ANTI-INFLAMMATORY EFFECTS OF OZONE IN HUMAN MELANOMA CELLS AND ITS MODULATION OF TUMOUR MICROENVIRONMENT.

ANTI-INFLAMMATORY EFFECTS OF OZONE IN HUMAN MELANOMA CELLS AND ITS MODULATION OF TUMOUR MICROENVIRONMENT.

Ozone therapy is an effective medical treatment for different diseases like mucositis, psoriasis, acute pain, neurovascular diseases and cancer. Emerging evidence indicates that ozone, a strong oxidant, could effectively improve organ ischemia-reperfusion, herniated disks and skin ulcers in clinical model with interesting anti-inflammatory properties through inhibition of NF- κB activation in acute and chronic disease. The aim of this study is based on the study of the biological effects of ozone in human melanoma cancer cells in order to investigate about its possible use in association to common therapy. Specifically, human melanoma cells were pre exposed or not to pro-inflammatory condition (Lipopolysaccharides) and administration of ozone at different concentration was performed in order to evaluate different biological parameters; cell viability, Measurement of Mitochondrial Matrix Potential (MMP), Evaluation of p65-Nfkb and changes in secretion of interleukins and growth factors involved in melanoma growth, survival and chemo-resistance: IL-1, IL-8, IL-6, TNF-α, IL-9, TGF-β, IL-19, VEGF, MMP-2, MMP-9 and IL-17 by ELISA methods. Results obtained shown ability of ozone to decrease cell viability up to 75% compared to control after 24h of incubation and inhibit all interleukins analyzed and involved in melanoma cell survival and drug resistance. Ozone at 50 µg/ml also decrease of 60% the activation of the pro-inflammatory mediator p65Nfkb and inhibit the Nitric oxide production under pro-inflammatory condition. However, taking the several limitations of this study, further biological preclinical investigations are being carried out in order to understand the potential of ozone as possible adjuvant agent in therapy of melanoma.

 

Ozonetherapy is an effective medical treatment for different diseases likemucositis, psoriasis, acute pain, neurovascular diseases and cancer. Emergingevidence indicates that ozone, a strong oxidant, could effectively improveorgan ischemia-reperfusion, herniated disks and skin ulcers in clinical modelwith interesting anti-inflammatory properties through inhibition of NF-κBactivation in acute and chronic disease. The aim of this study is based on thestudy of the biological effects of ozone in human melanoma cancer cells inorder to investigate about its possible use in association to common therapy.Specifically, human melanoma cells were preexposed or not to pro-inflammatory condition (Lipopolysaccharides) and administration of ozone at different concentration was performed in order to evaluate different biological parameters; cell viability, Measurement of Mitochondrial Matrix Potential(MMP), Evaluation of p65-Nfkb and changes in secretion of interleukins and growth factors involved in melanoma growth, survival and chemo-resistance: IL-1, IL-8,IL-6, TNF-α, IL-9, TGF-β, IL-19, VEGF, MMP-2, MMP-9 and IL-17 by ELISA methods.Results obtained shown ability of ozone to decrease cell viability up to 75%compared to control after 24h of incubation and inhibit all interleukinsanalyzed and involved in melanoma cell survival and drug resistance. Ozone at50 µg/ml also decrease of 60% the activation of the pro-inflammatory mediatorp65Nfkb and inhibit the Nitric oxide production under pro-inflammatorycondition. However, taking the several limitations of this study, furtherbiological preclinical investigations are being carried out in order tounderstand the potential of ozone as possible adjuvant agent in therapy ofmelanoma. Introduction:- In the last years, the use of ozone as an alternativetool for management of several diseases was sharply increased (Tylicki et al.,2004). Ozone therapy is a nonconventional form of medicine that has been usedsuccessfully in the treatment of ischemic disorders like chronic middle eardeafness and tuberculosis are two diseases well treated by using ozone (QuainJ,R.,1940 ; Stoker G., 1902). In the field of oncology, the use of ozone waswell known only to stimulate tissue oxygenation with possible implicationsin VEGF-oncogens pathway affections (Clavo B et al., 2004) ; in fact, despitebeing administered over a very short period, ozone therapy improved oxygenationin hypoxic tumors in same clinical cases (Clavo B et al., 2004). From amolecular point of view, ozone has several putative mechanisms of actioninvolving nuclear factor-erythroid 2-related factor 2 (Nrf2), a nucleartranscriptional factor with interesting anticancer and protective actionsagainst neurodegenerative diseases like Alzheimer’s and Parkinson’s diseases(Sagaiand V et al., 2011). Specifically, the activation of Nrf2 performed byozone could increase the activity of several tumor suppressor proteins likeSOD, catalase (CAT), GSH, GPx, GSH-S-transferase (GSTr), NADPHquinine-oxidoreductase 1 (NQO1), and heat shock protein 70 (HSP70). Theseeffects are substantially comparable to those observed with the use of samenutraceuticals having a great interest in CAM, in particular related tobreast cancer chemoprevention ( Vecchione R et al., 2016 ; Yucel et al., 2011). One of the most important factors involved in cancer growth, survival, andresistance to many chemotherapeutics is the anoxic microenvironment; from aclinical point of view, tumor hypoxia is an independent prognostic factor foradvanced cancer progression ( Nordsmark et al., 2004) in fact patients withhypoxic tumors have significantly lower overall survival or disease-freesurvival ( Simonetti V et al., 2018). One of the most interesting properties ofozone therapy is obviously the impact on the tissue oxygenation; in fact, asrecently well demonstrated in a pilot study, ozone therapy could increaseoxygenation in several hypoxic tumor tissues and it could be useful as possibleadjuvant in chemo-radiotherapy regimens (Clavo B et al., 2004) . Despite therecent clinical studies related to ozone therapy, more biological andbiochemical studies are required in order to understand the limitations and thepossible adverse effects related to its use in

human. We recently publisheddata related to anti-inflammatory effects of ozone in human colon cancer cellsand its possible adjuvant effect in combination with conventionalchemotherapies like cisplatin and 5-FluoroUracil ( Simonetti V et al., 2018).Here we analyzed the multiple effects of different ozone doses on humanmelanoma microenvironment by studying affections on several interleukins andgrowth factors under pro-inflammatory conditions understanding its possible andputative role in the complementary management of melanoma. Materials and Methods:- Cell Viability:- The cytotoxicity of ozone was evaluated on the human skinmelanoma cell line CHL-1 (purchased from the American Type Culture Collection )looking at their mitochondrial dehydrogenase activity by means of a modifiedMTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] method accordingto the manufacturer’s instructions (Dojindo Molecular Technologies Inc.,Rockville, MD). The CHL-1 cells were grown in Dulbecco’s modified Eagle’smedium (DMEM; Gibco; Thermo Fisher Scientific, Inc) supplemented with 10% FBSand 1% penicillin-streptomycin, and incubated at 37°C in a humidifiedatmosphere containing 5% CO2. After 24 h of appropriate growth, we tested in fullmedium the effects of ozone (from 10 up to 50 μg/mL) produced with Multiossigenmachinery, type Medical 99 IR) following the same procedure reported inliterature ( Mary VM et al., 2015) and by our group ( Simonetti V et al., 2018)by a method that avoided cells from direct expose to ozone. After treatments,melanoma cells were then incubated for 2,6,12 and 24 h under standardconditions. At the end of the incubation period the cells were washed threetimes with PBS at pH 7.4 and incubated with 100 μl of a MTT solution (0.5 mg/mlin cell culture medium) for 4 h at 37°C. The absorbance readings were acquiredat a wavelength of 450 nm with the Tecan Infinite M200 plate reader using I -controlsoftware. The relative cell viability (%) was calculated by the formula [A]test/[A] control × 100, where “[A] test” is the absorbance of the test sample and”[A] control” is the absorbance of the control cells incubated solely withinculture medium. After evaluating cell cytotoxicity, the total protein contentwas measured by using the Micro BCA protein assay kit (Pierce). Briefly, thecells were washed with ice-cold PBS and incubated for 15 min in 150 μl celllysis buffer (0.5% v/v Triton X-100 in PBS), to which 150 μL of Micro BCAprotein assay kit reagent (prepared following the instructions of themanufacturer) was added. The absorbance at 562 nm was finally measured on aplate reader. The cytotoxicity measurements were then normalized by the amountof total protein content in each well.

DESCARCATI TOT MATERIALUL ANTI-INFLAMMATORY-EFFECTS-OF-OZONE-IN-HUMAN-MELANOMA-CELLS-AND-ITS-MODULATION-OF-TUMOUR-MICROENVIRONMENT (1)

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