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Background/Aim. Cyclosporine (CyA) therapeutic drug monitoring (TDM) through the measurement of drug concentration in blood two hours after the administration (C2), and/or according to the calculated value of the area under the concentration - time curve during the first four hours following administration (AUC0−4) shows favourable correlation with clinical manifestations in patients with kidney transplantation (Tx). The aim of this study was to analyze clinical efficiency and usability of TDM CyA through C2 and AUC0-4 in the group of our kidney transplanted patients during the first 24 months following Tx. Methods. The study included 50 patients who had undergone kidney Tx using living donors at the Clinic of Nephrology Military Medical Academy, from 1996 to 2003. The first group (group C2) consisted of 25 patients in whom CyA dose was adjusted according to the target C2 and AUC0−4 (calculated by the regression formula based on C1, C2 and C3), while the second group (group C0) consisted of 25 "historical" patients in whom the dose of this drug was adjusted according to C0. Results. On the 6th day the average daily dose of CyA in the group C2 was 10.1±0.8 mg/kg, while in the group C0 it was 7.6±1.6 (p < 0.05). One month following the Tx, daily drug doses were quite similar in the two observed groups (6.2 mg/kg in C0 and 6.6 mg/kg in group C2, p = NS). In the group C2, target C2/AUC 0−4 (C2 1 700 ng/ml, AUC0−4 4 400 ng·h/ml) on the sixth day was achieved in 36.3%, and on day 14 in 76% of the patients. The target AUC 0−4, in relation with C2, in each observed time interval was reached in the higher number of patients. Maximum CyA concentrations in the group C2 were registered 2 hours following the administration (C2), when compared with the concentrations registered after the first and the third hour (C1 and C3). In relation with C1 and C3, C2 concentration correlated most favorably with AUC 0−4, both on the 6th (r = 0.85) and on the 9th day (r = 0.87). During the first three months following the Tx, in the group C0, 10 episodes (40%) of acute cell rejection (AR) were registered, while in the group C2, two episodes (8%, p = 0.07) were registered; in the observed period covering the first two years, a total of 13 (52%) AR episodes in the group C0 and 5 AR episodes (20%) in the group C2 (p = 0.03) were registered. All of five episodes of steroid resistant AR were registered in the group C0. In the group C2, all five patients with AR had lower C2 during AR: the average C2 at the moment of AR was 933.8 ng/ml, and in the patients without rejections was 1 364.2 ng/ml (p = 0.008). In the same group, the average C0 at the moment of AR was 263.2 ng/ml, and 240.0 ng/ml (p = 0,486) in the patients without AR. In the C0 group, average C0 concentration at the moment of AR was 227.1 ng/ml, while in the patients without AR it was 227.7 ng/ml (p = 0.95). Totally 68% of the patients showed signs of acute CyA nephrotoxicity during the first year in the group C2, and 52% in the group C0 (p = 0.38). In seven patients (28%) of the group C2 and six patients of the group C0 (24%, p = 0.96) in the first two years following Tx, administration of CyA was interrupted due to nephrotoxicity. Overall graft function was good in both groups during the period of two years. One graft was lost in the group C0 due to chronic allograft nephropathy. The patients in the group C2 had better early and the same late graft function. Five patients in the group C2 who did not reach the target C2/AUC during the first 30 days, did not have more AR or worse graft function, comparing with the patients who reached the target concentrations. Conclusion. In the patients with CyA TDM through with the C2 and AUC0−4., AR frequency was considerably lower, and AR episodes had a milder flow than in those with CyA TDM through the C0. The drug concetration in blood two hours after administration (C2) was a good predictor of acute graft rejection, while C0 failed to point to the patients with the insufficient drug concentration. Higher drug doses were administered in the group C2 during the first month following Tx, and these patients did not show significantly higher frequency of acute nephrotoxicity and more frequent requirement of the drug use interruption. Graft function in both groups was good during the period of two years. CyA dose determination through C2 and AUC0-4 is efficient TDM method, relatively simple for use in day to day clinical practice.

Background/Aim. Any forms of mercury have toxic action on the majority of organs, especially kidneys. The major source of professional exposure to mercury are departments for the production of chlorine which use mercury as catode. The aim of the study was to prove that chronic exposure to elementary mercury low concentrations could cause asymptomatic damages of the kidneys. Methods. A total of 40 workers from the factory "HIP Petrohemija" Pančevo, of the mean age 45±8 years, who were exposed to the effects of mercury for more than 20 years within the production procedure, and 20 workers from the factory "Panonijaplast" Pančevo, of the mean age 44±7 years, who were not exposed to mercury nor to other nephrotoxic agents, were submitted to laboratory analysis, renal function testing, and determination of mercury concentration in urine. Mercury concentration was also measured in the air of working premises of the factory. Results. The performed measurements confirmed that the concentrations of mercury at any tested working place in the Department of Electrolysis were not more than the maximally permitted concentration for an 8-hour exposition. In the exposed group (40 examinees) 75% of the examinees had mercury in urine in the concentration < 0.1 μmol/l, while in 25% of them it was 0.1-0.75 μmol/l. In the control group (20 examinees) all of the examinees showed to have < 0.1 mol/l mercury in urine. There was determined a positive corelation between the concentration of mercury in urine and the value of β2-microglobulin (p < 0.05), as well as between the corrcentration of mercury in urine and γGT activity (p < 0.05), and between the concentration of mercury in urine and the value of retinol-binding protein (p < 0.01). Conclusion. In 25% of the examinees excretion of mercury was significantly higher than in the control group. The frequency of asymptomatic renal tubular lesions and dysfunction of moderate extent were found to be higher in the exposed group than in the control one.

Background/Aim. Idiopathic retroperitoneal fibrosis (IRF) is an uncommon disease characterized by a retroperitoneal fibrotic tissue that often involve the ureters, leading to the obstructive nephropathy and variable impairment of renal function. Findings strongly suggest an autoimmune etiology. Surgery, medical treatment with immunosuppressive drugs, or a combination of both are proposed. The optimal treatment has not been established yet. The aim of this study was to present our experience with combined immunosuppressive therapy of IRF, steroids (S) and mycophenolate mofetil (MMF). Methods. We prospectively followed four patients with IRF from January 2004 to December 2006. Three patients had an active disease with bilateral hydronephrosis. In the two of them acute renal failure was presented, and ureteral catheters were inserted in one in order to manage ureteral obstruction. One patient has came to our unit with a relapse of IRF and incipient chronic renal failure after the prior therapy with ureterolysis and immunosuppressive drugs (azathioprine and tamoxifen). All patients received steroids and MMF. Two patients were treated with intravenous methylprednisolone pulses (250 mg each), for three consecutive days, followed by oral prednisone 0.5 mg/kg/day. The other two patients received oral prednisone at the same dose. Prednisone was gradually tappered to a maintenance dose of 10 mg/kg/day. Simultaneously, all patients received MMF, initially 1 g/day with the increase to 2 g/day. Results. After four weeks of the therapy all symptoms disappeared, as well as a hydronephrosis with a decrease of erythrocyte sedimentation rate and Creactive protein (CRP) to normal level in all patients. Three patients remain in remission untill the end of the follow up. One patient had a relapse because of stopping taking the therapy after six months. He was treated by oral prednisone 0.5 mg/kg/day, which was gradually decreased. After twelve weeks hydronephrosis disappeared and CRP returns to the normal level. Conclusion. The combination of steroids and mycophenolate mofetil led to the remission of IRF with a strong and quick immunosuppressive effect. It also provided avoiding the long-term use of high steroid dose and surgical procedures. .

Background: One of the most important strategies in research and development of new anticancer agents is the tumor-specific induction of apoptosis. The effects of semisynthetic derivative of vitamin E, (α-TOS, D-α-tocopheryl succinate), appear to be largely restricted to malignant cells. Methods: We investigated the in vivo effects of intraperitoneally administered α-TOS on vitality of Ehrlich ascites carcinoma cells (EAC) in mice, as well as the influence of α-TOS on specific activity of enzymes involved in antioxidative mechanisms in EAC cells. Results: According to our results, the intraperitoneal application of α-TOS induces the decrease of the EAC vitality, and the statistically significant alteration of the glutathione-dependent enzyme activity in EAC cells. Conclusion: We may conclude that α-TOS is an important micronutrient, with significant impact on vitality and metabolism of malignant cells.

Background. Multiple myeloma is a plasmaproliferative disease characterized by the uncontrolled proliferation of a pathogenic plasma cell clone engaged in the production of monoclonal immunoglobulin. This condition affects the bone marrow, but it can be manifested in any other organ or tissue. The urinary bladder involvement is extremely rare. Case report. We reported a 70-year-old male with the history of multiple myeloma, receiving chemotherapy containing melphalan and prednisone (MP). Two years after the treatment, there was a renal failure associated with oligoanuria, hematuria and bilateral hydronephrosis. The urine cytology tests revealed the atypical cells, so was suspected obstructive uropathy to be caused by urothelium cancer. However, only upon the cystoscopy and biopsy performed on the urinary bladder mass, plasmacytoid infiltration diagnosis was confirmed. This extremely rare variant was presented throughout the illness period and proved to be resistant to the administered chemotherapy. Conclusion. When renal failure associated with hematuria and bilateral hydronephrosis is presented in a patient with multiple myeloma, this unusual and rare extramedular localization should be also considered.

Background. One of the critical limitations for the administration of the chemotherapy is the toxicity affecting normal tissue. The main target organs for 5-fluorouracil (5-FU) toxicity in humans and experimental animals are the gastrointestinal tract, bone marrow, and skin. The cytotoxic effects of antimetabolite chemotherapy are based on their role as substrates for the same transport processes and enzymes involved in anabolism and catabolism as the natural substrates. The main goal of our study was to analyze the dose-dependent antiproliferative effects of 5-FU on intestinal mucosa, enterotoxic potential of 5-FU in experimental animals and to test possible protective role of glutamine. Methods. In our study, we used Sprague Dawley rats. The control group of rats included 50 animals, while the groups where either 5-fluorouracil (5-FU) alone or 5-FU and glutamine were administered included 200 animals. All experimental animals were further stratified according to the experimental model (25 animals in each of 8 experimental subgroups of animals). The 5-FU was administered by intraperitoneal application in single dose of 0, 100, 200, 300, and 400 mg of 5-FU per kg of body weight. Water solution of 1% glutamine was prepared daily and administered orally, in volume of 200 ml, for 7 days continuously, after the 7th day of 5-FU administration. Experimental animals were sacrificed 7 days after the administration of 5-FU. The isolation of enterocytes was performed according to the method of Kralovansky et al. In cell homogenate obtained by described method, we determined the protein content using the Biuret method and the DNA content using the Burton reagent. The activities of enzymes alkaline phosphatase (ALP), glutathione S-transferase (GST), glutathione reductase (GR), and glutathione peroxidase (GPX) were determined by kinetic method. All paraffin samples of the small intestine were stained by haematoxiline and eosine(HE method). All the experiments were done in duplicate and analyzed by standard statistical methods. All the experiments were done in duplicate and analyzed by standard statistical methods. Results: Our results of enterotoxicity induced by intraperitonealy administered 5-FU showed statistically significant decrease of DNA content in small intestine samples of experimental animals, decrease in activity of intestinal alkaline phosphatase enzyme and the increase in glutathione-dependent enzymes. The glutamine supplementation reduced 5-FU intestinal toxicity. Conclusion: Intestinal alkaline phosphatase is a good marker of the dose-dependent enterotoxicity induced by 5-fluorouracil.

Background/Aim. The efficacy and biocompatibility of hemodialysis have a singnificant impact on dialysis patient morbidity and mortality rate. The aim of our study was to compare the efficacy and biocompatibility of different hemodialysis modalities in our patients. Methods. A total of 55 patients were included in the study, and on the basis of dialysis modality, they were divided in four groups: group I - postdilution on-line hemodiafiltration (n = 15), group II - bicarbonate high-flux polysulphone hemodialysis (n = 15), group III - bicarbonate low-flux polysulphone hemodialysis (n = 15), and groupe IV - bicarbonate cuprophane hemodialysis (n = 10). The efficacy was evaluated on the basis of urea reduction rate (URR), urea Kt/V index and serum β2-microglobuline reduction rate, and the biocompatibility was evaluated on the basis of the leukocyte count fall during the first fifteen minutes of dialysis session, and of the serum C-reactive protein (CRP) level. Results. The highest mean URR was achieved in the group I (70.53 ± 6.49 %), and it was significantly higher in comparison with the average URR in the group IV (54.8 ± 6.35%) (p = 0.001). The average value of urea Kt/V index in the group I (1.48 ± 0.22) was significantly higher in comparison with the average value in the groupe II 1.30 ± 0.22 (p < 0.05), group III (1.05 ± 0.22), and group IV (0.98 + 0.22) (p = 0.001). Serum β2-microglobuline reduction rate was 68.93 ± 8.25% in the group I, and 58.86 ± 7.98% in the groupe II (p = 0.01). During the first 15 minutes of hemodialysis the leukocyte number was decreased by 12.57 ± 9.35% in the group I, 13.61 ± 9.64% in the group II, 18.3 ± 13.24 in the group III and 62.3 ± 15.4 in the group IV, on average. The mean serum level of CRP was 9.4 ± 6.47 mg/l in the group IV, and less than 3.5 mg/l in the group I of the patients (p = 0.001). Conclusion. Postdilution on-line hemodiafiltration in comparison with standard hemodialysis provided the more effective elimination of small and middle uremic toxins molecules and a significantly higher degree of biocompatibility. The patients treated with standard hemodialysis frequently do not achieve the minimal value of urea Kt/V index prescribed by National Kidney Foundation - Dialysis Outcomes Quality Inatiatives standards. These patients also have significantly higher serum CRP values which suggest the state of chronic microinflammation.

BACKGROUND: Rapidly proliferating crypt cells of the intestinal epithelium, the precursors of the mature enterocytes, are extremely sensitive to the effects of cytostatic agents. We investigated the effects of the methotrexate on rat intestinal mucosa in order to get the information on biochemical indicators of intestinal damage. METHODS: Biochemical parameters were investigated in isolated intestinal mucosa of Sprague-Dawley rats, previously treated with methotrexate by intraperitoneal administration. Glutamine was dissolved in water and administered orally. RESULTS: The activity of glutaminase and alkaline phosphatase showed the enzymatic response to different doses of methotrexate. The activity of both enzymes was significantly lower in the mucosa of treated animals, compared to control group. CONCLUSION: Minimal mucosal damage and regeneration time is dose dependent and influenced by the dosage schedule of antitumor therapy.

The author analyses the present situation and the prospects of internal and regional stability and security in the countries of the European South East. He says that we have now some peace and some stability and security in the region. The peace is fragile and more the state of no-war than stable peace. The fact remains that the self-sustaining regional stability remains a good distance away. The sources of regional instability are in uneliminated consequences of the recent wars and in the fact that nowhere in the region the complete discontinuity with the policies, ideologies, way of thinking and institutional set-up inherited from previous regimes has been realized. The region needs the coherent approach and active participation of the international community and its military presence in Bosnia, Kosovo and Macedonia for years to come. Let's not delude ourselves: if the troops were withdrawn today who could tell what would happen tomorrow.

Introduction. Hemorrhagic fever with renal syndrome (HFRS) is acute infective multisystemic disease followed by febrility, hemorrhages and acute renal insufficiency. Bleeding in the anterior pituitary lobe leading to tissue necrosis occurs in acute stage of severe clinical forms of HFRS, while atrophy of the anterior pituitary lobe with diminution of the gland function occurs after recovery stage. Case report. We presented a patient with the development of chronic renal insufficiency and hypopituitarism as complication that had been diagnosed six years after Hantavirus infection. Magnetic resonance of the pituitary gland revealed atrophy and empty sella turcica. Conclusion. Regarding frequency of this viral infection and its endemic character in some parts of our country partial and/or complete loss of pituitary function should be considered during the late stage of HFRS.

The estrogen receptor-α (ER-α) is a key driver of breast cancer (BC) and the ER-antagonist, tamoxifen, is a central pillar of BC treatment. However, cross-talk between ER-α, other hormone and growth factor receptors enables development of de novo resistance to tamoxifen. Herein, we mechanistically dissect the activity of a new class of anti-cancer agents that inhibit multiple growth factor receptors and down-stream signaling for the treatment of ER-positive BC. Using RNA sequencing and comprehensive protein expression analysis, we examined the activity of di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT) and di-2-pyridylketone-4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC), on the expression and activation of hormone and growth factor receptors, co-factors, and key resistance pathways in ER-α-positive BC. DpC differentially regulated 106 estrogen-response genes, and this was linked to decreased mRNA levels of 4 central hormone receptors involved in BC pathogenesis, namely ER, progesterone receptor (PR), androgen receptor (AR), and prolactin receptor (PRL-R). Mechanistic investigation demonstrated that due to DpC and Dp44mT binding metal ions, these agents caused a pronounced decrease in ER-α, AR, PR, and PRL-R protein expression. DpC and Dp44mT also inhibited activation and down-stream signaling of the epidermal growth factor (EGF) family receptors, and expression of co-factors that promote ER-α transcriptional activity, including SRC3, NF-κB p65, and SP1. In vivo, DpC was highly tolerable and effectively inhibited ER-α-positive BC growth. Through bespoke, non-hormonal, multi-modal mechanisms, Dp44mT and DpC decrease the expression of PR, AR, PRL-R, and tyrosine kinases that act with ER-α to promote BC, constituting an innovative therapeutic approach., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Pancreatic cancer (PaC) is a highly aggressive disease, with poor response to current treatments and 5-year survival rates of 10-15%. PaC progression is facilitated by its interaction with the complex and multifaceted tumor microenvironment (TME). In the TME, cancer cells and surrounding stromal cells constantly communicate with each other via the secretion and uptake of factors including cytokines, chemokines, growth factors, metabolites, and extracellular vesicles (EVs), reshaping the landscape of PaC. Recent studies demonstrated that the metastasis suppressor N-myc downstream regulated 1 (NDRG1) not only inhibits oncogenic signaling pathways in PaC cells but also alters the communication between PaC cells and the surrounding stroma. In fact, NDRG1 was found to influence the secretome of PaC cells, alter cancer cell metabolism, and interfere with intracellular trafficking and intercellular communication between PaC cells and surrounding fibroblasts. This review will present recent advancements in understanding the role of NDRG1 in PaC progression, with a focus on how this molecule influences PaC-stroma communication and its potential for re-shaping the PaC TME.

Galectin 3 plays a significant role in the development of chronic renal failure, particularly end-stage renal disease (ESRD). The aim of our study was to investigate the association between Gal-3 and biochemical parameters and primary disease in ESRD patients, by exploring the polymorphisms LGALS3 rs4644, rs4652, and rs11125. A total of 108 ESRD patients and 38 healthy controls were enrolled in the study. Genotyping of LGALS3 gene rs4644, rs4652, and rs11125 polymorphisms was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). By multivariate logistic regression analysis, we found that LGALS3 rs4644 CC and rs4652 AA genotypes were significantly associated with a higher risk for lower hemoglobin, higher level of parathyroid hormone, and also occurrence of diabetes mellitus and arterial hypertension. The CAA haplotype was significantly more common in patients with diabetes, low hemoglobin level, and normal PTH level. It has been observed as well that the ACT haplotype was more common in patients with low glomerular filtration, low PTH, and normal hemoglobin level. We found that the LGALS3 rs4644 and rs4652 gene polymorphism may be involved in the pathogenesis and appearance of complications in ESRD patients and thus could be considered a new genetic risk factor in this population.

Background: We aimed to examine the quality of life of children who engage in regular physical activities., Methods: We conducted a cross-sectional study with intersectional 301 children (182 boys and 119 girls) involved in regular sporting activities and the control group of 100 children (67 boys and 33 girls), not involved in extracurricular sports. Children in both groups were from 8 to 13 years, attending elementary school, and without any associated illnesses. The quality of life (QOL) was assessed using the Quality of Life questionnaire Kidscreen-27 version for parents. For comparison of groups, we used χ2 test, and for comparison of mean values among groups we used ANOVA test., Results: More boys were engaged in sport than girls and the choice of sport is gender-dependent ( P <0.01). Boys preferred collective sports, while girls were more oriented towards individual sports ( P <0.01). A significant statistical difference in the mean values of the socializing dimension was given to children who trained football ( P =0.04) and basketball ( P =0.02). In children engaged in volleyball, a statistical difference in the mean values was observed in all dimensions of the questionnaire. In children who trained water polo, a statistically significant difference in the mean values for dimensions of health ( P <0.01), mood ( P <0.01) and friendship ( P =0.01) was seen., Conclusion: KIDSCREEN scores were significantly higher in the examined group than in the control group, with a statistically significant difference between scores for different sports. Children involved in regular extracurricular physical activities had better QOL., Competing Interests: Conflict of interest All authors declare no support from any organization for the submitted work; no financial relationships with any organizations that might have an interest in the submitted work; no other relationships or activities that could appear to have influenced the submitted work. The authors declare that they have no conflict of interest., (Copyright © 2022 Arsenovic et al. Published by Tehran University of Medical Sciences.)

Background: The purpose of this study was to compare home and office BP in the adjustment of antihypertensive treatment. Methods: This study was an open, prospective, noninterventional, multicenter clinical trial that occurred between July 2019 and February 2020, in 34 cities in the territory of the Republic of Serbia, which monitored 1581 participants for 6 months. Depending on the used blood pressure monitoring method used, all patients were divided into control (office BP monitoring) and experimental (home BP telemonitoring) groups. We collected anamnestic data and data about systolic blood pressure (SP), in mmHg, diastolic blood pressure (DP), in mmHg, and heart rate (HR), in beats/minute, from all patients. Results: SP values were significantly different at baseline, and at the second, third, and fourth visits between the two tested groups. Home and office BP decreased significantly (p < 0.000) during the 6-month follow-up. We observed a statistically significant influence of the presence of diabetes mellitus and dyslipidemia on the dynamics of differences between SP monitoring values. Conclusions: Our study suggests that novel technologies in BP monitoring can be excellent alternatives for BP assessment in hypertensive patients with other cardiovascular risk factors such as diabetes and dyslipidemia.

The aim of the study was to examine the effect of 4-week supplementation of Alixir 400 PROTECT® (Standardized Aronia L. Melanocarpa Extract Extract-SAE) on clinical and biochemical parameters in patients with confirmed metabolic syndrome (MetS). This study was designed as a prospective open-label clinical case-series study with 28 days of follow-up with cases selected and followed during the period from February 1, 2018 to November 2019. The study included 143 male and female patients with MetS who were subjected to SAE. SAE supplementation significantly altered SP, BP as well as HR values. After 2 weeks, CHOL levels significantly decreased in the fMetS-DM group compared to the baseline values in this group, while the LDL levels significantly decreased in the fMetS group. Triglycerides significantly decreased only after 4 weeks of SAE treatment in diabetic groups of patients (fMetS-DM and mMetS-DM) compared to the baseline, while in non-diabetic groups this marker was not significantly altered. Increased polyphenols or SAE consumption is correlated with a positive effect on body weight, total cholesterol, low and high-density lipoproteins, blood pressure and glycemia. Increasing consumption of polyphenol-rich foods could be a promising strategy to reduce cardiovascular risk.

Pancreatic cancer (PaCa) is characterized by dense stroma that hinders treatment efficacy, with pancreatic stellate cells (PSCs) being a major contributor to this stromal barrier and PaCa progression. Activated PSCs release hepatocyte growth factor (HGF) and insulin-like growth factor (IGF-1) that induce PaCa proliferation, metastasis and resistance to chemotherapy. We demonstrate for the first time that the metastasis suppressor, N-myc downstream regulated gene 1 (NDRG1), is a potent inhibitor of the PaCa-PSC cross-talk, leading to inhibition of HGF and IGF-1 signaling. NDRG1 also potently reduced the key driver of PaCa metastasis, namely GLI1, leading to reduced PSC-mediated cell migration. The novel clinically trialed anticancer agent, di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC), which upregulates NDRG1, potently de-sensitized PaCa cells to ligands secreted by activated PSCs. DpC and NDRG1 also inhibited the PaCa-mediated activation of PSCs via inhibition of sonic hedgehog (SHH) signaling. In vivo, DpC markedly reduced PaCa tumor growth and metastasis more avidly than the standard chemotherapy for this disease, gemcitabine. Uniquely, DpC was selectively cytotoxic against PaCa cells, while "re-programming" PSCs to an inactive state, decreasing collagen deposition and desmoplasia. Thus, targeting NDRG1 can effectively break the oncogenic cycle of PaCa-PSC bi-directional cross-talk to overcome PaCa desmoplasia and improve therapeutic outcomes., (© 2021 Federation of American Societies for Experimental Biology.)

Objectives: This study has been aimed at evaluating knowledge, attitudes and behaviour of university students regarding the usage of antibiotics focusing on differences between medical and nonmedical students., Methods: This cross-sectional questionnaire-based study was performed on 800 students of the University of Novi Sad, Serbia., Results: The identified predictors of adequate antibiotic knowledge were enrolled in medical program, higher grade average and appropriate use of antibiotics during last infection. Multivariate regression identified following predictors of self-medication: being medical student (B = 0.715; p < 0.001), more frequent (B = 0.628; p < 0.001) and irregular (B = 0.584; p = 0.001) antibiotic use, a family member engaged in medical profession (B = 0.789; p < 0.001), living in dormitory (B = 0.679; p = 0.007) or rented apartment (B = 0.621; p = 0.002), using antibiotics until symptoms resolved (B = 1.616; p < 0.001) or until the bottle was finished (B = 0.628; p < 0.001) during the last infection., Conclusions: Although a high number of students showed adequate knowledge about antibiotics, numerous misconceptions were recorded, including self-medication. Further educational interventions are necessary to improve their understanding, perceptions as well as their behaviour towards antibiotic use.

N-myc downstream regulated gene-1 (NDRG1) is a potent metastasis suppressor that is regulated by hypoxia, metal ions including iron, the free radical nitric oxide (NO . ), and various stress stimuli. This intriguing molecule exhibits diverse functions in cancer, inhibiting epithelial-mesenchymal transition (EMT), cell migration and angiogenesis by modulation of a plethora of oncogenes via cellular signaling. Thus, pharmacological targeting of NDRG1 signaling in cancer is a promising therapeutic strategy. Of note, novel anti-tumor agents of the di-2-pyridylketone thiosemicarbazone series, which exert the "double punch" mechanism by binding metal ions to form redox-active complexes, have been demonstrated to markedly up-regulate NDRG1 expression in cancer cells. This review describes the mechanisms underlying NDRG1 modulation by the thiosemicarbazones and the diverse effects NDRG1 exerts in cancer. As a major induction mechanism, iron depletion appears critical, with NO . also inducing NDRG1 through its ability to bind iron and generate dinitrosyl-dithiol iron complexes, which are then effluxed from cells. Apart from its potent anti-metastatic role, several studies have reported a pro-oncogenic role of NDRG1 in a number of cancer-types. Hence, it has been suggested that NDRG1 plays pleiotropic roles depending on the cancer-type. The molecular mechanism(s) underlying NDRG1 pleiotropy remain elusive, but are linked to differential regulation of WNT signaling and potentially differential interaction with the tumor suppressor, PTEN. This review discusses NDRG1 induction mechanisms by metal ions and NO . and both the anti- and possible pro-oncogenic functions of NDRG1 in multiple cancer-types and compares the opposite effects this protein exerts on cancer progression., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The androgen receptor (AR) is a major driver of prostate cancer (PCa) and a key therapeutic target for AR inhibitors (ie, Enzalutamide). However, Enzalutamide only inhibits androgen-dependent AR signaling, enabling intrinsic AR activation via androgen-independent pathways, leading to aggressive castration-resistant PCa (CRPC). We investigated the ability of novel anti-cancer agents, Dp44mT and DpC, to overcome androgen resistance. The effect of Dp44mT and DpC on androgen-dependent and independent AR signaling was assessed in androgen-dependent and -independent PCa cells using 2D- and 3D-tissue culture. The clinically trialed DpC was then examined in vivo and compared to Enzalutamide. These agents uniquely promote AR proteasomal degradation and inhibit AR transcription in PCa cells via the upregulation of c-Jun, potently reducing the AR target, prostate-specific antigen (PSA). These agents also inhibited the activation of key molecules in both androgen-dependent and independent AR signaling (ie, EGFR, MAPK, PI3K), which promote CRPC. The clinically trialed DpC also significantly inhibited PCa tumor growth, AR, and PSA expression in vivo, being more potent than Enzalutamide. DpC is a promising candidate for a unique, structurally distinct generation of AR inhibitors that simultaneously target both androgen-dependent and independent arms of AR signaling. No other therapies exhibit such comprehensive and potent AR suppression, which is critical for overcoming the development of androgen resistance., (© 2020 Federation of American Societies for Experimental Biology.)

Objectives: As an important public health concern, antimicrobial resistance (AMR) is related to lack of knowledge among healthcare professionals. Since the Global Action Plan on AMR highlights the importance of training all healthcare professionals, it is essential to focus our attention on the education related to judicious antimicrobial use. The current study was the first attempt in southeastern Europe to quantify the knowledge about antimicrobial usage and biosecurity measure among veterinary students., Methods: This questionnaire-based study was performed between April and May of 2019 on 213 veterinary students of the University of Novi Sad, Serbia and the University of Zagreb, Croatia., Results: Veterinary students appeared to be little aware of antimicrobial use in veterinary medicine contribution to overall AMR since only 56.8% have chosen strong contribution as the answer. Of the students surveyed, only 22.1%/35.7% of them strongly agreed/agreed that the amount of teaching time for pharmacology was about right. Students who denied having good knowledge of the pharmacology of antimicrobials showed higher knowledge about systemic use of antimicrobials in different clinical scenarios (p = 0.002). High importance of some antimicrobials for human medicine was not recognized by surveyed students. Only 8.5% of them identified gentamicin correctly, as first-line therapy. Students expected to graduate later were more likely to identify the importance of rating antimicrobials correctly than those who thought they would graduate earlier (p = 0.002). More than half of students gave correct answer at scenario regarding a dog with recurrent pyoderma by choosing culture and susceptibility (C & S) testing. Our students who think they will graduate sooner have higher knowledge level on C & S testing sample submission for range of clinical scenarios (p = 0.004). Moreover, appropriate use of PPE (personal protective equipment) procedure and biosecurity measure were reported for two thirds of our students in case of only for two clinical scenarios., Conclusion: This study reveals that among veterinary students from Croatia and Serbia improved undergraduate education is needed on the AMR with emphasis on antimicrobial stewardship (AMS) and appropriate biosecurity., Competing Interests: All authors declare that they have no conflict of interest.

Background and Purpose: Breast cancer is the leading cause of death in women worldwide, with resistance to current therapeutic strategies, including tamoxifen, causing major clinical challenges and leading to more aggressive and metastatic disease. To address this, novel strategies that can inhibit the mechanisms responsible for tamoxifen resistance need to be assessed., Experimental Approach: We examined the effect of the novel, clinically-trialled, thiosemicarbazone anti-cancer agent, DpC, and its potential as a combination therapy with the clinically used estrogen receptor (ER) antagonist, tamoxifen, using both tamoxifen-resistant and -sensitive, human breast cancer cells (MDA-MB-453, MDA-MB-231 and MCF-7) in 2D and 3D cell-culture. Synergy was assessed using the Chou-Talalay method. The molecular and anti-proliferative effects of these agents and their combination was examined via Western blot, immunofluorescence and colony formation assays., Key Results: Combinations of tamoxifen with DpC were highly synergistic, leading to potent inhibition of cell proliferation, colony formation, and ER-α transcriptional activity. The combination also more efficiently reduced major molecular drivers of proliferation of tamoxifen-resistant cells, including c-Myc, cyclin D1, and p-AKT, while up-regulating the cell cycle inhibitor, p27, and inhibiting oncogenic phosphorylation of ER-α at Ser167. Assessing these effects using 3D cell culture further confirmed the greater effects of DpC combined with tamoxifen in reducing ER-α expression, and that of the proliferation marker, Ki-67, in both tamoxifen-sensitive and -resistant MCF-7 spheroids., Conclusions and Implications: These studies demonstrate that the synergistic combination of DpC with tamoxifen could be a promising new therapeutic strategy to overcome tamoxifen resistance in ER-positive breast cancer., (© 2020 The British Pharmacological Society.)

Considering the role of proto-oncogene c-Met (c-Met) in oncogenesis, we examined the effects of the metastasis suppressor, N-myc downstream-regulated gene-1 (NDRG1), and two NDRG1-inducing thiosemicarbazone-based agents, Dp44mT and DpC, on c-Met expression in DU145 and Huh7 cells. NDRG1 silencing without Dp44mT and DpC up-regulated c-Met expression, demonstrating that NDRG1 modulates c-Met levels. Dp44mT and DpC up-regulated NDRG1 by an iron-dependent mechanism and decreased c-Met levels, c-Met phosphorylation, and phosphorylation of its downstream effector, GRB2-associated binding protein 1 (GAB1). However, incubation with Dp44mT and DpC after NDRG1 silencing or silencing of the receptor tyrosine kinase inhibitor, mitogen-inducible gene 6 ( MIG6 ), decreased c-Met and its phosphorylation, suggesting NDRG1- and MIG6-independent mechanism(s). Lysosomal inhibitors rescued the Dp44mT- and DpC-mediated c-Met down-regulation in DU145 cells. Confocal microscopy revealed that lysosomotropic agents and the thiosemicarbazones significantly increased co-localization between c-Met and lysosomal-associated membrane protein 2 (LAMP2). Moreover, generation of c-Met C-terminal fragment (CTF) and its intracellular domain (ICD) suggested metalloprotease-mediated cleavage. In fact, Dp44mT increased c-Met CTF while decreasing the ICD. Dp44mT and a γ-secretase inhibitor increased cellular c-Met CTF levels, suggesting that Dp44mT induces c-Met CTF levels by increasing metalloprotease activity. The broad metalloprotease inhibitors, EDTA and batimastat, partially prevented Dp44mT-mediated down-regulation of c-Met. In contrast, the ADAM inhibitor, TIMP metallopeptidase inhibitor 3 (TIMP-3), had no such effect, suggesting c-Met cleavage by another metalloprotease. Notably, Dp44mT did not induce extracellular c-Met shedding that could decrease c-Met levels. In summary, the thiosemicarbazones Dp44mT and DpC effectively inhibit oncogenic c-Met through lysosomal degradation and metalloprotease-mediated cleavage., (© 2020 Park et al.)

Although primary prostate cancer is largely curable, progression to metastatic disease is associated with very poor prognosis. E6AP is an E3 ubiquitin ligase and a transcriptional co-factor involved in normal prostate development. E6AP drives prostate cancer when overexpressed. Our study exposed a role for E6AP in the promotion of metastatic phenotype in prostate cells. We revealed that elevated levels of E6AP in primary prostate cancer correlate with regional metastasis and demonstrated that E6AP promotes acquisition of mesenchymal features, migration potential, and ability for anchorage-independent growth. We identified the metastasis suppressor NDRG1 as a target of E6AP and showed it is key in E6AP induction of mesenchymal phenotype. We showed that treatment of prostate cancer cells with pharmacological agents upregulated NDRG1 expression suppressed E6AP-induced cell migration. We propose that the E6AP-NDRG1 axis is an attractive therapeutic target for the treatment of E6AP-driven metastatic prostate cancer., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Risk behaviors among young people are a major social and public health issue. This study aims to assess the impact of a life-skills-based prevention program (called Mission Papillagou) on self-esteem, well-being, and risk behaviors among adolescents., Method: In a two-arm controlled study involving 520 school pupils aged between 10 and 15 years old, participants taking part in the prevention program (the Papillagou group) were compared to pupils who did not take part (the control group). Two assessment sessions were performed, one at baseline, and one after either the Mission Papillagou program (Papillagou group) or usual lessons (control group). Participants self-reported on their self-esteem, well-being, behaviors, interests and opinions., Results: The Mission Papillagou program significantly improved Self-Esteem scores (ηρ 2  = .035). Well-being (Cramér's V = .14) and mood ("feeling of depression": Cramér's V = .503; "feeling hopelessness about the future": Cramér's V = .357; "waking up at night": Cramér's V = .343) also improved in the Papillagou group compared to the control group. Regarding risk behaviors, the prevention program produced a decrease in the frequency of insults (Cramér's V = .267) and rumor-spreading (Cramér's V = .440), and a change of opinion toward the possibility of smoking an electronic cigarette in the future (Cramér's V = .372)., Conclusion: This study suggests that life-skills-based risk prevention programs are effective.

A series of eight bis(thiosemicarbazone) ligands and 16 of their respective copper(II) and zinc(II) complexes containing a combination of hydrogen, methyl, pyridyl, phenyl, and/or ethyl substituents at the diimine position of the ligand backbone were synthesized and characterized. The objective of this study was to identify the structure-activity relationships within a series of analogues with different substituents at the diimine position of the backbone and at the terminal N atom. The Cu(II) complexes Cu(GTSM 2 ), Cu(GTSCM), Cu(PyTSM 2 ), Cu(EMTSM 2 ) and Cu(PGTSM 2 ) demonstrated a distorted square planar geometry, while the Zn(II) complexes Zn(ATSM 2 )(DMSO), Zn(PyTSM 2 )(DMSO), and Zn(PGTSM 2 )(H 2 O) formed a distorted square pyramidal geometry. Cyclic voltammetry showed that the Cu(II) complexes display quasi-reversible electrochemistry. Of the agents, Cu(II) glyoxal bis(4,4-dimethyl-3-thiosemicarbazone) [Cu(GTSM 2 )] and Cu(II) diacetyl bis(4,4-dimethyl-3-thiosemicarbazone) [Cu(ATSM 2 )] demonstrated the greatest antiproliferative activity against tumor cells. Substitutions at the diimine position and at the terminal N atom with hydrophobic moieties markedly decreased their antiproliferative activity. Complexation of the bis(thiosemicarbazones) with Zn(II) generally decreased their antiproliferative activity, suggesting the Zn(II) complex did not act as a chaperone to deliver the ligand intracellularly, in contrast to similar bis(thiosemicarbazone) cobalt(III) complexes [King et al. Inorg. Chem. 2017 , 56, 6609-6623]. However, five of the eight bis(thiosemicarbazone) Cu(II) complexes maintained or increased their antiproliferative activity, relative to the ligand alone, and a mechanism of Cu-induced oxidative stress is suggested. Surprisingly, relative to normoxic growth conditions, hypoxia that is found in the tumor microenvironment decreased the antiproliferative efficacy of most bis(thiosemicarbazones) and their copper complexes. This was independent of the potential hypoxia-selectivity mediated by Cu(II/I) redox potentials. These results provide structure-activity relationships useful for the rational design of bis(thiosemicarbazone) anticancer agents.

Competing Interests: The authors report no conflict of interest.

The metastasis suppressor, N-myc downstream-regulated gene-1 (NDRG1), plays multifaceted roles in inhibiting oncogenic signaling and can suppress the epithelial mesenchymal transition (EMT), a key step in metastasis. In this investigation, NDRG1 inhibited the oncogenic effects of transforming growth factor-β (TGF-β) in PANC-1 pancreatic cancer cells, promoting expression and co-localization of E-cadherin and β-catenin at the cell membrane. A similar effect of NDRG1 at supporting E-cadherin and β-catenin co-localization at the cell membrane was also demonstrated for HT-29 colon and CFPAC-1 pancreatic cancer cells. The increase in E-cadherin in PANC-1 cells in response to NDRG1 was mediated by the reduction of three transcriptional repressors of E-cadherin, namely SNAIL, SLUG and ZEB1. To dissect the mechanisms how NDRG1 inhibits nuclear SNAIL, SLUG and ZEB1, we assessed involvement of the nuclear factor-κB (NF-κB) pathway, as its aberrant activation contributes to the EMT. Interestingly, NDRG1 comprehensively inhibited oncogenic NF-κB signaling at multiple sites in this pathway, suppressing NEMO, Iĸĸα and IĸBα expression, as well as reducing the activating phosphorylation of Iĸĸα/β and IĸBα. NDRG1 also reduced the levels, nuclear co-localization and DNA-binding activity of NF-κB p65. Further, Iĸĸα, which integrates NF-κB and TGF-β signaling to upregulate ZEB1, SNAIL and SLUG, was identified as an NDRG1 target. Considering this, therapies targeting NDRG1 could be a new strategy to inhibit metastasis, and as such, we examined novel anticancer agents, namely di-2-pyridylketone thiosemicarbazones, which upregulate NDRG1. These agents downregulated SNAIL, SLUG and ZEB1 in vitro and in vivo using a PANC-1 tumor xenograft model, demonstrating their marked potential., (© The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background: Angiogenesis is essential for tumor growth, proliferation and metastasis. Tumor-related angiogenesis is complex and involves multiple signaling pathways. Controlling angiogenesis is a promising strategy for limiting cancer progression., Scope of Review: Several receptor tyrosine kinases influence the angiogenic response via multiple signaling molecules and pathways. Understanding the functional interaction of kinases in the angiogenic process and development of resistance to kinase inhibition is essential for future successful therapeutic strategies., Major Conclusions: Strategies that target receptor tyrosine kinases and other tumor microenvironment factors simultaneously, or sequentially, are required for achieving an efficient and robust anti-angiogenic response., General Significance: Understanding the molecular mechanism of angiogenesis has improved, and has led, to the clinical development and approval of anti-angiogenic drugs. While many patients have benefited from these agents, their limited efficacy and the development of resistance remains a challenge. This review highlights current therapies and challenges associated with targeting angiogenesis in cancer., (Copyright © 2019 Elsevier B.V. All rights reserved.)

N-myc downstream regulated gene 1 (NDRG1) is an intriguing metastasis suppressor protein, which plays an important role in suppressing multiple oncogenic signaling pathways. Interestingly, multiple isoforms of NDRG1 have been identified, although the molecular mechanisms involved in their generation remains elusive. Herein, we demonstrate the role of two mechanisms involving autophagic and proteasomal machinery as part of an intricate system to generate different NDRG1 isoforms. Examining multiple pancreatic cancer cell-types using immunoblotting demonstrated three major isoforms of NDRG1 at approximately 41-, 46- and 47-kDa. The top NDRG1 band at 47-kDa was shown to be processed by the proteasome, followed by autophagic metabolism of the middle NDRG1 band at 46-kDa. The role of the proteasomal and autophagic pathways in NDRG1 processing was further confirmed by co-localization analysis of confocal images using PSMD9 and LC3 as classical markers of these respective pathways. All NDRG1 isoforms were demonstrated to be, at least in part, phosphorylated forms of the protein. Inhibition of two well-characterized upstream kinases of NDRG1, namely GSK3β and SGK1, resulted in decreased levels of the top NDRG1 band. Studies demonstrated that inhibition of GSK3β decreased levels of the top 47-kDa NDRG1 band, independent of its kinase activity, and this effect was not mediated via the proteasomal pathway. In contrast, the decrease in the top NDRG1 band at 47-kDa after SGK1 inhibition, was due to suppression of its kinase activity. Overall, these studies elucidated the complex and intricate regulatory pathways involving both proteasomal and autophagic processing of the metastasis suppressor protein, NDRG1., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The metastasis suppressor, N-Myc downstream-regulated gene-1 (NDRG1) inhibits a plethora of oncogenic signaling pathways by down-regulating the epidermal growth factor receptor (EGFR). Herein, we examined the mechanism involved in NDRG1-mediated EGFR down-regulation. NDRG1 overexpression potently increased the levels of mitogen-inducible gene 6 (MIG6), which inhibits EGFR and facilitates its lysosomal processing and degradation. Conversely, silencing NDRG1 in multiple human cancer cell types decreased MIG6 expression, demonstrating the regulatory role of NDRG1. Further, NDRG1 overexpression facilitated MIG6-EGFR association in the cytoplasm, possibly explaining the significantly ( p <0.001) increased half-life of MIG6 from 1.6 ± 0.2 h under control conditions to 7.9 ± 0.4 h after NDRG1 overexpression. The increased MIG6 levels enhanced EGFR co-localization with the late endosome/lysosomal marker, lysosomal-associated membrane protein 2 (LAMP2). An increase in EGFR levels after MIG6 silencing was particularly apparent when NDRG1 was overexpressed, suggesting a role for MIG6 in NDRG1-mediated down-regulation of EGFR. Silencing phosphatase and tensin homolog ( PTEN ), which facilitates early to late endosome maturation, decreased MIG6, and also increased EGFR levels in both the presence and absence of NDRG1 overexpression. These results suggest a role for PTEN in regulating MIG6 expression. Anti-tumor drugs of the di-2-pyridylketone thiosemicarbazone class that activate NDRG1 expression also potently increased MIG6 and induced its cytosolic co-localization with NDRG1. This was accompanied by a decrease in activated and total EGFR levels and its redistribution to late endosomes/lysosomes. In conclusion, NDRG1 promotes EGFR down-regulation through the EGFR inhibitor MIG6, which leads to late endosomal/lysosomal processing of EGFR., (© 2019 Menezes et al.)

Significance: Nuclear factor kappa B (NF-κB) signaling is essential under physiologically relevant conditions. However, aberrant activation of this pathway plays a pertinent role in tumorigenesis and contributes to resistance. Recent Advances: The importance of the NF-κB pathway means that its targeting must be specific to avoid side effects. For many currently used therapeutics and those under development, the ability to generate reactive oxygen species (ROS) is a promising strategy., Critical Issues: As cancer cells exhibit greater ROS levels than their normal counterparts, they are more sensitive to additional ROS, which may be a potential therapeutic niche. It is known that ROS are involved in (i) the activation of NF-κB signaling, when in sublethal amounts; and (ii) high levels induce cytotoxicity resulting in apoptosis. Indeed, ROS-induced cytotoxicity is valuable for its capabilities in killing cancer cells, but establishing the potency of ROS for effective inhibition of NF-κB signaling is necessary. Indeed, some cancer treatments, currently used, activate NF-κB and may stimulate oncogenesis and confer resistance., Future Directions: Thus, combinatorial approaches using ROS-generating agents alongside conventional therapeutics may prove an effective tactic to reduce NF-κB activity to kill cancer cells. One strategy is the use of thiosemicarbazones, which form redox-active metal complexes that generate high ROS levels to deliver potent antitumor activity. These agents also upregulate the metastasis suppressor, N-myc downstream regulated gene 1 (NDRG1), which functions as an NF-κB signaling inhibitor. It is proposed that targeting NF-κB signaling may proffer a new therapeutic niche to improve the efficacy of anticancer regimens.

Iron (Fe) has become an important target for the development of anti-cancer therapeutics with a number of Fe chelators entering human clinical trials for advanced and resistant cancer. An important aspect of the activity of these compounds is their multiple molecular targets, including those that play roles in arresting the cell cycle, such as the cyclin-dependent kinase inhibitor, p21. At present, the exact mechanism by which Fe chelators regulate p21 expression remains unclear. However, recent studies indicate the ability of chelators to up-regulate p21 at the mRNA level was dependent on the chelator and cell-type investigated. Analysis of the p21 promoter identified that the Sp1-3-binding site played a significant role in the activation of p21 transcription by Fe chelators. Furthermore, there was increased Sp1/ER-α and Sp1/c-Jun complex formation in melanoma cells, suggesting these complexes were involved in p21 promoter activation. Elucidating the mechanisms involved in the regulation of p21 expression in response to Fe chelator treatment in neoplastic cells will further clarify how these agents achieve their anti-tumor activity. It will also enhance our understanding of the complex roles p21 may play in neoplastic cells and lead to the development of more effective and specific anti-cancer therapies., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Metals are vital cellular elements necessary for multiple indispensable biological processes of living organisms, including energy transduction and cell proliferation. Interestingly, alterations in metal levels and also changes in the expression of proteins involved in metal metabolism have been demonstrated in a variety of cancers. Considering this and the important role of metals for cell growth, the development of drugs that sequester metals has become an attractive target for the development of novel anti-cancer agents. Interest in this field has surged with the design and development of new generations of chelators of the thiosemicarbazone class. These ligands have shown potent anticancer and anti-metastatic activity in vitro and in vivo. Due to their efficacy and safe toxicological assessment, some of these agents have recently entered multi-center clinical trials as therapeutics for advanced and resistant tumors. This review highlights the role and changes in homeostasis of metals in cancer and emphasizes the pre-clinical development and clinical assessment of metal ion-binding agents, namely, thiosemicarbazones, as antitumor agents., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

The metastasis suppressor, N-myc downstream regulated gene-1 (NDRG1), exhibits pleiotropic activity, inhibiting metastasis of various tumor-types, while being correlated with metastasis in others. Notably, NDRG1 phosphorylation and cleavage are associated with its function, although it is unclear if these modifications occur universally, or selectively, in different cancer cell-types and if it contributes to its pleiotropy. Considering the suggested DNA repair role of nuclear NDRG1, the effects of the above post-translational modifications on its nuclear localization was examined. Herein, the full-length (FL) and truncated (T) NDRG1 isoforms were detected using a C-terminus-directed antibody, while only the FL isoform was identified using an N-terminus-directed antibody. For the first time, we demonstrate that the expression of the NDRG1 FL and T forms occurs in all cancer cell-types examined, as does its phosphorylation (p-NDRG1) at Ser330 and Thr346. The FL isoform localized highly in the nucleus compared to the T isoform. Moreover, p-NDRG1 (Ser330) was also markedly localized in the nucleus, while p-NDRG1 (Thr346) was predominantly cytoplasmic in all cell-types. These results indicate the N-terminus region and phosphorylation at Ser330 could be crucial for NDRG1 nuclear localization and function. PTEN silencing indicated that p-NDRG1 (Thr346) could be regulated differentially in different tumor cell-types, indicating PTEN may be involved in the mechanism(s) underlying the pleiotropic activity of NDRG1. Finally, therapeutics of the di-2-pyridylketone thiosemicarbazone class increased nuclear NDRG1 isoforms (FL and T) detected by the C-terminus-directed antibody in HepG2 cells, while having no significant effect in PC3 cells, indicating differential activity depending on the cell-type., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Detailed shape analysis of cells is important to better understand the physiological mechanisms of toxins and determine their effects on cell morphology. This study aimed to develop a procedure for accurate morphological analysis of cell shape and use it as a tool to estimate toxin activity. With the aim of optimizing the method of cell morphology analysis, we determined the influence of ostreolysin A and pleurotolysin B complex (OlyA/PlyB) on the morphology of murine neuronal NG108-15 cells. A computational method was introduced and successfully applied to quantify morphological attributes of the NG108-15 cell line before and after 30 and 60 min exposure to OlyA/PlyB using confocal microscopy. The modified circularity measure [Formula: see text] for shape analysis was applied, which defines the degree to which the shape of the neuron differs from a perfect circle. It enables better detection of small changes in the shape of cells, making the outcome easily detectable numerically. Additionally, we analyzed the influence of OlyA/PlyB on the cell area, allowing us to detect the cells with blebs. This is important because the formation of plasma membrane protrusions such as blebs often reflects cell injury that leads to necrotic cell death. In summary, we offer a novel analytical method of neuronal cell shape analysis and its correlation with the toxic effects of the pore-forming OlyA/PlyB toxin in situ.

Background: The cyclin-dependent kinase inhibitor, p21, is well known for its role in cell cycle arrest. Novel anti-cancer agents that deplete iron pools demonstrate marked anti-tumor activity and are also active in regulating p21 expression. These agents induce p21 mRNA levels independently of the tumor suppressor, p53, and differentially regulate p21 protein expression depending on the cell-type. Several chelators, including an analogue of the potent anti-tumor agent, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT), have entered clinical trials, and thus, their molecular mechanism of action is crucial to assess. Hence, this investigation examined how several iron chelators transcriptionally regulate p21., Methods: Promoter-deletion constructs; luciferase assays; RT-PCR; western analysis; gene silencing; co-immunoprecipitation., Results: The transcriptional regulation of the p21 promoter by iron chelators was demonstrated to be dependent on the chelator and cell-type examined. The potent anti-cancer chelator, Dp44mT, induced p21 promoter activity in SK-MEL-28 melanoma cells, but not in MCF-7 breast cancer cells. Further analysis of the p21 promoter identified a 50-bp region between -104 and -56-bp that was required for Dp44mT-induced activation in SK-MEL-28 cells. This region contained several Sp1-binding sites and mutational analysis of this region revealed the Sp1-3-binding site played a significant role in Dp44mT-induced activation of p21. Further, co-immunoprecipitation demonstrated that Dp44mT induced a marked increase in the interactions between Sp1 and the transcription factors, estrogen receptor-α and c-Jun., Conclusions and General Significance: Dp44mT-induced p21 promoter activation via the Sp1-3-binding site and increased Sp1/ER-α and Sp1/c-Jun complex formation in SK-MEL-28 cells, suggesting these complexes were involved in p21 promoter activation., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The iron-regulated metastasis suppressor N-myc downstream-regulated gene 1 (NDRG1) has been shown to inhibit numerous oncogenic signaling pathways in cancer cells. Recent findings have demonstrated that NDRG1 inhibits the ErbB family of receptors, which function as key inducers of carcinogenesis. NDRG1 attenuates ErbB signaling by inhibiting formation of epidermal growth factor receptor (EGFR)/human epidermal growth factor receptor 2 (HER2) and HER2/HER3 heterodimers and by down-regulating EGFR via a mechanism involving its degradation. Understanding the complex interplay between NDRG1, iron, and ErbB signaling is vital for identifying novel, more effective targets for cancer therapy., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Tumor necrosis factor α (TNF α ) plays a vital role in cancer progression as it is associated with inflammation and promotion of cancer angiogenesis and metastasis. The effects of TNF α are mediated by its downstream target, the oncogene lysine-rich CEACAM1 coisolated protein (LYRIC, also known as metadherin or astrocyte elevated gene-1). LYRIC plays an important role in activating the nuclear factor-ĸB (NF- κ B) signaling pathway, which controls multiple cellular processes, including proliferation, apoptosis, migration, etc. In contrast, the metastasis suppressor N-myc downstream regulated gene 1 (NDRG1) has the opposite effect on the NF- κ B pathway, being able to inhibit NF- κ B activation and reduce angiogenesis, proliferation, migration, and cancer cell invasion. These potent anticancer properties make NDRG1 an ideal therapeutic target. Indeed, a novel class of thiosemicarbazone anticancer agents that target this molecule has been developed; the lead agent, di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone, has recently entered clinical trials for advanced and resistant cancers. To further elucidate the interaction between NDRG1 and oncogenic signaling, this study for the first time assessed the effects of NDRG1 on the tumorigenic properties of TNF α and its downstream target, LYRIC. We have demonstrated that NDRG1 inhibits the TNF α -mediated epithelial-to-mesenchymal transition. Further, NDRG1 also potently inhibited LYRIC expression, with a negative feedback loop existing between these two molecules. Examining the mechanism involved, we demonstrated that NDRG1 inhibited phosphatidylinositol 3-kinase/AKT signaling, leading to reduced levels of the LYRIC transcriptional activator, c-Myc. Finally, we demonstrated that novel thiosemicarbazones that upregulate NDRG1 also inhibit LYRIC expression, further highlighting their marked potential for cancer treatment., (Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.)

Metastasis is currently the leading cause of cancer related death and is the most feared and difficult to treat outcome for cancer patients. This complex process is regulated by a plethora of signaling pathways and molecules that control cell proliferation, invasion, motility and adhesion. Many of these vital processes that enable metastasis to occur are influenced by metals, which play crucial roles in the function of numerous proteins and enzymes. Importantly, an excess of essential metals such as iron and copper is often associated with carcinogenesis and metastatic disease. As such, metals have emerged as promising and viable therapeutic targets for a new generation of anti-cancer and anti-metastatic agents. Further, the unique properties of metals, including their abilities to redox cycle or to mimic other metals, can also be utilized to more effectively target aggressive and metastatic cancer cells. This review will provide an overview of the role that metals play in the metastatic progression of cancer and the development of novel therapies that either target or utilize metal ions as part of their mechanism of action to inhibit metastasis., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Nitric oxide (NO) is integral to macrophage cytotoxicity against tumors due to its ability to induce iron release from cancer cells. However, the mechanism for how activated macrophages protect themselves from endogenous NO remains unknown. We previously demonstrated by using tumor cells that glutathione S-transferase P1 (GSTP1) sequesters NO as dinitrosyl-dithiol iron complexes (DNICs) and inhibits NO-mediated iron release from cells via the transporter multidrug resistance protein 1 (MRP1/ABCC1). These prior studies also showed that MRP1 and GSTP1 protect tumor cells against NO cytotoxicity, which parallels their roles in defending cancer cells from cytotoxic drugs. Considering this, and because GSTP1 and MRP1 are up-regulated during macrophage activation, this investigation examined whether this NO storage/transport system protects macrophages against endogenous NO cytotoxicity in two well characterized macrophage cell types (J774 and RAW 264.7). MRP1 expression markedly increased upon macrophage activation, and the role of MRP1 in NO-induced 59 Fe release was demonstrated by Mrp1 siRNA and the MRP1 inhibitor, MK571, which inhibited NO-mediated iron efflux. Furthermore, Mrp1 silencing increased DNIC accumulation in macrophages, indicating a role for MRP1 in transporting DNICs out of cells. In addition, macrophage 59 Fe release was enhanced by silencing Gstp1, suggesting GSTP1 was responsible for DNIC binding/storage. Viability studies demonstrated that GSTP1 and MRP1 protect activated macrophages from NO cytotoxicity. This was confirmed by silencing nuclear factor-erythroid 2-related factor 2 (Nrf2), which decreased MRP1 and GSTP1 expression, concomitant with reduced 59 Fe release and macrophage survival. Together, these results demonstrate a mechanism by which macrophages protect themselves against NO cytotoxicity., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Progesterone receptor membrane component 1 (PGRMC1) is a multi-functional protein with a heme-binding moiety related to that of cytochrome b 5 , which is a putative progesterone receptor. The recently solved PGRMC1 structure revealed that heme-binding involves coordination by a tyrosinate ion at Y113, and induces dimerization which is stabilized by hydrophobic stacking of heme on adjacent monomers. Dimerization is required for association with cytochrome P450 (cyP450) enzymes, which mediates chemoresistance to doxorubicin and may be responsible for PGRMC1's anti-apoptotic activity. Here we review the multiple attested involvement of PGRMC1 in diverse functions, including regulation of cytochrome P450, steroidogenesis, vesicle trafficking, progesterone signaling and mitotic spindle and cell cycle regulation. Its wide range of biological functions is attested to particularly by its emerging association with cancer and progesterone-responsive female reproductive tissues. PGRMC1 exhibits all the hallmarks of a higher order nexus signal integration hub protein. It appears capable of acting as a detector that integrates information from kinase/phosphatase pathways with heme and CO levels and probably redox status., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: Neuroblastoma is a relatively common and highly belligerent childhood tumor with poor prognosis by current therapeutic approaches. A novel anti-cancer agent of the di-2-pyridylketone thiosemicarbazone series, namely di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT), demonstrates promising anti-tumor activity. Recently, a second-generation analogue, namely di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC), has entered multi-center clinical trials for the treatment of advanced and resistant tumors. The current aim was to examine if these novel agents were effective against aggressive neuroblastoma in vitro and in vivo and to assess their mechanism of action., Methods: Neuroblastoma cancer cells as well as immortalized normal cells were used to assess the efficacy and selectivity of DpC in vitro. An orthotopic SK-N-LP/Luciferase xenograft model was used in nude mice to assess the efficacy of DpC in vivo. Apoptosis in tumors was confirmed by Annexin V/PI flow cytometry and H&E staining., Results: DpC demonstrated more potent cytotoxicity than Dp44mT against neuroblastoma cells in a dose- and time-dependent manner. DpC significantly increased levels of phosphorylated JNK, neuroglobin, cytoglobin, and cleaved caspase 3 and 9, while decreasing IkBα levels in vitro. The contribution of JNK, NF-ĸB, and caspase signaling/activity to the anti-tumor activity of DpC was verified by selective inhibitors of these pathways. After 3 weeks of treatment, tumor growth in mice was significantly (p < 0.05) reduced by DpC (4 mg/kg/day) given intravenously and the agent was well tolerated. Xenograft tissues showed significantly higher expression of neuroglobin, cytoglobin, caspase 3, and tumor necrosis factor-α (TNFα) levels and a slight decrease in interleukin-10 (IL-10)., Conclusions: DpC was found to be highly potent against neuroblastoma, demonstrating its potential as a novel therapeutic for this disease. The ability of DpC to increase TNFα in tumors could also promote the endogenous immune response to mediate enhanced cancer cell apoptosis.