ROP GTPases with a geranylgeranylation motif modulate alkaloid biosynthesis in Catharanthus roseus.

Rho of Plant (ROP) GTPases function as molecular switches that control signaling processes essential for growth, development, and defense. However, their role in specialized metabolism is poorly understood. Previously, we demonstrated that inhibition of protein geranylgeranyl transferase (PGGT-I) negatively impacts the biosynthesis of monoterpenoid indole alkaloids (MIA) in Madagascar periwinkle (Catharanthus roseus), indicating the involvement of prenylated proteins in signaling. Here, we show through biochemical, molecular, and in planta approaches that specific geranylgeranylated ROPs modulate C. roseus MIA biosynthesis. Among the six C. roseus ROP GTPases (CrROPs), only CrROP3 and CrROP5, having a C-terminal CSIL motif, were specifically prenylated by PGGT-I. Additionally, their transcripts showed higher expression in most parts than other CrROPs. Protein-protein interaction studies revealed that CrROP3 and CrROP5, but not ΔCrROP3, ΔCrROP5, and CrROP2 lacking the CSIL motif, interacted with CrPGGT-I. Further, CrROP3 and CrROP5 exhibited nuclear localization, whereas CrROP2 was localized to the plasma membrane. In planta functional studies revealed that silencing of CrROP3 and CrROP5 negatively affected MIA biosynthesis, while their overexpression upregulated MIA formation. In contrast, silencing and overexpression of CrROP2 had no effect on MIA biosynthesis. Moreover, overexpression of ΔCrROP3 and ΔCrROP5 mutants devoid of sequence coding for the CSIL motif failed to enhance MIA biosynthesis. These results implicate that CrROP3 and CrROP5 have a positive regulatory role on MIA biosynthesis and thus shed light on how geranylgeranylated ROP GTPases mediate the modulation of specialized metabolism in C. roseus.

Atypical histological presentation of bone regeneration after insertion of cryoprotected allogeneic bone graf / Presentación histológica atípica de la regeneración ósea después de la inserción de un injerto óseo alogénico crioprotegido

Background: To evaluate bone regenerative capacity of cryoprotected corticocancellous allogeneic bone graftperformed in type II and III post-extraction sockets for ridge preservation after twelve weeks in-vivo.Material and Methods: Twenty-seven type II or III bony-walled extraction sockets (mandible and maxilla) wereselected for this study. Following atraumatic tooth-extraction a cryoprotected corticocancellous allogeneic bonegraft material and a resorbable porcine-derived collagen membrane were used for ridge preservation. Duringre-entry surgery at approximately 12 weeks, bone core biopsies were obtained using a 3.2 mm trephine drill andsamples were histologically processed and subjected to qualitative and quantitative histomorphometric analysis.Quantitative data was analyzed using a general linear mixed model with results presented as mean values with thecorresponding 95% confidence interval values. Results: Healing without incident and ridge preservation allowed for the placement of dental implants after 12 weeksin 25 out of the 27 treated socket sites. Analyses yielded an average of ~21.0±7% of old/native bone, ~17±5.5% ofnewly regenerated bone (total of ~38±12.8% for all bone), 0.23±0.14% of new bone presenting with nucleating siteswithin the matrix, ~52±5.12% of soft tissue, and 3.6±2.09% of damaged bone. The average regenerated bone wasstatistically analogous to that of old/native bone (p=0.355). Furthermore, an atypical histological pattern of boneregeneration was observed, with newly formed bone exhibiting “infiltration-like” behavior and with new bone nucle-ating sites observed within the demineralized bone matrix.Conclusions: Cryoprotected corticocancellous allogeneic bone-graft demonstrated osteoconductive, osteoinductive,and osteogenic properties, yielding unique healing patterns which does warrant further investigation.(AU)

Implications of Undocumented Status for Latinx Families During the COVID-19 Pandemic: A Call to Action.

BACKGROUND: A disproportionate number of COVID-19 cases and deaths have been reported among Latinxs in the U.S. Among those most affected by the pandemic are marginalized families, including those that are undocumented and mixed-status, in which some, but not all members are undocumented. Undocumented and mixed-status families face multiple and chronic daily stressors that compromised their health and wellbeing. Salient stressors faced by undocumented Latinx families include poverty, social disadvantage, discrimination, dangerous living and working conditions, and limited access to healthcare. These stressors are frequently compounded with trauma, fear of detention, deportation, and family separation. PURPOSE: Informed by the literature and insights from our community-based work to address the health needs of undocumented and mixed status Latinx families during the pandemic, this paper uses a social determinants of health lens to present a narrative summary that highlights four primary psychosocial stressors faced by these families and their implications for mental health. DISCUSSION: These include stressors pertaining to (a) anti-immigrant rhetoric and actions; (b) family stressors and disruptions in family dynamics; (c) economic changes and financial losses; and (c) limited access to healthcare. Implications of the aforesaid stressors on the mental health of undocumented families and youth are also discussed. In addition, recommendations are provided for the provision of mental health services, best practices, and resources from a strengths-based approach.

Identification of a second 16-hydroxytabersonine-O-methyltransferase suggests an evolutionary relationship between alkaloid and flavonoid metabolisms in Catharanthus roseus.

The medicinal plant Catharanthus roseus biosynthesizes many important drugs for human health, including the anticancer monoterpene indole alkaloids (MIAs) vinblastine and vincristine. Over the past decades, the continuous increase in pharmaceutical demand has prompted several research groups to characterize MIA biosynthetic pathways for considering future metabolic engineering processes of supply. In line with previous work suggesting that diversification can potentially occur at various steps along the vindoline branch, we were here interested in investigating the involvement of distinct isoforms of tabersonine-16-O-methyltransferase (16OMT) which plays a pivotal role in the MIA biosynthetic pathway. By combining homology searches based on the previously characterized 16OMT1, phylogenetic analyses, functional assays in yeast, and biochemical and in planta characterizations, we identified a second isoform of 16OMT, referred to as 16OMT2. 16OMT2 appears to be a multifunctional enzyme working on both MIA and flavonoid substrates, suggesting that a constrained evolution of the enzyme for accommodating the MIA substrate has probably occurred to favor the apparition of 16OMT2 from an ancestral specific flavonoid-O-methyltransferase. Since 16OMT1 and 16OMT2 displays a high sequence identity and similar kinetic parameters for 16-hydroxytabersonine, we postulate that 16OMT1 may result from a later 16OMT2 gene duplication accompanied by a continuous neofunctionalization leading to an almost complete loss of flavonoid O-methyltransferase activity. Overall, these results participate in increasing our knowledge on the evolutionary processes that have likely led to enzyme co-optation for MIA synthesis.

"No one left behind": A social determinant of health lens to the wellbeing of undocumented immigrants.

Undocumented and mixed-status families, in which some, but not all members have an undocumented immigration legal status, often face multiple and chronic stressors that influence their health. Compounded by trauma and limited opportunities, the aforementioned stressors negatively impact the physical and mental health of the undocumented and mixed-status population. This article uses a social determinants of health (SDOH) framework, examining five key domains--economic stability, education, healthcare, the social and community context, and the neighborhood and built environment. Undocumented immigrants face challenging living, social and work environments, have less economic stability, and have restricted access to opportunities. The aforesaid challenges place undocumented and mixed-status families at risk. Strategies are discussed to improve the health outcomes of this vulnerable population.

A Rapid and Efficient Vacuum-Based Agroinfiltration Protocol for Transient Gene Overexpression in Leaves of Catharanthus roseus.

Functional genomics analyses in planta can be hampered in non-model plants that are recalcitrant to the genetic transformation such as the medicinal plant Catharanthus roseus (Apocynaceae). No stable transformation and regeneration of plantlets have been achieved with a high efficiency in this plant to date. In addition, while virus-mediated transient gene silencing has been reported a decade ago in C. roseus, tools for transient overexpression remain scarce. Here, we describe an efficient and reliable methodology for transiently overexpressing any gene of interest in C. roseus leaves. This protocol combines a vacuum-based Agroinfiltration approach and the high translational efficiency of a deconstructed virus-based binary vector (pEAQ-HT). The described methodology is robust, easy to perform, and results in high amount of transient expression in C. roseus. This protocol is expected to serve as valuable tool to enhance the in planta characterization of gene functions or even transiently knock-in novel enzymatic activities.

Implementing a Departure Lounge: A Strategy to Improve Patient Flow and the Discharge Process.

This column presents the development of a Departure Lounge at Cedars Sinai Medical Center as a mechanism to assist in addressing capacity constraints. Departure lounges have been presented as an option to improve hospital throughput by providing a safe space for discharged patients to wait once medical and nursing care has been completed.

Tonoplast and Peroxisome Targeting of γ-tocopherol N-methyltransferase Homologs Involved in the Synthesis of Monoterpene Indole Alkaloids.

Many plant species from the Apocynaceae, Loganiaceae and Rubiaceae families evolved a specialized metabolism leading to the synthesis of a broad palette of monoterpene indole alkaloids (MIAs). These compounds are believed to constitute a cornerstone of the plant chemical arsenal but above all several MIAs display pharmacological properties that have been exploited for decades by humans to treat various diseases. It is established that MIAs are produced in planta due to complex biosynthetic pathways engaging a multitude of specialized enzymes but also a complex tissue and subcellular organization. In this context, N-methyltransferases (NMTs) represent an important family of enzymes indispensable for MIA biosynthesis but their characterization has always remained challenging. In particular, little is known about the subcellular localization of NMTs in MIA-producing plants. Here, we performed an extensive analysis on the subcellular localization of NMTs from four distinct medicinal plants but also experimentally validated that two putative NMTs from Catharanthus roseus exhibit NMT activity. Apart from providing unprecedented data regarding the targeting of these enzymes in planta, our results point out an additional layer of complexity to the subcellular organization of the MIA biosynthetic pathway by introducing tonoplast and peroxisome as new actors of the final steps of MIA biosynthesis.

Optimization of Tabersonine Methoxylation to Increase Vindoline Precursor Synthesis in Yeast Cell Factories.

Plant specialized metabolites are widely used in the pharmaceutical industry, including the monoterpene indole alkaloids (MIAs) vinblastine and vincristine, which both display anticancer activity. Both compounds can be obtained through the chemical condensation of their precursors vindoline and catharanthine extracted from leaves of the Madagascar periwinkle. However, the extensive use of these molecules in chemotherapy increases precursor demand and results in recurrent shortages, explaining why the development of alternative production approaches, such microbial cell factories, is mandatory. In this context, the precursor-directed biosynthesis of vindoline from tabersonine in yeast-expressing heterologous biosynthetic genes is of particular interest but has not reached high production scales to date. To circumvent production bottlenecks, the metabolic flux was channeled towards the MIA of interest by modulating the copy number of the first two genes of the vindoline biosynthetic pathway, namely tabersonine 16-hydroxylase and tabersonine-16-O-methyltransferase. Increasing gene copies resulted in an optimized methoxylation of tabersonine and overcame the competition for tabersonine access with the third enzyme of the pathway, tabersonine 3-oxygenase, which exhibits a high substrate promiscuity. Through this approach, we successfully created a yeast strain that produces the fourth biosynthetic intermediate of vindoline without accumulation of other intermediates or undesired side-products. This optimization will probably pave the way towards the future development of yeast cell factories to produce vindoline at an industrial scale.

Enhanced bioproduction of anticancer precursor vindoline by yeast cell factories.

The pharmaceutical industry faces a growing demand and recurrent shortages in many anticancer plant drugs given their extensive use in human chemotherapy. Efficient alternative strategies of supply of these natural products such as bioproduction by microorganisms are needed to ensure stable and massive manufacturing. Here, we developed and optimized yeast cell factories efficiently converting tabersonine to vindoline, a precursor of the major anticancer alkaloids vinblastine and vincristine. First, fine-tuning of heterologous gene copies restrained side metabolites synthesis towards vindoline production. Tabersonine to vindoline bioconversion was further enhanced through a rational medium optimization (pH, composition) and a sequential feeding strategy. Finally, a vindoline titre of 266 mg l-1 (88% yield) was reached in an optimized fed-batch bioreactor. This precursor-directed synthesis of vindoline thus paves the way towards future industrial bioproduction through the valorization of abundant tabersonine resources.

Alternative splicing creates a pseudo-strictosidine ß-d-glucosidase modulating alkaloid synthesis in Catharanthus roseus.

Deglycosylation is a key step in the activation of specialized metabolites involved in plant defense mechanisms. This reaction is notably catalyzed by ß-glucosidases of the glycosyl hydrolase 1 (GH1) family such as strictosidine ß-d-glucosidase (SGD) from Catharanthus roseus. SGD catalyzes the deglycosylation of strictosidine, forming a highly reactive aglycone involved in the synthesis of cytotoxic monoterpene indole alkaloids (MIAs) and in the crosslinking of aggressor proteins. By exploring C. roseus transcriptomic resources, we identified an alternative splicing event of the SGD gene leading to the formation of a shorter isoform of this enzyme (shSGD) that lacks the last 71-residues and whose transcript ratio with SGD ranges from 1.7% up to 42.8%, depending on organs and conditions. Whereas it completely lacks ß-glucosidase activity, shSGD interacts with SGD and causes the disruption of SGD multimers. Such disorganization drastically inhibits SGD activity and impacts downstream MIA synthesis. In addition, shSGD disrupts the metabolic channeling of downstream biosynthetic steps by hampering the recruitment of tetrahydroalstonine synthase in cell nuclei. shSGD thus corresponds to a pseudo-enzyme acting as a regulator of MIA biosynthesis. These data shed light on a peculiar control mechanism of ß-glucosidase multimerization, an organization common to many defensive GH1 members.

Hormonal Carcinogenesis in Canine Mammary Cancer: Molecular Mechanisms of Estradiol Involved in Malignant Progression.

Mammary cancer is a frequent neoplasia in female dogs, in which most important risk factors are hormonal. Sexual hormones as estradiol play an important role in mammary carcinogenesis, being able to induce carcinogenic initiation, promotion and progression. However, the molecular mechanisms involved are incompletely understood. Estradiol is synthesized mainly in the ovaries, nevertheless, high concentrations of estradiol and some of its hormonal precursors have also been described in malignant mammary tumor tissue. The mechanisms of action of estradiol include the classic genomic effects that modulate gene transcription, and non-genomic effects, which trigger quick effects after estradiol binds to its specific receptors. These responses modulate various intracellular signaling pathways, triggering post-translational modification of several proteins. This review will discuss the well-known underlying mechanisms associated with the action of estradiol in the malignant progression of canine mammary tumors.

Identifying Genes Involved in alkaloid Biosynthesis in Vinca minor Through Transcriptomics and Gene Co-Expression Analysis.

The lesser periwinkle Vinca minor accumulates numerous monoterpene indole alkaloids (MIAs) including the vasodilator vincamine. While the biosynthetic pathway of MIAs has been largely elucidated in other Apocynaceae such as Catharanthus roseus, the counterpart in V. minor remains mostly unknown, especially for reactions leading to MIAs specific to this plant. As a consequence, we generated a comprehensive V. minor transcriptome elaborated from eight distinct samples including roots, old and young leaves exposed to low or high light exposure conditions. This optimized resource exhibits an improved completeness compared to already published ones. Through homology-based searches using C. roseus genes as bait, we predicted candidate genes for all common steps of the MIA pathway as illustrated by the cloning of a tabersonine/vincadifformine 16-O-methyltransferase (Vm16OMT) isoform. The functional validation of this enzyme revealed its capacity of methylating 16-hydroxylated derivatives of tabersonine, vincadifformine and lochnericine with a Km 0.94 ± 0.06 µM for 16-hydroxytabersonine. Furthermore, by combining expression of fusions with yellow fluorescent proteins and interaction assays, we established that Vm16OMT is located in the cytosol and forms homodimers. Finally, a gene co-expression network was performed to identify candidate genes of the missing V. minor biosynthetic steps to guide MIA pathway elucidation.

A Biolistic-Mediated Virus-Induced Gene Silencing in Apocynaceae to Map Biosynthetic Pathways of Alkaloids.

Monoterpene indole alkaloids (MIAs) are specialized metabolites synthesized in many plants of the Apocynaceae family including Catharanthus roseus and Rauvolfia sp. MIAs are part of the chemical arsenal that plants evolved to face pet and herbivore attacks, and their high biological activities also confer pharmaceutical properties exploited in human pharmacopeia. Developing robust and straightforward tools to elucidate each step of MIA biosynthetic pathways thus constitutes a prerequisite to the understanding of Apocynaceae defense mechanisms and to the exploitation of MIA cytotoxicity through their production by metabolic engineering. While protocols of virus-induced gene silencing (VIGS) based on Agrobacterium-based transformation have emerged, the recalcitrance of Apocynaceae to this type of transformation prompted us to develop an universal procedure of VIGS vector inoculation. Such procedure relies on the delivery of the transforming plasmids through a particle bombardment performed using a biolistic device and offers the possibility to overcome host specificity to silence genes in any plant species. Using silencing of geissoschizine oxidase as an example, we described the main steps of this biolistic mediated VIGS in C. roseus and R. tetraphylla.

In vitro Treatment of a Murine Mammary Adenocarcinoma Cell Line with Recombinant Trypanosoma cruzi Calreticulin Promotes Immunogenicity and Phagocytosis.

American Trypanosomiasis, a parasitic disease produced by Trypanosoma cruzi (T. cruzi), endemic in Latin America, infects about 6 million people. During the chronic stage of the infection, approximately 30% of infected people will develop Chagas Disease, the clinical manifestation. Few decades ago it was reported that, during the chronic stage, the parasite interferes with the development of solid tumors. However, the identification of parasite molecules responsible for such effects remained elusive. Years later, we described T.cruzi Calreticulin (TcCalr), an endoplasmic reticulum resident chaperone that infective trypomastigotes translocate to the parasite exterior, where it displays anticomplement activities. Most likely, at least some of these activities are related with the antitumor properties of TcCalr, as shown in in vitro, ex vivo, in ovum, and in vivo models. In this context we, we have seen that in vivo subcutaneous peritumoral inoculation of rTcCalr enhances local infiltration of T cells and slows tumor development. Based on these precedents, we propose that in vitro treatment of a mammary adenocarcinoma (TA3 cell line) with rTcCalr, will enhance tumor immunogenicity. In agreement with this proposal, we have shown that: i). rTcCalr binds to TA3 cells in a concentration-dependent fashion, ii). C1q binds to TA3 cells in an rTcCalr-dependent fashion, confirmed by the reversion attained using anti-TcS (a central TcCalr domain that binds C1) F(ab')2 antibody fragments, iii). incubation of TA3 cells with rTcCalr, promotes cell phagocytosis by murine macrophages and, iv). rTcCalr decreases the membrane expression of MHC class II, m-Dectin-1, Galectin-9 and PD-L1, while increasing the expression of Rae-1γ. In synthesis, herein we show that in vitro treatment of a murine mammary adenocarcinoma with rTcCalr enhances phagocytosis and modulates the expression of a variety of membrane molecules that correlates with increased tumor immunogenicity.

Diferencias en la competencia motriz percibida de una población infantil según la región geográfica / Differences in the perceived motor competence of children according to the geographical region

Esta investigación tuvo como propósito comparar la competencia motriz percibida en una población infantil (M ± DE 7.96 ± 0.74 años de edad), de dos regiones geográficas diferentes de un mismo país (rural y urbano); además se examinó, las diferencias entre niños y niñas. En total 55 escolares, de los cuales 15 niños y 17 niñas pertenecían a la región geográfica rural y 15 niños y 8 niñas pertenecían a la urbana. Se evaluó la competencia motriz percibida utilizando Pictorial Scale for Perceived Movement Skill Competence for Young Children. Después de aplicar análisis de varianza de 2 vías para grupos independientes (zona X sexo), se encontró que hay diferencias estadísticamente significativas entre niños y niñas en manipulación percibida, independientemente la región geográfica, siendo los niños los que presentan mayor manipulación percibida. No se encontró interacción estadísticamente significativa en la locomoción percibida, la manipulación percibida, el desempeño motor grueso percibido y la competencia motriz percibida, según la región geográfica y sexo. Se concluye que sin importar la región geográfica los niños tienen mayor manipulación percibida que las niñas. La región en la que vivían las poblaciones infantiles medidas, no influye en la competencia motriz percibida. Se recomienda en futuras investigaciones descriptivas o experimentales, utilizar y analizar esta prueba de percepción, de manera completa, debido a que se está usando de manera global y se podrían profundizar aspectos relevantes sobre este tema

The purpose of this research was to compare the perceived motor competence in children (mean age 7.96 ± 0.74 yrs.), from two different geographical regions (rural and urban), also the differences between boys and girls were examined. A total of 55 children of which 15 boys and 17 girls lived in the rural geographical region and 15 boys and 8 girls lived in the urban one. Perceived motor competence was assessed using Pictorial Scale for Perceived Movement Skills Competence for Young Children. A two-way ANOVA indicated that there are statistically significant differences between boys and girls in perceived object control, regardless of geographical region, with boys presenting greater perceived object control than girls, and there was no statistically significant interaction in perceived locomotion, perceived object control, perceived gross motor competence and perceived motor competence, according to the geographic region and sex. In conclusion, regardless of the geographical region, boys have higher perceived object control than girls; the region in which the children lived, did not influences the perceived motor competence. It is recommended, for future descriptive or experimental research to use and analyze this perception test, in a complete way, because it is being used globally and relevant aspects of this topic could be extended

New Insight into HPts as Hubs in Poplar Cytokinin and Osmosensing Multistep Phosphorelays: Cytokinin Pathway Uses Specific HPts.

We have previously identified proteins in poplar which belong to an osmosensing (OS) signaling pathway, called a multistep phosphorelay (MSP). The MSP comprises histidine-aspartate kinases (HK), which act as membrane receptors; histidine phosphotransfer (HPt) proteins, which act as phosphorelay proteins; and response regulators (RR), some of which act as transcription factors. In this study, we identified the HK proteins homologous to the Arabidopsis cytokinin (CK) receptors, which are first partners in the poplar cytokinin MSP, and focused on specificity of these two MSPs (CK and OS), which seem to share the same pool of HPt proteins. Firstly, we isolated five CK HKs from poplar which are homologous to Arabidopsis AHK2, AHK3, and AHK4, namely, HK2, HK3a, HK3b, HK4a, HK4b. These HKs were shown to be functional kinases, as observed in a functional complementation of a yeast HK deleted strain. Moreover, one of these HKs, HK4a, was shown to have kinase activity dependent on the presence of CK. Exhaustive interaction tests between these five CK HKs and the 10 HPts characterized in poplar were performed using two-hybrid and BiFC experiments. The resulting partnership was compared to that previously identified between putative osmosensors HK1a/1b and HPt proteins. Finally, in planta coexpression analysis of genes encoding these potential partners revealed that almost all HPts are coexpressed with CK HKs in four different poplar organs. Overall, these results allowed us to unravel the common and specific partnerships existing between OS and CK MSP in Populus.

Possibilidades de intervenção do psicólogo escolar na educação inclusiva / Possibilities for intervention of the school psychologist in inclusive education

Este artigo resulta de uma pesquisa de Iniciação Científica que objetivou fazer um mapeamento de ações na área da Psicologia Escolar Educacional de práticas inclusivas dentro das escolas e que, efetivamente, promovam o desenvolvimento dos sujeitos. Investigamos as práticas realizadas nas escolas com base nos conhecimentos da Psicologia e que se mostram efetivas no que concerne à promoção da inclusão na perspectiva teórico-metodológica da Psicologia Histórico-cultural. A pesquisa é de natureza bibliográfica e abordagem qualitativa, se propondo a fazer um mapeamento com análise crítica de práticas de inclusão objetivando a comparação de informações. Os resultados apontam que apesar da importância das ações mapeadas estas são ainda incipientes, com muitas lacunas a serem preenchidas por educadores e psicólogos para práticas realmente inclusivas.

This paper presents a scientific initiation study aimed at mapping actions in the area of ​​Educational School Psychology for inclusive practices that can effectively promote the development of the subjects. We investigate the practices adopted in schools based on the knowledge and principles of psychology and that are effective in promoting the inclusion within the theoretical-methodological perspective of Historical-Cultural Psychology. The research has a bibliographic and a qualitative approach proposing to make a roadmap and a critical analysis of inclusion practices in order to compare the information obtained. The results point out that, despite the importance of such actions, they are still incipient, with many gaps to be filled by educators and psychologists for truly inclusive practices.

27-hydroxycholesterol and the expression of three estrogen-sensitive proteins in MCF7 cells.

The principal aim of this study was to analyze in estrogen receptor-positive MCF7 cells the response of three estrogen-dependent proteins to 27-hydroxycholesterol (27OHC), a major circulating cholesterol metabolite. Immunofluorescence, immunoblotting and immunogold labelling analyses of MCF7 cells exposed for up to 72 h to 2 nM estradiol (E2) or to 2 µM 27OHC demonstrated similar responses in the expression of MnSOD and ERß compared to the non-stimulated cells. Thus, the results confirm 27OHC's function as a novel selective estrogen receptor modulator (SERM). The epithelial to mesenchymal transition (EMT), observed in MCF7 cells stimulated for longer than 48 h with 2 µM 27OHC, was accompanied by lower immunoreactive levels of nuclear FOXM1 in comparison to E2-treated cells. The results presented in this study are discussed taking into consideration the relationship of hypercholesterolemia, 27OHC production, ROS synthesis and macrophage infiltration, potentially occurring in obese patients with ERα-positive, infiltrated mammary tumors.

27-hydroxycholesterol induces the transition of MCF7 cells into a mesenchymal phenotype.

A decrease in the expression of E-cadherin and ß-catenin, paralleling the loss of adherens junction complex, was observed in MCF7 cells exposed for longer than 48 h to 2 µM 27-hydroxycholesterol (27OHC), indicating an epithelial-mesenchymal transition (EMT). Upon removal of 27OHC from the culture medium, the cells released by the exposure of 72 h to the oxysterol grew as loosely packed cell groups. In these cells, accumulation of E-cadherin and ß-catenin in the cytoplasm and the prolonged expression of epidermal growth factor receptor 2 (EGFR2/neu) in the plasma membrane were observed, suggesting that the acquired phenotype was related to the expression of this tyrosine kinase-growth factor receptor. The results presented here are discussed on the basis of the claimed relationship between 27OHC, hypercholesterolemia, macrophage infiltration and therapy-resistant ERα+ breast cancer incidence.