A Fast and Simple Fluorometric Method to Detect Cell Death in 3D Intestinal Organoids

Bode, Konstantin; Mueller, Stefanie; Schweinlin, Matthias; Metzger, Marco; Brunner, Thomas

doi:10.2144/btn-2019-0023

Cited by 30 publications

(32 citation statements)

References 29 publications

(26 reference statements)

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“…Before measurement, the staining medium was replaced with fresh Neurobasal Medium. Hoechst 33342 is a cell-permeant nuclear counterstain emitting blue fluorescence when bound to DNA, and PI is a membrane impermeant nuclear dye that emits red fluorescence in dead cells, as previously described [ 17 ]. The fluorescence of stained SH-SY5Y cells was measured using excitation/emission wavelengths for Hoechst 33342 350/461 nm and 535/617 nm for PI, respectively, by SpectraMax iD3 Multi-Mode Microplate Reader with SoftMaxPro7.1Setup Software (Molecular Devices, LLC.).…”

Section: Methodsmentioning

confidence: 99%

The Effects of Alpha-Linolenic Acid on the Secretory Activity of Astrocytes and β Amyloid-Associated Neurodegeneration in Differentiated SH-SY5Y Cells: Alpha-Linolenic Acid Protects the SH-SY5Y cells against β Amyloid Toxicity

Litwiniuk

Domańska

Chmielowska

et al. 2020

Oxidative Medicine and Cellular Longevity

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Alzheimer’s disease (AD) is the most common neurodegenerative disorder. Amyloid β- (Aβ-) induced mitochondrial dysfunction may be a primary process triggering all the cascades of events that lead to AD. Therefore, identification of natural factors and endogenous mechanisms that protect neurons against Aβ toxicity is needed. In the current study, we investigated whether alpha-linolenic acid (ALA), as a natural product, would increase insulin and IGF-I (insulin-like growth factor I) release from astrocytes. Moreover, we explored the protective effect of astrocytes-derived insulin/IGF-I on Aβ-induced neurotoxicity, with special attention paid to their impact on mitochondrial function of differentiated SH-SY5Y cells. The results showed that ALA induced insulin and IGF-I secretion from astrocytes. Our findings demonstrated that astrocyte-derived insulin/insulin-like growth factor I protects differentiated SH-SY5Y cells against Aβ1-42-induced cell death. Moreover, pretreatment with conditioned medium (CM) and ALA-preactivated CM (ALA-CM) protected the SH-SY5Y cells against Aβ1-42-induced mitochondrial dysfunction by reducing the depolarization of the mitochondrial membrane, increasing mitochondrial biogenesis, restoring the balance between fusion and fission processes, and regulation of mitophagy and autophagy processes. Our study suggested that astrocyte-derived insulin/insulin-like growth factor I suppresses Aβ1-42-induced cytotoxicity in the SH-SY5Y cells by protecting against mitochondrial dysfunction. Moreover, the neuroprotective effects of CM were intensified by preactivation with ALA.

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Section: Methodsmentioning

confidence: 99%

The Effects of Alpha-Linolenic Acid on the Secretory Activity of Astrocytes and β Amyloid-Associated Neurodegeneration in Differentiated SH-SY5Y Cells: Alpha-Linolenic Acid Protects the SH-SY5Y cells against β Amyloid Toxicity

Litwiniuk

Domańska

Chmielowska

et al. 2020

Oxidative Medicine and Cellular Longevity

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“…Moreover, neither 3d2 nor SR1848 inhibitor had direct cytotoxic effects in RAW 264.7 cells, as analyzed by AnnexinV staining and flow cytometry ( Fig. 4c), or by the propidium iodide (PI)/Hoechst dye-stained ratio (H33342) 20 (Fig. 4d).…”

Section: Lrh-1 Activity Regulates Mitochondrial Metabolism In Macrophmentioning

confidence: 99%

Pharmacological LRH-1/Nr5a2 inhibition limits pro-inflammatory cytokine production in macrophages and associated experimental hepatitis

et al. 2020

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Liver receptor homolog-1 (LRH-1, Nr5a2) is an orphan nuclear receptor mainly expressed in tissues of endodermal origin, where its physiological role has been extensively studied. LRH-1 has been implicated in liver cell differentiation and proliferation, as well as glucose, lipid, and bile acid metabolism. In addition, increasing evidence highlights its role in immunoregulatory processes via glucocorticoid synthesis in the intestinal epithelium. Although the direct function of LRH-1 in immune cells is fairly elucidated, a role of LRH-1 in the regulation of macrophage differentiation has been recently reported. In this study, we aimed to investigate the role of LRH-1 in the regulation of pro-inflammatory cytokine production in macrophages. Our data demonstrate that pharmacological inhibition, along with LRH-1 knockdown, significantly reduced the lipopolysaccharide (LPS)-induced production of pro-inflammatory cytokines in the macrophage line RAW 264.7 cells, as well as in primary murine macrophages. This inhibitory effect was found to be independent of defects of LRH-1-regulated cell proliferation or toxic effects of the LRH-1 inhibitors. In contrast, LRH-1 inhibition reduced the mitochondrial ATP production and metabolism of macrophages through downregulation of the LRH-1 targets glucokinase and glutminase-2, and thus impairing the LPS-induced macrophage activation. Interestingly, in vivo pharmacological inhibition of LRH-1 also resulted in reduced tumor necrosis factor (TNF) production and associated decreased liver damage in a macrophage-and TNF-dependent mouse model of hepatitis. Noteworthy, despite hepatocytes expressing high levels of LRH-1, pharmacological inhibition of LRH-1 per se did not cause any obvious liver damage. Therefore, this study proposes LRH-1 as an emerging therapeutic target in the treatment of inflammatory disorders, especially where macrophages and cytokines critically decide the extent of inflammation.

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“…Mouse intestinal organoids were generated and cultured as described in [54]. Colonic tumors were induced in C57BL/6 mice by the AOM/DSS model [55], and tumoroids were generated and cultured as described previously [56].…”

Section: Generation Of Murine Intestinal Organoids and Colonic Tumoroidsmentioning

confidence: 99%

Thiazolides promote G1 cell cycle arrest in colorectal cancer cells by targeting the mitochondrial respiratory chain

et al. 2019

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Systemic toxicity and tumor cell resistance still limit the efଏcacy of chemotherapy in colorectal cancer. Therefore, alternative treatments are desperately needed. The thiazolide Nitazoxanide (NTZ) is an FDA-approved drug for the treatment of parasite-mediated infectious diarrhea with a favorable safety proଏle. Interestingly, NTZ and the thiazolide RM4819-its bromo-derivative lacking antibiotic activity-are also promising candidates for cancer treatment. Yet the exact anticancer mechanism(s) of these compounds still remains unclear. In this study, we systematically investigated RM4819 and NTZ in 2D and 3D colorectal cancer culture systems. Both compounds strongly inhibited proliferation of colon carcinoma cell lines by promoting G1 phase cell cycle arrest. Thiazolide-induced cell cycle arrest was independent of the p53/p21 axis, but was mediated by inhibition of protein translation via the mTOR/c-Myc/p27 pathway, likely caused by inhibition of mitochondrial respiration. While both thiazolides demonstrated mitochondrial uncoupling activity, only RM4819 inhibited the mitochondrial respiratory chain complex III. Interestingly, thiazolides also potently inhibited the growth of murine colonic tumoroids in a comparable manner with cisplatin, while in contrast to cisplatin thiazolides did not affect the growth of primary intestinal organoids. Thus, thiazolides appear to have a tumor-selective antiproliferative activity, which offers new perspectives in the treatment of colorectal cancer.

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A Fast and Simple Fluorometric Method to Detect Cell Death in 3D Intestinal Organoids

Cited by 30 publications

References 29 publications

The Effects of Alpha-Linolenic Acid on the Secretory Activity of Astrocytes and β Amyloid-Associated Neurodegeneration in Differentiated SH-SY5Y Cells: Alpha-Linolenic Acid Protects the SH-SY5Y cells against β Amyloid Toxicity

The Effects of Alpha-Linolenic Acid on the Secretory Activity of Astrocytes and β Amyloid-Associated Neurodegeneration in Differentiated SH-SY5Y Cells: Alpha-Linolenic Acid Protects the SH-SY5Y cells against β Amyloid Toxicity

Pharmacological LRH-1/Nr5a2 inhibition limits pro-inflammatory cytokine production in macrophages and associated experimental hepatitis

Thiazolides promote G1 cell cycle arrest in colorectal cancer cells by targeting the mitochondrial respiratory chain

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