Primary Mammary Epithelial Cells Research Articles

Excessive supply of glucose elicits an NF-κB2-dependent glycolysis in lactating goat mammary glands.

During lactation, an improper glucose supply often threatens mammary gland (MG) health. However, information is limited on the metabolic trajectories and molecules that regulate lactating MGs with an excessive glucose supply. Based on the network analysis of transcriptome and microRNAs, we found that the oversupply of glucose-induced severe glucose metabolic disorders in MGs of lactating goats, shifting lactose synthesis to acute fermentative glycolysis which caused increased flux of glucose metabolism into lactate. Moreover, NF-κB2 played a key role in regulating glycolysis, exhibiting a metabolic shift when MGs had an excessive supply of glucose. In primary mammary epithelial cells, fermentative glycolysis, and intracellular concentration of reactive oxygen species (ROS) were reduced by ganoderic acid A through blocking NF-κB2, while activation of NF-κB2 with phorbol myristate acetate (PMA) upregulated fermentative glycolysis and increased cellular ROS accumulation under excessive glucose. Thus, we established an NF-κB2-targeting method to reform the metabolic shift toward glycolysis caused by glucose oversupply by integrating NF-κB2 blockade and intracellular ROS scavenging.

Investigation on the suitability of milk-derived primary bovine mammary epithelial cells grown on permeable membrane supports as an in vitro model for lactation.

This study aimed to establish an in vitro model for lipid synthesis in primary bovine mammary epithelial cells (pbMECs) extracted from milk and cultured on Transwell permeable supports (TW culture). The suitability of these cells as a functional model for lactation was assessed by measuring κ-casein (CSN3) and diacylglycerol acyl transferase 1 (DGAT1) gene expression, the presence of intracellular lipid droplets, and the concentration of triacylglycerol in the cell lysates. The functionality of the milk-derived pbMECs cultured under lactogenic conditions, with and without oleic acid supplementation, was evaluated by comparing the cells grown on Transwell supports to cells grown on an extracellular matrix (ECM) gel (3D culture) or a plastic surface (2D culture). Furthermore, the functionality of milk-derived cells was compared to pbMECs obtained from bovine mammary tissue. Here, we show that in both tissue and milk-derived pbMECs, 3D culture offered the most suitable in vitro environment and led to increased levels of CSN3 and DGAT1 gene expression along with increased intracellular triacylglycerol content. The TW culture conditions also resulted in increased DGAT1 gene expression compared to the 2D conditions and milk-derived pbMECs cultured on TW inserts showed the highest viability compared to cells grown under 2D or 3D treatments. However, this was not observed for tissue-derived pbMECs, suggesting that TW culture may offer a beneficial environment specifically for milk-derived cells. We suggest that with further optimization of the culture conditions, TW culture may present a suitable model for the study of milk lipid synthesis in pbMECs.

Inhibition of SYK kinase does not confer a pro-proliferative or pro-invasive phenotype in breast epithelium or breast cancer cells.

SYK has been reported to possess both tumour promotor and repressor activities and deletion has been linked to a pro-proliferative / pro-invasive phenotype in breast tumours. It is unclear whether this is a consequence of protein deletion or loss of kinase activity. The SYK inhibitor, BI 1002494, caused no increase in proliferation in breast cancer cells or primary mammary epithelial cells in 2D or 3D cultures, nor changes in proliferation (CD1/2, CDK4, PCNA, Ki67) or invadopodia markers (MMP14, PARP, phospho-vimentin Ser56). BI 1002494 did not alter SYK protein expression. There was no change in phenotype observed in 3D cultures after addition of BI 1002494. Thirteen weeks of treatment with BI 1002494 resulted in no ductal branching or cellular proliferation in the mammary glands of mice. An in silico genetic analysis in breast tumour samples revealed no evidence that SYK has a typical tumour suppressor gene profile such as focal deletion, inactivating mutations or lower expression levels. Furthermore, SYK mutations were not associated with reduction in survival and disease-free period in breast cancer patients. In conclusion, small molecule inhibition of the kinase function of SYK does not contribute to a typical tumour suppressor profile.

Role of peroxisome proliferator-activated receptor-α on the synthesis of monounsaturated fatty acids in goat mammary epithelial cells.

A key member of the nuclear receptor superfamily is the peroxisome proliferator-activated receptor alpha (PPARA) isoform, which in nonruminants is closely associated with fatty acid oxidation. Whether PPARA plays a role in milk fatty acid synthesis in ruminants is unknown. The main objective of the present study was to use primary goat mammary epithelial cells (GMEC) to activate PPARA via the agonist WY-14643 (WY) or to silence it via transfection of small-interfering RNA (siRNA). Three copies of the peroxisome proliferator-activated receptor response element (PPRE) contained in a luciferase reporter vector were transfected into GMEC followed by incubation with WY at 0, 10, 20, 30, 50, or 100 µM. A dose of 50 µM WY was most effective at activating PPRE without influencing PPARA mRNA abundance. Transfecting siRNA targeting PPARA decreased its mRNA abundance to 20% and protein level to 50% of basal levels. Use of WY upregulated FASN, SCD1, ACSL1, DGAT1, FABP4, and CD36 (1.1-, 1.5-, 2-, 1.4-, 1.5-, and 5-fold, respectively), but downregulated DGAT2 and PGC1A (-20% and -40%, respectively) abundance. In contrast, triacylglycerol concentration decreased and the content and desaturation index of C16:1 and C18:1 increased. Thus, activation of PPARA via WY appeared to channel fatty acids away from esterification. Knockdown of PPARA via siRNA downregulated ACACA, SCD1, AGPAT6, CD36, HSL, and SREBF1 (-43%, -67%, -16%, -56%, -26%, and -29%, respectively), but upregulated ACSL1, DGAT2, FABP3, and PGC1A (2-, 1.4-, 1.3-, and 2.5-fold, respectively) mRNA abundance. A decrease in the content and desaturation index of C16:1 and C18:1 coupled with an increase in triacylglycerol content accompanied those effects at the mRNA level. Overall, data suggest that PPARA could promote the synthesis of MUFA in GMEC through its effects on mRNA abundance of genes related to fatty acid synthesis, oxidation, transport, and triacylglycerol synthesis.

Abstract P3-03-11: Notch inhibition promotes triple negative breast cancer growth

Abstract Notch1 and Transforming Growth Factor-beta (TGF-β) signaling pathways in cancer have a paradoxical roles as they were shown to have both tumor suppressive and tumor promoting activities. The interaction between these two signaling pathways in cancer progression was addressed in a systematic way. In these studies, we utilized primary human mammary epithelial cells (HMEC) and various triple negative breast cancer (TNBC) cells and examined the molecular cross-talk between TGF-β and Notch signaling pathways using biochemical, molecular and cell biology techniques. First, we noticed lower expression of Notch1 in most of the TNBC cells compared to normal cells. Second, most of these TNBC cells overexpressed TMEPAI (transmembrane prostate androgen induced) protein, which was shown to switch TGF-β from a tumor suppressor to a tumor promoter role in breast cancer. Surprisingly, treatment of TNBC cells with Notch inhibitors increased their growth rate both in the absence or presence of TGF-β. Moreover, Notch inhibition relieved the growth suppression by TGF-β in normal cells. Both normal and TNBC cells showed increased growth in the presence of Notch inhibitors with decreased TGF-β signaling activity. Normal cells that express very low levels of TMEPAI both at mRNA and protein levels, overexpressed TMEPAI protein in the presence of various Notch inhibitors, suggesting that Notch negatively regulates TMEPAI expression. Correspondingly, knockdown of TMEPAI in TNBC cell lines showed increased expression of Notch1 and Jagged 1 suggesting a negative regulation of Notch1 by TMEPAI. Consistent with these observations, TMEPAI, a known binding partner of E3 ligases, causes increased turnover of Notch1 protein. In fact, over expression of active Notch1 in MDA-MB-231 cells inhibited tumor cell growth both in vitro and in vivo. Our results clearly suggest that a crosstalk exists between TGF-β and Notch1 signaling through TMEPAI. Therefore, our studies clearly indicate that use of Notch inhibitors is contraindicated in triple negative breast cancer patients. Citation Format: Pothana Saikumar, Prajjal K Singha, Manjeri A Venkatachalam. Notch inhibition promotes triple negative breast cancer growth [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-03-11.

A P53-Independent DNA Damage Response Suppresses Oncogenic Proliferation and Genome Instability

The Mre11-Rad50-Nbs1 complex is a DNA double-strand break sensor that mediates a tumor-suppressive DNA damage response (DDR) in cells undergoing oncogenic stress, yet the mechanisms underlying this effect are poorly understood. Using a genetically inducible primary mammary epithelial cell model, we demonstrate that Mre11 suppresses proliferation and DNA damage induced by diverse oncogenic drivers through a p53-independent mechanism. Breast tumorigenesis models engineered to express a hypomorphic Mre11 allele exhibit increased levels of oncogene-induced DNA damage, R-loop accumulation, and chromosomal instability with a characteristic copy number loss phenotype. Mre11 complex dysfunction is identified in a subset of human triple-negative breast cancers and is associated with increased sensitivity to DNA-damaging therapy and inhibitors of ataxia telangiectasia and Rad3 related (ATR) and poly (ADP-ribose) polymerase (PARP). Thus, deficiencies in the Mre11-dependent DDR drive proliferation and genome instability patterns in p53-deficient breast cancers and represent an opportunity for therapeutic exploitation.

Role of Toll-like receptor 2 against Streptococcus uberis infection in primary mouse mammary epithelial cells

Mammary epithelial cells (MECs) play an important role against Streptococcus uberis infection which is one of the main causes of bovine mastitis and a potential threat to human health. Toll-like receptors (TLRs) and their mediated signaling pathways are critical in both innate and infection responses, yet their roles in anti-S. uberis infection in MECs remains poorly defined. In this work we investigated the regulatory mechanisms of TLR2 in inflammatory responses, where WT and TLR2−/− mice were euthanized at 15–18 days gestation, and mammary gland tissues were collected aseptically. The mouse MECs (MMECs) were isolated by combined digestion with type I collagenase, hyaluronidase and trypsin. We challenged MMECs with S. uberis and quantified antioxidant capacity as well as reactive oxygen species (ROS), proinflammatory cytokines and cell damage at different times. The loss of TLR2 function in MMECs results in more serious cell damage, increased cell adhesion, and significantly decreased ROS and mitochondrial ROS (mROS) with bactericidal function in response to S. uberis infection. Moreover, it was observed that the antioxidant capacity declined, and the production of TLR2-mediated cytokines (except CXC ligand 15) also were reduced. We demonstrated that TLR2 can mediate cellular anti-infective processes in MMECs by regulating the production of ROS and mROS and the secretion of cytokines. The results suggest an unpredicted role of TLR2 in MMECs in response to S. uberis infection.

Loss of SIM2s inhibits RAD51 binding and leads to unresolved replication stress

BackgroundMutations in genes associated with homologous recombination (HR) increase an individual’s risk of developing triple-negative breast cancer (TNBC). Although known for their role in repairing dsDNA breaks, HR repair elements also stabilize and restart stalled replication forks. Essential to these functions are RAD51 and its paralogs, each of which has a unique role in preventing replication fork collapse and restart. However, progress toward understanding the regulation of these factors has been slow. With such a pivotal role in the maintenance of genomic integrity, furthering our understanding of this pathway through the discovery of new factors involved in HR is important. Recently, we showed that singleminded-2s (SIM2s) is stabilized in response to dsDNA breaks and is required for effective HR.MethodsInitial analysis of the effect loss of SIM2s has on replication stress resolution was conducted using DNA combing assays in established breast cancer cell lines. Further analysis was conducted via immunostaining to determine the effect loss of SIM2s has on factor recruitment. In vivo confirmation was achieved through the use of a mammary epithelial cell conditional knockout mouse model before SIM2s’ role in RAD51 recruitment was determined by immunoblotting.ResultsHere, we show loss of SIM2s decreases replication fork stability, leading to fork collapse in response to genotoxic stress. Furthermore, loss of SIM2s results in aberrant separation of sister chromatids during mitosis, which has been previously shown to result in chromosomal fragmentation and aneuploidy. Interestingly, loss of SIM2s was shown to result in failure of RAD51 to localize to sites of replication stress in both breast cancer cell lines and primary mammary epithelial cells. Finally, we observed SIM2 is stabilized in response to genotoxic stress and interacts with RAD51, which is necessary for RAD51-DNA binding.ConclusionsTogether, these results show a role for SIM2s in the resolution of replication stress and further characterize the necessity of SIM2s for effective RAD51 loading in response to DNA damage or stress, ultimately promoting genomic integrity and thus preventing the accumulation of cancer-promoting mutations.

Quantification of Self-renewal in Murine Mammosphere Cultures.

The mammary gland is characterized by extensive regeneration capacity, as it goes through massive hormonal changes throughout the life cycle of a female. The role of mammary stem cells (MaSCs) is widely studied both in the physiological/developmental context and with regards to breast carcinogenesis. In this aspect, ex vivo studies focused on MaSC properties are highly sought after. Mammosphere cultures represent a surrogate of organ formation and have become a valuable tool for both basic and translational research. Here, we present a detailed protocol for the generation of murine primary mammosphere cultures and the quantitation of MaSC growth properties. The protocol includes mammary gland collection and digestion, isolation of primary mammary epithelial cells (MECs), establishment of primary mammosphere cultures, serial passaging, quantitation of mammosphere growth parameters and interpretation of the results. As an example, we present the effect of low-level constitutive Myc expression on normal MECs leading to increased self-renewal and proliferation.

Exogenous ERα Expression in the Mammary Epithelium Decreases Over Time and Does Not Contribute to p53-Deficient Mammary Tumor Formation in Mice.

Approximately 75% of all breast cancers express the nuclear hormone receptor estrogen receptor α (ERα). However, the majority of mammary tumors from genetically engineered mouse models (GEMMs) are ERα-negative. To model ERα-positive breast cancer in mice, we exogenously introduced expression of mouse and human ERα in an existing GEMM of p53-deficient breast cancer. After initial ERα expression during mammary gland development, expression was reduced or lost in adult glands and p53-deficient mammary tumors. Chromatin immunoprecipitation (ChIP)-sequencing analysis of primary mouse mammary epithelial cells (MMECs) derived from these models, in which expression of the ERα constructs was induced in vitro, confirmed interaction of ERα with the DNA. In human breast and endometrial cancer, and also in healthy breast tissue, DNA binding of ERα is facilitated by the pioneer factor FOXA1. Surprisingly, the ERα binding sites identified in primary MMECs, but also in mouse mammary gland and uterus, showed an high enrichment of ERE motifs, but were devoid of Forkhead motifs. Furthermore, exogenous introduction of FOXA1 and GATA3 in ERα-expressing MMECs was not sufficient to promote ERα-responsiveness of these cells. Together, this suggests that species-specific differences in pioneer factor usage between mouse and human are dictated by the DNA sequence, resulting in ERα-dependencies in mice that are not FOXA1 driven. These species-specific differences in ERα-biology may limit the utility of mice for in vivo modeling of ERα-positive breast cancer.

Methionine and arginine supplementation alter inflammatory and oxidative stress responses during lipopolysaccharide challenge in bovine mammary epithelial cells in vitro

Mastitis, inflammation of the udder, is one of the most common diseases hampering milk yield of dairy cows. Methionine (Met) and arginine (Arg) are key nutrients with potential to regulate inflammation and oxidative stress. The aim of this study was to evaluate the effect of increased supply of Met and Arg on mRNA and protein abundance associated with innate immune response and redox balance during lipopolysaccharide (LPS) stimulation in primary bovine mammary epithelial cells (BMEC). Primary BMEC (n = 4 replicates per treatment) were pre-incubated for 12 h in media with the following amino acid combinations: ideal profile of amino acids (control; Con), increased Met supply (incMet), increased Arg supply (incArg), and increased supply of Met and Arg (incMetArg). Subsequently, cells were challenged with or without LPS (1 µg/mL) and incubated for 6 h. Data were analyzed as a 2 × 2 × 2 factorial using the MIXED procedure of SAS 9.4 (SAS Institute Inc., Cary, NC). The downregulation of SLC36A1 and SLC7A1 mRNA abundance induced by LPS was attenuated in the incArg cultures. Although challenge with LPS led to lower abundance of proteins related to the antioxidant response (NFE2L2, NQO1, GPX1), lower levels of ATG7, and lower mRNA abundance of GPX3, we found little effect in cultures with incMet or incArg. Cultures with incMet, incArg, or incMetArg led to attenuation of the upregulation of SOD2 and NOS2 induced by LPS. Abundance of phosphorylated p65 (RELA) was greater after LPS stimulation, but the response was attenuated in cultures with incMet. The greater ratio of pRELA to total RELA in responses to LPS was also attenuated in cultures with incMetArg. The greater mRNA abundance of the proinflammatory cytokine IL1B induced by LPS was attenuated in cultures with incMet, and the same trend induced by LPS on CXCL2 was also alleviated in cultures with incArg. Overall, the data suggest that greater supply of Met and Arg alleviated the proinflammatory responses triggered by LPS through controlling the abundance of proinflammatory cytokines and chemokines and activity of NF-κB. Little benefit on oxidative stress induced by LPS challenge in BMEC was detected with greater supply of Met and Arg.

Productive Infection of Mouse Mammary Glands and Human Mammary Epithelial Cells by Zika Virus.

Zika virus (ZIKV) belongs to the large category of arboviruses. Surprisingly, several human-to-human transmissions of ZIKV have been notified, either following sexual intercourse or from the mother to fetus during pregnancy. Importantly, high viral loads have been detected in the human breast milk of infected mothers, and the existence of breastfeeding as a new mode of mother-to-child transmission of ZIKV was recently hypothesized. However, the maternal origin of infectious particles in breast milk is currently unknown. Here, we show that ZIKV disseminates to the mammary glands of infected mice after both systemic and local exposure with differential kinetics. Ex vivo, we demonstrate that primary human mammary epithelial cells were sensitive and permissive to ZIKV infection in this study. Moreover, by using in vitro models, we prove that mammary luminal- and myoepithelial-phenotype cell lines are both able to produce important virus progeny after ZIKV exposure. Our data suggest that the dissemination of ZIKV to the mammary glands and subsequent infection of the mammary epithelium could be one mechanism of viral excretion in human breast milk.

Chi-miR-143-3p Promotes Apoptosis of Mammary Gland Epithelial Cells from Dairy Goats by Targeting Ndfip1

The mammary gland is an important organ for lactation in dairy goats. Mammary gland development and lactation functions are primarily regulated by natural hormones and certain crucial regulatory factors. Nedd4 family-interacting protein 1 (Ndfip1) can specifically bind to neural precursor cell-expressed, developmentally downregulated protein 4 (Nedd4) family members to participate in ubiquitination, which in turn regulates a range of biological processes in the body. However, the effects of Ndfip1 expression regulation at the post-transcriptional level on the development of mammary gland cells have not been previously reported. To study the regulation of Ndfip1 at post-transcriptional level, the overexpression and interference vectors of Ndfip1 were constructed, and co-transfected into the primary mammary gland epithelial cells cultured in vitro with miR-143 mimics and inhibitor. Dual luciferase reporter gene system, real-time quantitative polymerase chain reaction, western blotting, cholecystokinin octapeptide assays, and flow cytometry were used to identify their regulation and function. As a result, Ndfip1 was targeted and regulated by miR-143, which influences the development of mammary gland epithelial cells in dairy goats cultured in vitro. This study will lay an experimental foundation for further understanding the functions of Ndfip1 and miR-143.

ASCT2 Is Involved in SARS-Mediated β-Casein Synthesis of Bovine Mammary Epithelial Cells with Methionine Supply.

The methionine (Met) uptake into mammary cells depends upon the corresponding amino acid (AA) transporters, which play a regulatory role in the mammary protein production beyond transport. Our previous studies have identified that seryl-tRNA synthetase (SARS) could be a novel mediator to regulate essential AA-stimulated casein synthesis in primary bovine mammary epithelial cells (BMECs). However, the regulatory mechanisms of Met in milk protein production in dairy cows remain further clarified. Here, we aimed to investigate the effects of Met on milk protein synthesis in BMECs and explore the underlying mechanism. The effects of Met on the AA transporter, casein synthesis, and the related signaling pathway were evaluated in the BMECs treated with 0.6 mM Met for 6 h combined with or without the inhibition of AA transporter (ASCT2, a neutral AA transporter) activity by the corresponding inhibitor (GPNA). Besides, the effects of SARS on the cells were mainly evaluated in the BMECs treated with 0.6 mM Met for 6 h together with or without SARS knockdown by RNAi interference. The gene expression of AA transporters and pathway-related genes were analyzed by the real-time quantitative polymerase chain reaction method, and the protein expression of related proteins were determined by the western blot assay. Results showed that 0.6 mM Met remarkably enhanced cell growth and β-casein synthesis compared to the supply of other Met concentrations. Among 13 amino acid transporters, 0.6 mM Met highly increased ASCT2 expression. This Met-stimulated ASCT2 expression and the enhanced mammary intracellular Met uptake were both decreased by the addition of 500 μM GPNA, an inhibitor of ASCT2. In the presence of 0.6 mM Met, the inhibition of ASCT2 activity (by GPNA) and SARS expression (by RNAi) both reduced β-casein synthesis. Additionally, 0.6 mM Met increased the gene expression of mTOR, S6K1, 4EBP1, and Akt; in contrast, the inhibition of ASCT2 by GPNA lowered the gene expression of these four genes. Collectively, this work suggests that ASCT2 is involved in the SARS-mediated Met stimulation of β-casein synthesis through enhancing mammary Met uptake and activating the mTOR signaling pathway in BMECs.

Large-Scale Analysis of the Mycoplasma bovis Genome Identified Non-essential, Adhesion- and Virulence-Related Genes.

Mycoplasma bovis is an important pathogen of cattle causing bovine mycoplasmosis. Clinical manifestations are numerous, but pneumonia, mastitis, and arthritis cases are mainly reported. Currently, no efficient vaccine is available and antibiotic treatments are not always satisfactory. The design of new, efficient prophylactic and therapeutic approaches requires a better understanding of the molecular mechanisms responsible for M. bovis pathogenicity. Random transposon mutagenesis has been widely used in Mycoplasma species to identify potential gene functions. Such an approach can also be used to screen genomes and search for essential and non-essential genes for growth. Here, we generated a random transposon mutant library of M. bovis strain JF4278 containing approximately 4000 independent insertion sites. We then coupled high-throughput screening of this mutant library to transposon sequencing and bioinformatic analysis to identify M. bovis non-essential, adhesion- and virulence-related genes. Three hundred and fifty-two genes of M. bovis were assigned as essential for growth in rich medium. Among the remaining non-essential genes, putative virulence-related factors were subsequently identified. The complete mutant library was screened for adhesion using primary bovine mammary gland epithelial cells. Data from this assay resulted in a list of conditional-essential genes with putative adhesion-related functions by identifying non-essential genes for growth that are essential for host cell-adhesion. By individually assessing the adhesion capacity of six selected mutants, two previously unknown factors and the adhesin TrmFO were associated with a reduced adhesion phenotype. Overall, our study (i) uncovers new, putative virulence-related genes; (ii) offers a list of putative adhesion-related factors; and (iii) provides valuable information for vaccine design and for exploring M. bovis biology, pathogenesis, and host-interaction.

Effects of varying extracellular amino acid profile on intracellular free amino acid concentrations and cell signaling in primary mammary epithelial cells

Extracellular amino acid profiles affect intracellular AA concentrations and profile as well as signaling proteins that regulate protein translation rates. The objective of this study was to assess whether various extracellular AA profiles and varied ratios of Lys to Met would increase the phosphorylation of signaling proteins related to protein metabolism. Six AA profiles, reflecting Dulbecco's modified Eagle's medium (DMEM), blood meal (BM), corn gluten meal (CGM), casein (CAS), plasma of lactating cows (PLA), and a negative control (NEG) represented the first factor (F1), and the ratio of Lys to Met (unaltered or set to 3:1) was the second factor (F2). Treatments were arranged in a 6 × 2 factorial manner, resulting in 12 treatments that were replicated 4 times. The total AA masses for all treatments were set to 659 mg/L (63% of DMEM) except NEG (0 mg/L). Confluent mammary epithelial cells were exposed to treatment media for 80 min (SD = 7.4). Intracellular concentrations of 17 AA were changed according to F1. The Met and Leu percent of total intracellular AA mass, as an example, varied from 0.58 (PLA) to 6.94 (NEG, +F2) for Met and 0.05 (NEG, -F2) to 4.63 (CGM, +F2) for Leu. Overall, balancing for Lys and Met at a 3:1 ratio increased intracellular concentrations of Lys and Met by 54 and 71%, respectively. Within the mechanistic target of rapamycin (mTOR) pathway, phosphorylation of mTOR (Ser2448), ribosomal protein S6 (Ser235/236), and eukaryotic initiation factor 4E binding protein 1 (Thr37/46) (4EBP1) were increased by all 5 AA profiles compared with the NEG control. We found no differences in phosphorylation state among the 5 AA profiles, indicating lack of sensitivity to various AA profiles. This lack of sensitivity between AA profiles might also be due to assay imprecision or other experimental limitations. Only phosphorylation of 4EBP1 was increased for F2. Phosphorylation of eukaryotic initiation factor 2 α subunit (Ser51) was unaffected by either F1 or F2 factors. Regression analyses indicated that intracellular concentrations of Met, Thr, Ile, and Leu predicted phosphorylation of mTOR-related proteins with adequate precision and accuracy, suggesting that multiple EAA dictate regulation, regardless of AA ratios. Changes in extracellular AA profiles translated to modified intracellular AA profiles, and no single profile uniquely stimulated phosphorylation of the mTOR pathway-related proteins.

Targeted Killing of Monocytes/Macrophages and Myeloid Leukemia Cells with Pro-Apoptotic Peptides.

Several cells of myeloid origin, such as monocytes and macrophages are involved in various human disorders, including cancer and inflammatory diseases. Hence, they represent attractive therapeutic targets. Here we developed three lytic hybrid peptides, by fusing a monocyte- and macrophage-binding peptide to pro-apoptotic peptides, and investigated their killing potency on blood monocytes, macrophages, and leukemia cells. We first showed that the targeting NW peptide is effective for depleting monocytes from whole peripheral blood mononuclear cells (PBMCs). Incubating the cells with biotin-conjugated NW peptide, and the subsequent capture on streptavidin-conjugated magnetic beads, depleted monocytes from the PBMCs. The NW peptide also depleted myeloid leukemia blasts from patient PBMCs. The treatment of the PBMCs with the lytic hybrid NW-KLA peptide killed monocytes, but not lymphocytes and primary mammary epithelial cells. Additionally, the fusion peptide exhibited a potent toxicity against macrophages and leukemia cells. The free lytic KLA peptide did not affect cells. Similarly, a second lytic hybrid peptide killed macrophages, leukemia cell lines, and blood leukemia blasts from patients with acute and chronic myeloid leukemia. The IC50 towards target cells were in the low macromolar range (4–12 µM). Overall, the data indicate that the NW peptide could be a potential drug delivery agent for monocytes, macrophages, and leukemia cells. Moreover, the engineered lytic hybrid peptides acting alone, or in combination with other therapeutic agents, might benefit many cancer patients and overcome drug resistance.

Plasticity of patient-matched normal mammary epithelial cells is dependent on autologous adipose-derived stem cells

Due to the increasing clinical application of adipose-derived stem cells (ADSC), e.g. lipotransfer for breast reconstruction, this study aimed to gain novel insights regarding ADSC influence on breast tissue remodeling and determine patient-dependent factors affecting lipotransfer as well as begin to address its oncological risks. The ADSC secretome was analyzed from five normal breast reduction patients and contained elevated levels of growth factors, cytokines and proteins mediating invasion. ADSC/ADSC secretomes were tested for their influence on the function of primary mammary epithelial cells, and tumor epithelial cells using cell culture assays. ADSC/ADSC secretomes significantly stimulated proliferation, transmigration and 3D-invasion of primary normal and tumor epithelial cells. IL-6 significantly induced an EMT and invasion. The ADSC secretome significantly upregulated normal epithelial cell gene expression including MMPs and ECM receptors. Our study supports that ADSC and its secretome promote favorable conditions for normal breast tissue remodeling by changing the microenvironment. and may also be important regarding residual breast cancer cells following surgery.

Effects of Free Fatty Acids with Different Chain Lengths and Degrees of Saturability on the Milk Fat Synthesis in Primary Cultured Bovine Mammary Epithelial Cells.

How short-chain fatty acids (FAs) affect cell membrane morphology and milk fat biosynthesis in mammary epithelial cells (MECs) is yet unclear. This study investigated the primary bovine MEC response to different FAs. We observed that the cell surface ultrastructures were influenced by chain length and degree of saturability of FAs. The CD36, FATP1, and FABP3 gene expression was affected independent of the type of FA. FASN, LPIN1, PPARα, and PPARγ transcripts were more sensitive to the short-chain FAs (acetic and β-hydroxybutyric acids). Furthermore, short-chain FAs inclined to regulate FA degradation-, elongation-, and metabolism-associated pathways, while long-chain FAs (stearic and trans-10,cis-12 conjugated linolenic acids) modulated extracellular matrix-receptor interaction-, transcriptional misregulation-, microRNA-, and ribosome biogenesis-related pathways. However, triacylglycerol accumulation in the cytoplasm was not changed by all of the FAs. Overall, FAs with different chain lengths and degrees of saturability could differentially alter primary bovine MEC cell morphology and influence protein profiles involved in milk fat synthesis pathways.

Bta-miR-34b regulates milk fat biosynthesis by targeting mRNA decapping enzyme 1A (DCP1A) in cultured bovine mammary epithelial cells1.

Milk fat is a main nutritional component of milk, and it has become one of the important traits of dairy cow breeding. Recently, there is increasing evidence that microRNAs (miRNA) play significant roles in the process of milk fat synthesis in the mammary gland. Primary bovine mammary epithelial cells (BMEC) were harvested from midlactation cows and cultured in DMEM/F-12 medium with 10% fetal bovine serum, 100 units/mL penicillin, 100 µg/mL streptomycin, 5 µg/mL bovine insulin, 1 µg/mL hydrocortisone, and 2 µg/mL bovine prolactin. We found that miR-34b mimic transfection in BMEC reduced the content of intracellular triacylglycerol (TAG) and lipid droplet accumulation via triacylglycerol assay and Oil Red O staining; meanwhile, overexpression of miR-34b inhibited mRNA expression of lipid metabolism-related genes such as peroxisome proliferator-activated receptor gamma (PPARγ), fatty acid synthase (FASN), fatty acid binding protein 4 (FABP4), and CCAAT enhancer binding protein alpha (C/EBPα). Whereas miR-34b inhibitor resulted in completely opposite results. Furthermore, q-PCR and western blot analysis revealed the mRNA and protein expression levels of DCP1A were downregulated in miR-34b mimic transfection group and upregulated in miR-34b inhibitor group. Moreover, luciferase reporter assays verified that DCP1A was the direct target of miR-34b and DCP1A gene silencing in BMEC-inhibited TAG accumulation and suppressed lipid droplet formation. In conclusion, these findings revealed a novel miR-34b-DCP1A axis that has a significant role in regulating milk fat synthesis and suggested that miR-34b may be used to improve the beneficial ingredients in milk.