A practical perspective on how to develop, implement, execute, and reproduce high-resolution respirometry experiments: The physiologist's guide to an Oroboros O2k.

The development of high-resolution respirometry (HRR) has greatly expanded the analytical scope to study mitochondrial respiratory control relative to specific tissue/cell types across various metabolic states. Specifically, the Oroboros Oxygraph 2000 (O2k) is a common tool for measuring rates of mitochondrial respiration and is the focus of this perspective. The O2k platform is amenable for answering numerous bioenergetic questions. However, inherent variability with HRR-derived data, both within and amongst users, can impede progress in bioenergetics research. Therefore, we advocate for several vital considerations when planning and conducting O2k experiments to ultimately enhance transparency and reproducibility across laboratories. In this perspective, we offer guidance for best practices of mitochondrial preparation, protocol selection, and measures to increase reproducibility. The goal of this perspective is to propagate the use of the O2k, enhance reliability and validity for both new and experienced O2k users, and provide a reference for peer reviewers.

Phytochemical compound PB125 attenuates skeletal muscle mitochondrial dysfunction and impaired proteostasis in a model of musculoskeletal decline.

Impaired mitochondrial function and disrupted proteostasis contribute to musculoskeletal dysfunction. However, few interventions simultaneously target these two drivers to prevent musculoskeletal decline. Nuclear factor erythroid 2-related factor 2 (Nrf2) activates a transcriptional programme promoting cytoprotection, metabolism, and proteostasis. We hypothesized daily treatment with a purported Nrf2 activator, PB125, in Hartley guinea pigs, a model of musculoskeletal decline, would attenuate the progression of skeletal muscle mitochondrial dysfunction and impaired proteostasis and preserve musculoskeletal function. We treated 2- and 5-month-old male and female Hartley guinea pigs for 3 and 10 months, respectively, with the phytochemical compound PB125. Longitudinal assessments of voluntary mobility were measured using Any-MazeTM open-field enclosure monitoring. Cumulative skeletal muscle protein synthesis rates were measured using deuterium oxide over the final 30 days of treatment. Mitochondrial oxygen consumption in soleus muscles was measured using high resolution respirometry. In both sexes, PB125 (1) increased electron transfer system capacity; (2) attenuated the disease/age-related decline in coupled and uncoupled mitochondrial respiration; and (3) attenuated declines in protein synthesis in the myofibrillar, mitochondrial and cytosolic subfractions of the soleus. These effects were not associated with statistically significant prolonged maintenance of voluntary mobility in guinea pigs. Collectively, treatment with PB125 contributed to maintenance of skeletal muscle mitochondrial respiration and proteostasis in a pre-clinical model of musculoskeletal decline. Further investigation is necessary to determine if these documented effects of PB125 are also accompanied by slowed progression of other aspects of musculoskeletal dysfunction. KEY POINTS: Aside from exercise, there are no effective interventions for musculoskeletal decline, which begins in the fifth decade of life and contributes to disability and cardiometabolic diseases. Targeting both mitochondrial dysfunction and impaired protein homeostasis (proteostasis), which contribute to the age and disease process, may mitigate the progressive decline in overall musculoskeletal function (e.g. gait, strength). A potential intervention to target disease drivers is to stimulate nuclear factor erythroid 2-related factor 2 (Nrf2) activation, which leads to the transcription of genes responsible for redox homeostasis, proteome maintenance and mitochondrial energetics. Here, we tested a purported phytochemical Nrf2 activator, PB125, to improve mitochondrial function and proteostasis in male and female Hartley guinea pigs, which are a model for musculoskeletal ageing. PB125 improved mitochondrial respiration and attenuated disease- and age-related declines in skeletal muscle protein synthesis, a component of proteostasis, in both male and female Hartley guinea pigs.

SMYD1a protects the heart from ischemic injury by regulating OPA1-mediated cristae remodeling and supercomplex formation.

SMYD1, a striated muscle-specific lysine methyltransferase, was originally shown to play a key role in embryonic cardiac development but more recently we demonstrated that loss of Smyd1 in the murine adult heart leads to cardiac hypertrophy and failure. However, the effects of SMYD1 overexpression in the heart and its molecular function in the cardiomyocyte in response to ischemic stress are unknown. In this study, we show that inducible, cardiomyocyte-specific overexpression of SMYD1a in mice protects the heart from ischemic injury as seen by a > 50% reduction in infarct size and decreased myocyte cell death. We also demonstrate that attenuated pathological remodeling is a result of enhanced mitochondrial respiration efficiency, which is driven by increased mitochondrial cristae formation and stabilization of respiratory chain supercomplexes within the cristae. These morphological changes occur concomitant with increased OPA1 expression, a known driver of cristae morphology and supercomplex formation. Together, these analyses identify OPA1 as a novel downstream target of SMYD1a whereby cardiomyocytes upregulate energy efficiency to dynamically adapt to the energy demands of the cell. In addition, these findings highlight a new epigenetic mechanism by which SMYD1a regulates mitochondrial energetics and functions to protect the heart from ischemic injury.

Structural and Spectroscopic Study of New Copper(II) and Zinc(II) Complexes of Coumarin Oxyacetate Ligands and Determination of Their Antimicrobial Activity.

Tackling antimicrobial resistance is of increasing concern in a post-pandemic world where overuse of antibiotics has increased the threat of another pandemic caused by antimicrobial-resistant pathogens. Derivatives of coumarins, a naturally occurring bioactive compound, and its metal complexes have proven therapeutic potential as antimicrobial agents and in this study a series of copper(II) and zinc(II) complexes of coumarin oxyacetate ligands were synthesised and characterised by spectroscopic techniques (IR, 1H, 13C NMR, UV-Vis) and by X-ray crystallography for two of the zinc complexes. The experimental spectroscopic data were then interpreted on the basis of molecular structure modelling and subsequent spectra simulation using the density functional theory method to identify the coordination mode in solution for the metal ions in the complexes. Interestingly, the solid-state coordination environment of the zinc complexes is in good agreement with the simulated solution state, which has not been the case in our previous studies of these ligands when coordinated to silver(I). Previous studies had indicated excellent antimicrobial activity for Ag(I) analogues of these ligands and related copper and zinc complexes of coumarin-derived ligands, but in this study none of the complexes displayed antimicrobial activity against the clinically relevant methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa and Candida albicans.

EFHD1 ablation inhibits cardiac mitoflash activation and protects cardiomyocytes from ischemia.

Altered levels of intracellular calcium (Ca2+) are a highly prevalent feature in different forms of cardiac injury, producing changes in contractility, arrhythmias, and mitochondrial dysfunction. In cardiac ischemia-reperfusion injury, mitochondrial Ca2+ overload leads to pathological production of reactive oxygen species (ROS), activates the permeability transition, and cardiomyocyte death. Here we investigated the cardiac phenotype caused by deletion of EF-hand domain-containing protein D1 (Efhd1-/-), a Ca2+-binding mitochondrial protein whose function is poorly understood. Efhd1-/- mice are viable and have no adverse cardiac phenotypes. They feature reductions in basal ROS levels and mitoflash events, both important precursors for mitochondrial injury, though cardiac mitochondria have normal susceptibility to Ca2+ overload. Notably, we also find that Efhd1-/- mice and their cardiomyocytes are resistant to hypoxic injury.

Rasagiline for amyotrophic lateral sclerosis: A randomized, controlled trial.

INTRODUCTION: Rasagiline is a monoamine oxidase B (MAO-B) inhibitor with possible neuroprotective effects in patients with amyotrophic lateral sclerosis (ALS). METHODS: We performed a randomized, double-blind, placebo-controlled trial of 80 ALS participants with enrichment of the placebo group with historical controls (n = 177) at 10 centers in the United States. Participants were randomized in a 3:1 ratio to 2 mg/day rasagiline or placebo. The primary outcome was average slope of decline on the ALS Functional Rating Scale-Revised (ALSFRS-R). Secondary measures included slow vital capacity, survival, mitochondrial and molecular biomarkers, and adverse-event reporting. RESULTS: There was no difference in the average 12-month ALSFRS-R slope between rasagiline and the mixed placebo and historical control cohorts. Rasagiline did not show signs of drug-target engagement in urine and blood biomarkers. Rasagiline was well tolerated with no serious adverse events. DISCUSSION: Rasagiline did not alter disease progression compared with controls over 12 months of treatment. Muscle Nerve 59:201-207, 2019.

Bullous Pemphigoid Arising in Lower Leg Vein Graft Incision Site.

Bullous pemphigoid is a blistering disorder which can be idiopathic or arise secondary to drugs or trauma; however, blistering arising within surgical scars is rare. We present a patient with no prior skin history who developed blistering in his left calf vein harvesting scar soon after coronary artery bypass surgery.

Subungual Congenital Nevus with Recurrent Nevus Phenomenon.

Melanonychia is uncommon. We report the first case of histopathologic recurrence of a completely excised subungual congenital nevus that presented as congenital melanonychia.

Interaction of anticancer reduced Schiff base coumarin derivatives with human serum albumin investigated by fluorescence quenching and molecular modeling.

The specific binding of five reduced Schiff base derived 7-amino-coumarin compounds with antitumor activity to human serum albumin, the principal binding protein of blood, was studied by fluorescence spectroscopy. Their conditional binding constants were computed and the reversible binding at the Sudlow's site I was found to be strong (KD∼0.03-2.09 µM). Based on the data albumin can provide a depot for the compounds and is responsible for their biodistribution and transport processes. The experimental data is complemented by protein-ligand docking calculations for two representatives which support the observations. The proton dissociation constants of the compounds were also determined by UV-Vis spectrophotometric and fluorometric titrations to obtain the actual charges and distribution of the species in the various protonation states at physiological pH.

Non-transgenic guinea pig strains exhibit divergent age-related changes in hippocampal mitochondrial respiration.

AIM: Alzheimer's disease (AD) is the most common form of dementia. However, while 150+ animal models of AD exist, drug translation from preclinical models to humans for treatment usually fails. One factor contributing to low translation is likely the absence of neurodegenerative models that also encompass the multi-morbidities of human aging. We previously demonstrated that, in comparison to the PigmEnTed (PET) guinea pig strain which models "typical" brain aging, the Hartley strain develops hallmarks of AD like aging humans. Hartleys also exhibit age-related impairments in cartilage and skeletal muscle. Impaired mitochondrial respiration is one driver of both cellular aging and AD. In humans with cognitive decline, diminished skeletal muscle and brain respiratory control occurs in parallel. We previously reported age-related declines in skeletal muscle mitochondrial respiration in Hartleys. It is unknown if there is concomitant mitochondrial dysfunction in the brain. METHODS: Therefore, we assessed hippocampal mitochondrial respiration in 5- and 12-month Hartley and PET guinea pigs using high-resolution respirometry. RESULTS: At 12 months, PETs had higher complex I supported mitochondrial respiration paralleling their increase in body mass compared to 5 months PETs. Hartleys were also heavier at 12 months compared to 5 months but did not have higher complex I respiration. Compared to 5 months Hartleys, 12 months Hartleys had lower complex I mitochondrial efficiency and compensatory increases in mitochondrial proteins collectively suggesting mitochondrial dysfunction with age. CONCLUSIONS: Therefore, Hartleys might be a relevant model to test promising therapies targeting mitochondria to slow brain aging and AD progression.

Sedentary behavior in mice induces metabolic inflexibility by suppressing skeletal muscle pyruvate metabolism.

Carbohydrates and lipids provide the majority of substrates to fuel mitochondrial oxidative phosphorylation. Metabolic inflexibility, defined as an impaired ability to switch between these fuels, is implicated in a number of metabolic diseases. Here, we explore the mechanism by which physical inactivity promotes metabolic inflexibility in skeletal muscle. We developed a mouse model of sedentariness, small mouse cage (SMC), that, unlike other classic models of disuse in mice, faithfully recapitulated metabolic responses that occur in humans. Bioenergetic phenotyping of skeletal muscle mitochondria displayed metabolic inflexibility induced by physical inactivity, demonstrated by a reduction in pyruvate-stimulated respiration (JO2) in the absence of a change in palmitate-stimulated JO2. Pyruvate resistance in these mitochondria was likely driven by a decrease in phosphatidylethanolamine (PE) abundance in the mitochondrial membrane. Reduction in mitochondrial PE by heterozygous deletion of phosphatidylserine decarboxylase (PSD) was sufficient to induce metabolic inflexibility measured at the whole-body level, as well as at the level of skeletal muscle mitochondria. Low mitochondrial PE in C2C12 myotubes was sufficient to increase glucose flux toward lactate. We further implicate that resistance to pyruvate metabolism is due to attenuated mitochondrial entry via mitochondrial pyruvate carrier (MPC). These findings suggest a mechanism by which mitochondrial PE directly regulates MPC activity to modulate metabolic flexibility in mice.

Cardiac gene therapy treats diabetic cardiomyopathy and lowers blood glucose.

Diabetic cardiomyopathy, an increasingly global epidemic and a major cause of heart failure with preserved ejection fraction (HFpEF), is associated with hyperglycemia, insulin resistance, and intracardiomyocyte calcium mishandling. Here we identify that, in db/db mice with type 2 diabetes-induced HFpEF, abnormal remodeling of cardiomyocyte transverse-tubule microdomains occurs with downregulation of the membrane scaffolding protein cardiac bridging integrator 1 (cBIN1). Transduction of cBIN1 by AAV9 gene therapy can restore transverse-tubule microdomains to normalize intracellular distribution of calcium-handling proteins and, surprisingly, glucose transporter 4 (GLUT4). Cardiac proteomics revealed that AAV9-cBIN1 normalized components of calcium handling and GLUT4 translocation machineries. Functional studies further identified that AAV9-cBIN1 normalized insulin-dependent glucose uptake in diabetic cardiomyocytes. Phenotypically, AAV9-cBIN1 rescued cardiac lusitropy, improved exercise intolerance, and ameliorated hyperglycemia in diabetic mice. Restoration of transverse-tubule microdomains can improve cardiac function in the setting of diabetic cardiomyopathy and can also improve systemic glycemic control.

Evaluation of the ICDP-UEMS Dermatopathology Examination.

A detailed analysis of the results of the international annual International Committee for Dermatopathology-Union Européene des Médecins Specialistes dermatopathology examination was undertaken to identify clues for further improvement. The analysis covered 5 consecutive years (2006-2010) and involved a total of 860 questions (591 common questions and 269 uncommon questions) and 181 participants. It focused on the overall performance of the participants, the performance per part of the examination (theoretical or practical), the performance per format of question (multiple choice or open), the performance per dermatopathological topic, and the performance per professional background (dermatologist or pathologist). The overall performance of the participants was high (on average 75% correct answers in 2006 and 85% correct answers in the subsequent years). In the theoretical part of the examination, the topics of vascular diseases and lichenoid dermatoses scored better than the average of all topics, and the topics of cutaneous lymphoproliferative diseases and melanocytic disorders scored worse. In the first practical part (interpretation of images), dermatologists outperformed pathologists, especially on providing a diagnosis (open question format) of clinical images. In the second practical part (microscopical examination), the topics of vascular diseases, granulomatous diseases, including necrobiotic and degenerative and metabolic diseases scored better than the average of all topics, and the topic of infectious diseases scored worse. The results of this detailed analysis provide an excellent feedback to the examination committee that will be used to consider the adjustment of parts and/or topics of the examination that showed a deviant performance by the participants. In addition, it is recommended to give more attention to the postgraduate education of certain dermatopathological topics, including cutaneous lymphoproliferative diseases, melanocytic disorders, and infectious diseases.

Nontransgenic Guinea Pig Strains Exhibit Hallmarks of Human Brain Aging and Alzheimer's Disease.

Older age is the primary risk factor for most chronic diseases, including Alzheimer's disease (AD). Current preclinical models to study brain aging and AD are mainly transgenic and harbor mutations intended to mirror brain pathologies associated with human brain aging/AD (eg, by increasing production of the amyloid precursor protein, amyloid beta [Aß], and/or phosphorylated tau, all of which are key pathological mediators of AD). Although these models may provide insight on pathophysiological processes in AD, none completely recapitulate the disease and its strong age-dependence, and there has been limited success in translating preclinical results and treatments to humans. Here, we describe 2 nontransgenic guinea pig (GP) models, a standard PigmEnTed (PET) strain, and lesser-studied Dunkin-Hartley (DH) strain, that may naturally mimic key features of brain aging and AD in humans. We show that brain aging in PET GP is transcriptomically similar to human brain aging, whereas older DH brains are transcriptomically more similar to human AD. Both strains/models also exhibit increased neurofilament light chain (NFL, a marker of neuronal damage) with aging, and DH animals display greater S100 calcium-binding protein B (S100ß), ionized calcium-binding adapter molecule 1 (Iba1), and Aß and phosphorylated tau-which are all important markers of neuroinflammation-associated AD. Collectively, our results suggest that both the PET and DH GP may be useful, nontransgenic models to study brain aging and AD, respectively.

A family with Fabry disease diagnosed by a single angiokeratoma.

This case presents a 39-year-old gentleman with a single angiokeratoma on the abdomen. Because of a family history of early onset cardiac disease, testing for Fabry disease was performed and a mis-sense mutation (A143T) in the Fabry gene confirmed the diagnosis. The unusual aspect of this case is that the patient otherwise had normal health. His only detectable abnormality was a high serum creatinine at 116 mmol/L. Two further affected males and four carrier females were detected on family screening. We tested a further five patients with a single angiokeratoma for Fabry disease. In the five tested though, no suggestive personal or family history was given for any of the patients and no further cases were detected. This case highlights the need for vigilance within dermatology clinics to consider Fabry disease even if a solitary angiokeratoma is the only presenting feature. Some patients do display a milder phenotype and thus a detailed family history should always be taken. As in this case, a solitary angiokeratoma and a suspicious family history may be the only clue. Because enzyme replacement therapy is now available, the potential benefits for the patient and their family are high.

The Dunkin Hartley Guinea Pig Is a Model of Primary Osteoarthritis That Also Exhibits Early Onset Myofiber Remodeling That Resembles Human Musculoskeletal Aging.

Skeletal muscle dysfunction, articular cartilage degeneration, and bone loss occur essentially in parallel during aging. Mechanisms contributing to this systemic musculoskeletal decline remain incompletely understood, limiting progress toward developing effective therapeutics. Because the progression of human musculoskeletal aging is slow, researchers rely on rodent models to identify mechanisms and test interventions. The Dunkin Hartley guinea pig is an outbred strain that begins developing primary osteoarthritis by 4 months of age with a progression and pathology similar to aging humans. The purpose of this study was to determine if skeletal muscle remodeling during the progression of osteoarthritis in these guinea pigs resembles musculoskeletal aging in humans. We compared Dunkin Hartley guinea pigs to Strain 13 guinea pigs, which develop osteoarthritis much later in the lifespan. We measured myofiber type and size, muscle density, and long-term fractional protein synthesis rates of the gastrocnemius and soleus muscles in 5, 9, and 15-month-old guinea pigs. There was an age-related decline in skeletal muscle density, a greater proportion of smaller myofibers, and a decline in type II concomitant with a rise in type I myofibers in the gastrocnemius muscles from Dunkin Hartley guinea pigs only. These changes were accompanied by age-related declines in myofibrillar and mitochondrial protein synthesis in the gastrocnemius and soleus. Collectively, these findings suggest Dunkin Hartley guinea pigs experience myofiber remodeling alongside the progression of osteoarthritis, consistent with human musculoskeletal aging. Thus, Dunkin Hartley guinea pigs may be a model to advance discovery and therapeutic development for human musculoskeletal aging.

Aminolaevulinic acid diffusion characteristics in 'in vitro' normal human skin and actinic keratosis: implications for topical photodynamic therapy.

BACKGROUND: The response rate of aminolaevulinic acid (ALA)-based photodynamic therapy (PDT) in certain subtypes of actinic keratosis (AK), such as hypertrophic and hyperkeratotic lesions, is variable, an effect attributable to a supposed lack of ALA penetration. A detailed and depth-related profile of spatial ALA permeation in AK following drug administration would lead to a greater understanding of concentrations achievable before protoporphyrin IX biosynthesis and subsequent PDT. METHODS: ALA penetration through excised normal human skin (NS) and AK lesions was evaluated using a cryostatic sectioning technique and radio-isotope counting following drug delivery using a novel, bioadhesive patch, loaded with 19, 38 or 50 mg/cm(2) ALA. RESULTS: Distinct differences in ALA concentration with respect to depth between AK and NS samples were shown, particularly within the superficial layers of the tissue structure, down to a depth of 1.0 mm. Patch application times were shown to influence ALA concentrations in tissue, but there was no clear correlation between ALA penetration in AK lesions taken from different body locations and from patients of different age. Similarly, the thickness of stratum corneum was not related to the ALA distribution profiles. CONCLUSIONS: Sizable variation in ALA concentration was a prominent feature of profiles through AK lesions, which may explain the variation of observed protoporphyrin IX production seen in the clinical implementation of AK PDT. That said, the results of this study show sufficient ALA penetration to a depth of 1.0 mm, which should be satisfactory for successful treatment of the majority of non-hyperkeratotic, hypertrophic AK using patch-based delivery methods.

In vitro anti-tumour and cyto-selective effects of coumarin-3-carboxylic acid and three of its hydroxylated derivatives, along with their silver-based complexes, using human epithelial carcinoma cell lines.

The chemotherapeutic potential of coumarin-3-carboxylic acid (C-3-COOH) and a series of three hydroxylated coumarin-3-carboxylic acid ligands, namely 6-hydroxy-coumarin-3-carboxylic acid (6-OH-C-3-COOH), 7-hydroxy-coumarin-3-carboxylic acid (7-OH-C-3-COOH) and 8-hydroxy-coumarin-3-carboxylic acid (8-OH-C-3-COOH), along with their corresponding silver-based complexes, namely 6-hydroxycoumarin-3-carboxylatosilver (6-OH-C-COO-Ag), 7-hydroxycoumarin-3-carboxylatosilver (7-OH-C-COO-Ag) and 8-hydroxycoumarin-3-carboxylatosilver (8-OH-C-COO-Ag), was determined using two human-derived carcinoma (A-498 and Hep-G2), along with two non-carcinoma human-derived cell lines (CHANG and HK-2). All of the ligands and their silver complexes induced a concentration-dependent cytotoxic effect. Furthermore, hydroxylation of C-3-COOH and its subsequent complexation with silver led to the production of a series of compounds with dramatically enhanced cytotoxicity, with 6-OH-C-3-COO-Ag having the greatest activity. Additionally, all of the metal-based complexes were selectively cytotoxic to both carcinoma-derived cell lines, relative to normal renal and hepatic cells. In comparative studies with cisplatin, and based on the IC(50) values obtained with Hep-G2 cells, it appeared that the coumarin-silver complexes were between 2 and 5.5 times more cytotoxic than cisplatin. All of the coumarin-silver complexes inhibited DNA synthesis, which did not appear to be mediated through intercalation. Furthermore, results obtained from Ames tests showed that all of the test agents and their phase I metabolites were non-mutagenic. Taken together, these findings suggest that both hydroxylation particularly in the 6th position and complexation with silver, served to significantly augment the cytotoxic properties of C-3-COOH, to yield a compound which acts as a cyto-selective agent, as it is a significant killer of cancer, relative to normal cells. We suggest that this group of compounds may have a therapeutic role to play in the successful treatment and management of cancer in man.

A study of the role of apoptotic cell death and cell cycle events mediating the mechanism of action of 6-hydroxycoumarin-3-carboxylatosilver in human malignant hepatic cells.

Previously our research group has studied the anti-proliferative effects of a series of hydroxylated derivatives and silver (I) complexes of coumarin-3-carboxylic acid (C-3-COOH) using two human-derived carcinoma cell lines (A-498 and Hep-G2). Results obtained suggested that both hydroxylation and complexation with silver served to significantly augment the cytotoxic properties of C-3-COOH, to yield a compound, namely 6-hydroxycoumarin-3-carboxylatosilver (6-OH-C-COO-Ag) which could act as a potent and cyto-selective agent, capable of killing cancer cells, and with limited toxicity to cells derived from normal tissue. Here we seek to expand on these findings by probing the molecular mechanism underlying this effect. Results from cytological staining clearly illustrated cellular changes consistent with the induction of apoptotic cell death and which occurred 24 h post-drug-treatment. Additionally, electrophoretic analysis of genomic DNA showed the presence of a ladder pattern, characteristic of apoptotic cell death. This result was subsequently confirmed using a selection of biochemical assays, where increased activity of pro-apoptotic caspases 3 and 9, and increased cleavage of poly(ADP-ribose)-polymerase protein (PARP) were observed. This result was further underpinned by the appearance of a sub-G(1) peak, representing hypo-diploid cells, using flow cytometric analysis. Furthermore, 6-OH-C-COO-Ag was seen to function through an alteration in the percentage of cells entering the G(0)/G(1) phase of cell cycle. Consequently, 6-OH-C-COO-Ag has been shown to a more potent and selective anti-cancer agent than cisplatin, capable of altering key biochemical events leading to the execution of apoptotic cell death as early as 24 h post-treatment, suggesting that it may represent a novel therapeutic agent for the safe and effective treatment of cancer in man.

Apoptotic cell death: a possible key event in mediating the in vitro anti-proliferative effect of a novel copper(II) complex, [Cu(4-Mecdoa)(phen)(2)] (phen=phenanthroline, 4-Mecdoa=4-methylcoumarin-6,7-dioxactetate), in human malignant cancer cells.

The central objective of the current study was to investigate the potential in vitro anti-proliferative effect of the parent ligand, 4-methylcoumarin-6,7-dioxyacyeic acid (4-MecdoaH(2)), and its copper (II) complex, bis(phenanthroline4-methylcoumarin-6,7-dioxacetatocopper(II) ([Cu(4-Mecdoa)(phen)(2)]) using four human model cell lines. In addition, selected mechanistic studies were carried out using the most sensitive of the four cell lines. Results obtained show that the complex could alter proliferation of both human neoplastic renal (A-498) and hepatic (HepG2) cells. Furthermore, non-neoplastic hepatic (CHANG) cells appeared to be less sensitive. However, this effect was not duplicated with non-neoplastic renal (HK-2) cells, a profile shared by cisplatin. The observed anti-proliferative effect appeared to be dose-and time-dependent, and could be attributed to the complex, rather than any of the free components i.e. the 1,10-phenanthroline or coumarin ligand, or the simple metal salt. Furthermore, the complex was shown to decrease DNA synthesis, but did not intercalate with it. Based on IC(50) values, [Cu(4-Mecdoa)(phen)(2)] was shown to be almost 12 times more potent than cisplatin. Moreover, there was no evidence that P-glycoprotein-mediated multi-drug resistance was likely to decrease anti-proliferative activity. Cytological stains, analysis of genomic DNA, and biochemical assays [caspase-3 and -9 and cleaved poly(ADP-ribose)-polymerase protein], showed that cell death could switch between apoptosis and necrosis, and this effect appeared to be concentration-dependent. Additionally, flow cytometric analysis showed that the complex functioned through an alteration in cell cycle progression. Taken together, [Cu(4-Mecdoa)(phen)(2)] has been shown to be a more potent anti-proliferative agent than either the ligand or cisplatin, and is capable of altering key biochemical events leading to the execution of apoptotic and/or necrotic cell death, suggesting that it is worthy of further investigation.