1. Mechanistic Studies on the Anti-tumor Effects of Indirubin-3‘-oxime on Human Neuroblastoma Cells K.N. Leung Food and Nutritional Sciences and Biochemistry Programmes School of Life Sciences The Chinese University of Hong Kong 5 th Asia-Pacific Summit on Cancer Therapy July 20-22, 2015 Brisbane, Australia

2. Contents Contents  Introduction Human Neuroblastoma (NB) and Indirubin-3‘-oxime (I3M)  Anti-tumor Effects of Indirubin-3‘-oxime on Human Neuroblastoma Cells  Mechanistic Studies and Molecular Action Mechanisms of Indirubin-3‘-oxime  Conclusions and Future Perspectives

3.  An extracranial tumor derived from primitive cells of the sympathetic nervous system  The most common and deadly solid tumor of childhood  Accounts for 8 - 10% of all childhood cancers and ~ 15% of all childhood cancer-related deaths Chest Bones around the eyes or orbits Abdomen Symptoms ： What is Neuroblastoma?

4. Etiology of Neuroblastoma Risk factors: Risk factors:  Occupation  Smoking  Alcohol consumption  Use of hormones and fertility drugs  Maternal use of hair dye  Use of medicinal drugs during pregnancy pregnancy Protective factors:  Folic acid supplements  Vitamin supplements

5.  N-myc amplification state: a key factor in the genesis of human neuroblastoma  Chromosomal abnormalities  Mutations in the PHOX2B and ALK genes  Disruption of cell division cycle, apoptosis and other signaling pathways Molecular Pathogenesis of Neuroblastoma

6. Treatment of Neuroblastoma Treatment of Neuroblastoma >90% Surgery alone 70% - 90% Surgery and Chemotherapy ~30% Multimodality therapy: Chemotherapy Surgery Radiation therapy Stem cell transplantation Differentiation therapy Immunotherapy Intermediate risk High risk Low risk Cure rate

7. Indirubin-3'-oxime (I3M) Indirubin-3'-oxime (I3M) (A cell permeable derivative of indirubin ) Isatis tinctoriaPolygonum tinctoriumIsatis indigoticaStrobilanthes cusia Indirubin is an indole alkaloid isolated from the dried roots (Banlangen) of medicinal indigo plants

8. Indirubin- 3'-oxime Anti-angiogenic activity Anti-angiogenic activity Anti-viral activity Anti-viral activity Anti-inflammatory activity Anti-inflammatory activity Inhibitor of CDKs and GSK-3  Inhibitor of CDKs and GSK-3  Anti-tumor activity Biological and Pharmacological Activities of Indirubin-3'-oxime (I3M)

9. Indirubin-3'-oxime Inhibited the Growth of LA-N-1, SK-N-DZ and SH-SY5Y Human Neuroblastoma Cells MTT assay NB cells IC 50 (  M) at 48 h LA-N-1 3.27 ± 0.9 SK-N-DZ SH-SY5Y 5.4 ± 0.28 18.15± 1.91

10. Effect of I3M on the Viability of Normal Cells

11. I3M Failed to Induce Apoptosis in LA-N-1 cells ** P< 0.01

12. I3M Arrested LA-N-1 Cells at G 0 /G 1 Phase ** P< 0.01; *** P< 0.001

13. Mitochondria and Cell Cycle hMTERF4 Induces cell cycle arrest at G1 phase Promotes cell proliferation In HeLa cells ： human cervical carcinoma In Drosophila Mutation in the cytochrome oxidase subunit Va ATP production Cell cycle arrest at G1 phase Pdsw, a subunit of mitochondrial complex I Reactive oxygen species Cyclin E CDKI p27 Kip1 Blocked in G1-S transition

14.  Estrogen-related receptor  and  (ERR  and ERR  ) are constitutively active nuclear hormone receptors.  They are thought to regulate mitochondrial biogenesis and oxidative phosphorylation together with their coactivators peroxisome proliferator-activated receptor  coactivator-1  and -1  (PGC-1  and PGC-1  ). (Sonoda J et al. 2008) ERRs and PGC-1s

15. I3M Selectively Reduced Mitochondrial Regulators (ERR  and PGC-1  in LA-N-1 cells ** P< 0.01; *** P< 0.001

16. I3M Reduced Mitochondrial Regulated Gene Expression in LA-N-1 cells Tfam SOD1 SOD2 ATP5b ** P< 0.01 *** P< 0.001 Mito. Transcription Factor A ATP synthase Superoxide dismutase 2

17. I3M Caused Mitochondrial Dysfunction in LA-N-1 Cells Mitochondrial Mass Mitochondrial ROS Level Mitochondrial Membrane Potential ** P< 0.01 *** P< 0.001

18. I3M effects on LA-N-1 cells ERR  PGC-1  Mitochondrial mass Mitochondrial membrane potential Reactive oxygen species CDK2, Cyclin E p27 Kip1 Cell growth inhibition Cell cycle arrest at G0/G1 phase

19. Anti-angiogenic Activities of I3M on Human Microvascular Endothelial HMEC-1 cells I3M Angiogenic factors Ang-1 and MMP2 Angiogenic factors Ang-1 and MMP2 p-VEGFR2 p-MEK1/2, p-ERK1/2 p-AKT, p-GSK3β Endothelial cell proliferation, migration, and tube formation Angiogenesis

20. I3M Reduced the Matrigel Plug In vivo Angiogenesis *** p< 0.001

21. Conclusions and Future Perspectives  Indirubin-3’-oxime (I3M) is a potential therapeutic agent for high risk neuroblastoma with N-myc amplification  I3M possesses anti-angiogenic activities, both in vitro and in vivo  In vivo studies of I3M in animal models (anti-tumor efficacy and acute and chronic toxicities)  Any synergistic effects when used in combinations with other chemotherapeutic drugs or natural products

22. Acknowledgements Dr. Selena LIAO Xuemei (CUHK) Dr. Simon LIU Wai-nam (CUHK) Ms. Ada KONG Lai-ping (CUHK) Prof. N.K. MAK (HKBU) Prof. N.S. WONG (HKBU)

23. Thank you for your attention!