Ultrastructural analysis of LUT-treated MET4 cells using transmission electron microscopy showed increased presence of autophagosomes filled with cargo. An increase in autophagosome accumulation may result either from increased autophagosome formation or from the blockage of autophagic degradation after fusion of the autophagosome with a lysosome. The tandem-tagged LC3 construct, mRFP-GFP-LC3, is used to distinguish early and late autophagosomes. The GFP-tag will be quenched quickly in the acidic environment of the autolysosome, leaving only the mRFPtag detectable. Figure 4B clearly shows the accumulation of mRFP punctae in the absence of green fluorescence in LUTtreated MET4 cells, indicating an increased autolysosome formation and enhanced autophagic flux. Autophagic flux can also be evaluated by decreased p62 protein levels, since p62 acts as an autophagosomal cargo receptor for ubiquitinated proteins, which is degraded in the autolysosome. Indeed, addition of LUT to MET4 cells decreased p62 levels, albeit only at higher LUT concentrations, suggesting that LUT increases the autophagic flux. Levels of LC3-II, which is incorporated in the outer and inner membrane of the autophagosomes, decreased as well upon treatment with 50 mM of LUT which is possibly due to the massive formation of autolysosomes and the simultaneous induction of apoptosis. In agreement, blocking lysosomal degradation using chloroquine rescued LC3-II and p62 breakdown. Interestingly, caspase inhibition by ZVAD-fmk also increased the level of LC3-II and p62, suggesting that caspase signaling may modulate the autophagic process. In conclusion, our data GDC-0879 indicate that LUT induced autophagy in the metastatic MET4 cells. Since autophagy, and more specifically formation of autolysosomes, was increased in MET4 cells upon treatment with LUT, we hypothesized that blockage of late phase autophagy might make the MET4 cells more susceptible to LUT-induced apoptosis. We therefore used the late phase autophagy inhibitor chloroquine in combination with LUT and evaluated apoptosis induction. Treatment of MET4 cells with CQ and LUT, resulted in a significant increased induction of apoptosis and in enhanced caspase-3 and Parp cleavage compared to treatment with LUT alone. Inhibition of autophagy at an early stage using 3-methyl adenine simultaneously with CQ and LUT rescued partially the apoptosis-inducing effect of CQ. In this study, we showed for the first time that the promising anticarcinogenic flavonoid Luteolin decreased AKT/ mTOR signaling and in parallel induced caspase-dependent cell death in both primary and metastatic cutaneous squamous cell WY 14643 carcinoma cells. LUT-induced apoptosis was selective for cancer cells as normal keratinocytes were resistant to treatment with LUT.