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Pretreatment with Gemcitabine/5-Fluorouracil Enhances the Cytotoxicity of Trastuzumab to HER2-Negative Human Gallbladder Cancer Cells In Vitro and In Vivo.

ABSTRACT: The effects of standard clinical therapies including surgery and chemotherapy are poor in advanced gallbladder cancer (GBC). There are a few reported cases of human epidermal growth factor receptor 2 (HER2)-positive GBC that responded well to trastuzumab. But trastuzumab has not yet been used to treat HER2-negative GBC. In this study, we investigated the cytotoxic effects of different combined therapies with trastuzumab and gemcitabine and/or 5-fluorouracil on HER2-negative GBC cell lines in vitro and in vivo. Trastuzumab alone showed almost no cytotoxicity to GBC cells with originally low HER2 gene amplification. Sequential therapy with chemotherapy followed by trastuzumab showed superiority over reverse sequential chemotherapy (P<0.05), concurrent combined chemotherapy (P<0.05), chemotherapy alone (P<0.05), and trastuzumab alone (P<0.05) in terms of cytotoxicity. Sequential therapy with chemotherapy followed by trastuzumab nearly completely inhibited cell viability in HER2-negative GBC cells. Similar results were observed with regard to apoptosis. Western blot analysis showed that gemcitabine/5-fluorouracil increased the expressions of total and phosphorylated forms of HER2, thus enhancing the cytotoxicity of trastuzumab. In vivo study verified the results of in vitro study by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assay and immunohistochemical analysis. Moreover, not only the lightest tumor bearing but also the best survival state was detected in sequential therapy with chemotherapy followed by trastuzumab group compared with other groups. Our in vivo and in vitro data suggest that sequential therapy with gemcitabine/5-fluorouracil followed by trastuzumab represents a novel and promising therapeutic strategy against HER2-negative GBC. The upregulation of phosphorylated HER2 and phosphorylated-AKT induced by gemcitabine/5-fluorouracil treatment shows that HER2/AKT pathway is triggered.

SUBMITTER: Wang W

PROVIDER: S-EPMC6452559 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Pretreatment with Gemcitabine/5-Fluorouracil Enhances the Cytotoxicity of Trastuzumab to HER2-Negative Human Gallbladder Cancer Cells In Vitro and In Vivo.

Wang Wei W Hu Zhenhua Z Huang Yu Y Zheng Huilin H Sun Qiang Q Yang Qifan Q Zhang Yuan Y Zhang Linshi L Wang Weilin W

BioMed research international 20190325

The effects of standard clinical therapies including surgery and chemotherapy are poor in advanced gallbladder cancer (GBC). There are a few reported cases of human epidermal growth factor receptor 2 (HER2)-positive GBC that responded well to trastuzumab. But trastuzumab has not yet been used to treat HER2-negative GBC. In this study, we investigated the cytotoxic effects of different combined therapies with trastuzumab and gemcitabine and/or 5-fluorouracil on HER2-negative GBC cell lines in vit ...[more]

PMID: 31019975

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Project description:BACKGROUND: Advanced biliary tract carcinomas (BTCs) have poor prognosis and limited therapeutic options. Therefore, it is crucial to combine standard therapies with molecular targeting. In this study EGFR, HER2, and their molecular transducers were analysed in terms of mutations, amplifications and over-expression in a BTC case series. Furthermore, we tested the efficacy of drugs targeting these molecules, as single agents or in combination with gemcitabine, the standard therapeutic agent against BTC. METHODS: Immunohistochemistry, FISH and mutational analysis were performed on 49 BTC samples of intrahepatic (ICCs), extrahepatic (ECCs), and gallbladder (GBCs) origin. The effect on cell proliferation of different EGFR/HER2 pathway inhibitors as single agents or in combination with gemcitabine was investigated on BTC cell lines. Western blot analyses were performed to investigate molecular mechanisms of targeted drugs. RESULTS: EGFR is expressed in 100% of ICCs, 52.6% of ECCs, and in 38.5% of GBCs. P-MAPK and p-Akt are highly expressed in ICCs (>58% of samples), and to a lower extent in ECCs and GBCs (<46%), indicating EGFR pathway activation. HER2 is overexpressed in 10% of GBCs (with genomic amplification), and 26.3% of ECCs (half of which has genomic amplification). EGFR or its signal transducers are mutated in 26.5% of cases: 4 samples bear mutations of PI3K (8.2%), 3 cases (6.1%) in K-RAS, 4 (8.2%) in B-RAF, and 2 cases (4.1%) in PTEN, but no loss of PTEN expression is detected. EGI-1 cell line is highly sensitive to gemcitabine, TFK1 and TGBC1-TKB cell lines are responsive and HuH28 cell line is resistant. In EGI-1 cells, combination with gefitinib further increases the antiproliferative effect of gemcitabine. In TFK1 and TGBC1-TKB cells, the efficacy of gemcitabine is increased with addiction of sorafenib and everolimus. In TGBC1-TKB cells, lapatinib also has a synergic effect with gemcitabine. HuH28 becomes responsive if treated in combination with erlotinib. Moreover, HuH28 cells are sensitive to lapatinib as a single agent. Molecular mechanisms were confirmed by western blot analysis. CONCLUSION: These data demonstrate that EGFR and HER2 pathways are suitable therapeutic targets for BTCs. The combination of gemcitabine with drugs targeting these pathways gives encouraging results and further clinical studies could be warranted.

Dataset Information

Pretreatment with Gemcitabine/5-Fluorouracil Enhances the Cytotoxicity of Trastuzumab to HER2-Negative Human Gallbladder Cancer Cells In Vitro and In Vivo.

Publications

Pretreatment with Gemcitabine/5-Fluorouracil Enhances the Cytotoxicity of Trastuzumab to HER2-Negative Human Gallbladder Cancer Cells In Vitro and In Vivo.

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