Mitochondrial nicotinic acetylcholine receptors form complexes with Bax upon apoptosis induction

Nicotinic acetylcholine receptors (nAChRs) mediate fast synaptic transmission in muscles and autonomic ganglia and regulate cytokine and neurotransmitter release in the brain and nonexcitable cells. The nAChRs expressed in the outer membrane of mitochondria control the early events of mitochondria-driven apoptosis like cytochrome c release by affecting intramitochondrial kinase pathways. However, the mechanisms through which nAChRs influence mitochondrial permeability remain obscure. Previously we demonstrated that mitochondrial nAChRs interact with voltage-dependent anion channels (VDAC) involved in forming the pore in mitochondria membrane. Here we put an aim to explore the connection of nAChRs to pro-apoptotic protein Bax and its changes in the course of apoptosis induction. By using molecular modeling in silico, it was shown that both Bax and VDAC bind within the 4th transmembrane portion of nAChR subunits. Experimentally, α7 nAChR-Bax and α7 nAChR-VDAC complexes were identified by sandwich ELISA in mitochondria isolated from astrocytoma U373 cells. Stimulating apoptosis of U373 cells by 1μM H2O2 disrupted α7-VDAC complexes and favored formation of α7-Bax complexes. α7-selective agonist PNU282987 and type 2 positive allosteric modulator PNU120596 disrupted α7-Bax and returned α7 nAChR to complex with VDAC. It is concluded that mitochondrial nAChRs regulate apoptosis-induced mitochondrial channel formation by modulating the interplay of apoptosis-related proteins in mitochondria outer membrane.

Effect of PGK1 on radiosensitivity through regulation of CIN and CFL1 in human glioma cells.

e14631 Background: Phosphoglycerate kinase 1 (PGK1) has been detected overexpressed in many malignancies and usually correlated with increased tumorigenesis and poor survival. Our previous study revealed that PGK1 significantly up-regulated and positively correlated with cofilin-1 (CFL1) expression in radioresistant astrocytomas samples. However, the further mechanism remains unknown. Methods: In the present study, functional interactions among PGK1, chronophin (CIN) and CFL1 were analyzed by co-immunoprecipitation and immunofluorescence experiments. Transfection of shRNA and pcDNA3.1-plasmid were respectively utilized to silence and overexpress PGK1 or CIN in both normal glioma cells (U251 and U373) and estabilshed radioresistant (RR-U251 and RR-U373) cells. The effect of PGK1 on CIN and CFL1 expression levels was investigated with Western-blot method; while the effect of PGK1 or CIN on radiosensitivity of glioma cells was evaluated using cell viability, migration and invasion assays after irradiation. Results: Cell proliferation, migration and invasion abilities significantly increased in RR-U251 and RR-U373 cells compared with those of normal cells, indicating the successful establishment of RR-glioma cell models. Laser confocal experiment in vitro showed the colocalization of PGK1 and CIN. Meanwhile, PGK1 specifically and directly combined with CIN, while did not interplay with CFL1. Elevated PGK1 expression level was observed in radioresistant cells compared with that of normal cells. The western blot analysis showed that CFL1 and CIN levels were positively correlated with the PGK1 expression in both normal and radioresistant cells. The proliferation, migration and invasion capabilities significantly decreased in PGK1-silenced and CIN-silenced glioma cells, indicating the improvement of the radiosensitivity of radioresistant cells. Conclusions: Our research implied the effect of CIN-mediated PGK1 regulation on CFL1 level, thereby altering the migration and invasion abilities in nomal and notably radioresistant glioma cells. These findings clarify a novel underlying mechanism of radioresistance in glioma cells and provide valid basis for further explorations concerning radiotherapy.

Effect of SSH1/SSH2 expression changes by shRNA in glioma cell lines on response to radiotherapy and cofilin-1 reactivation.

e13505 Background: Radiotherapy has become the most important treatment for malignant glioma following surgery. However, development of radioresistance in glioma cells limits therapeutic efficacy. The slingshot (SSH) family of phosphatases is a potent regulator of Cofilin-1 activation. Methods: We investigate the role of SSH1(slingshot protein phosphatase 1) and SSH2 in radioresistance via using shRNA to block SSH1/2 expression in U251 and U373 cells as well as established radioresistant U251 (RR-U251) and U373 (RR-U373) cells. Results: We found that both SSH1 and SSH2-shRNA efficiently sensitized glioma cells to radiation with a sensitization enhancement ratio (SER) of 1.01-1.73. In SSH1-silenced cells, the cell viability, migration, and invasion abilities following radiation were remarkably reduced and radiation induced cell apoptosis was markedly enhanced compared with control cells. While in SSH2-silenced cells, the alterations were not as significant. Furthermore, the result of Western-blot suggested that radiosensization of SSH1/SSH2 silencing was mediated by inhibiting reactivation of phosphorylated CFL-1. Conclusions: Our study demonstrated that SSH1 and SSH2 are valid radiosensitizing targets in not only normal glioma cells but radioresistant lines, suggesting a novel therapeutic strategy to improve the efficacy of radiotherapy in patients with glioma.

Evidence for the Inhibition by Temozolomide, an Imidazotetrazine Family Alkylator, of Intermediate-Conductance Ca2+-Activated K+ Channels in Glioma Cells

Background: Temozolomide (TMZ), an oral alkylator of the imidazotetrazine family, is used to treat glioma. Whether this drug has any ionic effects in glioma cells remains largely unclear. Methods: With the aid of patch-clamp technology, we investigated the effects of TMZ on the ionic currents in U373 glioma cells. The mRNA expression of KCNN4 (KCa3.1) in U373 glioma cells and TMZ's effect on K+ currents in these KCNN4 siRNA-transfected U373 cells were investigated. Results: In whole-cell recordings, TMZ decreased the amplitude of voltage-dependent K+ currents (IK) in U373 cells. TMZ-induced IK inhibition was reversed by ionomycin or 1-ethyl-2-benzimidazolinone (1-EBIO). In cell-attached configuration, TMZ concentration-dependently reduced the activity of intermediate-conductance Ca2+-activated K+ (IKCa) channels with an IC50 value of 9.2 µM. Chlorzoxazone or 1-EBIO counteracted the TMZ-induced inhibition of IKCa channels. Although TMZ was unable to modify single-channel conductance, its inhibition of IKCa channels was weakly voltage-dependent and accompanied by a significant prolongation in the slow component of mean closed time. However, neitherlarge-conductance Ca2+-activated (BKCa) nor inwardly rectifying K+ (Kir) channels were affected by TMZ. In current-clamp mode, TMZ depolarized the cell membrane and 1-EBIO reversed TMZ-induced depolarization. TMZ had no effect on IK in KCNN4 siRNA-transfected U373 cells. Conclusion: In addition to the DNA damage it does, its inhibitory effect on IKCa channels accompanied by membrane depolarization could be an important mechanism underlying TMZ-induced antineoplastic actions.

Asymmetric formation of coated pits on dorsal and ventral surfaces at the leading edges of motile cells and on protrusions of immobile cells

Clathrin/AP2-coated vesicles are the principal endocytic carriers originating at the plasma membrane. In the experiments reported here, we used spinning-disk confocal and lattice light-sheet microscopy to study the assembly dynamics of coated pits on the dorsal and ventral membranes of migrating U373 glioblastoma cells stably expressing AP2 tagged with enhanced green fluorescence (AP2-EGFP) and on lateral protrusions from immobile SUM159 breast carcinoma cells, gene-edited to express AP2-EGFP. On U373 cells, coated pits initiated on the dorsal membrane at the front of the lamellipodium and at the approximate boundary between the lamellipodium and lamella and continued to grow as they were swept back toward the cell body; coated pits were absent from the corresponding ventral membrane. We observed a similar dorsal/ventral asymmetry on membrane protrusions from SUM159 cells. Stationary coated pits formed and budded on the remainder of the dorsal and ventral surfaces of both types of cells. These observations support a previously proposed model that invokes net membrane deposition at the leading edge due to an imbalance between the endocytic and exocytic membrane flow at the front of a migrating cell.

Progesterone Induces the Growth and Infiltration of Human Astrocytoma Cells Implanted in the Cerebral Cortex of the Rat

Progesterone (P4) promotes cell proliferation in several types of cancer, including brain tumors such as astrocytomas, the most common and aggressive primary intracerebral neoplasm in humans. In this work, we studied the effects of P4and its intracellular receptor antagonist, RU486, on growth and infiltration of U373 cells derived from a human astrocytoma grade III, implanted in the motor cortex of adult male rats, using two treatment schemes. In the first one, fifteen days after cells implantation, rats were daily subcutaneously treated with vehicle (propylene glycol, 160 μL), P4(1 mg), RU486 (5 mg), or P4+ RU486 (1 mg and 5 mg, resp.) for 21 days. In the second one, treatments started 8 weeks after cells implantation and lasted for 14 days. In both schemes we found that P4significantly increased the tumor area as compared with the rest of the treatments, whereas RU486 blocked P4effects. All rats treated with P4showed tumor infiltration, while 28.6% and 42.9% of the animals treated with RU486 and P4+ RU486, respectively, presented it. Our data suggest that P4promotes growth and migration of human astrocytoma cells implanted in the motor cortex of the rat through the interaction with its intracellular receptor.