Extracts from the Perilla frutescens var. (Odash.) Kudo leaves have anti-tumor effects on the breast cancer cell via suppression of the YAP activity
- 주제(키워드) Hippo pathway , Perilla
- 발행기관 아주대학교
- 지도교수 모정순
- 발행년도 2020
- 학위수여년월 2020. 8
- 학위명 석사
- 학과 및 전공 일반대학원 의생명과학과
- 실제URI http://www.dcollection.net/handler/ajou/000000030307
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
The Hippo signaling pathway plays a critical role in cell proliferation, apoptosis, differentiation, and development. It has emerged as a key signal transduction pathway that regulates various biological functions in many different types of tumors. Yes-associated protein (YAP) and WW domain-containing transcription regulator protein (TAZ) are the major effectors of the Hippo signaling pathway, which have been observed in many types of cancer. Furthermore, functional dysregulation of the Hippo signaling pathway enhances the oncogenic properties of YAP and TAZ and promotes cancer development. Recently, the use of complementary and alternative medicine is rapidly increasing in Asia and Western countries. Perilla frutescens var. acuta (Odash.) Kudo leaves, a common Korean medical plant, is known to possess anti-oxidant, anti-allergic, anti-inflammatory, and tumor-preventing properties against several types of cancer. In this study, we aimed to evaluate the biological mechanisms of the anti-tumor effect of P. frutescens leaf extract (PLE) via the Hippo signaling pathway in breast cancer cells. Using the MTT assay, cell viability in breast cancer cells was reduced by PLE. Following treatment with PLE, YAP/TAZ protein levels were reduced and the biomarkers cleaved caspase-3 and poly ADP ribose polymerase (PARP) levels increased in MDA-MB-231 and BT549 cells, which are triple negative breast cancer cell lines. To elucidate the molecular mechanism of PLE on the regulation of YAP activity, we investigated the expression of YAP after PLE treatment. PLE enhanced the phosphorylation of YAP/TAZ. Furthermore, PLE suppresses YAP/TAZ transcriptional activity through the disruption of the YAP/TAZ-TEAD complex. Moreover, this activity was substantially suppressed in large tumor suppressor kinase 1/2 (LATS1/2) depletion cells compared to LATS1/2 wild-type cells. The migration rate of breast cancer cells was suppressed by PLE treatment in a time-dependent manner. This ability was weakened in cells expressing TEAD1△C-YAP (AD), which is a constitutively active form of the YAP fusion protein, suggesting that the migration inhibition effect of PLE is achieved by regulating YAP activity. Collectively, PLE diminishes growth of breast cancer cells via regulation of the Hippo signaling pathway in a LATS1/2-dependent manner, which suggests that PLE can be used as a therapeutic medical herb for breast cancer.
more목차
I. INTRODUCTION 1
II. MATERIAL AND METHODS 4
A. Cell culture and transfection 4
B. Preparation of Perilla frutescens extracts(PLE) 4
C. Reagents and antibodies 4
D. High performance liquid chromatography(HPLC) 5
E. Western blot and immunoprecipitation 5
F. Luciferase assay 6
G. RNA isolation and real-time quantitative polymerase chain reaction (RT-qPCR) 6
H. Retrovirus infection and stable cell lines 7
I. Wound healing assay 7
J. Cell viability assay 8
K. Clonogenic growth assay 8
L. Generation of knock-out cells 9
M. Statistical analysis 9
III. RESULTS 10
A. Identification of rosmarinic acid in Perilla frutescens by HPLC-UV analysis 10
B. Effects of PLE on cytotoxicity leads to cell apoptosis in MDA-MB-231 and BT549 TNBC 13
C. PLE increases YAP phosphorylation and reduces YAP/TAZ-TEAD-mediated transcriptional activity 16
D. PLE induces YAP phosphorylation via LATS1/2-dependent mechanism, but not MST1/2 20
E. PLE blocks cell proliferation and migration of human breast cancer cells by repressing YAP activity 23
IV. DISCUSSION 26
V. REFERENCE 29
