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

Simonetti V1,2, Franzini M2,3, Iaffaioli RV4, Pandolfi S1,2, Valdenassi L1,2 and Quagliariello V4.

1. “Kaos” ONLUS Foundation, Turin, Italy.

2. Oxygen-Ozone Therapy Scientific Society (SIOOT), Gorle, Italy.

3. University of Pavia, Pavia, Italy

4. Oncology Department, Istituto Nazionale Tumori, IRCCS, Fondazione G. Pascale, Naples, Italy.

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.

Ozone therapy is an effective medical treatment for different diseases like mucositis, psoriasis, acute pain, neuro vascular diseases and cancer. Emerging evidence indicates that ozone, a strong oxidant, could effectively improve organ ischemia-re perfusion, herniated disks and skin ulcers in clinical model with interesting anti-inflammatory  properties through inhibition of NF-κBactivation 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 interleukinsanalyzed and involved in melanoma cell survival and drug resistance. Ozone at 50 µg/ml also decrease of 60% the activation of the pro-inflammatory mediatorp65Nfkb and inhibit the Nitric oxide  production under pro-inflammatory condition. However, taking the several limitations of this study, further biological pre clinical investigations are being carried out in order to understand the potential of ozone as possible adjuvant agent in therapy of melanoma.

Introduction

In the last years, the use of ozone as an alternative tool for management of several diseases was sharply increased (Tylicki et al.,2004). Ozone therapy is a non conventional form of medicine that has been used successfully in the treatment of ischemic disorders like chronic middle ear deafness and tuberculosis are two diseases well treated by using ozone (Quain J,R.,1940 ; Stoker G., 1902). In the field of oncology, the use of ozone was well known only to   stimulate tissue oxygenation with possible implication sin VEGF-oncogens pathway affections (Clavo B et al., 2004) ; in fact, despite being administered over a very short period, ozone therapy improved oxygenation in hypoxic tumors in same clinical cases (Clavo B et al., 2004). From a molecular point of view, ozone has several putative mechanisms of action involving nuclear factor-erythroid 2-related factor 2 (Nrf2), a nuclear transcriptional factor with interesting anticancer and protective actions against neuro degenerative diseases like Alzheimer’s and Parkinson’s diseases(Sagaiand V et al., 2011). Specifically, the activation of Nrf2 performed by ozone could increase the activity of several tumor suppressor proteins like SOD, catalase (CAT), GSH, GPx, GSH-S-transferase (GSTr), NADPHquinine-oxidoreductase 1 (NQO1), and heat shock protein 70 (HSP70). These effects are substantially comparable to those observed with the use of same nutraceuticals having a great interest in CAM, in  particular related to breast cancer chemo prevention ( Vecchione R et al., 2016 ; Yucel et al., 2011). One of the most important factors involved in cancer growth, survival, and resistance to many chemo therapeutics is the anoxic micro environment; from a clinical point of view, tumor hypoxia is an independent prognostic factor for advanced cancer progression ( Nordsmark et al., 2004) in fact patients with hypoxic tumors have significantly lower overall survival or disease-free survival ( Simonetti V et al., 2018). One of the most interesting properties of ozone therapy is obviously the impact on the tissue oxygenation; in fact, as recently well demonstrated in a pilot study, ozone therapy could increase oxygenation in several hypoxic tumor tissues and it could be useful as possible adjuvant in chemo-radiotherapy regimens (Clavo B et al., 2004) . Despite the recent clinical studies related to ozone therapy, more biological and biochemical studies are required in order to understand the limitations and the possible adverse effects related to its use in human. We recently published data related to anti-inflammatory effects of ozone in human colon cancer cells and its possible adjuvant effect in combination with conventional chemo therapies like cisplatin and 5-FluoroUracil ( Simonetti V et al., 2018).Here we analyzed the multiple effects of different ozone doses on human melanoma micro environment by studying affections on several interleukins and growth factors under pro-inflammatory conditions understanding its possible and putative role in the complementary management of melanoma.  

Materials and Methods:- Cell Viability:

The cytotoxicity of ozone was evaluated on the human skin melanoma 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 according to 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 humidified atmosphere containing 5% CO2. After 24 h of appropriate growth, we tested in full medium the effects of ozone (from 10 up to 50 μg/mL) produced with Multiossigen machinery, type Medical 99 IR) following the same procedure reported in literature ( 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 standard conditions. At the end of the incubation period the cells were washed three times with PBS at pH 7.4 and incubated with 100 μl of a MTT solution (0.5 mg/ml in cell culture medium) for 4 h at 37°C. The absorbance readings were acquired at a wavelength of 450 nm with the Tecan Infinite M200 plate reader using I -control software. 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 within culture medium. After evaluating cell cytotoxicity, the total protein content was 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 the manufacturer) was added. The absorbance at 562 nm was finally measured on aplate reader. The cytotoxicity measurements were then normalized by the amount of total protein content in each well.

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

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