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Introduction

ErbB2 (or HER2) overexpression is found in 25–30% of breast cancer and 4–50% of gastric cancer cases (1–3). Trastuzumab is an anti-ErbB2 (human) antibody which is used for the clinical treatment of ErbB2-amplified metastatic breast, metastatic gastric and gastro-esophageal junction cancer (4,5). However, ~70% of patients with cancer do not respond to trastuzumab, and most of trastuzumab-responsive patients develop resistance to trastuzumab within 1 year of treatment initiation (6–8).

Despite cancer progression on ErbB2-directed therapies (9–12), ErbB2 remains considered as a valid therapeutic target. Pertuzumab is another anti-ErbB2 (human) antibody, that binds to an epitope of ErbB2 different from trastuzumab (9,13). Among the strategies to overcome trastuzumab resistance, the combination of pertuzumab and trastuzumab has provided clinical benefits (9,13). However, the objective response rate is 24.2% and the complete response rate is ~8% (9). Therefore, there is an urgent need to develop novel strategies to overcome trastuzumab resistance.

It is well established that pertuzumab recognizes domain II of ErbB2, while trastuzumab binds to domain IV. When cells are stimulated with the ErbB3 ligand, pertuzumab efficiently inhibits the formation of the ErbB2-ErbB3 complex (14), whereas the same effect is not observed with trastuzumab (14). Notably, in the absence of the ErbB3 ligand, the extent of ErbB2-ErbB3 complex formation is markedly decreased with trastuzumab treatment, whereas pertuzumab induces a very minor decrease in the formation of the ErbB2-ErbB3 complex (15). The combination of trastuzumab and pertuzumab provides a complementary mechanism of action that synergistically inhibits the proliferation of ErbB2-overexpressing breast cancer cell lines, both in vitro and in vivo (16,17). Trastuzumab predominantly interferes with ligand-independent ErbB2-ErbB3 complex formation, whereas pertuzumab inhibits ligand-induced ErbB2 heterodimerization (14,15). The clinical success of the combination of pertuzumab and trastuzumab may be partially explained by the ability to inhibit ErbB2 heterodimerization more thoroughly.

In previous studies, an ErbB2 domain I-specific human antibody, H2-18, was developed, which exhibited a more potent antitumor activity than trastuzumab and pertuzumab, either alone or in combination, in trastuzumab-resistant breast and gastric cancer cells (13,18). H2-18 functions by potently inducing programmed cell death (PCD), a different mechanism of action from either trastuzumab or pertuzumab (13,18). Therefore, it was speculated that the two anti-ErbB2 antibodies, H2-18 and trastuzumab, which have different mechanisms of action, may also achieve a synergistic effect on the inhibition of trastuzumab-resistant cancer.

In the present study, the in vivo and in vitro antitumor capability of H2-18 plus trastuzumab in the trastuzumab-sensitive gastric cancer cell line, NCI-N87, and trastuzumab-resistant gastric cancer cell line, NCI-N87-TraRT, was investigated. Additionally, the antitumor effect of H2-18 plus trastuzumab was compared with that of pertuzumab plus trastuzumab.

Materials and methods

Antibodies

The H2-18 antibody was expressed and purified using a method as previously described (19). The recombinant antibody was purified by affinity chromatography on Protein A Sepharose (GE Healthcare). The purified antibodies were analyzed via 10% SDS-PAGE under non-reducing and reducing conditions, followed by Coomassie Brilliant Blue staining. Under reducing conditions, the H2-18 antibody yielded two protein bands with a molecular mass of ~50 kDa (heavy chain) and ~25 kDa (light chain), respectively (Fig. S1). The SDS-PAGE analysis under non-reducing conditions exhibited a single band at ~150 kDa for the H2-18 antibody (Fig. S1).

The anti-ErbB2 antibodies trastuzumab and pertuzumab and the anti-CD20 antibody rituximab were expressed and purified by a similar method described in previous studies (20–22). The drug concentrations used in the present experiments and the ratio of anti-ErbB2 antibodies in antibody combinations were based on previous experiments (13,23).

Cell lines and mice

NCI-N87 is an ErbB2-amplified human gastric cancer cell line. BT-474 is an ErbB2-amplified human breast cancer cell line. All the cell lines were obtained from the American Type Culture Collection and were routinely cultured in DMEM (Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Thermo Fisher Scientific, Inc.), 100 U/ml penicillin, and 100 µg/m streptomycin (Gibco; Thermo Fisher Scientific, Inc.) at 37°C in a humidified incubator with 5% CO2. NCI-N87 cells were treated consecutively with trastuzumab (10 µg/ml) for 2 years to obtain a trastuzumab-resistant subline cell line, termed NCI-N87-TraRT. The cells were authenticated by morphologic and isoenzyme analyses, various times during the study period. They were routinely checked for mycoplasma contamination using Hoechst staining, which was consistently found to be negative. The BALB/c nude mice were obtained from the Shanghai Experimental Animal Center of the Chinese Academy of Sciences. The cages with food and water were changed twice a week, and the mice were fed ad libitum. The targeted conditions for the animal room environment and photo period were as follows: Temperature, 20–26°C; humidity, 40–70%; light/dark cycle, 12/12 h. All the animals were treated according to the guidelines of the Committee on Animals of the Second Military Medical University (Shanghai, China). The study was approved by the Committee on Animals of the Second Military Medical University.

Detection of ErbB2 expression

BT-474, NCI-N87 and NCI-N87-TraRT cells were treated with anti-ErbB2 primary antibodies (trastuzumab, 10 µg/ml) or control anti-CD20 antibodies (rituximab, 10 µg/ml) for 1 h on ice at 4°C. After washing 3 times, secondary goat anti-human IgG H&L (FITC) (1:100; cat. no., ab6854; Abcam) were added and incubated with cells for 40 min at 0°C. After washing 3 times, the cells were resuspended in PBS, measured using a FACSCalibur flow cytometer (Becton, Dickinson and Company), then analyzed using FlowJo v7.6.1 software (FlowJo LLC).

Cell proliferation experiments

The cells were seeded in a 96-well plate at a density of 4×103 cells/well in a humidified chamber at 37°C with 5% CO2. After attachment, cells were treated with control IgG (5 µg/ml), trastuzumab (5 µg/ml), pertuzumab (5 µg/ml), H2-18 (5 µg/ml), trastuzumab plus pertuzumab (5 µg/ml for both), trastuzumab plus H2-18 (5 µg/ml for both) or trastuzumab plus pertuzumab plus H2-18 (5 µg/ml for each) at 37°C in the humidified incubator with 5% CO2 for 5 days. The medium was changed every 2 days. Finally, the cells were incubated with Cell Counting Kit-8 (CCK-8) reagent for 60–90 min and the cell proliferation was determined according to the manufacturer's instructions (Dojindo Molecular Technologies, Inc.).

Analysis of single and combined drug effects

Cells were seeded in a 96-well plate at a density of 4×103 cells/well. After attachment, the cells were treated with a range of concentrations of the aforementioned antibodies for 5 days. The media was refreshed every 2 days. Finally, the CCK-8 assay was used to assess the cell proliferation inhibition rate. The cell proliferation inhibition rate was calculated as follows: [1-(treated cells/untreated cells)] ×100%. Combination index (CI) values were calculated using the Chou-Talalay method (24). CI values <1.0 represented drug synergy, CI values=1.0 represented drug addition and CI values >1.0 indicated drug antagonism.

Reactive oxygen species (ROS) detection

The 2′,7′-dichlorofluorescin diacetate (DCFH-DA; Sigma-Aldrich; Merck KGaA) was used to detect ROS. Cells were seeded in a flat-bottomed 24-well plate at a density of 1×105 cells/well. After a 4-h incubation at 37°C with control IgG (5 µg/ml), trastuzumab (5 µg/ml), pertuzumab (5 µg/ml), H2-18 (5 µg/ml), trastuzumab plus pertuzumab (5 µg/ml for both), trastuzumab plus H2-18 (5 µg/ml for both) or trastuzumab plus pertuzumab plus H2-18 (5 µg/ml for each), the cells were incubated with 10 µM DCFH-DA for 20 min at 37°C. After washing twice with PBS, the fluorescence level of the cells treated with DCFH-DA was measured using a FACSCalibur flow cytometer (Becton, Dickinson and Company) and analyzed with FlowJo v7.6.1 software (FlowJo LLC).

Immunoblotting

Cells were seeded in a 24-well plate at a density of 1×105 cells/well in a humidified atmosphere at 37°C with 5% CO2. After attachment, the cells were incubated with the aforementioned drugs. Subsequently, cells were lysed in RIPA buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate and 0.1% SDS) containing a protease inhibitor cocktail (cat. no. 11836153001; Roche Diagnostics) and a phosphatase inhibitor cocktail (cat. no. P5726; Sigma-Aldrich; Merck KGaA). Protein concentrations were quantified using a BCA Protein Assay kit (cat. no. 23225; Pierce: Thermo Fisher Scientific, Inc.). Equal amounts of protein (15 µg/lane) were separated via 10% SDS-PAGE and transferred to PVDF membranes. After blocking with 5% BSA (cat. no. A1933; Sigma-Aldrich; Merck KGaA) for 1 h at 37°C, the membranes were immunoblotted with antibodies against HER2 (cat. no. 4290s), phosphorylated (p)-HER2 (cat. no. 2243s), HER3 (cat. no. 12708s), p-HER3 (cat. no. 2842s), AKT (cat. no. 9272s), p-AKT (cat. no. 2965s), ERK1/2 (cat. no. 9102s), p-ERK1/2 (cat. no. 9106s), JNK (cat. no. 9252s), p-JNK (cat. no. 9251s), c-jun (cat. no. 9165s), p-c-jun (cat. no. 2361s), and GAPDH (1:2,000; cat. no. cat. no.5174S) (all from Cell Signaling Technology, Inc.) overnight at 4°C. After washing with PBS-Tween 20 (0.05%), the membranes were immunoblotted with anti-rabbit IgG HRP-conjugated secondary antibody (1:3,000; cat. no. 7074s; Cell Signaling Technology, Inc.) at room temperature for 1 h. Finally, the ECL reagents (Plus-ECL; PerkinElmer, Inc.) or chemiluminescence reagents (EMD Millipore) were used to detect the proteins. Each band was quantified using TanonImage software (v1.0; Tanon Science and Technology Co., Ltd.).

Cell death assay

Cells were seeded at a density of 1×105 cells/well in a flat-bottomed 24-well plate in the DMEM supplemented with 10% FBS (Thermo Fisher Scientific, Inc.), 100 U/ml penicillin, and 100 µg/ml streptomycin (Gibco; Thermo Fisher Scientific, Inc.) at 37°C in a humidified incubator with 5% CO2. After 24 h of attachment at 37°C, cells were treated with control IgG (5 µg/ml), trastuzumab (5 µg/ml), pertuzumab (5 µg/ml), H2-18 (5 µg/ml), trastuzumab (5 µg/ml) plus pertuzumab (5 µg/ml), trastuzumab (5 µg/ml) plus H2-18 (5 µg/ml) and trastuzumab (5 µg/ml) plus pertuzumab (5 µg/ml) plus H2-18 (5 µg/ml). After 24 h of treatment at 37°C, the cells were harvested and incubated with 5 µl 488-conjugated annexin-V and 0.1 µl PI (both from Tianjin Sungene Biotech Co., Ltd) per well for 15 min at room temperature. Finally, the cells were measured using a FACSCalibur flow cytometer (Becton, Dickinson and Company) and analyzed using FlowJo 7.6.1 software (FlowJo LLC).

Colony formation assay

Cells were seeded at a density of 800 cells/well in a 6-well plate. After 24 h of attachment at 37°C, the cells were treated with control IgG (10 µg/ml), trastuzumab (10 µg/ml), pertuzumab (10 µg/ml), H2-18 (10 µg/ml), trastuzumab (10 µg/ml) plus pertuzumab (10 µg/ml), trastuzumab (10 µg/ml) plus H2-18 (10 µg/ml), and trastuzumab (10 µg/ml) plus pertuzumab (10 µg/ml) plus H2-18 (10 µg/ml) for 5 days at 37°C. The medium was changed every 3–4 days. After 5 days of treatment, the medium was replaced with fresh RPMI 1640 medium and cultured for another 12 days at 37°C. Finally, the colonies were fixed with 4% paraformaldehyde at room temperature for 15 min and then stained with 0.1% crystal violet at room temperature for ~10 min. The number of stained colonies containing >50 cells were recorded under a light microscope (×40).

In vivo animal experiments

NCI-N87 or NCI-N87-TraRT cells were resuspended in PBS and implanted into the right mammary fat pad of 78 6-week-old female BALB/c nude mice (1×107 cells/mouse; 78 mice in total; weight, 17–20 g; in the NCI-N87 model 7 mice per cohort, 6 cohorts in total; in the NCI-N87-TraRT model 6 mice per cohort, 6 cohorts in total). When the average volume of tumors reached 100 mm3, the mice were randomly grouped into cohorts. The mice were injected intravenously with control IgG (10 mg/kg), trastuzumab (10 mg/kg), H2-18 (10 mg/kg), trastuzumab (10 mg/kg) plus pertuzumab (10 mg/kg), trastuzumab (10 mg/kg) plus H2-18 (10 mg/kg) and trastuzumab (10 mg/kg) plus pertuzumab (10 mg/kg) plus H2-18 (10 mg/kg), twice a week for 3 weeks. Tumors were measured with digital calipers twice a week and the volumes were calculated using the following formula: Volume (mm3)=length × (width)2/2. The duration of the experiment was 39 days. The following humane endpoints were used: Loss of significant body mass (emaciated); obvious body weight loss >20% of initial body weight; animals could not get to adequate food or water; cachexia appearance was found; and tumor volume reached ≥2,000 mm3. All the 78 nude mice were euthanized at the end of the experiment or when they had reached these humane endpoints. The mice were sacrificed by CO2 inhalation (with a flow rate of 30%/min).

Statistical analysis

SPSS v19.0 software (SPSS, Inc.) was used for the analysis. Normality tests were conducted before data analysis. One-way ANOVA with Tukey's multiple comparison post-hoc test was used to identify significant differences among multiple groups, unless otherwise indicated. The in vivo animal experiments were analyzed using Kruskal-Wallis test with Dunn's post-hoc test. The data are presented as the mean ± SD (n=3). P<0.05 was considered to indicate a statistically significant difference.

Results

Addition of H2-18 to trastuzumab enhances its inhibitory effect on cell proliferation of ErbB2-overexpressing cancer cell lines

The trastuzumab-resistant cancer cell line, NCI-N87-TraRT, was derived from the trastuzumab-sensitive cancer cell line, NCI-N87 (13). It has been reported that NCI-N87 and BT-474 are high-ErbB2-expressing cell lines (25,26). Flow cytometry was used to examine ErbB2 expression in the cancer cell lines, NCI-N87, NCI-N87-TraRT and BT-474. The results revealed that ErbB2 expression in NCI-N87-TraRT cells was significantly higher compared with that in the NCI-N87 cells (Fig. 1A). Compared with that in the ErbB2-amplified BT-474 cells, NCI-N87 and NCI-N87-TraRT cells exhibited higher levels of ErbB2 receptor (Fig. 1A). The protein expression levels of ErbB2 in these cells were also confirmed using western blotting (Fig. 1B).

Figure 1. Addition of H2-18 to trastuzumab enhances the inhibitory effect on cell proliferation of ErbB2-overexpressing cancer cell lines. (A) Flow cytometry examining the expression levels of ErbB2 in the human gastric cancer cell lines NCI-N87 and NCI-N87-TraRT and the human breast cancer cell line, BT-474. (B) Immunoblots examining ErbB2 protein expression in NCI-N87, NCI-N87-TraRT and BT-474 cells. (C) Cell Counting Kit-8 assay was used to compare cell proliferation of the gastric cancer NCI-N87 and NCI-N87-TraRT cell lines upon the indicated treatments. *P<0.05; **P<0.01; ***P<0.001. NEG, negative control; T, trastuzumab; P, pertuzumab; H, H2-18.

Subsequently, CCK-8 assays were used to determine the cell proliferation inhibitory effect of trastuzumab, pertuzumab, H2-18, trastuzumab plus pertuzumab, trastuzumab plus H2-18 and the combination of H2-18, trastuzumab and pertuzumab on NCI-N87 and NCI-N87-TraRT cells. In both ErbB2-overexpressing cancer cell lines, NCI-N87 and NCI-N87-TraRT, trastuzumab plus pertuzumab exhibited a significantly more potent inhibitory effect than trastuzumab, pertuzumab or H2-18 alone (Fig. 1C). Notably, trastuzumab plus H2-18 inhibited cell proliferation more effectively than trastuzumab plus pertuzumab (Fig. 1C). Moreover, the combination of H2-18, trastuzumab and pertuzumab exhibited the maximal inhibitory effect among all of the monoclonal antibody (mAb) combinations tested (Fig. 1C).

H2-18 and trastuzumab synergistically inhibits the cell proliferation of both trastuzumab-sensitive and trastuzumab-resistant cancer cells

The ErbB2-overexpressing trastuzumab-sensitive cancer cell line, NCI-N87, and the trastuzumab-resistant cancer cell line, NCI-N87-TraRT, were treated with various concentrations of H2-18 alone, trastuzumab alone or trastuzumab plus H2-18. The results revealed that H2-18 inhibited cell proliferation in a dose-dependent manner in both cell lines (Fig. 2A and B). The method described by Chou and Talalay was used to analyze the data. Synergy was defined as a CI value (at ED90, ED75 or ED50) <1.0, antagonism as a CI value >1.0 and additivity as a CI value of 1.0. The results demonstrated that in both cell lines, H2-18 and trastuzumab inhibited the in vitro cell proliferation synergistically (Fig. 2A and B). In addition, NCI-N87 and NCI-N87-TraRT cells were treated with various concentrations of H2-18 alone, trastuzumab plus pertuzumab and the combination of H2-18, trastuzumab and pertuzumab. Similar results were observed (Fig. 2A and B), with the combination of H2-18 with trastuzumab plus pertuzumab synergistically inhibiting the in vitro proliferation of NCI-N87 and NCI-N87-TraRT cells (Fig. 2A and B).

Figure 2. H2-18 and trastuzumab synergistically inhibit the proliferation of both trastuzumab-sensitive and trastuzumab-resistant cancer cells. Cell Counting Kit-8 assays were used to compare the proliferation of (A) NCI-N87 cells and (B) NCI-N87-TraRT cells upon the indicated treatments. CI values were calculated using the Chou-Talalay method. Drug synergy, addition and antagonism were defined as CI values <1.0, =1.0 or >1.0, respectively. The experiments were performed at least three times. CI, combination index; T, trastuzumab; P, pertuzumab; H, H2-18; ED, effective dose; Fa, given effect; Dm, dose of drugs that exhibit a 50% inhibition; m, slope; r, regression coefficient.

Trastuzumab plus H2-18 exhibits a more potent inhibitory effect on colony formation compared with trastuzumab plus pertuzumab in NCI-N87-TraRT cells

In the trastuzumab-sensitive NCI-N87 cell line, trastuzumab plus H2-18 exhibited a similar inhibitory effect on the colony forming ability as that of trastuzumab plus pertuzumab; however, in trastuzumab-resistant NCI-N87-TraRT cells, trastuzumab plus H2-18 significantly decreased the formation of colonies more effectively than trastuzumab plus pertuzumab (Fig. 3). Notably, the combination of H2-18, trastuzumab and pertuzumab inhibited colony formation more potently than trastuzumab plus pertuzumab (Fig. 3).

Figure 3. Trastuzumab plus H2-18 exhibits a more potent inhibitory effect on colony formation compared with trastuzumab plus pertuzumab in trastuzumab-resistant NCI-N87-TraRT cells. Representative images of colony formation assays in NCI-N87 and NCI-N87-TraRT cells with the indicated treatments are presented. The data are presented as the mean ± SD. *P<0.05; **P<0.01; ***P<0.001. NEG, negative control; T, trastuzumab; P, pertuzumab; H, H2-18.

Trastuzumab plus H2-18 exhibits a greater PCD inducing ability than trastuzumab plus pertuzumab

In both NCI-N87-TraRT and NCI-N87 cell lines, trastuzumab, pertuzumab and trastuzumab plus pertuzumab did not effectively induce cell death, whereas H2-18, trastuzumab plus H2-18 and the combination of H2-18, trastuzumab and pertuzumab significantly increased cell death (Fig. 4). However, trastuzumab plus H2-18 and the combination of H2-18, trastuzumab and pertuzumab did not induce more annexin V+ and PI+ cells than H2-18 alone (Fig. 4).

Figure 4. Trastuzumab plus H2-18 exhibits a greater programmed cell death inducing ability compared with trastuzumab plus pertuzumab. Cell death induced by trastuzumab, pertuzumab, trastuzumab plus pertuzumab, H2-18, trastuzumab plus H2-18 and trastuzumab plus pertuzumab plus H2-18 was examined by flow cytometry using an Annexin V/PI double-staining kit in the gastric cancer NCI-N87 and NCI-N87-TraRT cell lines. The data are presented as the mean ± SD. *P<0.05; **P<0.01; ***P<0.001. NEG, negative control; T, trastuzumab; P, pertuzumab; H, H2-18.

Trastuzumab plus H2-18 inhibits in vivo tumor growth more potently than trastuzumab plus pertuzumab

In the NCI-N87 tumor xenografts, trastuzumab plus H2-18 exhibited a more potent inhibitory effect on tumor growth compared with that in the mice treated with trastuzumab or H2-18 alone, and the combination of H2-18, trastuzumab and pertuzumab further augmented this inhibitory effect (Fig. 5). In the NCI-N87-TraRT tumor xenografts, trastuzumab alone did not effectively inhibit tumor growth, whereas H2-18 alone was similar to trastuzumab plus pertuzumab in terms of the inhibitory effect on tumor growth (Fig. 5). Trastuzumab plus H2-18 exhibited a more potent ability to inhibit tumor growth than trastuzumab plus pertuzumab (Fig. 5). Trastuzumab plus H2-18 and pertuzumab exhibited a more potent inhibitory effect on tumor growth compared with that in the mice treated with H2-18 and trastuzumab plus H2-18 (Fig. 5).

Figure 5. Trastuzumab plus H2-18 inhibits in vivo tumor growth more potently than trastuzumab plus pertuzumab. Tumor volume of NCI-N87 and NCI-N87-TraRT gastric tumor xenografts after treatment with control IgG (10 mg/kg, twice a week), trastuzumab (10 mg/kg, twice a week), trastuzumab (10 mg/kg, twice a week) plus pertuzumab (10 mg/kg, twice a week), H2-18 (10 mg/kg, twice a week), trastuzumab (10 mg/kg, twice a week) plus H2-18 (10 mg/kg, twice a week) and trastuzumab (10 mg/kg, twice a week) plus pertuzumab (10 mg/kg, twice a week) plus H2-18 (10 mg/kg, twice a week). The data are presented as the mean ± SEM. *P<0.05 and **P<0.01 analyzed by Kruskal-Wallis test. T, trastuzumab; P, pertuzumab; H, H2-18.

Trastuzumab plus H2-18 significantly induces ROS production compared with trastuzumab plus pertuzumab

In both the NCI-N87-TraRT and NCI-N87 cell lines, H2-18, trastuzumab plus H2-18 and the combination of H2-18, trastuzumab and pertuzumab significantly induced ROS production compared with that in the cells treated with trastuzumab and pertuzumab alone and with trastuzumab plus pertuzumab (Fig. 6).

Figure 6. Trastuzumab plus H2-18 activates ROS production in both NCI-N87 and NCI-N87-TraRT cell lines. 2′,7′-dichlorofluorescin diacetate was detected by flow cytometry to measure the levels of ROS production in NCI-N87 and NCI-N87-TraRT cells treated with trastuzumab, pertuzumab, trastuzumab plus pertuzumab, H2-18, trastuzumab plus H2-18 and trastuzumab plus pertuzumab plus H2-18 for 4 h. The data are presented as the mean ± SD (n=3). *P<0.05; **P<0.01. NEG, negative control; T, trastuzumab; P, pertuzumab; H, H2-18; ROS, reactive oxygen species.

Trastuzumab plus H2-18 activates the ROS-JNK-c-jun signaling pathway in both NCI-N87 and NCI-N87-TraRT cell lines

As reported in a previous study, H2-18 induces programmed cell death through activating the RIP1-ROS-JNK-c-jun signaling pathway (18). Compared with trastuzumab plus pertuzumab, trastuzumab plus H2-18 effectively activated the phosphorylation of both JNK and c-jun in NCI-N87 cells and NCI-N87-TraRT cells (Fig. 7). The combination of H2-18, trastuzumab and pertuzumab exhibited a similar effect on the upregulation of p-JNK and p-c-jun to that of trastuzumab plus H2-18 (Fig. 7).

Figure 7. Trastuzumab plus H2-18 activates the ROS-JNK-c-jun signaling pathway and inhibits ErbB2 signaling pathways in both NCI-N87 and NCI-N87-TraRT cell lines. (A) Immunoblots examining the phosphorylation of HER2, HER3, AKT, ERK1/2, JNK and c-jun in NCI-N87 and NCI-N87-TraRT cells treated with trastuzumab, pertuzumab, trastuzumab plus pertuzumab, H2-18, trastuzumab plus H2-18 and trastuzumab plus pertuzumab plus H2-18 for 4 h. Quantification of the phosphorylation of HER3, AKT, ERK, JNK and c-jun in (B) NCI-N87 and (C) NCI-N87-TraRT cells following the indicated drug treatments. The data are presented as the mean ± SD. *P<0.05; **P<0.01; ***P<0.001. NEG, negative control; T, trastuzumab; P, pertuzumab; H, H2-18; p-, phosphorylated.

Trastuzumab plus H2-18 inhibits ErbB2 signaling pathways in both NCI-N87 and NCI-N87-TraRT cell lines

Western blotting was used to determine the status of ErbB2 signaling pathways in the NCI-N87 and NCI-N87-TraRT cells treated with the various mAb combinations. The results revealed that in NCI-N87 cells, all of the mAb combinations exhibited a more potent ability to inhibit the expression levels of p-HER3, p-AKT and p-ERK compared with any of the mAbs alone (Fig. 7A and B). Trastuzumab plus H2-18 inhibited p-AKT expression more effectively than trastuzumab plus pertuzumab, whereas trastuzumab plus H2-18 and trastuzumab plus pertuzumab exhibited a similar effect on the inhibition of p-HER3 and p-ERK expression (Fig. 7A and B). In the NCI-N87-TraRT cells, the ability of trastuzumab plus H2-18 to inhibit p-HER3, p-AKT and p-ERK expression was similar to that of trastuzumab plus pertuzumab (Fig. 7A and C). The combination of H2-18, trastuzumab and pertuzumab exhibited a significantly more potent effect on the inhibition of p-HER3, p-AKT and p-ERK expression compared with that in cells treated with trastuzumab plus pertuzumab in both the NCI-N87 and NCI-N87-TraRT cell lines (Fig. 7A-C). In addition, treatment with all the combinations of the mAbs or alone had no significant effect on the levels of HER2 and pHER2 (Fig. 7).

Discussion

The limited antitumor effect of trastuzumab, as well as the common occurrence of trastuzumab resistance, has driven numerous studies to develop new trastuzumab-based strategies (27,28). Previous studies have demonstrated that trastuzumab only partially inhibits ErbB2-dimer formation; ErbB2 heterodimerization may still initiate signaling events that confer resistance when ErbB2 is inhibited by trastuzumab (29,30). The combination of the two anti-ErbB2 antibodies, pertuzumab and trastuzumab, which have a complementary mechanism of action, has been reported to overcome trastuzumab resistance (9). However, the response rate of trastuzumab plus pertuzumab is currently unsatisfactory. The final results of a phase 3 study, JACOB, revealed that the addition of pertuzumab to trastuzumab for chemotherapy did not significantly improve OS in patients with HER2+ metastatic gastric or gastro-oesophageal junction cancer (31,32). Therefore, further studies are required to investigate novel agents for the treatment of gastric cancer.

A trastuzumab-resistant NCI-N87-TraRT cell line derived from trastuzumab-sensitive NCI-N87 cells has been developed after two years of trastuzumab treatment (13). In the present study, the growth inhibition rate of NCI-N87-TraRT cells was very low, <20% when treated with trastuzumab, while the growth inhibition rate of the parental NCI-N87 cell line was ~40%.

An ErbB2 domain I-specific antibody, H2-18, has been previously described (18). It is well-known that trastuzumab and pertuzumab function by blocking ErbB2 dimerization and inhibiting the activation of the main downstream signaling pathways of ErbB2: PI3K/AKT and MAPK/ERK signaling pathways (19). However, neither trastuzumab nor pertuzumab significantly induced cell death in the present study. In contrast to trastuzumab and pertuzumab, H2-18 has a unique ability to overcome trastuzumab resistance both in vitro and in vivo (18). The main mechanism of action underlying the ability of H2-18 to overcome trastuzumab resistance is to induce programmed cell death through the activation of the RIP1-ROS-JNK-c-Jun signaling pathway (18). In the present study, it was found that H2-18 plus trastuzumab was more effective than pertuzumab plus trastuzumab in inhibiting cell proliferation in both the trastuzumab-sensitive NCI-N87 cells and trastuzumab-resistant NCI-N87-TraRT cells. Moreover, H2-18 and trastuzumab exhibited a synergistic antitumor effect and a more potent ability to inhibit the growth of NCI-N87-TraRT tumors than pertuzumab plus trastuzumab. Further experiments demonstrated that trastuzumab plus H2-18 decreased the formation of colonies more effectively than trastuzumab plus pertuzumab in trastuzumab-resistant NCI-N87-TraRT cells. Notably, H2-18 plus trastuzumab exhibited a more potent ability to induce cell death than pertuzumab plus trastuzumab. The expression levels of p-HER2, p-HER3, p-AKT and p-ERK in the NCI-N87-TraRT cells treated with trastuzumab plus H2-18 were similar to those in NCI-N87-TraT cells treated with trastuzumab plus pertuzumab. However, in contrast to trastuzumab plus pertuzumab, trastuzumab plus H2-18 effectively activated the phosphorylation of both JNK and c-jun in NCI-N87 cells and NCI-N87-TraRT cells. Thus, the superior antitumor efficacy of H2-18 plus trastuzumab over pertuzumab plus trastuzumab may be attributable to its enhanced ability to inhibit colony formation and to induce cell death.

In addition to H2-18 plus trastuzumab, the present study investigated the antitumor effects of the combination of H2-18, pertuzumab and trastuzumab. It is known that trastuzumab, pertuzumab and H2-18 recognize different epitopes of the ErbB2 molecule. Trastuzumab recognizes domain IV of ErbB2, while pertuzumab binds with domain II and H2-18 targets domain I. These anti-ErbB2 antibodies have different mechanisms of action on the molecule (18). The present results revealed that H2-18 plus pertuzumab plus trastuzumab provided the most potent antitumor effect compared with all of the other mAbs alone, as well as all the other combinations of mAbs, in both N87 and NCI-N87-TraRT cell lines. Therefore, the present study suggested that combination therapy of these various anti-ErbB2 antibodies may provide a novel strategy for the treatment of ErbB2-amplified cancer. However, there were several limitations in the present study, including the lack of safety and toxicity studies, the lack of further mechanistic studies and the lack of use of inhibitors of the JNK and c-jun signaling pathways. Thus, further studies are required in the future.

In summary, the present study investigated the antitumor effect of the combination of H2-18 and trastuzumab, as well as their associated mechanisms of action. The present data indicated that H2-18 plus trastuzumab exhibited a superior antitumor effect over pertuzumab plus trastuzumab in trastuzumab-resistant cancer cells. The strong antitumor efficacy of H2-18 plus trastuzumab may be attributable to a number of factors, including a more effective inhibition of colony formation and more potent induction of cell death. The present data suggested that H2-18 plus trastuzumab may have the potential to be an effective strategy to circumvent trastuzumab resistance in ErbB2-overexpressing cancer.

Supplementary Material

Supporting Data

Acknowledgements

Not applicable.

Funding

The present study was supported by the National Natural Science Foundation of China (grant nos. 81572996, 81830052, 81573004 and 81773275), the Construction Project of Shanghai Key Laboratory of Molecular Imaging (grant no. 18DZ2260400), the Shanghai Municipal Education Commission Class II Plateau Disciplinary Construction Program of Medical Technology of Shanghai University of Medicine and Health Sciences, (2018–2020), the Shanghai Health and Family Planning Commission Foundation Youth Project (grant no. 20164Y0272), the Medical Science and Technology Project of Zhejiang Province (grant no. 2020391513), the Top-level Clinical Discipline Project of Shanghai Pudong (grant no. PWYgf2018-04) and the Pudong New District Health and Family Planning Commission Youth Science and Technology Project (grant no. PW2016B-4).

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors' contributions

CW, LW and YM performed the experiments. CW, LW, BL, BZ, YS, YM, JD, LC and BL analyzed the data. LW and BL wrote the manuscript. The authenticity of data was confirmed by CW, LW and BL. All authors read and approved the final manuscript.

Ethics approval and consent to participate

All the animals were treated according to the guidelines of the Committee on Animals of the Second Military Medical University (Shanghai, China). The study was approved by the Committee on Animals of the Second Military Medical University.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References