Chemosensitization by phenothiazines in human lung cancer cells: impaired resolution of γH2AX and increased oxidative stress elicit apoptosis associated with lysosomal expansion and intense vacuolation

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

Molecular factors relevant to the radiosensitivity of human tumours

Oncogenes, activated copies of normal cellular genes, are involved in carcinogenesis and have been suggested to be determinants of cellular radiosensitivity (RS). Mutations and gene rearrangement of c-Ha-RAS and c-MYC oncogenes were analysed in 70 biopsies and 51 blood samples from the same patients and compared with RS estimated by the clinical tumour response and by response to irradiation of tumour samples using subrenal capsule assay. We did not find a correlation between the alterations of c-Ha-RAS and/or c-MYC and RS. DNA is considered to be a target for ionizing radiation. Efficiency of repair of DNA double-strand breaks (DSBs) has been suggested to be a determinant of cellular RS. Two human squamous cell carcinoma cell lines showing different RSs did not differ with regard to induction levels of DNA DSBs. However, the resistant cell line showed a faster rate of the first part of the repair process. The radioresistant cell line had a higher activity of the DNA dependent protein kinase (DNA-PK), an enzyme which is a component of the DNA DSB repair apparatus called non-homologous end joining - NHEJ, compared with the more radiosensitive cell line. In order to study DNA DSB and SSB repair processes in more detail, an in vitro endjoining assay based on utilization of bacterial plasmid DNA, was developed. Using this method we showed that Ku protein, the function of which is considered to be limited to NHEJ, stimulates the repair of SSBs. The result therefore points towards the possibility of sharing of the same enzyme by two DNA repair pathways. Ku protein did not protect DNA against nuclease degradation but it did compete with the process of nuclease degradation by accelerating the repair process. Using the same system, we compared the efficiency of repair of SSBs generated by low (X-ray)- and high (nitrogen ions)- LET (linear energy transfer) radiations. High-LET irradiation has been shown to result in a higher proportion of locally multiply damaged sites (LMDSs) compared to low-LET irradiation. LMDSs are DNA lesions that are thought to be least repairable. However, in our system, X-ray and nitrogen iongenerated SSBs were repaired with the same efficiency. The DNA is constantly inflicted by DNA damage that the cellular repair system copes with most likely in competition with nuclease activities. The constitutive level of DNA single-strand breaks (SSBs) correlated with RS in four squamous cell carcinoma cell lines (including the two described above). As imparity in DNA replication is a major source of SSBs during normal cell metabolism, DNA polymerase (pol) and DNA ligase activities were measured. DNA pol activity increased gradually with increasing RS. The ligase activity of the most radiosensitive cell line was lowest compared to the three other cell lines. Our data indicate that low ligase activity may depend on a high activity of nucleases in the cells. The results suggest that spontaneous level of SSBs may be predictable for cellular RS. The high level of SSBs may be a result of deficiency in handling of strand breaks, which consists of an imbalance between the processes that lead to introduction of DNA strand breaks and the DNA strand break repair process