Quantitative spectrofluorometric assay detecting nuclear condensation and fragmentation in intact cells

Majtnerová, Pavlína; Čapek, Jan; Petira, Filip; Handl, Jiří; Roušar, Tomáš

doi:10.1038/s41598-021-91380-3

Cited by 13 publications

(5 citation statements)

References 67 publications

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“…GPR56-KD clearly caused VDAC1 mistargeting to the cell surface, as revealed by co-staining with Na + /K + ATPase in EndoC βH1 cells ( Figure 6 D,E). GPR56-KD also resulted in an increased intensity of nuclear Hoechst staining, indicating an increased apoptotic rate [ 32 ] ( Figure 6 F). Interestingly, GPR56-KD ( Supplementary Figure S2A–C ) in EndoC βH1 cells attenuated both GSIS and glucose-induced increase in cAMP when the cells were incubated for 60 min at 20 mM glucose, while basal insulin release or cAMP content was not influenced ( Figure 6 G,H).…”

Section: Resultsmentioning

confidence: 99%

Ablation of GPR56 Causes β-Cell Dysfunction by ATP Loss through Mistargeting of Mitochondrial VDAC1 to the Plasma Membrane

et al. 2023

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The activation of G Protein-Coupled Receptor 56 (GPR56), also referred to as Adhesion G-Protein-Coupled Ceceptor G1 (ADGRG1), by Collagen Type III (Coll III) prompts cell growth, proliferation, and survival, among other attributes. We investigated the signaling cascades mediating this functional effect in relation to the mitochondrial outer membrane voltage-dependent anion Channel-1 (VDAC1) expression in pancreatic β-cells. GPR56KD attenuated the Coll III-induced suppression of P70S6K, JNK, AKT, NFκB, STAT3, and STAT5 phosphorylation/activity in INS-1 cells cultured at 20 mM glucose (glucotoxicity) for 72 h. GPR56-KD also increased Chrebp, Txnip, and Vdac1 while decreasing Vdac2 mRNA expression. In GPR56-KD islet β-cells, Vdac1 was co-localized with SNAP-25, demonstrating its plasma membrane translocation. This resulted in ATP loss, reduced cAMP production and impaired glucose-stimulated insulin secretion (GSIS) in INS-1 and human EndoC βH1 cells. The latter defects were reversed by an acute inhibition of VDAC1 with an antibody or the VDAC1 inhibitor VBIT-4. We demonstrate that Coll III potentiates GSIS by increasing cAMP and preserving β-cell functionality under glucotoxic conditions in a GPR56-dependent manner by attenuating the inflammatory response. These results emphasize GPR56 and VDAC1 as drug targets in conditions with impaired β-cell function.

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Section: Resultsmentioning

confidence: 99%

Ablation of GPR56 Causes β-Cell Dysfunction by ATP Loss through Mistargeting of Mitochondrial VDAC1 to the Plasma Membrane

et al. 2023

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“…Condensation and nuclear fragmentation are considered typical events of the cell death, especially in late apoptosis [56], together with morphological changes, cell shrinkage and the formation of apoptotic bodies in plasmatic membrane [56]. From the events of nuclear fragmentation evidenced in the results, it is possible suggest that this effect is one of the mechanisms involved in the cytotoxic activity that [6]-Shogaol presents, typical events of the apoptotic cell death process, and reported in the literature as apoptotic nuclear morphology stage I and II to chromatin condensation and nuclear fragmentation events, respectively [27,33].…”

Section: Discussionmentioning

confidence: 99%

[6]-Shogaol Induces Apoptosis of Murine Bladder Cancer Cells

Nina Nina,

Robeldo,

Almada da Silva

et al. 2024

Cell Physiol Biochem

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Background/Aims: Bladder cancer is considered one of the most aggressive neoplasms due to its recurrence and progression profile, and even with the improvement in diagnosis and treatment methods, the mortality rate has not shown a declining trend in recent decades. From this perspective, the search and development of more effective and safer therapeutic alternatives are necessary. Phytochemicals are excellent sources of active principles with therapeutic potential. [6]-Shogaol is a phenolic compound extracted from the ginger rhizomes that has shown antitumor effects in a wide variety of cancer models. However, there is no record in the literature of studies reporting these effects in models of bladder cancer. Thus, this study aimed to investigate the in vitro cytotoxic and pro-apoptotic potential of [6]-Shogaol against murine bladder cancer urothelial cells (MB49). Methods: The cytotoxic effects of [6]-Shogaol on cell viability (MTT method), cell morphology (light microscopy), alteration of proliferative processes (clonogenic assay), oxidative stress pathway (levels of reactive oxygen species) and the induction of apoptotic events (flow cytometry and high-resolution epifluorescence imaging) were evaluated in murine urothelial bladder cancer cell lines (MB49), relative to non-tumor murine fibroblasts (L929). Results: The results showed that [6]-Shogaol was able to induce concentration-dependent cytotoxic effects, which compromised cell viability, exhibiting an inhibitory concentration of 50% of cells (IC50) of 146.8 µM for MB49 tumor cells and 236.0 µM for L929 non-tumor fibroblasts. In addition to inhibiting and altering the proliferative processes if colony formation, it presented pro-apoptotic activity identified through a quantitative analysis and the observation of apoptotic phenotypes, events apparently mediated by the induction of nuclear fragmentation. Conclusion: The data presented suggest that [6]-Shogaol has a higher concentration-dependent cytotoxic and apoptosis-inducing potential in MB49 cells than in L929 fibroblasts. These results may contribute to the development of therapeutic alternatives for bladder cancer.

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“…The immunofluorescence assay was performed in HaCaT, A375, and MCF-7 cells treated for 48 h with the highest concentration of LP (10 10 CFU/mL); the selection was based on cell viability results. The cells were labeled by Hoechst 33342 nuclear staining assay (Thermo Fisher Scientific) and Beta-Actin Mouse Monoclonal Antibody (Product MA5-15739, Thermo Fisher Scientific) [ 17 ]. The protocol applied for the immunofluorescence staining was established according to the manufacturer’s recommendation and adapted to our laboratory conditions, as follows: (i) treatment of cultured cells with 10 10 CFU/mL LP for 48 h; (ii) cell fixation with 4% paraformaldehyde for 15 min; (iii) cell permeabilization with 0.25% Triton™ X-100 for 10 min, and blocking with 5% BSA for 1 h at room temperature; (iv) incubation with 2 µg/mL Beta-Actin Antibody in 0.1% BSA for 3 h at room temperature; (v) primary antibody labeling with 1:2000 Alexa Fluor 488 Goat Anti-Mouse Secondary Antibody (Product A28175, Thermo Fisher Scientific) for 45 min at room temperature; (vi) addition of Hoechst staining solution (5 μg/mL), and 10 min incubation at room temperature and in the dark; (vii) 3x washing steps with PBS.…”

Section: Methodsmentioning

confidence: 99%

Lactiplantibacillus plantarum Induces Apoptosis in Melanoma and Breast Cancer Cells

Budu,

Mioc,

Soica

et al. 2024

Microorganisms

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Despite the notable advancements witnessed in the past decade in medical and health research domain, cancer remains a prominent global cause of mortality. Moreover, the conventional treatments employed to combat this disease have been found to considerably compromise the quality of life experienced by patients due to its severe side effects. Recent in vitro studies revealed encouraging findings on the potential beneficial effects of probiotics as adjuvants of anticancer therapy, and even as possible agents for the prevention and treatment of various types of malignancies. From this standpoint, the primary objective of this work was to investigate the anticancer properties of Lactiplantibacillus plantarum (LP) and elucidate its underlying mechanism of action. In order to investigate this matter, several doses of LP (ranging from 105 to 1010 CFU/mL) were examined in relation to melanoma cancer cell lines (A375) and breast cancer cell line (MCF-7). The cell viability findings, which were substantiated by morphological investigations and annexin V/PI assay, indicated that LP exerted inhibitory effects on cellular activity and triggered apoptosis. Additionally, upon further investigation into its mechanism, it was observed through the apoptosis assay and Western blot analysis that the administration of LP resulted in an elevation of pro-apoptotic BAX protein levels and an upregulation of cleaved poly-ADP-ribose polymerase (PARP) protein expression. Conversely, the levels of anti-apoptotic Bcl-2 protein were found to decrease in the A375 and MCF-7 cell lines. These findings provide insight into the pro-apoptotic mechanism of action of LP in these specific cell lines.

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Quantitative spectrofluorometric assay detecting nuclear condensation and fragmentation in intact cells

Cited by 13 publications

References 67 publications

Ablation of GPR56 Causes β-Cell Dysfunction by ATP Loss through Mistargeting of Mitochondrial VDAC1 to the Plasma Membrane

Ablation of GPR56 Causes β-Cell Dysfunction by ATP Loss through Mistargeting of Mitochondrial VDAC1 to the Plasma Membrane

[6]-Shogaol Induces Apoptosis of Murine Bladder Cancer Cells

Lactiplantibacillus plantarum Induces Apoptosis in Melanoma and Breast Cancer Cells

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