Açaí (Euterpe oleracea Mart.) Seed Oil Exerts a Cytotoxic Role over Colorectal Cancer Cells: Insights of Annexin A2 Regulation and Molecular Modeling.

Açaí, Euterpe oleracea Mart., is a native plant from the Amazonian and is rich in several phytochemicals with anti-tumor activities. The aim was to analyze the effects of açaí seed oil on colorectal adenocarcinoma (ADC) cells. In vitro analyses were performed on CACO-2, HCT-116, and HT-29 cell lines. The strains were treated with açaí seed oil for 24, 48, and 72 h, and cell viability, death, and morphology were analyzed. Molecular docking was performed to evaluate the interaction between the major compounds in açaí seed oil and Annexin A2. The viability assay showed the cytotoxic effect of the oil in colorectal adenocarcinoma cells. Acai seed oil induced increased apoptosis in CACO-2 and HCT-116 cells and interfered with the cell cycle. Western blotting showed an increased expression of LC3-B, suggestive of autophagy, and Annexin A2, an apoptosis regulatory protein. Molecular docking confirmed the interaction of major fatty acids with Annexin A2, suggesting a role of açaí seed oil in modulating Annexin A2 expression in these cancer cell lines. Our results suggest the anti-tumor potential of açaí seed oil in colorectal adenocarcinoma cells and contribute to the development of an active drug from a known natural product.

Proteomic Analysis of HCC-1954 and MCF-7 Cell Lines Highlights Crosstalk between αv and ß1 Integrins, E-Cadherin and HER-2.

Overexpression of human epidermal growth factor receptor-2 (HER-2) occurs in 20% of all breast cancer subtypes, especially those that present the worst prognostic outcome through a very invasive and aggressive tumour. HCC-1954 (HER-2+) is a highly invasive, metastatic cell line, whereas MCF-7 is mildly aggressive and non-invasive. We investigated membrane proteins from both cell lines that could have a pivotal biological significance in metastasis. Membrane protein enrichment for HCC-1954 and MCF-7 proteomic analysis was performed. The samples were analysed and quantified by mass spectrometry. High abundance membrane proteins were confirmed by Western blot, immunofluorescence, and flow cytometry. Protein interaction prediction and correlations with the Cancer Genome Atlas (TCGA) patient data were conducted by bioinformatic analysis. In addition, ß1 integrin expression was analysed by Western blot in cells upon trastuzumab treatment. The comparison between HCC-1954 and MCF-7 membrane-enriched proteins revealed that proteins involved in cytoskeleton organisation, such as HER-2, αv and ß1 integrins, E-cadherin, and CD166 were more abundant in HCC-1954. ß1 integrin membrane expression was higher in the HCC-1954 cell line resistant after trastuzumab treatment. TCGA data analysis showed a trend toward a positive correlation between HER-2 and ß1 integrin in HER-2+ breast cancer patients. Differences in protein profile and abundance reflected distinctive capabilities for aggressiveness and invasiveness between HCC-1954 and MCF-7 cell line phenotypes. The higher membrane ß1 integrin expression after trastuzumab treatment in the HCC-1954 cell line emphasised the need for investigating the contribution of ß1 integrin modulation and its effect on the mechanism of trastuzumab resistance.

Crosstalk between PI3K/Akt and Wnt/ß-catenin pathways promote colorectal cancer progression regardless of mutational status.

The PI3K/Akt and Wnt/ß-catenin pathways play an important role in the acquisition of the malignant phenotype in cancer. However, there are few data regarding the role of the interplay between both pathways in colorectal cancer (CRC) progression. The mutational status and the clinicopathological characteristics of PI3K/Akt and Wnt/ß-catenin pathways were accessed by bioinformatic analysis whereas that the impact of the interplay between the activity of both pathways to explain tumorigenic potential was performed in vitro using IGF-1 and Wnt3a treatments in CRC cell models. The mutational status of these pathways did not influence the survival of CRC patients, but an association between clinicopathological characteristics in patients with mutations in one, but not in both pathways was observed. A potentiating effect on the activation of both pathways and enhanced cellular migration and proliferation was observed when both pathways were activated simultaneously with IGF-1 and Wnt3a. In addition, these effects were hindered after pretreatment with LY294002, a specific PI3K inhibitor, suggesting some dependence between these two signaling cascades. Our findings show that, regardless of mutational status, there is an interplay between the activity of PI3K/Akt and Wnt/ß-catenin pathways that contributes to events related to CRC progression and that the reversal of such events using a PI3K inhibitor highlights the value of targeting these pathways for potential directed therapies in CRC patients.

Docosahexaenoic acid promotes cell cycle arrest and decreases proliferation through WNT/ß-catenin modulation in colorectal cancer cells exposed to γ-radiation.

The effects of radiation are known to be potentiated by N-3 polyunsaturated fatty acids, which modulate several signaling pathways, but the molecular mechanisms through which these fatty acids enhance the anticancer effects of irradiation in colorectal cancer (CRC) treatment remain poorly elucidated. Here, we aimed to ascertain whether the fatty acid docosahexaenoic acid (DHA) exerts a modulating effect on the response elicited by radiation treatment (RT). Two CRC cell lines, Caco-2 and HT-29, were exposed to RT, DHA, or both (DHA + RT) for various times, and then cell viability, proliferation, and clonogenicity were assessed. Moreover, cell cycle, apoptosis, and necrosis were analyzed using flow cytometry, and the involvement of WNT/ß-catenin signaling was investigated by immunofluorescence to determine nuclear ß-catenin, GSK3ß phosphorylation status, and TCF/LEF-activity reporter. DHA and RT applied separately diminished the viability of both HT-29 and Caco-2 cells, and DHA + RT caused a further reduction in proliferation mainly in HT-29 cells, particularly in terms of colony formation. Concomitantly, our results verified cell cycle arrest in G0/G1 phase, a reduction of cyclin D1 expression, and a decrease in GSK3ß phosphorylation after the combined treatment. Furthermore, immunofluorescence quantification revealed that nuclear ß-catenin was increased in RT-exposed cells, but this effect was abrogated in cells exposed to DHA + RT, and the results of TCF/LEF-activity assays confirmed that DHA attenuated the increase in nuclear ß-catenin activity induced by irradiation. Our finding shows that DHA applied in combination with RT enhanced the antitumor effects of irradiation on CRC cells, and that the underlying mechanism involved the WNT/ß-catenin pathway. © 2018 BioFactors, 45(1):24-34, 2019.

Cofilin-1 signaling mediates epithelial-mesenchymal transition by promoting actin cytoskeleton reorganization and cell-cell adhesion regulation in colorectal cancer cells.

Colorectal cancer (CRC) is frequently a lethal disease because of metastasis. Actin cytoskeletal rearrangement is an essential step in cell migration during activation of the epithelial-mesenchymal transition (EMT) program, which is associated with metastatic properties of cancer cells. Cofilin-1 protein modulates actin dynamics by promoting actin treadmilling, thereby driving membrane protrusion and cell migration and invasion. However, the role of cofilin-1 during EMT in CRC is unknown. Here, we show that cofilin-1 and p-cofilin-1 have distinct subcellular distribution in EMT cells, as determined by super-resolution microscopy images, indicating distinct roles in different areas of cells. Silenced cofilin-1 cells treated with TGF-ß (siCofilin-1/TGF-ß) evaded p-LIMK2-p-cofilin-1 status, leading to recovery of E-cadherin and claudin-3 at the cell-cell contact and their respective protein levels, actin reorganization, and decreased mesenchymal protein level. Furthermore, siCofilin-1/TGF-ß cells exhibited decreased migration and invasion rates as well as MMP-2 and -9 activity and augmented focal adhesion size. The expression of an inactive phospho-cofilin-1 mimetic (S3E) reduced E-cadherin and claudin-3 in cell-cell contacts, reduced their protein levels, and increased vimentin protein. Based on our findings, we suggest that cofilin-1 is crucial to switching from epithelial to mesenchymal-like morphology and cell migration and invasion by regulating actin cytoskeleton organization through activation of RhoA-LIMK2-cofilin-1 signaling, impacting the cell-cell adhesion organization of colon cancer cells in EMT.

Brain prolactin is involved in stress-induced REM sleep rebound.

REM sleep rebound is a common behavioural response to some stressors and represents an adaptive coping strategy. Animals submitted to multiple, intermittent, footshock stress (FS) sessions during 96h of REM sleep deprivation (REMSD) display increased REM sleep rebound (when compared to the only REMSD ones, without FS), which is correlated to high plasma prolactin levels. To investigate whether brain prolactin plays a role in stress-induced REM sleep rebound two experiments were carried out. In experiment 1, rats were either not sleep-deprived (NSD) or submitted to 96h of REMSD associated or not to FS and brains were evaluated for PRL immunoreactivity (PRL-ir) and determination of PRL concentrations in the lateral hypothalamus and dorsal raphe nucleus. In experiment 2, rats were implanted with cannulas in the dorsal raphe nucleus for prolactin infusion and were sleep-recorded. REMSD associated with FS increased PRL-ir and content in the lateral hypothalamus and all manipulations increased prolactin content in the dorsal raphe nucleus compared to the NSD group. Prolactin infusion in the dorsal raphe nucleus increased the time and length of REM sleep episodes 3h after the infusion until the end of the light phase of the day cycle. Based on these results we concluded that brain prolactin is a major mediator of stress-induced REMS. The effect of PRL infusion in the dorsal raphe nucleus is discussed in light of the existence of a bidirectional relationship between this hormone and serotonin as regulators of stress-induced REM sleep rebound.

Papel da anexina A2 na progressão do câncer colorretal / [Role of annexin A2 in the progression of colorectal cancer]

Anexinas são proteínas ligantes de fosfolipídeos dependente de cálcio que exercem funções celulares como organização do citoesqueleto, transporte de íons e sinalização celular. Por estarem relacionadas com a organização de actina e com a proliferação celular;; alterações na sua expressão podem ser implicadas na tumorigênese de diferentes tipos de câncer. A anexina A2 (ANXA2), promove a proliferação, migração e invasão celular quando superexpressa. Em estudo recente, sugeriu-­se a utilização da anexina A2 como biomarcador do desenvolvimento do câncer colorretal (CCR). Contudo, existem poucas evidências sobre como a ANXA2 é regulada e seu papel na modulação de vias de sinalização é pouco conhecido. O objetivo desse estudo foi a elucidação do papel da ANXA2 em eventos relacionados com a progressão do CCR. Análise da expressão de ANXA2 em amostras de pacientes de CCR revelou aumento da expressão no tecido tumoral, corroborados pela análise do banco de dados do TCGA e por nossos resultados de IHC indicando relação da ANXA2 com estadios mais avançados e marcação diferencial na metástase. Foram realizados ensaios in vitro para avaliar os níveis de expressão e fosforilação (resíduo Y23) da proteína anexina A2, em linhagens celulares de adenocarcinoma de cólon (Caco-­2, HT-­29 e HCT-­116), através de imunoblotting. Para a avaliação do papel da ANXA2 na progressão tumoral, submetemos células HT-­29, silenciada ou não para a ANXA2, ao tratamento com TGF-­ß , avaliando o potencial proliferativo, migratório e invasivo destas células. Os resultados mostram que as células Caco-­2 apresentam um nível de fosforilação da ANXA2 menor do que os níveis observados nas células HT-­29 e HCT-­116, as quais apresentaram níveis similares de fosforilação desta proteína. Para avaliação de eventos relacionados com a EMT, foram analisados marcadores epiteliais e mesenquimas por meio de imunoblotting e imunofluorescência. Mediante tratamento com TGF-­ß , a linhagem HT-­29 exibiu alterações morfológicas características com o desenvolvimento da EMT, assim como redução de E-­caderina e aumento na expressão de marcadores mesenquimais como a vimentina. Houve aumento de expressão total de ANXA2, da sua forma fosforilada e relocalização da superfície celular para o citoplasma na linhagem HT-­29. Concomitantemente ao tratamento com TGF-­ß observou-­se a perda dos contatos intercelulares mediada pela internalização da E-­caderina e ANXA2. A análise de microscopia de iluminação estruturada confirmou a colocalização de ambas proteinas intracelularmente, assim como a transfecção de mutantes sítios específicos para a ANXA2 provou a sua interação com E-­caderina. O ensaio de proliferação mostrou que o TGF-­ß e o silenciamento de ANXA2 são anti-­proliferativos. O silenciamento causou redução na migração (Wound Healing), independente do aumento migratório causado pelo TGF-­ß . A indução da EMT por TGF-­ß levou ao aumento na capacidade invasiva destas células, aumento que não ocorreu nas células silenciadas para a ANXA2 ou tratadas com STA21 (inibidor de STAT3). O uso deste inibidor, STA21 e de PP2 (inibidor de Src) impediram a EMT nas células tratadas com TGF-­ß. Concluimos que a ANXA2 atua na manutenção das junções celulares mediadas pela E-­caderina;; do aumento da capacidade invasiva, características do processo de EMT, via Src/ANXA2/STAT3 e também na proliferação e migração celular.

EphA4-mediated signaling regulates the aggressive phenotype of irradiation survivor colorectal cancer cells.

Radiotherapy is widely used for advanced rectal tumors. However, tumor recurrence after this treatment tends to be more aggressive and is associated with a poor prognosis. Uncovering the molecular mechanism that controls this recurrence is essential for developing new therapeutic applications. In the present study, we demonstrated that radiation increases the EphA4 activation level of the survivor progeny of colorectal cancer cells submitted to this treatment and that such activation promoted the internalization of a complex E-cadherin-EphA4, inducing cell-cell adhesion disruption. Moreover, EphA4 knockdown in the progeny of irradiated cells reduced the migratory and invasive potentials and metalloprotease activity induced by irradiation. Finally, we demonstrated that the cell migration and invasion potential were regulated by AKT and ERK1/2 signaling, with the ERK1/2 activity being dependent on EphA4. In summary, our study demonstrates that these signaling pathways could be responsible for the therapeutic failure, thereby promoting local invasion and metastasis in rectal cancer after radiotherapy. We also postulate that EphA4 is a potential therapeutic target for colorectal cancer treatment.

Alterations of the apical junctional complex and actin cytoskeleton and their role in colorectal cancer progression.

Colorectal cancer represents the fourth highest mortality rate among cancer types worldwide. An understanding of the molecular mechanisms that regulate their progression can prevents or reduces mortality due to this disease. Epithelial cells present an apical junctional complex connected to the actin cytoskeleton, which maintains the dynamic properties of this complex, tissue architecture and cell homeostasis. Several studies have indicated that apical junctional complex alterations and actin cytoskeleton disorganization play a critical role in epithelial cancer progression. However, few studies have examined the existence of an interrelation between these 2 components, particularly in colorectal cancer. This review discusses the recent progress toward elucidating the role of alterations of apical junctional complex constituents and of modifications of actin cytoskeleton organization and discusses how these events are interlinked to modulate cellular responses related to colorectal cancer progression toward successful metastasis.