Gap junctions enhance the antiproliferative effect of microrna-124-3p in glioblastoma cells

OBJECTIVE MicroR NA(miR NA)holds promise as a novel therapeutic tool for cancer treatment.However,the transfection efficiency of current delivery systems represents a bottleneck for clinical applications.Here,we demonstrate that gap junctions mediate an augmentative effect on the antiproliferation mediated by mi R-124-3p in U87 and C6 glioblastoma cells.METHODS The functional inhibition of gap junctions using either si RNA or pharmacological inhibition eliminated the mi R-124-3p-mediated antiproliferation,whereas the enhancement of gap junctions with retinoic acid treatment augmented this mi R-124-3p-mediated antiproliferation.A similar effect was observed in glioblastoma xenograft models.RESULTS More importantly,patch clamp and co-culture assays demonstrated the transmission of mi R-124-3p through gap junction channels into adjacent cells.In further exploring the impact of gap junction-mediated transport of mi R-124-3p on mi R-124-3p target pathways,we found that mi R-124-3p inhibited glioblastoma cell growth in part by decreasing the protein expression of cyclindependent kinase 6,leading to cel cycle arrest at the G0/G1phase;moreover,pharmacological regulation of gap junctions affected this cell cycle arrest.CONCLUSION Our results indicate that the″bystander″effects of functional gap junctions composed of connexin 43 enhance the antitumor effect of mi R-124-3p in glioblastoma cells by transferring mi R-124-3p to adjacent cells,thereby enhancing G0/G1cell cycle arrest.These observations provide a new guiding strategy for the clinical application of mi RNA therapy in tumor treatment.

Apoptosis of Glioblastoma U251 Cells Induced by Carmustine Combined All-trans Retinoic Acid via Regulating Cyclin E and p27kip 1

The effect and mechanism of carmustine（BCNU） combined with all-trans retinoic acid（ATRA） on the apoptosis of human glioblastoma U251 cells were investigated by means of 3-（4,5-dimethylthiazol-2-yl）-2,5-diphe- nyltetrazolium bromide（MTT） assay, flow cytometry, reverse transcription-polymerase chain reaction（RT-PCR） and Western blot analysis. The results show that BCNU or ATRA shows time- and dose-dependent inhibition effects on human glioblastoma U251 cells and the combination of BCNU with ATRA shows an synergistic inhibition effect on human glioblastoma U251 cells, and the combined BCNU and ATRA can significantly inhibit the proliferation of human glioblastoma U251 cells, and induce the apoptosis of them, making the cells arrest in the stage of G1 phase, the stage of S and G2 phases decline, the rate of the apoptosis of human glioblastoma U251 cells increase, the corresponding mRNA expression of cyclin E and cyclin-dependent kinase 2（CDK2） downregulated and the correspon- ding mRNA expression of p27kip 1 unregulated. In addition, the combined BCNU and ATRA reduced the protein expression of nuclear factor kappa B（NF-κB）. Taken together, these results suggest that the treatment of human glioblastoma U251 cells with a combination application of ATRA and BCNU can exert synergistic effect, the course of this kind of combination chemotherapy may likely be associated with multiple molecular mechanisms for apoptosis, furthermore, the cyclin E and p27kip 1 should be considered as novel targets for controlling the growth of glioblastoma cells.

Emerging targets for glioblastoma stem cell therapy

Glioblastoma multiforme（GBM）,designated as World Health Organization（WHO）grade IV astrocytoma,is a lethal and therapy-resistant brain cancer comprised of several tumor cell subpopulations,including GBM stem cells（GSCs）which are believed to contribute to tumor recurrence following initial response to therapies.Emerging evidence demonstrates that GBM tumors are initiated from GSCs.The development and use of novel therapies including small molecule inhibitors of specific proteins in signaling pathways that regulate sternness,proliferation and migration of GSCs,immunotherapy,and non-coding microRNAs may provide better means of treating GBM.Identification and characterization of GSC-specific signaling pathways would be necessary to identify specific therapeutic targets which may lead to the development of more efficient therapies selectively targeting GSCs.Several signaling pathways including mTOR,AKT,maternal embryonic leucine zipper kinase（MELK）,NOTCH1 and Wnt/β-catenin as well as expression of cancer stem cell markers CD133,CD44,Oct4,Sox2,Nanog,and ALDHlA1 maintain GSC properties.Moreover,the data published in the Cancer Genome Atlas（TCGA）specifically demonstrated the activated PI3K/AKT/mTOR pathway in GBM tumorigenesis.Studying such pathways may help to understand GSC biology and lead to the development of potential therapeutic interventions to render them more sensitive to chemotherapy and radiation therapy.Furthemore,recent demonstration of dedifferentiation of GBM cell lines into CSC-like cells prove that any successful therapeutic agent or combination of drugs for GBM therapy must eliminate not only GSCs,but the differentiated GBM cells and the entire bulk of tumor cells.

Glioblastoma chemoresistance:roles of the mitochondrial melatonergic pathway

Treatment-resistance is common in glioblastoma(GBM)and the glioblastoma stem-like cells(GSC)from which they arise.Current treatment options are generally regarded as very poor and this arises from a poor conceptualization of the biological underpinnings of GBM/GSC and of the plasticity that these cells are capable of utilizing in response to different treatments.A number of studies indicate melatonin to have utility in the management of GBM/GSC,both per se and when adjunctive to chemotherapy.Recent work shows melatonin to be produced in mitochondria,with the mitochondrial melatonergic pathway proposed to be a crucial factor in driving the wide array of changes in intra-and inter-cellular processes,as well as receptors that can be evident in the cells of the GBM/GSC microenvironment.Variations in the enzymatic conversion of N-acetylserotonin(NAS)to melatonin may be especially important in GSC,as NAS can activate the tyrosine receptor kinase B to increase GSC survival and proliferation.Consequently,variations in the NAS/melatonin ratio may have contrasting effects on GBM/GSC survival.It is proposed that mitochondrial communication across cell types in the tumour microenvironment is strongly driven by the need to carefully control the mitochondrial melatonergic pathways across cell types,with a number of intra-and inter-cellular processes occurring as a consequence of the need to carefully regulate the NAS/melatonin ratio.This better integrates previously disparate data on GBM/GSC as well as providing clear future research and treatment options.