tram-34 and Glioma

Nanocarriers are delivery platforms of drugs, peptides, nucleic acids and other therapeutic molecules that are indicated for severe human diseases. Gliomas are the most frequent type of brain tumor, with glioblastoma being the most common and malignant type. The current state of glioma treatment requires innovative approaches that will lead to efficient and safe therapies. Advanced nanosystems and stimuli-responsive materials are available and well-studied technologies that may contribute to this effort. The present study deals with the development of functional chimeric nanocarriers composed of a phospholipid and a diblock copolymer, for the incorporation, delivery and pH-responsive release of the antiglioma agent TRAM-34 inside glioblastoma cells. Nanocarrier analysis included light scattering, protein incubation and electron microscopy, and fluorescence anisotropy and thermal analysis techniques were also applied. Biological assays were carried out in order to evaluate the nanocarrier nanotoxicity in vitro and in vivo, as well as to evaluate antiglioma activity. The nanosystems were able to successfully manifest functional properties under pH conditions, and their biocompatibility and cellular internalization were also evident. The chimeric nanoplatforms presented herein have shown promise for biomedical applications so far and should be further studied in terms of their ability to deliver TRAM-34 and other therapeutic molecules to glioblastoma cells.

Topics: Apoptosis; Cell Proliferation; Drug Carriers; Drug Delivery Systems; Glioma; Humans; Hydrogen-Ion Concentration; Liposomes; Nanoparticles; Polymers; Pyrazoles; Tumor Cells, Cultured

Among the strategies adopted by glioma to successfully invade the brain parenchyma is turning the infiltrating microglia/macrophages (M/MΦ) into allies, by shifting them toward an anti-inflammatory, pro-tumor phenotype. Both glioma and infiltrating M/MΦ cells express the Ca(2+)-activated K(+) channel (KCa3.1), and the inhibition of KCa3.1 activity on glioma cells reduces tumor infiltration in the healthy brain parenchyma. We wondered whether KCa3.1 inhibition could prevent the acquisition of a pro-tumor phenotype by M/MΦ cells, thus contributing to reduce glioma development. With this aim, we studied microglia cultured in glioma-conditioned medium or treated with IL-4, as well as M/MΦ cells acutely isolated from glioma-bearing mice and from human glioma biopsies. Under these different conditions, M/MΦ were always polarized toward an anti-inflammatory state, and preventing KCa3.1 activation by 1-[(2-Chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34), we observed a switch toward a pro-inflammatory, antitumor phenotype. We identified FAK and PI3K/AKT as the molecular mechanisms involved in this phenotype switch, activated in sequence after KCa3.1. Anti-inflammatory M/MΦ have higher expression levels of KCa3.1 mRNA (kcnn4) that are reduced by KCa3.1 inhibition. In line with these findings, TRAM-34 treatment, in vivo, significantly reduced the size of tumors in glioma-bearing mice. Our data indicate that KCa3.1 channels are involved in the inhibitory effects exerted by the glioma microenvironment on infiltrating M/MΦ, suggesting a possible role as therapeutic targets in glioma.

Topics: Animals; Brain; Cell Line, Tumor; Chromones; Focal Adhesion Kinase 1; Glioma; Humans; Interleukin-4; Intermediate-Conductance Calcium-Activated Potassium Channels; Macrophages; Male; Mice; Mice, Inbred C57BL; Microglia; Morpholines; Phagocytosis; Phenotype; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Pyrazoles; RNA, Messenger

Malignant gliomas are among the most frequent and aggressive cerebral tumors, characterized by high proliferative and invasive indexes. Standard therapy for patients, after surgery and radiotherapy, consists of temozolomide (TMZ), a methylating agent that blocks tumor cell proliferation. Currently, there are no therapies aimed at reducing tumor cell invasion. Ion channels are candidate molecular targets involved in glioma cell migration and infiltration into the brain parenchyma. In this paper we demonstrate that: i) blockade of the calcium-activated potassium channel KCa3.1 with TRAM-34 has co-adjuvant effects with TMZ, reducing GL261 glioma cell migration, invasion and colony forming activity, increasing apoptosis, and forcing cells to pass the G2/M cell cycle phase, likely through cdc2 de-phosphorylation; ii) KCa3.1 silencing potentiates the inhibitory effect of TMZ on glioma cell viability; iii) the combination of TMZ/TRAM-34 attenuates the toxic effects of glioma conditioned medium on neuronal cultures, through a microglia dependent mechanism since the effect is abolished by clodronate-induced microglia killing; iv) TMZ/TRAM-34 co-treatment increases the number of apoptotic tumor cells, and the mean survival time in a syngeneic mouse glioma model (C57BL6 mice implanted with GL261 cells); v) TMZ/TRAM-34 co-treatment reduces cell viability of GBM cells and cancer stem cells (CSC) freshly isolated from patients.Taken together, these data suggest a new therapeutic approach for malignant glioma, targeting both glioma cell proliferating and migration, and demonstrate that TMZ/TRAM-34 co-treatment affects both glioma cells and infiltrating microglia, resulting in an overall reduction of tumor cell progression.

Topics: Animals; Antineoplastic Agents, Alkylating; Apoptosis; Brain Neoplasms; CDC2 Protein Kinase; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Dacarbazine; Drug Synergism; Drug Therapy, Combination; G2 Phase Cell Cycle Checkpoints; Glioma; Humans; Intermediate-Conductance Calcium-Activated Potassium Channels; Kaplan-Meier Estimate; Mice; Mice, Inbred C57BL; Microglia; Neoplasms, Experimental; Neoplastic Stem Cells; Phosphorylation; Primary Cell Culture; Pyrazoles; Temozolomide

Ca(2+)-activated K(+) channels, such as BK and IK channels, have been proposed to fulfill pivotal functions in neoplastic transformation, malignant progression, and brain infiltration of glioblastoma cells. Here, the ionizing radiation (IR) effect of IK K(+) channel targeting was tested in human glioblastoma cells. IK channels were inhibited pharmacologically by TRAM-34 or genetically by knockdown, cells were irradiated with 6 MV photons and IK channel activity, Ca(2+) signaling, cell cycling, residual double-strand breaks, and clonogenic survival were determined. In addition, the radiosensitizing effect of TRAM-34 was analyzed in vivo in ectopic tumors. Moreover, The Cancer Genome Atlas (TCGA) was queried to expose the dependence of IK mRNA abundance on overall survival (OS) of patients with glioma. Results indicate that radiation increased the activity of IK channels, modified Ca(2+) signaling, and induced a G2-M cell-cycle arrest. TRAM-34 decreased the IR-induced accumulation in G2-M arrest and increased the number of γH2AX foci post-IR, suggesting that TRAM-34 mediated an increase of residual DNA double-strand breaks. Mechanistically, IK knockdown abolished the TRAM-34 effects indicating the IK specificity of TRAM-34. Finally, TRAM-34 radiosensitized ectopic glioblastoma in vivo and high IK mRNA abundance associated with shorter patient OS in low-grade glioma and glioblastoma.. Together, these data support a cell-cycle regulatory function for IK K(+) channels, and combined therapy using IK channel targeting and radiation is a new strategy for anti-glioblastoma therapy.

Topics: Animals; Calcium; Cell Line, Tumor; Combined Modality Therapy; Dacarbazine; Disease-Free Survival; Female; G2 Phase Cell Cycle Checkpoints; Gene Knockdown Techniques; Glioblastoma; Glioma; Histones; Humans; Intermediate-Conductance Calcium-Activated Potassium Channels; Mice; Potassium Channel Blockers; Pyrazoles; Radiation-Sensitizing Agents; RNA, Messenger; Signal Transduction; Temozolomide

Oxaliplatin (OXAL) is a third-generation organoplatinum which is effective against advanced cancer cells including glioma cells. How this agent and other related compounds interacts with ion channels in glioma cells is poorly understood. OXAL (100 µM) suppressed the amplitude of whole-cell K+ currents (I(K)); and, either DCEBIO or ionomycin significantly reversed OXAL-mediated inhibition of I(K) in human 13-06-MG glioma cells. In OXAL-treated cells, TRAM-34 did not suppress I(K) amplitude in these cells. The intermediate-conductance Ca(2+)-activated K+ (IK(Ca)) channels subject to activation by DCEBIO and to inhibition by TRAM-34 or clotrimazole were functionally expressed in these cells. Unlike cisplatin, OXAL decreased the probability of IK(Ca)-channel openings in a concentration-dependent manner with an IC50 value of 67 µM. No significant change in single-channel conductance of IK(Ca) channels in the presence of OXAL was demonstrated. Neither large-conductance Ca(2+)-activated K+ channels nor inwardly rectifying K+ currents in these cells were affected in the presence of OXAL. OXAL also suppressed the proliferation and migration of 13-06-MG cells in a concentration- and time-dependent manner. OXAL reduced IK(Ca)-channel activity in LoVo colorectal cancer cells. Taken together, the inhibition by OXAL of IK(Ca) channels would conceivably be an important mechanism through which it acts on the functional activities of glioma cells occurring in vivo.

Topics: Antineoplastic Agents; Cell Line, Tumor; Cell Movement; Cell Proliferation; Clotrimazole; Glioma; Humans; Intermediate-Conductance Calcium-Activated Potassium Channels; Membrane Potentials; Organoplatinum Compounds; Oxaliplatin; Patch-Clamp Techniques; Platinum; Pyrazoles; RNA, Messenger