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. 2018 Nov 27:9:1392.
doi: 10.3389/fphar.2018.01392. eCollection 2018.

Osthole Synergizes With HER2 Inhibitor, Trastuzumab in HER2-Overexpressed N87 Gastric Cancer by Inducing Apoptosis and Inhibition of AKT-MAPK Pathway

Yun Yang  1   2   3 , Feng Ren  1 , Ziyin Tian  1 , Wei Song  4 , Binfeng Cheng  5 , Zhiwei Feng  1
Affiliations

Osthole Synergizes With HER2 Inhibitor, Trastuzumab in HER2-Overexpressed N87 Gastric Cancer by Inducing Apoptosis and Inhibition of AKT-MAPK Pathway

Yun Yang et al. Front Pharmacol. .

Abstract

Background and Purpose: Although trastuzumab has shown considerable activity in the treatment of HER2-positive breast and gastric cancers, a significant proportion of patients do not respond to trastuzumab. Recent studies revealed that osthole, an active coumarin isolated from Cnidium monnieri (L.) Cusson possesses potent anti-tumor activity. Here, we for the first time investigated the anti-tumor activity of trastuzumab in combination with osthole in HER2-overexpressing cancers. Materials and Methods: N87 and SK-BR-3 cell lines, which were HER2-overexpressing cancer cells were used in our study. Cell Counting Kit-8 (CCK-8) assay was utilized to test the inhibitory effects of trastuzumab plus osthole. Combination index (CI) values were calculated using the Chou-Talalay method. Fluorescence-Activated Cell Sorter (FACS) assay was used to examine the cell cycle change and apoptosis upon combinatorial treatment. N87 tumor xenografts were established to evaluate in vivo effects of trastuzumab plus osthole. In addition, molecular mechanisms were analyzed by Western blot in vitro and in vivo. Results: As shown in our study, osthole alone exhibited effective anti-tumor activity against HER2-overexpressed N87 gastric cancer cells and SK-BR-3 breast cancer cells, which may be attributed to cell cycle arrest on G2/M phase and apoptosis. More importantly, our data demonstrated that trastuzumab plus osthole was much more potent than either agent alone in inhibiting the growth of N87 cancer cells in vitro and in vivo, which may be partly explained by the enhanced apoptosis upon the combinatorial treatment. Besides these, we also observed a significant decrease on the phosphorylation of AKT and MAPK in N87 cells when treated with trastuzumab plus osthole compared to either agent alone. Further data from N87 tumor xenografts revealed that trastuzumab plus osthole exerted their synergistic effects mainly on AKT signaling pathway. Conclusion: Collectively, these results support the clinical development of combination osthole with trastuzumab for the treatment of HER2-overexpressed gastric cancer.

Keywords: AKT; apoptosis; gastric cancer; osthole; trastuzumab.

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Figures

FIGURE 1
FIGURE 1
Osthole inhibited the growth of N87 and SK-BR-3 cells and induced cell cycle arrest and apoptosis. (A) CCK-8 assay evaluating cell growth of N87 and SK-BR-3 cells upon treatment with increasing concentration of osthole for 48 h. (B) Cell cycle analysis of N87 cells following 40 μM osthole treatment for 0, 6, and 12 h by flow cytometry. (C) Effects of osthole on cell cycle of SK-BR-3 cells. (D) N87 and SK-BR-3 cells were treated with 40 μM osthole for 30 h and cleaved Caspase-3 and Bcl-2 were examined by Western blot.
FIGURE 2
FIGURE 2
Osthole and trastuzumab synergistically inhibited the in vitro growth of N87 cells. (A) The inhibitory effects of osthole and trastuzumab combinatorial treatment against N87 cells for 48 h. (B) The inhibitory effects of osthole and trastuzumab combinatorial treatment against SK-BR-3 cells for 48 h. (C) The synergistic effect of trastuzumab in combination with osthole on the growth of N87 cell line. Combination index (CI) values were calculated at the drug concentration of trastuzumab (3.125 μg/mL) plus osthole (6.25 μM), trastuzumab (12.5 μg/mL) plus osthole (25 μM), trastuzumab (50 μg/mL) plus osthole (100 μM) using the Chou-Talalay method. Drug synergy, addition, and antagonism are defined by C.I. values less than 1.0, equal to 1.0, or greater than 1.0, respectively. Data show the mean ± SD (three independent experiments); ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. ns, no significant difference.
FIGURE 3
FIGURE 3
Trastuzumab enhanced osthole-induced apoptosis, which may partly explained the synergistic anti-tumor effect of trastuzumab in combination with osthole. (A) Induction of apoptosis of N87 cells after control IgG (10 μg/mL), osthole (40 μM), trastuzumab (10 μg/mL) or the combinatorial treatment for 30 h. Apoptosis was measured by flow cytometry. (B) Statistical analysis of the percentages of the apoptotic cells. Data was shown with mean ± SD. (C) Cell cycle and apoptosis related protein (CyclinB1, cleaved Caspase-3, Bax or Bcl-2) was examined in N87 cells when treated with control IgG (10 μg/mL), osthole (40 μM), trastuzumab (10 μg/mL), or the combination for 30 h. (D) Quantification of Western blot signal intensity analysis is expressed relative to the β-actin loading control by using Image J software. Data show the mean ± SD (three independent experiments); ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.
FIGURE 4
FIGURE 4
Trastuzumab in combination with osthole blocked AKT pathway in a synergistic manner. (A) Immunoblots assessing AKT and MAPK signaling in the N87 cell lines upon treatment with control IgG (10 μg/mL), trastuzumab (10 μg/mL), osthole (40 μM), or trastuzumab (10 μg/mL) plus osthole (40 μM) for 30 h. Data are representative of three independent experiments. (B) Quantification of Western blot signal intensity analysis is expressed relative to the β-actin loading control by using Image J software. Data show the mean ± SD (three independent experiments); ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.
FIGURE 5
FIGURE 5
Trastuzumab plus osthole combinatorial treatment inhibits the growth of N87 cancer cells in vivo. (A) Tumor volume of N87 xenografts after injection with control IgG (15 mg/kg), Trastuzumab (15 mg/kg), Osthole (100 mg/kg), or Trastuzumab (15 mg/kg) plus Osthole (100 mg/kg). (B) On day 16 post first injection, xenograft tumors from each group were removed and photographed. Representative tumors in each group were shown. (C) After xenograft tumors were removed, these tumors were weighted. (D) Effects of agents on tumor-bearing mice body weight were determined using N87 tumor-bearing nude mice. Mice were weighed at regular intervals during the whole period to monitor unspecific toxicity. Data are shown as mean ± SD. (n = 6 mice, each group); ∗∗p < 0.01; ∗∗∗p < 0.001.
FIGURE 6
FIGURE 6
Trastuzumab in combination with osthole inhibited AKT signaling pathway in vivo. (A) Tumor tissues isolated from N87 xenografts upon treatment with control IgG (15 mg/kg), Trastuzumab (15 mg/kg), Osthole (100 mg/kg), Sor Trastuzumab (15 mg/kg) plus Osthole (100 mg/kg) were then subjected to Western blot to detect the expression of AKT, p-AKT, MAPK and p-AMPK. Data are representative of three independent experiments. (B) Quantification of Western blot signal intensity analysis is expressed relative to the -actin loading control by using Image J software. Data show the mean ± SD (three independent experiments); ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.
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