RESUMEN

The goal of proteomics experiments is to identify proteins to observe changes in cellular processes and diseases. One challenge in proteomics is the removal of contaminants following protein extraction, which can limit protein identifications. Single-pot, solid-phase-enhanced sample preparation (SP3) is a cleanup technique in which proteins are captured on carboxylate-modified particles through a proposed hydrophilic-interaction-liquid-chromatography (HILIC)-like mechanism. Recent results have suggested that proteins are captured in SP3 due to a protein-aggregation mechanism. Solvent precipitation, single-pot, solid-phase-enhanced sample preparation (SP4) is a newer cleanup technique that employs protein aggregation to capture proteins without modified particles. We hypothesize that differences in capture mechanisms of SP3 and SP4 affect which proteins are identified by each cleanup technique. Herein, we assess the proteins identified and enriched using SP3 versus SP4 for MCF7 subcellular fractions and correlate protein capture in each method to protein hydrophobicity. Our results indicate that SP3 captures more hydrophilic proteins through a combination of HILIC-like and protein-aggregation mechanisms, while SP4 captures more hydrophobic proteins through a protein-aggregation mechanism. Ultimately, we demonstrate that protein-capture mechanisms are distinct, and the selection of a cleanup technique that yields high proteome coverage is dependent on protein-sample hydrophobicity. Data has been deposited into MassIVE (MSV000094130) and ProteomeXchange (PXD049965).

Asunto(s)

RESUMEN

The goal of proteomics experiments is to identify proteins to observe changes in cellular processes and diseases. One challenge in proteomics is the removal of contaminants following protein extraction, which can limit protein identification. Single-pot, solid-phase-enhanced sample preparation (SP3) is a clean-up technique in which proteins are captured on carboxylate-modified particles through a proposed hydrophilic-interaction-liquid-chromatography (HILIC)-like mechanism. However, recent results have suggested that proteins are captured in SP3 due to a protein-aggregation mechanism. Thus, solvent precipitation, single-pot, solid-phase-enhanced sample preparation (SP4) is a newer clean-up technique that employs protein-aggregation to capture proteins without modified particles. SP4 has previously enriched low-solubility proteins, though differences in protein capture could affect which proteins are detected and identified. We hypothesize that the mechanisms of capture for SP3 and SP4 are distinct. Herein, we assess the proteins identified and enriched using SP3 versus SP4 for MCF7 subcellular fractions and correlate protein capture in each method to protein hydrophobicity. Our results indicate that SP3 captures more hydrophilic proteins through a combination of HILIC-like and protein-aggregation mechanisms, while SP4 captures more hydrophobic proteins through a protein-aggregation mechanism. From these results, we recommend clean-up techniques based on protein-sample hydrophobicity to yield high proteome coverage in biological samples.

RESUMEN

Biological nanoparticles, such as bacterial outer membrane vesicles (OMVs), are routinely characterized through transmission electron microscopy (TEM). In this study, we report a novel method to prepare OMVs for TEM imaging. To preserve vesicular shape and structure, we developed a dual fixation protocol involving osmium tetroxide incubation prior to negative staining with uranyl acetate. Combining osmium tetroxide with uranyl acetate resulted in preservation of sub-50 nm vesicles and improved morphological stability, enhancing characterization of lipid-based nanoparticles by TEM.

Asunto(s)

RESUMEN

Covering: 1995 to 2022Tumors possess both genetic and phenotypic heterogeneity leading to the survival of subpopulations post-treatment. The term cancer stem cells (CSCs) describes a subpopulation that is resistant to many types of chemotherapy and which also possess enhanced migratory and anchorage-independent growth capabilities. These cells are enriched in residual tumor material post-treatment and can serve as the seed for future tumor re-growth, at both primary and metastatic sites. Elimination of CSCs is a key goal in enhancing cancer treatment and may be aided by application of natural products in conjunction with conventional treatments. In this review, we highlight molecular features of CSCs and discuss synthesis, structure-activity relationships, derivatization, and effects of six natural products with anti-CSC activity.

Asunto(s)

RESUMEN

Epithelial-mesenchymal transition (EMT) and its reverse mesenchymal-epithelial transition (MET) are critical during embryonic development, wound healing and cancer metastasis. While phenotypic changes during short-term EMT induction are reversible, long-term EMT induction has been often associated with irreversibility. Here, we show that phenotypic changes seen in MCF10A cells upon long-term EMT induction by TGFß need not be irreversible, but have relatively longer time scales of reversibility than those seen in short-term induction. Next, using a phenomenological mathematical model to account for the chromatin-mediated epigenetic silencing of the miR-200 family by ZEB family, we highlight how the epigenetic memory gained during long-term EMT induction can slow the recovery to the epithelial state post-TGFß withdrawal. Our results suggest that epigenetic modifiers can govern the extent and time scale of EMT reversibility and advise caution against labelling phenotypic changes seen in long-term EMT induction as 'irreversible'.

Asunto(s)

RESUMEN

Epithelial-mesenchymal transition (EMT) and its reversal, mesenchymal-epithelial transition (MET) drive tissue reorganization critical for early development. In carcinomas, processing through EMT, MET, or partial states promotes migration, invasion, dormancy, and metastatic colonization. As a reversible process, EMT is inherently regulated at epigenetic and epigenomic levels. To understand the epigenomic nature of reversible EMT and its partial states, we characterized chromatin accessibility dynamics, transcriptomic output, protein expression, and cellular phenotypes during stepwise reversible EMT. We find that the chromatin insulating protein machinery, including CTCF, is suppressed and re-expressed, coincident with broad alterations in chromatin accessibility, during EMT/MET, and is lower in triple-negative breast cancer cell lines with EMT features. Through an analysis of chromatin accessibility using ATAC-seq, we identify that early phases of EMT are characterized by enrichment for AP-1 family member binding motifs, but also by a diminished enrichment for CTCF binding motifs. Through a loss-of-function analysis, we demonstrate that the suppression of CTCF alters cellular plasticity, strengthening the epithelial phenotype via the upregulation of epithelial markers E-cadherin/CDH1 and downregulation of N-cadherin/CDH2. Conversely, the upregulation of CTCF leads to the upregulation of EMT gene expression and an increase in mesenchymal traits. These findings are indicative of a role of CTCF in regulating epithelial-mesenchymal plasticity and gene expression.

RESUMEN

Triple-negative breast cancers affect thousands of women in the United States and disproportionately drive mortality from breast cancer. MicroRNAs are small, non-coding RNAs that negatively regulate gene expression post-transcriptionally by inhibiting target mRNA translation or by promoting mRNA degradation. We have identified that miRNA-203, silenced by epithelial-mesenchymal transition (EMT), is a tumor suppressor and can promote differentiation of breast cancer stem cells. In this study, we tested the ability of liposomal delivery of miR-203 to reverse aspects of breast cancer pathogenesis using breast cancer and EMT cell lines. We show that translationally relevant methods for increasing miR-203 abundance within a target tissue affects cellular properties associated with cancer progression. While stable miR-203 expression suppresses LASP1 and survivin, nanoliposomal delivery suppresses BMI1, indicating that suppression of distinct mRNA target profiles can lead to loss of cancer cell migration.

RESUMEN

Alterations in diversity and function of the gut microbiome are associated with concomitant changes in immune response, including chronic inflammation. Chronic inflammation is a major risk factor for colorectal cancer (CRC). An important component of the inflammatory response system are the toll-like receptors (TLRs). TLRs are capable of sensing microbial components, including nucleic acids, lipopolysaccharides and peptidoglycans, as well as bacterial outer membrane vesicles (OMV). OMVs can be decorated with or carry as cargo these TLR activating factors. These microbial factors can either promote tolerance or activate signaling pathways leading to chronic inflammation. Herein we discuss the role of the microbiome and the OMVs that originate from intestinal bacteria in promoting chronic inflammation and the development of colitis-associated CRC. We also discuss the contribution of TLRs in mediating the microbiome-inflammation axis and subsequent cancer development. Understanding the role of the microbiome and its secretory factors in TLR response may lead to the development of better cancer therapeutics.

Asunto(s)

RESUMEN

The epithelial-mesenchymal transition (EMT) imparts properties of cancer stem-like cells, including resistance to frequently used chemotherapies, necessitating the identification of molecules that induce cell death specifically in stem-like cells with EMT properties. Herein, we demonstrate that breast cancer cells enriched for EMT features are more sensitive to cytotoxicity induced by ophiobolin A (OpA), a sesterterpenoid natural product. Using a model of experimentally induced EMT in human mammary epithelial (HMLE) cells, we show that EMT is both necessary and sufficient for OpA sensitivity. Moreover prolonged, sub-cytotoxic exposure to OpA is sufficient to suppress EMT-imparted CSC features including sphere formation and resistance to doxorubicin. In vivo growth of CSC-rich mammary cell tumors, is suppressed by OpA treatment. These data identify a driver of EMT-driven cytotoxicity with significant potential for use either in combination with standard chemotherapy or for tumors enriched for EMT features.

Asunto(s)

RESUMEN

We are glad to share with you our eighth Journal Club and to highlight some of the most interesting papers published recently [...].

RESUMEN

Staurosporine is among the most potent naturally occurring kinase inhibitors isolated to date and has served as a lead compound for numerous drug development efforts in several therapeutic areas. Herein we report that C-H borylation chemistry provides access to analogs of staurosporine that were previously inaccessible to medicinal chemists who, in the past four decades, have prepared over 1000 semisynthetic staurosporine analogs.

RESUMEN

Pharmacophore-directed retrosynthesis applied to ophiobolin A led to bicyclic derivatives that were synthesized and display anticancer activity. Key features of the ultimate defensive synthetic strategy include a Michael addition/facially selective protonation sequence to set the critical C6 stereocenter and a ring-closing metathesis to form the cyclooctene. Cytotoxicity assays toward a breast cancer cell line (MDA-MB-231) confirm the anticipated importance of structural complexity for selectivity (vs MCF10A cells) while C3 variations modulate stability.

Asunto(s)

RESUMEN

Properly timed addition and removal of histone 3 lysine 27 tri-methylation (H3K27me3) is critical for enabling proper differentiation throughout all stages of development and, likewise, can guide carcinoma cells into altered differentiation states which correspond to poor prognoses and treatment evasion. In early embryonic stages, H3K27me3 is invoked to silence genes and restrict cell fate. Not surprisingly, mutation or altered functionality in the enzymes that regulate this pathway results in aberrant methylation or demethylation that can lead to malignancy. Likewise, changes in expression or activity of these enzymes impact cellular plasticity, metastasis, and treatment evasion. This review focuses on current knowledge regarding methylation and de-methylation of H3K27 in cancer initiation and cancer cell plasticity.

RESUMEN

BACKGROUND: The term triple-negative breast cancer (TNBC) is used to describe breast cancers without expression of estrogen receptor, progesterone receptor or HER2 amplification. To advance targeted treatment options for TNBC, it is critical that the subtypes within this classification be described in regard to their characteristic biology and gene expression. The Cancer Genome Atlas (TCGA) dataset provides not only clinical and mRNA expression data but also expression data for microRNAs. RESULTS: In this study, we applied the Lehmann classifier to TCGA-derived TNBC cases which also contained microRNA expression data and derived subtype-specific microRNA expression patterns. Subsequent analyses integrated known and predicted microRNA-mRNA regulatory nodes as well as patient survival data to identify key networks. Notably, basal-like 1 (BL1) TNBCs were distinguished from basal-like 2 TNBCs through up-regulation of members of the miR-17-92 cluster of microRNAs and suppression of several known miR-17-92 targets including inositol polyphosphate 4-phosphatase type II, INPP4B. CONCLUSIONS: These data demonstrate TNBC subtype-specific microRNA and target mRNA expression which may be applied to future biomarker and therapeutic development studies.

Asunto(s)

RESUMEN

Fragile X syndrome (FXS) is a neurodevelopmental disorder caused by a single genetic mutation in the Fmr1 gene, serving as the largest genetic cause of intellectual disability. Trinucleotide expansion mutations in Fmr1 result in silencing and hypermethylation of the gene, preventing synthesis of the RNA binding protein Fragile X mental retardation protein which functions as a translational repressor. Abnormal immune responses have been demonstrated to play a role in FXS pathophysiology, however, whether these alterations impact how those with FXS respond to an immune insult behaviorally is not entirely known. In the current study, we examine how Fmr1 knockout (KO) and wild type (WT) mice respond to the innate immune stimulus lipopolysaccharide (LPS), both on a molecular and behavioral level, to determine if Fmr1 mutations impact the normal physiological response to an immune insult. In response to LPS, Fmr1 KO mice had elevated hippocampal IL-1ß and IL-6 mRNA levels 4 h post-treatment compared to WT mice, with no differences detected in any cytokines at baseline or between genotypes 24 h post-LPS administration. Fmr1 KO mice also had upregulated hippocampal BDNF gene expression 4 h post-treatment compared to WT mice, which was not dependent on LPS administration. There were no differences in hippocampal protein expression between genotypes in microglia (Iba1) or astrocyte (GFAP) reactivity. Further, both genotypes displayed the typical sickness response following LPS stimulation, demonstrated by a significant reduction in food burrowed by LPS-treated mice in a burrowing task. Additional investigation is critical to determine if the transient increases in cytokine expression could lead to long-term changes in downstream molecular signaling in FXS.

Asunto(s)

RESUMEN

Introduction: The use of liposomes as a drug delivery carrier (DDC) for the treatment of various diseases, especially cancer, is rapidly increasing, requiring more stringent synthesis, formulation, and preservation techniques to bolster safety and efficacy. Liposomes otherwise referred to as phospholipid vesicles are self-assembled colloidal particles. When formed in either the micrometer or nanometer size range, they are ideal candidates as DDC because of their biological availability, performance, activity, and compatibility. Defining and addressing the critical quality attributes (CQAs) along the pharmaceutical production scale will enable a higher level of quality control for reproducibility. More specifically, understanding the CQAs of nanoliposomes that dictate its homogeneity and stability has the potential to widen applications in biomedical science. Methods: To this end, we designed a study that aimed to define synthesis, characterization, formulation (encapsulation), preservation, and cargo delivery and trafficking as the major components within a target product profile for nanoliposomes. A series of synthetic schemes were employed to measure physicochemical properties relevant to nanomaterial drug product development, including concentration gradients, probe versus bath sonication, and storage temperature measured by microscopy (electron and light) and dynamic light scattering. Results: Concentration was found to be a vital CQA as reducing concentrations resulted in nanometer-sized liposomes of <350 nm. Liposomes were loaded with microRNA and fluorescence spectroscopy was used to determine loading efficacy and stability over time. Lyophilization was used to create a dry powder formulation that was then assessed for stability for 6 months. Lastly, breast cancer cell lines were used to ensure efficacy of microRNA delivery and localization. Conclusion: We conclude that microRNA can be loaded into nanometer-sized liposomes, preserved for months in a dried form, and maintain encapsulation after extended time periods in storage.

Asunto(s)

RESUMEN

BACKGROUND: Triple-negative breast cancers (TNBCs), which lack receptors for estrogen, progesterone, and amplification of epidermal growth factor receptor 2, are highly aggressive. Consequently, patients diagnosed with TNBCs have reduced overall and disease-free survival rates compared to patients with other subtypes of breast cancer. TNBCs are characterized by the presence of cancer cells with mesenchymal properties, indicating that the epithelial to mesenchymal transition (EMT) plays a major role in the progression of this disease. The EMT program has also been implicated in chemoresistance, tumor recurrence, and induction of cancer stem cell (CSC) properties. Currently, there are no targeted therapies for TNBC, and hence, it is critical to identify the novel targets to treat TNBC. METHODS: A library of compounds was screened for their ability to inhibit EMT in cells with mesenchymal phenotype as assessed using the previously described Z-cad reporters. Of the several drugs tested, GSK3ß inhibitors were identified as EMT inhibitors. The effects of GSK3ß inhibitors on the properties of TNBC cells with a mesenchymal phenotype were assessed using qRT-PCR, flow cytometry, western blot, mammosphere, and migration and cell viability assays. Publicly available datasets also were analyzed to examine if the expression of GSK3ß correlates with the overall survival of breast cancer patients. RESULTS: We identified a GSK3ß inhibitor, BIO, in a drug screen as one of the most potent inhibitors of EMT. BIO and two other GSK3ß inhibitors, TWS119 and LiCl, also decreased the expression of mesenchymal markers in several different cell lines with a mesenchymal phenotype. Further, inhibition of GSK3ß reduced EMT-related migratory properties of cells with mesenchymal properties. To determine if GSK3ß inhibitors target mesenchymal-like cells by affecting the CSC population, we employed mammosphere assays and profiled the stem cell-related cell surface marker CD44+/24- in cells after exposure to GSK3ß inhibitors. We found that GSK3ß inhibitors indeed decreased the CSC properties of cell types with mesenchymal properties. We treated cells with epithelial and mesenchymal properties with GSK3ß inhibitors and found that GSK3ß inhibitors selectively kill cells with mesenchymal attributes while sparing cells with epithelial properties. We analyzed patient data to identify genes predictive of poor clinical outcome that could serve as novel therapeutic targets for TNBC. The Wnt signaling pathway is critical to EMT, but among the various factors known to be involved in Wnt signaling, only the higher expression of GSK3ß correlated with poorer overall patient survival. CONCLUSIONS: Taken together, our data demonstrate that GSK3ß is a potential target for TNBCs and suggest that GSK3ß inhibitors could serve as selective inhibitors of EMT and CSC properties for the treatment of a subset of aggressive TNBC. GSK3ß inhibitors should be tested for use in combination with standard-of-care drugs in preclinical TNBC models.

Asunto(s)

RESUMEN

The hypercalins are dearomatized acylphloroglucinols with a pendant complex cyclopentane ring that exhibit activity against several cancer cell lines. We report the first total synthesis of (+)-hypercalin C employing a convergent strategy that enabled the dissection of the essential structural features required for the observed anticancer activity. A strategic disconnection involving an unusual C sp3 -C sp2 Suzuki-Miyaura coupling with an α-bromo enolether also revealed an unexpected C-H activation. This strategy targeted designed analogues along the synthetic route to address particular biological questions. These results support the hypothesis that hypercalin C may act as a proton shuttle with the dearomatized acylphloroglucinol moiety being essential for this activity.

Asunto(s)

RESUMEN

We are delighted to share with you our sixth Journal Club and highlight some of the most interesting papers published recently [...].