1. The relationship between autophagy ＆ apoptosis IMI CONFIDENTIAL Gu Chunbo 2014.09.29

2. www.xianimi.com 2 IMI CONFIDENTIAL Outline 1 Introduction of autophagy 2 The interaction between autophagy and apoptosis 3 A latest research of interaction of autophagy and apoptosis

3. www.xianimi.com 3 IMI CONFIDENTIAL 1 Introduction of autophagy Definition: Autophagy, a cellular process of "self-eating" by which intracellular components (organelles and macromolecules) are degraded within the lysosome, is an evolutionarily conserved response to various stresses. Autophagy is associated with numerous patho-physiological conditions, and dysregulation of autophagy contributes to the pathogenesis of a variety of human diseases including cancer.

4. www.xianimi.com IMI CONFIDENTIAL 4 1 Introduction of autophagy The classification of autophagy: macroautophagy, microautophagy and chaperone-mediated autophagy (CMA)

5. www.xianimi.com IMI CONFIDENTIAL 5 1 Introduction of autophagy Note: 1.Part of the cell itself is digested (self-eating) in autophagy in the presence of inducing factors, but rare in the normal cells. 2.It is a rapid process: formation of autophagosome in 8 min, and complete degradation in 2 h. 3.Autophagy is conservative in Eukarya and different types of cells including cancer cells, which is beneficial to cell survival. 4.Dual actions of autophagy in the genesis and development of cancer. a) in the different tumor cells b) impacted by different external factors in the same type of cells. c) enhanced autophagy in the early stage of tumor; weakened autophagy in the development phase of tumor. d) two-tier roles in the single tumor cell and the whole tumor.

6. www.xianimi.com IMI CONFIDENTIAL 6 2. The interaction between autophagy and apoptosis The relationship of autophagy and apoptosis is summarized below: Both can induce cell death. a) they lead to cell death independently (no interaction) b) autophagy is in the upstream of apoptosis pathway c) autophagy is inhibited by apoptosis. When apoptosis is suppressed, autophagy is induced to skill cells. Autophagy inhibits apoptosis by degrading impaired proteins and DNA. Autophagy promotes apoptosis. Autophagy itself can not induce cell death, but can provide energy for formation of apoptosome and phagocytosis.

7. www.xianimi.com IMI CONFIDENTIAL 7 Molecular mechanism for interaction of autophagy and apoptosis: Atg5: the dipolymer of Atg5 and Atg12 play a vital role in the prolong of bilayer membrane of autophagosome; The products degraded by calpain can act on Bcl-XL in mitochondria, inducing apoptosis by activating Caspase. Bcl-2: It is well known that Bcl-2 is a protein that inhibits apoptosis. A recent study found that Bcl-2 can inhibit the expression of Beclin-1 after coupling with Beclin-1, resulting in weaken level of autophagy. Separating Bcl-2 from Beclin-1 promotes autophagy and inhibits apoptosis. 2. The interaction between autophagy and apoptosis

8. www.xianimi.com IMI CONFIDENTIAL 8 p53: p53 is a common anti-oncogene, which exerts lethal effects by enhancing apoptosis at transcription level. A study indicated that p53 intensifies autophagy by acting on autophagy-related gene. Another study showed that it is in cytoplasm but not cell nucleus that p53 dampen-downs autophagy. DAPK (death-associated protein kinase): we know that the combination of Beclin-1 and Bcl-2 attenuates autophagy. However, activated DAPK can dissociate Bcl-2 from Beclin-1 by phosphorylation at 119-Thr, enhancing autophagy. On the other hand, DAPK up-regulates p53, leading to apoptosis. 2. The interaction between autophagy and apoptosis

9. www.xianimi.com 3. A latest research about autophagy and apoptosis IMI CONFIDENTIAL 9

10. www.xianimi.com Background IMI CONFIDENTIAL 10 Apoptosis, autophagy, necrosis and cellular senescence are key responses of cells that were exposed to genotoxicants. Temozolomide (TMZ) is the first line chemotherapeutic drug, which induces several DNA adducts among which the minor adduct O6-methylguanine (O6-MeG) is the mocytotoxic lesion, if not repaired by O6-methylguanine-DNA methyltransferase (MGMT). The Types of DNA damage triggering these responses and their interrelationship are largely unknown. Here the authors studied these responses in glioma cells treated with the methylating agent temozolomide (TMZ)

11. www.xianimi.com Results IMI CONFIDENTIAL 11 Figure 1. TMZ induces autophagy in LN-229 cells.

12. www.xianimi.com Results Figure 2. TMZ-induced autophagy is MGMT dependent 12

13. www.xianimi.com IMI CONFIDENTIAL 13 Figure 3. TMZ-induced autophagy is MMR and HR dependent.

14. www.xianimi.com IMI CONFIDENTIAL 14 Figure 4. TMZ- induced autophagy comes earlier than apoptosis and inhibits it.

15. www.xianimi.com IMI CONFIDENTIAL 15 Figure 6. TMZ induces senescence in LN-229 cells, which is dependent on autophagy

16. www.xianimi.com IMI CONFIDENTIAL 16 Thanks for your patience!