Nitric Oxide Chemical Donor Affects the Early Phases of In Vitro Wound Healing Process

Torre, Cristina La; Cinque, Benedetta; Lombardi, Francesca; Miconi, Gianfranca; Palumbo, Paola; Evtoski, Zoran; Placidi, Giuseppe; Fanini, D.; Cimini, Annamaria; Benedetti, Elisabetta; Giuliani, Michael J.; Cifone, Maria Grazia

doi:10.1002/jcp.25331

Cited by 18 publications

(13 citation statements)

References 34 publications

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“…As previously described [ 13 ], for the in vitro monolayer wound healing assay, cell cultures were allowed to proliferate until ~90% confluence was reached, then DMEM was removed from the well and cell monolayers were scratched using a 200 μl pipet tip to create a uniform cell-free wound area with reproducible width of wounding (0.7 mm width). Debris was removed from the culture by gently washing with sterile PBS.…”

Section: Methodsmentioning

confidence: 99%

Production Conditions Affect the In Vitro Anti-Tumoral Effects of a High Concentration Multi-Strain Probiotic Preparation

et al. 2016

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A careful selection of the probiotic agent, standardization of the dose and detailed characterization of the beneficial effects are essential when considering use of a probiotic for the dietary management of serious diseases. However, changes in the manufacturing processes, equipment or facilities can result in differences in the product itself due to the live nature of probiotics. The need to reconfirm safety and/or efficacy for any probiotic product made at a different factory is therefore mandatory. Recently, under the brand VSL#3®, a formulation produced by a manufacturer different from the previous one, has been commercialized in some European countries (the UK and Holland). VSL#3 is a high concentration multi-strain preparation which has been recognized by the main Gastroenterology Associations for the dietary management of pouchitis as well as ulcerative colitis. We have compared the “original” VSL#3 produced in USA with the “newfound” VSL#3 produced in Italy. According to our results, the “newfound” VSL#3 has 130–150% more “dead bacteria” compared to the “original” product, raising concerns for the well-known association between dead microbes with adverse effects. The abilities of bacterial lysates from the two formulations to influence in vitro viability and proliferation of different tumor cell lines also resulted different. The repair of previously scratched monolayers of various adherent tumor cell lines (i.e. HT1080, and Caco-2 cells) was inhibited more significantly by the “original” VSL#3 when compared to the “newfound” VSL#3. Tumor cell cycle profile, in particular cell cycle arrest and apoptotic death of the cancer cells, further confirms that the “original” VSL#3 has a better functional profile than the “newfound” VSL#3, at least in in vitro. Our data stress the importance of the production conditions for the “newfound” VSL#3 considering that this product is intended to be used for the dietary management of patients with very serious diseases, such as chronic inflammatory bowel diseases.

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Section: Methodsmentioning

confidence: 99%

Production Conditions Affect the In Vitro Anti-Tumoral Effects of a High Concentration Multi-Strain Probiotic Preparation

et al. 2016

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“…Representative images from microscopic observations are also shown ( Figure 4B). As expected, in accordance with our previous findings [39], the pre-treatment with AG strongly affected the physiological repair of control monolayer and significantly prevented the stimulating effect of all these bacterial samples in terms of monolayer repair rate expressed as % re-epithelialization vs. relative T0. To verify the statistical relationship between the percentages of wound closure, NOS2 expression, and nitrite levels, Pearson's correlation test was used.…”

Section: Nos2 Expression and Nitrite Levelssupporting

confidence: 92%

“…The ability of the bacterial samples to regulate NOS2 activity was analyzed by measuring nitrite levels in supernatants of scratched HaCaT monolayers pre-treated with or without the selective NOS2 inhibitor, AG ( Figure 3A). Pre-treatment with AG prevented the increase of nitrite generation due to NOS activity induced by the scratch of control monolayer (p < 0.05), thus confirming our previous findings [39]. The treatment with S. thermophilus, L. plantarum, and L. acidophilus samples, able to up-modulate NOS2 expression, induced a significant increase of nitrite levels in culture medium versus control, even if at different extent (S. thermophilus, p < 0.01; L. plantarum, p < 0.0001; L. acidophilus p < 0.05).…”

Section: Nos2 Expression and Nitrite Levelssupporting

confidence: 90%

“…As previously described [39], for the in vitro wound healing assay, HaCaT cells were cultured in 6-well microplates under normal culture conditions to reach~90% confluence, then DMEM was removed from the wells and cell monolayers were scratched using a 200 µL pipet tip to create a uniform cell-free wound area with reproducible width of wounding. Debris was removed from the culture by gently washing with sterile PBS.…”

Section: In Vitro Wound Healing Modelmentioning

confidence: 99%

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Soluble Fraction from Lysates of Selected Probiotic Strains Differently Influences Re-Epithelialization of HaCaT Scratched Monolayer Through a Mechanism Involving Nitric Oxide Synthase 2

et al. 2019

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A growing body of evidence supports the use of probiotics in the treatment of several skin conditions, including wounds. Even if in vitro and in vivo studies have highlighted the pro-healing effects of some probiotic bacteria, the underlying mechanisms are still not fully defined. The current investigation aimed to determine the re-epithelialization potential of the soluble fraction from lysate of seven different probiotic strains belonging to different genera (i.e., Streptococcus, Lactobacillus, and Bifidobacterium) on in vitro physically wounded HaCaT monolayer model. The results suggested that the soluble fraction of S. thermophilus, L. plantarum, and L. acidophilus promoted the re-epithelialization of scratched HaCaT monolayers, whereas those from B. longum, B. infantis, and B. breve significantly inhibited the process. On the other hand, L. bulgaricus showed no significant effect on in vitro wound repair. The mechanisms underlying the pro- or anti-healing properties of selected bacterial strains strictly and positively correlated with their ability to modulate nitric oxide synthase 2 (NOS2) expression and activity. Accordingly, the pre-treatment with aminoguanidine (AG), a specific inhibitor of NOS2 activity, abrogated the pro-healing effects of S. thermophilus, L. plantarum, and L. acidophilus.

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“…The effect of 1400W on glioma cell proliferation and migration was assessed using wound-healing assay, as previously described [ 62 ]. U-87 MG and T98G cells were plated at 6 × 10 4 /cm 2 in six-well plates and cultured until reaching confluence.…”

Section: Methodsmentioning

confidence: 99%

Involvement of NOS2 Activity on Human Glioma Cell Growth, Clonogenic Potential, and Neurosphere Generation

Palumbo

Lombardi

Siragusa

et al. 2018

IJMS

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Aberrant nitric oxide synthase 2 (NOS2) expression has been suggested as an interesting therapeutic target that is being implicated as a component of the molecular profile of several human malignant tumors, including glioblastoma, which is the most aggressive brain tumor with limited therapeutic options and poor prognosis. The aim of the present work was to evaluate the effect of 1400W, a specific NOS2 inhibitor, on human glioma cells in terms of clonogenic potential, proliferation, migration rate, and neurosphere generation ability. NOS2 expression was determined by Western blotting. Nitric oxide (NO) production was measured through nitrite level determination. The trypan blue exclusion test and the plate colony formation assay were performed to evaluate cell proliferation and clonogenic potential. Cell proliferation and migration ability was assessed by the in vitro wound-healing assay. Neurosphere generation in a specific stemcell medium was investigated. NOS2 was confirmed to be expressed in both the glioma cell line and a human glioma primary culture, and overexpressed in relative derived neurospheres. Experiments that aimed to evaluate the influence of 1400W on U-87 MG, T98G (glioblastoma cell lines) and primary glioma cells sustained the crucial role played by NOS2 in proliferation, colony formation, migration, and neurosphere generation, thus supporting the emerging relevance of a NOS2/NO system as a prognostic factor for glioma malignancy and recurrence.

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Nitric Oxide Chemical Donor Affects the Early Phases of In Vitro Wound Healing Process

Cited by 18 publications

References 34 publications

Production Conditions Affect the In Vitro Anti-Tumoral Effects of a High Concentration Multi-Strain Probiotic Preparation

Production Conditions Affect the In Vitro Anti-Tumoral Effects of a High Concentration Multi-Strain Probiotic Preparation

Soluble Fraction from Lysates of Selected Probiotic Strains Differently Influences Re-Epithelialization of HaCaT Scratched Monolayer Through a Mechanism Involving Nitric Oxide Synthase 2

Involvement of NOS2 Activity on Human Glioma Cell Growth, Clonogenic Potential, and Neurosphere Generation

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