Theanine from tea and its semi-synthetic derivative TBrC suppress human cervical cancer growth and migration by inhibiting EGFR/Met-Akt/NF-κB signaling

doi:10.1016/j.ejphar.2016.09.007

European Journal of Pharmacology

Volume 791, 15 November 2016, Pages 297-307

https://doi.org/10.1016/j.ejphar.2016.09.007 Get rights and content

Abstract

Cervical cancer is the third most prevalent cancer among women worldwide. Theanine from tea and its derivatives show some anticancer activities. However, the role of theanine and its derivatives against human cervical cancer and the molecular mechanisms of action remain unclear. Thus, in this study, we aim to investigate the anticancer activities and underlying mechanisms of theanine and a theanine derivative, ethyl 6-bromocoumarin-3- carboxylyl L-theanine (TBrC), against human cervical cancer. In vitro and in vivo assays for cancer cell growth and migration have confirmed the inhibition of the cell growth and migration by TBrC and theanine in highly-metastatic human cervical cancer. TBrC displays much stronger activity than theanine on inhibition of the cell growth and migration as well as induction of apoptosis and regulation of related protein expressions in the human cervical cancer cells. TBrC and theanine greatly reduced endogenous and exogenous factors-stimulated cell migration and completely repressed HGF- and EGF+HGF-activated EGFR/Met-Akt/NF-κB signaling by reducing the phosphorylation and expressions of EGFR, Met, Akt, and NF-κB in cervical cancer cells. The enhancer of zeste homolog 2 (EZH2) knockdown decreased the cancer cell migration and NF-κB expression. The NF-κB knockdown reduced the cancer cell migration. TBrC and theanine reduced the EZH2 expression by more than 80%. In addition, TBrC and theanine significantly suppressed human cervical tumor growth in tumor-bearing nude mice without toxicity to the mice. Our results suggest that TBrC and theanine may have the potentials of the therapeutic and/or adjuvant therapeutic application in the treatment of human cervical cancer.

Introduction

Cervical cancer is the third most prevalent cancer among women worldwide. According to GLOBOCAN statistics on cervical cancer (Ferlay et al., 2010), there were approximately 529,000 new cases and 275,000 deaths in 2008, of which more than 85% occurred in developing countries. Epidemiologic and molecular studies support that persistent infections with high-risk types of human papillomavirus (HPV) are essential but not exclusive prerequisites for cervical carcinogenesis (Lazo, 1999). For cervical cancer, there are few effective therapeutic options for patients who progressed after first-line chemotherapy. Metastasis is the main cause of morbidity and mortality in the thousands upon thousands of patients with cervical cancer. The endless proliferation and migration of cancer cells are the important steps of cervical cancer metastasis. Clearly, an agent which could efficiently inhibit proliferation and migration would suppress cancer progression and metastasis and thus could reduce mortality.

Theanine as a characteristic amino acid found in tea has been widely used for many years. It is a safe food additive without limitation to its dose. Some experimental results showed the anticancer activities of theanine (Sugiyama and Sadzuka, 1998, Sugiyama et al., 1999, Zhang et al., 2001, Zhang et al., 2002, Liu et al., 2009). Our previous study indicated that theanine and the sera from rats treated with theanine significantly repressed the in vitro and ex vivo invasion of rat hepatoma cells (Zhang et al., 2001) and the in vivo growth of rat hepatoma (Zhang et al., 2002). We also reported that theanine repressed the growth and migration of lung cancer cells (Liu et al., 2009). To develop new effective compounds to inhibit cancer, we previously synthesized some theanine derivatives such as ethyl 6-fluorocoumarin-3-carboxylyl L-theanine (TFC), ethyl 6-nitrocoumarin-3-carboxylyl L-theanine (TNC), and ethyl 6-bromocoumarin-3-carboxylyl L-theanine (TBrC) by using theanine from green tea, and confirmed that the theanine derivatives significantly suppressed the growth of the highly-metastatic mouse Lewis lung carcinoma (LLC) cells without toxicity to normal cells (Zhang et al., 2014; Ji et al., 2014;). In order to further explore the anticancer activity of theanine and TBrC and their molecular mechanisms of action against highly metastatic cervical cancer, here we have studied the effects of theanine from tea and TBrC on cell growth, migration, and tumor growth as well as the relevant signaling pathways in human cervical cancer cell lines HeLa and CaSki with papillomavirus (HPV) types 16 and 18 sequences.

Section snippets

Chemicals and antibodies

The primary antibodies against human p-Akt (Ser473), Akt, p-NF-κB p-65 (Ser536), NF-κB (p-65), p-EGFR (Tyr1068), EGFR, p53, p-Met (Tyr1234/1235), Met, β-tubulin, GAPDH, EGF and HGF and the hourseradish peroxidase-conjugated second antibodies were obtained from Cell Signaling Technology Inc. (Danvers, MA, USA). The primary antibodies against human cyclin D1, caspase-3, PARP-1, Bcl-2, Bax, cytochrome c, EZH2 (ENX-1), and β-actin and the hourseradish peroxidase-conjugated second antibodies were

Effects of TBrC and theanine on the cell growth and migration in human cervical cancer

The chemical structures of theanine and TBrC are shown in Fig. 1A. We first compared the in vitro anticancer activity of TBrC with its parent compound theanine on the growth inhibition of human cervical cancer cell lines HeLa and CaSki. In the preliminary experiments, theanine and TBrC at 16 µM did not show significant inhibitory effect on the cell growth of the human cervical cells but had significant inhibition of the cancer cell migration after 6–24 h treatment of the cancer cells with

Discussion

In this study, we have assessed the anticancer activities of theanine derivative TBrC by comparing with theanine against human cervical cancer cells in vitro and in vivo. TBrC and theanine can significantly inhibit the in vitro growth (Figs. 1B and C) and migration (Figs. 1D and E) in HeLa and CaSki cells. TBrC has displayed much stronger activity than theanine on inhibition of the in vitro growth and migration in HeLa and CaSki cells. In addition, TBrC and theanine can induce apoptosis in HeLa

Acknowledgments

This work is supported in part by Grant (863) from the Ministry of Science and Technology of the People's Republic of China to G.Z (2012AA020206), from the Ministry of Human Resources and Social Security of the People's Republic of China to G.Z (No.200812), Projects of Yantai University to GZ (YD2006;YD201401), Project from the National Natural Science Foundation of China to GZ (No. 30973553), projects sponsored by ‘Yantai Double Hundred Talent Plan’ to G.Z (YT201506), and Grants from the

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Molecular and cellular pharmacologyTheanine from tea and its semi-synthetic derivative TBrC suppress human cervical cancer growth and migration by inhibiting EGFR/Met-Akt/NF-κB signaling

Abstract

Introduction

Section snippets

Chemicals and antibodies

Effects of TBrC and theanine on the cell growth and migration in human cervical cancer

Discussion

Acknowledgments

Eur. J. Pharmacol.

Curr. Opin. Genet. Dev.

Cancer Lett.

Drug Resist. Updat.

Targeting the hepatocyte growth factor-cMET axis in cancer therapy

J. Clin. Oncol.

Expression of P-Akt, NFkappaB and their correlation with human papillomavirus infection in cervical carcinoma

Eur. J. Gynaecol. Oncol.

Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008

Int. J. Cancer

Suppression of proliferation and migration in highly-metastatic lung cancer cells as well as tumor growth by a new synthesized compound TBrC and its molecular mechanisms of action

Cytotechnology

Effects of bufalin on the proliferation of human lung cancer cells and its molecular mechanisms of action

Cytotechnology

EZH2 is a marker of aggressive breast cancer and promotes neoplastic transformation of breast epithelial cells

Proc. Natl. Acad. Sci. USA

The molecular genetics of cervical carcinoma

Br. J. Cancer

Effects of theanine on growth of human lung cancer and leukemia cells as well as migration and invasion of human lung cancer cells

Cytotechnology

Inhibitory effects of norcantharidin against human lung cancer cell growth and migration

Cytotechnology

Apoptosis initiated by Bcl-2-regulated caspase activation independently of the cytochrome c/Apaf-1/caspase-9 apoptosome

Nature

Expression of the enhancer of zeste homolog 2 is correlated with poor prognosis in human gastric cancer

Cancer Sci.

Anti-tumor activity of the combination of cetuximab, an anti-EGFR blocking monoclonal antibody and ZD6474, an inhibitor of VEGFR and EGFR tyrosine kinases

Cell Physiol.

Molecular and cellular pharmacology
Theanine from tea and its semi-synthetic derivative TBrC suppress human cervical cancer growth and migration by inhibiting EGFR/Met-Akt/NF-κB signaling