1. Stimulation of barrier functions by
digitoxin in NCI-H460 cells
Hosam A. Elbaz1, Reem El Dawud2, Todd A. Stueckle1,3, Liying Wang3, Yon Rojanasakul1, and
Cerasela Zoica Dinu2
1Department of Basic Pharmaceutical Sciences, West Virginia University,
2Department of Chemical Engineering, West Virginia University,
3National Institute for Occupational Safety and Health
Funding: WVNano, NSF/SEED (EPS-1003907 )
WVU School of Pharmacy

2. Introduction
Wang et al., 2010
Cell cycle arrest
Elbaz et al., in press

3. Objective and Hypothesis
• Objective:
– To study the cytotoxic effects of digitoxin in real-time using
electrical impedance technology.
• Hypothesis:
– Digitoxin will induce morphological changes in NCI-H460
cell monolayer over time.
– Morphology changes correlates with digitoxin’s cytotoxic
effect on NCI-H460 cells.

4. Electric cell-substrate impedance sensing
(ECIS)
http://www.biophysics.com/ecis-theory.php
Z= (R2 + Xc
2) 0.5
Zmodel (f) = f {Cm, α, Rb}
• Rb : barrier function of the cell layer
• α : current flow beneath the cell
• Cm : cell membrane capacitance
[Giaever, I. and Keese, C.R., PNAS 81,
3761 (1991)]

5. Digitoxin at 20 nM increases the resistance of
the cell monolayer formed by NCI-H460 cells
Control
20 nM
40 nM
80 nM

6. Several possibilities can explain the increased
resistance due to 20 nM digitoxin
Increased cell proliferation Cell swelling Increased cell adhesion (α)
Affected cell membrane capacitance (CM)
Increased barrier function (Rb)

7. 20 nM
40 nM 80 nM
0 nM
50 µm
Cell growth does not account for increased
resistance of NCI-H460 cell monolayer
*
*
*
*
C
A
Visual inspection of NCI-H460 cells showed
morphological changes
B
20 nM
Elbaz et al., in press
50 µm
50 µm 50 µm

8. Cell adhesion does not account for increased
resistance of NCI-H460 cell monolayer
A) After 24 hours of exposure
*
B) After 48 hours of exposure
*
C) Real-time change in cell adhesion
***
*
*
α: current flow beneath the cell
D)

9. Cell membrane capacitance does not account
for increased resistance
B) After 48 hours of exposure
*
*
C) Real-time change in CM capacitance
*
*
*
A) After 24 hours of exposure
Cm : cell membrane capacitance
D)

10. Barrier function in NCI-H460 cells correlates
with ZO-1 localization to cell edges
6 hours 12 hours 24 hours
A
*
B
Rb : barrier function of the cell layer
D

11. Digitoxin affects barrier function in NCI-H460
cells B) After 48 hours of exposure
*
*
*
*
*
*
*
*
C) Real-time change in barrier function
A) After 24 hours of exposure
*
Rb : barrier function of the cell layer
D)

12. Increased tight junction signaling potentially inhibits
cell proliferation
ZO-1
Recruiting ZONAB from
nucleus
CDK4  in the
nucleus
Cell proliferation 
Digitoxin
A
*
*
C
Digitoxin (nM)
0 20 40
CDK4
β-actin
B

13. Conclusions
• Digitoxin increases the resistance of NCI-H460 cell monolayer
at 20 nM.
• Digitoxin affects neither cell adhesion nor cell membrane
capacitance at 20 nM.
• Digitoxin stimulates tight junctions in non-small cell lung
cancer cells at 20 nM.
• Digitoxin inhibits cell proliferation and CDK4 expression at 20
nM.
• Stimulation of tight junctions by digitoxin correlates with its
antiproliferative activity and CDK4 degradation.