Design, synthesis and cytotoxic evaluation of peptoid analogs of an anticancer active triazolylpeptidyl penicillin

Aim: Encouraged by the antitumor activity exhibited by triazolylpeptidyl penicillins, we decided to synthesize and evaluate a library of peptoid analogs. Results: The replacement of the dipeptide unit of the reference compound, TAP7f, was investigated. In addition, the effect of the triazole linking group on the biological activity of these new derivatives was evaluated, exchanging it with a glycine spacer. The cytotoxic effect of the library compounds was determined in the B16-F0 cell line and compared with the effects on normal murine mammary gland cells. Conclusion: Among the tested compounds, peptoid 4e exhibited the highest antiproliferative activity.

Comprehensive multi-omics analysis uncovers a group of TGF-β-regulated genes among lncRNA EPR direct transcriptional targets

Long non-coding RNAs (lncRNAs) can affect multiple layers of gene expression to control crucial cellular functions. We have previously demonstrated that the lncRNA EPR, by controlling gene expression at different levels, affects cell proliferation and migration in cultured mammary gland cells and impairs breast tumor formation in an orthotopic transplant model in mice. Here, we used ChIRP-Seq to identify EPR binding sites on chromatin of NMuMG mammary gland cells overexpressing EPR and identified its trans binding sites in the genome. Then, with the purpose of relating EPR/chromatin interactions to the reshaping of the epitranscriptome landscape, we profiled histone activation marks at promoter/enhancer regions by ChIP-Seq. Finally, we integrated data derived from ChIRP-Seq, ChIP-Seq as well as RNA-Seq in a comprehensive analysis and we selected a group of bona fide direct transcriptional targets of EPR. Among them, we identified a subset of EPR targets whose expression is controlled by TGF-β with one of them—Arrdc3—being able to modulate Epithelial to Mesenchymal Transition. This experimental framework allowed us to correlate lncRNA/chromatin interactions with the real outcome of gene expression and to start defining the gene network regulated by EPR as a component of the TGF-β pathway.

Examining metastatic behavior within 3D bioprinted vasculature for the validation of a 3D computational flow model

Understanding the dynamics of circulating tumor cell (CTC) behavior within the vasculature has remained an elusive goal in cancer biology. To elucidate the contribution of hydrodynamics in determining sites of CTC vascular colonization, the physical forces affecting these cells must be evaluated in a highly controlled manner. To this end, we have bioprinted endothelialized vascular beds and perfused these constructs with metastatic mammary gland cells under physiological flow rates. By pairing these in vitro devices with an advanced computational flow model, we found that the bioprinted analog was readily capable of evaluating the accuracy and integrated complexity of a computational flow model, while also highlighting the discrete contribution of hydrodynamics in vascular colonization. This intersection of these two technologies, bioprinting and computational simulation, is a key demonstration in the establishment of an experimentation pipeline for the understanding of complex biophysical events.

Nuclear role for human Argonaute-1 as an estrogen-dependent transcription coactivator

In mammals, argonaute (AGO) proteins have been characterized for their roles in small RNA–mediated posttranscriptional and also in transcriptional gene silencing. Here, we report a different role for AGO1 in estradiol-triggered transcriptional activation in human cells. We show that in MCF-7 mammary gland cells, AGO1 associates with transcriptional enhancers of estrogen receptor α (ERα) and that this association is up-regulated by treating the cells with estrogen (E2), displaying a positive correlation with the activation of these enhancers. Moreover, we show that AGO1 interacts with ERα and that this interaction is also increased by E2 treatment, but occurs in the absence of RNA. We show that AGO1 acts positively as a coactivator in estradiol-triggered transcription regulation by promoting ERα binding to its enhancers. Consistently, AGO1 depletion decreases long-range contacts between ERα enhancers and their target promoters. Our results point to a role of AGO1 in transcriptional regulation in human cells that is independent from small RNA binding.

CHARACTERISTICS OF MAJOR HISTOCOMPATIBILITY COMPLEX EXPRESSION AND CYTOKINE PROFILES IN SPONTANEOUS CANINE TUMOURS

Recent studies have reported gene therapy as an alternative treatment for canine’s cancer. Fifty-three samples from canine patients were obtained and the expression of MHC classes I and II and CD-markers in tumour cells, subpopulations of tumour-infiltrating lymphocytes (TILs), peripheral blood lymphocytes (PBLs) through flow cytometry and tumour cell cytokines were examined through real-time reverse transcription polymerase chain reaction. These data were compared by using Biomarker Patterns Software. Mammary gland tumours (MGTs) showed low MHC I expression that can be correlated with tumour malignancy, with cut-off values of 72.58% (MGT, [Formula: see text]% and non-MGT, [Formula: see text]%). IL-8, showing a positive correlation with angiogenesis, expression in MGTs was significantly high and IL-12 expression in complex and tubulopapillary carcinomas were significantly lower than that in healthy mammary gland cells ([Formula: see text]). PBLs of dogs with MGTs had fewer T cells, particularly T helper cells, and B cells and more non-T, non-B cells, particularly malignant MGTs, than did PBLs of healthy dogs. Among all surface markers and cytokines detected, high CD3 TIL was significantly correlated with a more favourable prognosis. Cytokines, such as IL-8, -12, and -15, can be the indicators of therapeutic targets because of their specific expression patterns in specific MGT subtypes.

In vitro culture of ovine mammary gland cells expressing beta-lactoglobulin and beta-casein

A expressão in vitro de proteínas do leite e essencial para explorar as células da glândula mamaria como um modelo biológico. A desagregação tecidual via enzimática e amplamente utilizada para o estabelecimento cultivo de células mamarias. No entanto, seu efeito a longo prazo no cultivo de células da glândula mamaria ovina ainda não e bem elucidado. Este estudo tem como objetivo comparar dois métodos de dissociação tecidual, mecânico/enzimático e mecânico, para estabelecer cultivo celular de glândula mamaria ovina. A indução da diferenciação celular, por adição de soro de ovelha lactante ou soro fetal bovino, foi avaliada pelos níveis de expressão de proteínas do leite (beta-lactoglobulina, alpha s1-caseina e beta-caseína). Células mecanicamente dissociadas foram positivamente marcadas para a presença de citoqueratina 8.13, marcador para células epiteliais mamarias. Essas células são as responsáveis pela produção das proteínas do leite e são pouco marcadas para a presença de vimentina, marcador para células de origem mesenquimal. Já as células obtidas da dissociação mecânica/ enzimática foram positivamente marcadas para presença de vimentina. A maior velocidade de crescimento (células/hora) foi observado para o grupo com dissociação mecânica cultivado em meio com 10% de soro fetal bovino. As células obtidas tanto da dissociação mecânica quanto mecânica/enzimática foram capazes de expressar beta-lactoglobulina e beta-caseína, mas não alfa s1-caseina. A expressão relativa de beta-lactoglobulina não foi afetada pelo método de dissociação ou meio de cultivo. A expressão relativa da beta-caseína foi negativamente regulada para células mecanicamente dissociadas e cultivadas na presença de soro de ovelha lactante (P = 0,019). A expressão relativa da beta-caseína também foi negativamente regulada para células dissociadas de forma mecânica/enzimática e cultivadas com soro fetal bovino (P = 0,021). Nas condições do presente estudo, concluímos que o método de dissociação mecânica seguido pelo cultivo em meio com 10% de soro fetal bovino foi o método mais eficiente para induzir a expressão mRNA de proteínas do leite por células epiteliais mamarias ovinas in vitro.

Differential effects of Pax3 on expression of polysialyltransferases STX and PST in TGF-β-treated normal murine mammary gland cells

Glycosylation of certain proteins at the mammalian cell surface is an essential event in carcinogenesis. Sialylation, one type of glycosylation, can act on multiple cell-behaviors, such as migration, growth, and malignant invasion. Two polysialyltransferases, ST8Sia II (STX) and ST8Sia IV (PST), are responsible for synthesis of polysialic acid on neural cell adhesion molecule. We showed previously that STX and PST are oppositely expressed in normal murine mammary gland cells undergoing transforming growth factor-β-induced epithelial-mesenchymal transition. The molecular basis for regulation of STX and PST remained unclear. In the present study, we observed that transcription factor Pax3 upregulates STX expression, downregulates PST expression, and modulates upregulated expression of PSA, which attaches primarily to neural cell adhesion molecule to form PSA-NCAM. Overexpression of Pax3 in normal murine mammary gland cells transformed the expression of epithelial-mesenchymal transition markers E-cadherin and N-cadherin, and significantly promoted cell migration, but had no effect on cell proliferation.