CROSSTALK BETWEEN AUTOPHAGY AND APOPTOSIS IN CD437-INDUCED А549 LUNG CARCINOMA CELL DEATH

Abstract

Introduction. Synthetic retinoids CD437, an agonist of the gamma retinoic acid receptor (RARγ), not only induces growth arrest, but in contrast to retinoid acid, it also induces RARγ-independent apoptosis in many tumor cells through a unique mechanism that is independent of the retinoic acid receptor-mediated pathway. The aim of the study was to study the relationship between apoptosis and autophagy in CD437-induced cell death. Materials and methods. In this study we used 2D-culturing of lung carcinoma cells A549, immunocytochemistry, flow cytometry and fluorescence microscopy. Results. CD437 at concentrations between 0.2 and 5.0 μM increased the number of PI-positive cells in A549 lung cancer cells. The retinoid at concentrations close to IC 50 reduced the cell population in the G2/M-phase and arrest cell cycle in the S-phase. CD437 dose-dependent increased the number of apoptotic cells in the presence of non-cytotoxic concentrations of wortmanin, an irreversible inhibitor of anti-stress kinasе PI-3K, and LY 200192, a reversible inhibitor. CD437 also activated the biogenesis of autophagosomes, and there was a dose-dependent increase in the fluorescence intensity of monodansilkadaverine, a marker of autophagy. However, accumulation of LC-3B was not observed with an increase of CD437 concentration from 0.1 to 5.0 μM suggesting that the fusion between autophagic vacuoles and lysosomes was inhibited. zVAD-fmk, an irreversible caspase inhibitor, did not restore autophagy in A549 cells, and LC-3B levels did not change significantly with the increasing of CD437 concentration, indicating that CD437 was involved in autophagosome-lysosome fusion. When cells grew with non-cytotoxic concentrations of chlorokine, a late stage autophagy inhibitor, there were virtually no living cells at CD437 concentrations close to IC 50 . The additive effect of CD437 and chlorokine in inducing A549 cell death confirms that CD437 involved in fusion between autophagosomes and lysosomes required for final catabolism of autophagic material. Conclusions. The data obtained indicate that CD437 induced a failure of cytoprotective function of autophagy and apoptosis, that raise the question of combined therapy of CD437 with cytotoxic drugs in the treatment of lung carcinoma