Areca nut extract induced oxidative stress and upregulated hypoxia inducing factor le

ading to autophagy in oral cancer cells.

Hsuan-Hsuan Lu,1,† Shou-Yen Kao,1,3 Tsung-Yun Liu,4 Shou-Tien Liu,1 Wei-Pang Huang,2 Kuo-Wei Chang1,3,*and Shu-Chun Lin1,3,*

Autophagy 6:6, 725-737; August 16, 2010

指導老師：鄭伯智老師、林宏榮老師 學生：黃美淑 (N99H0003)

Introduction

Areca nut

Areca nut ： Group I carcinogen to humans (IARC, 2004). The pathogenetic impact of areca on oral epithelial cells was still unclear. The malignant transformation of OSCC wa

s tightly associated with multiple risk factors, areca (betel) chewing was the most important environmental factor.

Jeng et al ., 2001 ； Sundqvist et al ., 1989; Jeng et al ., 1994

ROS （ Reactive oxygen species ）H2O2 、• O2- 、• OH …

Low levels ： these species may function in cell signalling processes.

High levels ： may damage cellular macromolecules (such as DNA and RNA) and participate in apoptosis (programmed cell death).

MAPK pathway

MKK3/6

（ ROS 、 oxidate stress ）

MAPK pathway

＝ MAPK phosphataseNF-κB

NF-κB activation

HIF-1αPrevious studies indicated that ROS stabilized HIF-1α.

Jung SN et al .2008

Respiratory Research 2009, 10:23

HIF-1α

Autophagy

Type I programmed cell death (PCD) ： Apoptosis.

Type II programmed cell death ： Autophagy.Self-digesting mechanism involved in the removal of cytosolic constituents.

Kondo Y, Kondo S. 2006 ； Singletary K, et al ., 2008

Has an important role to play in the cell’s response to stresses.

Autophagy

Autophagy

Aim

Autophagy

?

Materials and Methods

cell culture

OC3 SAS OECM-1

Non-tumourigenic OSCC cell line with wild-type p53 activit

y

A tumourigenic

OSCC cell line with

wild-type p53 activity

Non-tumourigenic

OSCC cell line with

a p53 missense

mutation

Reagents

Areca nut extract

Ripe arece nuts

Blockers ： NAC 、 Na3VO4 、 SB203580 、 Tiron 、 U0126 、 3-MA

Methods MTT assayMTT assay ：： Cell viability Flow cytometry Plasmid, virus, transfection and infection. HIF and NFκB transactivation activity assay. HIF and NFκB transactivation activity assay. ROS detection. Western blot analysis. Electrophoretic mobility shift assay (EMSA). Acridine orange stain and fluorescence microscopy. Confocal fluorescent microscopic detection. Electron microscopy (EM). Statistics ： ANOVA analysis （ p < 0.05 ）

Results

Fig1 A. Cell cycle analysis

With 10 μg/ml and 20 μg/ml ANE treatment in SAS and OC3 cells for 24 h.

Fig 1.ANE induced ROS via NFκB pathway.

Fig 1.ANE induced ROS via NFκB pathway.

Transfection pFLAG-IκBα-S32S36A

Fig 1.ANE-induced ROS was NFκB-dependent in SAS cells.

【 antioxidant 】

【 NF-κB 】

Left upper ： EMSA analysis of NFκB activity.

Left lower ： western analysis.

Quantification of NFκB activity【 antioxidant 】

ANE induced p38 activation and MKP-1 expression viaROS signaling.

Fig 2. ANE was shown to activate MAPKs in OSCC cell lines. Lin SC et al ., 2005

Internal control

Internal control

Internal control

Fig 2.ANE induced p38 activation and MKP-1 expression via ROS signaling.

【 ROS】

ROS blockers

ROS blockers

FIG 3.ANE induced autophagy in OSCC cells.

dose- and time-dependent

Grey zones, the percentage of cells in the I and II quadrants.

Arcridine orange

Fig 3.ANE induced autophagy in OSCC cells.

SAS cellOC3 cell

Fig 3.ANE induced autophagy in OSCC cellsElectron microscopy

(a) Control cells; (b–d), ANE-treated cells

autophagosomes

(b)Left ： SAS cell ； Right ： OC3 cell

Fig 4.AN-induced autophagy through ROS, p38 and MKP-1.

Fig 5.ANE treatment upregulated HIF-1α and induced autophagy through ROS genesis.

Fig 5.ANE upregulated HIF-1α and induced autophagy.

Exogenous MKK6, MKP-1 and HI F-1α expression induced LC3-II accumulation.

Fig 5.Exogenous MKK6, MKP-1 and HIF-1α expression induced LC3-II accumulation.

Fig 5.Exogenous MKK6, MKP-1 and HIF-1α expression induced LC3-II accumulation.

SAS cell

Fig 6. ANE treatment upregulated HIF-1α and induced autophagy through ROS genesis.

Fig 6. ANE treatment upregulated HIF-1α and induced autophagy through ROS genesis.

Fig 6. ANE treatment upregulated HIF-1α and induced autophagy through ROS genesis.

control

Fig 7. Blockage of ANE-induced autophagy provoked apoptosis in SAS and OC3 cells.

MTT assay

Discussion

Autophagy may play a protective roleAutophagy may play a protective role

against infection by intracellular against infection by intracellular

pathogens or may inhibit ROS- mediatedpathogens or may inhibit ROS- mediated

apoptosis.apoptosis.

AutophagyAutophagy also contributes to the

development of disease in some

situations. Cheng Y, et al ., 2009

How autophagy and apoptosis

interconnect ？

Conclusion

AutophagyHIF-1α

Thank you for attention