Search Results (32)

Physiological changes in animals induced by environmental shifts in aquatic ecosystems can be studied using fish cell lines derived from vulnerable species. Therefore, we investigated how environmental shifts—specifically, an increase of 5 °C in temperature—impact the physiology of the cell line CMAfin1 derived from maraena whitefish (Coregonus maraena). Cellular growth, morphology, and metabolic responses were examined under two growth conditions: a control temperature of 20 °C and an elevated temperature of 25 °C. Using trypan blue staining, automated cell counting, phase contrast microscopy, and actin staining, we observed morphological changes in the cells. Metabolic functions were assessed using a Seahorse XFe96 Flux Analyzer, focusing on the bioenergetic capacities of mitochondrial respiration and glycolytic activity. Hyperthermia resulted in faster growth rates but reduced cell size in the CMAfin1 cell line. The cells’ metabolic activity (mitochondrial respiration and glycolytic activity) was inhibited, leading to a quiescent energy state. Our findings indicate reduced motility and altered intercellular communication at higher temperatures. The results highlight the potential of in vitro models to study environmental stress on fish physiology and emphasize the value of fish cell lines for understanding metabolic responses. Full article

Background: Combination therapies for cancer have gained considerable attention due to their potential for enhancing therapeutic efficacy and decreasing drug resistance. Introducing nanodrug delivery systems in this context may further improve the therapy due to targeted delivery, improved drug stability, sustained drug release, and prevention of rapid clearance from circulation. This study evaluates the combinatorial effects of two cytotoxic drugs, cabazitaxel (CBZ) and RSL3 (RAS-selective lethal 3), in free form as well as encapsulated within poly(2-ethyl butyl cyanoacrylate) (PEBCA) nanoparticles (NPs) in breast cancer cell lines. Methods: Cell proliferation was assessed using IncuCyte technology, and synergistic drug effects were determined with SynergyFinder Plus. Cell viability was measured with the MTT assay. Additionally, we investigated whether the combinatorial effects were reflected in alterations of metabolic activity or reactive oxygen species (ROS) production using Seahorse technology and the CM-H₂DCFDA assay, respectively. Results: The data presented reveal, for the first time, that CBZ and RSL3 exhibit synergistically or additively combinatorial effects on various breast cancer cell lines. The pattern of cytotoxic effects was consistent, whether the drugs were in free form or encapsulated in NPs. Moreover, the combinatorial effects were not observed to be associated with early changes in metabolic activity or ROS production. Conclusion: This study highlights the potential of CBZ and RSL3 in combinatorial nanomedicine as they may act synergistically. Further studies are warranted to better understand the mechanisms behind these combinatorial effects. Full article

Colorectal cancer (CRC) is the second deadliest cancer in the Western world. Increased body weight, a diet rich in red meat and alcohol, as well as a sedentary lifestyle, are all involved in sporadic CRC pathogenesis. Since current CRC therapies show several side effects, there is a need to find new and more effective therapeutic approaches, allowing conventional drug dosages and toxicity to be reduced. Gratiola officinalis alcoholic extract was characterized by LC-MS and its effect investigated on a healthy colon mucosa cell line and on different colorectal cancer cell lines. Cell viability, apoptosis and cell cycle progression were evaluated through flow cytometry; energy production and glycolysis were investigated using Seahorse technology, while cancer markers were analyzed through Western blotting. The untargeted metabolomics analysis of G. officinalis alcoholic extract revealed glycosides of different polyphenols and glycosides of cucurbitane-type triterpenes. This extract showed a stronger impact on CRC cell line viability compared to healthy colon cells. In the E705 CRC cell line, it induced cell apoptosis and caused the downregulation of glycolysis, inhibiting cell proliferation. On the other hand, SW480 CRC cells treated with G. officinalis extract showed G₂/M cell cycle arrest. This work shows that G. officinalis extract can reduce glycolysis and promote cell cycle arrest in CRC cells, suggesting that G. officinalis could represent a novel player in the prevention and treatment of CRC. Full article

Oxidative phosphorylation and glycolysis are the main ATP-generating pathways in cell metabolism. The balance between these two pathways is frequently altered to carry out cell-specific activities in response to stimuli involving activation, proliferation, or differentiation. Despite being a useful tool for researching metabolic profiles in real time in relatively small numbers of cancer cells, the main Agilent Seahorse XF Pro Analyzer (Agilent Technologies, Santa Clara, CA, USA) guideline is currently not fully detailed in the distinction between suspensions vs. adherent cancer cells. This article provides step-by-step protocols for profiling metabolic fluxes in suspension vs. adherent cancer cells via Seahorse technology, including adjustments for normalisation of data on the basis of the number of viable cells or the total protein content. Owing to the adaptations of plates, reagents, cell count, and protein quantification, it is possible to (i) analyse both adherent and suspension cells with a single instrument; (ii) conduct all experiments in 96-well plates, thus using fewer cells, media, and reagents; (iii) determine the effect of a drug or compound directly on cell metabolism; (iv) normalise data on the basis of the number of viable cells or the total protein content via a spectrophotometer; and (v) achieve notable savings in cost and time. Full article

LAH, an acetogenin from the Annonaceae family, has demonstrated antitumor activity in several cancer cell lines and in vivo models, where it reduced the tumor size and induced programmed cell death. We focused on the effects of LAH on mitochondrial dynamics, mTOR signaling, autophagy, and apoptosis in colorectal cancer (CRC) cells to explore its anticancer potential. Methods: CRC cells were treated with LAH, and its effects on mitochondrial respiration and glycolysis were measured using Seahorse XF technology. The changes in mitochondrial dynamics were observed through fluorescent imaging, while Western blot analysis was used to examine key autophagy and apoptosis markers. Results: LAH significantly inhibited mitochondrial complex I activity, inducing ATP depletion and a compensatory increase in glycolysis. This disruption caused mitochondrial fragmentation, a trigger for autophagy, as shown by increased LC3-II expression and mTOR suppression. Apoptosis was also confirmed through the cleavage of caspase-3, contributing to reduced cancer cell viability. Conclusions: LAH’s anticancer effects in CRC cells are driven by its disruption of mitochondrial function, triggering both autophagy and apoptosis. These findings highlight its potential as a therapeutic compound for further exploration in cancer treatment. Full article

The high incidence of epithelial malignancies in HIV-1 infected individuals is associated with co-infection with oncogenic viruses, such as high-risk human papillomaviruses (HR HPVs), mostly HPV16. The molecular mechanisms underlying the HIV-1-associated increase in epithelial malignancies are not fully understood. A collaboration between HIV-1 and HR HPVs in the malignant transformation of epithelial cells has long been anticipated. Here, we delineated the effects of HIV-1 reverse transcriptase on the in vitro and in vivo properties of HPV16-infected cervical cancer cells. A human cervical carcinoma cell line infected with HPV16 (Ca Ski) was made to express HIV-1 reverse transcriptase (RT) by lentiviral transduction. The levels of the mRNA of the E6 isoforms and of the factors characteristic to the epithelial/mesenchymal transition were assessed by real-time RT-PCR. The parameters of glycolysis and mitochondrial respiration were determined using Seahorse technology. RT expressing Ca Ski subclones were assessed for the capacity to form tumors in nude mice. RT expression increased the expression of the E6*I isoform, modulated the expression of E-CADHERIN and VIMENTIN, indicating the presence of a hybrid epithelial/mesenchymal phenotype, enhanced glycolysis, and inhibited mitochondrial respiration. In addition, the expression of RT induced phenotypic alterations impacting cell motility, clonogenic activity, and the capacity of Ca Ski cells to form tumors in nude mice. These findings suggest that HIV-RT, a multifunctional protein, affects HPV16-induced oncogenesis, which is achieved through modulation of the expression of the E6 oncoprotein. These results highlight a complex interplay between HIV antigens and HPV oncoproteins potentiating the malignant transformation of epithelial cells. Full article

Seahorses are valuable species for their use in traditional Chinese medicine, as well as for the aquarium trade as ornamentals and curiosities. To balance market demand and reduce pressure on wild populations, many countries have undertaken commercial seahorse cultivation. Skeletal development plays a crucial role in fish fry culture, affecting external morphology, feeding, and movement. This study investigated the ontogeny allometry, timing, and progression of skeletal development in H. abdominalis from DAB (day after birth) 1 to DAB 100 under mass-scale captive breeding conditions in north China. The results of this study revealed the growth rate was significantly increased between DAB 30 and DAB 54. Allometry analysis revealed that in the early stage, the head, trunk, and tail demonstrated almost isometric growth. However, in the later stage, the head and trunk exhibited negative isometric growth, whereas the tail displayed positive isometric growth. Skeletal staining results showed that newborn seahorses do not have ossified bones until DAB 11 (SL 28.14 ± 2.94 mm). Ossification was primarily observed in the jaw region and the tubular nasal structure of the cranium, which indicated the importance of the early development of feeding organs. The initial formation of ossified vertebral columns was observed at DAB 13 (SL 26.48 ± 0.63 mm), with the complete ossification of all vertebrae occurring by DAB 45 (SL 54.87 ± 4.70 mm). Furthermore, the cranium, rings, and plates were all fully ossified by DAB 30. Ossification of the fins began at DAB 23 (SL 31.27 ± 4.05 mm). However, neither of them were fully ossified by DAB 100. The pelvic fin and the complete structure of the caudal fin were not observed, possibly because of caudal fin ray structure degeneration within the pouch. In addition, no skeletal deformities were observed in all the tested samples. The results of this study provide valuable information on the developmental biology of H. abdominalis, enriching our understanding of their growth and offering insights for optimizing fish fry breeding technologies. Full article

Background: The microtubule protein inhibitor C118P shows excellent anti-breast cancer effects. However, the potential targets and mechanisms of C118P in breast cancer remain unknown. Methods: Real-time cellular analysis (RTCA) was used to detect cell viability. Apoptosis and the cell cycle were detected by flow cytometry. Computer docking simulations, surface plasmon resonance (SPR) technology, and microscale thermophoresis (MST) were conducted to study the interaction between C118P and alanine-serine-cysteine transporter 2 (ASCT2). Seahorse XF technology was used to measure the basal oxygen consumption rate (OCR). The effect of C118P in the adipose microenvironment was explored using a co-culture model of adipocytes and breast cancer cells and mouse cytokine chip. Results: C118P inhibited proliferation, potentiated apoptosis, and induced G2/M cell cycle arrest in breast cancer cells. Notably, ASCT2 was validated as a C118P target through reverse docking, SPR, and MST. C118P suppressed glutamine metabolism and mediated autophagy via ASCT2. Similar results were obtained in the adipocyte–breast cancer microenvironment. Adipose-derived interleukin-6 (IL-6) promoted the proliferation of breast cancer cells by enhancing glutamine metabolism via ASCT2. C118P inhibited the upregulation of ASCT2 by inhibiting the effect of IL-6 in co-cultures. Conclusion: C118P exerts an antitumour effect against breast cancer via the glutamine transporter ASCT2. Full article

Mitochondria are responsible for ATP synthesis through oxidative phosphorylation in cells. However, there are limited data on the influence of mitochondrial mass (MM) in the adequate assessment of cellular stress assay (CSA) results in human peripheral blood mononuclear cells (PBMCs). Therefore, the aim of this study was to determine MM in PBMCS and assess its influence on the results of CSA measurements. Blood samples were collected and sent to the laboratory for MM and CSA measurements during different seasons of the year. The mitochondrial mass was determined based on the mtDNA:nDNA ratio in PBMCs using quantitative real-time PCR (qRT-PCR). CSA was measured using Seahorse technology. The MM was significantly lower during summer and autumn compared to winter and spring (p < 0.0001). On the contrary, we found that the maximal respiration per mitochondrion (MP) was significantly higher in summer and autumn compared to winter and spring (p < 0.0001). The estimated effect of MM on mitochondrial performance was −0.002 pmol/min/mitochondrion (p < 0.0001) and a correlation coefficient (r) of −0.612. Similarly, MM was negatively correlated with maximal respiration (r = −0.12) and spare capacity (in % r = −0.05, in pmol/min r = −0.11). In conclusion, this study reveals that MM changes significantly with seasons and is negatively correlated with CSA parameters and MP. Our findings indicate that the mitochondrial mass is a key parameter for determination of mitochondrial fitness. Therefore, we recommend the determination of MM during the measurement of CSA parameters for the correct interpretation and assessment of mitochondrial function. Full article

Background: The metabolic state of pulmonary artery smooth muscle cells (PASMCs) from patients with pulmonary arterial hypertension (PAH) is not well understood. In this study, we examined the balance between glycolysis and mitochondrial respiration in non-PAH-PASMCs and PAH-PASMCs under normoxia and hypoxia. Methods: We investigated the enzymes involved in glycolysis and mitochondrial respiration, and studied the two major energy-yielding pathways (glycolysis and mitochondrial respiration) by measuring extracellular acidification rate (ECAR) and cellular oxygen consumption rate (OCR) using the Seahorse extracellular flux technology. Results: Under both normoxia and hypoxia, the mRNA and protein levels of pyruvate dehydrogenase kinase 1 and pyruvate dehydrogenase were increased in PAH-PASMCs compared with non-PAH-PASMCs. The mRNA and protein levels of lactate dehydrogenase, as well as the intracellular lactate concentration, were also increased in PAH-PASMCs compared with non-PAH-PASMCs under normoxia. However, these were not significantly increased in PAH-PASMCs compared with non-PAH-PASMCs under hypoxia. Under normoxia, ATP production was significantly lower in PAH-PASMCs (59 ± 5 pmol/min) than in non-PAH-PASMCs (70 ± 10 pmol/min). On the other hand, ATP production was significantly higher in PAH-PASMCs (31 ± 5 pmol/min) than in non-PAH-PASMCs (14 ± 3 pmol/min) under hypoxia. Conclusions: There is an underlying change in the metabolic strategy to generate ATP production under the challenge of hypoxia. Full article

The United States Food and Drug Administration Adverse Event Reporting System (FAERS) logged 27,140 rhabdomyolysis cases from 2004 to 31 March 2020. We used FAERS to identify 14 drugs frequently reported in 6583 rhabdomyolysis cases and to investigate whether mitochondrial toxicity is a common pathway of drug-induced rhabdomyolysis by these drugs. Preliminary screening for mitochondrial toxicity was performed using the acute metabolic switch assay, which is adapted here for use in murine L6 cells. Fenofibrate, risperidone, pregabalin, propofol, and simvastatin lactone drugs were identified as mitotoxic and underwent further investigation, using real-time respirometry (Seahorse Technology) to provide more detail on the mechanism of mitochondrial-induced toxicity. To confirm the human relevance of the findings, fenofibrate and risperidone were evaluated in primary human skeletal muscle-derived cells (HSKMDC), using the acute metabolic switch assay and real-time respirometry, which confirmed this designation, although the toxic effects on the mitochondria were more pronounced in HSKMDC. Overall, these studies demonstrate that the L6 model of acute modification may find utility as an initial, cost-effective screen for identifying potential myotoxicants with relevance to humans and, importantly, that drug-induced mitochondrial dysfunction may be a common mechanism shared by some drugs that induce myotoxicity. Full article

Oesophageal adenocarcinoma (OAC) is a poor prognosis cancer with limited response rates to current treatment modalities and has a strong link to obesity. To better elucidate the role of visceral adiposity in this disease state, a full metabolic profile combined with analysis of secreted pro-inflammatory cytokines, metabolites, and lipid profiles were assessed in human ex vivo adipose tissue explants from obese and non-obese OAC patients. These data were then related to extensive clinical data including obesity status, metabolic dysfunction, previous treatment exposure, and tumour regression grades. Real-time energy metabolism profiles were assessed using the seahorse technology. Adipose explant conditioned media was screened using multiplex ELISA to assess secreted levels of 54 pro-inflammatory mediators. Targeted secreted metabolite and lipid profiles were analysed using Ultra-High-Performance Liquid Chromatography coupled with Mass Spectrometry. Adipose tissue explants and matched clinical data were collected from OAC patients (n = 32). Compared to visceral fat from non-obese patients (n = 16), visceral fat explants from obese OAC patients (n = 16) had significantly elevated oxidative phosphorylation metabolism profiles and an increase in Eotaxin-3, IL-17A, IL-17D, IL-3, MCP-1, and MDC and altered secretions of glutamine associated metabolites. Adipose explants from patients with metabolic dysfunction correlated with increased oxidative phosphorylation metabolism, and increases in IL-5, IL-7, SAA, VEGF-C, triacylglycerides, and metabolites compared with metabolically healthy patients. Adipose explants generated from patients who had previously received neo-adjuvant chemotherapy (n = 14) showed elevated secretions of pro-inflammatory mediators, IL-12p40, IL-1$\mathsf{\alpha }$, IL-22, and TNF-$\mathsf{\beta }$ and a decreased expression of triacylglycerides. Furthermore, decreased secreted levels of triacylglycerides were also observed in the adipose secretome of patients who received the chemotherapy-only regimen FLOT compared with patients who received no neo-adjuvant treatment or chemo-radiotherapy regimen CROSS. For those patients who showed the poorest response to currently available treatments, their adipose tissue was associated with higher glycolytic metabolism compared to patients who had good treatment responses. This study demonstrates that the adipose secretome in OAC patients is enriched with mediators that could prime the tumour microenvironment to aid tumour progression and attenuate responses to conventional cancer treatments, an effect which appears to be augmented by obesity and metabolic dysfunction and exposure to different treatment regimes. Full article

Neutral lipid storage disease type M (NLSD-M) is an ultra-rare, autosomal recessive disorder that causes severe skeletal and cardiac muscle damage and lipid accumulation in all body tissues. In this hereditary pathology, the defective action of the adipose triglyceride lipase (ATGL) enzyme induces the enlargement of cytoplasmic lipid droplets and reduction in the detachment of mono- (MG) and diglycerides (DG). Although the pathogenesis of muscle fiber necrosis is unknown, some studies have shown alterations in cellular energy production, probably because MG and DG, the substrates of Krebs cycle, are less available. No tests have been tried with medium-chain fatty acid molecules to evaluate the anaplerotic effect in NLSD cells. In this study, we evaluated the in vitro effect of triheptanoin (Dojolvi^®), a highly purified chemical triglyceride with seven carbon atoms, in fibroblasts obtained from five NLSD-M patients. Glycolytic and mitochondrial functions were determined by Seahorse XF Agylent Technology, and cellular viability and triglyceride content were measured through colorimetric assays. After the addition of triheptanoin, we observed an increase in glycolysis and mitochondrial respiration in all patients compared with healthy controls. These preliminary results show that triheptanoin is able to induce an anaplerotic effect in NLSD-M fibroblasts, paving the way towards new therapeutic strategies. Full article

Although impaired mitochondrial function has been proposed as a hallmark of multiple sclerosis (MS) disease, few studies focus on the mitochondria of immune cells. We aimed to compare the mitochondrial function of the peripheral blood mononuclear cells (PBMCs) from MS patients with (M+) and without (M−) lipid-specific oligoclonal immunoglobulin M bands (LS-OCMB), and healthydonors (HD). We conducted an exploratory cross-sectional study with 19 untreated MS patients (M+ = 9 and M− = 10) and 17 HDs. Mitochondrial superoxide anion production and mitochondrial mass in PBMCs were assessed without and with phytohemagglutinin by flow cytometry. The PBMCs’ mitochondrial function was analyzed using Seahorse technology. Superoxide anion production corrected by the mitochondrial mass was higher in MS patients compared with HDs (p = 0.011). Mitochondrial function from M+ patients showed some impairments compared with M− patients. Without stimulus, we observed higher proton leak (p = 0.041) but lower coupling efficiency (p = 0.041) in M+ patients; and under stimulation, lower metabolic potential ECAR (p = 0.011), and lower stressed OCR/ECAR in the same patients. Exclusively among M+ patients, we described a higher mitochondrial dysfunction in the oldest ones. The mitochondrial impairments found in the PBMCs from MS patients, specifically in M+ patients, could help to better understand the disease’s physiopathology. Full article

Cellular stress is central to the understanding of pathological mechanisms and the development of new therapeutic strategies and serves as a biomarker for disease progression in neurodegeneration, diabetes, cancer, cardiovascular and other chronic diseases. The common cellular stress assay (CSA) based on Seahorse technology in peripheral blood mononuclear cells (PBMCs) shows inconsistent results, which prevents its use as a biomarker for the progression of chronic diseases. Therefore, the aim of this study was to investigate potential factors that affect the CSA in PBMCs. We measured the CSA parameters in PBMCs from study participants and compared the results according to the potential factors, namely, the PBMC isolation method, age, seasonal variation and the gender of the study participants. PBMCs were isolated by OptiPrep^® and Robosep^TM-S methods. PBMCs isolated with the OptiPrep method showed much higher extracellular acidification and higher respiration compared to Robosep-isolated cells. Moreover, OptiPrep-isolated cells showed a higher number of outliers for the proton production rate (PPR) and a high respiratory quotient, indicating impurities with other cells, such as platelets, and technical inconsistencies. PBMCs from older individuals showed higher maximal respiration, spare capacity and extracellular acidification than younger participants. Additionally, in winter, maximal respiration and spare capacity decreased. From spring until early autumn, spare capacity and maximal respiration continuously increased. Elderly males also showed higher basal respiration, spare capacity and extracellular acidification than females. In conclusion, the findings of this study clearly demonstrate that the results of CSA parameters measured in PBMCs are influenced by the PBMC isolation method, age, seasonal variation and gender. Therefore, we recommend that researchers and physicians properly interpret the results of CSA parameters in PBMCs by considering these factors. It is important to use separate CSA evaluation standards based on the isolation method, age, gender and season-dependent factors. To assess the cellular stress situation in PBMCs, both extracellular acidification and mitochondrial respiration should be taken into account. Further study of additional factors, such as mitochondrial mass, should be conducted to improve the measurement of CSA parameters for the assessment of the real mitochondrial fitness. Full article