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

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 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.

Electric cell-substrate impedance sensing (ECIS)

http://www.biophysics.com/ecis-theory.php

Z= (R2 + Xc2) 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)]

Digitoxin at 20 nM increases the resistance of the cell monolayer formed by NCI-H460 cells

Control

20 nM

40 nM

80 nM

Au electrode

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)

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 press50 µm

50 µm 50 µm

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)

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)

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

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)

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 40CDK4

β-actin

B

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.