Background: Recent studies have reported that an elevated intracellular glutathione (GSH) level is associated with resistance of non-small cell lung cancer (NSCLC) cell lines to cisplatin (CDDP). It is well-known that GSH is widely used in the clinic as a hepatoprotective agent. However, whether exogenous GSH can affect the sensitivity of NSCLC cells to CDDP remains unclear. The aim of this study is to evaluate the role of exogenous GSH in the resistance of A549 cells to CDDP.
Methods: The effect of GSH and CDDP on the proliferation of A549 cells was analyzed by MTT assay. Subsequent experiments were conducted in A549 cells divided into four groups: control group (untreated cells), GSH group (treated with 120 μg/ml GSH for 48 h), CDDP group (treated with 10 μg/ml CDDP for 48 h) and CDDP+GSH group (treated with 10 μg/ml CDDP+120 μg/ml GSH for 48 h). Apoptosis was detected by flow cytometry. Light microscopy, fluorescence microscopy and electron microscopy were performed to study morphologic and ultrastructural differences among the four groups of cells. Intracellular GSH level and γ-GCS expression were determined by immunohistochemistry (IHC). Cellular platinum uptake was assessed by inductively coupled plasma mass spectrometry (ICP-MS). Quantitative RT-PCR analysis was performed to measure the expression of caspase3, caspase9, bax, bcl-2 and MDR-1. Western blot analysis was conducted to examine the protein levels of GST-π, MRP-1 and P-gp.
Results: Growth inhibition and apoptosis were reduced in A549 cells in the CDDP+GSH group compared to those in the CDDP group 48 h post-treatment. Alterations in cellular morphology and ultrastructure, as well as typical characteristics of apoptosis, were observed. Intracellular GSH and γ-GCS levels were elevated by exogenous administration of GSH; in contrast, cellular platinum concentration fell rapidly. Relative to the CDDP group, the CDDP+GSH group exhibited 47.92%, 47.82% and 63.75% downregulation in caspase3, caspase9 and bax mRNA expression, respectively, and a 2.17-fold increase in bcl-2 mRNA level. In addition, there were 1.58-fold and 2.67-fold increases in the level of GST-π and MRP-1, respectively; however, the changes in MDR-1 and P-gp levels were not statistically significant.
Conclusions: Our data demonstrated that exogenous GSH used as hepatinica in the clinic could induce resistance of A549 cells to CDDP by inhibiting apoptosis, elevating cellular GSH levels, inactivating the mitochondria-mediated signaling pathway, and increasing the expression of GST-π, γ-GCS and MRP1 to increase CDDP efflux.
Keywords: A549 cells; CDDP; GSH; apoptosis; platinum concentration.