Combined therapy with delta-aminolevulinic acid/photodynamic therapy and EGF-R inhibitor Gefitinib in non small cell lung carcinoma cell lines

Abstract

Non-small-cell lung cancer (NSCLC), accounts for approximately 80% of all lung tumors, that are a leading cause of cancer-related mortality in the world. Several protocols of monotherapy have been attempted with some success in a limited number and selected cases with several delusion and even failures. Combination therapy, may present some advantages and should be considered as a potentially promising approach. Although it has been proposed in certain conditions, yet it has not fully exploited. We have conducted an in vitro study on two non small cell lung carcinoma cell lines, namely A549 and H1299, that differ for the status of p53 protein (wid type and null respectively). We proposed a therapeutic approach based on the combination of anti EGFR drug (Gefitinib) and PDT (ALA-PDT). The combination is potentially efficient since the drug and PDT appear to target different cell growth pathways. The occurrence of simultaneous damages to different sites may improve the efficiency in killing the target cells. Gefitinib, is a synthetic EGF receptor inhibitor that blocks EGFR signal transduction pathways, responsible of the promotion of cancer cell proliferation. Photodynamic therapy (PDT) is a cancer treatment whose principal cytotoxicity is mediated by the production of reactive oxygen species that efficiently damage sub-cellular components. We demonstrated that specific combinations of Gefitinib and PDT treatment exert synergistic effect on cell growth inhibition of A549 and H1299 cells, and above all it strongly deranges the mitochondrial activity and cell proliferation. Interesting enough, such combinations contained Gefitinib at concentrations below the canonical effective value, so that the possible noxious side effects associated with the drug can be minimized. Having analyzed several molecular pathways upon individual and combined treatments, we observed that the two cell lines respond not identically at molecular levels especially as cell cycle and nuclear involvement are concerned. In particular PDT treatment induced S-phase block in H1299 and DNA damage. The latter observation was strengthened by the activation of damage sensor-proteins (ATM, ATR) plus the recruitment of histone H2AX. A549 cells did not perform similarly. Tentatively, this different behaviuor can be ascribed to the different status of the p53 protein in the two cell lines. In contrast, the behaviour of NF-B pathway, does not significantly differ in both cell lines. In fact the activation of NF-B transcription factor activity did not change following treatment with Gefitinib, was slight reduced following PDT, while in combined conditions a significant reduction in NF-B was observed. This decrease was determined by the the PDT-mediated inhibition of the proteasome, another recognized target in cancer therapy. In our systems we observed that the proteasome inhibition was primarily caused by PDT treatment, but it was efficiently sustained by Gefitinib. This fact explains the efficiency of the combined therapy