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Stimulation of barrier functions by
digitoxin in NCI-H460 cells
Hosam A. Elbaz1, Reem El Dawud2, Todd A. Stueckle1,3, Liyi...
Introduction
Wang et al., 2010
Cell cycle arrest
Elbaz et al., in press
Objective and Hypothesis
• Objective:
– To study the cytotoxic effects of digitoxin in real-time using
electrical impedanc...
Electric cell-substrate impedance sensing
(ECIS)
http://www.biophysics.com/ecis-theory.php
Z= (R2 + Xc
2) 0.5
Zmodel (f) =...
Digitoxin at 20 nM increases the resistance of
the cell monolayer formed by NCI-H460 cells
Control
20 nM
40 nM
80 nM
Several possibilities can explain the increased
resistance due to 20 nM digitoxin
Increased cell proliferation Cell swelli...
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
...
Cell adhesion does not account for increased
resistance of NCI-H460 cell monolayer
A) After 24 hours of exposure
*
B) Afte...
Cell membrane capacitance does not account
for increased resistance
B) After 48 hours of exposure
*
*
C) Real-time change ...
Barrier function in NCI-H460 cells correlates
with ZO-1 localization to cell edges
6 hours 12 hours 24 hours
A
*
B
Rb : ba...
Digitoxin affects barrier function in NCI-H460
cells B) After 48 hours of exposure
*
*
*
*
*
*
*
*
C) Real-time change in ...
Increased tight junction signaling potentially inhibits
cell proliferation
ZO-1
Recruiting ZONAB from
nucleus
CDK4  in th...
Conclusions
• Digitoxin increases the resistance of NCI-H460 cell monolayer
at 20 nM.
• Digitoxin affects neither cell adh...
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Duquesne AAPS 10-20-11

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Duquesne AAPS 10-20-11

  1. 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. 2. Introduction Wang et al., 2010 Cell cycle arrest Elbaz et al., in press
  3. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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.

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