Effects of ω-3 PUFA-enriched diet on growth parameters in a syngenic murine model of breast adenocarcinoma: a possible role for estrogen receptor alpha

Background: Breast cancer (BC) is the most common tumour among women and 75% of BC are estrogen receptor (ER)dependent. In particular, ERα promotes tumour growth, while ERβ has an anti-proliferative effect [1]. Epidemiological data have linked ω-3 polyunsaturated fatty acid (PUFA) consumption to lower incidence of BC and several experimental studies showed the anti-proliferative effects of ω-3 fish oil in different tumour models [2,3]. Chia seed oil is rich in α-linolenic acid (ALA 18:3 ω-3), while corn oil is rich in linoleic acid (LA 18:2 ω-6), precursors of eicosapentaenoic acid (EPA) and arachidonic acid (AA), respectively. Based on substrate availability, these FAs give rise to different eicosanoid signatures with opposite effects in cancer [4]. ω-3 PUFAs generate both anti-inflammatory prostanoids and reactive oxygen species (ROS), which in turn could affect NF-κB. Indeed, NF-κB belongs to a family of transcription factors with a key role in inflammation and oxidative stress, but its role in tumour development is still controversial [5]. The aim of the study was to determine possible processes that are activated by dietary lipids regulating BC growth and metastasis.Fil: Vara Messler, Marianela. Universidad Nacional de Córdoba. Facultad de Ciencias Médicas. Instituto de Biología Celular; Argentina.Fil: Vara Messler, Marianela. Universidad de Padova. Departamento de Ciencias Farmacéuticas y Farmacológicas; Italia.Fil: Pasqualini, María Eugenia. Universidad Nacional de Córdoba. Facultad de Ciencias Médicas. Instituto de Biología Celular; Argentina.Fil: Comba, Andrea. Universidad Nacional de Córdoba. Facultad de Ciencias Médicas. Instituto de Biología Celular. Cátedra de Biología, Histología y Embriología; Argentina.Fil:Comba, Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ciencias de la Salud; Argentina.Fil: Toniolo, Alicia. Universidad de Padova. Departamento de Ciencias Farmacéuticas y Farmacológicas; Italia.Fil: Trenti, Annalisa. Universidad de Padova. Departamento de Ciencias Farmacéuticas y Farmacológicas; Italia.Fil: Quiroga, Patricia. Universidad Nacional de Córdoba. Facultad de Ciencias Médicas. Instituto de Biología Celular; Argentina.Fil: Valentich, Mirta Ana. Universidad Nacional de Córdoba. Facultad de Ciencias Médicas. Instituto de Biología Celular; Argentina.Fil: Valentich, Mirta Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ciencias de la Salud; Argentina.Fil: Bolego, Chiara. Universidad de Padova. Departamento de Ciencias Farmacéuticas y Farmacológicas; Italia.Otras Ciencias de la Salu

Dynamic Interplay between Pericytes and Endothelial Cells during Sprouting Angiogenesis

Vascular physiology relies on the concerted dynamics of several cell types, including pericytes, endothelial, and vascular smooth muscle cells. The interactions between such cell types are inherently dynamic and are not easily described with static, fixed, experimental approaches. Pericytes are mural cells that support vascular development, remodeling, and homeostasis, and are involved in a number of pathological situations including cancer. The dynamic interplay between pericytes and endothelial cells is at the basis of vascular physiology and few experimental tools exist to properly describe and study it. Here we employ a previously developed ex vivo murine aortic explant to study the formation of new blood capillary-like structures close to physiological situation. We develop several mouse models to culture, identify, characterize, and follow simultaneously single endothelial cells and pericytes during angiogenesis. We employ microscopy and image analysis to dissect the interactions between cell types and the process of cellular recruitment on the newly forming vessel. We find that pericytes are recruited on the developing sprout by proliferation, migrate independently from endothelial cells, and can proliferate on the growing capillary. Our results help elucidating several relevant mechanisms of interactions between endothelial cells and pericytes

Eicosapentaenoic acid prevents salt sensitivity in diabetic rats and decreases oxidative stress

Objectives: Salt sensitivity (SS) is associated with increased cardiovascular risk in patients with Type 2 diabetes mellitus (T2-DM) due to an increase in renal oxidation. ω-3 polyunsaturated fatty acids have shown antioxidant effects, but a typical Western diet contains limited content. In particular, ω-3 polyunsaturated fatty acids are able to activate nuclear factor erythroid 2-related factor 2 (Nrf-2) to prevent diabetes mellitus–related complications by mitigating oxidative stress. Therefore, we hypothesized that eicosapentaenoic acid (EPA; ω-3) modulates SS in rats with T2-DM by decreasing renal oxidative stress via Nrf-2 activation and enhancing the antiinflammatory response via interleukin (IL) 6 modulation. Methods: Three-month-old male rats (n = 40) were fed with a Normal Na-diet (NNaD) and randomly selected into four groups: Healthy Wistar nondiabetic rats (Wi), diabetic controls (eSS), arachidonic acid-treated eSS (AA; ω-6), and EPA-treated eSS (ω-3). After 1 year, rats were placed in metabolic cages for 7 d and fed a NNaD, followed by a 7-d period with a High Na-diet (HNaD). Systolic blood pressure, body weight, serum IL-6 and reactive oxygen species (ROS) levels were determined at the end of each 7-d period. Glycated hemoglobin (HbA1c), triacylglycerol, creatinine, and cholesterol levels were determined. ROS levels and Nrf-2 expression in kidney lysates were also assayed. Histologic changes were evaluated. A t test or analysis of variance was used for the statistical analysis. Results: After a HNaD, systolic blood pressure increased in both the control eSS and AA groups, but not in the EPA and Wi groups. However, HbA1c levels remained unchanged by the treatments, which suggests that the observed beneficial effect was independent of HbA1c levels. The IL-6 levels were higher in the eSS and AA groups, but remained unaltered in EPA and Wi rats after a HNaD diet. Interestingly, EPA protected against serum ROS in rats fed the HNaD, whereas AA did not. In kidney lysates, ROS decreased significantly in the EPA group compared with the eSS group, and Nrf-2 expression was consistently higher compared with the AA and eSS groups. Diabetic rats presented focal segmental sclerosis, adherence to Bowman capsule, and mild-to-moderate interstitial fibrosis. EPA and AA treatment prevented kidney damage. Conclusions: An adequate ω3-to-ω6 ratio prevents SS in diabetic rats by a mechanism that is independent of glucose metabolism but associated with the prevention of renal oxidative stress generation. These data suggest that EPA antioxidant properties may prevent the development of hypertension or kidney damage.Fil: Vara Messler, Marianela. Università di Torino; Italia. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Mukdsi, Jorge Humberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Ciencias de la Salud. Universidad Nacional de Córdoba. Instituto de Investigaciones en Ciencias de la Salud; ArgentinaFil: Osieki, Natalia I.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Ciencias de la Salud. Universidad Nacional de Córdoba. Instituto de Investigaciones en Ciencias de la Salud; Argentina. Universidad Nacional de Córdoba. Facultad de Medicina. Instituto de Biología Celular; ArgentinaFil: Benizio, Evangelina Leticia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Ciencias de la Salud. Universidad Nacional de Córdoba. Instituto de Investigaciones en Ciencias de la Salud; ArgentinaFil: Repossi Marquez, Pablo Gaston. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Ciencias de la Salud. Universidad Nacional de Córdoba. Instituto de Investigaciones en Ciencias de la Salud; ArgentinaFil: Ajayi, Ebenezer Idowu O. Osun State University; Nigeria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; ArgentinaFil: Garcia, Nestor Horacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Ciencias de la Salud. Universidad Nacional de Córdoba. Instituto de Investigaciones en Ciencias de la Salud; Argentin

The role of omega-3 PUFA-enriched diet and DHA metabolites derived from aspirin-acetylated COX-2 on tumor growth and angiogenesis.

Background: Breast cancer (BC) is the most frequent cancer occurring in women. Epidemiological data have linked omega-3 polyunsaturated fatty acids (n-3 PUFAs) consumption to lower incidence of BC and several experimental studies showed the anti-proliferative effects of n-3 PUFAs in different BC models. Chia oil is rich in alpha-linolenic acid (ALA, 18:3 n-3), while corn oil is rich in linoleic acid (LA, 18:2 n-6). Angiogenesis is a tightly regulated process involving endothelial cells (ECs) proliferation, migration and tube formation. Docosahexaenoic acid (DHA, 22:6 n-3) a downstream metabolite from ALA, has been demonstrated to regulate cancer-related angiogenesis, while arachidonic acid (AA, 20:4 n-6) promotes angiogenesis. In addition, aspirin (ASA) has antineoplastic effects that are mediated at least in part by metabolites derived from acetylated COX-2. The study was aimed to determine a possible role of dietary n-3 PUFAs on BC growth and, in particular, the effect of DHA metabolites from ASA-acetylated COX-2 in the angiogenic process. Methods: 40 BALB/c mice were fed 1) a Chia Oil (ChO)-rich (n-3), or 2) a Corn Oil (CO)-rich diet (n-6). Afterwards, mice were inoculated with mouse BC cells (LM3) and tumour growth parameters were recorded after 45 days. Mitotic and apoptotic figures, as well angiogenesis in tumour sections were assessed by immunohistochemistry. Human endothelial cells (ECs) were treated with DHA or AA (1-100 microM) for different times in the presence or absence of ASA (50 microM). Selected experiments were performed with 17R-HDHA (100 nM – 3 microM), a DHA metabolite derived from acetylated COX-2. Endogenous production of 17R-HDHA by HUVECs was analyzed by Liquid chromatography-tandem mass spectrometry. EC viability was analyzed with the MTT assay. The angiogenetic capability was evaluated using a) the wound healing assay, b) a chemotaxis chamber, and c) matrigel. Results: After 45 days tumour incidence was higher in CO-fed compared with ChO-fed mice (100 vs 85%, p<0.05). Tumor weight and volume as well as metastasis number were lower, whereas tumor latency time was longer in ChO-fed mice. Compared to the control group, a higher number of apoptotic bodies, a lower number of mitosis and vessels were consistently observed in the ChO-fed mice group. Given these in vivo findings, we next studied whether n-3 PUFAs had direct anti-angiogenic actions on human ECs. Cells were viable when treated with up to 50 microM DHA or AA in the presence or absence of ASA for 24h. Challenging the cells with 17R-HDHA for 24h did not affect cell viability. EC migration, as evaluated by wound healing assay, was significantly decreased in cells pretreated for 24h with 30 microM, but not 10 microM DHA compared to control. Interestingly, 10 microM DHA in the presence of 50 microM ASA significantly inhibited ECs migration (-36%±5.4%, p<0.001) already after 24h. By contrast, AA did not affect EC migration at any concentration tested (1-30 microM) in the presence or absence of ASA. Consistently, ECs treated with DHA+ASA produced higher amounts of 17R-HDHA with respect to cells treated with DHA alone. Pretreatment with 17R-HDHA (1-3 microM, 24h) decreased EC migration (-12%±4.1% and -28%±1.9%, p<0.05 and p<0.01, respectively) evaluated by the wound healing assay. Additionally, 17R- HDHA (300 nM, 1 or 3 microM, 6h) reduced EC migration as evaluated in a micro chemotaxis chamber (-20%±1.4%, -30%±4.0% and -65%±3.5%, p<0.01 and p<0.001, respectively), while DHA did not show any effect. Furthermore, 17R-HDHA inhibited EC tube formation after a 6h treatment. In particular, nodes, meshes and mesh area were significantly lower in 10 microM DHA-treated cells in the presence of 50 microM ASA, as well as in 17R-HDHA (300 nM-3 microM)-treated cells. Conclusion: In vivo and ex vivo data point to a central role of n-3 PUFAs in BC growth. In vitro data demonstrate that aspirin-acetylated COX-2 enhances the anti-angiogenic effects of DHA and may have a role in the setting of tumors.Background: Il tumore alla mammella (TM) è il tumore più frequente che si verifica nelle donne. Dati epidemiologici hanno collegato l’assunzione degli acidi grassi polinsaturi omega-3 (n-3 PUFA) con la minore incidenza di TM e diversi studi sperimentali hanno dimostrato gli effetti anti-proliferativi di n-3 PUFA in diversi modelli di TM. L’olio di chia è ricco di acido alfa-linolenico (ALA, 18:3 n-3), mentre l'olio di mais è ricco di acido linoleico (LA, 18:2 n-6). L'angiogenesi è un processo strettamente regolato che coinvolge la proliferazione, la migrazione e la formazione di capillari da parte delle cellule endoteliali (CE). L’acido docosaesaenoico (DHA, 22:6 n-3), un metabolita a valle dell’ALA, è stato dimostrato essere in grado di regolare l'angiogenesi correlatea al tumore, mentre l'acido arachidonico (AA, 20:4 n-6) promuove l'angiogenesi. Inoltre, l'aspirina (ASA) ha effetti antineoplastici che sono mediati, almeno in parte, da metaboliti derivati dall’acetilazione della COX-2. Lo scopo dello studio è stato quello di determinare il ruolo degli n-3 PUFA sulla crescita del un tumore alla mammella e, in particolare, di valutare l'effetto di metaboliti derivati dalla COX-2 acetilata in presenza di DHA nell'angiogenesi. Metodi: 40 topi BALB/c sono stati nutriti con 1) una dieta ricca di n-3 PUFA contenente il 10% di olio di chia (ChO); o 2) una dieta ricca d n-6 PUFA contenente il 10% d’olio mais (CO). In seguito, i topi sono stati inoculati con una linea di cellule tumorali (LM3) per indurre il tumore primario ed i parametri di crescita tumorale sono stati registrati dopo 45 giorni. Nelle sezioni di tessuto tumorale sono state valutate le cellule mitotiche ed apoptotiche e l'angiogenesi mediante immunoistochimica. Gli esperimenti in vitro sono stati condotti con cellule endoteliali umane (HUVECs). Le HUVECs sono state trattate con DHA o AA (1-100 microM) per diversi tempi in presenza o assenza di ASA (50 microM). Inoltre, alcuni esperimenti sono stati effettuati con 17R-HDHA (100 nM – 3 microM), un metabolita derivato dalla COX-2 acetilata in presenza di DHA. La produzione endogena di 17R-HDHA da parte delle HUVECs è stata analizzata mediante spettrometria di massa. La citotossicitá dei composti è stata valutata con il test MTT. La capacità angiogenica delle HUVECs è stata valutata utilizzando a) il saggio della chiusura della ferita, b) la chemiotassi in camera di di Boyden e c) la formazione dei capillari con il saggio di matrigel. Risultati: Dopo 45 giorni l'incidenza tumorale era più alta nei topi sottoposti a dieta con olio di mais (CO) rispetto a quelli alimentati con la dieta chia (ChO) (100 vs 85%, p <0.05). Il peso tumorale ed il volume del tumore, così come il numero delle metastasi erano più bassi nei topi che avevano ricevuto la dieta ChO, mentre il tempo di latenza del tumore è risultato più lungo nei topi alimentati con la dieta ChO. Inoltre, nel gruppo di topi sottoposti a dieta ChO sono state osservateun maggior numero di cellule apoptotiche, un minor numero di cellule mitotiche cosí come un minor numero di vasi rispetto al gruppo di controllo (CO). Alla luce di questi risultati in vivo, abbiamo studiato se n-3 PUFA avevano attivitá anti-angiogenica diretta sulle CE umane. Non abbiamo osservato variazioni nella vitalità delle cellule endoteliali trattate con DHA fino a 50 microM o AA, in presenza o assenza di ASA per 24h, cosí come nelle cellule trattate con 17R-HDHA. La migrazione delle CE valutata mediante il saggio di chiusura della ferita, è significativamente diminuita rispetto al controllo nelle cellule pretrattate per 24 ore con 30 microM DHA, ma non in quelle pretrattate con 10 microM DHA. Tuttavia, è interessante sottolineare che 10 microM DHA in presenza di 50 microM ASA ha inibito significativamente la migrazione delle CE (-36%±5.4%, p<0.001) già dopo 24 ore. Per contro, AA in presenza o assenza di ASA non ha influenzato la migrazione cellulare a nessuna delle concentrazioni valutate (1-30 microM). In linea con questi risultati, le HUVECs trattate con DHA in presenza d’ASA ha prodotto una maggiore quantità di 17R-HDHA rispetto alle cellule trattate con solo DHA. Inoltre, il pretrattamento per 24h con 17R-HDHA (1-3 microM) ha diminuito la migrazione delle cellule endoteliali valutata con il test di chiusura della ferita (-12%±4.1% and -28%±1.9%, p<0.05 e p<0.01, rispettivamente). Inoltre, 17R-HDHA (300 nM, 1-3 microM per 6 ore) ha ridotto la migrazione cellulare valutata in una camera di chemiotassi (-20%±1.4%, -30%±4.0% and -65%±3.5%, p<0.01 e p<0.001, rispettivamente), mentre DHA da solo non ha mostrato alcun effetto. Inoltre, 17R-HDHA ha diminuito la formazione di strutture capillaro-simili dopo 6 ore. In particolare, valutando diversi parametri, si è visto che i nodi, le maglie e la zona delimitata dalle maglie erano significativamente più bassi nelle cellule trattate con 10 microM DHA in presenza di 50 microM ASA, così come in quelle cellule trattate con 17R-HDHA (300 nM - 3 microM). Conclusione: i risultati in vivo in un modello murino di tumore al seno, mettono in luce un ruolo degli n-3 PUFA nella crescita tumorale. I risultati in vitro dimostrano che l’acetilazione della COX-2 da parte dell’aspirina porta alla produzione di metaboliti che potenziano gli effetti anti-angiogenici di DHA e possono avere un ruolo nel tumore

The role of methylglyoxal stress in developing acquired resistance to cetuximab in quadruple wild-type colorectal cancer

Colorectal cancer (CRC) ranks as the third most frequently diagnosed cancer, and the limited efficacy of current treatments often leads to frequent relapses. Cetuximab (CTX) stands as the most used anti-EGFR targeted therapy for CRC. Mutation of oncogenes is commonly associated with the development of resistance to CTX. However, patients with CRC classified as "quadruple wild type" (4WT), characterized by the absence of mutations in KRAS, NRAS, BRAF, and PIK3CA, also exhibit a resistance to this therapy. The dependency of cancer cells on glycolysis promotes the initiation and progression of tumors. Methylglyoxal (MG), an inevitable by-product of glycolysis, induces glycation on proteins, lipids and DNA. We have previously shown that the depletion of glyoxalase 1 (GLO1), the main MG detoxifying enzyme, induces an endogenous MG stress that enhances growth and metastatic capacity. We observed that GLO1-depleted CRC cells develop resistance to CTX. This project endeavours to elucidate the molecular mechanisms, driven by MG stress, that lead to the development of CTX resistance in 4WT cells. The use of CRC resistant clones, generated after long term challenge with CTX, let us conclude that MG stress was not a major feature of resistant cells. Therefore, we next focused on the generation of persistent CRC cells, enriched after short challenge with CTX, to better position MG stress during the process of the acquisition of resistance. Persistent cells demonstrated resistance to CTX that was evidenced by a gain in AKT and ERK activation, efficient escape from apoptosis when compared with parental cells. Nevertheless, persistence was not associated with MG stress as shown by unchanged free MG/MG protein adducts levels and GLO1 activity. Knowing that the sole induction of MG stress in 4WT CRC cells render them resistant to CTX resistance, we next used GLO1-depleted cells to understand how MG triggers acquired resistance. Upon MG stress and CTX challenge, resistant cells displayed enhanced EGFR activation and persistent activation of ERK/MEK pathway when compared with control cells. Taken together, these findings suggest that MG stress may facilitate cellular resistance to anti-EGFR therapy. Ongoing experiments will help demonstrating the use of MG scavengers to potentially resensitize resistant CRC cells to CTX

Acquired resistance to EGFR-targeted therapy in quadruple wild-type colorectal cancer: role of methylglyoxal stress

Colorectal cancer (CRC) is the third most common cancer diagnosed and the lack of effectiveness of current therapies leads to frequent relapses. Monoclonal antibodies cetuximab and panitumumab are the most used anti-EGFR targeted therapy in CRC. Genetic alterations, such as activating mutations, are commonly associated with a lack of response to anti-EGFR therapy. Strikingly, patients with CRC diagnosed as "quadruple wild type" (unmutated for KRAS, NRAS, BRAF and PIK3CA) also show a lack of response to this therapy. Reliance of cancer cells on glycolysis promotes tumorigenesis and malignant progression. Methylglyoxal (MG) is an unavoidable glycolytic by-product that glycates proteins, lipids, and DNA. MG stress results from the imbalance between MG production and its enzymatic detoxification by glyoxalase 1 (GLO1). Upon MG stress, cancer cells enhanced their growth and metastatic potential. In our hands, induction of MG stress in quadruple WT cells rendered them resistant to cetuximab. In this project, we aim to explore MG stress-driven molecular mechanisms underlying the acquisition of cetuximab resistance in quadruple WT CRC. Indeed, such resistant tumours could benefit from the targeting of MG stress using potent MG scavengers, such as carnosine and metformin. We have previously shown that LIM1215 human quadruple WT CRC cells undergo a glycolytic switch associated with MG stress upon the acquisition of cetuximab resistance. GLO1-depleted LIM1215 cells showed an increased viability when challenged with cetuximab. Consistently, NOD-SCID mice sub-cutaneously injected with GLO1-depleted LIM1215 cells developed cetuximab resistant tumours. We aim to use patient-derived xenograft and organoids generated from quadruple WT CRC patients to demonstrate: (1) the occurrence of MG stress and (2) the potential anti-cancer effects of MG scavengers. Mechanistically, we will consider the mutagenic potential of MG on specific oncogenic drivers of CRC, and/or the glycation of specific proteins, such as transcription factors and signalling pathway effectors, known to be implicated in CRC initiation and progression. In summary, this study will highlight the importance of the glycolytic switch in resistance to EGFR targeted therapy in quadruple WT CRC