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

1. 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 指導老師：鄭伯智老師、林宏榮老師 學生：黃美淑(N99H0003)

2. Introduction

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 was tightly associated with multiple riskfactors,areca (betel) chewingwas the most important environmental factor. Jeng et al ., 2001； Sundqvist et al ., 1989; Jeng et al ., 1994

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).

6. MAPK pathway （ROS、oxidate stress ） MKK3/6

7. MAPK pathway ＝MAPK phosphatase NF-κB

8. NF-κB activation

9. HIF-1α • Previous studies indicated that ROS stabilized HIF-1α. • Jung SN et al .2008

10. HIF-1α Respiratory Research 2009, 10:23

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.

12. Autophagy

13. Autophagy

14. Aim ? Autophagy

15. Materials and Methods

16. cell culture OC3 SAS OECM-1 Non-tumourigenic OSCC cell line with wild-type p53 activity Non-tumourigenic OSCC cell line with a p53 missense mutation A tumourigenic OSCC cell line with wild-type p53 activity

17. Reagents Ripe arece nuts Areca nut extract Blockers：NAC、Na3VO4、 SB203580、 Tiron、 U0126 、 3-MA

18. Methods • MTT 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）

19. Results

20. Fig1 A. Cell cycle analysis With 10 μg/ml and 20 μg/ml ANE treatment in SAS and OC3 cells for 24 h.

21. Fig 1.ANE induced ROS via NFκB pathway.

22. Fig 1.ANE induced ROS via NFκB pathway. Transfection pFLAG-IκBα-S32S36A

23. Fig 1.ANE-induced ROS was NFκB-dependent in SAS cells. 【 antioxidant 】 Quantification of NFκB activity 【 antioxidant 】 【NF-κB】 Left upper：EMSA analysis of NFκB activity. Left lower：western analysis.

24. ANE induced p38 activation and MKP-1 expression viaROS signaling. Internal control Internal control Internal control Fig 2. ANE was shown to activate MAPKs in OSCC cell lines. Lin SC et al ., 2005

25. Fig 2.ANE induced p38 activation and MKP-1 expression via ROS signaling. 【ROS】 ROS blockers ROS blockers

26. FIG 3.ANE induced autophagy in OSCC cells. dose- and time-dependent Arcridine orange Grey zones, the percentage of cells in the I and II quadrants.

27. Fig 3.ANE induced autophagy in OSCC cells. SAS cell OC3 cell

28. Fig 3.ANE induced autophagy in OSCC cells Electron microscopy autophagosomes (a) Control cells; (b–d), ANE-treated cells (b)Left：SAS cell；Right：OC3 cell

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

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

31. Fig 5.ANE upregulated HIF-1α and induced autophagy. Exogenous MKK6, MKP-1 and HI F-1α expression induced LC3-II accumulation.

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

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

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

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

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

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

38. Discussion

39. Autophagy may play a protective role against infection by intracellular pathogens or may inhibit ROS- mediated apoptosis. • Autophagy also contributes to the development of disease in some situations. Cheng Y, et al ., 2009

40. How autophagy and apoptosis interconnect？

41. Conclusion

42. Autophagy HIF-1α

43. Thank you for attention