tacrolimus and Glioblastoma

Poor response to current treatments for glioblastoma has been attributed to the presence of glioblastoma stem-like cells (GSCs). GSCs are able to expel antitumor drugs to the extracellular medium using the multidrug resistance-associated protein 1 (MRP1) transporter. Tacrolimus (FK506) has been identified as an MRP1 regulator in differentiated glioblastoma (GBM) cells (non-GSCs); however, the effect of FK506 on GSCs is currently unknown. The objective of the following research is to evaluate the effect of FK506 on the MRP1-related chemo-resistant phenotype of GSCs. For this, U87MG and C6 glioma cell lines were used to generate non-GSCs and GSCs. mRNA and MRP1-positive cells were evaluated by RT-qPCR and flow cytometry, respectively. A Carboxyfluorescein Diacetate (CFDA)-retention assay was performed to evaluate the MRP1 activity. Apoptosis and MTT assays were employed to evaluate the cytotoxic effects of FK506 plus Vincristine (MRP1 substrate). GSC-derived subcutaneous tumors were generated to evaluate the in vivo effect of FK506/Vincristine treatment. No differences in transcript levels and positive cells for MRP1 were observed in FK506-treated cells. Lesser cell viability, increased apoptosis, and CFDA-retention in the FK506/Vincristine-treated cells were observed. In vivo, the FK506/Vincristine treatment decreased the tumor size as well as ki67, Glial Fibrillary Acidic Protein (GFAP), and nestin expression. We conclude that FK506 confers a chemo-sensitive phenotype to MRP1-drug substrate in GSCs.

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Brain Neoplasms; Cell Line, Tumor; Drug Resistance, Neoplasm; Glioblastoma; Humans; Male; Multidrug Resistance-Associated Proteins; Neoplastic Stem Cells; Phenotype; Rats, Sprague-Dawley; Tacrolimus; Vincristine

Tryptophan catabolism is increasingly recognized as a key and druggable molecular mechanism active in cancer, immune, and glioneural cells and involved in the modulation of antitumor immunity, autoimmunity and glioneural function. In addition to the pivotal rate limiting enzyme indoleamine-2,3-dioxygenase, expression of tryptophan-2,3-dioxygenase (TDO) has recently been described as an alternative pathway responsible for constitutive tryptophan degradation in malignant gliomas and other types of cancer. In addition, TDO has been implicated as a key regulator of neurotoxicity involved in neurodegenerative diseases and ageing. The pathways regulating TDO expression, however, are largely unknown. Here, a siRNA-based transcription factor profiling in human glioblastoma cells revealed that the expression of human TDO is suppressed by endogenous glucocorticoid signaling. Similarly, treatment of glioblastoma cells with the synthetic glucocorticoid dexamethasone led to a reduction of TDO expression and activity in vitro and in vivo. TDO inhibition was dependent on the immunophilin FKBP52, whose FK1 domain physically interacted with the glucocorticoid receptor as demonstrated by bimolecular fluorescence complementation and in situ proximity ligation assays. Accordingly, gene expression profile analyses revealed negative correlation of FKBP52 and TDO in glial and neural tumors and in normal brain. Knockdown of FKBP52 and treatment with the FK-binding immunosuppressant FK506 enhanced TDO expression and activity in glioblastoma cells. In summary, we identify a novel steroid-responsive FKBP52-dependent pathway suppressing the expression and activity of TDO, a central and rate-limiting enzyme in tryptophan metabolism, in human gliomas.

Topics: Aging; Animals; Cell Line, Tumor; Dexamethasone; Glioblastoma; Humans; Mice; Signal Transduction; Tacrolimus; Tacrolimus Binding Proteins; Tryptophan; Tryptophan Oxygenase

Members of the nuclear factor of activated T cells (NFAT) family have been identified as regulators of oncogenic transformation in several human malignancies. A prominent member of this family, NFAT1, is associated with tumor cell survival, apoptosis, migration and invasion. Here, we investigated the role of NFAT1 in glioma cells. In 111 clinical samples, microarray analysis demonstrated that NFAT1 was over-expressed in glioblastoma multiforme (GBM), compared with low-grade gliomas, a result confirmed by RT-PCR in 24 clinical samples and in the U87 and U251 cell lines. Immunohistochemistry and immunofluorescence stain indicated that over-expressed NFAT1 was mainly located in the nucleus, where it acted as a transcription factor. After treatment with the NFAT antagonist cyclosporin A (CsA) and FK506, levels of NFAT1 in the nuclei of U87 GBM cells were dramatically reduced. The invasive potential of U87 cells was reduced by the same treatment, as well as by inhibition of NFAT1 expression using small hairpin RNA. Proliferation of U87 cells was unaffected by CsA, FK506 and NFAT1 shRNA transfection. Clustering analysis and Pearson correlation analysis of microarray data showed that the expression of NFAT1 correlated with the expression of the invasion-related genes cyclooxygenase-2 (COX-2), matrix metalloproteinase-7 (MMP-7) and MMP-9, a result confirmed by in vitro analysis. These findings demonstrate that NFAT1 contributes to the invasive potential but not the proliferation of GBM cells, and suggest that CsA may find application as an adjuvant in combined treatment strategies for GBM.

Topics: Biopsy; Brain Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Nucleus; Cell Proliferation; Cyclooxygenase 2; Cyclosporine; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Matrix Metalloproteinase 7; Matrix Metalloproteinase 9; Neoplasm Invasiveness; NFATC Transcription Factors; Oligonucleotide Array Sequence Analysis; RNA, Small Interfering; Tacrolimus

Glioblastoma multiforme (GBM) is the most aggressive of brain tumors and is extremely insensitive to anticancer drugs. Studies have attributed the ABC transporter Mrp1 (ABCC1, multiple-drug resistance protein 1) with conferring chemoresistance in this tumor by extrusion of a wide spectrum of anticancer drugs. Therefore it is crucial to search for and investigate inhibitors of Mrp1 activity in GBM cells, particularly those that could be safe as chemosensitizers to anticancer drugs in clinical studies. We find that in primary cultured or T98G GBM cells exposed to therapeutic plasma concentrations of FK506 (tacrolimus), the expression of Mrp1 was decreased in a dose-dependent manner. The activity of this transporter, measured by CFDA fluorescent substrate extrusion, decreased significantly in primary cultured GBM cells on exposure to FK506 at concentrations of 15 ng/ml. When GBM cells were exposed to anticancer drugs vincristine, etoposide or taxol, cell viability was not affected. However when the anticancer drugs were assayed in combination with FK506, cell viability was significantly decreased by as much as 50% in GBM primary culture. We conclude that FK506 could be a valuable tool for chemosensitization of GBM cells, offering a possible improvement to the current poor therapy available for high-grade human gliomas.

Topics: Antineoplastic Agents; Brain Neoplasms; Cell Line, Tumor; Drug Resistance, Neoplasm; Glioblastoma; Humans; Immunosuppressive Agents; Multidrug Resistance-Associated Proteins; Tacrolimus