1. Development of novel therapeutic agents for neuroblastoma through iron and cholesterol metabolisms study
Sang Y. Lee, Ph.D. Assistant Professor Department of Neurosurgery Penn State College of Medicine

2. Neuroblastoma
Neuroendocrine tumor arising from any neural crest element of the sympathetic nervous system
Approximately 10% of all childhood cancers. Each year, approximately new cases of NB occur in the US
The second most common solid cancer in children
The most common cancer in infants (20~50% of infant cancers)
Account for 15% of childhood cancer mortality

3. Treatment of Neuroblastoma
Based on risk categories
Low risk patients --- frequently be observed w/o any treatment at all or cured with surgery alone
Intermediate risk patients --- surgery and chemotherapy
High risk patients --- intensive chemotherapy, surgery, radiation therapy, bone marrow/hematopoietic stem cell transplantation, biological based therapy with 13-cis-retinoic acid (isotretinoin or Accutane), and monoclonal antibody therapy (chimeric antibody)
Cure rate
Low risk patients %
Intermediate risk patients %
High risk patients %

4. Hypothesis & rationale
Cancer cells need more iron and cholesterol than normal cells for their rapid growth and building block of cell membrane
C282Y HFE expressing NB and drug resistant NB cells have altered iron and cholesterol concentrations, due to altered expression of proteins needed for their metabolism

5. HFE genotype of NB cell lines
H63D
C282Y
Drug resistance
BE(2)-C
wildtype
Yes
IMR-32
SH-SY5Y
SK-N-AS
homo
SK-N-DZ
SK-N-F1
SK-N-MC
CHLA-136
CHLA-171
Cell line
claimed
cell type
Contaminating
cell line
Actual
misidentification reported by
SK-N-MC
neuroblastoma
unknown
sarcoma (Ewing's)
Staege et al. 2004

6. Iron level of NB cells A. B. C. ** ** *** ** ***
HFE mutant/SH-SY5Y cells have higher iron B.
*** C.
CHLA-171 NB cells have higher iron at early times

7. Cholesterol level of NB cell linesA. B. **
***
*** **
CHLA-171 NB cells have higher cholesterol at early culture period
C282Y/SH-SY5Y cells showed increased cholesterol 7

8. Effect of HFE siRNA on the cholesterol level
CHLA-171 NB cells (1 day) **
***
off-target effect

9. Effect of cholesterol inhibitors in the NB cells* *

10. Effect of serum stress in the NB cells

10% or 20% FBS % FBS
CHLA-171 cells are more sensitive to serum stress
than other NB cells

11. Effect of simvastatin on the NB s.c. tumor model
Control group
Simvastatin treated group
Tumor weight
Average/SE: 2.70.7 gram
Tumor weight
Average/SE: 1.60.5 gram

12. Conclusions
Iron and cholesterol in NB cells during the time they were cultured
Iron and  cholesterol in HFE stably transfected NB cells (Association between HFE expression level and cholesterol level?)
Iron level affects cholesterol level of NB cells
Drug resistant NB cells (SK-N-AS, CHLA-171) are sensitive to simvastatin
CHLA-171 cells are sensitive to the serum stress
Mild anti-tumor effect at 20 mg/kg simvastatin (3x/week, oral gavage) in SK-N-AS sc NB tumor model