Lovastatin cytotoxicity induced alone or in combination with gefitinib is mediated by apoptosis induction.

<p><i>(A)</i> Western blot analysis was used to determine the onset of apoptosis to 10 µM lovastatin, 10 µM gefitinib and their combination through visualization of cleaved Poly ADP-ribose Polymerase (c-PARP), a hallmark of cellular apoptosis. c-PARP was induced with both agents, however, their combination displayed a more rapid and robust response (readily detected in 12 hrs). <i>(B)</i> LC3 is a major constituent of the autophagosome, and during autophagy, the cytoplasmic form (LC3-I) is processed and recruited to the autophagosomes where LC3-II is generated. In the treatments outlined above in the SCC25 cells, only the 10 µM gefitinib treatment induced significant conversion to the LC3-II isoform that was not enhanced by its combination with 10 µM lovastatin. <i>(C)</i> The hallmark of autophagic activation is the formation of cellular autophagosome punctae containing LC3-II. Fluorescent microscopy employing exogenously expressed LC3-GFP chimeric protein showed that fewer than 2% of cells at 48 hr treatment with 10 µM lovastatin displayed LC3-GFP expression in a punctae-staining pattern resembling putative autophagosomes.