autophagy 6:6, 725-737; august 16, 2010
DESCRIPTION
Areca nut extract induced oxidative stress and upregulated hypoxia inducing factor leading 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. - PowerPoint PPT PresentationTRANSCRIPT
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