Radiol Oncol 2002; 36(2): 172-3. 

The effect of E-64 and monoclonal antibodies on proliferative and 
invasive activity of ras transformed human breast epithelial cell line 
MCF10A neoT tested in in vitro assays 

Aleš Premzl1 and Janko Kos2 

1Jožef Stefan Institute, Department of Biochemistry and Molecular Biology, Ljubljana, Slovenia; 
2Krka, d.d., Research and Development Division, Department of Biochemical Research and Drug 
Design, Ljubljana, Slovenia 

Introduction 

The characteristic of malignant tumours is 
their potential to invade normal host tissue 
and to metastasize to distant organs.1,2 Tumour 
cell invasion of the extracellular matrix 
(ECM) is described3 as a multi-step process. 
First, tumour cells need to be attached to the 
components of the ECM. Next step is the local 
degradation of ECM components, followed 
by the migration of the tumour cell through 
locally modified matrix. Invasive behaviour 
of tumour cells requires proteolytic activity 
associated with the concerted action of 
various intra and/or extracellular proteinases, 
including a cysteine proteinase cathepsin B. 
Cathepsin B was found localized on the surface 
of different brain and breast cancer cells, 
including MCF10A neoT.4 Another prerequisite 
for tumour cells to form distant metastases 
is tumour cell proliferation.2 

The effect of E-64, an irreversible cysteine 
proteinase inhibitor and various monoclonal 
antibodies raised to human cathepsin B, was 
tested on ras transformed human breast epithelial 
cell line MCF10A neoT, using in vitro 
proliferation and invasion assays. 

Materials and methods 

Hybridoma technique, first demonstrated by 
Köhler and Millstein in 19755, was used to pre


pare mouse anti-cathepsin B monoclonal antibodies. 
Recombinant cathepsin B was used for 
immunization of Balb/c mice. For in vitro proliferation 
and invasion assays we used MAbs, 
derived from two clones of hybridoma cells. 

The MCF10A neo T, ras transformed human 
breast epithelial cell line was used in the 
in vitro assays. The cells were cultured in 
DMEM/F12 (1:1), with 5 % foetal calf serum 
(FCS) and supplemented with insulin, epidermal 
growth factor, hydrocortisone and antibiotics 
at 37 °C and 5 % CO2 in humidified 
atmosphere, to about 70-80 % confluency. The 
assays were performed using the same 
DMEM/F12 medium, however, FCS used was 
purified on the affinity chromatography column 
using immobilized papain. 

The MTT-cell proliferation assay6 was used 
to assess possible influence of inhibitor and 
antibodies on tumour cell proliferation. It is a 
quantitative colorimetric assay based on cleavage 
of the tetrazolium salt MTT (3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyl tetrazolium 
bromide) into water insoluble, dark-blue formazan 
crystals by the mitochondrial enzyme 
succinate-dehydrogenase.7 Assays were performed 
in quadruplicates. 

To study the effects of MAbs and E-64 on 
tumour cell invasion, Costar Transwells with 
12 mm polycarbonate filters and 12 µm pore 
size were used. Filters were coated with Matrigel 
(Becton Dickinson).8 Assays were performed 
in triplicates. 


Premzl A et al. / E-64 antibodies and cell proliferation 

Results and conclusions 

In the proliferation assay, MAbs derived from 
clone A and E-64 didn’t show any effect in the 
concentration range tested (2-0,01 µM and 
100-0,1 µM). MAbs derived from clone B expressed 
a slight anti-proliferative effect in a 
dose-dependent manner (2-0,01 µM). 

Results of in vitro invasion assay show a 
strong inhibitory effect expressed on the invasion 
of MCF10A neoT cells by E-64. MAbs 
derived from clone B had no effect, whereas 
MAbs from clone A expressed a 20 % inhibition 
compared to control at 1 µM concentration. 


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Radiol Oncol 2002; 36(2): 172-3.