Synergistic effects of combined platelet-activating factor receptor and epidermal growth factor receptor targeting in ovarian cancer cells

The ovarian cancer cell lines CAOV-3 and SKOV-3 (purchased from the Cell Bank of the Chinese Academy of Science, Shanghai, China) were cultured at 37°C in a humidified 5% CO₂ atmosphere in RPMI-1640 medium with 10% fetal calf serum (Gibco, Invitrogen, Carlsbad, CA), 100 IU/ml penicillin G, and 100 mg/ml streptomycin sulfate (Sigma-Aldrich, St. Louis, MO). AG1478 (EGFR inhibitor)[24] and WEB2086 (PAFR antagonist)[25] were purchased from Sigma-Aldrich (St Louis, MO). A Cell Counting Kit 8 (CCK8) was purchased from Dojindo Molecular Technologies, Inc. (Kumamoto, Japan), and the Alexa Fluor 488 annexin V/Dead Cell Apoptosis Kit was obtained Invitrogen (Carlsbad, CA). Rabbit polyclonal antibodies directed against PAFR, cleaved-caspase3, cleaved-PARP, phospho/total- EGFR, phospho/total- β-arrestin2, phospho/total- P70S6K, phospho/total- AKT, phospho/total- 4EBP1, and phospho/total- ERK were used in this study. All of these antibodies were purchased from Cell Signaling Technology Co. Mouse monoclonal antibodies directed against actin were also used (Sigma, Missouri, USA).

Cellular extracts were prepared in modified radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris–HCl pH 7.4, 1% NP-40, 0.25% Na-deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, and protease inhibitor cocktail). The protein concentrations of the cellular extracts were measured using a Bio-Rad protein assay kit. The cellular extracts were subjected to SDS-PAGE, and the proteins were transferred to PVDF membranes. After blocking for 1 h at room temperature in 5% BSA, the blots were probed with the primary antibody at a 1:1000 dilution and incubated overnight at 4°C. Subsequently, the blots were washed three times and incubated for 1 h at room temperature with a 1:10000 dilution of the secondary peroxidase-conjugated antibodies. Following three washes, the immunoreactive bands were detected using electrochemiluminescence (ECL).

Immunocytochemistry was used to detect the expression of EGFR and PAFR. After fixation with 4% paraformaldehyde, cells were incubated with an anti-EGFR (1:50) or -PAFR (1:50) antibody overnight, and then incubated with peroxidase-conjugated anti-rabbit IgG for 30 min. The staining reaction was performed with diaminobenzidine. The cells were counter-stained with hematoxylin to detect nuclei, and imaged using light microscopy (Olympus, Tokyo, Japan).

Cells were seeded in 96-well plates and treated with different doses of WEB2086, AG1478, or both for 72 hours. Cell proliferation was measured with the CCK-8 assay. A 10-μL aliquot of CCK-8 solution was added to 100 μL medium in each well for 1–4 hours, and the absorbance was measured at 450 nm. The percentage of cell viability was determined relative to the control. Each experiment was performed in six replicate wells for each drug concentration. The IC50 values were calculated with the SPSS software using the bliss method.

Cell invasion activity assay were conducted with an 8 μm matrigel invasion chamber (Corning, NY). Experiments were carried out according to the manufacture’s protocal. Briefly, each well insert was coated with 100 μl mixture matrigel: serum-free medium, followed by incubation at 37°C for 4 hours. Cells were then trypsin digested and transferred into top matrigel wells (10⁵ cancer cells per well) with WEB2086 (0.1 mM), AG1478 (10 μM) or both. 600 μl of 10% FBS media was added to the bottom of the chamber and incubated for 48 hours in the invasion assay. Invasion activity was assessed by the number of cells that crossed the matrigel and filter membrane. Cell numbers were counted under a light microscope and presented as percentage compared to the controls. Experiments were performed twice, at least three repeats for each treatment.

Apoptosis was detected by flow cytometry via the examination of altered plasma membrane phospholipid packing by the lipophilic dye Annexin V as described elsewhere. Briefly, cells were treated with inhibitors and harvested at 24 hours after treatment. The cells were washed twice with PBS and then resuspended in binding buffer at a concentration of 1 × 10⁶ cells/mL according to the manufacturer’s instructions. Thereafter, 5 μL of Annexin V-FITC and 1 μL of propidium iodide were added to 100 μL of cell suspension and incubated for 20 min at room temperature in the dark. After adding 400 μL of binding buffer, the labeled cells were counted by flow cytometry within 1 hour. All early apoptotic cells (Annexin V-positive, propidium iodide -negative), necrotic/late apoptotic cells (double positive), and living cells (double negative) were detected using a FACSCalibur flow cytometer and subsequently analyzed by Cell Quest software. For the cell cycle analyses, treated cells were fixed in 70% ethanol and stored at -20°C overnight; the cells were labeled with propidium iodide (50 μg/ml) and RNase (100 μg/ml) for 30 min before flow cytometry analysis.

Female athymic nu/nu mice (4 to 6 weeks old) were obtained from the Laboratory Animal Center of the Shanghai Institutes For Biological Sciences of the Chinese Academy of Sciences. All animal studies were conducted in strict accordance with protocols approved by the Ethics Committee for Animal Experimentation of Fudan University. A total of 2 × 10⁶ CAOV-3 cells was injected into the flanks of Female athymic nu/nu mice. When established tumors of approximately 75 mm³ in Diameter were detected, the mice were randomly divided into four groups (8 mice/group), and subjected to various treatments. The PAFR antagonist WEB2086 was intraperitoneally injected at 5 mg/kg every three days for 2 weeks, and the EGFR inhibitor AG1478 was intraperitoneally injected at 10 mg/kg every three days for 2 weeks. After 3 weeks, all of the mice were killed, and the tumors were excised and weighed. The tumor size for the xenografts was determined using a caliper, and the volume was calculated as = length × width²/2, where the width is the smallest measurement and the length is the longest measurement.

Synergy between WEB2086 and AG1478 was determined by isobologram analysis using CalcuSyn software (Biosoft, Cambridge, UK), which analyzes the data from proliferation assays to determine the interaction between equipotent drug combinations[26]. The combination index values <1, =1 and >1 indicate synergy, additivity and antagonism, respectively.

As we previously reported that inhibition of PAFR decrased ovarian cancer cell proliferation and the EGFR inhibition has shown promise in clinical trials in various types of cancers, including ovarian cancer, we asked whether simultaneously targeting the two receptors would have synergistic effects. The inhibitory effects of combined use of the PAFR antagonist WEB2086 and the EGFR inhibitor AG1478 were tested in two ovarian cancer cell lines, CAOV-3 and SKOV-3 cells. We first examined the basal levels of PAFR and EGFR in both cells. As shown in Figure 1A and B, both cells express PAFR and EGFR, while the expression levels of PAFR and EGFR were higher in CAOV-3 cells compared with that in SKOV-3 cells. To determine whether WEB2086 and AG1478 exhibit a combined effect in ovarian cancer cells, we examined the effect of individual and combination treatment with WEB2086 and AG1478 after 72-h exposure using the CCK-8 assay. To ensure that the contributing effects from each drug was equivalent, IC50 for each was determined and serial dilutions were generated based on the IC50 of each drug providing 1:1 equipotent WEB2086/AG1478 ratio. We found that both compounds inhibited cell growth in a dose-dependent manner in CAOV-3 and SKOV-3 cells (Figure 1C). To determine whether WEB2086 and AG1478 interact synergistically, isobologram analysis was performed. This analysis provides a CI value that measures the degree of interaction between two or mor drug, where a CI < 1 and a CI > 1 indicates synergism and antagonism, respectively. A CI of 0.49 and 0.58 was indentified for CAOV-3 and SKOV-3 cells, when the effective dose (ED) of both agents inhibited cell viability by 50% (Figure 1D). As shown in Figure 1E, inrespective of high cytotoxicity (ED90) or low cytotoxicity (ED25), the CI values was remained below 1, indicating that synergism occurs independently of the equipotency levels of WEB2086 and AG1478. Our results demonstrate that WEB2086 and AG1478 exhibit a synergistic interaction in ovarian cancer cells, particularly in CAOV-3 cells.

To test whether the combined targeting of PAFR and EGFR resulted in enhanced growth inhibition compared with the single inhibition, half of the IC50 for each drug was used to treat ovarian cancer cells, followed by the CCK-8 assay. As shown in Figure 2A and B, the combined inhibition of both PAFR and EGFR resulted in significantly enhanced growth inhibition compared with either treatment alone over a 72-hour exposure. The results in CAOV-3 and SKOV-3 cell lines confirmed a strong inhibitory effect by adding WEB2086 to AG1478.

We previously reported that PAF promots ovarian cancer cell invasion. In addition to cell growth, we examined the effects on cell invasion of combined PAFR and EGFR targeting in ovarian cancer cells. As shown in Figure 2C and D, although WEB2086 or AG1478 alone decreased ovarian cancer cell invasion, combined targeting significantly enhanced the effect when compared with either treatment alone.

To determine whether the increased antiproliferative effect was due to increased apoptosis and/or cell cycle alterations, we examined apoptosis by Annexin V analysis following WEB2086 and AG1478 treatment. The number of apoptotic cells was quantified (Figure 3A and D). As shown in Figure 3B and E, a flow cytometric analysis of CAOV-3 and SKOV-3 cells revealed that both WEB2086 and AG1478 increased the number of apoptotic cells compared to that observed in the untreated cells; additionally, the combined targeting significantly enhanced CAOV-3 and SKOV-3 cell apoptosis to 71.43% and 78.81%, respectively. These results were confirmed by a western blot analysis. Both WEB2086 and AG1478 were able to induce the cleavage of 113-kDa poly-ADP-ribose polymerase (PARP) to the 89-kDa fragment and induce cleaved caspase-3 in CAOV-3 and SKOV-3 cells, and the combination of WEB2086 and AG1478 was accompanied by increased expression of cleaved PARP and caspase-3 (Figure 3C and F). As shown in Figure 4, a significant increase in G0/G1-phase cells after treatment with WEB2086 combined with AG1478 when compared with the single treatment in CAOV-3 and SKOV-3 cells was observed. This cell cycle delay was also accompanied by a decreased percentage of S-phase cells.

Enhanced antitumor effects were observed by combined PAFR and EGFR targeting, which indicates that intracellular signaling pathway crosstalk may occur between the activated PAFR and EGFR pathways. We next evaluated the effects of activated PAFR and EGFR on the expression of selected proteins and their activated forms, which are known to be important steps in prosurvival and proliferation pathways in ovarian cancer cells. Because mTOR pathway regulates cell growth via the phosphorylation of eukaryotic initiation factor 4E-binding protein 1 (4EBP1) and ribosomal protein S6 kinase (P70S6K), we assessed the activation of the mTOR pathway by determining the phosphorylation of P70S6K and 4EBP1. CAOV-3 cells were stimulated with PAF (from 0.1 to 1000 nM) or EGF (from 0.1 to 20 ng/ml), for 24 hours. As shown in Figure 5, both PAF and EGF evoked increased levels of activated P70S6K and 4EBP1, without affecting the total amount of P70S6K and 4EBP1. AKT and ERK phosphorylation was also increased upon stimulation with PAF and EGF, respectively. However, the levels of phosphorylated β-arrestin2 were increased by PAF stimulation, but not by EGF stimulation in CAOV-3 cells.

As we have verified that both activated PAFR and EGFR can evoke the increased phosphorylation levels of P70S6K, 4EBP1, AKT and MAPK, we next determine whether the synergistic growth inhibition effects obtained by the combination of the PAFR antagonist WEB2086 and the EGFR inhibitor AG1478, were due to a more effective inhibition of intracellular signaling, and we also analysis the effect of WEB2086 and/or AG1478 treatment on the expression level of EGFR and PAFR in both cell lines. Western blot analyses were performed on proteins from CAOV-3 and SKOV-3 cells treated with 10 μM of AG1478, 0.1 mM WEB2086, or a combination with both, treatment was conducted for 24 hours. As shown in Figure 6, the combined PAFR and EGFR targeting decreased the phosphorylation levels of EGFR and β-arrestin2 compared with AG1478 or WEB2086 treatment alone, without affecting the total amount of EGFR and PAFR. β-arrestin2 has been shown to be coupled with the activated PAFR and, therefore, the phosphorylation of β-arrestin2 indicates the PAFR activation. Our results suggest that bidirectional crosstalk may occur between PAFR and EGFR. In addition, treatment with WEB2086 in combination with AG1478 resulted in a more pronounced decrease in the levels of protein phosphorylation (p-ERK, p-AKT). The combined treatment also affected mTOR signaling, as suggested by the sustained inhibition of P70S6K phosphorylation. These results suggest that combined therapy targeting PAFR and EGFR augments antitumor efficacy by inhibiting specific downstream signaling proteins.

We finally investigated the in vivo antitumor activity of combined PAFR and EGFR targeting in nude mice bearing CAOV-3 cells that were grown subcutaneously as tumor xenografts. As shown in Figure 7, either AG1478 or WEB2086 could inhibit tumor growth compared with the control group (p < 0.001). Compared with those two treatments, the combination of AG1478 and WEB2086 led to the most significant inhibition of tumor growth (p < 0.001) at day 21 post-treatment. The combined treatment of the two drugs was well tolerated, as no obvious side effects were observed in the mice treated with AG1478 and/or WEB2086.