Patchouli alcohol induces autophagy in human lung adenocarcinoma cells A549 via increasing

Cancer proliferation

Introduction

Lung cancer is a kind of malignant tumors that seriously endangers human health.In2015,among all kinds of malignant tumors,the incidence of lung cancer in males and females in China ranked the first and second, respectively.The mortality rate ranked first in the world [1,2].According to its pathological features,lung cancer is divided into non-small cell lung cancer (NSCLC)and small cell lung cancer(SCLC).The NSCLC accounts for about80%-90%of lung cancer

cases.Early lung cancer is mainly treated with surgery, but over70%of patients have been already in the locally advanced or advanced stage at the time of diagnosis,and the prognosis is extremely poor[3,4]. Traditional application of chemotherapy has limited therapeutic effects,and the effective rate is only 20-30%[5].Furthermore,it also causes certain damage to the normal cells,which brings about various adverse reactions and seriously affects the quality of life of the patients[6].Therefore,looking for new anti-tumor drugs and exploring their anti-cancer mechanisms have become a hot spot in current cancer research. Autophagy,also known as type II programmed cell death,is a process in which cells undergo self-digestion and absorption.It was characterized by autophagosomes that coat the organelles in the cytoplasm.Autophagy is common in eukaryotic cells,which has dual roles of both protecting and killing tumor cells[7].Therefore, research on cell autophagy is important to explain the occurrence and development of tumor.Patchouli is a commonly used Chinese herb for phlegm-dampness elimination.It is pungent in flavor and warm in nature, and enters into spleen,stomach and lung meridians.It has the functions of turbidity dispelling,vomiting stopping and summer-heat clearing.Modern research shows that Patchouli mainly contains volatile oil, flavonoids and trace elements,which have the functions of protecting the gastrointestinal tract,killing pathogenic microorganisms and tumors and modulating immunity[8].Patchouli is used to treat lung cancer with phlegm-dampness accumulation[9],which fully embodies the academic thought of"Treating Disease According to Its Origin".Patchouli is pungent in flavor and warm in nature,which can dispel the phlegm-dampness.It enters into the spleen,stomach and lung meridians,which can dry the source of sputum. Therefore,it treats both appearance and root of diseases.

Patchouli alcohol(PA)is the main active compound of patchouli oil[10],which is commonly used as the quality control standard for patchouli.PA is a tricyclic sesquiterpene compound(C15H26O)with a molecular weight of222.37.The structural formula is shown in Figure1.In recent years,it has been discovered that PA has a significant anti-tumor effect[11].It can effectively inhibit the proliferation of human prostate cancer PC3cells and induce its apoptosis[12].PA is also able to inhibit the proliferation of human lung cancer cell line A549[13].Therefore,in this study, human lung cancer A549cells were used as the research object to observe the induction of autophagy by PA,and to explore the role of autophagy in inhibiting the proliferation of lung cancer cell A549.

Materials and methods

Experimental materials

Reagents Dimethyl sulfoxide(DMSO)was purchased from Sigma(USA).RPMI1640medium,streptomycin double antibody,and fetal bovine serum were purchased from Gibco(USA).Western blot kit,protease inhibitor, and the cell lysate were purchased from Haimen Biyuntian Co.,Ltd.(China).GAPDH antibody and horseradish peroxidase-labeled goat anti-rabbit IgG, rabbit anti-human microtubule-associated protein1light chain3(MAP1-LC3,LC3)and p62protein antibody were purchased from CST company. Chemiluminescence kit was purchased from Millipore (USA).LC3double fluorescent lentivirus system (stubRFP-sensGFP-LC3Lentivirus)was prepared by Shanghai Jikai Gene Chemical Technology Co.,Ltd (China).

Cell line and its culture Human lung cancer cell line A549was purchased from the US ATCC cell preservation library.They were cultured in RPMI1640 medium containing10%fetal bovine serum in a humidified,5%CO2,37°C incubator.

Drugs PA with a purity of98%was purchased from Sigma(USA).It was dissolved in DMSO at20g/L as the stocking solution and was diluted with culture medium.

Main instruments Cell incubator,Thermo Company (USA);microplate reader,Bio-Rad(USA);high-speed refrigerated centrifuge,Eppendorf(Germany);vertical electrophoresis instrument and semi-dry film transfer instrument,Bio-Rad(USA);Imaging Analyzer, Shanghai Tianneng Company(China);Fluorescence Microscope,Olympus Corporation(Japan). Lentivirus infection and construction of A549-mRFP-GFP-LC3cell line

A549cells in the logarithmic growth phase were seeded in96-well plates with5x103cells per well.The Figure1Molecular structure of PA