Abstract
ObjectivesGiven 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 methodsThe 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.ResultsTN‐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.ConclusionsOur 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.
Highlights
Autophagy is an evolutionarily conserved process of salvaging cellular constituents
We have revealed that diminished autophagic flux in TN-16–treated cells is associated with induction of apoptosis and these two mechanisms influence each other
We investigated whether TN-16–dependent cell growth inhibition is associated with apoptosis
Summary
Autophagy is an evolutionarily conserved process of salvaging cellular constituents. It serves as an alternative mechanism of energy production during metabolic stress. By chemical inhibitors, is usually achieved by targeting either at early phase of phagophore formation (eg, 3-MA, LY294002, Wortmannin) or at late phase of autophagolysosomal proteolysis (eg, Bafilomycin A1, CQ, HCQ). Autophagosomes, formed in cytosol, move along the microtubule track to transport cellular cargo towards lysosomes at perinuclear region, and disruption of tubulin network results in suppression of autophagic process.[11] By targeting this essential step of autophagic process, microtubule destabilizing agents indirectly inhibit autophagy by preventing autophagosome-lysosome fusion. We have revealed that diminished autophagic flux in TN-16–treated cells is associated with induction of apoptosis and these two mechanisms influence each other
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