Changes In Sodium Pump Expression Dictate The Effects Of Ouabain On Cell Growth

In this thesis,we used kidney epithelial cells (LLC-PK1).breast cancer cells(BT20) and prostate cancer cells (DU145) to compare the cell growth andNa/K-ATPase expression levels.The results showed that ouabain differentiallyregulates cell growth in these three cell lines.It stimulated LLC-PK1 cellsproliferation at 10 to 50 nM,but induced cell growth inhibition at the sameconcentrations in BT20 cells and DU145 cells.Meanwhile,treatment with lowconcentration of ouabain up-regulated the Na/K-ATPase expression in LLC-PK1cells but dramatically decreased Na/K-ATPase amount in BT20 cells and DU145cells.Collectively,we show that ouabain-induced cell growth regulation isintrinsically coupled to changes in the cellular amount of Na⁺/K⁺-ATPase via thePI3K/Akt/mTOR pathway.Ouabain increases the endocytosis and degradation ofNa⁺/K⁺-ATPase in LLC-PK1,human breast (BT20) and prostate (DU145) cancercells.However,ouabain stimulates the PI3K/Akt/mTOR pathway andconsequently up-regulates the expression of Na⁺/K⁺-ATPase in LLC-PK1,but notBT20 and DU145 cells.This up-regulation is sufficient to replete the plasmamembrane pool of Na⁺/K⁺-ATPase and stimulate cell growth in LLC-pK1 cells.Onthe other hand,ouabain causes a gradual depletion of Na⁺/K⁺-ATPase,andconsequently an increased expression of cell cycle inhibitor p21^cip and cell growthinhibition in BT20 and DU 145 cells.Consistently,we observe that siRNA-mediated knockdown of Na⁺/K⁺-ATPase is sufficient to induce the expression of p21^cip andslow the growth of LLC-PK1 cells.Moreover,this knockdown converts thegrowth stimulatory effect of ouabain to growth inhibition in LLC-PK1 cells.Mechanistically,both Src and caveolin-1 are required for ouabain-inducedactivation of Akt and up-regulation of Na⁺/K⁺-ATPase.Furthermore,inhibition ofthe PI3K/Akt/mTOR pathway by rapamycin completely blocks ouabain-inducedexpression of Na⁺/K⁺-ATPase,and converts ouabain-induced growth stimulation togrowth inhibition in LLC-PK1 cells.Taken together,we conclude that changes inthe expression of Na⁺/K⁺-ATPase dictate the growth regulatory effects of ouabainon cells.We also suggest that the expression of Na⁺/K⁺-ATPase may be targeted fordeveloping new anti-cancer therapeutics.