Role of Na+, K+-ATPase alpha1 subunit in the intracellular accumulation of cisplatin.

The present study was undertaken to identify what regulates intracellular cisplatin (CDDP) accumulation and what changes in membrane fraction of CDDDP-resistant cell line. The CDDP-resistant rat hepatoma cell line, H4-II-E/CDDP, shows a significant decrease in intracellular platinum accumulation compared with parental H4-II-E cells, although there was no difference in the efflux of CDDP between these two cell lines. In this study, we examined the contribution of functional change in active transport to the CDDP resistance of H4-II-E/CDDP cells. Compared with the resistant cells, platinum accumulation in the parental cells was clearly decreased by low temperature or ATP depletion. In addition, the Na+, K+-ATPase inhibitor ouabain and the K+ channel inhibitor tetraethylammonium decreased platinum accumulation in parental cells but did not change the accumulation in resistant cells. Amphotericin B, an antifungal agent, increased the intracellular platinum accumulation in resistant cells to the same level as in parent cells. Western blot analysis demonstrated that the Na+, K+-ATPase alpha1 subunit was reduced in resistant cells compared with the parental cells, although there was no difference in the expression of the beta1 subunit between the two cell lines. Furthermore, the Na+, K+-ATPase alpha1 subunit of H4-II-E was decreased following a 24-h exposure to CDDP. These results suggest that Na+, K+-ATPase-dependent active transport of CDDP does not occur in resistant cells, and, furthermore, our findings provide the first evidence that the Na+, K+-ATPase alpha1 subunit plays an important role in the transport of CDDP.

Continuous exposure to low-dose cisplatin and apoptosis.

A high concentration of cisplatin induces apoptosis in many tumor cell lines. Whether cisplatin induces apoptosis even in a controlled release formulation has not been determined. We therefore studied the relationship between the dosing regimen of cisplatin and the induction of apoptosis in rat hepatoma AH-109A cells. A colorimetric assay was used to quantify cell proliferation and viability, and caspase activity was determined using an exogenous fluorogenic peptide substrate. When delivered as a single dose, cisplatin caused a dose-dependent inhibition of AH-109A growth and enhancement of caspase-3 activity. Also, DNA laddering was detected in cells that had elevated caspase-3 activity. However, caspase-3 activity was low and DNA laddering and a sub-G1 population were not detected when cells were treated with a combination of cisplatin and the caspase inhibitor Z-VAD-FMK. These results suggest that cisplatin is cytotoxic in AH-109A cells because it induces apoptosis. We next examined intermittent exposure to cisplatin to estimate the effects of continuous exposure by a controlled release formulation. Cisplatin was divided into equal parts and was added intermittently into the medium resulting in the same final concentration as the single dose. The individual additions alone were not cytotoxic, but all of the doses together had a similar cytotoxic effect as a single exposure of cisplatin. The intermittent exposure resulted caspase-3 activity even higher than a single dose. These findings indicate that cisplatin induces apoptosis in AH-109A cells when delivered continuously even at the concentration that alone have no activity.