Application of Electron Paramagnetic Resonance Spectroscopy to Examine Free Radicals in Melanin Polymers and the Human Melanoma Malignum Cells

The studies of free radicals in melanin and the human melanoma malignum cells by an X-band (9.3 GHz) electron paramagnetic resonance (EPR) spectroscopy were presented. The original results were compared with those published earlier. The aim of this work was application of the advanced spectral analysis to determine free radical properties in melanin biopolymers obtained from different melanotic tumor cells and free radicals existing in the human melanoma cells. Magnetic spin-lattice interactions in melanin samples were tested. The evolution of lineshape of tumor cells with increasing of microwave power was determined to confirm their complex free radical system. The useful shape parameters were proposed. The shape of melanotic tumor cells was analyzed. EPR spectra of free radicals in the melanin isolated from different tumor cells measured in the wide range of microwave power were analyzed. The melanins were obtained from the control tumor cells and the cells cultured with the several antitumor substances. The usefulness of the electron paramagnetic resonance spectroscopy was confirmed

Application of EPR spectroscopy in qualitative and quantitative examinations of paramagnetic centers in melanin

Spektroskopia elektronowego rezonansu paramagnetycznego (electron paramagnetic resonance – EPR) jest metodą przydatną w biologii i medycynie do badania substancji paramagnetycznych, ich roli w procesach chorobowych oraz terapii. Celem pracy jest przedstawienie podstaw fizycznych spektroskopii EPR oraz dokonanie przeglądu zastosowań metody EPR do badań jakościowych i ilościowych centrów paramagnetycznych melanin. Omówiono możliwości spek-troskopii EPR i procedury eksperymentalne stosowane do wyznaczenia rodzajów centrów paramagnetycznych wystę-pujących w melaninach syntetycznych oraz w biopolimerach melaninowych. Parametrem spektroskopowym przydat-nym do określenia rodzaju centrów paramagnetycznych jest współczynnik rozszczepienia spektroskopowego g, który zależy od lokalizacji niesparowanego elektronu w cząsteczce. W melaninach występują o-semichinonowe wolne rodniki o spinie S = 1/2 oraz birodniki o spinie S = 1. Wolne rodniki i birodniki można odróżnić spektroskopowo poprzez analizy wpływu temperatury pomiaru na intensywność integralną linii EPR. Koncentracja centrów paramagnetycznych w melaninie jest proporcjonalna do intensywności integralnej widma EPR. Przedstawiono wpływ paramagnetycznych i diamagnetycznych jonów metali oraz tlenu na koncentrację centrów paramagnetycznych w melaninie. Dokonano przeglądu publikacji dotyczących wpływu substancji leczniczych na koncentrację centrów paramagnetycznych w melaninie. Przedstawiono przydatność spektroskopii EPR w identyfikowaniu melaniny w próbkach biologicznych, m.in. komór-kach nowotworowych, bakteriach i grzybach.Electron paramagnetic resonance (EPR) spectroscopy is a method useful in biology and medicine to examine paramagnetic substances, their role in disease processes and therapy. The aim of this review work is to present the physical foundations of EPR spectroscopy and to review the applications of the EPR method for the qualitative and quantitative research on paramagnetic centers in melanin. The possibilities of EPR spectroscopy and experimental procedures applied to determine the types of paramagnetic centers existing in synthetic melanin and in melanin biopolymers are discussed. A useful spectroscopic parameter to determine the type of paramagnetic centers is the spectroscopic cleavage coefficient g, which depends on the location of the unpaired electron in the molecule. o-Semiquinone free radicals with spin S = 1/2 and biradicals with spin S = 1, exist in melanin. Free radicals and biradicals can be distinguished spectroscopically by analysing the influence of temperature on the integral intensity of EPR lines. The concentration of paramagnetic centers in melanin is proportional to the intensity of the integral EPR spectrum. The influence of paramagnetic and diamagnetic metal ions, and oxygen on the concentration of paramagnetic centers in melanin is presented. The publications on the influence of medicinal substances on the concentration of paramagnetic centers in tumor cells were reviewed. The usefulness of EPR spectroscopy in identifying melanin in biological samples, among others, cancer cells, bacteria, and fungi, is presented

The role of epigenetics in the pathogenesis of melanoma

Epigenetics represents the mechanisms that influence the regulation and modification of the expression of genetic material not related to the alterations in DNA sequences. These mechanisms include both DNA methylation and histone modifications. In the present article, we review current views on the role of aberrations of DNA hyper- and hypomethylation processes and the acetylation of histones, associated with genes that control the cell cycle, cell differentiation, DNA repair, apoptosis, cell signaling, angiogenesis, metabolism of xenobiotics and invasion, in the pathogenesis of melanoma. In addition, new strategies for treatment of melanoma associated with epigenetics are presented.Przez pojęcie epigenetyka należy rozumieć mechanizmy wpływające na regulację i modyfi kację ekspresji materiału genetycznego, jednocześnie niezmieniające sekwencji nukleotydów. Mechanizmy te obejmują zarówno metylację DNA, jak i modyfikacje histonów. W artykule dokonano przeglądu aktualnych poglądów dotyczących zaburzeń procesów hiperihipometylacji DNA oraz acetylacji histonów w patogenezie czerniaka, związanych z genami kontrolującymi cykl komórkowy, różnicowanie, naprawę DNA, apoptozę, sygnalizację komórkową, angiogenezę, metabolizm ksenobiotyków i powstawanie przerzutów. Ponadto przedstawiono nowe strategie leczenia czerniaka związane z epigenetyką

Zastosowanie pirolitycznej metylacji w oznaczaniu profi lu krótkołańcuchowych kwasów tłuszczowych w wybranych preparatach farmaceutycznych techniką GC/MS

Lactic Acid Bacteria (LAB) belong to the normal fl ora of human alimentary tract and vaginal epithelium. The main products of LAB metabolism are short chain fatty acids (SCFA), which are known to have signifi cant infl uence on human health. The aim of this study was to examine the suitability of direct pyrolytic methylation in GC/MS profi ling of SCFA in the selected pharmaceutical preparations of LAB. For method optimization, standard SCFA samples consisting of acetic, propionic, butyric and lactic acids were pyrolyzed under various temperature conditions in the presence of methanolic solution of tetramethylammonium hydroxide (TMAH) as a derivatizing reagent. It was demonstrated that pyrolytic derivatization of standard SCFA to methyl esters was the most effi cient with the use 10% TMAH and the pyrolytic fi laments with Curie temperature of 480°C, when the pyrolysis cell was kept at 150°C. It was shown that the method is suitable for GC/MS qualitative analysis of SCFA profi le in the pharmaceutical preparations that contain LAB in lyophilized form.Bakterie kwasu mlekowego (Lactic Acid Bacteria – LAB) należą do naturalnej flory bakteryjnej zasiedlającej przewód pokarmowy oraz nabłonek pochwy człowieka. Głównym produktem metabolizmu LAB są krótkołańcuchowe kwasy tłuszczowe (short-chain fatty acids – SCFA) wywierające istotny wpływ na zdrowie człowieka. W pracy poddano ocenie przydatność techniki bezpośredniej metylacji pirolitycznej w oznaczaniu profi lu SCFA w wybranych preparatach farmaceutycznych LAB techniką GC/MS. W celu optymalizacji metody, wzorcowe próbki SCFA zawierające kwas octowy, propionowy, masłowy i mlekowy poddano pirolizie w obecności metanolowego roztworu wodorotlenku tetrametyloamoniowego (TMAH) w różnych warunkach temperaturowych. Stwierdzono, że derywatyzacja analizowanych kwasów tłuszczowych do estrów metylowych zachodziła z największą wydajnością pod wpływem 10% TMAH, gdy zastosowano druty pirolityczne o temperaturze Curie 480°C, a komorę pirolizera utrzymywano w temperaturze 150°C. Wykazano, że zoptymalizowana metoda jest przydatna w prowadzonej techniką GC/MS analizie jakościowej profi lu SCFA w wybranych preparatach farmaceutycznych zawierających LAB w formie liofi lizatu

Photon Upconversion in Small Molecules

Upconversion (UC) is a process that describes the emission of shorter-wavelength light compared to that of the excitation source. Thus, UC is also referred to as anti-Stokes emission because the excitation wavelength is longer than the emission wavelength. UC materials are used in many fields, from electronics to medicine. The objective of using UC in medical research is to synthesize upconversion nanoparticles (UCNPs) composed of a lanthanide core with a coating of adsorbed dye that will generate fluorescence after excitation with near-infrared light to illuminate deep tissue. Emission occurs in the visible and UV range, and excitation mainly in the near-infrared spectrum. UC is observed for lanthanide ions due to the arrangement of their energy levels resulting from f-f electronic transitions. Organic compounds and transition metal ions are also able to form the UC process. Biocompatible UCNPs are designed to absorb infrared light and emit visible light in the UC process. Fluorescent dyes are adsorbed to UCNPs and employed in PDT to achieve deeper tissue effects upon irradiation with infrared light. Fluorescent UCNPs afford selectivity as they may be activated only by illumination of an area of diseased tissue, such as a tumor, with infrared light and are by themselves atoxic in the absence of infrared light. UCNP constructs can be monitored as to their location in the body and uptake by cancer cells, aiding in evaluation of exact doses required to treat the targeted cancer. In this paper, we review current research in UC studies and UCNP development

Fisetin, a Potent Anticancer Flavonol Exhibiting Cytotoxic Activity against Neoplastic Malignant Cells and Cancerous Conditions: A Scoping, Comprehensive Review

Diet plays a crucial role in homeostasis maintenance. Plants and spices containing flavonoids have been widely used in traditional medicine for thousands of years. Flavonols present in our diet may prevent cancer initiation, promotion and progression by modulating important enzymes and receptors in signal transduction pathways related to proliferation, differentiation, apoptosis, inflammation, angiogenesis, metastasis and reversal of multidrug resistance. The anticancer activity of fisetin has been widely documented in numerous in vitro and in vivo studies. This review summarizes the worldwide, evidence-based research on the activity of fisetin toward various types of cancerous conditions, while describing the chemopreventive and therapeutic effects, molecular targets and mechanisms that contribute to the observed anticancer activity of fisetin. In addition, this review synthesized the results from preclinical studies on the use of fisetin as an anticancer agent. Based on the available literature, it might be suggested that fisetin has a bioactive potential to become a complementary drug in the prevention and treatment of cancerous conditions. However, more in-depth research is required to validate current data, so that this compound or its derivatives can enter the clinical trial phase

Evaluation of Melanogenesis in A-375 Cells in the Presence of DMSO and Analysis of Pyrolytic Profile of Isolated Melanin

The increase of a skin malignant melanoma (melanoma malignum) incidence in the world has been observed in recent years. The tumour, especially in advanced stadium with metastases, is highly resistant to conventional treatment. One of the strategies is to modulate melanogenesis using chemical compounds. In this study, the processes of differentiation and melanogenesis induced by dimethylsulfoxide (DMSO) in human melanoma cells (A-375) were investigated. Natural melanin isolated from A-375 melanoma cell line treated with 0.3% DMSO was analyzed by pyrolysis-gas chromatography-mass spectrometry (Py-GC/MS) method. The products derived from pheomelanin have not been stated in the pyrolytic profile of analyzed melanin. Within all products derived from eumelanins, 1,2-benzenediol has been predominated. It has been shown that in the melanoma cells stimulated with 0.3% and 1% DMSO, the increase of transcriptional activity of the tyrosinase gene took place. It was accompanied by the rise of tyrosinase activity and an accumulation of melanin in the cells. The better knowledge about the structure of melanins can contribute to establish the uniform criteria of malignant melanoma morbidity risk

Antiproliferative and Cytotoxic Properties of Propynoyl Betulin Derivatives against Human Ovarian Cancer Cells: In Vitro Studies

Due to the incidence of ovarian cancer (OC) and the limitations of available therapeutic strategies, it is necessary to search for novel therapeutic solutions. The aim of this study was to evaluate the cytotoxic effect of betulin 1 and its propynoyl derivatives 2–6 against ovarian cancer cells (SK-OV-3, OVCAR-3) and normal myofibroblasts (18Co). Paclitaxel was used as the reference compound. The propynoyl derivatives 2–6 exhibited stronger antiproliferative and cytotoxic activities compared to betulin 1. In both ovarian cancer cell lines, the most potent compound was 28-propynoylbetulin 2. In the case of compound 2, the calculated IC50 values were 0.2 µM for the SK-OV-3 cells and 0.19 µM for the OVCAR-3 cells. Under the same culture conditions, the calculated IC50 values for compound 6 were 0.26 µM and 0.59 µM, respectively. It was observed that cells treated with compounds 2 and 6 caused a decrease in the potential of the mitochondrial membrane and a significant change in cell morphology. Betulin 1, a diol from the group of pentacyclic triterpenes, has a confirmed wide spectrum of biological effects, including a significant anticancer effect. It is characterized by low bioavailability, which can be improved by introducing changes to its structure. The results showed that chemical modifications of betulin 1 only at position C-28 with the propynoyl group (compound 2) and additionally at position C-3 with the phosphate group (compound 3) or at C-29 with the phosphonate group (compound 6) allowed us to obtain compounds with greater cytotoxic activity than their parent compounds, which could be used to develop novel therapeutic systems effective in the treatment of ovarian cancer

Anticancer Activity of the Acetylenic Derivative of Betulin Phosphate Involves Induction of Necrotic-Like Death in Breast Cancer Cells In Vitro

Betulin (BT) is a natural pentacyclic lupane-type triterpene exhibiting anticancer activity. Betulin derivatives bearing propynoyloxy and phosphate groups were prepared in an effort to improve the availability and efficacy of the drug. In this study, a comparative assessment of the in vitro anticancer activity of betulin and its four derivatives was carried out using two human breast cancer cell lines: SK-BR-3 and MCF-7. In both studied cell lines, 30-diethoxyphosphoryl-28-propynoylbetulin (compound 4) turned out to be the most powerful inhibitor of growth and inducer of cellular death. Detailed examination of that derivative pertained to the mechanisms underlying its anticancer action. Treatment with compound 4 decreased DNA synthesis and up-regulated p21WAF1/Cip1 mRNA and protein levels in both cell lines. On the other hand, that derivative caused a significant increase in cell death, as evidenced by increased lactate dehydrogenase (LDH) release and ethidium homodimer uptake. Shortly after the compound addition, an increased generation of reactive oxygen species and loss of mitochondrial membrane potential were detected. The activation of caspase-3 and fragmentation of genomic DNA suggested an apoptotic type of cell death. However, analysis of cellular morphology did not reveal any nuclear features typical of apoptosis. Despite necrosis-like morphology, dead cells exhibited activation of the cascade of caspases. These observations have led to the conclusion that compound 4 pushed cells to undergo a form of necrotic-like regulated cell demise