Kinase inhibitors: 20 years of success and many new challenges and recent trends in their patents.

INTRODUCTION: Protein kinases (PKs) play key roles in cellular signaling and regulation cascades and therefore are listed among the most investigated enzymes with the intent to develop drugs that are able to modulate their catalytic features. Specifically, PKs are involved in chronic diseases of large impact in the society such as cancers and neurodegeneration. Since the approval of Fasudil for the management of cerebral vasospasm, frantic efforts are currently ongoing for the development of selective PK-modulating agents. AREAS COVERED: A selection of the most relevant patents in the European Patent Office for biomedical innovation and/or industrial development covering the years 2020-2023 on PK modulators either of the antibody and small-molecule type is reported. In addition to the examined patents, we also reported the contributions claiming the use of antibody-targeted PKs for lab bench identification kits. EXPERT OPINION: The field of PK modulators for biomedical purposes is particularly crowded with contributions, making it rich in valuable information for the development of potential drugs. An emerging frontier is represented by PK activators that aims to complement the use of PK inhibitors with the final intent of finely adjusting any PK-related disruption responsible for triggering any disease.

Comprehensive analysis of different solvent extracts of Ferula communis L. fruit reveals phenolic compounds and their biological properties via in vitro and in silico assays.

Although giant fennel is recognized as a "superfood" rich in phytochemicals with antioxidant activity, research into the antibacterial properties of its fruits has been relatively limited, compared to studies involving the root and aerial parts of the plant. In this study, seven solvents-acetone, methanol, ethanol, ethyl acetate, chloroform, water, and hexane-were used to extract the chemical constituents of the fruit of giant fennel (Ferula communis), a species of flowering plant in the carrot family Apiaceae. Specific attributes of these extracts were investigated using in silico simulations and in vitro bioassays. High-performance liquid chromatography equipped with a diode-array detector (HPLC-DAD) identified 15 compounds in giant fennel extract, with p-coumaric acid, 3-hydroxybenzoic acid, sinapic acid, and syringic acid being dominant. Among the solvents tested, ethanol demonstrated superior antioxidant activity and phenolic and flavonoid contents. F. communis extracts showed advanced inhibition of gram-negative pathogens (Escherichia coli and Proteus mirabilis) and variable antifungal activity against tested strains. Molecular docking simulations assessed the antioxidative, antibacterial, and antifungal properties of F. communis, facilitating innovative therapeutic development through predicted compound-protein interactions. In conclusion, the results validate the ethnomedicinal use and potential of F. communis. This highlights its significance in natural product research and ethnopharmacology.

Prevalence and association of hyperuricemia with liver function in Saudi Arabia: a large cross-sectional study.

BACKGROUND: Hyperuricemia is linked to an increased risk of various chronic diseases, but data on the prevalence and association of hyperuricemia with liver function in Saudi Arabia are scarce. OBJECTIVES: Evaluate the prevalence, association, and risk measures of hyperuricemia and liver function in the Saudi population. DESIGN: Retrospective, cross-sectional analysis. SETTING: Database on large portion of Saudi population. PATIENTS AND METHODS: Laboratory data, age, and gender of the studied subjects were collected from Al Borg Diagnostics. Subjects were stratified, based on their uric acid (UA) levels, into three groups: hypouricemic, normouricemic, and hyperuricemic. The association of UA with liver enzymes was examined in all three groups. MAIN OUTCOME MEASURES: Association of serum UA levels with alanine transaminase (ALT), aspartate transferase (AST), alkaline phosphatase (ALP), and total bilirubin (TB). SAMPLE SIZE: 13 314 subjects. RESULTS: Our study showed that the prevalence of hyperuricemia in the Saudi population is 17.3% (20.3% in males and 15.1% in females). We also found a positive correlation between ALT, AST, and TB with UA levels. The risk of being hyperuricemic was significantly increased in individuals with elevated ALT, AST, and TB. Individuals with elevated ALT, AST, and total TB had a higher chance of having hyperuricemia than those with normal activity. Notably, ALT, AST, and TB had good discriminating capacity for hyperuricemia. CONCLUSIONS: Hyperuricemia is highly prevalent in the Saudi population and is associated with compromised liver function. However, further studies are needed to elucidate the mechanisms underlying these findings in large prospective cohort studies in different populations. LIMITATIONS: Lack of data on other potential confounding variables.

Effects of unpredictable chronic mild stress on the cellular redox state and mitochondrial energy homeostasis in rat adipose tissue: A comprehensive metabolic study.

Unpredictable chronic mild stress (UCMS) leads to variable metabolic effects. Oxidative stress (OS) of adipose tissue (AT) and mitochondrial energy homeostasis is little investigated. This work studied the effects of UCMS on OS and the antioxidant/redox status in AT and mitochondrial energy homeostasis in rats. Twenty-four male Wistar rats (180-220 g) were divided into two equal groups; the normal control (NC) group and the UCMS group which were exposed to various stresses for 28 days. An indirect calorimetry machine was used to measure volumes of respiratory gases (VO2 & VCO2 ), total energy expenditure (TEE), and food intake (FI). The AT depots were collected, weighed, and used for measuring activities and gene expression of key antioxidant enzymes (GPx1, SOD, CAT, GR, GCL, and GS), OS marker levels including superoxide anion (SA), peroxynitrite radical (PON), nitric oxide (NO), hydrogen peroxide (H2 O2 ), lipid peroxides (LPO), t-protein carbonyl content (PCC), and reduced/oxidized glutathione levels (GSH, GSSG). Additionally, AT mitochondrial fractions were used to determine the activities of the tricarboxylic acid cycle (TCA) cycle enzymes (CS, α-KGDH, ICDH, SDH, MDH), respiratory chain complexes I-III, II-III, IV, the nicotinamide coenzymes NAD+ , NADH, and ATP/ADP levels. Compared with the NC group, the UCMS group showed very significantly increased OS marker levels, lowered antioxidant enzyme activities and gene expression, as well as lowered TCA cycle and respiratory chain activity and NAD+ , NADH, and ATP levels (p < .001 for all comparisons). Besides, the UCMS group had lowered TEE and insignificant FI and weight gain. In conclusion, AT of the UCMS-subjected rats showed a state of disturbed redox balance linked to disrupted energy homeostasis producing augmentation of AT.

A Retrospective Analysis of the Association of Neutrophil-Lymphocyte Ratio (NLR) with Anemia in the Saudi Population.

Background: The link between inflammation and anemia is well established but fluctuations in the emerging inflammatory index, neutrophil-lymphocyte ratio (NLR), in anemic subjects remain ambiguous. The purpose of this study is to address the prevailing knowledge gaps regarding the association of NLR with anemia in the Saudi population. Methods: Laboratory results of NLR, C-reactive protein (CRP), and hemoglobin for 14,261 subjects were obtained from Al Borg Diagnostics and retrospectively analyzed. Means, risk measures, and the diagnostic performance of NLR for anemia were examined in age- and gender-wise comparisons. Results: NLR was significantly elevated in anemic individuals and those with high NLR had a significantly lower Hb concentration. Moreover, elevated NLR was more prevalent in anemic subjects (PR: 1.87, 95% CI: 1.46-2.40, p < 0.0001) and carried a greater risk for the condition (OR: 1.91, 95% CI: 1.47-2.48, p < 0.0001) as did CRP. These observations demonstrated distinct age- and gender-specific patterns. However, both parameters were of no value in the diagnosis of anemia as seen from receiver operating characteristic curves. Conclusions: Altogether, these findings indicate that elevated NLR is associated with anemia, which suggests its usefulness for monitoring rather than diagnosing anemia associated with inflammation in Saudi subjects. Further examination of this association in longitudinal studies is needed.

Evaluation of psychological distress is essential for patients with locally advanced breast cancer prior to neoadjuvant chemotherapy: baseline findings from cohort study.

BACKGROUND: patients with locally advanced breast cancer (LABC) are often treated by neoadjuvant chemotherapy (NACT). This study aims to determine the prevalence of psychological distress and their sociodemographic and clinical factors in patients recently diagnosed with LABC and before NACT. METHODS: A total of 209 LABC patients without metastatic localization were recruited between 2021 and 2022 in the oncology hospital at Fez. A structured questionnaire and the Hospital Anxiety and Depression Scale were used. A descriptive analysis and linear model were performed. RESULTS: a mean age of participants was 47.43 ± 9.45 years. The prevalence of depression, anxiety and psychological distress among participants was 59.62% (95% CI: 52.61-33.34), 47.85% (95% CI: 40.91-54.85), and 65.07 (95% CI: 58.19-71.52) respectively. Depression was associated to age (< 50 years) (AOR = 2.19; 95% CI = 1.13-4.23) and health insurance (AOR = 3.64; 95% CI: 1.18-11.26). Anxiety was associated to age (< 50) (AOR = 2.21; 95% CI: 1.18-4.13) and right breast cancer (AOA = 2.01; 95% CI: 1.11-3.65). Psychological distress was associated to chronic illness (AOR = 2.78; 95% CI: 1.32-5.85) and lymph node status (AOR = 2.39; 95% CI: 1.26-4.57). CONCLUSIONS: Based on the high prevalence of depression and anxiety it appears opportune to treat psychological distress among LABC patient candidates for NACT. Each psychological intervention should take into account sociodemographic and clinical factors found associated in our study. Psychological therapeutic interventions are crucial for LABC patients as early as the time of diagnosis and through subsequent steps in NACT to improve their overall mental health.

Inflammatory Cytokines, Redox Status, and Cardiovascular Diseases Risk after Weight Loss via Bariatric Surgery and Lifestyle Intervention.

Background and Objectives: Obesity is a chronic inflammatory condition and is considered a major risk factor for cardiovascular disease (CVD). The effects of obesity management via sleeve gastrectomy (SG) and lifestyle intervention (LS) on inflammatory cytokines, redox status, and CVD risk were studied in this work. Materials and Methods: A total of 92 participants (18 to 60 years old) with obesity (BMI ≥ 35 kg/m2 were divided into two groups: the bariatric surgery (BS) group (n = 30), and the LS group (n = 62). According to the achievement of 7% weight loss after 6 months, the participants were allocated to either the BS group, the weight loss (WL) group, or the weight resistance (WR) group. Assessments were performed for body composition (by bioelectric impedance), inflammatory markers (by ELISA kits), oxidative stress (OS), antioxidants (by spectrophotometry), and CVD risk (by the Framingham risk score (FRS) and lifetime atherosclerotic cardiovascular disease risk (ASCVD)). Measurements were taken before and after six months of either SG or LS (500 kcal deficit balanced diet, physical activity, and behavioral modification). Results: At the final assessment, only 18 participants in the BS group, 14 participants in the WL group, and 24 participants in the WR group remained. The loss in fat mass (FM) and weight loss were greatest in the BS group (p < 0.0001). Levels of IL-6, TNF-a, MCP-1, CRP, and OS indicators were significantly reduced in the BS and WL groups. The WR group had significant change only in MCP-1 and CRP. Significant reductions in the CVD risk in the WL and BS groups were detected only when using FRS rather than ASCVD. The FM loss correlated inversely with FRS-BMI and ASCVD in the BS group, whereas in the WL group, FM loss correlated only with ASCVD. Conclusions: BS produced superior weight and fat mass loss. However, both BS and LS produced a similar reduction in the inflammatory cytokines, relief of OS indicators, and enhancement of antioxidant capacity, and consequently reduced the CVD risk.

Volatile Constituents in Essential Oil from Leaves of Withania adpressa Coss. Ex Exhibit Potent Antioxidant and Antimicrobial Properties against Clinically-Relevant Pathogens.

Withania adpressa Coss. ex is a plant used in traditional medications. Antioxidant, antibacterial, and antifungal properties of the essential oil from leaves of Withania adpressa Coss ex. (EOW) were investigated. EOW was extracted using a Clevenger apparatus, and its volatile compounds were characterized by GC-MS. Antioxidant potency was determined using DPPH, FRAP, and TAC assays. Antibacterial effects were determined vs. Escherichia coli, Klebsiella pneumonia, Staphylococcus aureus, and Streptococcus pneumonia; while its antifungal efficacy was determined vs. Candida albicans, Aspergillus flavus, Aspergillus niger, and Fusarium oxysporum using the disc diffusion and minimum inhibitory concentration bioassays. A chromatographic analysis showed that EOW contained eight phytochemical compounds constituting 99.14% of the total mass of oil. Caryophyllene (24.74%), Longifolene (21.37%), δ-Cadinene (19.08%), and Carene (14.86%) were predominant compounds in EOW. The concentrations required to inhibit 50% of free radical (IC50) values of antioxidant activities of EOW were 0.031 ± 0.006 mg/mL (DPPH), 0.011 ± 0.003 mg/mL (FRAP), and 846.25 ± 1.07 mg AAE/g (TAC). Inhibition zone diameters of EOW vs. bacteria were 18.11 ± 0.5 mm (E. coli), 17.10 ± 0.42 mm (S. aureus), 12.13 ± 0.31 mm (K. pneumoniae), and 11.09 ± 0.47 mm (S. pneumoniae), while MIC values were 51 ± 3, 47 ± 5, 46 ± 3 and 31 ± 1 µg/mL, respectively. Inhibition zone diameters of EOW vs. fungi were 31.32 ± 1.32, 29.00 ± 1.5, 27.63 ± 2.10, and 24.51 ± s1.07 mm for A. flavus, C. albicans, F. oxysporum, and A. niger, respectively. MIC values were 8.41 ± 0.40, 28.04 ± 0.26, 9.05 ± 0.76, and 22.26 ± 0.55 µg/mL, respectively. Importantly, the highest dose of EOW (1 mg/mL) showed negligible (~5%) cytotoxicity against MCF-12, a normal human epithelial cell line derived from the mammary gland, thus underscoring its wide safety and selectivity against tested microbes. To sum it up, EOW has exhibited promising antioxidant and antimicrobial properties, which suggests potential to abrogate antibiotic resistance.

Extracts of Brocchia cinerea (Delile) Vis Exhibit In Vivo Wound Healing, Anti-Inflammatory and Analgesic Activities, and Other In Vitro Therapeutic Effects.

The plant Brocchia cinerea (Delile) (B. cinerea) has many uses in traditional pharmacology. Aqueous (BCAE) and ethanolic extracts (BCEE) obtained from the aerial parts can be used as an alternative to some synthetic drugs. In vitro, DPPH, FRAP and TAC are three tests used to measure antioxidant efficacy. Antibacterial activities were determined against one Gram positive and two Gram negative strains of bacteria. The analgesic power was evaluated in vivo using the abdominal contortion model in mice, while carrageenan-induced edema in rats was the model chosen for the anti-inflammatory test; wound healing was evaluated in an experimental second degree burn model. The results of the phytochemical analysis showed that BCEE had the greatest content of polyphenols (21.06 mg AGE/g extract), flavonoids (10.43 mg QE/g extract) and tannins (24.05 mg TAE/g extract). HPLC-DAD reveals the high content of gallic acid, quercetin and caffeic acid in extracts. BCEE has a strong antiradical potency against DPPH (IC50 = 0.14 mg/mL) and a medium iron reducing activity (EC50 = 0.24 mg/mL), while BCAE inhibited the growth of the antibiotic resistant bacterium, P. aeruginosa (MIC = 10 mg/mL). BCAE also exhibited significant pharmacological effects and analgesic efficacy (55.81% inhibition 55.64% for the standard used) and the re-epithelialization of wounds, with 96.91% against 98.60% for the standard. These results confirm the validity of the traditional applications of this plant and its potential as a model to develop analogous drugs.

Blood indices of omega-3 and omega-6 polyunsaturated fatty acids are altered in hyperglycemia.

Polyunsaturated fatty acids (PUFAs) may favorably influence the risk and clinical course of diabetes mellitus (DM). In particular, eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and arachidonic acid (AA) alleviate oxidative injury and insulin resistance characteristic of DM. Uncertainty still remains, however, as to the composition and proportions of blood PUFAs in relation to fasting blood glucose levels. This study, thus, aims to examine the patterns of blood PUFA indices in normoglycemic (NG) and hyperglycemic (HG) Saudi subjects. Age, gender, FA profiles, and laboratory records of 143 subjects collected from September 2014 to March 2018 were retrospectively analyzed. Means, prevalence rates, associations, risk measures, and the diagnostic accuracy of PUFAs were determined. HG subjects had significantly lower AA (0.70%, 95% CI: 0.59-0.80% vs 0.46%, 95% CI: 0.38-0.53%) and higher EPA/AA ratio (0.36, 95% CI: 0.30-0.42 vs 0.69, 95% CI: 0.61-0.77). Gender-wise comparisons revealed that ώ-6/ώ-3 ratio was the only PUFA index significantly elevated in HG males (0.36, 95% CI: 0.26-0.45 vs 5.68, 95% CI: 4.98-6.38) while both DHA (2.91%, 95% CI: 2.54-3.29% vs 3.37%, 95% CI: 3.13-3.60%) and ώ-3 index (3.1%, 95% CI: 2.70-3.49% vs 3.63%, 95% CI: 3.38-3.88%) were significantly elevated in HG females. Furthermore, reduced AA and elevated EPA/AA ratio were more prevalent in HG subjects (26.53 vs 28.72 and 30.61 vs 38.29, respectively) and exhibited the highest diagnostic accuracy for HG among all PUFA indices. Altogether, our study revealed that distinct, gender-specific blood PUFA indices are differentially regulated in HG subjects which may be valuable for DM management.

Origanum majorana L. polyphenols: in vivo antiepileptic effect, in silico evaluation of their bioavailability, and interaction with the NMDA receptor.

Introduction: Epilepsy is a chronic brain disease characterized by repeated seizures and caused by excessive glutamate receptor activation. Many plants are traditionally used in the treatment of this disease. This study aimed to evaluate the bioavailability of a polyphenolic extract obtained from Origanum majorana L. (OMP) leaves, as well as its antiepileptic activity and its potential mechanism of action. Methods: We have developed and validated a simple, rapid, and accurate stability-indicating reversed-phase liquid chromatographic method for the simultaneous determination of caffeine and quercetin in rat plasma. The OMP antiepileptic effect was evaluated with pilocarpine-induced seizures, and a docking method was used to determine the possible interaction between caffeic acid and quercetin with the N-methyl-D-aspartate (NMDA) receptor. Results and Discussion: Both compounds tested showed low bioavailability in unchanged form. However, the tested extract showed an anticonvulsant effect due to the considerably delayed onset of seizures in the pilocarpine model at a dose of 100 mg/kg. The molecular docking proved a high-affinity interaction between the caffeic acid and quercetin with the NMDA receptor. Taken together, OLP polyphenols demonstrated good antiepileptic activity, probably due to the interaction of quercetin, caffeic acid, or their metabolites with the NMDA receptor.

Monocyte-Lymphocyte Ratio and Dysglycemia: A Retrospective, Cross-Sectional Study of the Saudi Population.

Background: Abnormalities in fasting blood glucose (FBG) resulting in hypoglycemia (OG), impaired fasting glycemia (IFG), or hyperglycemia (HG) arise from disordered metabolic regulation caused in part by inflammation. To date, there is a dearth of evidence regarding the clinical utility of the monocyte−lymphocyte ratio (MLR), an emerging inflammatory index, in the management of dysglycemia. Methods: This retrospective, cross-sectional study explored MLR fluctuations as a function of glycemic control in 14,173 Saudi subjects. Data collected from 11 August 2014 to 18 July 2020 were retrieved from Al-Borg Medical Laboratories. Medians were compared by Mann−Whitney U or Kruskal−Wallis tests and the prevalence, relative risk (RR), and odds ratio (OR) were calculated. Results: MLR was significantly elevated in IFG (p < 0.0001) and HG (p < 0.05) groups compared to the normoglycemia (NG) group, and individuals with elevated MLR (>0.191) had significantly increased FBG (p < 0.001). The risk of IFG (RR = 1.12, 95% CI: 1.06−1.19, p < 0.0002) and HG (RR = 1.10, 95% CI: 1.01−1.20, p < 0.0216) was significantly increased if MLR was elevated, and individuals with elevated MLR were 1.17 times more likely to have IFG (OR = 1.17, 95% CI: 1.08−1.26, p < 0.0002) and 1.13 times more likely to have HG (OR = 1.13, 95% CI: 1.02−1.24, p < 0.0216). Conclusion: Elevated MLR is correlated with and carries a greater risk for IFG and HG. However, large prospective cohort studies are needed to establish the temporal relationship between MLR and FBG and to examine the prognostic value of this novel marker.

Apoptosis-mediated anti-proliferative activity of Calligonum comosum against human breast cancer cells, and molecular docking of its major polyphenolics to Caspase-3.

Due to poor diagnosis breast cancer in women has emerged as the most common cause of death disease in developing countries. Medicinal plants have been used for thousands of years and can be useful in healthcare, especially in developing countries. Ethanol extracts of leaves of fire bush or arta (Calligonum comosum; EECC), exhibited significant anticancer potencies against two breast cancer cell lines, MCF-7 and MDA 231. These in vitro effects of EECC indicated potential anticancer activities that were determined to be specific since minimal toxicity was recorded against MCF-12, a non-cancerous breast cell line used as a reference. EECC also induced cell cycle arrest in MCF-7 and MDA 231 as revealed by the increased proportions of sub-G1 cells. Fluorescence-activated cell sorter analysis (FACS), utilizing double staining by annexin V-FITC/propidium iodide, revealed that the observed cytotoxic effects were mediated via apoptosis and necrosis. FACS measurement of thegreater in fluorescence intensity, linked with oxidation of DCFH to DCF, revealed that apoptosis was attributable to production of free radicals. EECC-mediated apoptosis was further validated by observation of up-regulation in the "executioner" enzyme, caspase 3. The current findings reveal that EECC exhibits significant, selective cytotoxicity to breast cancer cells, that proceeds via the generation of ROS, which culminates in apoptosis. The anti-proliferative effects of EECC weres further verified by use of a structure-based, virtual screening between its major bioactive polyphenolic constituents and the apoptosis executioner marker enzyme, caspase-3. Based on their glide score values against the active site of caspase 3, some phyto-constituents present in EECC, such as DL-alpha-tocopherol and campesterol, exhibited distinctive, drug-like potential with no predicted toxicity to non-target cells. Taken together, the usefulness of natural phenolic and flavonoid compounds contained in Calligonum comosum were suggested to be potent anticancer agents.

Isolated and Combined Effect of Age and Gender on Neutrophil-Lymphocyte Ratio in the Hyperglycemic Saudi Population.

Inflammation is pivotal to the pathogenesis of diabetes mellitus (DM), but pathological alterations of the neutrophil−lymphocyte ratio (NLR), an emerging inflammatory index in DM management, remains understudied. The aim of this study is to examine the relationship between NLR and glycemic control in the Saudi population. Gender, age, WBC count, and fasting blood glucose (FBG) were obtained from Al-Borg Medical Laboratories for 14,205 subjects. Means, prevalence, risk measures, and the diagnostic accuracy of elevated NLR and hyperglycemia (HG) were evaluated. Subjects with elevated NLR (>3) had significantly higher FBG (105.10 ± 0.33 vs. 114.0 ± 2.81) and NLR was significantly elevated in impaired fasting glycemia (IFG; 1.21 ± 0.01 vs. 1.25 ± 0.01) and HG (1.21 ± 0.01 vs. 1.39 ± 0.02). Elevations of NLR in HG but not in IFG persisted across all age groups except young males and elderly females. The prevalence of elevated NLR in hyperglycemic subjects was 4.12% compared to 2.16% in subjects with normal FBG. HG was more prevalent in subjects with elevated NLR (17.33% vs. 12.46%) who had a relative risk (RR) of 1.68 (95% CI = 1.38−2.06, p < 0.0001) and an odds ratio (OR) of 1.94 (95% CI = 1.48−2.56, p < 0.0001) to be hyperglycemic. Nevertheless, NLR failed to discriminate individuals with normal FBG from those with HG based on ROC curve analysis. Pathological fluctuations in NLR may serve as supportive evidence in DM management.

Biochemical and molecular assessment of selenium forms for the alleviation of oxidative stress in senescent human fibroblasts.

Selenium enhances the cellular antioxidant capacity and alleviates oxidative stress. We investigated the transcriptional and enzymatic activities of selenium-dependent glutathione peroxidase 1 and thioredoxin reductase 1 (TrxR1), and levels of glutathione, hydrogen peroxide, lipid peroxides, and protein carbonyls in primary passage 5 (P5) and senescent passage 25 (P25) and 30 (P30) fibroblasts. Cells were incubated in either standard Dulbecco growth medium (CM1) containing normal plasma selenium levels (0.8 µmol/l), or in CM2, CM3, and CM4 containing 3 µmol/l (5 µmol/l for TrxR1) sodium selenite, L-hydroxyselenomethionine, or Se-methylselenocysteine, respectively. Gene transcripts and activities of both investigated enzymes as well as the levels of reduced glutathione were significantly increased in CM2-, CM3-, and CM4-incubated senescent P25 and P35 cells compared against those incubated in CM1. In congruence, although all oxidative stress parameters including oxidized glutathione were significantly lower in CM2-, CM3-, and CM4-incubated senescent cells compared against those incubated in CM1, such reductions were of significantly higher magnitude in CM3 and CM4 cells compared against those in CM2. In conclusion, organic L-hydroxyselenomethionine and Se-methylselenocysteine are equally more potent at alleviating oxidative stress in senescent cells than inorganic sodium selenite, and thus could be beneficial for use in elderly subjects and those with oxidative stress-associated disease.

Comprehensive investigations of key mitochondrial metabolic changes in senescent human fibroblasts.

There is a paucity of detailed data related to the effect of senescence on the mitochondrial antioxidant capacity and redox state of senescent human cells. Activities of TCA cycle enzymes, respiratory chain complexes, hydrogen peroxide (H2O2), superoxide anions (SA), lipid peroxides (LPO), protein carbonyl content (PCC), thioredoxin reductase 2 (TrxR2), superoxide dismutase 2 (SOD2), glutathione peroxidase 1 (GPx1), glutathione reductase (GR), reduced glutathione (GSH), and oxidized glutathione (GSSG), along with levels of nicotinamide cofactors and ATP content were measured in young and senescent human foreskin fibroblasts. Primary and senescent cultures were biochemically identified by monitoring the augmented cellular activities of key glycolytic enzymes including phosphofructokinase, lactate dehydrogenase, and glycogen phosphorylase, and accumulation of H2O2, SA, LPO, PCC, and GSSG. Citrate synthase, aconitase, α-ketoglutarate dehydrogenase, succinate dehydrogenase, malate dehydrogenase, isocitrate dehydrogenase, and complex I-III, IIIII, and IV activities were significantly diminished in P25 and P35 cells compared to P5 cells. This was accompanied by significant accumulation of mitochondrial H2O2, SA, LPO, and PCC, along with increased transcriptional and enzymatic activities of TrxR2, SOD2, GPx1, and GR. Notably, the GSH/GSSG ratio was significantly reduced whereas NAD+/NADH and NADP+/NADPH ratios were significantly elevated. Metabolic exhaustion was also evident in senescent cells underscored by the severely diminished ATP/ADP ratio. Profound oxidative stress may contribute, at least in part, to senescence pointing at a potential protective role of antioxidants in aging-associated disease.

Physcion Induces Hemolysis and Premature Phosphatidylserine Externalization in Human Erythrocytes.

The prevalence of cancer-associated anemia (CIA) is high, and the mechanisms governing its development remain poorly understood. Eryptosis, the suicidal cell death of red blood cells (RBCs), may account for CIA as it is triggered by clinically approved chemotherapeutics including cisplatin and paclitaxel. Physcion (PSN), an anthraquinone extracted from rhubarb and other plants, has shown great promise as an anticancer agent. However, the potential toxicity of PSN to RBCs remains elusive. RBCs were isolated from heparinized blood, and incubated with 10-100 µM of PSN for 24 h at 37 °C. Hemolysis was photometrically calculated from hemoglobin concentration in the medium at 405 nm, while flow cytometry was employed to investigate cardinal markers of eryptosis. Phosphatidylserine (PS) exposure was detected by Annexin-V-fluorescein isothiocyanate (FITC), intracellular calcium by Fluo4/AM, cellular volume from forward scatter (FSC), and oxidative stress by 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA). PSN induced overt hemolysis at 50 and 100 µM which was not mediated through calcium influx, protein kinase C, casein kinase 1α, or receptor-interacting protein 1. Moreover, PSN caused significant increase in Annexin-V-FITC and Fluo4 fluorescence with no appreciable influence on FSC or DCF values. Accordingly, PSN stimulates premature eryptosis characterized by PS externalization and intracellular calcium overload without cell shrinkage or oxidative damage. In conclusion, this report shows, for the first time, that PSN is cytotoxic to RBCs by inducing hemolysis and programmed cell death which may limit its success as a chemotherapeutic agent.

Stimulation of calcium influx and CK1α by NF-κB antagonist [6]-Gingerol reprograms red blood cell longevity.

Chemotherapy-induced anemia (CIA) is a major obstacle in cancer management. Although the mechanisms governing CIA are poorly understood, recent efforts have identified suicidal erythrocyte (red blood cell, RBC) death as a possible cause of CIA. [6]-Gingerol (GNG), a polyphenol extracted from Zingiber officinale plant, exhibits a wide array of biological activities including antimicrobial, antioxidant, anti-inflammatory, immunomodulatory, and anticancer activities, in vitro and in vivo. However, the potential toxicity of GNG to human RBCs remains unexplored. RBCs from heparinized blood were isolated by centrifugation and exposed to antitumor concentrations (10-100 µM) of GNG for 24 hr at 37°C. Hemolysis was calculated from hemoglobin leakage in the supernatant (λmax  = 405 nm), while cytofluorometric analysis of eryptosis employed Annexin-V-FITC to detect phosphatidylserine (PS) exposure, forward scatter (FSC) to estimate cell volume, Fluo4/AM to measure calcium activity, and H2 DCFDA to assess oxidative stress. Moreover, zVAD(OMe)-FMK, SB203580, necrostatin-2, staurosporin, and D4476 were used to identify signaling pathways responsive to GNG. GNG induced significant hemolysis at 100 µM, independently of extracellular calcium, and increased Annexin-V-FITC fluorescence that was thoroughly abrogated without extracellular calcium. GNG also enhanced Fluo4 fluorescence and reduced FSC, but had no significant effect on DCF fluorescence. Importantly, the presence of D4476 significantly attenuated GNG-induced hemolysis. In conclusion, GNG stimulates premature RBC death characterized by loss of membrane asymmetry, elevated cytosolic calcium, cell shrinkage, and casein kinase 1α activation. Blocking the activity of calcium channels or CK1α may, therefore, ameliorate the toxic effects of GNG on RBCs. PRACTICAL APPLICATIONS: This report presents a safety assessment of GNG as a chemotherapeutic agent and highlights the novel toxicity of GNG to human RBCs. Our findings provide novel insights that may lead to more efficient utilization of GNG in chemotherapy. Specifically, our data revealed the involvement of calcium channels and casein kinase 1α in mediating GNG-induced premature RBC death, and, therefore, inverse agonists or inhibitors of either pathway may be used as pharmaceutical adjuvants to attenuate the toxic effects of GNG.

Synthesis and Bioactivity Assessment of Novel Spiro Pyrazole-Oxindole Congeners Exhibiting Potent and Selective in vitro Anticancer Effects.

The present work aims to design and synthesize novel series of spiro pyrazole-3,3'-oxindoles analogues and investigate their bioactivity as antioxidant and antimicrobial agents, as well as antiproliferative potency against selected human cancerous cell lines (i.e., breast, MCF-7; colon, HCT-116 and liver, HepG-2) relative to healthy noncancerous control skin fibroblast cells (BJ-1). The mechanism of their cytotoxic activity has been also examined by immunoassaying the levels of key anti- and proapoptotic protein markers. The analytical and spectral data of the all synthesized target congeners were compatible with their structures. Synthesized compounds showed diverse moderate to powerful antimicrobial and antioxidant activities. Results of MTT assay revealed that seven synthesized compounds (i.e., 11a, 11b, 12a, 12b, 13b, 13c and 13h) particularly exhibited significant cytotoxicity against the three cancerous cell lines under investigation. Ranges of IC50 values obtained were 5.7-21.3 and 5.8-37.4 µg/mL against HCT-116 and MCF-7, respectively; which is 3.8 and 6.5-fold (based on the least IC50 values) more significant relative to the reference chemotherapeutic drug doxorubicin. In HepG-2 cells, the analogue 13h the highest cytotoxicity with IC50 value of 19.2µg/mL relative to doxorubicin (IC50 = 21.6µg/mL). The observed cytotoxicity was specific to cancerous cells, as evidenced by the minimal toxicity in the noncancerous control skin-fibroblast cells. ELISA results indicated that the observed antiproliferative effect against examined cancer cell lines is mediated via engaging the activation of apoptosis as illustrated by the significant increase in proapoptotic protein markers (p53, bax and caspase-3) and reduction in the antiapoptotic marker bcl-2. Taken together, results of the present study emphasize the potential of spiro pyrazole-oxindole analogues as valuable candidate anticancer agents against human cancer cells.