3. College of Pharmacy, Keimyung Univ.
Programmed Necrosis
Friend?
Foe?
College of Pharmacy, Keimyung Univ.
Young Sik Cho

4. Life or Death

5. What if programmed cell death might be blocked?
Cell Responses to Stress
Proliferation
Differentiation
Senescence
What if programmed cell death might be blocked?

6. Apoptosis
Necrosis
Yasuhara et al., JHC 2003; 51:
Normal

7. APOPTOSIS VS. NECROSIS (I)
Programmed Cell Death
Uncontrolled, Accidental Death
Naturally occurred
Caused by external factor
Beneficial effects
Detrimental effects

8. APOPTOSIS VS. NECROSIS (II)
Cell morphology
Shrinkage
Swelling
Membrane integrity
Preservation
Loss
DNA
Fragmentation
Random digestion
Caspase activation
Caspase dependent
No involvement of caspase
RIP1 requirement
Not necessary
Required
Immune system
No inflammatory response
Significant inflammatory response
Apoptosis
Necrosis

9. Programmed Necrosis Extensive network of genes involved
Executed by regulated mechanism
Non-Apoptotic Cell Death
Caspase independent Cell Death
Necroptosis
Necrostatin-1, specific inhibitor of PN
Ischemic brain injury, myocardial infarction and septic shock

10. NECROSIS vs. PROGRAMMED NECROSIS
NecroX
Mitochondria ROS scavenger
Inhibits chemical-induced necrosis
LG 생명과학
RIP1 specific inhibitor
Protects from TNF-induced PN
Junying Yuan (HMS)

11. Putative Necrosis-inducing agents
DNA alkylating
agent
Heavy metal
Shikonin
Death Receptors
Nec-1

12. Tumor Necrosis Factor alpha
Manipulation of Cell Death
Tumor Necrosis Factor alpha
(TNF α)
185 AA, 17 kDa forms homotrimer
Signal transduction through binding
TNF  to TNFR
Pleiotropic inflammatory cytokine
Survival
CHX
zVAD
TNF alpha
Nec-1
Apoptosis
Necrosis

13. TNF-mediated cell death via two sequential complexDD
RIP1
TRAF2 DD
TNFR-1
RIP1 DD DD
TRADD
TRAF2
RIP1 DD DD
C-8&FADD
NFkB
Survival
Necrosis
Apoptosis
Micheau and Tschopp, Cell, 2003

14. Programmed necrosis regulator
Topics for discussion DD
Unknown
RIP1 P P
Programmed necrosis regulator
Physiological role of Programmed Necrosis?

15. Identification of RIP3 as a hit for regulation of PN
Silencing genes
TNF 
Apoptosis PN
Survival

16. Receptor interacting protein kinase 3 (RIPK3)
RIP family of serine/threonine protein kinases
Unique C-terminal domain distinct from other RIP family members
Component of TNF R-I signaling complex
Induce apoptosis and weakly activate the NFkB transcription factor

17. RIP1 and RIP3 are specifically required for PN,
but not apoptosis

18. RIP3 -/- MEFs are resistant to TNF-induced PN,
but not apoptosis
Apoptosis
Necrosis

19. The Kinase and RHIM domains of RIP3 are crucial
for programmed necrosis

20. Pronecrotic complex formation under condition of PN
Nec-1: Specific inhibitor of RIP1

21. Apoptosis Programmed Necrosis RIP3 as a regulator of cell death TNFα

22. Role of RIP3 for TNF-stimulated PN when caspase inactivatedB
Uninfected C
VV-NS
WO/CHX
zVAD surrogate
VV-TNF

23. RIP3-dependent PN & Inflammation during VV infection
Fat
Liver

24. Viral titers in tissues and survival plot of VV-infected
RIP3+/+ and RIP3 -/- mice

25. Model of the role of RIP3-dependent PN
in the innate immune defense against VV infections

26. Programmed necrosis: Innate immune response
Take-Home Message P DD
RIP3
RIP1 P P
Receptor interacting protein-3 (RIP3) as a regulator of programmed necrosis
Programmed necrosis: Innate immune response

28. Physiological consequences of Programmed Necrosis
Diseases
Host defensive machinery
Stroke
Heart attack
Sepsis

29. RIP3 as a therapeutic target?

30. Interface between apoptosis and programmed necrosis
Galluzzi & Kromer, Cell 2008, 135:
Wang et al., Cell 2012, 148,

31. Drug development targeting pronecrotic complex
RIP1 DD
RIP3 P ① ②
RIP1-RIP3 interaction
Kinase inhibtion
Protein substrate ③

32. Chemicals against programmed necrosis
Mol Structure
EC50 (µM)
Apoptosis
TNF
zVAD I
2.8 - II
5.4
3.6
III
6.0 IV
5.0
2.0 V
3.0 VI
4.8
VII
12.0
VIII
6.2
0.6 XI
0.1
0.3
1.0
-: not protected

33. Chemical-induced Programmed Necrosis
Ongoing Project
Chemical-induced Programmed Necrosis
Dissection of Programmed Necrosis
Cell Host & Microbe 7, , 2010

34. Chemical-Induced Necrosis
Shikonin (SKN)
IP : RIP3
WB : RIP1
Nec-1
10 uM
TNF-induced PN
50 uM
CHX -
WB : RIP3
+ SKN
CONT.

35. Differential roles of RIP1 and RIP3 for programmed necrosis
(B)
mRIP1/mRIP3
Mock
mRIP1
mRIP3
siRNA
RIP3
RIP1
(C)
β-actin
L929 cell line
Fibrosarcoma cell line
Caspases not activated
Gene silencing

36. Caspase Necrosis zVAD TNFα Nec I
Proposed model for distinctive role s of RIP1 and RIP3
Caspase
zVAD
TNFα DD
Nec I
RIP3
RIP1
Necrosis

37. 세포사멸 통합 시스템 구축 세포 괴사 자살 자가 포식 심근경식 뇌경색 폐혈성 쇼크 항암 치료 퇴행성 뇌질환 자가면역 질환
당뇨병

38. Therapeutic Use of Programmed Necrosis for cancer
Chemotherapy
BCR-ABL positive leukemia by Gleevec (imatinib)
DNA alkylating agent
Photosensitising molecules
Host-Microbes: To kill or To be killed
Virus
Intracellular bacteria
Acute and Chronic Diseases
Neurodegenerative disease
Septic shock
Pancreatitis

39. Summary of clinical trials for the extrinsic
apoptotic pathway
Christina et al., Am J Cancer Res 2011, 1: 43-61

40. Summary of clinical trials for the autophagy pathway
Christina et al., Am J Cancer Res 2011, 1: 43-61

41. Harnessing Cell Death Pathways for Cancer Therapy
Boosting apoptotic sensitization to chemotherapy or radiotherapy
Harnessing alternative cell death: Drug resistance
Autophagy
Necrosis
PDT

42. PARP activity switches the cell fate
DNA repair
PARP
Glucose
2 Pyruvate
2NAD+
2NADH+
2pyruvate
CO2+H2O
ADP
ATP
NAD
Alkylating agent
Ionizing radiation
Amino acids
Fatty acids
Macro-molecular
synthesis
Oxidative
Phosphorylation
Proliferation Cells
Programmed Necrosis
Vegetative cells
Cell survival and

43. Acknowledgement KMU UMASS Seung Yeon Park Ji Hyun Bae Francis Chan
David Moquin
Sreerupa Challa
Melissa Guildford
UMASS
Seung Yeon Park
Ji Hyun Bae
KMU

44. “빛을 여는 계명 약대”