in this research paper ,researchers found a new therapeutic drug that is SULFASALAZINE for the treatment of neuroblastoma that has a action on SEPIATERIN REDUCTASE

1. EFFECT OF SULFASALAZINE ON
HUMAN NEUROBLASTOMA :
ANALYSIS OF SEPIAPTERIN REDUCTASE
AS A NEW THERAPEUTIC TARGET
NAME : RUCHI RANI
ROLL NO. : 20
CLASS : M.Sc BIOTECHNOLOGY, IIIrd SEMESTER

3. NEUROBLASTOMA
 Neuroblastoma is an aggressive childhood
malignancy in children upto 5 yr of age.
 It is a rare disease in which a solid tumor is
formed by special nerve cells called
neuroblasts
 Normally, these immature cells grow and
mature into functioning nerve cells. But in
neuroblastoma, they become cancer cells
instead
 NB most commonly starts in the tissue of
the adrenal glands
 Like other cancers, NB can metastasize to
other parts of the body, such as the lymph
nodes, skin, liver, and bones
 In a few cases, the tendency to get this type
of cancer can be passed down from a parent
to a child (familial type), but most cases of
NB (98%) aren't inherited (sporadic type)
 It happens almost exclusively in infants and
children, and is slightly more common in
boys than in girls

4. POLYAMINE
BIOSYNTHESISS
 Polyamines are certain biogenic di-, tri-,
and tetra-amines which are derived from
amino acids. In general, the compounds
putrescine, spermine, and spermidine,
sometimes also cadaverine are summarized
as polyamines
By SAMDC
Contribute to cell
migration and
metastasis
Addition of DFMO
Inhibit enzyme ODC
Decrease cell
proliferation
POLYAMINE BIOSYNTHESIS
PA Production
Increases In NB
PA Depletion SPERMIDINE
..
Koomoa DL, Borsics T, Feith DJ, Coleman CC, Wallick CJ,
Gamper I, et al. Inhibition of S- adenosylmethionine decarboxylase
by inhibitor SAM486A connects polyamine metabolism with p53-
Mdm2-Akt/protein kinase B regulation and apoptosis in
neuroblastoma. Mol Cancer Ther. 2009;8(7):2067–75

5. SULFASALAZINE
 Sulfasalazine is FDA approved
salicyclate based anti- inflammatory
and immune - modulatory drug
 Used to treat bowel inflammation
in patients with ulcerative colitis and
Crohn’s disease and also indicated
for use in rheumatoid arthritis
 SSZ identified to inhibit sepiapterin
reductase (SPR) , an enzyme in the
biosynthesis of tetrahydrobiopterin.
 From the early research , it is well
known that reduction in SPR
expression significantly reduced
native Ornithine decarboxylase
(ODC) enzyme activity and impeded
the proliferation of NB
 They first report the cellular effects of
SSZ on NB tumor cells, due to
inhibition of SPR
 They also demonstrate the relevance
of SPR in human NB tumors and
show the combination of SSZ with
DFMO produces synergistic anti-
proliferative effects
SPR
SSZ

6. SPR AND ODC INTERACTION
SPR
ODC
Lange I, Geerts D, Feith DJ, Mocz G, Koster J, Bachmann AS.
Novel interaction of ornithine decarboxylase with sepiapterin
reductase regulates neuroblastoma cell proliferation. J Mol Biol.
2014;426(2):332–46.

7. METHODOLOGY

8. MAMMALIAN CELL CULTURE
ISOLATION AND CULTURE OF NB CELL LINES-
 Isolate human NB cell line SK-N-Be , LAN -5 and SK-N-H
 Culture in RPMI medium supplemented with 10% heat inactivated FBS and then mixture
was supplemented with antibiotics
 Incubated at 37°C for 3 h
ANALYSIS
WHEN NO SSZ IN CELL CULTURE -
 Cell viability assay by formazan
 Analysis of SPR mRNA expression in NB by using these parameters :-
A) Age Group
B) MYCN Amplification
C) INSS Stage
 SPR –ODC mRNA expression correlation in NB by using these parameters:-
A) Age Group
B) MYCN Amplification
C) INSS Stage

9. ANALYSIS
WHEN SSZ IN CELL CULTURE –
SSZ and DMSO solution were diluted in culture medium and then analysis –
 Effect of SSZ on the viability of NB cells lines-
A) SK-N-Be(2)
B) LAN-5
C) SK-N-SH
 Synergism of SSZ and DMFO combination treatment in NB cells by using
……….Isobologram analysis
 Determination of protein – ligand binding of SSZ to SPR

10. RESULT and
discussion

11. CELL VIABILITY TEST BY FORMAZAN
Figure 1. Cell viability test of NB cell in
……………………. RPMI medium by Formazan.
Figure 2. Cell plate showing NB cell ……
……..Growth in Formazan .
SK-N-Be(2))
SK-N-Be(2)
b
Time

12. ANALYSIS OF SPR mRNA EXPRESSION
Figure 3. SPR mRNA expression
correlation with neuroblastoma clinical
parameters-
a) Age Group
b) MYCN Amplification
c) INSS Stage

13. Spr–odc mrna expression
.
Figure 4. SPR expression correlation with ODC expression in NB : Visual
representation of SPR and ODC expression in NB tumor samples, ranked
horizontally from left to right according to their SPR expression.

14. effect of SSZ ON CELL VIABILITY
Figure 5. Effect of SSZ on the viability of NB cells using the MTS cell viability assay. NB cell
lines SK-N-BE(2)c, SK-N-SH and LAN-5 were treated with increasing concentration of SSZ
for 48 h. Dose dependent inhibition of cell viability was observed.

15. synergism between ssz and dfmo
SK-N-Be(2)c LAN-5
IC50 4.007mM 5.788mM
SK-N-Be(2)c LAN-5
IC50 133.1µM 337.2µM
Figure 6. Analysis for SSZ and DFMO in NB.NB cell lines were used to determine the inhibitory concentration at …
.. which 50% of cells are dead (IC50) after 48h of treatment with –
a) SSZ and b) DFMO
c) Combined cytotoxicity of SSZ and DFMO using the IC50 values from a and b

16. 3D structure of binding of ssz to spr
Figure 7. Binding of SSZ to SPR –
a) SPR dimer front view . Both chains bind SSZ independently;
b) SPR dimer in complex with NADP+;
c) SPR monomer close up front view of the SSZ binding pocket;
d) Overlay view of SSZ and NADP+ binding sites
e) SPR monomer close up front view of the NADP+ binding pocket;

17. .
The results of NB cell experiment show that SSZ has a detrimental effect on
NB cells in in vitro culture and shows synergy with DFMO treatment which is
encouraging
 Considering the low toxicity of DFMO and its current use in NB clinical
trials, a combination with the equally low toxic and clinically evaluated SSZ
appears a good lead for future clinical studies
The use of SSZ in combination with DFMO for further experiments, and
possible prioritization as novel therapy for the treatment of NB patients
The identification of the molecular pathways that are activated in response to
SSZ action will need further studies

18. REFeRENCES

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decarboxylase with sepiapterin reductase regulates neuroblastoma cell proliferation. J Mol Biol.
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adenosylmethionine decarboxylase by inhibitor SAM486A connects polyamine metabolism with p53-
Mdm2-Akt/protein kinase B regulation and apoptosis in neuroblastoma. Mol Cancer Ther.;8(7):2067–
75.
7) Casero RA Jr, Marton LJ(2007): Targeting polyamine metabolism and function in cancer and other
hyperproliferative diseases. Nat Rev Drug Discov, 6(5):373-390.

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