Disulfiram targets cancer stem-like cells and reverses resistance and cross-resistance in acquired paclitaxel-resistant triple-negative breast cancer cells.

BACKGROUND: Triple-negative breast cancer (TNBC) has significantly worse prognosis. Acquired chemoresistance remains the major cause of therapeutic failure of TNBC. In clinic, the relapsed TNBC is commonly pan-resistant to various drugs with completely different resistant mechanisms. Investigation of the mechanisms and development of new drugs to target pan-chemoresistance will potentially improve the therapeutic outcomes of TNBC patients. METHODS: In this study, 1-(4,5-Dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT), combination index (CI)-isobologram, western blot, ALDEFLUOR analysis, clonogenic assay and immunocytochemistry were used. RESULTS: The chemoresistant MDA-MB-231PAC10 cells are highly cross-resistant to paclitaxel (PAC), cisplatin (CDDP), docetaxel and doxorubicin. The MDA-MB-231PAC10 cells are quiescent with significantly longer doubling time (64.9 vs 31.7 h). This may be caused by high expression of p21(Waf1). The MDA-MB-231PAC10 cells express high aldehyde dehydrogenase (ALDH) activity and a panel of embryonic stem cell-related proteins, for example, Oct4, Sox2, Nanog and nuclealisation of HIF2α and NF-κBp65. We have previously reported that disulfiram (DS), an antialcoholism drug, targets cancer stem cells (CSCs) and enhances cytotoxicity of anticancer drugs. Disulfiram abolished CSC characters and completely reversed PAC and CDDP resistance in MDA-MB-231PAC10 cells. CONCLUSION: Cancer stem cells may be responsible for acquired pan-chemoresistance. As a drug used in clinic, DS may be repurposed as a CSC inhibitor to reverse the acquired pan-chemoresistance.

Cytotoxic effect of disulfiram/copper on human glioblastoma cell lines and ALDH-positive cancer-stem-like cells.

BACKGROUND: Glioblastoma multiforme (GBM) cells are resistant to anticancer drugs. Cancer stem cells (CSCs) are a key mediator of chemoresistance. We have reported that disulfiram (DS), an aldehyde dehydrogenase (ALDH) inhibitor, targets breast CSC-like cells. In this study, the effect of DS and combination of DS and gemcitabine (dFdC) on GBM cells and GBM stem-like cells was investigated. METHODS: 1-(4,5-Dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT), combination index (CI)-isobologram, western blot, luciferase reporter gene assay, electrophoretic mobility-shift assay and ALDH analysis were used in this study. RESULTS: Disulfiram is cytotoxic in GBM cell lines in a copper (Cu)-dependent manner. Disulfiram/copper enhances the cytotoxicity of dFdC. Combination index-isobologram analysis indicates a synergistic effect between DS/Cu and dFdC. Disulfiram/copper induces reactive oxygen species (ROS), activates JNK and p38 pathways and inhibits nuclear factor-kappa B activity in GBM cell lines. Disulfiram/copper may trigger intrinsic apoptotic pathway via modulation of the Bcl2 family. Disulfiram/copper abolishes stem-like cell population in GBM cell lines. CONCLUSION: Our findings indicate that the cytotoxicity of DS/Cu and the enhancing effect of DS/Cu on the cytotoxicity of dFdC in GBM stem-like cells may be caused by induction of ROS and inhibition of both ALDH and the NFkB pathway. Both DS and dFdC can traverse the blood-brain barrier. Further study may lead them into GBM chemotherapy.

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 IC(50) 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 CD24(Low)/CD44(High) 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.

Astrocytoma derived short-term cell cultures retain molecular signatures characteristic of the tumour in situ.

The heterogeneity of tumours and uncertainties surrounding derived short-term cell cultures and established cell lines fundamentally challenge the research and understanding of tumour growth and development. When tumour cells are cultured, changes are inevitably induced due to the artificial growth conditions. Several recent studies have questioned how representative established cell lines or derived short-term cell cultures are of the tumour in situ. We have characterised gene expression changes induced by short-term culture in astrocytoma in order to determine whether derived short-term cell cultures are representative of the tumour in situ. In comparison to the majority of studies, paired biopsies and derived short-term cultures were investigated to reduce the effects of long-term culture and inter-tumour variability when comparing biopsies and derived cultures from tumours with the same histology from different individuals. We have used the Affymetrix GeneChip U133A to generate gene expression profiles of 6 paediatric pilocytic astrocytoma (PA) biopsies and derived short-term cell cultures and 3 adult glioblastoma multiforme (GBM) biopsies and derived short-term cultures. Significant differential gene expression is induced by short-term culture. However, when the biopsy and derived short-term cell culture samples were grouped according to tumour type (PA and GBM) a molecular signature of 608 genes showed significant differential expression between the groups. This gene cohort can distinguish PA and GBM tumours, regardless of the sample source, suggesting that astrocytoma derived short-term cultures do retain key aspects of the global tumour expression profile and are representative of the tumour in situ. Furthermore, these genes are involved in pathways and functions characteristic of adult GBM including VEGF signalling, hypoxia and TP53 signalling.

Pretreatment prediction of the chemotherapeutic response of human glioma cell cultures using nuclear magnetic resonance spectroscopy and artificial neural networks.

Both tumor metabolism and its response to cytotoxic drugs are intrinsic properties of tumor cells. It is therefore likely that there is a relationship between the two properties, however subtle and complex, wherein the metabolic characteristics of tumor cells can reflect the inherent response (resistance or sensitivity) of these cells to cytotoxic drugs. We used artificial neural network analysis to show that it is possible to distinguish, prior to treatment, between drug-resistant and drug-sensitive human glioma cell cultures from their metabolic profiles, as given by high-resolution proton nuclear magnetic resonance spectra of the cell extracts, and to predict their cellular response to the chemotherapeutic drug 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea in vitro. The results suggest that neural network analysis of tumor nuclear magnetic resonance spectra has potential as a prognostic tool for determining treatment of gliomas, ultimately noninvasively, and may be used to provide information about the metabolic pathways involved in drug response that may be helpful in developing novel treatments for these tumors.

Adenovirus-mediated expression of HSV1-TK or Fas ligand induces cell death in primary human glioma-derived cell cultures that are resistant to the chemotherapeutic agent CCNU.

Due to minimal treatment success with surgery, radiotherapy, and chemotherapy, the aim of this study was to test the therapeutic potential of gene therapy for the treatment of glioblastoma multiforme (GBM). We have quantitatively analyzed two gene therapy approaches using short-term human glioma cell cultures derived from surgical biopsies (designated IN859, IN1612, IN2045, IN1760, and IN1265) and compared the results of gene therapy with the chemosensitivity of the same cells. All of the glioma cell cultures tested were susceptible to recombinant adenovirus (RAd)-mediated infection. Expression of herpes simplex virus type 1-thymidine kinase (RAd128), followed by ganciclovir treatment, induced apoptosis in all of the glioma cell cultures studied, including three that are resistant to the chemotherapeutic drug 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU). Expression of murine Fas ligand (RAdhCMV-mFasL) also induced cell death in four of the five cell cultures studied. One cell culture that was resistant to CCNU was also resistant to apoptosis induced by mFasL expression. These results suggest that sensitivity to chemotherapeutic agents does not necessarily correlate with the sensitivity to gene therapy treatments. RAds expressing therapeutic gene products in human glioma cell cultures are able to induce apoptosis even in some cells that are resistant to a commonly used chemotherapeutic agent. Therefore, RAd-mediated gene transfer could be a good candidate to further develop gene therapy for the treatment of GBM.

Chemosensitivity in childhood brain tumours in vitro: evidence of differential sensitivity to lomustine (CCNU) and vincristine.

The aim of this study was to examine the range of sensitivity of a panel of short-term cultures derived from different types of malignant childhood brain tumours including medulloblastoma, ependymoma and glioblastoma multiforme to three cytotoxic drugs, lomustine (CCNU), vincristine (VCR) and procarbazine (PCB). Sensitivity was assessed using a modification of the dimethylthiazolyl-2,5-diphenyl tetrazolium bromide (MTT) assay. Short-term cell lines derived from ependymomas were considerably more resistant to VCR than other types of childhood brain tumours, while cultures derived from supratentorial primitive neuroectodermal tumour (PNET) displayed marked sensitivity to both lomustine and VCR. Cultures from ependymomas, medulloblastoma and astrocytic gliomas had similar sensitivity to lomustine and PCB as cultures derived from adult malignant astrocytoma.

Heterogeneity of radiosensitivity in a human glioma cell line.

Sixteen clones were isolated from an early-passage human glioma cell line (IN859) and have been found to show variation in several biological characteristics including DNA content, modal chromosome number, and morphology. In addition, heterogeneity of radiosensitivity was detected: the doses that gave a surviving fraction of 0.01 varied by a factor of approximately 1.5. The most sensitive (clone 6) and the most resistant (clone 9) clones were selected for further study; their surviving fractions at 2Gy (SF2) were 0.37 and 0.64, respectively. When compared at a fixed radiation dose the sensitive clone surprisingly demonstrated greater split-dose recovery than the resistant clone; it also showed greater low dose-rate sparing.

Radiosensitivity, recovery and dose-rate effect in three human glioma cell lines.

The results of radiotherapy in the treatment of high-grade gliomas are disappointing. In this study three recently established cell lines from high-grade human gliomas have been found to exhibit a sensitivity that is at the resistant end of the spectrum of radiosensitivities seen in human tumour cells generally. The results support the view that inherent cellular radioresistance may be an important cause of failure in this disease. All three cell lines showed an increase in survival when the radiation dose rate was reduced. In split-dose experiments, recovery was found to increase with dose in a manner consistent with the predictions of the linear-quadratic equation.

Three cell lines from a spontaneous murine astrocytoma show variation in astrocytic differentiation.

Three cell lines originally derived from a spontaneous, transplantable, murine astrocytoma have been maintained in vitro. Immunofluorescence confirmed their astrocytic nature while electron microscopy revealed differences in the number and ratio of 10 nm filaments and 24 nm microtubules in the 3 lines; a feature related to the degree of astrocytic differentiation.

Cell lines (VMDk) derived from a spontaneous murine astrocytoma. Morphological and immunocytochemical characterization.

Three cell lines (VMDk) derived from a spontaneous, murine astrocytoma, which produce tumours when injected either subcutaneously or intracranially into syngeneic mice, have been examined in vitro. Ultrastructurally, the cells show astrocytic features but each line differs in its degree of differentiation. Treatment with both dexamethasone and dibutyryl adenosine 3',5'-cyclic monophosphate (dbcAMP) increases intracytoplasmic differentiation and causes surface structural changes. The addition of dbcAMP also induces a statistically significant increase in the length and number of cell processes. All three cell lines express the astrocyte-specific markers, glial fibrillary acidic protein (GFAP) and glutamine synthetase (GS), by indirect immunofluorescence, with two lines showing an increased propensity to stain for GFAP following dbcAMP treatment. The cell surface antigen fibronectin is also detected in all lines. Thus these VMDK cell lines exhibit both the morphological and antigenic characteristics of astrocytes and respond to dexamethasone and dbcAMP and may be used to provide a suitable in vivo-in vitro model system for the study of astrocytoma.

Tumorigenicity of cell lines (VMDk) derived from a spontaneous murine astrocytoma. Histology, fine structure and immunocytochemistry of tumours.

Three cell lines (VMDk), derived from a spontaneous murine astrocytoma, which exhibit both morphological and antigenic characteristics of astrocytes in vitro (Pilkington et al. 1983), have been injected intracerebrally and subcutaneously into syngeneic mice at a range of concentrations in order to assess the number of cells required to produce the highest yield of tumours with the shortest possible latent period. Light- and electron-microscopical studies of the tumours confirmed their glial nature, however immunocytochemical staining for the astrocyte-specific markers glial fibrillary acidic protein (GFAP) and glutamine synthetase (GS) and for vimentin suggest that the tumours are composed mainly of poorly-differentiated neoplastic glial cells. Although the best-differentiated of the three cell lines failed to produce tumours, an invasive transplantable astrocytoma model has, nevertheless, been characterised fully and can now be used to study the effects of various therapeutic regimens.

Alterations in the cellular immune response of patients with cerebral glioma, benign intracranial tumour, and spontaneous subarachnoid haemorrhage measured in vitro by the leucocyte migration inhibition test.

Leucocyte migration inhibition in response to ubiquitous antigens was studied in 104 patients as an in vitro indicator of cell-mediated immunity. Patients with cerebral glioma, benign intracranial tumours, and subarachnoid haemorrhage demonstrated impaired inhibition of leucocyte migration compared with control subjects. The greatest impairment occurred in patients with subarachnoid haemorrhage, while the least impairment was seen in patients with glioma. Significant rises in inhibition of leucocyte migration in response to antigen preparations from glioma and normal brain were seen in the early post-operative period in patients with glioma and subarachnoid haemorrhage. Impaired cellular immunity, together with sensitivity of lymphocytes to brain-derived antigens, are features of cerebral disease in general and not specific for glioma.

Chronic low-dose exposure of sodium nitrite in VM-strain mice: central nervous system changes.

1. There is suggestive evidence that nitrite may be a causative factor in cerebral glioma. 2. To test this hypothesis we selected the VM mouse strain, known for its susceptibility to spontaneous glioma formation, and exposed 300 animals to 0.2% sodium nitrite in their drinking water. One hundred of this group were exposed both in utero and throughout their adult lives. The remaining 200 animals received nitrite from the time of weaning. A further 200 mice were used as controls and received distilled water. 3. All animals were maintained until their natural death and were then subjected to autopsy and routine histological examination. 4. There was no excess of nervous system tumours in the experimental groups.

In vitro evaluation of cancer-specific NF-kappaB-CEA enhancer-promoter system for 5-fluorouracil prodrug gene therapy in colon cancer cell lines.

Nuclear factor-kappa B (NF-kappaB) is a transcription factor with high transcriptional activity in cancer cells. In this study, we developed a novel enhancer-promoter system, kappaB4-CEA205, in which the basal carcinoembryonic antigen (CEA) promoter sequence (CEA205) was placed downstream of the four tandem-linked NF-kappaB DNA-binding sites (kappaB4). In combination with a kappaB4 enhancer, the transcriptional activity of the CEA promoter was significantly enhanced (three- to eight-fold) in cancer cell lines but not in normal cells. In cancer cell lines, the transcriptional activity of kappaB4-CEA205 was comparable with that of the SV40 promoter. We also constructed vectors in which the thymidine phosphorylase (TP) cDNA was under the control of CEA205, kappaB4, kappaB4-CEA205 and CMV promoters, respectively. TP protein and enzyme activity were detected at comparable levels in kappaB4-CEA205- and CMV-driven TP cDNA-transfected cancer cell lines (H630 and RKO). The kappaB4-TP and CEA205-TP-transfected cell lines, respectively, only demonstrated negligible and low levels of TP protein and enzyme activity. Both CMV- and kappaB4-CEA205-driven TP cDNA transiently transfected cells were 8- to 10-fold sensitised to 5-fluorouracil (5-FU) prodrug, 5'-deoxy-5-fluorouradine (5'-DFUR), in contrast to only 1.5- to 2-fold sensitised by the kappaB4- and CEA205-driven TP cDNA-transfected cells. The bystander killing effect of CMV- and kappaB4-CEA205-driven TP cDNA-transfected cells was comparable. This is the first report that indicates that the NF-kappaB DNA-binding site could be used as a novel cancer-specific enhancer to improve cancer-specific promoter activity in gene-directed enzyme prodrug therapy.

Scintillation autofluorographic assessment of isotope uptake in human glioma cells grown on microtitration plates using sodium salicylate.

We describe a simple method for detecting [35S]methionine-labeled protein in fixed human astrocytoma cells grown in 96-well microtitration plates using a modified scintillation autofluorographic method. Following isotopic labeling, cells are fixed in situ and a solution of salicylic acid in methanol is dried onto the cell layer. The fluorographic image is detected using blue-sensitive X-ray film attached to the base of the plate which, following development, can be quantitated using a scanning densitometer. The relationship between cell number and optical density is linear, and there is a close correlation between the dose-response curves generated by this method and alternative isotopic detection methods and cell counting. This assay provides a suitable alternative to the use of potentially toxic scintillation fluids based on organic solvents like toluene or xylene in chemosensitivity testing of human brain tumors.

Biology and genetics of malignant brain tumours.

Conventional therapies such as surgery, radiotherapy and, to a lesser extent, chemotherapy have produced significant increases in survival in patients with some types of brain tumours such as medulloblastoma. However, in many other types of brain tumour in both adults and children, the effect of these modalities has been more modest. A thorough understanding of the biology of malignant brain tumours is likely to provide the background for the development of new leads that might be amenable to therapeutic exploitation. This review examines some aspects of glioma biology that have been reported in the past 12 months, and which might be translated into clinical application.

Response of short-term cultures derived from human malignant glioma to aziridinylbenzoquinone, etoposide and doxorubicin: an in vitro phase II trial.

The relative resistance of malignant glioma to chemotherapy makes the identification of new cytotoxic drugs critically important. The use of short-term cultures derived from these tumors to screen drugs at doses that can be attained within human intracranial tumors provides a model system that should be capable of identifying effective drugs suitable for clinical evaluation. The sensitivity of a panel of short-term cultures derived from 22 malignant astrocytoma and four malignant oligodendroglioma was assessed to aziridinylbenzoquinone (AZQ), etoposide and doxorubicin (DOX) using a [(35)S] methione uptake assay. The ID(50) of each culture was compared to the levels of drug which could be achieved in the tumor using standard doses. There was marked heterogeneity between cultures in response to each drug. Whilst there was no evidence that cultures derived from grade III astrocytoma were more sensitive to any of the drugs than cultures derived from grade IV astrocytoma, cultures derived from oligodendroglioma tended to be more sensitive to the alkylating agent AZQ, but not to either of the other drugs. The sensitivity of these short-term cultures at concentrations that can be achieved in situ corresponded well with the clinical efficacy of AZQ and etoposide. Although DOX appeared to be toxic to human gliomas cells in vitro, its limited penetration into the intact brain would seem to preclude its use i.v., but it is likely to be effective if local drug delivery techniques could be employed. The study suggests that short-term cultures derived from malignant glioma should be used to screen investigational agents for potential clinical efficacy.

The in-vitro chemosensitivity of three cell lines derived from the VM/DK spontaneous murine astrocytoma.

Three cell lines, VM/Dk P497 P540 and P560 derived from the VM spontaneous murine astrocytoma have previously been fully characterised and found to differ in their degree of astrocytic differentiation. The in vitro chemosensitivity of the three lines has been investigated using the 35S-methionine uptake assay. Differential chemosensitivity was found to exist between the cell lines. The pattern of chemosensitivity in relation to astrocytic differentiation was complex but the least differentiated cell line, P497, tended to be the least chemosensitive.