scholarly journals Magnesium Absorption in Intestinal Cells: Evidence of Cross-Talk between EGF and TRPM6 and Novel Implications for Cetuximab Therapy

Nutrients ◽  
2020 ◽  
Vol 12 (11) ◽  
pp. 3277
Author(s):  
Giuseppe Pietropaolo ◽  
Daniela Pugliese ◽  
Alessandro Armuzzi ◽  
Luisa Guidi ◽  
Antonio Gasbarrini ◽  
...  
Keyword(s):  
Growth Factor Receptor ◽  
Human Colon ◽  
Nutritional Intervention ◽  
Intestinal Cells ◽  
Epithelial Growth Factor ◽  
Dependent Inhibition ◽  
Vitro Model ◽  
Egf Signaling ◽  
Cetuximab Therapy

Hypomagnesemia is very commonly observed in cancer patients, most frequently in association with therapy with cetuximab (CTX), a monoclonal antibody targeting the epithelial growth factor receptor (EGFR). CTX-induced hypomagnesemia has been ascribed to renal magnesium (Mg) wasting. Here, we sought to clarify whether CTX may also influence intestinal Mg absorption and if Mg supplementation may interfere with CTX activity. We used human colon carcinoma CaCo-2 cells as an in vitro model to study the mechanisms underlying Mg transport and CTX activity. Our findings demonstrate that TRPM6 is the key channel that mediates Mg influx in intestinal cells and that EGF stimulates such influx; consequently, CTX downregulates TRPM6-mediated Mg influx by interfering with EGF signaling. Moreover, we show that Mg supplementation does not modify either the CTX IC50 or CTX-dependent inhibition of ERK1/2 phosphorylation. Our results suggest that reduced Mg absorption in the intestine may contribute to the severe hypomagnesemia that occurs in CTX-treated patients, and Mg supplementation may represent a safe and effective nutritional intervention to restore Mg status without impairing the CTX efficacy.

scholarly journals Anticancer Activity of Triazolo-Thiadiazole Derivatives and Inhibition of AKT1 and AKT2 Activation

Pharmaceutics ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 493
Author(s):  
Dimitrios T. Trafalis ◽  
Sofia Sagredou ◽  
Panayiotis Dalezis ◽  
Maria Voura ◽  
Stella Fountoulaki ◽  
...  
Keyword(s):  
Anticancer Activity ◽  
Human Colon ◽  
Tumor Xenograft ◽  
Atp Binding Site ◽  
Anticancer Properties ◽  
Extensive Range ◽  
Dependent Inhibition ◽  
Ht 29

The fusion of 1,2,4-triazole and 1,3,4-thiadiazole rings results in a class of heterocycles compounds with an extensive range of pharmacological properties. A series of 1,2,4-triazolo[3,4-b]-1,2,4-thiadiazoles was synthesized and tested for its enzyme inhibition potential and anticancer activity. The results show that 1,2,4-triazolo[3,4-b]-1,2,4-thiadiazoles display potent anticancer properties in vitro against a panel of cancer cells and in vivo efficacy in HT-29 human colon tumor xenograft in CB17 severe combined immunodeficient (SCID) mice. Preliminary mechanistic studies revealed that KA25 and KA39 exhibit time- and concentration-dependent inhibition of Akt Ser-473 phosphorylation. Molecular modeling experiments indicated that 1,2,4-triazolo[3,4-b]-1,2,4-thiadiazoles bind well to the ATP binding site in Akt1 and Akt2. The low acute toxicity combined with in vitro and in vivo anticancer activity render triazolo[3,4-b]thiadiazoles KA25, KA26, and KA39 promising cancer therapeutic agents.

scholarly journals The use of an in-vitro batch fermentation (human colon) model for investigating mechanisms of TMA production from choline, l-carnitine and related precursors by the human gut microbiota

2021 ◽  
Author(s):  
Priscilla Day-Walsh ◽  
Emad Shehata ◽  
Shikha Saha ◽  
George M. Savva ◽  
Barbora Nemeckova ◽  
...  
Keyword(s):  
Gut Microbiota ◽  
Metabolic Diseases ◽  
Human Colon ◽  
Human Studies ◽  
Bacterial Strains ◽  
Increased Risk ◽  
Carnitine Metabolism ◽  
Registration Number ◽  
Vitro Model

Abstract Purpose Plasma trimethylamine-N-oxide (TMAO) levels have been shown to correlate with increased risk of metabolic diseases including cardiovascular diseases. TMAO exposure predominantly occurs as a consequence of gut microbiota-dependent trimethylamine (TMA) production from dietary substrates including choline, carnitine and betaine, which is then converted to TMAO in the liver. Reducing microbial TMA production is likely to be the most effective and sustainable approach to overcoming TMAO burden in humans. Current models for studying microbial TMA production have numerous weaknesses including the cost and length of human studies, differences in TMA(O) metabolism in animal models and the risk of failing to replicate multi-enzyme/multi-strain pathways when using isolated bacterial strains. The purpose of this research was to investigate TMA production from dietary precursors in an in-vitro model of the human colon. Methods TMA production from choline, l-carnitine, betaine and γ-butyrobetaine was studied over 24–48 h using an in-vitro human colon model with metabolite quantification performed using LC–MS. Results Choline was metabolised via the direct choline TMA-lyase route but not the indirect choline–betaine-TMA route, conversion of l-carnitine to TMA was slower than that of choline and involves the formation of the intermediate γ-BB, whereas the Rieske-type monooxygenase/reductase pathway for l-carnitine metabolism to TMA was negligible. The rate of TMA production from precursors was choline > carnitine > betaine > γ-BB. 3,3-Dimethyl-1-butanol (DMB) had no effect on the conversion of choline to TMA. Conclusion The metabolic routes for microbial TMA production in the colon model are consistent with observations from human studies. Thus, this model is suitable for studying gut microbiota metabolism of TMA and for screening potential therapeutic targets that aim to attenuate TMA production by the gut microbiota. Trial registration number NCT02653001 (http://www.clinicaltrials.gov), registered 12 Jan 2016.

scholarly journals Chemokine Ligand 20: A Signal for Leukocyte Recruitment During Human Ovulation?

Endocrinology ◽  
2015 ◽  
Vol 156 (9) ◽  
pp. 3358-3369 ◽  
Author(s):  
Linah Al-Alem ◽  
Muraly Puttabyatappa ◽  
Kathy Rosewell ◽  
Mats Brännström ◽  
James Akin ◽  
...  
Keyword(s):  
In Vitro Model ◽  
Leukocyte Migration ◽  
Human Ovary ◽  
Epithelial Growth Factor ◽  
Chemokine Ligand ◽  
Vitro Model ◽  
Ovulatory Process ◽  
Epithelial Growth

Ovulation is one of the cornerstones of female fertility. Disruption of the ovulatory process results in infertility, which affects approximately 10% of couples. Using a unique model in which the dominant follicle is collected across the periovulatory period in women, we have identified a leukocyte chemoattractant, chemokine ligand 20 (CCL20), in the human ovary. CCL20 mRNA is massively induced after an in vivo human chorionic gonadotropin (hCG) stimulus in granulosa (>10 000-fold) and theca (>4000-fold) cells collected during the early ovulatory (12–18 h) and late ovulatory (18–34 h) periods after hCG administration. Because the LH surge sets in motion an inflammatory reaction characterized by an influx of leukocytes and CCL20 is known to recruit leukocytes in other systems, the composition of ovarian leukocytes (CD45+) containing the CCL20 receptor CCR6 was determined immediately prior to ovulation. CD45+/CCR6+ cells were primarily natural killer cells (41%) along with B cells (12%), T cells (11%), neutrophils (10%), and monocytes (9%). Importantly, exogenous CCL20 stimulated ovarian leukocyte migration 59% within 90 minutes. Due to the difficulties in obtaining human follicles, an in vitro model was developed using granulosa-lutein cells to explore CCL20 regulation. CCL20 expression increased 40-fold within 6 hours after hCG, was regulated partially by the epithelial growth factor pathway, and was positively correlated with progesterone production. These results demonstrate that hCG dramatically increases CCL20 expression in the human ovary, that ovarian leukocytes contain the CCL20 receptor, and that CCL20 stimulates leukocyte migration. Our findings raise the prospect that CCL20 may aid in the final ovulatory events and contribute to fertility in women.

Comparative methods for fecal sample storage to preserve gut microbial structure and function in an in vitro model of the human colon

2020 ◽  
Vol 104 (23) ◽  
pp. 10233-10247
Author(s):  
Charlotte Deschamps ◽  
Elora Fournier ◽  
Ophélie Uriot ◽  
Frédérique Lajoie ◽  
Cécile Verdier ◽  
...  
Keyword(s):  
Fecal Sample ◽  
In Vitro Model ◽  
Human Colon ◽  
Structure And Function ◽  
Comparative Methods ◽  
Sample Storage ◽  
Microbial Structure ◽  
Vitro Model ◽  
And Function

Influence of Bacillus subtilis C-3102 on microbiota in a dynamic in vitro model of the gastrointestinal tract simulating human conditions

2012 ◽  
Vol 3 (3) ◽  
pp. 229-236 ◽  
Author(s):  
M. Hatanaka ◽  
Y. Nakamura ◽  
A.J.H. Maathuis ◽  
K. Venema ◽  
I. Murota ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Gastrointestinal Tract ◽  
In Vitro Model ◽  
Germination Rate ◽  
Human Colon ◽  
Micro Array ◽  
Vitro Model ◽  
Oral Doses ◽  
The Impact

Survival and germination rate of Bacillus subtilis C-3102 spores were investigated in a stomach and small intestine model (TIM-1), while the impact of C-3102 cells that had passed through TIM-1 on human colon microbiota was evaluated in a model of the large intestine (TIM-2). The survival of C-3102 spores in TIM-1 was 99%; 8% of the spores had germinated. Effluent of TIM-1 was subsequently introduced into TIM-2 and a micro-array platform was employed to assess changes in the microbiota composition. The effluent, which contained germinated C-3102 cells, increased some Bifidobacterium species and decreased some Clostridium groups. These changes were greater compared to those obtained by adding C-3102 spores directly to TIM-2. The present study suggests that oral doses of B. subtilis C-3102 spores have the potential to modulate the human colon microbiota. This effect may be caused by germination of the spores in the gastrointestinal tract.

scholarly journals Interleukin-1beta (IL-1beta) and IL-6 modulate insulin-like growth factor-binding protein (IGFBP) secretion in colon cancer epithelial (Caco-2) cells

2003 ◽  
Vol 179 (3) ◽  
pp. 405-415 ◽  
Author(s):  
ME Street ◽  
F Miraki-Moud ◽  
IR Sanderson ◽  
MO Savage ◽  
G Giovannelli ◽  
...  
Keyword(s):  
Human Colon ◽  
Band Intensity ◽  
Total Protein Content ◽  
Igf Binding Proteins ◽  
Undifferentiated State ◽  
Igf I ◽  
Igf Binding ◽  
Interleukin 1Beta ◽  
Vitro Model

Chronic inflammation is characterised by modifications in cytokine concentrations, whereas growth is mainly dependent on the GH-IGF axis. IGF-I bioavailability is modulated by a family of IGF-binding proteins (IGFBPs). The aim of the present study was to evaluate the interactions among interleukin-1beta (IL-1beta), IL-6 and IGFBP secretion by intestinal cells to assess whether cytokines modulate IGFBP secretion, and in turn IGF-I and IGF-II bioavailability. The human colon carcinoma derived cell line Caco-2 was used as an in vitro model for its capacity to differentiate spontaneously. Experiments were carried out on day 4 (undifferentiated state) and day 14 (differentiated state) after plating. Carcinoembryonic antigen (CEA) was used as a marker of differentiation and increased in the conditioned media (CM) from days 4 to 14 (0.2+/-0.01 ng/ml per 10(5) cells vs 3.3+/-0.2 ng/ml per 10(5) cells, P<0.05). IGFBP-2 and IGFBP-4 secretion decreased concomitantly. Cells were stimulated with IL-1beta and IL-6 at 1, 10 and 50 ng/ml, and with IL-1beta and IL-6 in combination at the same dose of 1 and 10 ng/ml. IGF-I at 50 ng/ml was used as a control. Caco-2 cells expressed and secreted mainly IGFBP-2 and IGFBP-4 into the CM. On day 4, IL-1beta (1 ng/ml) and IL-6 (10 and 50 ng/ml) reduced IGFBP-2 by 29+/-8%, and by 32+/-9 and 38+/-8% respectively (P<0.05). IGFBP-4 was also reduced by IL-1beta at 1 and 50 ng/ml (-14+/-4% and -46+/-11% vs serum free medium (SFM) respectively, P<0.05), and IL-6 at 50 ng/ml (-46+/-15%, P<0.05). Both IGFBP-2 and IGFBP-4 were reduced by IL-1beta and IL-6 in combination at 1 and 10 ng/ml (P<0.05). On day 14, IGFBP-2 band intensity was reduced at 10 ng/ml of IL-1beta (-22+/-15% vs SFM, P<0.05) and at 50 ng/ml of both cytokines (-33%+/-8% and -13%+/-13% vs baseline respectively, P<0.05). IGFBP-4 band intensity decreased with 10 and 50 ng/ml of IL-1beta (-35+/-11% and -46+/-15% vs SFM respectively) and IL-6 (-36%+/-10% and -46+/-15% vs SFM respectively). IL-1beta and IL-6 in combination at 1 and 10 ng/ml reduced both IGFBP-2 and IGFBP-4.In conclusion, IGFBP-2 and IGFBP-4 secretion in CM decreased with Caco-2 cell differentiation. IGFBP-2 and IGFBP-4 were significantly decreased by IL-1beta and IL-6 treatment in both the undifferentiated and differentiated state. Furthermore, these cytokines increased cell proliferation whereas total protein content was significantly reduced only at the higher concentrations of IL-6 and IL-1beta. These findings suggest that interleukins modulate the IGF-IGFBP system in Caco-2 cells in vitro.

scholarly journals PD-L2 glycosylation promotes immune evasion and predicts anti-EGFR efficacy

2021 ◽  
Vol 9 (10) ◽  
pp. e002699
Author(s):  
Yiqi Xu ◽  
Zhenyue Gao ◽  
Ruxin Hu ◽  
Yuqing Wang ◽  
Yuhong Wang ◽  
...  
Keyword(s):  
Immune Evasion ◽  
Cytotoxic T Lymphocyte ◽  
Growth Factor Receptor ◽  
Stat3 Signaling ◽  
Programmed Death ◽  
Lymphocyte Activity ◽  
Epidermal Growth ◽  
Glycosylation Inhibition ◽  
Cetuximab Therapy

BackgroundCombination therapy has been explored for advanced head and neck squamous cell carcinoma (HNSCC) owing to the limited efficacy of anti-epidermal growth factor receptor (EGFR) therapy. Increased expression and glycosylation of immune checkpoint molecules in tumors are responsible for cetuximab therapy refractoriness. The role of programmed death ligand 2 (PD-L2), a ligand of PD-1, in the immune function is unclear. Here, we examined the regulatory mechanism of PD-L2 glycosylation and its role in antitumor immunity and cetuximab therapy.MethodsSingle-cell RNA sequencing and immunohistochemical staining were used to investigate PD-L2 expression in cetuximab-resistant/sensitive HNSCC tissues. The mechanism of PD-L2 glycosylation regulation was explored in vitro. The effects of PD-L2 glycosylation on immune evasion and cetuximab efficacy were verified in vitro and using mice bearing orthotopic SCC7 tumors.ResultsThe PD-L2 levels were elevated and N-glycosylated in patients with cetuximab-resistant HNSCC. Glycosylated PD-L2 formed a complex with EGFR, which resulted in the activation of EGFR/signal transducer and activator of transcription 3 (STAT3) signaling and decreased the cetuximab binding affinity to EGFR. The N-glycosyltransferase fucosyltransferase (FUT8), a transcriptional target of STAT3, was required for PD-L2 glycosylation. Moreover, glycosylation modification stabilized PD-L2 by blocking ubiquitin-dependent lysosomal degradation, which consequently promoted its binding to PD-1 and immune evasion. Inhibition of PD-L2 glycosylation using Stattic, a specific STAT3 inhibitor, or PD-L2 mutation blocking its binding to FUT8, increased cytotoxic T lymphocyte activity and augmented response to cetuximab.ConclusionsIncreased expression and glycosylation of PD-L2 in tumors are an important mechanism for cetuximab therapy refractoriness. Thus, the combination of PD-L2 glycosylation inhibition and cetuximab is a potential therapeutic strategy for cancer.

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