Cytotoxicity study of Novel polyamine conjugates in

i
rug resistant and drug sensitive breast tumour cell l

Rohit Shrivastava

Supervisor: Dr. Heather M.

Wallace
 Introduction

Biogenic aliphatic amines

Polycationic and water soluble

Polyamines
Putrescine, Spermidine,
Spermine and Cadaverine

Membrane stability

Cell differentiation and transformation

Functions
Cell cycle regulation

Influences stability and functions of DNA and RNA

 Polyamine biosynthetic pathway

Antizyme
L-Methionine L - o rn ith in e

SAMDC Ornithine Decarboxylase

Uptake
CO2 CO2

Decarboxylated S-adenosylmethionine Putrescine PAO

Spermidine Synthase N1-acetylspemidine Export

SSAT
Methylthio-adenosine
Spermidine
PAO
Spermine Synthase
SMO N1-acetylspermine

Methylthio-adenosine Spermine
SSAT

•
 Polyamine transport system

• Carrier mediated , energy

1. Receptor dependent
2. Membrane
mediated , temperature and saturable
transporter
endocytosis

• Structural tolerance towards substrates

• Polyamine Transporter
Mammalian
Regulated and selective process
•
not been isolated, cloned and characterized


1. Receptor mediated endocytosis

Theories for polyamine

transport
2. Membrane transporter

Image
Image
adapted
adapted
from
from
Soulet et , al
Belting .,2002
2002 ; Soulet et al.,2002
 Exploiting the polyamine transport system

Vector Cargo

Polyamines Anthracene

Ant 4

Ant 44

Ant 444
 Hypothesis

tes will be delivered into cells via uptake by polyamine transport system in in vitro models of

Experimental Aims

study the cytotoxicity of polyamine conjugates in breast cancer cells.

study the effect of polyamine conjugates on cell number and viability.
study the effect of polyamine conjugates on protein content of the breast cancer cells.
study the effect of polyamine conjugates on intracellular concentrations of putrescine, spermidine and s
 Methods used

Drug sensitive Drug resistant

In vitro model system
MCF-7 MCF-7 TAX 30

MDA MB 231 MDA MB 231 TAX 30

MTT cytotoxicity assay

Cell count determination

Techniques used
Modified Lowry assay

HPLC
 Results

IC50 values from MTT assay

MCF - 7 MCF -7 TAX 30

Doxorubicin 3.4 ± 0.15 (4) > 25 (3)
Ant 4 23 ± 0.31(4) 22.08 ± 0.36(3)
Ant 44 7.3 ± 0.11 (4) 11 ± 0.14 (3)
Ant 444 4.02 ± 0.1 (4) 8 ± 0.28 (3)

MDA MB 231 MDA MB 231 TAX 30

Doxorubicin 5.0 ± 0.2 (4) > 25 (5)
Ant 4 17.25 ± 0.43 (4) > 25 (4)
Ant 44 8.25 ± 0.1 (4) 21.07 ± 0.21 (4)
Ant 444 6.81 ± 0.11 (4) > 25 (4)

 Cells were seeded at a density of 2.4 x 104 cells/cm2on 96 well plate. Cells were allowed to
attach and then allowed to grow for 48 hours. After 48 hours medium was replaced with medium
containing drug or vehicle. Plates were then incubated for 48 hours and assayed as per the MTT
protocol. Number of independent experiments are indicated in the brackets.
 Effect of Anthracene-polyamine conjugates on cell growth

***
MCF-7 MCF-7 TAX 30

MDA TAX 30
MDA MB 231

Cells were seeded at a density of 2.4 x 104 cells/cm2 on 5 cm diameter plates in duplicate
and allowed to attach for 4 hours i.e. t = 0 at which plates were harvested. After 48 hours, medium
was replaced with medium containing 2.5 µM concentration of Ant compounds or vehicle. Cells were
harvested after 48 hours by trypsinization and counted using haemocytometer. Statistical analysis
was done using one way ANOVA with Dunnet’spost test. * p < 0.05. ** p < 0.01. *** p < 0.001.
 Conclusions

Order of Potency : Ant 444 > Ant 44 > Ant 4 except in MDA TAX 30 (Ant 44)

duction in cell count and protein content by Ant 444 except in MDA TAX 30 (Ant 44

dicates significant depletion of intracellular polyamines by Ant 444 & by Ant 44

Suggested future work

ODC enzyme stability assay

DNA damage - Comet assay; oxidative stress – glutathione assay.

 Acknowledgements

Dr. Heather M Wallace

Dr. Fiona Saunders, Radiah Abdul Ghani & Jun Li
Jessie, Petra and Janette
Denise Tosh (Tissue Culture training)
Gary Cameron (HPLC)