•Fractionated extracts from varieties of mango were analysed by mass spectrometry.•Gallic acid substituted compounds were the principal components observed.•Flavonoids and fatty acids and their ...glucosides were also detected.•Compounds present in mango peel and flesh are likely subject to genetic control.
In this study we determined the qualitative composition and distribution of phytochemicals in peel and flesh of fruits from four different varieties of mango using mass spectrometry profiling following fractionation of methanol extracts by preparative HPLC. Gallic acid substituted compounds, of diverse core structure, were characteristic of the phytochemicals extracted using this approach. Other principal compounds identified were from the quercetin family, the hydrolysable tannins and fatty acids and their derivatives. This work provides additional information regarding mango fruit phytochemical composition and its potential contribution to human health and nutrition. Compounds present in mango peel and flesh are likely subject to genetic control and this will be the subject of future studies.
•Some clinical breast cancers are associated with MCU overexpression.•MCU silencing did not alter cell death initiated with the Bcl-2 inhibitor ABT-263.•MCU silencing potentiated caspase-independent ...cell death initiated by ionomycin.•MCU silencing promoted ionomycin-mediated cell death without changes in bulk Ca2+.
The mitochondrial calcium uniporter (MCU) transports free ionic Ca2+ into the mitochondrial matrix. We assessed MCU expression in clinical breast cancer samples using microarray analysis and the consequences of MCU silencing in a breast cancer cell line. Our results indicate that estrogen receptor negative and basal-like breast cancers are characterized by elevated levels of MCU. Silencing of MCU expression in the basal-like MDA-MB-231 breast cancer cell line produced no change in proliferation or cell viability. However, distinct consequences of MCU silencing were seen on cell death pathways. Caspase-dependent cell death initiated by the Bcl-2 inhibitor ABT-263 was not altered by MCU silencing; whereas caspase-independent cell death induced by the calcium ionophore ionomycin was potentiated by MCU silencing. Measurement of cytosolic Ca2+ levels showed that the promotion of ionomycin-induced cell death by MCU silencing occurs independently of changes in bulk cytosolic Ca2+ levels. This study demonstrates that MCU overexpression is a feature of some breast cancers and that MCU overexpression may offer a survival advantage against some cell death pathways. MCU inhibitors may be a strategy to increase the effectiveness of therapies that act through the induction of caspase-independent cell death pathways in estrogen receptor negative and basal-like breast cancers.
The health and productivity of dairy goats continue to be impacted by gastrointestinal nematodes (GIN) and lungworms (LW). Eprinomectin (EPN) is frequently selected for treatment because it is ...generally effective and does not require a milk withdrawal period. However, some factors, such as lactation, can have an impact on EPN pharmacokinetics and potentially its efficacy. To evaluate whether this can alter the efficacy of Eprecis
2%, an eprinomectin injectable solution, a study was performed in lactating goats using the dose currently registered in cattle, sheep and goats (0.2 mg/kg).
This study was a blinded, randomized, controlled trial performed according to the VICH guidelines. Eighteen (18) worm-free lactating goats were included and experimentally challenged on day 28 with a mixed culture of infective gastrointestinal and lung nematode larvae (Haemonchus contortus, Trichostrongylus colubriformis, Teladorsagia circumcincta, Dictyocaulus filaria). At D-1, fecal samples were collected to confirm patent infection in all animals. On D0, the goats were randomly allocated into two groups of nine goats; group 1 was treated with Eprecis
2% at 0.2 mg/kg BW by subcutaneous injection, while group 2 remained untreated. Fecal samples for egg counts were collected from all animals on days 3, 5, 7, 9, 11 and 14. On D14, all goats were killed, and the abomasum, small intestine and lungs were removed, processed and subsampled to record the number and species of worms.
The treatment was well tolerated. After treatment, the arithmetic mean FEC decreased in the treated group and remained < 5 EPG until the end of the study, while the arithmetic mean FEC in the control group remained > 849.0 EPG. At D14, goats in the treated group had very limited or zero total worm counts, whereas all animals from the control group had a high worm burden. The measured efficacy was 100.0% against H. contortus and T. colubriformis, 99.9% against T. circumcincta and 98.0% against D. filaria.
Eprinomectin (Eprecis
, 20 mg/ml), administered at the label dose (0.2 mg/kg), is highly effective against gastrointestinal nematodes and lungworms in lactating goats.
Despite structural similarity with other tumor necrosis factor receptor superfamily (TNFRSF) members, the p75 neurotrophin receptor (p75
, TNFR16) mediates pleiotropic biological functions not shared ...with other TNFRs. The high level of p75
expression in the nervous system instead of immune cells, its utilization of co-receptors, and its interaction with soluble dimeric, rather than soluble or cell-tethered trimeric ligands are all characteristics which distinguish it from most other TNFRs. Here, we compare these attributes to other members of the TNFR superfamily. In addition, we describe the recent evolutionary adaptation in B7-1 (CD80), an immunoglobulin (Ig) superfamily member, which allows engagement to neuronally-expressed p75
. B7-1-mediated binding to p75
occurs in humans and other primates, but not lower mammals due to specific sequence changes that evolved recently in primate B7-1. This discovery highlights an additional mechanism by which p75
can respond to inflammatory cues and trigger synaptic elimination in the brain through engagement of B7-1, which was considered to be immune-restricted. These observations suggest p75
does share commonality with other immune co-modulatory TNFR family members, by responding to immunoregulatory cues. The evolution of primate B7-1 to bind and elicit p75
-mediated effects on neuronal morphology and function are discussed in relationship to immune-driven modulation of synaptic actions during injury or inflammation.
Epithelial-mesenchymal transition (EMT), a process implicated in cancer metastasis, is associated with the transcriptional regulation of members of the ATP-binding cassette superfamily of efflux ...pumps, and drug resistance in breast cancer cells. Epidermal growth factor (EGF)-induced EMT in MDA-MB-468 breast cancer cells is calcium signal dependent. In this study induction of EMT was shown to result in the transcriptional up-regulation of ATP-binding cassette, subfamily C, member 3 (ABCC3), a member of the ABC transporter superfamily, which has a recognized role in multidrug resistance. Buffering of cytosolic free calcium inhibited EGF-mediated ABCC3 increases, indicating a calcium-dependent mode of regulation. Silencing of TRPM7 (an ion channel involved in EMT associated vimentin induction) did not inhibit ABCC3 up-regulation. Silencing of the store operated calcium entry (SOCE) pathway components ORAI1 and STIM1 also did not alter ABCC3 induction by EGF. However, the calcium permeable ion channel transient receptor potential cation channel, subfamily C, member 1 (TRPC1) appears to contribute to the regulation of both basal and EGF-induced ABCC3 mRNA. Improved understanding of the relationship between calcium signaling, EMT and the regulation of genes important in therapeutic resistance may help identify novel therapeutic targets for breast cancer.
Triple-negative breast cancers (TNBC) are often associated with high relapse rates, despite treatment with chemotherapy agents such as doxorubicin. A better understanding of the signaling and ...molecular changes associated with doxorubicin may provide novel insights into strategies to enhance treatment efficacy. Calcium signaling is involved in many pathways influencing the efficacy of chemotherapy agents such as proliferation and cell death. However, there are a limited number of studies exploring the effect of doxorubicin on calcium signaling in TNBC. In this study, MDA-MB-231 triple-negative, basal breast cancer cells stably expressing the genetically-encoded calcium indicator GCaMP6m (GCaMP6m-MDA-MB-231) were used to define alterations in calcium signaling. The effects of doxorubicin in GCaMP6m-MDA-MB-231 cells were determined using live cell imaging and fluorescence microscopy. Changes in mRNA levels of specific calcium regulating proteins as a result of doxorubicin treatment were also assessed using real time qPCR. Doxorubicin (1 μM) produced alterations in intracellular calcium signaling, including enhancing the sensitivity of MDA-MB-231 cells to ATP stimulation and prolonging the recovery time after store-operated calcium entry. Upregulation in mRNA levels of ORAI1, TRPC1, SERCA1, IP3R2 and PMCA2 with doxorubicin 1 μM treatment was also observed. Doxorubicin treatment is associated with specific remodeling in calcium signaling in MDA-MB-231 cells, with associated changes in mRNA levels of specific calcium-regulating proteins.
•The effect of doxorubicin on Ca2+ signaling was assessed using GCaMP6m.•Doxorubicin enhanced the sensitivity of MDA-MB-231 cells to ATP stimulation.•Doxorubicin prolonged Ca2+ recovery time after store-operated Ca2+ entry.•An upregulation in mRNA levels of Ca2+ regulating proteins with doxorubicin was identified.
Although breast cancer cells often exhibit both abnormal AKT signaling and calcium signaling, the association between these two pathways is unclear. Using a combination of pharmacological tools, ...siRNA and CRISPR/Cas9 gene silencing techniques, we investigated the association between PTEN, AKT phosphorylation and calcium signaling in a basal breast cancer cell line. We found that siRNA-mediated PTEN silencing promotes AKT phosphorylation and calcium influx in MDA-MB-231 cells. This increase in AKT phosphorylation and calcium influx was phenocopied by the pharmacological AKT activator, SC79. The increased calcium influx associated with SC79 is inhibited by silencing AKT2, but not AKT1. This increase in calcium influx is suppressed when the store-operated calcium channel, ORAI1 is silenced. The results from this study open a novel avenue for therapeutic targeting of cancer cells with increased AKT activation. Given the association between ORAI1 and breast cancer, ORAI1 is a possible therapeutic target in cancers with abnormal AKT signaling.
The microenvironment plays a pivotal role in tumor cell proliferation, survival and migration. Invasive cancer cells face a new set of environmental challenges as they breach the basement membrane ...and colonize distant organs during the process of metastasis. Phenotypic switching, such as that which occurs during epithelial-mesenchymal transition (EMT), may be associated with a remodeling of cell surface receptors and thus altered responses to signals from the tumor microenvironment.
We assessed changes in intracellular Ca(2+) in cells loaded with Fluo-4 AM using a fluorometric imaging plate reader (FLIPR(TETRA)) and observed significant changes in the potency of ATP (EC(50) 0.175 µM (-EGF) versus 1.731 µM (+EGF), P<0.05), and the nature of the ATP-induced Ca(2+) transient, corresponding with a 10-fold increase in the mesenchymal marker vimentin (P<0.05). We observed no change in the sensitivity to PAR2-mediated Ca(2+) signaling, indicating that these alterations are not simply a consequence of changes in global Ca(2+) homeostasis. To determine whether changes in ATP-mediated Ca(2+) signaling are preceded by alterations in the transcriptional profile of purinergic receptors, we analyzed the expression of a panel of P2X ionotropic and P2Y metabotropic purinergic receptors using real-time RT-PCR and found significant and specific alterations in the suite of ATP-activated purinergic receptors during EGF-induced EMT in breast cancer cells. Our studies are the first to show that P2X(5) ionotropic receptors are enriched in the mesenchymal phenotype and that silencing of P2X(5) leads to a significant reduction (25%, P<0.05) in EGF-induced vimentin protein expression.
The acquisition of a new suite of cell surface purinergic receptors is a feature of EGF-mediated EMT in MDA-MB-468 breast cancer cells. Such changes may impart advantageous phenotypic traits and represent a novel mechanism for the targeting of cancer metastasis.
PDF
