Page 1
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)
Page 4
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
Page 5
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).
Page 6
MAPK pathway
MKK3/6
( ROS 、 oxidate stress )
Page 7
MAPK pathway
= MAPK phosphataseNF-κB
Page 9
HIF-1αPrevious studies indicated that ROS stabilized HIF-1α.
Jung SN et al .2008
Page 10
Respiratory Research 2009, 10:23
HIF-1α
Page 11
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.
Page 15
Materials and Methods
Page 16
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
Page 17
Reagents
Areca nut extract
Ripe arece nuts
Blockers : NAC 、 Na3VO4 、 SB203580 、 Tiron 、 U0126 、 3-MA
Page 18
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 )
Page 20
Fig1 A. Cell cycle analysis
With 10 μg/ml and 20 μg/ml ANE treatment in SAS and OC3 cells for 24 h.
Page 21
Fig 1.ANE induced ROS via NFκB pathway.
Page 22
Fig 1.ANE induced ROS via NFκB pathway.
Transfection pFLAG-IκBα-S32S36A
Page 23
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 】
Page 24
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
Page 25
Fig 2.ANE induced p38 activation and MKP-1 expression via ROS signaling.
【 ROS】
ROS blockers
ROS blockers
Page 26
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
Page 27
Fig 3.ANE induced autophagy in OSCC cells.
SAS cellOC3 cell
Page 28
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
Page 29
Fig 4.AN-induced autophagy through ROS, p38 and MKP-1.
Page 30
Fig 5.ANE treatment upregulated HIF-1α and induced autophagy through ROS genesis.
Page 31
Fig 5.ANE upregulated HIF-1α and induced autophagy.
Exogenous MKK6, MKP-1 and HI F-1α expression induced LC3-II accumulation.
Page 32
Fig 5.Exogenous MKK6, MKP-1 and HIF-1α expression induced LC3-II accumulation.
Page 33
Fig 5.Exogenous MKK6, MKP-1 and HIF-1α expression induced LC3-II accumulation.
SAS cell
Page 34
Fig 6. ANE treatment upregulated HIF-1α and induced autophagy through ROS genesis.
Page 35
Fig 6. ANE treatment upregulated HIF-1α and induced autophagy through ROS genesis.
Page 36
Fig 6. ANE treatment upregulated HIF-1α and induced autophagy through ROS genesis.
control
Page 37
Fig 7. Blockage of ANE-induced autophagy provoked apoptosis in SAS and OC3 cells.
MTT assay
Page 39
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
Page 40
How autophagy and apoptosis
interconnect ?
Page 43
Thank you for attention