Silencing FXYD3 protein enhances cytotoxicity effect of doxorubicin and gamma irradiation in human breast cancer cells

Easy access to the Na⁺-K⁺ pump in the cell surface membrane
and the critical dependence of cell survival on the pump has
made it is an attractive therapeutic target in cancer. Use of
cardiac glycosides have been explored but has turned out to have
limited utility due cardiac toxicity of the drugs. As an alternative
we have examined if targeting FXYD proteins that associate
closely with the Na⁺-K⁺ pump molecular complex might be useful.
FXYD3 is of particular interest because it is markedly overexpressed
in several common cancers and we have shown that
several FXYD proteins, including FXYD3, are critical for reversal
of glutathionylation of the β1 Na⁺-K⁺ pump subunit, an oxidative
modification that inhibits pump activity.
We hypothesized that FXYD3 protein overexpression protects
pump function against inhibition by the high levels of oxidative
stress in cancer cells typically encounter and a reduction in
FXYD3 expression levels would sensitize cells to chemotherapy
and radiotherapy that largely induce cell kill by increasing
oxidative stress.
We exposed MCF-7 human breast cancer cells to FXYD3 siRNA
for 48 hours to knock down FXYD3. This reduced FXYD3
mRNA/protein expression and Na⁺-K⁺ ATPase activity to 50 % of
expression and activity in untreated control cells. Exposure of
MCF-7 cells to 1 μM doxorubicin (Dox) reduced cell viability by 20
% after 24 hours. FXYD3 knock down alone had no significant
effect on viability measured two days after transfection but it
amplified Dox-induced cell kill and apoptosis. The combination of
FXYD3 siRNA and Dox increased glutathionylation of β1 Na⁺-
K⁺ pump subunit and reduced Na⁺-K⁺ ATPase activity. Exposure
of MCF-7 control cells to gamma irradiation had no significant
effect on cell viability after 24 hours. However, it induced a
decrease in viability in cells subjected to FXYD3 knock down.
FXYD3 knock down also increased irradiation-induced apoptosis.
target for cancer therapies.