BACKGROUND/AIMS: The main focus of this investigation is to identify deleterious single nucleotide polymorphisms (SNPs) located in the BRCA2 gene through in silico approach, thereby,providing an understanding of potentia...BACKGROUND/AIMS: The main focus of this investigation is to identify deleterious single nucleotide polymorphisms (SNPs) located in the BRCA2 gene through in silico approach, thereby,providing an understanding of potential consequences regarding the susceptibility to breast cancer. METHODS: The GenomAD database was used to identify SNPs. To determine the potential adverse consequences, our study employed various prediction tools, including SIFT, PolyPhen, PredictSNP, SNAP2, PhD-SNP, and ClinVar. The pathogenicity associated with the deleterious snSNPs was evaluated bu MutPred and Fathmm. Additionally, I-Mutant and MuPro were used to assess the stability, followed by conservation and protein-protein interaction analysis using robust computational tools. The 3D structure of BRCA2 protein was generated by SwissModel, followed by validation using PROCHECK and Errat. RESULTS: The GenomAD database was used to identify a total of 7, 921 SNPs, including 1940 missense SNPs. A set of 69 SNPs predicted by consensus to be damaging across all platforms was identified. Mutpred and Fathmm identified 48 and 38 SNPs, respectively to be associated with cancer. While I- Mutant and MuPro assays suggested 22 SNPs to decrease protein stability. Additionally, these 22 SNPs reside within highly conserved regions of the BRCA2 protein. Domain analysis, utilizing InterPro, pinpointed 18 deleterious mutations within crucial DNA binding domains and one in the BRC repeat region. CONCLUSION: This study establishes a foundation for future experimental validations and the creation of breast cancer-targeted treatment approaches.
Anomalous expression of potassium channels in cancer tissues is associated with several cancer hallmarks that support deregulated proliferation and tumor progression. Ion channels seem to influence cell proliferation; ho...Anomalous expression of potassium channels in cancer tissues is associated with several cancer hallmarks that support deregulated proliferation and tumor progression. Ion channels seem to influence cell proliferation; however, the crucial molecular mechanisms involved remain elusive. Some results show how extracellular mitogenic signals modulate ion channel activity through intracellular secondary messengers. It is relevant because we are beginning to understand how potassium channels can affect the proliferative capacity of cells, either in normal mitogen-dependent proliferation or in mitogen-unresponsive proliferation. Calciumdependent potassium channels have been implicated in cell cycle signaling in many cancerous cell lines. In particular, the so-called intermediate conductance KCa3.1 (IKCa) is reported to play a significant role in uncontrolled cell cycle signaling, among other malignant processes driven by cancer hallmarks. In addition to these features, this channel can be subjected to specific pharmacological regulation, making it a promising cornerstone for understanding the signaling behavior of several types of cancer and as a target for chemotherapeutic approaches. This review is dedicated to the connection of KCa3.1 activity, in canonical and non-canonical ways, to the cell cycle signaling, including the cooperation with calcium channels to generate calcium signals and its role as a mediator of proliferative signals.
BACKGROUND/AIMS: Unrestricted increased table salt (NaCl) intake is associated with oxidative stress and inflammation, leading to endothelial dysfunction and atherosclerosis. However, data on salt-induced immunomodulator...BACKGROUND/AIMS: Unrestricted increased table salt (NaCl) intake is associated with oxidative stress and inflammation, leading to endothelial dysfunction and atherosclerosis. However, data on salt-induced immunomodulatory effects in the earliest phase of salt loading are scarce. METHODS: In the present study, an animal model of short-term salt loading was employed, including male Sprague Dawley rats consuming a high-salt diet (HSD; 4% NaCl) or standard laboratory chow (low-salt; LSD; 0.4% NaCl) during a 7-day period. The contribution of angiotensin II (ANGII) suppression was tested by adding a group of rats on a high-salt diet receiving ANGII infusions Samples of peripheral blood/mesenteric lymph node leukocytes, brain blood vessels, and serum samples were processed for flow cytometry, quantitative real-time PCR, total proteome analysis, and multiplex immunoassay. RESULTS: Data analysis revealed the up-regulation of Il 6 gene in the microcirculation of high-salt-fed rats, accompanied by an increased serum level of TNF-alpha cytokine. The high-salt diet resulted in increased proportion of serum mono-unsaturated fatty acids and saturated fatty acids, reduced levels of linoleic (C18:2 ω-6) and α-linolenic (C18:3 ω-3) acid, and increased levels of palmitoleic acid (C16:1 ω-7). The high-salt diet had distinct, lymphoid compartment-specific effects on leukocyte subpopulations, which could be attributed to the increased expression of salt-sensitive SGK-1 kinase. Complete proteome analysis revealed high-salt-diet-induced vascular tissue remodeling and perturbations in energy metabolism. Interestingly, many of the observed effects were reversed by ANGII supplementation. CONCLUSION: Low-grade systemic inflammation induced by a HSD could be related to suppressed ANGII levels. The effects of HSD involved changes in Th17 and Treg cell distribution, vascular wall remodeling, and a shift in lipid and arachidonic acid metabolism.
BACKGROUND/AIMS: Endothelial cells (ECs) play a crucial role in various physiological processes, particularly those related to the cardiovascular system, but also those affecting the entire organism. The biology of ECs i...BACKGROUND/AIMS: Endothelial cells (ECs) play a crucial role in various physiological processes, particularly those related to the cardiovascular system, but also those affecting the entire organism. The biology of ECs is regulated by multiple biochemical stimuli and epigenetic drivers that govern gene expression. We investigated the angiogenic potential of ECs from a protein citrullination perspective, regulated by peptidyl-arginine deiminases (PADs) that modify histone and non-histone proteins. Although the involvement of PADs has been demonstrated in several physiological processes, inflammation-related disorders and cancer, their role in angiogenesis remains unclear. METHODS: To elucidate the role of PADs in endothelial angiogenesis, we used two human EC models: primary vein (HUVECs) and microvascular endothelial cells (HMEC-1). PADs activity was inhibited using irreversible inhibitors: BB-Cl-amidine, Cl-amidine and F-amidine. We analyzed all three steps of angiogenesis : proliferation, migration, and capillary-like tube formation, as well as secretory activities, gene expression and signaling in ECs. RESULTS: All used PAD inhibitors reduced the histone H3 citrullination (H3cit) mark, inhibited endothelial cell migration and capillary-like tube formation, and favored an angiostatic activity in HMEC-1 cells, by increasing PEDF (pigment epithelium-derived factor) and reducing VEGF (vascular endothelial growth factor) mRNA expression and protein secretion. Additionally, BB-Cl-amidine reduced the total activity of MMPs (Matrix metalloproteinases). The observed effects were underlined by the inhibition of Akt phosphorylation.>. CONCLUSION: Our findings suggest that pharmacological inhibitors of citrullination are promising therapeutic agents to target angiogenesis.
BACKGROUND/AIMS: Bladder cancer is considered one of the most aggressive neoplasms due to its recurrence and progression profile, and even with the improvement in diagnosis and treatment methods, the mortality rate has n...BACKGROUND/AIMS: Bladder cancer is considered one of the most aggressive neoplasms due to its recurrence and progression profile, and even with the improvement in diagnosis and treatment methods, the mortality rate has not shown a declining trend in recent decades. From this perspective, the search and development of more effective and safer therapeutic alternatives are necessary. Phytochemicals are excellent sources of active principles with therapeutic potential. [6]-Shogaol is a phenolic compound extracted from the ginger rhizomes that has shown antitumor effects in a wide variety of cancer models. However, there is no record in the literature of studies reporting these effects in models of bladder cancer. Thus, this study aimed to investigate the cytotoxic and pro-apoptotic potential of [6]-Shogaol against murine bladder cancer urothelial cells (MB49). METHODS: The cytotoxic effects of [6]-Shogaol on cell viability (MTT method), cell morphology (light microscopy), alteration of proliferative processes (clonogenic assay), oxidative stress pathway (levels of reactive oxygen species) and the induction of apoptotic events (flow cytometry and high-resolution epifluorescence imaging) were evaluated in murine urothelial bladder cancer cell lines (MB49), relative to non-tumor murine fibroblasts (L929). RESULTS: The results showed that [6]-Shogaol was able to induce concentration-dependent cytotoxic effects, which compromised cell viability, exhibiting an inhibitory concentration of 50% of cells (IC50) of 146.8 µM for MB49 tumor cells and 236.0 µM for L929 non-tumor fibroblasts. In addition to inhibiting and altering the proliferative processes if colony formation, it presented pro-apoptotic activity identified through a quantitative analysis and the observation of apoptotic phenotypes, events apparently mediated by the induction of nuclear fragmentation. CONCLUSION: The data presented suggest that [6]-Shogaol has a higher concentration-dependent cytotoxic and apoptosis-inducing potential in MB49 cells than in L929 fibroblasts. These results may contribute to the development of therapeutic alternatives for bladder cancer.
BACKGROUND/AIMS: Nitric oxide (NO) plays a dual role, acting as both an oxidant and a reducer, with various effects depending on its concentration and environment. Acute kidney injury's (AKI) pathogenesis observed in car...BACKGROUND/AIMS: Nitric oxide (NO) plays a dual role, acting as both an oxidant and a reducer, with various effects depending on its concentration and environment. Acute kidney injury's (AKI) pathogenesis observed in cardiorenal syndrome 3 (CRS 3) involves inflammatory responses and the production of reactive oxygen and nitrogen species. However, the role of NO on the development of CRS 3 is still not completely understood. The study aimed to mimic CRS 3 in vitro and investigate NO signaling and inflammatory molecules. METHODS: Thus, HEK293 cells were submitted to normoxia (NX) or hypoxia (HX) protocols for 16 h followed by 3 h of reoxygenation, treated or not with L-NAME. Conditionate medium by HEK293 was transferred to H9c2 for 24 h. Cellular viability was evaluated by MTT assay, real time PCR was used to analyze gene expression and NO content were evaluated in the intra and extracellular medium by amperimetry. RESULTS: Carbonic anhydrase 9 (CA9) expression increased 2.9-fold after hypoxia. Hypoxia reduced 18 % cell viability in HEK293 that was restored by L-NAME treatment. The sum of nitrite (NO2-) and S-nitrosothiol (S-NO) fractions in HEK293 cells showed a substantial decrease on NO intracellular content (38 %). Both IL-6 and IL-10 decreased in all groups compared to NX cells. Besides TNF-α and Bax/Bcl2 ratio increased in hypoxia (approximately 120-fold and 600-fold, respectively) and L-NAME restored this effect. Regarding H9c2 cells, the S-NO fractions showed a substantial decrease in extracellular content after HX (17%) that was not restored by L-NAME. IL-1β decreases in cardiac cells treated with conditioned medium from HX/L-NAME. CONCLUSION: In conclusion this study highlights the complex interplay of NO and inflammatory factors in hypoxia-induced renal and cardiac cell responses, with potential implications for cardiorenal syndrome.
BACKGROUND/AIMS: Ischemic reperfusion (I-R) injury is greatly influenced by the testicular torsion/detorsion process (TDP). In this instance, the anti-inflammatory properties of plateletrich plasma (PRP) combined with ta...BACKGROUND/AIMS: Ischemic reperfusion (I-R) injury is greatly influenced by the testicular torsion/detorsion process (TDP). In this instance, the anti-inflammatory properties of plateletrich plasma (PRP) combined with tadalafil (Td) significantly promote tissue healing in the I-R injury model. METHODS: Five groups of rats were created: the control group, the I-R group not receiving any therapy, the I-R group receiving a single dosage of Td (0.25 mg/kg, I.P.), the I-R group receiving a single dose of PRP (80 l, intratesticular), and the I-R group receiving both Td and PRP. Sperm morphology, motility, and histology were assessed. The levels of TNF-, BAX, antioxidant status, and testosterone were measured. Additionally, E-selectin expression was done. RESULTS: PRP reduced oxidative stress, inflammation, and apoptosis while also boosting testosterone levels, which alleviated I-R injury. Otherwise, PRP reduces E-selectin expression, which modifies the pathways that control endothelial function. Td also partially demonstrated its testicular-protective activity at the same time. CONCLUSION: PRP's proven anti-inflammatory, antioxidant, and antiapoptotic potentials make it a natural treatment for testicular harm caused by tadalafil. For the first time, it was demonstrated that PRP therapy restored the functionality of the vascular endothelium, specifically the control of E-selectin expression. Combining Td and PRP therapy may be a promising strategy for improving response to PDE5 inhibitors.
BACKGROUND/AIMS: Factors influencing gene expression through chemical modifications of histones may play an important role in the regulation of the autophagy process in cancers. RING1A or RING1B are responsible for the c...BACKGROUND/AIMS: Factors influencing gene expression through chemical modifications of histones may play an important role in the regulation of the autophagy process in cancers. RING1A or RING1B are responsible for the catalytical activity of Polycomb repressive complex 1 (PRC1) which monoubiquitylate histone H2A. The aim of the study was to determine the effect of the RING1A/B protein inhibition on the autophagy process in endometrial cancer cells and the anticancer effectiveness of RING1 inhibitor PRT4165 in combination with autophagy inhibitors. METHODS: The expression of autophagy genes and proteins were analyzed in endometrial cancer cells HEC-1A and Ishikawa grown in different glucose concentrations and treated with PRT4165. To assess the effectiveness of PRT4165 used alone or in combination with HCQ or Lys05, IC and the combination index (CI) were calculated. Flow cytometry method was used to estimate apoptotic cells after treatment. RESULTS: The results confirm the impact of RINGs on autophagy and apoptosis in endometrial cancer cells. PRT4165 inhibitor causes changes in the expression of ATG genes and autophagy markers and the effect depends on glucose concentration and cell types. However, the anticancer effectiveness of PRT4165 was lower when it was used in combination with autophagy inhibitors, suggesting that such a combination is not a promising anticancer strategy. CONCLUSION: The results indicate the importance of the RINGs in the process of autophagy and apoptosis. Further potentially more effective combinations of PRT4165 with autophagy modulators should be sought.
BACKGROUND/AIMS: Many questions in cancer biology remain unanswered. Perhaps the most important issues remaining to be addressed focus on the molecular basis of carcinogenesis. Today's cancer focus lies on genetics and g...BACKGROUND/AIMS: Many questions in cancer biology remain unanswered. Perhaps the most important issues remaining to be addressed focus on the molecular basis of carcinogenesis. Today's cancer focus lies on genetics and gene expression, which is unlikely to explain the true cause of most cancers or lead to a cure. METHODS: Earlier, we provided a plausible mechanism for this process, specifically, that most cancers develop in response to pathogenic stimuli that induce chronic inflammation, fibrosis, and remodeling of the cellular microenvironment. Collectively, these changes generate a precancerous niche (PCN) in which fibrosis and remodeling are ongoing secondary to persistent inflammation, followed by the deployment of a chronic stress escape strategy (CSES). If the CSES is unsuccessful, the cell undergoes a normal cell to cancer cell transformation (NCCT). RESULTS: Here, we highlight the critical role of fibroblasts as the first cells to undergo neoplastic transformation to a cancerous phenotype which is based on several critical findings. First, persistent disruption of homeostatic crosstalk increases lysyl oxidase activity and lysine oxidation which leads to increased collagen stiffness and decreased elasticity. If unresolved, chronic tissue stress will lead to an escape strategy that involves the recruitment of fibroblasts and fibrocytes from the bone marrow as well as cells undergoing an epithelial-mesenchymal transition (EMT). This yields a heterogeneous pool of cells that express both epithelial and mesenchymal markers and that will ultimately differentiate into cancer-associated fibroblasts (CAFs). Finally, CAFs undergo a mesenchymalepithelial transition (MET) and express epithelial markers that facilitate their integration into the target tissue. CONCLUSION: Here, we review the published findings that led us to this conclusion which is the most plausible answer to this critical question.
Cell senescence was considered an attribute of normal dividing cells, which distinguishing them from cancer cells that do not have a division limit. However, recent studies show that senescence could also occur in cancer...Cell senescence was considered an attribute of normal dividing cells, which distinguishing them from cancer cells that do not have a division limit. However, recent studies show that senescence could also occur in cancer cells. Cancer cell senescence could occur as a result of chemotherapy, radiation, inhibition of telomerase activity, induction of DNA damage, changes in the tumor microenvironment, regulation of senescence-related proteins, oxidative stress, inflammation, or epigenetic dysregulation. It seems that the induction of senescence in cancer cells could significantly affect the inhibition of tumor progression, but in some types of cancer, it can affect their invasive character. Furthermore, considering the therapeutic implications of this process, it is essential to consider the positive and negative aspects of cancer cell senescence. It is crucial to understand the molecular mechanisms that induce senescence under specific conditions, considering the potential hazards. In the future, the senescence of cancer cells may contribute to using this property in modern cancer treatment strategies.
BACKGROUND/AIMS: All body functions are activated, synchronized and controlled by a substantial, complex network, the nervous system. Upon injury, pathophysiology of the nerve injury proceeds through different paths. The...BACKGROUND/AIMS: All body functions are activated, synchronized and controlled by a substantial, complex network, the nervous system. Upon injury, pathophysiology of the nerve injury proceeds through different paths. The axon may undergo a degenerative retraction from the site of injury for a short distance unless the injury is near to the cell body, in which case it continues to the soma and undergoes retrograde neuronal degeneration. Otherwise, the distal section suffers from Wallerian degeneration, which is marked by axonal swelling, spheroids, and cytoskeleton degeneration. The objective of the study was to evaluate the potential of mesenchymal stem cell laden neural scaffold and insulin-like growth factor I (IGF-I) in nerve regeneration following sciatic nerve injury in a rat model. METHODS: The animals were anaesthetized and a cranio-lateral incision over left thigh was made. Sciatic nerve was exposed and crush injury was introduced for 90 seconds using haemostat at second locking position. The muscle and skin were sutured in routine fashion and thus the rat model of sciatic crush injury was prepared. The animal models were equally distributed into 5 different groups namely A, B, C, D and E and treated with phosphate buffer saline (PBS), carbon nanotubes based neural scaffold only, scaffold with IGF-I, stem cell laden scaffold and stem cell laden scaffold with IGF-I respectively. In vitro scaffold testing was performed. The nerve regeneration was assessed based on physico-neuronal, biochemical, histopathological examination, and relative expression of NRP-1, NRP-2 and GAP-43 and scanning electron microscopy. RESULTS: Sciatic nerve injury model with crush injury produced for 90 seconds was standardized and successfully used in this study. All the biochemical parameters were in normal range in all the groups indicating no scaffold related changes. Physico-neuronal, histopathological, relative gene expression and scanning electron microscopy observations revealed appreciable nerve regeneration in groups E and D, followed by C and B. Restricted to no regeneration was observed in group A. CONCLUSION: Carbon nanotubes based scaffold provided electro-conductivity for proper neuronal regeneration while rat bone marrow-derived mesenchymal stem cells were found to induce axonal sprouting, cellular transformation; whereas IGF-I induced stem cell differentiation, myelin synthesis, angiogenesis and muscle differentiation.
BACKGROUND/AIMS: Currently, it is proven that the cellular metabolism of nitric oxide is necessary to maintain optimal health and adaptation of the organism to the impact of various environmental factors. The aim of this...BACKGROUND/AIMS: Currently, it is proven that the cellular metabolism of nitric oxide is necessary to maintain optimal health and adaptation of the organism to the impact of various environmental factors. The aim of this work was to reveal the biological role of nitric oxide, its metabolic changes, and its mechanism of action in tissues under hypoxia, as well as the possibility of tissue metabolism correction through NO-dependent systems under the influence of Krebs cycle intermediates. METHODS: A systematic assessment of the effect of succinate (SC, 50 mg/kg b.w.) and α-ketoglutarate (KGL, 50 mg/kg b.w.) in the regulation of oxygendependent processes in rats (mitochondrial oxidative phosphorylation, microsomal oxidation, intensity of lipid peroxidation processes, and the state of the antioxidant defense system) depending on functional changes in nitric oxide production during hypoxia was evaluated. The state of the nitric oxide system was estimated spectrophotometrically by determination of the concentration of its stable nitrite anion metabolite (NO -). The levels of catecholamines were estimated from the content of epinephrine and norepinephrine using the differentially fluorescent method. The activity of cytochrome P450-dependent aminopyrine-N-demethylase was determined with the Nash reagent. RESULTS: Tissue hypoxia and metabolic disorders caused by this condition through changes in the content of catecholamines (epinephrine, norepinephrine, dopamine, DOPA) as well as the cholinesterase-related system (acetylcholine content and acetylcholinesterase activity) were the studied experimental parameters under acute hypoxia (AH, 7% O in N, 30 min). The activation of lipid peroxidation and oxidatively modified proteins and an increase in the epinephrine content in AH are associated with an increased role of SC and a decrease in KGL as substrates of oxidation in mitochondria. A more pronounced effect of exogenous KGL, compared to SC, on the content of nitrite anion as a stable metabolite of nitric oxide in the liver under acute hypoxia against the background of a decrease in the intensity of lipid peroxidation processes was revealed. The activation of SC-dependent mitochondrial oxidative processes caused by AH was found to decrease in animals after an intermittent hypoxia training (IHT) course. IHT (7% O in N, 15-min, 5 times daily, 14 days) prevented the activation of oxidative stress in tissues and blood after the AH impact and increased the efficiency of energy-related reactions in the functioning of hepatic mitochondria through increased oxidation of KGL. CONCLUSION: The studied effects of adaptation are mediated by an increase in the role of NO-dependent mechanisms, as assessed by changes in the pool of nitrates, nitrites, carbamides, and total polyamines.
BACKGROUND/AIMS: Microglial cells play a crucial role in the development of neuroinflammation in response to harmful stimuli, such as infection, ischemia or injury. Their chronic activation, however, is associated with a...BACKGROUND/AIMS: Microglial cells play a crucial role in the development of neuroinflammation in response to harmful stimuli, such as infection, ischemia or injury. Their chronic activation, however, is associated with a progression of neurodegenerative diseases. Therefore, looking for potential factors limiting microglial activation, the effect of docosahexaenoic acid (DHA) on the inflammatory response and TREM2-dependent phagocytic activity in microglia was investigated. METHODS: In LPS-induced primary microglia preincubated with DHA, or without preincubation the expression of ATF3 and TREM2 genes and TREM2, Syk, Akt proteins were determined by RT-PCR and WB, respectively. Cell viability was assayed by MTT and cytokine and chemokine expression was determined by the Proteome Profiler assay. Moreover, the phagocytic activity of microglia was assayed using immunofluorescence. RESULTS: We found that DHA significantly increased the expression of ATF3 , and decreased the levels of CINC-1, CINC-2αβ, CINC-3 chemokines, IL-1α and IL-1β cytokines, and ICAM-1 adhesion protein. Additionally, preincubation of microglia with DHA resulted in increased Src/Syk kinases activation associated with increased phagocytic microglia activity. CONCLUSION: These findings indicate that DHA efficiently inhibits ATF3-dependent release of proinflammatory mediators and enhances phagocytic activity of microglia. The study provides a new mechanism of DHA action in reactive microglia, which may help limit neuronal damage caused by the pro-inflammatory milieu in the brain.