Mechanistic Studies on the Anti-tumor Mechanistic Studies on the Anti-tumor

Effects of Indirubin-3‘-oxime on Effects of Indirubin-3‘-oxime on

Human Neuroblastoma CellsHuman Neuroblastoma CellsK.N. Leung

Food and Nutritional Sciences

and Biochemistry Programmes

School of Life SciencesThe Chinese University of Hong Kong

5th Asia-Pacific Summit onCancer TherapyCancer Therapy

July 20-22, 2015 Brisbane, Australia

ContentsContents

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

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?What is Neuroblastoma?

Etiology of NeuroblastomaEtiology 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

ProtectiveProtective factors: factors:

Folic acid supplements

Vitamin supplements

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 NeuroblastomaMolecular Pathogenesis of Neuroblastoma

Treatment of NeuroblastomaTreatment 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

Indirubin-3'-oxime (I3M) Indirubin-3'-oxime (I3M) (A cell permeable derivative of indirubin )

Isatis tinctoria Polygonum tinctorium Isatis indigotica Strobilanthes cusia

Indirubin is an indole alkaloid isolated from the dried roots (Banlangen) of medicinal indigo plants

Indirubin-Indirubin-3'-oxime3'-oxime

Anti-inflammatoryactivity

Anti-inflammatoryactivity

Anti-tumor activityAnti-tumor activity

Biological and Pharmacological Biological and Pharmacological Activities of Indirubin-3'-oxime (I3M)Activities of Indirubin-3'-oxime (I3M)

Indirubin-3'-oxime Inhibited the Growth of LA-N-1, SK-N-DZ and SH-SY5Y Human Neuroblastoma Cells

MTT assay

NB cells IC50 (M)

at 48 h

LA-N-1 3.27 ± 0.9

SK-N-DZ

SH-SY5Y

5.4 ± 0.28

18.15± 1.91

Effect of I3M on the Viability of Normal Cells

I3M Failed to Induce Apoptosis in LA-N-1 cells

** P< 0.01

I3M Arrested LA-N-1 Cells at G0/G1 Phase

** P< 0.01; *** P< 0.001

Mitochondria and Cell CycleMitochondria 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 p27Kip1

Blocked in G1-S transition

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-1sERRs and PGC-1s

I3M Selectively Reduced Mitochondrial Regulators (ERR and PGC-1 in LA-N-1 cells

** P< 0.01; *** P< 0.001

I3M Reduced Mitochondrial Regulated Gene Expression in LA-N-1 cells

Tfam SOD1

SOD2ATP5b

** P< 0.01*** P< 0.001

Mito. Transcription Factor A

ATP synthase Superoxide dismutase 2

I3M Caused Mitochondrial Dysfunction in LA-N-1 Cells

Mitochondrial MassMitochondrial

ROS LevelMitochondrial

Membrane Potential

** P< 0.01*** P< 0.001

I3M effects on LA-N-1 cells

ERR PGC-1

Mitochondrial massMitochondrial membrane potentialReactive oxygen species

CDK2, Cyclin E

p27Kip1

Cell growth inhibition

Cell cycle arrest at G0/G1 phase

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-VEGFR2p-VEGFR2

p-MEK1/2, p-ERK1/2p-MEK1/2, p-ERK1/2 p-AKT, p-GSK3βp-AKT, p-GSK3β

Endothelial cell proliferation, migration, and tube formationEndothelial cell proliferation, migration, and tube formation

AngiogenesisAngiogenesis

I3M Reduced the Matrigel Plug In vivo Angiogenesis

*** p< 0.001

Conclusions and Future PerspectivesConclusions 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

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)

Thank you for Thank you for your attention!your attention!