Disulfiram modulated ROS–MAPK and NFκB pathways and targeted breast cancer cells with cancer stem cell-like properties

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 μM), the IC50 concentrations of DS in BC cell lines were 200–500 nM. 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.

The development of drug resistance remains the major obstacle to the success of breast cancer (BC) chemotherapy. Drug-induced DNA damage triggers the expression of anti-apoptotic proteins, which confer drug resistance upon cancer. The NFkB is one of the major chemoresistance-related anti-apoptotic factors. Many human cancers including BC possess high levels of the constitutive NFkB activity, which can be further induced by some anticancer drugs. High NFkB activity links inflammation and tumourigenesis (Greten et al, 2004). Activated NFkB triggers a series of molecular reactions including up-regulation of anti-apoptotic protein-encoding genes (Dutta et al, 2006) that induce cancer chemoresistance. High NFkB activity has been identified in drug-resistant cancer cells and ectopic over-expression of NFkB can block anticancer drug-induced apoptosis (Wang et al, 1998(Wang et al, , 1999(Wang et al, , 2004. Previous studies in our laboratory demonstrate that 5-fluorouracil (5-FU)and gemcitabine (dFdC)-resistant cancer cell lines possess higher NFkB activity Guo et al, 2009). Overexpression of p50 and p65, the two subunits of NFkB, results in increased NFkB activity and induces 5-FU and dFdC resistance (Wang et al, 2004;Guo et al, 2009). Although NFkB is an attractive molecular target for therapeutic intervention, inhibition of NFkB alone can only induce limited cell death. The disappointing clinical trial outcomes from using NFkB inhibitor in treatment of metastatic BC patients Cresta et al, 2008) indicate that BC chemotherapy cannot be efficiently improved by only targeting NFkB pathway.
Reactive oxygen species (ROS) (Gupte and Mumper, 2009) are a group of oxygen-containing chemical species normally generated from mitochondrial respiratory chain reaction with reactive chemical properties. High ROS activity can damage DNA, protein and lipid membrane leading to apoptosis. In comparison with normal tissues, cancer cells generally possess high ROS activity (Fruehauf and Meyskens, 2007) and can tolerate higher levels of ROS. It has been suggested that further increasing ROS exposure induced by ROS-generating agents will exhaust the cellular antioxidant capacity, pushing cancer cells over the tolerated ROS threshold and leading to apoptosis (Lopez-Lazaro, 2007). Reactive oxygen species-induced apoptosis is highly reliant on persistent activation of pro-apoptotic MAPK pathways (cJun N-terminal kinases (JNKs) and p38) (Nakano et al, 2006) mainly through modulating the activities of mitochondrial pro-and anti-apoptotic proteins by phosphorylation events (Junttila et al, 2008). Many conventional anticancer drugs induce ROS generation and trigger cancer cell apoptosis via ROS -MAPK pathway. However, anticancer drug-induced ROS activation also triggers expression and activation of a number of anti-apoptotic factors including NFkB that dampen the ROS-induced cytotoxic effect (Nakano et al, 2006).
Owing to the cross-talk between NFkB and ROS -MAPK pathways, singly targeting either pathway may not be sufficient for inducing cancer cell killing. Therefore, identification of small molecules that simultaneously activate the ROS -MAPK proapoptotic pathway and block ROS-induced anti-apoptotic pathways may improve BC chemotherapy. Disulfiram (DS) is a commercially available anti-alcoholism drug (Johansson, 1992). We have previously demonstrated that DS inhibits NFkB activity and enhances 5-FU-and dFdC-induced apoptosis in drug-sensitive and -resistant colon cancer cell lines Guo et al, 2009). Disulfiram also potentiates the cytotoxicity of cyclophosphamide, cisplatin and radiation in vitro and protects normal cells in kidney, gut and bone marrow in vivo, while increasing the therapeutic index of a wide range of cytotoxic drugs (Evans et al, 1982;Hacker et al, 1982;Bodenner et al, 1986). The molecular anticancer mechanisms of DS are still largely unknown. The previous publications indicate that the anticancer effect of DS is copper (Cu) dependent (Nobel et al, 1995;Cen et al, 2002Cen et al, , 2004Chen et al, 2006). Copper has a crucial role in redox reactions and triggers generation of ROS in human cells. Disulfiram/copper is a strong ROS inducer (Nobel et al, 1995) and proteasome-NFkB pathway inhibitor (Chen et al, 2006). Combination of DS with Cu may target cancer cells by simultaneously tackling both ROS and NFkB.
Cancer derives from a very small fraction (1%) of cancer stem cells (CSCs) (Dalerba et al, 2007), which are relatively quiescent and express multidrug resistant and anti-apoptotic proteins (Marques et al, 2010;Storci et al, 2010). Conventional anticancer drugs target the proliferating and differentiated tumour bulk, but fail to eradicate the CSCs, which become the source of tumour recurrence. Aldehyde dehydrogenases (ALDHs) are functional markers of normal and breast cancer stem cells (BCSCs) (Ginestier et al, 2007;Marcato et al, 2011). It recently reported that targeting ALDH1A1 gene can target ovarian CSCs (Landen et al, 2009). Disulfiram is a specific inhibitor of ALDHs (Johansson, 1992;Lam et al, 1997). Therefore, it may also be an inhibitor of BCSCs.
This study demonstrated that in combination with physiological concentration of Cu, DS was highly cytotoxic to BCSCs and synergistically enhanced the cytotoxicity of paclitaxel (PAC) in BC cell lines.

Cell culture and cytotoxicity analysis
All cell lines were cultured in DMEM (Lonza, Wokingham, UK) supplemented with 10% FCS, 50 units ml -1 penicillin and 50 mg ml -1 streptomycin. For in vitro cytotoxicity assay, cells (5000 per well) were cultured overnight in 96-well flat-bottomed microtitre plates, then exposed to drugs for 72 h and subjected to a standard MTT assay (Plumb et al, 1989).

Analysis of the combinational effect of PAC and DS/Cu by combination index isobologram
Overnight cultured cells were exposed to various concentrations of PAC, DS þ Cu 1 mM or in combination of PAC and DS þ Cu 1 mM at a constant PAC:DS ratio of 62.5:1 for 72 h. The cells were then subjected to MTT analysis as described above. The combinational cytotoxicity of PAC and DS/Cu 1mM was determined using combination index (CI) isobologram analysed by CalcuSyn software (Biosoft, Cambridge, UK) (Chou and Talalay, 1984). The CI was determined by mutually exclusive equations.

Electrophoretic mobility-shift assays
Detection of NFkB-oligonucleotide complex was performed using a LightShift chemiluminescent electrophoretic mobility-shift assay kit (Pierce, Northumberland, UK). Briefly, nuclear protein (5 mg) was incubated with 20 fmol of biotin-labelled oligonucleotide for 20 min at room temperature in binding buffer. The specificity of the NFkB DNA binding was determined in competition reactions in which a 200-fold molar excess of unlabelled wild-type (5 0 -AGT TGA GGG GAC TTT CCC AGG C-3 0 ) or mutant (5 0 -AGT TGA TAT TAC TTT TAT AGG C-3 0 ) NFkB probes were added to the binding reaction. The signal was detected by chemiluminescent photography.

Flow cytometric analysis of DNA content
Cells (1 Â 10 6 ) were exposed to drugs and harvested by trypsinisation. The cells were fixed in 70% ethanol and then incubated with RNase A (100 mg ml -1 ) and propidium iodide (50 mg ml -1 ) for 30 min. The data from 10 000 cells of each sample were collected by FACS Scan (Becton Dickinson, Franklin Lakes, NJ, USA) and the DNA content analysed using CellQuest software (BD Biosciences, Oxford, UK).

Luciferase reporter gene assay
All the transfections were performed using Lipofectamine 2000 (Invitrogen) transfection reagent following the manufacturer's instructions. The cells were co-transfected with luciferase reporter vectors (pNFkB-Tal-Luc (BD Biosciences) and pGL3-Basic (Promega, Southampton, UK)) and an internal control, pSV40-Renilla (Promega). The luciferase activities were determined using Dual Luciferase Assay reagents (Promega) following the manufacturer's instructions. The luciferase activity in each well was normalised to pSV40-Renilla using the formula of Ln ¼ L/R (Ln, normalised luciferase activity; L, luciferase activity reading and R, Renilla activity reading). The Ln was further standardised by the transcriptional activity of the pGL3-Basic using the formula of RLU ¼ Ln NF k B /Ln Basic (RLU, relative luciferase unit).

Clonogenic assay
Cells (5 Â 10 4 per well in six-well plates) were exposed to designated concentration of DS/Cu 1 mM , PAC or PAC þ DS/Cu 1 mM for 24 h. The cells were collected and further cultured for 7 (MDA-MB-231 and MCF7) to 14 (T47D) days in six-well plates containing drug-free medium at a cell density of 2.5 Â 10 3 per well. Clonogenic cells were determined as those able to form a colony consisting of at least 50 cells.

Detection of ALDH-positive population
The ALDH-positive population in drug-treated BC cell lines was detected by ALDEFLUOR kit (StemCell Tech., Durham, NC, USA) following the supplier's instruction. The cells (2.5 Â 10 5 ) were analysed after staining in ALDH substrate containing assay buffer for 30 min at 371C. The negative control was treated with diethylaminobenzaldehyde (DEAB), a specific ALDH inhibitor.

Flow cytometric analysis of CD24 and CD44 expression
The adherent or mammosphere cells were trypsinised and passed through a 25G needle. The cells (2.5 Â 10 5 ) were incubated with CD24 and CD44 antibodies (BD Pharmingen, Oxford, UK) for 20 min at 41C. Unbound antibodies were washed off with 2% FCS HBSS (Sigma) and the cells (10 000 events) were examined no longer than 1 h after staining on a BD Facscalibur (Dorset, UK).

Statistical analysis
The data analysis was performed using Student's t-test and one-way ANOVA.

The cytotoxicity of DS in BC cells was Cu dependent
In CuCl 2 (1 mM)-supplemented medium, DS was highly cytotoxic to BC cell lines (IC 50_72h : 110 -476 nM; Figure 1A; Table 1). Disulfiram was also toxic to cancer cell lines in the complete medium without CuCl 2 supplement with higher IC 50s (456 -1100 nM; Figure 1A; Table 1). A biphasic effect was observed in two out of three BC cell lines. The cancer cells appeared to be protected at higher concentrations of DS. Disulfiram alone in serum-free medium (to rule out the influence of trace amount of Cu contained in FCS) or Cu alone was not toxic to BC cell lines even at a very high concentration (20 mM). The drug-induced morphological changes are shown in Figure 1B. The flow cytometric DNA content analysis demonstrated significant increase of apoptosis (sub-G1 population) in 72 h DS/Cu treated, but not other groups ( Figure 1C). The cleaved PARP protein, an indicator of caspase activation, was detected in DS/Cu-treated cells ( Figure 1D). Disulfiram/copper significantly inhibited Bcl2 and induced Bax expression; therefore, the Bax/Bcl2 ratio was increased in DS/Cu-treated cells ( Figure 1E).   .d.)). **Compared with PAC alone, significant difference (Po0.01, n ¼ 3). The cells were treated for 72 h. DS/Cu: DS in medium supplemented with 1 mM CuCl 2 ; DS -serum: DS in serum-free medium; DS+serum: DS in serum-containing medium.

DS/Cu synergistically enhanced the cytotoxicity of PAC in BC cell lines
In combination with DS/Cu, the cytotoxicity of PAC was significantly enhanced in BC cell lines (4 -16-fold) ( Figure 1F; Table 1). There was a very strong synergistic effect between DS/Cu and PAC over a wide range of concentrations (IC 50 -IC 90 ; Table 1). In contrast to the slight induction of apoptosis at low concentration of PAC alone (1 nM), the proportion of apoptotic cells was massively increased by DS/Cu (DS 100 -150 nM/Cu 1 mM) and PAC in combination ( Figure 1G).

ROS activation was responsible for DS/Cu-induced cytotoxicity
The Cu-dependent cytotoxicity of DS indicates that ROS may be the mediator for DS/Cu-induced apoptosis. Disulfiram/copper significantly induced ROS activity in BC cell lines (Po0.01), which was reversed by addition of an ROS inhibitor, NAC (Po0.01; Figure 2A). To determine the effect of ROS on DS/Cu-induced cell death, the cytotoxicity assay was performed with or without ROS inhibitor. As shown in Figure 2B, the DS/Cu-induced cytotoxicity was significantly reversed by addition of NAC in the culture (Po0.01). Figure 3A shows the effect of PAC, DS/Cu and PAC/DS/Cu on the activation of the JNK pathway. Total JNK protein expression was not affected by the above treatments. However, the expression of phosphorylated JNK, cJun and total cJun was persistently (up to 24 h) induced by DS/Cu and PAC/DS/Cu. In contrast, the expression of these proteins was not or only very mildly upregulated by PAC. High levels of phosphorylated p38 were also detected up to 24 h following DS/Cu and PAC/DS/Cu exposure ( Figure 3B). To determine the causal relationship between ROS and JNK, p38 pathways, BC cell lines were exposed to DS/Cu for 24 h with or without addition of NAC. N-acetyl-cysteine significantly inhibited or totally blocked DS/Cu-induced cJun and p38 phosphorylation ( Figure 3C). cJun N-terminal kinase and p38 are the major pathways responsible for ROS-induced apoptosis (McCubrey et al, 2006). Singly blocking JNK or p38 also reversed the DS/Cu-induced cytotoxicity, but at a significantly lower levels than NAC-induced ROS blocking (Po0.01; Figure 2B).

DS/Cu inhibited NFjB activity in BC cell lines
The NFkB is an ROS-induced transcription factor with strong antiapoptotic activity, which in turn dampens the pro-apoptotic effect of ROS (Nakano et al, 2006). Blockage of NFkB activation enhances ROS-induced cytotoxicity. Both PAC and DS/Cu inhibited NFkB DNA-binding activity in BC cell lines. The strongest inhibition was observed in the cells treated with PAC/DS/Cu in combination ( Figure 3D). The inhibition of NFkB transcriptional activity was also detected in PAC-, DS/Cu-and PAC/DS/Cu-treated cells by reporter gene assay ( Figure 3E).

DS/Cu inhibited the clonogenity in BC cell lines
Clonogenic assays (Franken et al, 2006) were performed to examine the ability of DS/Cu to induce 'reproductive death' in BC cells. After 16 h exposure to PAC (40 nM: 4 -18-fold higher than IC 50 concentration), DS (200 -250 nM: sub-IC 50 concentration)/ Cu 1 mM or PAC and DS/Cu in combination, the treated cells were collected and cultured in drug-free medium for 7 -14 days. The colony number was reduced by exposure to PAC, DS or Cu alone. The colony number in PAC-, DS-and Cu-treated groups was decreased, which was caused by slow growth of the surviving cells leading to the cell number in some colonies not reaching the counting threshold (50 cells). In contrast, the clonogenicity of BC cell lines was significantly inhibited by DS/Cu and totally eradicated by exposure to PAC plus DS/Cu ( Figure 4A).

DS/Cu targeted BCSCs
Furthermore, we examined the effect of different treatments on CSCs population in MDA-MB-231 and T47D cell lines. The mammosphere formation in both cell lines was completely blocked by exposure to DS 1 mM /Cu 1 mM or DS/Cu plus PAC 40 nM for 48 h, but not affected by PAC, DS or Cu alone ( Figure 4B). To determine the targeting effect of DS/Cu on CSCs, the BCSCs markers in drugtreated mammosphere cells were also analysed. Figure 4C demonstrates that the ALDH-positive population in mammospheres was significantly inhibited by DS/Cu, but not affected or even enriched by DS or Cu treatment. In order to determine the effect of DS/Cu on CSCs, the expression status of CD24 Low / CD44 High , another marker of BCSCs, was also examined. After 16 h exposure to different drugs, the CD24 Low /CD44 High population in the mammosphere cells was determined by flow cytometry. In comparison with the attached cells, the mammosphere population contained significantly higher percentage of CD24 Low /CD44 High BCSCs ( Figure 4D). The percentage of CD24 Low /CD44 High cells in mammosphere was significantly reduced following 16 h exposure to DS/Cu and PAC/DS/Cu, but not influenced by PAC, DS or Cu ( Figure 4D).

DISCUSSION
Disulfiram is a Food and Drug Administration-approved antialcoholism drug used in clinic with extensive available pre-clinical and clinical data (Eneanya et al, 1981). Our study demonstrated high cytotoxicity of DS to BC cell lines in a Cu-dependent manner. Using Cu to treat cancer has a long history (Hieger, 1926;Gupte and Mumper, 2009), but the intracellular transport of Cu is still one of the major hurdles for its clinical efficacy. The transport of Cu into cell is mediated and tightly controlled by the copper transporter, Ctr1. A derivative of DS, N,N-diethyldithiocarbamate (deDTC), binds to Cu forming a Cu(deDTC) 2 complex, which improves the intracellular trafficking of Cu and this is probably responsible for DS-induced apoptosis (Cen et al, 2004). Disulfiram can also penetrate into cancer cells to form Cu(deDTC) 2 with intracellular Cu. In comparison with normal tissues, many cancers including BC possess higher levels of Cu (two-to three-fold) (Mulay et al, 1971;Margalioth et al, 1983;Rizk and Sky-Peck, 1984), which may enable DS to target cancer cells selectively (Evans et al, 1982;Hacker et al, 1982;Bodenner et al, 1986;Chen et al, 2006;Iljin et al, 2009). In line with a previous report (Wickstrom et al, 2007), a biphasic cytotoxic effect of DS was observed in BC cell lines tested in complete medium without Cu supplement ( Figure 1A). Breast cancer cells were killed at low concentration, but revived at higher DS concentrations (B10 mM).
The mechanism of the biphasic effect remains unclear. A degradation product of DS may compete trace amounts of Cu, block formation of Cu(deDTC) 2 and inhibit transport of Cu into cells (Cen et al, 2004). We have previously reported that DS enhances the cytotoxicity of 5-FU and gemcitabine in colon and BC cell lines Guo et al, 2009). Here, we demonstrated synergistic cytotoxic effect of DS/Cu and PAC on BC cell lines. Previous studies demonstrate that in combination with Cu, DS induces ROS activity in melanoma cell lines (Cen et al, 2002;Morrison et al, 2010). The recent study from Dou's group demonstrates that gold -dithiocarbamato complexes strongly induce ROS and inhibit proteasome activity in BC cells (Zhang et al, 2010). In consistence with these results, our study showed that DS/Cu induced ROS activity, which was responsible for DS/ Cu-induced cytotoxicity in BC cell lines. The ROS-induced apoptosis is commonly mediated by the persistent activation of JNK and p38 MAPK pathways (Junttila et al, 2008). In our study, both JNK and p38 pathways were persistently (over 24 h) activated (phosphorylation of cJun and p38) by DS/Cu and blocked by NAC. cJun N-terminal kinase and p38 inhibitors reduced cytotoxicity of DS/Cu, although to a lesser degree than ROS inhibition. Therefore, ROS-activated JNK and p38 pathways were, at least partially, responsible for ROS-induced apoptosis. The persistent activation of JNK and p38 induces apoptosis via mitochondrial apoptotic pathways (Junttila et al, 2008). The DS/Cu-induced apoptosis was confirmed by DNA content and PARP protein cleavage. The expression of Bax and Bcl2 proteins was induced and suppressed by DS/Cu, respectively, leading to increased Bax/Bcl2 ratio. The elevated Bax/Bcl2 ratio indicated that the intrinsic apoptotic pathway may be involved in DS/Cu-induced apoptosis.
Owing to the high proliferative rate and energy requirement, cancer cells are under higher ROS stress than their normal counterparts. High levels of ROS can damage DNA, mitochondrial inner membrane and membrane phospholipids leading to apoptosis (Gupte and Mumper, 2009). However, ROS also activate a wide range of anti-apoptotic factors. The effect of ROS on cancer cells depends on the balance between ROS-induced pro-and antiapoptotic factors. The NFkB is one of the most important ROSinduced anti-apoptotic factors (Gloire et al, 2006). The NFkB activation in turn inhibits ROS and JNK, p38 activation and ultimately inhibits ROS-induced apoptosis. Breast cancer cell lines commonly possess high levels of constitutive NFkB activity (Wang et al, 2004;Guo et al, 2009;Xu et al, 2009). Consistent with previous publications Guo et al, 2009), DS/Cu inhibited NFkB activity in BC cell lines. This indicates that DS/Cu may induce apoptosis of BC cells by simultaneously inducing ROS generation and inhibiting ROS-NFkB pathway.
The effect of DS/Cu on the regeneration of minimal-residual cancer cells, the main source of cancer relapse after chemotherapy, was examined using a clonogenic assay, a gold measure to detect the cell 'reproductive death' after cytotoxic agent treatments (Franken et al, 2006). In contrast to the moderate inhibiting effect of PAC, DS and Cu on clonogenicity of BC cells, the colony formation was significantly reduced or completely eradicated by DS/Cu and PAC/DS/Cu, respectively ( Figure 4A).
Disulfiram/copper reverses cancer cell chemoresistance induced by a wide range of different mechanisms (Wang et al, 2001(Wang et al, , 2004Guo et al, 2009). It has been widely accepted that CSCs are responsible for tumour recurrence and may display significant resistance to different cytotoxic drugs (Liu and Wicha, 2010). The effect of DS/Cu on clonogenicity of BC cell lines prompted us to examine the effect of DS/Cu on BCSCs. Disulfiram is an inhibitor of ALDHs. Human ALDHs are a superfamily with 19 members involved in detoxifying a wide range of aldehydes to their corresponding weak carboxylic acids (Sladek, 2003); ALDH1A1 has been identified as a functional marker of several different types of CSCs including BCSCs (Ginestier et al, 2007;Alison et al, 2011). It recently reported that knockdown of ALDH1A1 expression using siRNA can target ovarian CSCs and potentiate cytotoxicity of taxane and platinum in vitro and in vivo (Landen et al, 2009 1.7 ± 0.1** 16.2 ± 0.5 17.3 ± 0.3 11.6 ± 0.1 1.1 ± 0.08 11.4 ± 0.3 Figure 4 Continued. marker of BCSCs. Therefore, ALDHs may be redundantly expressed in different cancer types and targeting one isoform may not be sufficient for CSCs targeting. Disulfiram is a strong inhibitor for both cytosol and mitochondrial ALDHs (Eneanya et al, 1981;Lam et al, 1997). Kast and Belda-Iniesta (2009) hypothesised that targeting ALDHs by DS may reverse chemoresistance in glioblastoma. Our study is the first report of using DS to target BCSCs. The ALDH þ VE population in BCSCs was significantly inhibited by DS/Cu. The ability of BC cell lines to form mammospheres was completely inhibited by 24 h exposure to DS/Cu or PAC/DS/Cu ( Figure 4B). The effect of DS/Cu on CSCs was also confirmed by the reduction of the CD24 Low /CD44 High population ( Figures 4C and D). The detailed molecular mechanisms underlying the effect of DS/Cu on BCSCs are unclear. Aldehyde dehydrogenases detoxify intracellular aldehydes, which can form adducts with glutathione, nucleic acids and amino acids leading to cell death (Marchitti et al, 2008). The high expression of ALDHs in CSCs may be protective. Mammalian cornea cells contain abundant ALDH, which has critical role in scavenging ROS and reduce UV-induced oxidative stress (Estey et al, 2007). Aldehyde dehydrogenase deficiency in central nervous system is associated with progressive neurodegeneration (Marchitti et al, 2007). Inhibition of NFkB pathway and induction of ROS result in reduction of stem-like properties in CSCs derived from pancreatic cancer and leukaemia (Greten et al, 2004;Jin et al, 2010;Rausch et al, 2010). Disulfiram/copper may target BCSCs by simultaneously inhibiting NFkB and activating ROS activity.