Harnessing the lysosome-dependent antitumor activity of phenothiazines in human small cell lung cancer.

Phenothiazines are a family of heterocyclic compounds whose clinical utility includes treatment of psychiatric disorders as well as chemotherapy-induced emesis. Various studies have demonstrated that these compounds possess cytotoxic activities in tumor cell lines of different origin. However, there is considerable confusion regarding the molecular basis of phenothiazine-induced cell death. Lung cancer (LC) remains one of the most prevalent and deadly malignancies worldwide despite considerable efforts in the development of treatment strategies, especially new targeted therapies. In this work, we evaluated the potential utility of phenothiazines in human LC. We show that phenothiazines as single treatment decreased cell viability and induced cell death preferentially in small cell lung carcinoma (SCLC) over non small cell lung carcinoma (NSCLC) cell lines. Sensitivity to phenothiazines was not correlated with induction of apoptosis but due to phenothiazine-induced lysosomal dysfunction. Interestingly, the higher susceptibility of SCLC cells to phenothiazine-induced cell death correlated with an intrinsically lower buffer capacity in response to disruption of lysosomal homeostasis. Importantly, this effect in SCLC occurred despite mutation in p53 and was not influenced by intrinsic sensitivity/resistance toward conventional chemotherapeutic agents. Our data thus uncovered a novel context-dependent activity of phenothiazines in SCLC and suggest that phenothiazines could be considered as a treatment regimen of this disease, however, extended cell line analyses as well as in vivo studies are needed to make such conclusion.

Resistance to DNA-damaging treatment in non-small cell lung cancer tumor-initiating cells involves reduced DNA-PK/ATM activation and diminished cell cycle arrest.

Increasing evidence suggests that tumor-initiating cells (TICs), also called cancer stem cells, are partly responsible for resistance to DNA-damaging treatment. Here we addressed if such a phenotype may contribute to radio- and cisplatin resistance in non-small cell lung cancer (NSCLC). We showed that four out of eight NSCLC cell lines (H125, A549, H1299 and H23) possess sphere-forming capacity when cultured in stem cell media and three of these display elevated levels of CD133. Indeed, sphere-forming NSCLC cells, hereafter called TICs, showed a reduced apoptotic response and increased survival after irradiation (IR), as compared with the corresponding bulk cell population. Decreased cytotoxicity and apoptotic signaling manifested by diminished poly (ADP-ribose) polymerase (PARP) cleavage and caspase 3 activity was also evident in TICs after cisplatin treatment. Neither radiation nor cisplatin resistance was due to quiescence as H125 TICs proliferated at a rate comparable to bulk cells. However, TICs displayed less pronounced G2 cell cycle arrest and S/G2-phase block after IR and cisplatin, respectively. Additionally, we confirmed a cisplatin-refractory phenotype of H125 TICs in vivo in a mouse xenograft model. We further examined TICs for altered expression or activation of DNA damage repair proteins as a way to explain their increased radio- and/or chemotherapy resistance. Indeed, we found that TICs exhibited increased basal γH2AX (H2A histone family, member X) expression and diminished DNA damage-induced phosphorylation of DNA-dependent protein kinase (DNA-PK), ataxia telangiectasia-mutated (ATM), Krüppel-associated protein 1 (KAP1) and monoubiquitination of Fanconi anemia, complementation group D2 (FANCD2). As a proof of principle, ATM inhibition in bulk cells increased their cisplatin resistance, as demonstrated by reduced PARP cleavage. In conclusion, we show that reduced apoptotic response, altered DNA repair signaling and cell cycle perturbations in NSCLC TICs are possible factors contributing to their therapy resistance, which may be exploited for DNA damage-sensitizing purposes.

Subcutaneous insulin B:9-23/IFA immunisation induces Tregs that control late-stage prediabetes in NOD mice through IL-10 and IFNgamma.

AIMS/HYPOTHESIS: Subcutaneous immunisation with the 9-23 amino acid region of the insulin B chain (B:9-23) in incomplete Freund's adjuvant (IFA) can protect the majority of 4- to 6-week-old prediabetic NOD mice and is currently in clinical trials. Here we analysed the effect of B:9-23/IFA immunisation at later stages of the disease and the underlying mechanisms. METHODS: NOD mice were immunised once s.c. with B:9-23/IFA at 5 or 9 weeks of age, or when blood glucose reached 10 mmol/l or higher. Diabetes incidence was followed in addition to variables such as regulatory T cell (Treg) induction, cytokine production (analysed by Elispot) and emergence of pathogenic CD8(+)/NRP-V7(+) cells. RESULTS: A single B:9-23/IFA immunisation protected the majority of NOD mice at advanced stages of insulitis, but not after blood glucose reached 13.9 mmol/l. It increased Treg numbers and lost its protective effect after IFNgamma or IL-10 neutralisation, but not in the absence of IL-4. CD4(+)CD25(+) and to a lesser extent IFNgamma-producing cells from mice protected by B:9-23/IFA induced tolerance upon transfer into new NOD animals, indicating that a dominant Treg-mediated effect was operational. Reduced numbers of CD8(+)/NRP-V7(+) memory T cells coincided with protection from the disease. CONCLUSIONS/INTERPRETATION: Protection from diabetes after B:9-23/IFA immunisation cannot be achieved once diabetes is fully established, but can be achieved at most prediabetic stages of the disease. Protection is mediated by Tregs that require IFNgamma and IL-10. These findings should provide important guidance for ongoing human trials, especially for the development of suitable T cell biomarkers.