The role of molecular mechanisms of neoangiogenesis as tumor markers in the treatment individualization of breast cancer patients

REZIME: Karcinom dojke je vodeći uzrok smrtnosti žena širom sveta kada su u pitanju maligne bolesti. Svetska zdravstvena organizacija je procenila da će u ovom veku svaka 8. žena na planeti oboleti od ove bolesti. Uprkos ostvarenom napretku u dijagnostici i lečenju, očigledan je visok morbiditet i mortalitet od karcinoma dojke, tako da su neophodni novi pristupi u lečenju ove bolesti. Činjenica je da su današnji onkološki protokoli previše kruti i aproksimativni i donekle zanemaruju osobenosti pacijenta i biologiju tumora svakog pacijenta. Stoga se teži individualizaciji/personalizaciji terapije, koja će odgovarati biološkom profilu svakog pacijenta ponaosob, što bi vodilo uvođenju novih i poboljšanju postojećih onkoloških protokola lečenja. Za to je neophodan multidisciplinarni pristup, u kome će sarađivati eksperti iz različitih oblasti, a koji uključuje data mining sisteme za obradu podataka i obećavajuće, ali nedovoljno istražene modalitete lečenja, kao što su elektroporacija, elektrohemioterapija i fitoterapija. Prvi deo istraživanja predstavlja prospektivnu studiju koja uključuje pacijente sa dijagnozom karcinoma dojke KC Kragujevac u petogodišnjem periodu praćenja. Tokom operacije koja se rutinski izvodi na Klinici za opštu i grudnu hirurgiju u KC Kragujevac uzimani su uzorci karcinoma dojke i peritumorskog tkiva, a potom su pored standardnih patohistoloških pregleda sprovedene i dodatne analize: određivanje koncentracije metaloproteinaze 9 (MMP- 9); ispitivanje genske ekspresije parametara neoangiogeneze VEGF-A, HIF-1, CXCL-12 i iNOS (Quantitative/Real Time PCR) i proteinske ekspresije imunofluorescentnom metodom (VEGF165b i CXCR-4). Drugi deo istraživanja, predstavlja in vitro ispitivanje novih vidova terapije- elektroporacije i elektrohemioterapije na karcinomskim (MDA-MB-231, MCF-7, SW-480, HCT-116) i zdravim (MRC-5, HUVEC i hAoSm) imortalizovanim ćelijskim linijama. Citotoksični efekti elektroporacije i elektrohemioterapije na ispitivanim ćelijskim linijama, praćeni su u realnom vremenu, primenom xCELLigence sistema (Real Time Cell Analysis-RTCA), kao i na osnovu analiziranja tipa ćelijske smrti akridin oranž/etidijum bromid mikroskopskom metodom. U cilju pronalaženja novih antineoplastičnih tretmana karcinoma dojke, ispitivano je antiinvazivno dejstvo ekstrakata listova invazivnih biljaka Doktorska disertacija Robinia pseudoacacia (L) i Amorpha fruticosa (L) na MRC-5 i MDA-MB-231 ćelijama, praćenjem relativne ekspresije MMP-9, VEGF-A, HIF-1α, CXCL-12 i iNOS gena (Quantitative/Real Time PCR metodom). Za data mining obradu podataka su korišćeni softveri (Machine Learning Techniques) koji su trenirani za konkretan problem predikcije recidiva i metastaza, kao najznačajnije prognostičke parametre ishoda bolesti.SUMMARY: Breast cancer is the leading cause of mortality related to cancer among women around the world. The World Health Organization estimated that in this century, every eighth woman on the planet will be affected by this disease. Despite the progress made in diagnosis and treatment, high morbidity and mortality from breast cancer is evident, so new approaches are needed in the treatment of this disease. The fact is that today's oncology protocols are too rigid and approximate and somewhat neglect the patient's specificity and biology of the tumor of each patient. It therefore seeks individualization / personalization of therapy, corresponding to the biological profile of each patient individually, which would lead to the introduction of the new and improvement of the existing oncological protocols. This requires a multidisciplinary approach, with collaboration of experts from different fields, which will include data mining systems for data processing as well as promising, but so far insufficiently investigated treatment modalities, such as electroporation, electrochemotherapy and phytotherapy. The first part of the study is a prospective study involving patients with breast cancer diagnosis at CC Kragujevac in a five-year follow-up period. During the surgery performed routinely at the Clinic for General and Chest Surgery in CC Kragujevac, samples of breast and peritumor tissue were taken, followed by the analyzes in addition to standard pathohistological examinations: determining the concentration of metalloproteinase 9 (MMP-9); gene expression of neoangiogenesis parameters of VEGF-A, HIF-1, CXCL-12 and iNOS (Quantitative / Real Time PCR) and protein expression using the immunofluorescence method (VEGF165b and CXCR-4). The second part of the study is an in vitro examination of new forms of therapy-electroporation and electrochemotherapy in cancer (MDA-MB- 231, MCF-7, SW-480, HCT-116) and healthy (MRC-5, HUVEC and hAoSm) immortalized cell lines. The cytotoxic effects of electroporation and electrochemotherapy on the examined cell lines were monitored in real time by using the xCELLigence system (Real Time Cell Analysis-RTCA) as well as by analyzing the cell-death by acridine orange/ethidium bromide microscopic method. In order to find new antineoplastic breast cancer treatments, the anti-invasive effects of leaf extracts of Robinia pseudoacacia (L) and Amorpha fruticosa (L) invading plants on MRC-5 and MDA-MB-231 cells were studied, followed by gene expression of MMP-9, VEGF-A, HIF-1α, CXCL-12, and iNOS (Quantitative / Real Time PCR method). For data mining, data processing was used (Machine Learning Techniques) trained for the specific problem of recurrence and metastasis prediction as the most important prognostic parameters of the disease outcome

TECHNOLOGICAL CHARACTERISTICS OF INTERSPECIFIC HYBRIDS BRONNER, MUSCARIS AND MORAVA IN BANJA LUKA REGION

Interspecific hybrids are increasingly finding their place in the assortment structure of modern viticulture, thanks to their notable resistance to diseases and adaptability to different climatic conditions. The aim of the research was to determine and compare the basic ampelographic and technological characteristics of the interspecific hybrids - Bronner, Muscaris and Morava with the conventional white wine variety Pinot Blanc, grown in the Banja Luka region (Bosnia and Herzegovina). The variety Muscaris stood out compared to the other analyzed varieties on the basis of the largest grape cluster weight (244.99 g), the weight of 100 grape berries (173.06 g), the weight of 100 grape skins (17.54 g) and the weight of seeds in100 grape berries (11.50 g). This variety also had the highest sugar content in grape juice (27.63 °Brix), followed by the Bronner variety (24.67 °Brix), which, according to the OIV categorization, ranks them among varieties with a very high sugar content. The Morava and Pinot Blanc varieties had a relatively similar sugar content (22.02 and 21.77 °Brix, respectively). The content of total polyphenols in the skins of Bronner variety grapes was statistically significantly higher (1876.73 mg/kg) compared to the other analyzed varieties, while a similar situation was observed in the seeds of the Pinot Blanc variety (2913.68 mg/kg). Based on the results, it was concluded that the variety Muscaris showed the best qualitative characteristics

Altered apoptosis and biotransformation signaling in HCT-116 colorectal carcinoma cells induced by Teucrium chamaedrys L. extract

The aim of this study was an investigation of pro-apoptotic activity of methanol extract from T. chamaedrys, a more detailed determination of the signal molecules activated in the process of apoptosis, and effects on mRNA expression of enzymes involved in biotransformation (CYP1A1 and GSTP1) and membrane transporter, MRP-2 in HCT-116 colorectal carcinoma cells. The results show pronounced proapoptotic activity of T. chamaedrys extract, due to activation of both extrinsic and intrinsic pathways. The death receptor associated signaling pathway was activated in HCT-116 following treatment by T. chamaedrys, via increased Fas receptor expression and activity of caspase 8. Activation of caspase 9 suggests that mitochondrial signalling also has an impact. The extract reduced mRNA expression of GSTP1 and MRP-2 genes, as one of the causes of multi drug resistance in cancer cells. Observed results offer the possibility for the use of T. chamaedrys extract in the context of cancer prevention and therapy.Publishe

Lipid Peroxidation and Antioxidant Supplementation in Neurodegenerative Diseases: A Review of Human Studies

Being characterized by progressive and severe damage in neuronal cells, neurodegenerative diseases (NDDs) are the major cause of disability and morbidity in the elderly, imposing a significant economic and social burden. As major components of the central nervous system, lipids play important roles in neural health and pathology. Disturbed lipid metabolism, particularly lipid peroxidation (LPO), is associated with the development of many NDDs, including Alzheimer's disease (AD), Parkinson's disease (PD), and amyotrophic lateral sclerosis (ALS), all of which show elevated levels of LPO products and LPO-modified proteins. Thus, the inhibition of neuronal oxidation might slow the progression and reduce the severity of NDD; natural antioxidants, such as polyphenols and antioxidant vitamins, seem to be the most promising agents. Here, we summarize current literature data that were derived from human studies on the effect of natural polyphenols and vitamins A, C, and E supplementation in patients with AD, PD, and ALS. Although these compounds may reduce the severity and slow the progression of NDD, research gaps remain in antioxidants supplementation in AD, PD, and ALS patients, which indicates that further human studies applying antioxidant supplementation in different forms of NDDs are urgently needed

The biological activity of Ocimum minimum L. flowers on redox status parameters in HCT-116 colorectal carcinoma cells

Medicinal plants have widely been used as a natural source of remedies for treating several diseases, including colorectal cancer. Ocimum minimum L. is a very important dietary plant used in traditional and modern medicine, due to its health beneficial effect realized by cytotoxic, proapoptotic, antioxidant/prooxidant, antiviral and antimicrobial activity. The biological activity of O. minimum flowers has been evaluated in HCT116 colorectal carcinoma cells through antiproliferative activity by MTT assay, pro-apoptotic activity by AO/EB and concentrations of redox status parameters (O2∙ and lipid peroxidation) by colorimetric methods. The protein expression of iNOS was analyzed by immunocytochemistry, while the antimigratory effect was measured by xCELLigence system. The treatment with O. minimum shows the antiproliferative, proapoptotic, impact on redox status parameters and antimigratory effect on HCT-116 cells. Based on obtained results, the pharmacological effect of O. minimum is evident against HCT-116 colorectal carcinoma cells, suggesting that this plant may be good start material for future anticancer therapy investigation.Publishe

Effects of royal jelly on energy status and expression of apoptosis and biotransformation genes in normal fibroblast and colon cancer cells

Royal jelly is natural bee product, traditionally used in medicine for antitumor, anti-inflammatory, antioxidant, antibiotic and many other beneficial properties. The aim of this study was to determine biological effects of royal jelly samples originating from Serbia on normal human fibroblast (MRC-5) and colorectal cancer (HCT-116 and SW-480) cells. MTT cell viability assay was used to determine cytotoxic activity, and NBT test was used for determination of superoxide anion radical concentration. Parameters of cell energy status were determined using LDH and ATP colorimetric methods. Relative expression of mRNA of apoptosis and biotransformation genes was monitored by qPCR method. Royal jelly affected cell viability, caused oxidative stress appearance and elevated parameters of energy status in cancer cell lines. The relative expression of genes whose proteins are included in biotransformation of xenobiotics were changed with notable suppression of CYP1A1, while increased expression of apoptosis genes was noted in tested cell lines. Royal jelly demonstrated cell selective effect and could be prospective in anticancer therapy.Publishe

Shelf-life study of osmodehydrated white cabbage packaged in modified atmosphere: A mathematical approach

Osmotic treatment (OT) is a process applied for drying of fruits and vegetables where the hypertonic solution is osmotic medium. White cabbage (cultivar „Futoški“) shelf-life analysis was conducted after OT in three different hypertonic solutions: a mixture of commercial sucrose and NaCl (S1), a mixture of S1 and molasses in the ratio 1:1 (S2) and. pure sugar beet molasses (85.4% dry matter) (S3). After the OT, cabbage samples were packed in high barrier bags in the modified atmosphere packaging (MAP). The composition of two mix-tures of used gas (40:60/CO2:N2 and 80:20/CO2:N2) was imported into bags. The samples were analysed for L-ascorbic acid content, pH, acidity and a total number of microorganisms and sensorial attributes during 90 days of storage in a refrigerator at 4-8 °C in defined time intervals. During the 90-day storage in the MAP, microbiological analysis showed that the number of microorganisms decreased during the storage in the MAP. The highest retention of ascorbic acid (27.35%) was observed in OT cabbage dehydrated in pure molasses solution and 80:20/CO2:N2 gas mixture after 90 days of storage. Sensory analysis showed that osmodehydrated cabbage for 20 days in S1, and 45 days for OT cabbage in solutions S2 and S3 had acceptable consumable characteristics

Elucidating the antibiofilm activity of Frangula emodin against Staphylococcus aureus biofilms

Aims Because the Staphylococcus aureus is one of the most well‐known pathogens associated with medical devices and nosocomial infections, the aim of the study was to examine antibiofilm potential of emodin against it. Methods and Results Antibacterial activity was examined through microdilution assay. Antibiofilm testing included crystal violet staining of biofilm biomass and morphology analysis by Atomic force microscopy (AFM). Furthermore, aerobic respiration was monitored using the Micro‐Oxymax respirometer. For investigation of gene expression qRT‐PCR was performed. Emodin demonstrated strong antibacterial activity and ability to inhibit biofilm formation of all tested strains. The effect on preformed biofilms was spotted in few strains. AFM revealed that emodin affects biofilm structure and roughness. Monitoring of respiration under emodin treatment in planktonic and biofilm form revealed that emodin influenced aerobic respiration. Moreover, qRT‐PCR showed that emodin modulates expression of icaA, icaD, srrA and srrB genes, as well as RNAIII, and that this activity was strain‐specific. Conclusion The results obtained in this study indicate the novel antibiofilm activity of emodin and its multiple pathways of action. Significance and Impact of Study This is the first study that examined pathways through which emodin expressed its antibiofilm activit

Patterns of herbivore damage, developmental stability, morphological and biochemical traits in female and male Mercurialis perennis in contrasting light habitats

Light environments can influence variation in plant morphology, development and susceptibility to herbivores. Our research interest was to investigate the patterns of herbivore damage and developmental stability in dioecious understory forb Mercurialis perennis in contrasting light habitats, located at 1700 m a.s.l. on Mt. Kopaonik. Male and female plants from two light habitats, open (a sun-exposed field) and shaded (a spruce forest) were examined with respect to: herbivore damage (percentage of leaf area loss), fluctuating asymetry (FA) as a measurement of developmental stability, plant morphological and, specifically, leaf size traits, as well as biochemical traits relating to nutritional quality and defence, taking into account the possible presence of intersexual differences. Our results show that herbivore damage was significantly higher in open habitat, as well as one out of four univariate FA indices and the multivariate index. Morphological and biochemical traits, apart from defensive compounds, had higher values in the shade, pointing to sun-exposed habitat being more stressful for this species. Intersexual differences were observed for foliar damage, defensive compounds (phenolics and tannins), all leaf size traits, total leaf area, and protein content. Contrasting light habitats affected most of the analysed traits. Both foliar damage and FA were higher in a more stressful habitat; within habitats, no positive correlations were found. Herbivore damage was significantly male biased in open habitat. The analysis of intersexual differences in developmental stability measured by leaf asymmetry levels provided no evidence that female plants were more sensitive to environmental stress.This is a post-peer-review, pre-copyedit version of an article published in Alpine Botany. The final authenticated version is available online at: [http://dx.doi.org/10.1007/s00035-018-0203-8