Evaluating the Effects of an Organic Extract from the Mediterranean Sponge Geodia cydonium on Human Breast Cancer Cell Lines.

Marine sponges are an excellent source of bioactive secondary metabolites for pharmacological applications. In the present study, we evaluated the chemistry, cytotoxicity and metabolomics of an organic extract from the Mediterranean marine sponge Geodia cydonium, collected in coastal waters of the Gulf of Naples. We identified an active fraction able to block proliferation of breast cancer cell lines MCF-7, MDA-MB231, and MDA-MB468 and to induce cellular apoptosis, whereas it was inactive on normal breast cells (MCF-10A). Metabolomic studies showed that this active fraction was able to interfere with amino acid metabolism, as well as to modulate glycolysis and glycosphingolipid metabolic pathways. In addition, the evaluation of the cytokinome profile on the polar fractions of three treated breast cancer cell lines (compared to untreated cells) demonstrated that this fraction induced a slight anti-inflammatory effect. Finally, the chemical entities present in this fraction were analyzed by liquid chromatography high resolution mass spectrometry combined with molecular networking.

Serum Cytokinome Profile Evaluation: A Tool to Define New Diagnostic and Prognostic Markers of Cancer Using Multiplexed Bead-Based Immunoassays.

In recent years, many researchers are focusing their attention on the link between inflammation and cancer. The inflammation is involved in the tumor development and suppression, by stimulating the immune response. In particular, the transition from chronic inflammation to cancer produces angiogenic and growth factors able to repair the tissue and to promote cancer cell survival, implantation, and growth. In this contest, the cytokines contribute to the development of these processes becoming active before and during the inflammatory process and playing an important function at the various disease levels. Thus, these proteins can represent specific markers of tumor development and progression. Therefore the "cytokinome" term is used to indicate the evaluation of cytokine pattern by using an "omics" approach. Newly, specific protein chips of considerable and improved sensitivity are being developed to determine simultaneously several and different cytokines. This can be achieved by a multiplex technology that, through the use of small amounts of serum or other fluids, is used to determine the presence and the levels of underrepresented cytokines. Since this method is an accurate, sensitive, and reproducible cytokine assay, it is already used in many different studies. Thus, this review focuses on the more latest aspects related to cytokinome profile evaluation in different cancers.

Differential Response of Two Human Breast Cancer Cell Lines to the Phenolic Extract from Flaxseed Oil.

Many studies have evidenced that the phenolic components from flaxseed (FS) oil have potential health benefits. The effect of the phenolic extract from FS oil has been evaluated on two human breast cancer cell lines, MCF7 and MDA-MB231, and on the human non-cancerous breast cell line, MCF10A, by SRB assay, cellular death, cell cycle, cell signaling, lipid peroxidation and expression of some key genes. We have evidenced that the extract shows anti-proliferative activity on MCF7 cells by inducing cellular apoptosis, increase of the percentage of cells in G0/G1 phase and of lipid peroxidation, activation of the H2AX signaling pathway, and upregulation of a six gene signature. On the other hand, on the MDA-MB2131 cells we verified only an anti-proliferative activity, a weak lipid peroxidation, the activation of the PI3K signaling pathway and an up-regulation of four genes. Overall these data suggest that the extract has both cytotoxic and pro-oxidant effects only on MCF7 cells, and can act as a metabolic probe, inducing differences in the gene expression. For this purpose, we have performed an interactomic analysis, highlighting the existing associations. From this approach, we show that the phenotypic difference between the two cell lines can be explained through their differential response to the phenolic extract.

Potential Anticancer Effects of Polyphenols from Chestnut Shell Extracts: Modulation of Cell Growth, and Cytokinomic and Metabolomic Profiles.

In this study, a hydroalcoholic chestnut shell extract was characterized and tested on six different human cell lines. Gallic, ellagic, and syringic acids were the most abundant non-condensed compounds in the chestnut extract, as determined by high performance liquid chromatography (HPLC). Tannins were mainly represented by condensed monomeric units of epigallocatechin and catechin/epicatechin. After 48 h of treatment, only the human hepatoblastoma HepG2 cells reached an inhibition corresponding to IC50 with an increase of apoptosis and mitochondrial depolarization. The cytokinome evaluation before and after treatment revealed that the vascular endothelial growth factor (VEGF) and the tumor necrosis factor (TNF)-α decreased after the treatment, suggesting a potential anti-angiogenic and anti-inflammatory effect of this extract. Moreover, the metabolome evaluation by ¹H-NMR evidenced that the polyphenols extracted from chestnut shell (PECS) treatment affected the levels of some amino acids and other metabolites. Overall, these data highlight the effects of biomolecules on cell proliferation, apoptosis, cell cycle and mitochondrial depolarization, and on cytokinomics and metabolomics profiles.

Anti-Inflammatory Effects of a Methanol Extract from the Marine Sponge Geodia cydonium on the Human Breast Cancer MCF-7 Cell Line.

Many research groups are working to find new possible anti-inflammatory molecules, and marine sponges represent a rich source of biologically active compounds with pharmacological applications. In the present study, we tested different concentrations of the methanol extract from the marine sponge, Geodia cydonium, on normal human breast epithelial cells (MCF-10A) and human breast cancer cells (MCF-7). Our results show that this extract has no cytotoxic effects on both cell lines whereas it induces a decrease in levels of VEGF and five proinflammatory cytokines (CCL2, CXCL8, CXCL10, IFN-γ, and TNF-α) only in MCF-7 cells in a dose-dependent manner, thereby indicating an anti-inflammatory effect. Moreover, interactomic analysis suggests that all six cytokines are involved in a network and are connected with some HUB nodes such as NF-kB subunits and ESR1 (estrogen receptor 1). We also report a decrease in the expression of two NFKB1 and c-Rel subunits by RT-qPCR experiments only in MCF-7 cells after extract treatment, confirming NF-kB inactivation. These data highlight the potential of G. cydonium for future drug discovery against major diseases, such as breast cancer.

Is serum Interleukin-17 associated with early atherosclerosis in obese patients?

Atherosclerosis is a chronic inflammatory process of the vessel walls, and CD4+ T-cells are peculiar to both human and murine atherosclerotic lesions. There is a recent line of research favoring hypothetic allergic mechanisms in the genesis of atherosclerosis and, consequently, coronary artery disease (CAD), among which Interleukin (IL)-17 appears to be a key cytokine regulating local tissue inflammation. The objective was to add a piece of information on the role of IL-17 in the genesis of atherosclerosis. Eighty obese patients with normal liver enzyme levels but presenting with ultrasonographic evidence of NAFLD formed the population of this cross-sectional study. Anthropometric measures, data on excess adiposity, metabolic profile, serum concentrations of IL-17, eotaxin-3, IL-8, and CCL4/MIP1ß, C-reactive protein, fibrinogen, ferritin, TNF-α, as well carotid intima-media thickness (IMT), a marker of atherosclerosis, and the main risk factors for CAD, such as blood pressure and smoking status, but also less determinant ones such as degree of NAFLD severity, Intramuscular Triglyceride storage and Resting Metabolic Rate were evaluated. Serum concentrations of Il-17 were detected as related to those of inflammatory cytokines, IL-6, IFN-γ and TNF-α. Furthermore, circulating levels of IL-17 were linked to those mirroring allergic process, IL-8, CCL4/MIP1ß and eotaxin. Early atherosclerosis, evidenced as increased IMT, was not associated with circulating IL-17 levels. At multiple regression,IMT was predicted, other than by age, by the amount of the visceral adiposity, expressed as visceral adipose tissue at ultrasonography, and by serum eotaxin. In conclusion, a strong relationship was found between the IL-17-related chemokine eotaxin and IMT. The association found between the amount of visceral fat and circulating levels of eotaxin on the one hand, and IMT on the other, could reinforce the hypothesis that IL-17, released by the visceral adipose tissue, induces eotaxin secretion via the smooth muscle cells present in the atheromatosus vessels.

Synergistic antitumor effect of Doxorubicin and tacrolimus (FK506) on hepatocellular carcinoma cell lines.

Hepatocellular carcinoma is the fifth most common cancer worldwide and shows a complex clinical course, poor response to pharmacological treatment, and a severe prognosis. Thus, the aim of this study was to investigate whether tacrolimus (FK506) has synergistic antitumor effects with doxorubicin on two human hepatocellular carcinoma cell lines, Huh7 and HepG2. Cell viability was analyzed by Sulforhodamine B assay and synergic effect was evaluated by the software CalcuSyn. Cell apoptosis was evaluated using Annexin V and Dead Cell assay. Apoptosis-related protein PARP-1 cleaved and autophagy-related protein expressions (Beclin-1 and LC3B) were measured by western blotting analysis. Cytokines concentration in cellular supernatants after treatments was studied by Bio-Plex assay. Interestingly the formulation with doxorubicin and tacrolimus induced higher cytotoxicity level on tumor cells than single treatment. Moreover, our results showed that the mechanisms involved were (i) a strong cell apoptosis induction, (ii) contemporaneous decrease of autophagy activation, understood as prosurvival process, and (iii) downregulation of proinflammatory cytokines. In conclusion, future studies could relate to the doxorubicin/tacrolimus combination effects in mice models bearing HCC in order to see if this formulation could be useful in HCC treatment.

Potential anti-inflammatory effects of the hydrophilic fraction of pomegranate (Punica granatum L.) seed oil on breast cancer cell lines.

In this work, we characterized conjugated linolenic acids (e.g., punicic acid) as the major components of the hydrophilic fraction (80% aqueous methanol extract) from pomegranate (Punica granatum L.) seed oil (PSO) and evaluated their anti-inflammatory potential on some human colon (HT29 and HCT116), liver (HepG2 and Huh7), breast (MCF-7 and MDA-MB-231) and prostate (DU145) cancer lines. Our results demonstrated that punicic acid and its congeners induce a significant decrease of cell viability for two breast cell lines with a related increase of the cell cycle G0/G1 phase respect to untreated cells. Moreover, the evaluation of a great panel of cytokines expressed by MCF-7 and MDA-MB-231 cells showed that the levels of VEGF and nine pro-inflammatory cytokines (IL-2, IL-6, IL-12, IL-17, IP-10, MIP-1α, MIP-1ß, MCP-1 and TNF-α) decreased in a dose dependent way with increasing amounts of the hydrophilic extracts of PSO, supporting the evidence of an anti-inflammatory effect. Taken together, the data herein suggest a potential synergistic cytotoxic, anti-inflammatory and anti-oxidant role of the polar compounds from PSO.

Dissimilar cytokine patterns in different human liver and colon cancer cell lines.

An accurate and simultaneous estimate of cellular levels of a large cytokine number is very useful to obtain information about an organ dysfunction leading to cancer because through the understanding of the evolution of cytokine patterns we can recognize and predict the disease progression. Cancer cell lines are commonly used to study the cancer microenvironment, to analyze their chemosensitivity and carcinogenesis as well as to test in vitro the effect of molecules, such as drugs or anti-oxidants, on the inflammation status and its progression. We noted that various cell lines commonly used as a model for studies on liver and colon cancer possess different patterns of cytokines. This aspect may generate data not comparable in laboratories using different cell lines; thus, to investigate the origin of these abnormalities we compared the cell lines HepG2 and Huh7, and HT-29 and HCT-116, for liver and colon cancer, respectively. In this context we have evaluated and compared the levels of cytokines, chemokines and growth factors in the supernatants of these cellular lines. Our aim was to identify what cytokines were significantly different correlating similarities and differences to the specific inflammation status of each cellular model of cancer.

Regulatory T cells, interleukin (IL)-6, IL-8, vascular endothelial growth factor (VEGF), CXCL10, CXCL11, epidermal growth factor (EGF) and hepatocyte growth factor (HGF) as surrogate markers of host immunity in patients with renal cell carcinoma.

OBJECTIVE: To identify a phenotype that could be informative and prognostic in patients with renal cell carcinoma (RCC) peripheral blood was evaluated for TH1, TH2, regulatory T cells (Tregs), natural killer (NK) and NKT cells and for cytokines/chemokines. PATIENTS AND METHODS: Peripheral blood from 77 patients with RCC and 40 healthy controls was evaluated by flow cytometry using monoclonal antibodies against CD4, CD25, FoxP3, CD45RA, CD45RO, CD152, CD184, CD279, CD3, CD16, CD56, CD161, CD158a, CD4, CD26, CD30, CD183 and CD184. A concomitant evaluation of 38 molecules was conducted in patients' serum using a multiplex biometric ELISA-based immunoassay. RESULTS: The number of NK cells CD3⁻/CD16⁺, CD3⁻/CD16⁺/CD161⁺ (NK) and CD3⁻/CD16⁺/CD161⁺/CD158a⁺ (NK- Kir 2+) was greater in the patients with RCC (P < 0.05); and the number of Treg cells CD4⁺/CD25(high+)/FOXP3⁺ and the subset CD4⁺/CD25(high+)/FOXP3⁺/CD45RA⁺ (naïve) and CD45R0⁺(memory) cells, were greater in the patients with RCC (P < 0.001). An increase in the following was observed in the serum of patients with RCC compared with healthy controls: interleukin (IL)-4, IL-6, IL-8, IL-10, G-CSF, CXCL10, CXCL11, hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF). According to Ingenuity Pathway Analysis (IPA), CXCL10, IL-6, IL-8, epidermal growth factor (EGF), HGF and VEGF were associated with a network that controls cellular movement, tissue development and cellular growth. Kaplan-Meier analysis for disease-free survival showed that high numbers of CD4⁺/CD25(high+)/FOXP3⁺/CD45RA⁺ (Treg naïve) and low numbers of CD3⁻/CD16⁺/CD161⁺/CD158a⁺ (NK-Kir+) cells predict short disease-free survival in patients with RCC. CONCLUSION: Concomitant evaluation of Treg (CD4⁺/CD25(high+)/FOXP3⁺ and CD4⁺/CD25(high+)/FOXP3⁺/CD45RA⁺) and of six soluble factors (IL-6, IL-8 ,VEGF, CXCL10, CXCL11, EGF, HGF) might be a surrogate marker of host immunity in patients with RCC.

Evaluation of selenite effects on selenoproteins and cytokinome in human hepatoma cell lines.

The need to explore new alternative therapeutic strategies and chemoprevention methods for hepatocellular carcinoma is growing significantly. Selenium is a trace element that plays a critical role in physiological processes, and is used in cancer chemoprevention. The aim of this work was to test in vitro the effect of sodium selenite on the human hepatoma cell lines, HepG2 and Huh7, to assess its effect on the expression of GPX1, SELK and SELENBP1 and also to evaluate its action on inflammation determinants such as cytokines. Our results show that: (i) the increase observed for the GPX1 and SELK expression is correlated with an increase in the sodium selenite concentration, also evidencing an inverse association between the levels of these two proteins and SELENBP1; (ii) the selenium concentrations evaluated in protein extracts increase in proportional way with the selenite concentrations used in the treatment, suggesting that other selenoproteins can also be modulated and should be evaluated in further studies, and (iii) some cytokines, VEGF and three pro-inflammatory cytokines, i.e., IL-6, IL-8, and IL-17, decreased with an increasing selenite concentration. Finally, interactomic studies show that GPX1 and SELK, and the four pro-inflammatory cytokines are functionally correlated evidencing a putative anti-inflammatory role for the selenite.

Structural and functional studies of the human selenium binding protein-1 and its involvement in hepatocellular carcinoma.

The reduced expression of human selenium binding protein-1 (SELENBP1) has been reported for some human cancers. In this work we have estimated a reduced SELENBP1 expression by immunohistochemistry for the first time also in liver tissues of patients with hepatocarcinoma (HCC). Since the structure-function relationships of SELENBP1 are unknown, we have performed computational and experimental studies to have insight on the structural features of this protein focusing our attention on the properties of cysteines to assess their ability to interact with selenium. We have performed CD studies on the purified protein, modeled its three-dimensional structure, studied the energetic stability of the protein by molecular dynamics simulations, and titrated the cysteines by DTNB (5,5'-dithiobis (2-nitrobenzoic acid). The secondary structure content evaluated by CD has been found similar to that of 3D model. Our studies demonstrate that (i) SELENBP1 is an alpha-beta protein with some loop regions characterized by the presence of intrinsically unordered segments, (ii) only one cysteine (Cys57) is enough exposed to solvent, located on a loop and surrounded by charged and hydrophobic residues, and can be the cysteine able to bind the selenium. Furthermore, during the molecular dynamics simulation at neutral pH the loop containing Cys57 opens and exposes this residue to solvent, confirming that it is the best candidate to bind the selenium. Experimentally we found that only one cysteine is titratable by DTNB. This supports the hypothesis that Cys57 is a residue functionally important and this may open new pharmacological perspectives.

Effects of lipoic acid, caffeic acid and a synthesized lipoyl-caffeic conjugate on human hepatoma cell lines.

Hepatocellular carcinoma (HCC) is among the most aggressive and fatal cancers. Its treatment with conventional chemotherapeutic agents is inefficient, due to several side effects linked to impaired organ function typical of liver diseases. Consequently, there exists a decisive requirement to explore possible alternative chemopreventive and therapeutic strategies. The use of dietary antioxidants and micronutrients has been proposed for HCC successful management. The aim of this work was to test in vitro the effects of lipoic acid, caffeic acid and a new synthesized lipoyl-caffeic conjugate on human hepatoma cell lines in order to assess their effect on tumor cell growth. The results of cytotoxicity assays at different times showed that the cell viability was directly proportional to the molecule concentrations and incubation times. Moreover, to evaluate the pro- or anti-inflammatory effects of these molecules, the cytokine concentrations were evaluated in treated and untreated cellular supernatants. The obtained cytokine pattern showed that, at the increasing of three molecules concentrations, three pro-inflammatory cytokines such as IL-1ß, IL-8 and TNF-α decreased whereas the anti-inflammatory cytokine such as IL-10 increased.

UbcH10 expression in human lymphomas.

AIMS: The UbcH10 ubiquitin-conjugating enzyme plays a key role in regulating mitosis completion. We have previously reported that UbcH10 overexpression is associated with aggressive thyroid, ovarian and breast carcinomas. The aim of this study was to investigate UbcH10 expression in human lymphomas. METHODS AND RESULTS: Cell lines and tissue samples of Hodgkin's lymphoma (HL) and of non-Hodgkin's lymphoma (NHL) were screened for UbcH10 expression at transcriptional and translational levels. UbcH10 expression was related to the grade of malignancy. In fact, it was low in indolent tumours and high in a variety of HL and NHL cell lines and in aggressive lymphomas. It was highest in Burkitt's lymphoma, as shown by quantitative real-time polymerase chain reaction and by tissue microarray immunohistochemistry. Flow cytometry of cell lines confirmed that UbcH10 expression is cell-cycle dependent, steadily increasing in S phase, peaking in G(2)/M phase and dramatically decreasing in G(0)/G(1) phases. We also showed that UbcH10 plays a relevant role in lymphoid cell proliferation, since blocking of its synthesis by RNA interference inhibited cell growth. CONCLUSIONS: Taken together, these results indicate that UbcH10 is a novel lymphoid proliferation marker encompassing the cell cycle window associated with exit from mitosis. Its overexpression in aggressive lymphomas suggests that UbcH10 could be a therapeutic target in this setting.

Structural analysis of human SEPHS2 protein, a selenocysteine machinery component, over-expressed in triple negative breast cancer.

Selenophosphate synthetase 2 (SEPHS2) synthesizes selenide and ATP into selenophosphate, the selenium donor for selenocysteine (Sec), which is cotranslationally incorporated into selenoproteins. The action and regulatory mechanisms of SEPHS2 as well as its role in carcinogenesis (especially breast cancer) remain ambiguous and need further clarification. Therefore, lacking an experimentally determined structure for SEPHS2, we first analyzed the physicochemical properties of its sequence, modeled its three-dimensional structure and studied its conformational behavior to identify the key residues (named HUB nodes) responsible for protein stability and to clarify the molecular mechanisms by which it induced its function. Bioinformatics analysis evidenced higher amplification frequencies of SEPHS2 in breast cancer than in other cancer types. Therefore, because triple negative breast cancer (TNBC) is biologically the most aggressive breast cancer subtype and its treatment represents a challenge due to the absence of well-defined molecular targets, we evaluated SEPHS2 expression in two TNBC cell lines and patient samples. We demonstrated mRNA and protein overexpression to be correlated with aggressiveness and malignant tumor grade, suggesting that this protein could potentially be considered a prognostic marker and/or therapeutic target for TNBC.

UbcH10 is overexpressed in malignant breast carcinomas.

Our group has recently demonstrated the overexpression of the UbcH10 gene in undifferentiated thyroid carcinomas. Subsequently, a clear correlation between UbcH10 overexpression and a reduced survival in ovarian carcinoma patients has been described indicating UbcH10 as a valid prognostic marker in this neoplastic disease. Here we have extended the analysis of the UbcH10 expression to neoplastic breast diseases. We demonstrated, by tissue micro-arrays immunohistochemical studies, a significant difference (p=0.0001) in the mean percentage of UbcH10 stained cells between benign (0.22%) and malignant (11.01%) neoplastic lesions. High UbcH10 expression was associated with intense Ki-67 staining (p=0.015) and ErbB2 positivity (p=0.092). The suppression of the ErbB2 expression in breast carcinoma cell lines induces a reduction of UbcH10 level. Consistently, the inhibition of breast carcinoma cell growth was achieved following the block of UbcH10 protein synthesis by RNA interference. Therefore, these results suggest the perspective of a therapy of aggressive breast carcinomas based on the suppression of the UbcH10 function.

Combining doxorubicin with a phenolic extract from flaxseed oil: Evaluation of the effect on two breast cancer cell lines.

Breast cancer is one of the most frequently diagnosed forms of cancer and different treatments are used to block its progression. However, it still represents a very common cause of death in women. Doxorubicin (Dox) is reported as an effective agent in breast cancer treatment nonetheless it induces many side­effects. For this reason, many laboratories are engaged in understanding how it is possible to decrease the drug concentration, considering that one of the possible solutions is to use drug synergy, combining it with natural substances. Recently we showed that a phenolic extract from flaxseed (FS) oil, named PEFSO, induced on MCF­7 cell line an increase of apoptosis with related modification of G0/G1 phase cell cycle, and the activation of signaling and pro­oxidant pathways. In this study we present data on the combined effect of Dox and PEFSO on two different breast cancer cell lines to define the conditions to use lower doses of this chemotherapeutic agent. We report the data relating to the ability of this mixture to induce cytotoxicity and apoptosis, cell cycle modification, mitochondrial membrane depolarization and activation of extrinsic and/or intrinsic apoptotic pathway.

The Cytokinome Profile in Patients with Hepatocellular Carcinoma and Type 2 Diabetes.

Understanding the dynamics of the complex interaction network of cytokines, defined as ''cytokinome'', can be useful to follow progression and evolution of hepatocellular carcinoma (HCC) from its early stages as well as to define therapeutic strategies. Recently we have evaluated the cytokinome profile in patients with type 2 diabetes (T2D) and/or chronic hepatitis C (CHC) infection and/or cirrhosis suggesting specific markers for the different stages of the diseases. Since T2D has been identified as one of the contributory cause of HCC, in this paper we examined the serum levels of cytokines, growth factors, chemokines, as well as of other cancer and diabetes biomarkers in a discovery cohort of patients with T2D, chronic hepatitis C (CHC) and/or CHC-related HCC comparing them with a healthy control group to define a profile of proteins able to characterize these patients, and to recognize the association between diabetes and HCC. The results have evidenced that the serum levels of some proteins are significantly and differently up-regulated in all the patients but they increased still more when HCC develops on the background of T2D. Our results were verified also using a separate validation cohort. Furthermore, significant correlations between clinical and laboratory data characterizing the various stages of this complex disease, have been found. In overall, our results highlighted that a large and simple omics approach, such as that of the cytokinome analysis, supplemented by common biochemical and clinical data, can give a complete picture able to improve the prognosis of the various stages of the disease progression. We have also demonstrated by means of interactomic analysis that our experimental results correlate positively with the general metabolic picture that is emerging in the literature for this complex multifactorial disease.

Vitamin C effect on mitoxantrone-induced cytotoxicity in human breast cancer cell lines.

In recent years the use of natural dietary antioxidants to minimize the cytotoxicity and the damage induced in normal tissues by antitumor agents is gaining consideration. In literature, it is reported that vitamin C exhibits some degree of antineoplastic activity whereas Mitoxantrone (MTZ) is a synthetic anti-cancer drug with significant clinical effectiveness in the treatment of human malignancies but with severe side effects. Therefore, we have investigated the effect of vitamin C alone or combined with MTZ on MDA-MB231 and MCF7 human breast cancer cell lines to analyze their dose-effect on the tumor cellular growth, cellular death, cell cycle and cell signaling. Our results have evidenced that there is a dose-dependence on the inhibition of the breast carcinoma cell lines, MCF7 and MDA-MB231, treated with vitamin C and MTZ. Moreover, their combination induces: i) a cytotoxic effect by apoptotic death, ii) a mild G2/M elongation and iii) H2AX and mild PI3K activation. Hence, the formulation of vitamin C with MTZ induces a higher cytotoxicity level on tumor cells compared to a disjointed treatment. We have also found that the vitamin C enhances the MTZ effect allowing the utilization of lower chemotherapic concentrations in comparison to the single treatments.

Ascorbic acid: its role in immune system and chronic inflammation diseases.

Ascorbic acid (AA), also known as vitamin C, was initially identified as the factor preventing the scurvy disease, and became very popular for its antioxidant properties. It is an important co-substrate of a large class of enzymes, and regulates gene expression by interacting with important transcription factors. AA is important in all stressful conditions that are linked to inflammatory processes and involve immunity. It has been known for decades that the persistence of an inflammatory stimulus is responsible for the onset of many diseases. AA is essential to stimulate the immune system by increasing the strength and protection of the organism. Therefore, its immunostimulant, antinflammatory, antiviral and antibacterial roles are well known, we have summarized its main functions in different types of diseases related to the immune system and chronic inflammation. We can conclude that AA, due to its effects and diversity of regulated pathways, is suitable for use in various fields of medicine including immunology, toxicology, radiobiology and others. AA is not preferable to be used as an isolated mode of treatment, but it can be co-applied as an adjuvant to regulate immunity, gene expression and other important physiological processes. However, we propose that future studies will take into consideration the research of new combinations of antioxidant natural substances and drugs.