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Inhibition of autophagic flux differently modulates cannabidiol-induced death in 2D and 3D glioblastoma cell cultures.

ABSTRACT: Radiotherapy combined with chemotherapy is the major treatment modality for human glioblastoma multiforme (GBM). GBMs eventually relapse after treatment and the average survival of GBM patients is less than two years. There is some evidence that cannabidiol (CBD) can induce cell death and increases the radiosensitivity of GBM by enhancing apoptosis. Beside initiation of death, CBD has been demonstrated as an inducer of autophagy. In the present study, we address the question whether CBD simultaneously induces a protective effect in GBM by upregulating autophagy. Addition of chloroquine that suppressed autophagic flux to 2D GBM cultures increased CBD-induced cell death, presenting proof for the protective autophagy. Blockage of autophagy upregulated radiation-induced cytotoxicity but only modestly affected the levels of cell death in CBD- or CBD/γ-irradiated 3D GBM cultures. Furthermore, CBD enhanced the pro-apoptotic activities of JNK1/2 and MAPK p38 signaling cascades while partially downregulated the pro-survival PI3K-AKT cascade, thereby changing a balance between cell death and survival. Suppression of JNK activation partially reduced CBD-induced cell death in 3D GBM cultures. In contrast, co-treatment of CBD-targeted cells with inhibitors of PI3K-AKT-NF-κB, IKK-NF-κB or JAK2-STAT3 pathways killed surviving GBM cells in both 2D and 3D cultures, potentially improving the therapeutic ratio of GBM.

SUBMITTER: Ivanov VN

PROVIDER: S-EPMC7021896 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Author Correction: Inhibition of autophagic flux differently modulates cannabidiol-induced death in 2D and 3D glioblastoma cell cultures.

Project description: Not available

| S-EPMC8440618 | biostudies-literature

WNK2 Inhibits Autophagic Flux in Human Glioblastoma Cell Line.

Project description:Autophagy is a cell-survival pathway with dual role in tumorigenesis, promoting either tumor survival or tumor death. WNK2 gene, a member of the WNK (with no lysine (K)) subfamily, acts as a tumor suppressor gene in gliomas, regulating cell migration and invasion; however, its role in autophagy process is poorly explored. The WNK2-methylated human glioblastoma cell line A172 WT (wild type) was compared to transfected clones A172 EV (empty vector), and A172 WNK2 (WNK2 overexpression) for the evaluation of autophagy using an inhibitor (bafilomycin A1-baf A1) and an inducer (everolimus) of autophagic flux. Western blot and immunofluorescence approaches were used to monitor autophagic markers, LC3A/B and SQSTM1/p62. A172 WNK2 cells presented a significant decrease in LC3B and p62 protein levels, and in LC3A/B ratio when compared with control cells, after treatment with baf A1 + everolimus, suggesting that WNK2 overexpression inhibits the autophagic flux in gliomas. The mTOR pathway was also evaluated under the same conditions, and the observed results suggest that the inhibition of autophagy mediated by WNK2 occurs through a mTOR-independent pathway. In conclusion, the evaluation of the autophagic process demonstrated that WNK2 inhibits the autophagic flux in glioblastoma cell line.

| S-EPMC7072831 | biostudies-literature

Rab7b modulates autophagic flux by interacting with Atg4B.

Project description:Autophagy (macroautophagy) is a highly conserved eukaryotic degradation pathway in which cytosolic components and organelles are sequestered by specialized autophagic membranes and degraded through the lysosomal system. The autophagic pathway maintains basal cellular homeostasis and helps cells adapt during stress; thus, defects in autophagy can cause detrimental effects. It is therefore crucial that autophagy is properly regulated. In this study, we show that the cysteine protease Atg4B, a key enzyme in autophagy that cleaves LC3, is an interactor of the small GTPase Rab7b. Indeed, Atg4B interacts and co-localizes with Rab7b on vesicles. Depletion of Rab7b increases autophagic flux as indicated by the increased size of autophagic structures as well as the magnitude of macroautophagic sequestration and degradation. Importantly, we demonstrate that Rab7b regulates LC3 processing by modulating Atg4B activity. Taken together, our findings reveal Rab7b as a novel negative regulator of autophagy through its interaction with Atg4B.

| S-EPMC5623852 | biostudies-other

The Differences in the Proteome Profile of Cannabidiol-Treated Skin Fibroblasts following UVA or UVB Irradiation in 2D and 3D Cell Cultures.

Project description:Cannabidiol (CBD), as the only phytocannabinoid that has no psychoactive effect, has both antioxidant and anti-inflammatory effects, and thus might be suggested as a cytoprotective compound against UV-induced metabolic changes in skin cells. Therefore, the aim of this study was to investigate the level of protective CBD activity by evaluating the proteomic profile of 2D and 3D cultured skin fibroblasts models following exposure to UVA and UVB radiation. The CBD cytoprotective effect against UV-induced damage in 2D and 3D cultured fibroblasts were different. The main alterations focus on the range of cell reaction and involved different proteins associated with various molecular functions. In the 2D cultured cells, following UV radiation, the major changes were associated with proteins involved in antioxidant response and inflammation, while, in the 3D cultured fibroblasts, CBD action against UV induced changes were mainly associated with the activation of signalling pathways. Therefore, the knowledge of the CBD action in a multilayer skin cells model allowed for the prediction of changes in cell-cell interactions and skin cell metabolism. Knowledge about the lower protective effect of CBD in 3D cultured fibroblasts should be taken into account during the design of UV light protection.

| S-EPMC6770406 | biostudies-literature

Epithelial-To-Mesenchymal Transition Markers and CD44 Isoforms Are Differently Expressed in 2D and 3D Cell Cultures of Prostate Cancer Cells.

Project description:Three-dimensional (3D) cell cultures allow the mimic of functions of living tissues andprovide key information encoded in tissue architecture. Considered the pivotal role of epithelial-tomesenchymaltransition (EMT) in carcinoma progression, including prostate cancer (PCa), weaimed at investigating the effect of the 3D arrangement on the expression of some key markers ofEMT in cultured human prostate cancer (PCa) cells, to better understand PCa cell behavior. PC3 andDU145 PCa cells were cultured in RPMI cell culture medium either in 2D-monolayers or in 3Dspheroids.The main EMT markers E-cadherin, N-cadherin, ?-smooth muscle actin (?SMA),vimentin, Snail, Slug, Twist and Zeb1 were evaluated by confocal microscopy, real-time PCR andWestern blot. Confocal microscopy revealed that E-cadherin was similarly expressed at the cellboundaries on the plasma membrane of PCa cells grown in 2D-monolayers, as well as in 3Dspheroids,but resulted up-regulated in 3D-spheroids, compared to 2D-monolayers, at the mRNAand protein level. Moreover, markers of the mesenchymal phenotype were expressed at very lowlevels in 3D-spheroids, suggesting important differences in the phenotype of PCa cells grown in 3Dspheroidsor in 2D-monolayers. Considered as a whole, our findings contribute to a clarification ofthe role of EMT in PCa and confirm that a 3D cell culture model could provide deeper insight intothe understanding of the biology of PCa.

| S-EPMC6406374 | biostudies-literature

Rotenone inhibits autophagic flux prior to inducing cell death.

Project description:Rotenone, which selectively inhibits mitochondrial complex I, induces oxidative stress, α-synuclein accumulation, and dopaminergic neuron death, principal pathological features of Parkinson's disease. The autophagy-lysosome pathway degrades damaged proteins and organelles for the intracellular maintenance of nutrient and energy balance. While it is known that rotenone causes autophagic vacuole accumulation, the mechanism by which this effect occurs has not been thoroughly investigated. Treatment of differentiated SH-SY5Y cells with rotenone (10 μM) induced the accumulation of autophagic vacuoles at 6 h and 24 h as indicated by Western blot analysis for microtubule associated protein-light chain 3-II (MAP-LC3-II). Assessment of autophagic flux at these time points indicated that autophagic vacuole accumulation resulted from a decrease in their effective lysosomal degradation, which was substantiated by increased levels of autophagy substrates p62 and α-synuclein. Inhibition of lysosomal degradation may be explained by the observed decrease in cellular ATP levels, which in turn may have caused the observed concomitant increase in acidic vesicle pH. The early (6 h) effects of rotenone on cellular energetics and autophagy-lysosome pathway function preceded the induction of cell death and apoptosis. These findings indicate that the classical mitochondrial toxin rotenone has a pronounced effect on macroautophagy completion that may contribute to its neurotoxic potential.

| S-EPMC3526971 | biostudies-other

Salinomycin induces autophagic cell death in salinomycin-sensitive melanoma cells through inhibition of autophagic flux.

Project description:Several literature has shown that salinomycin (Sal) is able to kill various types of cancer cells through different signaling pathways. However, its effect on melanoma has seldom been reported. We examined the anti-cancer efficacy of Sal in melanoma cell lines, and found six of eight cell lines were sensitive to Sal. Given the fact that the roles of Sal are diverse in different cancer types, we were eager to figure out the mechanism involved in the current study. We noticed the most sensitive line, SK-Mel-19, showed a typical morphological change after Sal treatment. The autophagy inhibitor, 3-MA, could effectively suppress Sal-induced cell death. It could also facilitate the increase of autophagic markers and reduce the turnover of autophagosomes, which resulted in an aberrant autophagic flux. On the other hand, Sal could stimulate endoplasmic reticulum stress and cause an accumulation of dysfunctional mitochondria. We also discovered a potential correlation between LC3B mRNA level and its sensitivity to Sal in 43 clinical melanoma samples. Overall, our results indicated that Sal could have multiple effect on melanoma cells and induce autophagic cell death in certain kinds of cells, which provided a new insight into the chemotherapy for melanoma.

| S-EPMC7595060 | biostudies-literature

Lovastatin Enhances Cytotoxicity of Temozolomide via Impairing Autophagic Flux in Glioblastoma Cells.

Project description:Drug resistance to temozolomide (TMZ) contributes to the majority of tumor recurrence and treatment failure in patients with glioblastoma multiforme (GBM). Autophagy has been reported to play a role in chemoresistance in various types of cancer, including GBM. The anticancer effect of statins is arousing great research interests and has been demonstrated to modulate autophagic function. In this study, we investigated the combinational effects of lovastatin and TMZ on treating U87 and U251 GBM cell lines. Cytotoxicity was measured by MTT and colony formation assays; apoptosis was measured by flow cytometry; the cellular autophagic function was detected by the EGFP-mRFP-LC3 reporter and western blot assay. The results showed that lovastatin might enhance the cytotoxicity of TMZ, increase the TMZ-induced cellular apoptosis, and impair the autophagic flux in GBM cells. Lovastatin triggered autophagy initiation possibly by inhibiting the Akt/mTOR signaling pathway. Moreover, lovastatin might impair the autophagosome-lysosome fusion machinery by suppressing LAMP2 and dynein. These results suggested that lovastatin could enhance the chemotherapy efficacy of TMZ in treating GBM cells. The mechanism may be associated with impaired autophagic flux and thereby the enhancement of cellular apoptosis. Combining TMZ with lovastatin could be a promising strategy for GBM treatment.

| S-EPMC6778891 | biostudies-literature

Miz1 is required to maintain autophagic flux

Project description:Miz1 is a zinc finger protein that regulates expression of cell cycle inhibitors as part of a complex with Myc. Cell cycle-independent functions of Miz1 are poorly understood. Here, we use a Nestin-Cre transgene to delete an essential domain of Miz1 in the central nervous system (Miz1M-NM-^TPOZNes). Miz1M-NM-^TPOZNes mice display cerebellar neurodegeneration characterized by the progressive loss of Purkinje cells. Chromatin immunoprecipitation sequencing and biochemical analyses show that Miz1 activates transcription upon binding to a non-palindromic sequence present in core promoters. Target genes of Miz1 encode regulators of autophagy and proteins involved in vesicular transport that are required for autophagy. Miz1M-NM-^TPOZ neuronal progenitors and fibroblasts show reduced autophagic flux. Consistently, polyubiquitinated proteins and p62/Sqtm1 accumulate in the cerebella of Miz1M-NM-^TPOZNes mice, characteristic features of defective autophagy. Our data suggest that Miz1 may link cell growth and ribosome biogenesis to the transcriptional regulation of vesicular transport and autophagy. ChIP-Seq with H190 and G18 on an Illumina Genome Analyzer IIx.

2013-09-27 | E-GEOD-48602 | biostudies-arrayexpress

Dynamic transcription factor activity profiling in 2D and 3D cell cultures.

Project description:Live-cell assays to measure cellular function performed within 3D cultures have the potential to elucidate the underlying processes behind disease progression and tissue formation. Cells cultured in 3D interact and remodel their microenvironment and can develop into complex structures. We have developed a transcription factor (TF) activity array that uses bioluminescence imaging (BLI) of lentiviral delivered luminescent reporter constructs that allows for the non-invasive imaging of TF activity in both 2D and 3D culture. Imaging can be applied repeatedly throughout culture to capture dynamic TF activity, though appropriate normalization is necessary. We investigated in-well normalization using Gaussia or Renilla luciferase, and external well normalization using firefly luciferase. Gaussia and Renilla luciferase were each unable to provide consistent normalization for long-term measurement of TF activity. However, external well normalization provided low variability and accounted for changes in cellular dynamics. Using external normalization, dynamic TF activities were quantified for five TFs. The array captured expected changes in TF activity to stimuli, however the array also provided dynamic profiles within 2D and 3D that have not been previously characterized. The development of the technology to dynamically track TF activity within cells cultured in both 2D and 3D can provide greater understanding of complex cellular processes.

| S-EPMC4073638 | biostudies-literature

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