Background:Previous studies indicate that disulfiram (DS), an anti-alcoholism drug, is cytotoxic to cancer cell lines and reverses anticancer drug resistance. Cancer stem cells (CSCs) are the major cause of chemoresistance leading to the failure of cancer chemotherapy. This study intended to examine the effect of DS on breast cancer stem cells (BCSCs).Methods:The effect of DS on BC cell lines and BCSCs was determined by MTT, western blot, CSCs culture and CSCs marker analysis.Results:Disulfiram was highly toxic to BC cell lines in vitro in a copper (Cu)-dependent manner. In Cu-containing medium (1 μ), the IC50 concentrations of DS in BC cell lines were 200–500 n. Disulfiram/copper significantly enhanced (3.7–15.5-fold) cytotoxicity of paclitaxel (PAC). Combination index isobologram analysis demonstrated a synergistic effect between DS/Cu and PAC. The increased Bax and Bcl2 protein expression ratio indicated that intrinsic apoptotic pathway may be involved in DS/Cu-induced apoptosis. Clonogenic assay showed DS/Cu-inhibited clonogenicity of BC cells. Mammosphere formation and the ALDH1+VE and CD24Low/CD44High CSCs population in mammospheres were significantly inhibited by exposure to DS/Cu for 24 h. Disulfiram/copper induced reactive oxygen species (ROS) generation and activated its downstream apoptosis-related cJun N-terminal kinase and p38 MAPK pathways. Meanwhile, the constitutive NFκB activity in BC cell lines was inhibited by DS/Cu.Conclusion:Disulfiram/copper inhibited BCSCs and enhanced cytotoxicity of PAC in BC cell lines. This may be caused by simultaneous induction of ROS and inhibition of NFκB.

Autophagy is an important cellular process that controls cells in a normal homeostatic state by recycling nutrients to maintain cellular energy levels for cell survival via the turnover of proteins and damaged organelles. However, persistent activation of autophagy can lead to excessive depletion of cellular organelles and essential proteins, leading to caspase-independent autophagic cell death. As such, inducing cell death through this autophagic mechanism could be an alternative approach to the treatment of cancers. Recently, we have identified a novel autophagic inducer, saikosaponin-d (Ssd), from a medicinal plant that induces autophagy in various types of cancer cells through the formation of autophagosomes as measured by GFP-LC3 puncta formation. By computational virtual docking analysis, biochemical assays and advanced live-cell imaging techniques, Ssd was shown to increase cytosolic calcium level via direct inhibition of sarcoplasmic/endoplasmic reticulum Ca2+ ATPase pump, leading to autophagy induction through the activation of the Ca2+/calmodulin-dependent kinase kinase–AMP-activated protein kinase–mammalian target of rapamycin pathway. In addition, Ssd treatment causes the disruption of calcium homeostasis, which induces endoplasmic reticulum stress as well as the unfolded protein responses pathway. Ssd also proved to be a potent cytotoxic agent in apoptosis-defective or apoptosis-resistant mouse embryonic fibroblast cells, which either lack caspases 3, 7 or 8 or had the Bax-Bak double knockout. These results provide a detailed understanding of the mechanism of action of Ssd, as a novel autophagic inducer, which has the potential of being developed into an anti-cancer agent for targeting apoptosis-resistant cancer cells.

Edited by Vladimir Skulachev

The interaction between Wilms tumor gene 1 (WT1) and the promoter region of the multidrug resistance-1 (MDR1) gene has been previously reported but the clinical significance of the coexpression of WT1 and MDR1 in acute lymphoblastic leukemia (ALL) is still largely unknown. In this study, the expression levels of WT1 and MDR1 mRNA in 57 adult ALL patients were simultaneously detected using multiplex fluorescence real-time quantitative polymerase chain reaction. The expression levels of WT1 and MDR1 in bone marrow samples of adult ALL patients were significantly higher than those in the normal samples (P<0.001), and in addition, the expression levels of WT1 and MDR1 mRNA were highly correlated (r(s)=0.404, P=0.002). According to the expression levels of these two genes, the patients in this study were subdivided into the following three groups: low-WT1/low-MDR1 (n=16), high-WT1 or high-MDR1 (n=24), high-WT1/high-MDR1 (n=17). There was no significant difference in response to induction therapy among these three cohorts (P=0.217). The overall first year relapse rate was 53.2% (25 out of 47 ALL patients). High-WT1/high-MDR1 mRNA expression was strongly associated with BCR-ABL expression and a higher tendency to be T-cell ALL type. In addition, high-WT1/high-MDR1 have a significantly higher relapse rate (P=0.048) and shorter disease free survival (P=0.016). Our data suggests that high-WT1/high-MDR1 levels of mRNA expression may be associated with relatively poorer outcomes in patients with ALL. Therefore, the expression of WT1 and MDR1 may provide useful information for clinical decision.

Gold surfaces were molecularly tailored with a saccharide binding motif capable of covalently adhering cells. This facilitated communication via the macrophage membrane with implications for understanding mammalian cell signalling.

Many anticancer drugs induce reactive oxygen species (ROS) in cancer cells. High ROS will activate JNK pathway and induce cancer cell apoptosis. Meanwhile, ROS also strongly induces NFκB activity which triggers expression of many antiapoptotic factors and inhibits ROS/JNK pathway therefore antagonises anticancer drug-induced programmed cell death. The fate of anticancer agent-treated cancer cells is highly determined by the cross-talk between NFκB and JNK pathways. In this study, we demonstrated that a clinically used anti-alcoholism drug, disulfiram (DS), simultaneously activated ROS-JNK pathway and inhibited NFκB. DS was cytotoxic to breast cancer cell lines in vitro. As a divalent metal ion chelator, the cytotoxic effect of DS on breast cancer cell lines was highly copper-dependent. In the medium containing physiological concentration of copper (Cu, 1µM) the IC50 concentrations of DS to breast cancer cell lines were 200 - 500nM. The chemosensitizing effect of DS on 3 anti-breast-cancer agents, gemecitabine (dFdC), doxorubicine (DOX) and paclitaxel (PAC), was determined in 3 breast cancer cell lines (MCF-7, MDA-MB-231 and T47D). In combination with DS/Cu, the cytotoxicity of dFdC, DOX and PAC was significantly enhanced (Table 1. DOX: 8 - 11-fold; dFdC: 1.2 - 4-fold; PAC: 4 - 10-fold). DS also reversed dFdC resistance in acquired dFdC resistant cell lines. CI-isobologram analysis demonstrated synergistic effect between DS and anticancer drugs. Flow cytometric analysis showed DS enhanced anticancer drug-induced apoptosis. DS enhanced anticancer drug-induced ROS activity. The phosphorylated c-Jun and JNK protein in breast cancer cell lines was significantly induced by exposure of cancer cells to DS/Cu complex. Transfection of NFκB p50 and p65 induced dFdC resistance in MCF7 cells. DS inhibited drug-induced IκBα degradation and NFκB activation. Our data suggested that DS may be developed as a chemosensitizer for BC chemotherapy. *Equal contributionTable 1.DS/Cu sensitised cytotoxicity of anticancer drugs to breast cancer cell linesTreatmentsDrug: DS/CuMCF7MDA-MB-231T47DDOX (nM) 440.5 (16.2)178.5 (11.9)160.0 (5.5)DOX + DS/Cu1:540.3 (9.3)22.0 (2.0)21.5 (3.1)dFdC (nM) 22.1 (3.4)12.3 (2.2)35.0 (4.3)dFdC + DS/Cu1:104.9 (0.5)11.3 (1.2)12.4 (0.7)PAC (nM) 4.3 (1.4)9.3 (0.7)2.6 (0.3)PAC + DS/Cu1:62.50.4 (0.1)0.6 (0.02)0.7 (0.1) Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5410.