John L. Darling

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.

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.

Gain of 1q and loss of 22 are the most common changes detected by comparative genomic hybridisation in paediatric ependymoma

Ependymomas are the third most common brain tumour in the paediatric population. Although cytogenetic and molecular analyses have pinpointed deletions of chromosomes 6q, 17, and 22 in a subset of tumours, definitive patterns of genetic aberrations have not been determined. In the present study, we analysed 40 ependymomas from paediatric patients for genomic loss or gain using comparative genomic hybridisation (CGH). Eighteen of the tumours (45%) had no detectable regions of imbalance. In the remaining cases, the most common copy number aberrations were loss of 22 (25% of tumours) and gain of 1q (20%). Three regions of high copy number amplification were noted at 1q24‐31 (three cases), 8q21‐23 (two cases), and 9p (one case). Although there was no association with the loss or gain of any chromosome arm or with benign versus anaplastic histologic characteristics, the incidence of gain of 7q and 9p and loss of 17 and 22 was significantly higher in recurrent versus primary tumours. This study has identified a number of chromosomal regions that may contain candidate genes involved in the development of different subgroups of ependymoma.

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 p21Waf1. 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.

Disulfiram/copper complex inhibiting NFκB activity and potentiating cytotoxic effect of gemcitabine on colon and breast cancer cell lines

Most of the gemcitabine (dFdC) resistant cell lines manifested high NFkappaB activity. The NFkappaB activity can be induced by dFdC and 5-FU exposure. The chemosensitizing effect of disulfiram (DS), an anti-alcoholism drug and NFkappaB inhibitor, and copper (Cu) on the chemoresistant cell lines was examined. The DS/Cu complex significantly enhanced the cytotoxicity of dFdC (resistant cells: 12.2-1085-fold) and completely reversed the dFdC resistance in the resitant cell lines. The dFdC-induced NFkappaB activity was markedly inhibited by DS/Cu complex. The data from this study indicated that DS may be used in clinic to improve the therapeutic effect of dFdC in breast and colon cancer patients.

Identification of extensive genomic loss and gain by comparative genomic hybridisation in malignant astrocytoma in children and young adults.

Although astrocytomas are the most common central nervous system tumours in all age groups, there is substantial evidence that tumours arising in young patients (< 25 years of age) do not have the same genetic abnormalities that are characteristic of tumours in older patients. Furthermore, novel, consistent changes have not been identified in astrocytomas in children and young adults. We analysed 13 malignant astrocytomas from young patients using comparative genomic hybridisation. Regions of genomic imbalance were identified in 10 cases. The most common recurrent copy number aberrations were loss of 16p (54% of cases), 17p (38%), 19p (38%), and 22 (38%) and gain on 2q (38%), 12q (38%), 13 (38%), 4q (31%), 5q (31%), and 8q (31%). Seven regions of high copy number amplification were observed at 8q21–22 (three cases), 7q22–23 (two cases), and 1p21–22, 2q22, 12q13‐pter, 12q15–21, and 13q11–14 (one case each). This study provides evidence of new characteristic chromosomal imbalances from which potential candidate genes involved in the development of malignant astrocytoma in children and young adults may be identified.

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. Cancer Gene Therapy (2001) 8, 589–598

Triptolide simultaneously induces reactive oxygen species, inhibits NF-κB activity and sensitizes 5-fluorouracil in colorectal cancer cell lines

The in vitro chemosensitizing effect of triptolide (PG490) on the cytotoxicity of 5-fluorouracil (5-FU) was determined in three colorectal cancer (CRC) cell lines. PG490 alone was toxic to CRC cell lines (IC(50)s: 1.39-5.51nM). The cytotoxicity of 5-FU to CRC cell lines was 4-15-fold sensitized by combining use of low concentration of PG490. CI-isobologram indicated that the effect of PG490 plus 5-FU was synergistic. PG490 and 5-FU treatment induced activated caspase 3 and Bax expression and inhibited Bcl-2 expression. PG490 simultaneously induced generation of reactive oxygen species (ROS) and inhibited NF-kappaB transcriptional activity.

Cell proliferation in serial biopsies through human malignant brain tumours: Measurement using Ki67 antibody labelling

Cell proliferation was assessed in brain tumours using the monoclonal antibody Ki67 which recognizes a nuclear antigen expressed by proliferating cells. Using a novel stereotactic biopsy procedure, serial 1 cm biopsies were taken along a trajectory through six malignant brain tumours. Specimens were also obtained from 10 other brain tumours during conventional surgery. The percentage of Ki67 positive cells was determined as a fraction of the total number of tumour cells present. The Ki67 index for anaplastic astrocytomas and glioblastomas was significantly higher (Ki67 index range 11-18%) than that for benign or low grade tumours. Significant variation in proliferation was measured along the biopsy track through individual tumours (e.g. 0-12.3%) which correlated well with histological appearance. The Ki67 indices of normal brain were very low. In general the Ki67 indices increased with increasing histological grade and also appear to be a useful indicator of the active tumour volume and margin. This method provides spatial information about tumour proliferation which may be used to decide between different treatments and relate to prognosis.

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.