for different incubation times (6, 12, 24 and 48 h) with SAV at concentrations of 1, 2, 5, 10, 20 and 40 ng/mL. The experiment was performed in triplicate, and the results are expressed as the mean percentage of viable cells ± SEM. Untreated (open bars) and SAV (10 ng/ml)-treated cells for 24 h were subjected to Trypan blue exclusion test. The experiment was performed in triplicate, and the results are expressed as the mean percentage of viable cell number ± SEM.
However, IGF-1-mediated phosphorylation of p38MAPK was similar in control untreated cells (285 ± 7.4) and SAV-treated cells (274 ± 7.8; Fig. 5B). Because antibodies directed against phospho-proteins vary in their sensitivity depending on the technique used, we confirmed our results using antibodies against pERK, pAKT and p38MAPK with western blot analysis. The levels of phosphorylated ERK, AKT and p38MAPK were normalized to the amounts of total ERK, AKT and p38MAPK, respectively. We observed that IGF-1-mediated phosphorylation of ERK and AKT was significantly diminished from 610 ± 12 and 751 ± 13, respectively, in untreated control cells to 60 ± 6.7 and 62 ± 7, respectively, in SAV-treated cells (p < 0.01; n = 6; Fig. 5C). In SAV-treated cells, IGF-1-induced p38MAPK phosphorylation was similar in untreated control cells (125 ± 7.8) and SAV-treated cells (120 ± 6.9).
4. Discussion Several studies have shown that different types of ant venom possess many pharmacological effects, such as the reduction of inflammation, pain relief, the inhibition of tumor growth, improving immunological function, liver protection and hepatitis treatment [4–6,14]. Because of their active substances, such as citral, ATP, histamine, growth hormone, testosterone and superoxide dismutase, ant venoms have been used as medicinal therapies [5]. The venoms of ants share some common proteins, but each species ap-
pears to have a number of unique components. Recent study has revealed the structure and importance of ant venoms [17]. Phenol-2, 4-bis[1,1-dimethylethyl] and trimethyl pyrazine are the main volatile components in the venom gland of samsum ants [18]. In this study, the anti-tumor activities of SAV on the human breast carcinoma cell line MCF-7 was investigated for the first time with promising results. SAV significantly affected cell viability of MCF-7 in a dose- and time-dependent manner, without affecting the viability of non-tumorigenic normal breast epithelial cells and normal PBMCs. Moreover, the percentages of apoptotic and, to a lesser extent of necrotic cells were significantly increased in SAV-treated MCF-7 cells. Our results are consistent with those of Goa and colleagues [19], who found that the venom of a spider (Macrothele raven) affected MCF-7 cell viability in a dose- and time-dependent manner. They also demonstrated by flow cytometry that the spider venom induced the apoptosis and necrosis of MCF-7 cells. Interestingly, SAV did not significantly induce neither apoptosis nor necrosis in both normal controls: an epithelial cell line, MCF-10 cells and PBMCs. The specificity of SAV mediating apoptosis of cancer cells without affecting their normal counterpart need further investigations for clarifying the underlying mechanisms. The insulin-like growth factor pathway has been shown to play a major role in cancer cell proliferation, survival and resistance to anti-cancer therapies in many human malignancies, including breast cancer. As a key signaling component of the IGF system, the IGF-1 receptor is the target of several investigational agents in clinical and preclinical development [20]. Targeting the IGF sig-
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Fig. 2. SAV-induced apoptosis of MCF-7 cells. MCF-7 cells were treated for 24 h with or without 10 ng/ml SAV. (A) MCF-7 cells were stained with Annexin V-FITC alone for early apoptosis detection and then analyzed by FACS. The percentage of early apoptotic cells is represented by Annexin V positive histograms. One representative experiment of three is shown. (B) After incubation of MCF-7 cells for 24 h with or without SAV, apoptotic and necrotic MCF-7 cells were distinguished using the Annexin V/PI kit according to manufacturer’s instructions, and the cells were analyzed by flow cytometry. The data presented in dot blots are as follows. UR indicates the percentage of necrotic cells (Annexin V and PI positive cells), and LR indicates the percentage of early apoptotic cells (Annexin V positive cells). The percentage of cells in each quadrant is shown. One representative experiment of three is shown. (C) Data from six independent experiments are expressed as the mean percentage of apoptotic and necrotic cells ± SEM in untreated control MCF-7 cells (open bars), SAV-treated MCF-7 cells (closed black bars), control MCF-10 cell (closed gray bars) and SAV-treated MCF-10 cells (hatched bars). ⁄p < 0.05 and ⁄⁄ p < 0.01.
naling pathway represents a promising strategy for the development of novel anti-cancer therapeutics. As a drug target, the IGF system has a number of appealing key features. The expression of IGF-1R, which is the major signal-transducing receptor of the pathway, appears to be necessary for malignant transformation in preclinical models of breast cancer [21]. Indeed, forced overexpression of IGF-1R not only increases the likelihood of tumor development but also accelerates the process in animal models [22,23]. Additionally, others have shown that IGF-1-deficient mice have a greatly reduced capacity to support tumor growth and metastasis [10]. Therefore, we attempted to investigate the effects of SAV on IGF-1 signaling; for example, we tested whether it impairs the organization of the cytoskeleton and the proliferation and maintenance of breast cancer cells. IGF-1 has been shown to promote cellular proliferation through multiple signaling pathways. Through their antiproliferative effects, inhibitors of the IGF-1R system may provide a number of clinically important benefits. For instance, maintenance therapy, which is aimed at suppressing the growth of residual subclinical disease, could have a
Fig. 3. SAV abrogates IGF-1-induced proliferation of MCF-7 cells. The ability of MCF-7 cells to proliferate spontaneously or in response to IGF-1 stimulation was evaluated after treatment with SAV using CFSE assays and flow cytometry. MCF-7 cells were treated for 24 h with or without 10 ng/ml SAV. CFSE-labeled cells were then left unstimulated (A) or stimulated with IGF-1 (B). Analysis of CFSE staining was performed after gating on viable cells. The percentage of proliferating cells (CFSE-lo) is indicated for each panel. One representative experiment of six is shown. (C) Data from 20 different experiments are expressed as the mean percentage of proliferating cells ± SEM, either without stimulation or in response to IGF-1 stimulation for untreated control cells (open bars) or SAV-treated cells (black bars). ⁄p < 0.05.
Fig. 4. Effects of SAV on IGF-1-mediated F-actin polymerization. MCF-7 cells were untreated (open circles) or treated with SAV for 24 h (black triangles). F-actin polymerization in response to IGF-1 was measured. The results are expressed as the percentage change in MFI (n = 6) ± SEM, as described in Section 2.
major impact if IGF signaling proves to be a critical factor, as suggested by prognostic data in patients with breast and ovarian can-
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Fig. 5. IGF-1-mediated PI3K/AKT and ERK signaling in MCF-7 cells impaired by treatment with SAV. Phosphorylation of ERK, AKT and p38MAPK, which are the main signaling pathways downstream of IGF-1, in MCF-7 cells was monitored during 24-h incubation in the presence or absence of SAV using Phosflow mAbs and flow cytometric analysis. (A) Phospho-ERK (pERK), pAKT and p38MAPK levels are displayed in untreated control cells (gray-filled histograms) and SAV-treated cells (open bold line histograms), while the isotype controls are represented by open dotted line histograms. One representative experiment of six in shown. (B) The results from six different independent experiments are expressed as the mean ± SEM of MFI values from untreated control cells (open bars) or SAV-treated cells (black bars). (C) To further confirm the impairment of IGF-1 signaling by SAV, after 24 h of incubation in the presence or absence of SAV, MCF-7 cells were stimulated with or without IGF-1, lysed and analyzed using Western blot analysis. Protein bands from one representative experiment out of the six performed are shown. The phosphorylation of ERK, AKT and p38MAPK was normalized to the total relevant protein on stripped blots. The results are expressed as the mean ± SEM of normalized phosphorylation values. ⁄⁄p < 0.01.
cer [24,25]. Our data demonstrate that SAV treatment which strongly decreases survival of MCF-7 cells also abrogates IGF-1mediated MCF-7 proliferation and actin polymerization. Actin polymerization is a critical event that determines the invasiveness of breast cancer cells, including MCF-7 cells [26]. One mechanism by which IGF-1-treated breast cancer cells have been shown to evade tamoxifen-induced apoptosis is through IGF-mediated activation of AKT. These results have revealed the importance of PI3 K/AKT signaling in the metastasis of breast cancer cells [27]. Therefore, defects in PI3 K/AKT signaling increase cellular susceptibility to apoptosis. Our results demonstrate that SAV abrogates the IGF-1-mediated phosphorylation of ERK and AKT, but not p38MAPK. Recent data have shown that ERK plays an important role in the resistance of MCF-7 cells to cell death, which demonstrates the importance of ERK signaling in the maintenance of breast cancer cells [28]. More importantly, a recent study has demonstrated that the suppression of IGF-1 receptor expression in MCF-7 cells causes a decrease in the growth rate and an increase in the rate of apoptosis by triggering the rapid phosphorylation of p38MAPK, which highlights the essential role of its signaling in the induction of apoptosis [29]. Insect-derived products have been widely used in folk medicine in many parts of the world since ancient times, and promising
treatments have been preliminarily studied experimentally. Combining insects with conventional treatments may provide further benefits [30]. Our data reveal a unique anti-tumor effect caused by the venom derived from the samsum ant. Therefore, SAV can be considered a new approach that enhances immunogenicity, reduces cell proliferation and increases apoptosis in cancer cells through the disruption of IGF-1 signaling. Although significant reductions in the phosphorylation of AKT and ERK, but not p38MAPK, were shown in this study, further research detailing the regulatory mechanisms underlying these events is needed. Further investigations should determine the mechanism by which SAV disrupts the action of IGF-1. For example, does SAV interfere with the binding of IGF-1 to its receptor IGF-1R or decrease IGF-1R membrane expression? In addition, other mechanisms must be taken into consideration. Studies focusing on the mechanism of SAV may be key to achieving effective anti-tumor immune responses that can be translated to clinical settings.
Competing interests None.
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Acknowledgments This work was supported by King Saud University, Saudi Arabia, through the Nobel Laureate Collaboration Project (No. NLCP-1/ 2009). Therefore, we thank Nobel Laureate Professor Gunter Blobel (Laboratory of Cell Biology, Howard Hughes Medical Institute, Rockefeller University, New York), the consultant on this project. References [1] C.A. Collingwood, Hymenoptera: Fam. Formicidae of Saudi Arabia, Fauna Saudi Arabia 7 (1985) 230–302. [2] M. Al-Shahwan, S. Al-Khenaizan, M. Al-Khalifa, Black [Samsum] ant induced anaphylaxis in Saudi Arabia, Saudi Med. J. 27 (2006) 1761–1763. [3] A. Potier, C. Lavigne, D. Chappard, J.L. Verret, A. Chevailler, B. Nicolie, et al., Cutaneous manifestations in Hymenoptera and Diptera anaphylaxis: relationship with basal serum tryptase, Clin. Exp. Allergy 39 (2009) 717–725. [4] R.D. Altman, D.R. Schultz, B. Collins-Yudiskas, J. Aldrich, P.I. Arnold, H.E. Brown, The effect of a partially purified fraction of ant venom in rheumatoid arthritis, Arthritis Rheum. 27 (1984) 277–285. [5] J. Kou, Y. Ni, N. Li, J. Wang, L. Liu, Z.H. Jiang, Analgesic and anti-inflammatory activities of total extract and individual fractions of Chinese medicinal ants Polyrhachis lamellidens, Biol. Pharm. Bull. 28 (2005) 176–180. [6] C.P. Wang, Y.L. Wu, Study on mechanism underlying the treatment of rheumatoid arthritis by Keshiling, Zhongguo Zhong Yao Za Zhi 31 (2006) 155–158. [7] D.K. Han, C.C. Haudenschild, M.K. Hong, B.T. Tinkle, M.B. Leon, G. Liau, Evidence for apoptosis in human atherogenesis and in a rat vascular injury model, Am. J. Pathol. 147 (1995) 267–277. [8] D. Kedrin, J. van Rheenen, L. Hernandez, J. Condeelis, J.E. Segall, Cell motility and cytoskeletal regulation in invasion and metastasis, J. Mammary Gland Biol. Neoplasia 12 (2007) 143–152. [9] T.D. Pollard, The cytoskeleton cellular motility and the reductionist agenda, Nature 17 (422) (2003) 741–745. [10] S. Yakar, D. Leroith, P. Brodt, The role of the growth hormone/insulin-like growth factor axis in tumor growth and progression: lessons from animal models, Cytokine Growth Factor Rev. 16 (2005) 407–420. [11] J.N. Lavoie, G. L’Allemain, A. Brunet, R. Müller, J. Pouysségur, Cyclin D1 expression is regulated positively by the p42/p44MAPK and negatively by the p38MAPK/HOGMAPK pathway, J. Biol. Chem. 23 (271) (1996) 20608–20616. [12] S.E. Dunn, R.A. Hardman, F.W. Kari, J.C. Barrett, Insulin-like growth factor 1 [IGF-1] alters drug sensitivity of HBL100 human breast cancer cells by inhibition of apoptosis induced by diverse anticancer drugs, Cancer Res. 157 (1997) 2687–2693. [13] J.L. Gooch, C.L. Van Den Berg, D. Yee, Insulin-like growth factor [IGF]-I rescues breast cancer cells from chemotherapy-induced cell death – proliferative and anti-apoptotic effects, Breast Cancer Res. Treat. 56 (1999) 1–10. [14] M. Dkhil, A. Abdel-Baki, S. Al-Quraishi, M. Al-Khalifa, Anti-inflammatory activity of the venom from samsumants Pachycondyla sennaarensis, Afr. J. Pharm. Pharmacol. 4 (2010) 115–118.
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