Effects of statins and farnesyl transferase inhibitors on ERK phosphorylation, apoptosis and cell viability in non-small lung cancer cells

Abstract

Objective: 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors (statins) can affect post-translational processes, thus being responsible for decreased farnesylation and geranylgeranylation of intracellular small G proteins such as Ras, Rho and Rac, essential for cell survival and proliferation. In this regard, recent in vitro and in vivo studies suggest a possible role for both statins and farnesyl transferase inhibitors in the treatment of malignancies. Within such a context, the aim of our study was to investigate effects of either simvastatin (at concentrations of 1, 15, and 30 μm) or the farnesyl transferase inhibitor R115777 (at concentrations of 0.1, 1, and 10 μm), on two cultures of human non-small lung cancer cells, adenocarcinoma (GLC-82) and squamous (CALU-1) cell lines. In particular, we evaluated actions of these two drugs on phosphorylation of the ERK1/2 group of mitogen-activated protein kinases and on apoptosis, plus on cell numbers and morphology.

Materials and methods: Western blotting was used to detect ERK phosphorylation, and to assess apoptosis by evaluating caspase-3 activation; apoptosis was also further assessed by terminal deoxynucleotidyl-mediated dUTP nick end labelling (TUNEL) assay. Cell counting was performed after trypan blue staining.

Results and conclusion: In both GLC-82 and CALU-1 cell lines, simvastatin and R115777 significantly reduced ERK phosphorylation; this effect, which reached the greatest intensity after 36 h treatment, was paralleled by a concomitant induction of apoptosis, documented by significant increase in both caspase-3 activation and TUNEL-positive cells, associated with a reduction in cell numbers. Our results thus suggest that simvastatin and R115777 may exert, in susceptible lung cancer cell phenotypes, a pro-apoptotic and anti-proliferative activity, which appears to be mediated by inhibition of the Ras/Raf/MEK/ERK signalling cascade.

Figure 1

Effects of different concentrations of simvastatin and R115777 on phosphorylated ERK1/2 (p‐ERK) and total ERK1/2 expression, evaluated by western blotting, on CALU‐1 cells (Panels a and b) and GLC‐82 cells (Panels c and d).

Figure 2

Effects of different concentrations of simvastatin and R115777 on active caspase‐3 expression, evaluated by western blotting on CALU‐1 cells (Panels a and b) and GLC‐82 cells (Panels c and d).

Figure 3

TUNEL assay on CALU‐1 cells (Panels a–c) and on GLC‐82 cells (Panels d–f) treated with simvastatin 30 μm (Panels b and e) or with R115777 10 μm (Panels c and f).

Figure 4

Effects of different concentrations of simvastatin (Panel a) and R115777 (Panel b) on CALU‐1 cell counts, expressed as confluence percentages. *P < 0.05; **P < 0.01.

Figure 5

Effects of different concentrations of simvastatin (Panel a) and R115777 (Panel b) on GLC‐82 cell counts, expressed as confluence percentages. *P < 0.05; **P < 0.01.

Figure 6

Effects of different concentrations of simvastatin (Panel a) and R115777 (Panel b) on CALU‐1 cell viability and morphology, visualized by light microscopy (40×); C: control.

Figure 7

Effects of different concentrations of simvastatin (Panel a) and R115777 (Panel b) on GLC‐82 cell viability and morphology, visualized by light microscopy (40×); C: control.