Objective   To explore the effect of Na⁺-K⁺-ATPase subunit expression on the proliferation and invasion of human colorectal cancer cell lines SW480 and SW620 and to determine the potential molecular mechanisms of ouabain anticancer therapeutics in human colorectal cancer.

    Methods   SW620 (Dukes C phase) and SW480 (Dukes B phase) cells were derived from the same patient and treated with or without a physiological concentration of 1 nM of ouabain. The proliferation of SW620 and SW480 cells was evaluated using an MTT reduction assay (i.e., 3-4, 5-dimethylthiahiazo -z-y1-3, 5-di-phenytetrazoliumromide). The invasion capacity was detected using a Transwell chamber. The Na⁺-K⁺-ATPase activity was measured using biochemistry and enzymology. The expression of the Na⁺-K⁺-ATPase α₁-, α₁-, and α₃-subunits were determined using real-time quantitative PCR and Western blot analysis.

    Results   The proliferation and invasion of SW620 cells significantly increased compared with those of SW480 cells (P < 0.05 to P < 0.01). The enzymatic activity of Na⁺-K⁺-ATPase remarkably decreased in SW620 cells (P < 0.001). Furthermore, in SW620 cells, the mRNA-level expression of Na⁺-K⁺-ATPase α3-subunits was significantly downregulated compared with that of the α₁- and α₂-subunits. However, the Na⁺-K⁺-ATPase α1-subunit expression was significantly upregulated (P < 0.01). Interestingly, the Na⁺-K⁺-ATPase α₃-subunit expression in SW480 cells was upregulated (P < 0.001). Nevertheless, the Na+, K+-ATPase α2-subunit expressions in the SW480 and SW620 cells were not significantly different (P> 0.05). In addition, the protein expression of the Na⁺-K⁺-ATPase α₁-, α₂-, and α₃-subunits were in line with the accompanying mRNA expression. A physiological concentration of 1 nM of ouabain, a Na⁺-K⁺-ATPase inhibitor, could significantly downregulate the protein-level expression of Na⁺-K⁺-ATPase α₁-subunits in SW620 and SW480 cells within 48 h, thus inhibiting the growth and decreasing the invasion capacity of SW620 and SW480 cells. Ouabain could increase Na⁺-K⁺-ATPase activity in SW620 cells. The Na⁺-K⁺-ATPase α₂- and α₃-subunit expression in the SW620 and SW480 cells treated withouabain for 48 h were not significantly different.

    Conclusion   The aberrant activity of Na⁺-K⁺-ATPase might involve tumor metastasis in human colorectal cancer, which is partly ascribed to the abnormal expression of the Na⁺-K⁺-ATPase α₁- and α₃-subunits. However, ouabain could effectively decrease the proliferation and invasion of SW480 and SW620 cells by downregulating the Na⁺-K⁺-ATPase α 1-subunits.