Role of Polyamines in Breast Cancer

and
Evaluation of cytotoxicity of novel
yamine conjugates on Breast cancer cell

Under the guidance of Dr . Heather M . Wall

Rohit Shrivastava
MSc Drug Development with Bio-
Breast cancer
• Breast cancer 2nd leading cause of death in
women today.
•
• Most common cancer affecting women.
•
• According to Am. Soc of Cancer, 465,000
women die of cancer annually.
•
• No single factor responsible for majority of
breast cancer. Some risk factors are-
 1. Increasing age
 2. Family history
 3. Previous breast cancer
What are Polyamines?
• Polyamines – chemically active aliphatic amines.
•
• Found in food and also produced by gut
microflora.
•
• Putrescine, Spermidine & Spermine - common
polyamines.
•
• At, physiological pH carry positive charge.
•
• Involved in cell growth, proliferation &
transformation.


• Also involved in several other processes like cell

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Polyamine metabolism
• Polyamines are derived from –dietary supply as
well as biosynthesis intracellularly.
•
• Biosynthesis of polyamine initiates with the
action of Ornithine Decarboxylase (ODC) on L-
ornithine.
•
• L-ornithine → Putrescine by ODC (rate limiting
enzyme).
•
• External stimuli- increases ODC expressed and
its activity.
•
• Antizyme , a protein inhibitor regulates the
Polyamine transport system (PTS)
• Polyamine transport system - import and export
of polyamine.
•
• Polyamine concentration decreases (↓) PTS
increases (↑) uptake.
•
• Transport- receptor mediated endocytosis
(energy dependent)
•
• Transporters
 - some cells have 1 transporter for all
polyamines.
 - some have 2 transporters (Putrescine ) and
for (Spermidine & Spermine)
 Role of polyamines in Breast cancer
• In breast cancer, polyamines:- 3-4 times of their normal
levels.
•
• Breast cancer cells- high concentration of acetyl polyamines
→ increased synthesis, decreased degradation, increased
uptake of polyamines.
•
• Polyamines have a stimulatory effect on the NF-κB-NRE
binding in breast cancer cells.


• ↑ Polyamine levels inhibits immune response against

tumour cells by blocking the action of NK cells.
•
• ODC gene-an oncogene, altered expression →invasiveness
and angiogenesis of tumour.
•
• A strong relationship exists between oestrogen, growth
Multi drug resistance (MDR)
• Resistance developed by cancer cells to multiple
chemotherapeutic drugs is termed as multi
drug resistance (MDR).
•
• MDR is linked to over expression of 3 proteins-
 1. P-glycoprotein (Pgp/MDR1)
 2. Multidrug resistance protein (MRP1/ABCC1)
 3. Breast cancer resistance protein (BCRP/
ABCG2)
•
 Other mechanism of MDR
• Drug resistant cancer cells have over expression
of Fatty acid synthase (FASN) →resistant to
Adriamycin and mitoxantrone
Inhibitors of Polyamine biosynthesis
and uptake
My Project
• Project aim:
 1. Assessment of cytotoxic effect of
polyamine conjugates (Ant 4,4).
 2. To study the effect of polyamine uptake
inhibitors on intracellular
polyamine concentration.


• Model systems used:

 Cell lines used are Breast cancer cell lines
– MCF-7, MCF-7 TAX 30, MDA-MB-231, MDA
TAX 30.
–
• Techniques used for assessing cytotoxicity:
Characteristics of Breast cancer
cell lines
•
• MCF-7 : Estrogen dependent hence have oestrogen
receptor (ER α positive) and are weakly invasive.
•
• MDA-MB-231: Estrogen independent hence do
not express estrogen receptor (ER α negative) and
phenotypically these are spindle shape highly
invasive and causes aggressive forms of cancer.
•
• MCF-7 TAX 30 & MDA-MB-231 TAX 30 : These are
drug resistant cell lines resistance to taxanes
(Docetaxel).


•
Effect of etoposide on MCF-7

Cytotoxicity of etoposide in MCF-7 cells

120

100
% of control (Viable cells)

80

60

40

20

0
0 20 40 60 80 100
Concentration of Etoposide (µM)

MCF-7 were seeded at a seeding density of 5.4 x 105 left for attachment
for 4 hours and 44 hours growth then etoposide was added for an
Exposure time of 48 hours, n=1. All values are mean values.
At 100 µM concentration 73.91 % cells were viable.
Effect of Etoposide on MDA
TAX 30
Cytotoxicity of Etoposide on MDA TAX 30

120

100
% of control (Viable cells)

80

60

40

20

0
0 20 40 60 80 100
Concentration of Etoposide (µM)

MDA TAX 30 were seeded at a seeding density of 5.4 x 105 left for attachment
for 4 hours and 44 hours growth then etoposide was added for an
Exposure time of 48 hours, n=1. All values are mean values.
At,100 µM concentration 74.16 % cells were viable.
Future Plans
• As MCF-7 haven’t reached IC50 . In
next MTT assay planned to increase
concentration up to 200 µM.
•
• Alternative drug for positive control
– Doxorubicin (0-25 µM)
•
• Test the effect of Etoposide and
Doxorubicin in all cell lines.