Microtubule disrupting agent-mediated inhibition of cancer cell growth is associated with blockade of autophagic flux and simultaneous induction of apoptosis

Abstract

Objectives: Given that autophagy inhibition is a feasible way to enhance sensitivity of cancer cells towards chemotherapeutic agents, identifying potent autophagy inhibitor has obvious clinical relevance. Here, we investigated ability of TN-16, a microtubule disrupting agent, on modulation of autophagic flux and its significance in promoting in vitro and in vivo cancer cell death. Materials and methods: The effect of TN-16 on cancer cell proliferation, cell division, autophagic process and apoptotic signalling was assessed by various biochemical (Western blot and SRB assay), morphological (TEM, SEM, confocal microscopy) and flowcytometric assays. In vivo anti-tumour efficacy of TN-16 was investigated in syngeneic mouse model of breast cancer. Results: TN-16 inhibited cancer cell proliferation by impairing late-stage autophagy and induction of apoptosis. Inhibition of autophagic flux was demonstrated by accumulation of autophagy-specific substrate p62 and lack of additional LC3-II turnover in the presence of lysosomotropic agent. The effect was validated by confocal micrographs showing diminished autophagosome-lysosome fusion. Further studies revealed that TN-16–mediated inhibition of autophagic flux promotes apoptotic cell death. Consistent with in vitro data, results of our in vivo study revealed that TN-16–mediated tumour growth suppression is associated with blockade of autophagic flux and enhanced apoptosis. Conclusions: Our data signify that TN-16 is a potent autophagy flux inhibitor and might be suitable for (pre-) clinical use as standard inhibitor of autophagy with anti-cancer activity.