three-dimensional-co-culture-method-for-study-indirect-interactions

Three-Dimensional Co-Culture Method for the Study of Indirect Interactions Between Different Cell Types Across a Reconstituted Basement Membrane

Three-dimensional (3D) co-culture is a rapidly evolving technique for researchers looking to accurately study cell-cell interactions using in vitro ...

Health Research Institute of the Balearic Islands (IdISBa) & University of the Balearic Islands

Instituto Salud Carlos III

Sirtuin 3 (SIRT3), the major deacetylase in mitochondria, plays a crucial role in modulating oxygen reactive species (ROS) and limiting the oxidative damage in cellular components. SIRT3 targets different enzymes which regulate mitochondrial metabolism and participate in ROS detoxification, such as the complexes of the respiratory chain, the isocitrate dehydrogenase, or the manganese superoxide dismutase. Thus, SIRT3 activity is essential in maintaining mitochondria homeostasis and has recently received great attention, as it is considered a fidelity protein for mitochondrial function. In some types of cancer, SIRT3 functions as a tumoral promoter, since it keeps ROS levels under a certain threshold compatible with cell viability and proliferation. On the contrary, other studies describe SIRT3 as a tumoral suppressor, as SIRT3 could trigger cell death under stress conditions. Thus, SIRT3 could have a dual role in cancer. In this regard, modulation of SIRT3 activity could be a new target to develop more personalized therapies against cancer.

SIRT3: Oncogene and Tumor Suppressor in Cancer.

Honeybees have an essential role in ecosystems pollinating wild flowers and cultivated crops, representing an important cultural and economic benefit for humans. Honeybee populations are decreasing over the last decade, due to multifactorial causes. The aim of this field study was to investigate the effects of the presence of the invasive species Vespa velutina, a bee predator, in oxidative stress parameters of honeybee workers. To achieve this objective, positive or negative apiaries for the presence of the V. velutina were selected. Five honeybees from six hives of each apiary were sampled in spring, summer and autumn, analysing a total of 233 samples. Analysis of mRNA expression of oxidative stress-related genes, catalase enzymatic activity and lipid peroxidation were performed. An increase in sod2, tpx3, trxR1, gtpx1, gstS1, coxI, cytC and if2mt genes expression, as well as a raise in catalase activity and lipid peroxidation were observed in V. velutina positive samples. Thus, here we present a new methodology to analyze the impact of the predation pressure of the invasive species V. velutina on honeybees under field conditions. In conclusion, the results obtained in this study indicate the negative impact of the presence of the yellow-legged hornet on honeybees' health and the activation of their antioxidant system to protect them against this biotic stressor. Moreover, the redox status they present could increase the susceptibility of honeybees, essential insects that currently receive many inputs of different stresses, to another stressor.

The impact of the invasive species Vespa velutina on honeybees: A new approach based on oxidative stress.

Antioxidant defences and oxidative stress are related to development, progression and malignancy of colorectal cancer. However, their role in early stages of cancer remains unknown. More and more recent studies have revealed that non-tumour adjacent tissue is not a normal tissue. Thus, our aim was to analyse protein levels of MnSOD (Manganese Superoxide Dismutase), acMnSOD (Acetylated Manganese superoxide Dismutase), SIRT3 (Sirtuin 3), CuZnSOD (Cupper Zinc Superoxide Dismutase), CAT (Catalase), GPx (Glutathione Peroxidase), and GRd (Glutathione Reductase) both in tumour and non-tumour adjacent tissue from colorectal cancer patients by western blot. Non-tumour adjacent tissue seemed to have higher levels of antioxidant enzymes that detoxify hydrogen peroxide compared to tumour tissue. In contrast, tumour tissue had higher levels of MnSOD and acMnSOD. Furthermore, most of the proteins analysed showed significant differences between stage I and II in both non-tumour adjacent and tumour tissue. This could indicate that antioxidant enzymes, especially MnSOD, play a crucial role in early stages of colorectal cancer in both tissues, so they could be analysed as novel biomarkers to improve colorectal cancer diagnosis.

Antioxidant enzymes change in different non-metastatic stages in tumoral and peritumoral tissues of colorectal cancer.

Honeybees are essential for the ecosystem maintenance and for plant production in agriculture. Glyphosate is a broad-spectrum systemic herbicide widely used in crops to control weeds and could affect honeybees' health in sublethal doses. Our aim was to study how sublethal doses of glyphosate affects to oxidative stress and mitochondrial homeostasis in honeybees. We exposed honeybees to glyphosate at 5 and 10 mg·l for 2 and 10 h for the gene expression analysis by reverse transcription polymerase chain reaction and for 48 and 72 h for the antioxidant enzymes activity and lipid peroxidation determination. We observed a general increase in antioxidant- and mitochondrial-related genes expression in honeybees after 2 h of exposition to glyphosate, as well as a rise in catalase and superoxide dismutase enzymatic activity after 48 h and an increment in lipid peroxidation adducts generation after 72 h. These results suggest a direct effect of glyphosate on honeybees' health, with an insufficient response of the antioxidant system to the generated oxidative stress, resulting in an increase in lipid peroxidation and, therefore, oxidative damage. Altogether, results obtained in this work demonstrate that sublethal treatments of glyphosate could directly affect honeybee individuals under laboratory conditions. Therefore, it is necessary to investigate alternatives to glyphosate to determine if they are less harmful to non-target organisms.

Sublethal doses of glyphosate modulates mitochondria and oxidative stress in honeybees by direct feeding.

Alkylphospholipids (APLs) have been studied as anticancer drugs that interfere with biological membranes without targeting DNA. Although their mechanism of action is not fully elucidated yet, it is known that they disrupt the intracellular trafficking of cholesterol and its metabolism. Here, we analyzed whether APLs could also interfere with mitochondrial function. For this purpose, we used HT29 colorectal cancer cells, derived from a primary tumor, and SW620 colorectal cancer cells, derived from a metastasis site. After treatment with the APLs miltefosine and perifosine, we analyzed various mitochondrial parameters, including mitochondrial mass, cardiolipin content, mitochondrial membrane potential, HO production, the levels of oxidative phosphorylation (OXPHOS) complexes, metabolic enzymes activity, the oxygen consumption rate, and the levels of apoptosis and autophagy markers. APLs, especially perifosine, increased mitochondrial mass while OXPHOS complexes levels were decreased without affecting the total oxygen consumption rate. Additionally, we observed an increase in pyruvate dehydrogenase (PDH) and isocitrate dehydrogenase (IDH) levels and a decrease in lactate dehydrogenase (LDH) activity, suggesting a metabolic rewiring induced by perifosine. These alterations led to higher mitochondrial membrane potential, which was potentiated by decreased uncoupling protein 2 (UCP2) levels and increased reactive oxygen species (ROS) production. Consequently, perifosine induced an imbalance in mitochondrial function, resulting in higher ROS production that ultimately impacted cellular viability.

Mitochondrial Functionality Is Regulated by Alkylphospholipids in Human Colon Cancer Cells.

Breast cancer is the most frequently diagnosed malignancy among women worldwide. Trace elements, such as selenium, play a crucial role in antioxidation and have gained interest in cancer research. This study aimed to assess the association between prediagnostic serum trace element concentrations and breast cancer risk, as well as their relationship with the expression of key antioxidant enzymes in leukocytes of ER-positive breast cancer patients.

Serum trace elements and expression of antioxidant-related genes in the detection of women with ER-positive breast cancer.

Oxaliplatin is commonly known as a successful chemotherapy for advanced colorectal cancer, improving patient survival and eradicating micro-metastases, but its use in early stages remains controversial. Mitochondria fuel energy-intensive programs such as cell migration, yet how oxaliplatin regulates the mitochondrial network in CRC - and how TP53 context shapes this - remains unclear. We investigated a matched pair of CRC cell lines from the same patient - SW480 (primary) and SW620 (lymph-node metastasis) - both harboring TP53-R273H mutation, to define differential responses in mitochondrial biogenesis, dynamics and respiration and the mechanisms underlying them. The results indicate that primary-derived colorectal cancer cell line increased cell migration, mitochondrial biogenesis, and mitochondrial respiration capacity in response to oxaliplatin through a new and firstly described gain-of-function (GOF) of p53-R273H. Additionally, in the primary-derived CRC line, oxaliplatin elicited fate heterogeneity - coexisting apoptotic and senescent fractions alongside an R273H-driven, bioenergetically primed migratory subpopulation - together with increased mitochondrial biogenesis and respiratory capacity; by contrast, the metastatic-derived line was more sensitive and displayed structural mitochondrial injury with reduced maximal respiration. More broadly, this work underscores the importance of p53 gain-of-function mutations in CRC: the same GOF (TP53-R273H) amplifies cell migration by coupling an enhanced mitochondrial biogenesis/OXPHOS program to motility. Oxaliplatin further accentuates this energetically primed, pre-metastatic state, arguing for mitochondrial-targeted combination strategies in early-stage CRC.

Hotspot mutant p53-R273H enhances mitochondrial biogenesis and cell migration in primary colorectal cancer in response to oxaliplatin.

Obesity contributes to worse outcomes in breast cancer, particularly in the luminal subtype, where cellular senescence could increase tumor aggressiveness. Parallelly, Estrogen Receptor Beta (ERβ) has emerged as an important mediator in the cellular response to obesity-associated inflammation that metformin could counteract. This study explores metformin's role in targeting senescence to mitigate obesity-induced tumor progression. Using the GSE189757 dataset, differentially expressed genes in obese versus lean luminal breast cancer patients were examined by in silico analysis. T47D, BT474, and MCF7 cell lines were treated with an obesity-related inflammatory cocktail (ELIT) and metformin. Protein levels, mRNA expression and functional assays assessed mitochondrial activity, oxidative stress, cell viability, and senescence-related markers. The role of ERβ was investigated through gene silencing, gene overexpression, and correlation studies using datasets. Experimental results show that ELIT exposure increased mitochondrial activity, oxidative stress, and senescence markers, particularly in T47D cells, effects that metformin mitigated. Metformin also reduced SASP-related gene expression, thereby limiting autocrine signaling effects on migration, mammosphere formation, and drug sensitivity. ERβ expression was observed as a potential modulator of ELIT-induced alterations, and metformin reduced its expression. Analysis of patient datasets revealed a positive correlation between ERβ gene (ESR2) expression and senescence-related markers in obese luminal breast cancer patients, particularly MCL1, BCL2L1, CCL2, and ICAM1. These findings indicate that ERβ exhibits a key role as mediator of obesity-induced tumor alterations by promoting senescence-related markers, and that metformin's ability to target ERβ offers a potential strategy to suppress senescence-driven malignancy and improve therapeutic outcomes of obese luminal breast cancer patients.

Article	Year
SIRT3: Oncogene and Tumor Suppressor in Cancer. Cancers\| PubMed ID: 28704962	2017
The impact of the invasive species Vespa velutina on honeybees: A new approach based on oxidative stress. The Science of the total environment\| PubMed ID: 31280152	2019
Antioxidant enzymes change in different non-metastatic stages in tumoral and peritumoral tissues of colorectal cancer. The international journal of biochemistry & cell biology\| PubMed ID: 31981728	2020
Sublethal doses of glyphosate modulates mitochondria and oxidative stress in honeybees by direct feeding. Archives of insect biochemistry and physiology\| PubMed ID: 37259187	2023
Mitochondrial Functionality Is Regulated by Alkylphospholipids in Human Colon Cancer Cells. Biology\| PubMed ID: 38132283	2023
Serum trace elements and expression of antioxidant-related genes in the detection of women with ER-positive breast cancer. Journal of trace elements in medicine and biology : organ of the Society for Minerals and Trace Elements (GMS)\| PubMed ID: 40972221	2025
Hotspot mutant p53-R273H enhances mitochondrial biogenesis and cell migration in primary colorectal cancer in response to oxaliplatin. Biochimica et biophysica acta. Molecular cell research\| PubMed ID: 41161608	2026
Metformin reduces senescence induced by obesity-related inflammation in luminal breast cancer through estrogen receptor beta modulation. Biochemical pharmacology\| PubMed ID: 41895619	2026

Jorge Sastre-Serra

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