Chemotherapy resistance poses severe limitations on the efficacy of anti-
cancer medications. Recently, the notion of using novel combinations of 'old' drugs for new indications has garnered significant interest. The potential of using
phenothiazines as chemosensitizers has been suggested earlier but so far our understanding of their molecular targets remains scant. The current study was designed to better define
phenothiazine-sensitive cellular processes in relation to chemosensitivity. We found that
phenothiazines shared the ability to delay γH2AX resolution in
DNA-damaged human
lung cancer cells. Accordingly, cells co-treated with
chemotherapy and
phenothiazines underwent protracted cell-cycle arrest followed by checkpoint escape that led to abnormal mitoses, secondary arrest and/or a form of apoptosis associated with increased endogenous oxidative stress and intense vacuolation. We provide evidence implicating lysosomal dysfunction as a key component of cell death in
phenothiazine co-treated cells, which also exhibited more typical hallmarks of apoptosis including the activation of both
caspase-dependent and -independent pathways. Finally, we demonstrated that vacuolation in
phenothiazine co-treated cells could be reduced by ROS scavengers or the
vacuolar ATPase inhibitor bafilomycin, leading to increased cell viability. Our data highlight the potential benefit of using
phenothiazines as chemosensitizers in
tumors that acquire molecular alterations rendering them insensitive to
caspase-mediated apoptosis.