Doxorubicin–transferrin conjugate alters mitochondrial homeostasis and energy metabolism in human breast cancer cells

Doxorubicin (DOX) is considered one of the most powerful chemotherapeutic agents but its clinical use has several limitations, including cardiomyopathy and cellular resistance to the drug. By using transferrin (Tf) as a drug carrier, however, the adverse effects of doxorubicin as well as drug resistance can be reduced. The main objective of this study was to determine the exact nature and extent to which mitochondrial function is influenced by DOX–Tf conjugate treatment, specifically in human breast adenocarcinoma cells. We assessed the potential of DOX–Tf conjugate as a drug delivery system, monitoring its cytotoxicity using the MTT assay and ATP measurements. Moreover, we measured the alterations of mitochondrial function and oxidative stress markers. The effect of DOX–Tf was the most pronounced in MDA-MB-231, triple-negative breast cancer cells, whereas non-cancer endothelial HUVEC-ST cells were more resistant to DOX–Tf conjugate than to free DOX treatment. A different sensitivity of two investigate breast cancer cell lines corresponded to the functionality of their cellular antioxidant systems and expression of estrogen receptors. Our data also revealed that conjugate treatment mediated free radical generation and altered the mitochondrial bioenergetics in breast cancer cells.This work was supported in part by Grant No. B1511000001026.02 of the University of Lodz, Poland

The role of oxidative and nitrosative stress and the tryptophan catabolites pathway in the pathogenesis of depression

W skład rozprawy doktorskiej wchodzi sześć publikacji: jedna przeglądowa i pięć doświadczalnych.Depresja jest najczęstszą chorobą psychiczną, dotykającą około 350 mln ludzi na całym Świecie. Dotychczasowe badania sugerują udział powiązanych ze sobą szlaków biochemicznych, tj.: stresu oksydacyjnego i nitracyjnego oraz nieprawidłowości szlaku katabolitów tryptofanu w rozwoju zaburzeń depresyjnych. Depresja związana jest z intensyfikacją procesów oksydacyjnych oraz niewydolnością systemów obrony antyoksydacyjnej. Co więcej, wykazano, że pacjenci z depresją cechują się obniżonym poziomem tryptofanu, a także zaburzeniami przemian tego egzogennego aminokwasu. W związku z tym celem niniejszej pracy było wyjaśnienie roli stresu oksydacyjnego i nitracyjnego oraz zaburzeń przebiegu szlaku katabolitów tryptofanu w molekularnym podłożu depresji. Materiał do badań stanowiły próbki genomowego DNA wyizolowane z krwi obwodowej pobranej od pacjentów ze zdiagnozowaną depresją i od osób z grupy kontrolnej, u których wykluczono obecność tej choroby oraz próbki genomowego DNA, RNA wyizolowane z krwi i struktur mózgowych oraz białka wyizolowanego ze struktur mózgowych szczurów poddanych procedurze chronicznego łagodnego stresu i terapii wenlafaksyną. Ludzki genomowy DNA wykorzystano do genotypowania polimorfizmów metodą łańcuchowej reakcji polimerazy z detekcją produktu specyficznego dla jednego allelu i/bądź drugiego allelu w czasie rzeczywistym Wyizolowane DNA, RNA oraz białko wykorzystano odpowiednio do analizy stopnia metylacji regionów promotorowych badanych genów, ekspresji na poziomie mRNA oraz białka. W tym celu zastosowano odpowiednio technikę łańcuchowej reakcji polimerazy z detekcją w czasie rzeczywistym (real-time PCR), analizy topnienia DNA o wysokiej rozdzielczości (MS-HRM, ang. methylation sensitive – high resolution melting) oraz Western Blot. Wyniki uzyskane w toku realizacji niniejszej pracy doktorskiej wskazują na udział stres oksydacyjnego i nitracyjnego, a także zaburzeń w przebiegu szlaku katabolitów tryptofanu w molekularnym mechanizmie rozwoju depresji.Grant Narodowego Centrum Nauki przyznany w ramach konkursu OPUS 10 nr 2015/19/B/NZ7/00410 „Rola procesu zapalnego, stresu oksydacyjnego i nitryzacyjnego, szlaku katabolitów tryptofanu oraz naprawy DNA przez wycinanie zasad w depresji i w mechanizmie działania leków przeciwdepresyjnych, w przedklinicznych i klinicznych badaniach in vitro i in vivo”; Kierownik – prof. dr hab. Tomasz Śliwiński

Variation of Genes Encoding Tryptophan Catabolites Pathway Enzymes in Stroke

The abnormal activation of the tryptophan catabolites pathway (TRYCATs) is observed in patients suffering from cerebrovascular disease, including stroke. A previous study confirmed that lower bioavailability of tryptophan for serotonin synthesis was characterized in the patients during the acute stroke phase. Interestingly, according to various studies, polymorphisms of the genes involved in the TRYCATs pathway may modulate the risk of stroke occurrence. Therefore, this study aimed to investigate the association between the occurrence of TPH1, TPH2, KAT1, KAT2 and IDO1 polymorphisms and the risk of stroke development.The following 10 polymorphisms of the genes encoding enzymes of the TRYCATs pathway were selected: c.804-7C > A (rs10488682), c.-1668T > A (rs623580), c.803+221C > A (rs1800532), c.-173A > T (rs1799913) – TPH1, c.-1449C > A (rs7963803), and c.-844G > T (rs4570625) – TPH2. c.*456G > A of KAT1 (rs10988134), c.975-7T > C of KAT2 (rs1480544), c.-1849C > A (rs3824259) and c. -1493G > C (rs10089084) of IDO1. The study was carried out on DNA isolated from the peripheral blood taken from 107 patients after a stroke and 107 healthy volunteers. All DNA samples were genotyped using TaqMan probes. The genotypes of eight studied polymorphisms modulated the risk of stroke. No significant difference in genotype and allele frequencies of the c.804-7C > A –TPH1 (rs10488682) and c.*456G > A – KAT1 (rs10988134) polymorphisms were found between patients and controls. Having performed haplotype and gen-gen analyses, it was possible to determine that patients after a stroke and controls differed in terms of the frequency of selected genotypes and haplotypes. Among the studied polymorphisms, eight SNPs were linked with stroke risk modulation. The results obtained confirmed our hypothesis regarding the involvement of the TRYCATs pathway in the pathogenesis of stroke

The Effect of Chronic Mild Stress and Escitalopram on the Expression and Methylation Levels of Genes Involved in the Oxidative and Nitrosative Stresses as Well as Tryptophan Catabolites Pathway in the Blood and Brain Structures

Previous studies suggest that depression may be associated with reactive oxygen species overproduction and disorders of the tryptophan catabolites pathway. Moreover, one-third of patients do not respond to conventional pharmacotherapy. Therefore, the study investigates the molecular effect of escitalopram on the expression of Cat, Gpx1/4, Nos1/2, Tph1/2, Ido1, Kmo, and Kynu and promoter methylation in the hippocampus, amygdala, cerebral cortex, and blood of rats exposed to CMS (chronic mild stress). The animals were exposed to CMS for two or seven weeks followed by escitalopram treatment for five weeks. The mRNA and protein expression of the genes were analysed using the TaqMan Gene Expression Assay and Western blotting, while the methylation was determined using methylation-sensitive high-resolution melting. The CMS caused an increase of Gpx1 and Nos1 mRNA expression in the hippocampus, which was normalised by escitalopram administration. Moreover, Tph1 and Tph2 mRNA expression in the cerebral cortex was increased in stressed rats after escitalopram therapy. The methylation status of the Cat promoter was decreased in the hippocampus and cerebral cortex of the rats after escitalopram therapy. The Gpx4 protein levels were decreased following escitalopram compared to the stressed/saline group. It appears that CMS and escitalopram influence the expression and methylation of the studied genes

Clinical Potential of Fruit in Bladder Cancer Prevention and Treatment

Bladder cancer (BC) is the most common tumor of the urinary system in the world. Moreover, despite using anticancer therapies, BC is also characterized by a high recurrence risk. Among numerous risk factors, cigarette smoking, occupational exposure to certain aromatic compounds, and genetic factors contribute most strongly to BC development. However, the epidemiological data to date suggests that diet quality may influence some carcinogenic factors of BC and, therefore, might have a preventative effect. Adequate consumption of selected fruits with scientifically proven properties, including pomegranates and cranberries, can significantly reduce the risk of developing BC, even in those at risk. Therefore, in this article, we aim to elucidate, using available literature, the role of fruits, including pomegranates, cranberries, citrus fruits, cactus pears, and apples, in BC prevention and treatment. Previous data indicate the role of compounds in the above-mentioned fruits in the modulation of the signaling pathways, including cell proliferation, cell growth, cell survival, and cell death

The role of oxidative and nitrative stress and condition inflammation in the molecular mechanism of development urolithiasis and bladder cancer.

Wśród chorób układu moczowego najczęściej diagnozowane są kamica moczowa i rak pęcherza moczowego (BC, ang. bladder cancer), które cechują się wysokim ryzykiem nawrotu i znaczącymi kosztami leczenia, co uzasadnia potrzebę ciągłego pogłębiania badań nad molekularnymi podstawami rozwoju tych chorób. Szczególne znaczenie może mieć zwrócenie uwagi na wspólny patomechanizm tych schorzeń, gdyż raporty epidemiologiczne wskazują na występowanie wielu podobieństw, a wśród nich, obok wysokiego odsetka nawrotu, na uwagę zasługują wspólne dla kamicy moczowej i BC czynniki ryzyka, takie jak płeć męska czy palenie tytoniu. Jednak, pomimo licznych badań nad czynnikami rozwoju tych chorób, które wskazują na kluczową rolę powiązanych ze sobą szlaków biochemicznych, takich jak stres oksydacyjny i nitracyjny oraz stan zapalny, wspólne podłoże molekularne obu schorzeń nadal pozostaje niewyjaśnione. Kamica moczowa jest chorobą przewlekłą, związaną z pojawieniem się w drogach moczowych złogów, które blokują przepływ moczu. Złogi te formułują się w konsekwencji wzrostu stężenia i krystalizacji nierozpuszczalnych składników moczu, w tym szczawianu wapania. W wyniku krystalizacji, szczawian wapnia może ulegać adhezji na powierzchni komórek kanalików nerkowych, a następnie może być internalizowany do komórek na drodze makropinocytozy w celu eliminacji. Produktami końcowymi degradacji szczawianów wapnia są wolne jony szczawianu i Ca2+, które zwiększają pulę wewnątrzkomórkowego wapnia. Przeładowanie komórek wapniem może prowadzić do dysfunkcji mitochondriów i nadprodukcji reaktywnych form tlenu (ROS, ang. reactive oxygen species), a w konsekwencji także indukować rozwój stanu zapalnego. Dotychczasowe wyniki wykazały, że pacjenci z kamicą moczową cechują się zwiększonym poziomem markerów peroksydacji lipidów (8-izoprostaglandyny F2 oraz malonylodialdehydu), a także cytokin prozapalnych (interleukiny 8) w moczu, surowicy i osoczu. Nadmiar ROS może również powodować uszkodzenia zarówno mtDNA jak i nDNA. W związku z tym, długotrwała nadprodukcja ROS i utrzymujący się stan zapalny obserwowane zwłaszcza u osób z nawracającymi atakami kolki nerkowej mogą przyczynić się do procesu kancerogenezy i rozwoju BC, który jest drugim najczęstszym nowotworem układu moczowo-płciowego. Podobnie jak w przypadku kamicy moczowej, także w przebiegu BC obserwuje się nasilenie procesów stresu oksydacyjnego i nitracyjnego. Badania przewidziane do realizacji w ramach planowanego projektu mają na celu zidentyfikowanie molekularnych podstaw istniejących zaburzeń w szlakach biochemicznych, które manifestują się stresem oksydacyjnym i nitracyjnym oraz stanem zapalnym w rozwoju kamicy moczowej i BC. W tym celu proponowane badania mają określić związek występowania wariantów polimorficznych (SNPs, ang. single nucleotide polymorphisms) genów SOD2 i NOS2 oraz IL-6 i IL-8, jak również poziomu ekspresji mRNA dla tych genów z rozwojem kamicy i BC. Wykazanie takiego związku pozwoli na wstępne wytypowanie potencjalnych biomarkerów prognostycznych, określających ryzyko rozwoju obu schorzeń. Planowane eksperymenty będą prowadzone we współpracy z Oddziałem Urologii Wojewódzkiego Szpitala Zespolonego im. M. Kacprzaka w Płocku. W ramach planowanego projektu zakłada się kwalifikację: 100 pacjentów ze zdiagnozowanym BC (w wieku 35-75 lat), 100 z kamicą moczową (w wieku 35-75 lat) hospitalizowanych na Oddziale Urologii WSZ w Płocku oraz homologicznej grupy 100 zdrowych ochotników na podstawie zgody Komisji Bioetycznej Uniwersytetu Łódzkiego nr 12/KBBN-UŁ/II/2020-21. Podczas procesu rekrutacji wśród uczestników przeprowadzona zostanie ankieta dotyczącą m.in.: wieku, płci, BMI, palenia tytoniu. Podstawowy materiał badawczy do realizacji zaplanowanych zadań stanowić będzie krew pełna pobrana z żyły łokciowej od każdego uczestnika badania Analiza SNPs będzie przeprowadzona na wyizolowanym z krwi pełnej DNA przy użyciu sond TaqMan™ SNP Genotyping Assay. Natomiast analiza ekspresji mRNA będzie przeprowadzona na wyizolowanym z krwi obwodowej RNA z zastosowaniem sond TaqMan™ Gene Expression Assay. Analiza wariantów polimorficznych przyczyni się do weryfikacji hipotezy zakładającej wspólny mechanizm rozwoju kamicy moczowej i BC. A określenie wpływu badanych polimorfizmów na poziom ekspresji genów może stanowić punkt wyjścia do opracowania markerów wczesnej identyfikacji pacjentów z grupy ryzyka rozwoju kamicy moczowej, a w konsekwencji również BC. Oczekuje się, że wyniki uzyskane w ramach proponowanego działania naukowego pozwolą na weryfikację hipotezy, zakładającej wspólny mechanizm rozwoju kamicy moczowej i BC, oparty na zaburzeniach funkcji synonimicznych czynników, zaangażowanych w stres oksydacyjny i nitracyjny oraz stan zapalny. Co więcej, badanie to umożliwi dokładniejsze zrozumienie patofizjologii kamicy moczowej i BC na poziomie molekularnym. W konsekwencji zdobyta wiedza będzie stanowiła podstawę do przeprowadzenia dalszych szeroko zakrojonych badań.Among the diseases of the urinary system, urolithiasis and bladder cancer (BC) are most often diagnosed, which are characterized by a high risk of recurrence and significant treatment costs, which justifies the need for continuous research into the molecular basis of the development of these diseases. It may be of particular importance to pay attention to the common pathomechanism of these diseases, as epidemiological reports indicate the occurrence of many similarities, and among them, apart from the high rate of recurrence, risk factors common for urolithiasis and BC, such as male sex or smoking, deserve attention. However, despite numerous studies on the factors of development of these diseases, which indicate the key role of interrelated biochemical pathways, such as oxidative and nitrative stress and inflammation, the common molecular basis of both diseases still remains unexplained. Urolithiasis is a chronic disease associated with the appearance of deposits in the urinary tract that blocks the flow of urine. These deposits are formed as a consequence of the increase in the concentration and crystallization of insoluble urine components, including calcium oxalate. As a result of crystallization, calcium oxalate can adhere to the surface of renal tubular cells and can then be internalized into the cells by macropinocytosis for elimination. The end products of calcium oxalate degradation are free oxalate and Ca2+ ions, which increase the pool of intracellular calcium. Overloading cells with calcium can lead to mitochondrial dysfunction and overproduction of reactive oxygen species (ROS), and consequently, also induce the development of inflammation. The results obtained so far have shown that patients with urolithiasis are characterized by increased levels of markers of lipid peroxidation (8-isoprostaglandin F2 and malondialdehyde), as well as pro-inflammatory cytokines (interleukins 8) in urine, serum and plasma. Excess ROS can also cause damage to both mtDNA and nDNA. Therefore, long-term overproduction of ROS and persistent inflammation observed especially in people with recurrent attacks of renal colic may contribute to the process of carcinogenesis and development of BC, which is the second most common cancer of the genitourinary system. As in the case of urolithiasis, intensification of oxidative and nitrative stress processes is also observed in the course of BC. The research to be carried out as part of the planned project is aimed at identifying the molecular basis of existing disorders in biochemical pathways, which are manifested by oxidative and nitration stress and inflammation in the development of urolithiasis and BC. To this end, the proposed studies are to determine the relationship between the occurrence of single nucleotide polymorphisms (SNPs) of the SOD2 and NOS2 and IL-6 and IL-8 genes, as well as the level of mRNA expression for these genes with the development of urolithiasis and BC. Demonstrating such a relationship will allow for the initial selection of potentially predictive biomarkers determining the risk of developing both diseases. The planned experiments will be conducted in cooperation with the Department of Urology of the Provincial Hospital of M. Kacprzak in Płock. The planned project assumes the qualification of 100 patients diagnosed with BC (aged 35-75), 100 with urolithiasis (aged 35-75) hospitalized at the Department of Urology of the WSZ in Płock and a homologous group of 100 healthy volunteers based on consent Bioethics Committee of the University of Lodz No. 12/KBBN-UŁ/II/2020-21. During the recruitment process, a survey will be conducted among the participants regarding, among others: age, gender, BMI, and smoking. The basic research material for the implementation of the planned tasks will be whole blood collected from the cubital vein from each participant of the study. SNPs analysis will be carried out on DNA isolated from whole blood using TaqMan™ SNP Genotyping Assay probes. The analysis of mRNA expression will be carried out on RNA isolated from peripheral blood using TaqMan™ Gene Expression Assay probes. The analysis of polymorphic variants will contribute to the verification of the hypothesis assuming a common mechanism for the development of urolithiasis and BC. Determining the impact of the studied polymorphisms on the level of gene expression may be a starting point for the development of markers for the early identification of patients at risk of developing urolithiasis, and consequently also BC. It is expected that the results obtained as part of the proposed scientific activity will allow us to verify the hypothesis assuming a common mechanism of urolithiasis and BC, based on dysfunctions of synonymous factors involved in oxidative and nitrative stress and inflammation. Moreover, this study will provide a better understanding of the pathophysiology of urolithiasis and BC at the molecular level. As a consequence, the acquired knowledge will form the basis for further extensive studies.Narodowe Centrum Nauki, MINIATURA 6, nr DEC-2022/06/X/NZ5/0033

Clinical Potential of Fruit in Bladder Cancer Prevention and Treatment

Bladder cancer (BC) is the most common tumor of the urinary system in the world. Moreover, despite using anticancer therapies, BC is also characterized by a high recurrence risk. Among numerous risk factors, cigarette smoking, occupational exposure to certain aromatic compounds, and genetic factors contribute most strongly to BC development. However, the epidemiological data to date suggests that diet quality may influence some carcinogenic factors of BC and, therefore, might have a preventative effect. Adequate consumption of selected fruits with scientifically proven properties, including pomegranates and cranberries, can significantly reduce the risk of developing BC, even in those at risk. Therefore, in this article, we aim to elucidate, using available literature, the role of fruits, including pomegranates, cranberries, citrus fruits, cactus pears, and apples, in BC prevention and treatment. Previous data indicate the role of compounds in the above-mentioned fruits in the modulation of the signaling pathways, including cell proliferation, cell growth, cell survival, and cell death

Probiotics in the Prevention of the Calcium Oxalate Urolithiasis

Nephrolithiasis ranks third among urological diseases in terms of prevalence, making up about 15% of cases. The continued increase in the incidence of nephrolithiasis is most probably due to changes in eating habits (high protein, sodium, and sugar diets) and lifestyle (reduced physical activity) in all developed countries. Some 80% of all kidney stones cases are oxalate urolithiasis, which is also characterized by the highest risk of recurrence. Frequent relapses of nephrolithiasis contribute to severe complications and high treatment costs. Unfortunately, there is no known effective way to prevent urolithiasis at present. In cases of diet-related urolithiasis, dietary changes may prevent recurrence. However, in some patients, the condition is unrelated to diet; in such cases, there is evidence to support the use of stone-related medications. Interestingly, a growing body of evidence indicates the potential of the microbiome to reduce the risk of developing renal colic. Previous studies have primarily focused on the use of Oxalobacterformigenes in patients with urolithiasis. Unfortunately, this bacterium is not an ideal probiotic due to its antibiotic sensitivity and low pH. Therefore, subsequent studies sought to find bacteria which are capable of oxalate degradation, focusing on well-known probiotics including Lactobacillus and Bifidobacterium strains, Eubacterium lentum, Enterococcus faecalis, and Escherichia coli

OXIDATIVE AND NITROSATIVE STRESS AS WELL AS THE TRYPTOPHAN CATABOLITES PATHWAY IN DEPRESSIVE DISORDERS

The aim of this paper is to elucidate the role of oxidative and nitrosative stress as well as the tryptophan catabolites pathway in the development of depression and the mechanism of action of antidepressant drugs, based on the available literature. According to the World Health Organization (WHO), an estimated 350 million people worldwide suffer from depression. The pathogenesis of depressive disorders has not been fully explained yet and several causes of this disease have been suggested. There is evidence for the involvement of several interconnected biochemical pathways, including oxidative and nitrosative stress as well as the tryptophan catabolites pathway. Studies to date indicate that patients with depression have lower levels of enzymatic and non-enzymatic elements of an antioxidant response and, at the same time, they display an increased amount of oxidative stress markers, when compared to healthy individuals. The development of depression is also associated with excessive activity of nitric oxide synthase. Furthermore, decreased levels of tryptophan and increased levels of its harmful catabolites, i.e. kynurenine and quinolinic acid, may lead to progression of the disease. Changes in these biochemical pathways can be used as risk factors for the development of depression and, in the future, they could be utilized as diagnostic biomarkers. Moreover, regulation of biochemical processes may contribute to the development of a new, effective and personalized antidepressant therapy

Variability, Expression, and Methylation of <i>IL-6</i> and <i>IL-8</i> Genes in Bladder Cancer Pathophysiology

Bladder cancer (BC) is the 10th most common form of cancer globally, but its complete aetiology is still unknown. Nevertheless, there is evidence that chronic inflammation plays a role in the development and progression of BC. Therefore, the presented study aimed to detect a potential association between selected single nucleotide polymorphisms (SNPs)—rs1800797 and rs2069845 in IL-6 and rs2227307 in IL-8—and BC development, as well as to identify the impact of BC on the level of expression and methylation of IL-6 and IL-8 promoters in PBMCs with the use of the TaqMan SNP genotyping assay, TaqMan gene expression assay, and methylation-sensitive high-resolution melting techniques. We did not find any association between the genotypes and combined genotypes of all studied polymorphisms and the occurrence of BC. However, we found that BC patients were characterised by decreased IL-6 and IL-8 mRNA expression levels compared to the controls. Additionally, the methylation status of the IL-6 promoter was higher in controls than in BC patients. Our findings suggest that inflammation may be involved in the development and progression of BC