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Dopamine is a member of the catecholamine family and is associated with multiple physiological functions. Together with its five receptor subtypes, dopamine is closely linked to neurological disorders such as schizophrenia, Parkinson's disease, depression, attention deficit-hyperactivity, and restless leg syndrome. Unfortunately, several dopamine receptor-based agonists used to treat some of these diseases cause nausea and vomiting as impending side-effects. The high degree of cross interactions of dopamine receptor ligands with many other targets including G-protein coupled receptors, transporters, enzymes, and ion-channels, add to the complexity of discovering new targets for the treatment of nausea and vomiting. Using activation status of signaling cascades as mechanism-based biomarkers to foresee drug sensitivity combined with the development of dopamine receptor-based biased agonists may hold great promise and seems as the next step in drug development for the treatment of such multifactorial diseases. In this review, we update the present knowledge on dopamine and dopamine receptors and their potential roles in nausea and vomiting. The pre- and clinical evidence provided in this review supports the implication of both dopamine and dopamine receptor agonists in the incidence of emesis. Besides the conventional dopaminergic antiemetic drugs, potential novel antiemetic targeting emetic protein signaling cascades may offer superior selectivity profile and potency.

With its five receptor subtypes (D1–5), dopamine is implicated in a myriad of neurological illnesses. Dopamine D2 receptor-based agonist therapy evokes nausea and vomiting. The signaling mechanisms by which dopamine D2 receptors evoke vomiting remains unknown. Phosphatidylinositol 3-kinases (PI3K)- and protein kinase C (PKC)-related signaling cascades stimulate vomiting post-injection of various emetogens in emetically competent animals. This study investigated potential mechanisms involved in dopamine D2 receptor-mediated vomiting using least shrews. We found that vomiting evoked by the selective dopamine D2 receptor agonist quinpirole (2 mg/kg, i.p.) was significantly suppressed by: i) a dopamine D2 preferring antagonist, sulpiride (s.c.); ii) a selective PI3K inhibitor, LY294002 (i.p.); iii) a PKCαβII inhibitor, GF109203X (i.p.); and iv) a selective inhibitor of extracellular signal-regulated protein kinase1/2 (ERK1/2), U0126 (i.p.). Quinpirole-evoked c-fos immunofluorescence in the nucleus tractus solitarius (NTS) was suppressed by pretreatment with sulpiride (8 mg/kg, s.c.). Western blot analysis of shrew brainstem emetic loci protein lysates revealed a significant and time-dependent increase in phosphorylation of Akt (protein kinase B (PKB)) at Ser473 following a 30-min exposure to quinpirole (2 mg/kg, i.p.). Pretreatment with effective antiemetic doses of sulpiride, LY294002, GF109203X, or U0126 significantly reduced quinpirole-stimulated phosphorylation of emesis-associated proteins including p-85PI3K, mTOR (Ser2448/2481), PKCαβII (Thr638/641), ERK1/2 (Thr202/204), and Akt (Ser473). Our results substantiate the implication of PI3K/mTOR/Akt and PI3K/PKCαβII/ERK1/2/Akt signaling pathways in dopamine D2 receptor-mediated vomiting. Potential novel antiemetics targeting emetic proteins associated with these signaling cascades may offer enhanced potency and/or efficacy against emesis.

Phosphaplatins are platinum-based antitumor compounds that, unlike other clinically utilized platinum drugs (i.e. cisplatin, carboplatin, and oxaliplatin), appear to target proteins rather than DNA. Because of their unique mode of action, phosphaplatins are promising drug candidates for cisplatin-resistant cancers. In this study, we discovered that Pt(II) and Pt(IV) phosphaplatins possess diverse antitumor properties. In addition to targeting apoptosis antigen (FAS) and proapoptotic gene products as described previously, phosphaplatins also target angiogenesis. We demonstrate that phosphaplatins inhibit human umbilical vein endothelial cell (HUVEC) migration and tube formation in vitro and suppress tumor angiogenesis and growth in immunodeficient mice that were inoculated with A2780 ovarian cancer cells in vivo. To provide insight into this novel antitumor mechanism, phosphaplatin-treated HUVECs were found to exhibit lower gene expression levels of vascular endothelial growth factors (VEGFs) and the VEGFR-2 receptor compared to untreated cells. Kinase inhibition studies suggest that phosphaplatins are inhibitors of VEGFR-2. In ligand exchange experiments using both Pt atomic absorption and 31P NMR spectroscopies, we show that phosphaplatins most likely bind to VEGFR-2 through metal-ligand coordination rather than electrostatic interactions. These studies enhance our understanding of the diverse and novel mechanisms of action of the phosphaplatin antitumor agents, which could potentially be used as chemotherapeutic agents against cisplatin-resistant cancers.

Pregnancy is characterized by an altered inflammatory profile, compared to the non-pregnant state with an adequate balance between pro-and anti-inflammatory cytokines needed for normal development. Cytokines are small secreted proteins expressed mainly in immunocompetent cells in the reproductive system. From early developmental stages onward, the secretory activity of placenta cells clearly contributes to increase local as well as systemic levels of cytokines. The placental production of cytokines may affect mother and fetus independently. In turn because of this unique position at the maternal fetal interface, the placenta is also exposed to the regulatory influence of cytokines from maternal and fetal circulations, and hence, may be affected by changes in any of these. Gestational diabetes mellitus (GDM) is associated with an overall alteration of the cytokine network. This review discusses the changes that occur in cytokines post GDM and their negative effects on maternal insulin and placental-fetal development.

Background/aim Phosphaplatin platinum (IV) (RRD4) complex has exceptional antitumor properties. The aim of this study was to investigate the effects and the mechanism of action of free and liposome-encapsulated RRD4 in breast cancer. Materials and methods Liposome-encapsulated RRD4 prepared by thin-film dehydration: hydration and free RRD4 were tested in vivo and in vitro against 4T1 breast cancer cells. Cell proliferation, migration and viability were determined. Tissue and cell production and expression of pigment epithelium-derived factor (PEDF) were assessed by ELISA and western blot. 4T1 cells treated with PEDF siRNA were evaluated for viability and apoptosis. Results RRD4 inhibited tumor growth and prevented distant metastasis. Liposome formulation enhanced this therapeutic benefit without increasing toxicity and prolonged RRD4 retention in tumor tissues. In vitro, RRD4 induced 4T1 apoptosis through up-regulation of FAS, BAX, and PUMA, and down-regulation of BCL2. RRD4 facilitates a FAS-intrinsic signaling mechanism. PEDF up-regulation represents another antitumor mechanism associated with this phosphaplatin compound. Discussion Free RRD4 or formulated into liposomes, are excellent candidates for adjuvant therapy against breast tumor growth and metastasis.

Δ9-THC suppresses cisplatin-induced vomiting through activation of cannabinoid CB1 receptors. Cisplatin-evoked emesis is predominantly due to release of serotonin and substance P (SP) in the gut and the brainstem which subsequently stimulate their corresponding 5-HT3-and neurokinin NK1-receptors to induce vomiting. Δ9-THC can inhibit vomiting caused either by the serotonin precursor 5-HTP, or the 5-HT3 receptor selective agonist, 2-methyserotonin. In the current study, we explored whether Δ9-THC and related CB1/CB2 receptor agonists (WIN55,212–2 and CP55,940) inhibit vomiting evoked by SP (50 mg/kg, i.p.) or the NK1 receptor selective agonist GR73632 (5 mg/kg, i.p.). Behavioral methods were employed to determine the antiemetic efficacy of cannabinoids in least shrews. Our results showed that administration of varying doses of Δ9-THC (i.p. or s.c.), WIN55,212–2 (i.p.), or CP55,940 (i.p.) caused significant suppression of SP-evoked vomiting in a dose-dependent manner. When tested against GR73632, Δ9-THC also dose-dependently reduced the evoked emesis. The antiemetic effect of Δ9-THC against SP-induced vomiting was prevented by low non-emetic doses of the CB1 receptor inverse-agonist/antagonist SR141716A (

Maternal nutrition during pregnancy has a pivotal role in the regulation of placental-fetal development and thereby affects the lifelong health and productivity of offspring. Suboptimal maternal nutrition yields low birth weight, with substantial effect on the short-term morbidity of the newborn. The placenta is the organ through which gases, nutrients, and wastes are exchanged between the maternal-fetal circulations. The size, morphology, and nutrient transfer capacity of the placenta determine the prenatal growth trajectory of the fetus to influence birth weight. Transplacental exchange depends on uterine, placental, and umbilical blood flow. Most important, maternal nutrition influences factors associated not only with placental homeostasis but also with optimal fetal development. This review associates fetal growth with maternal nutrition during pregnancy, placental growth and vascular development, and placental nutrient transport.

Maternal food restriction (FR) during pregnancy results in decreased body weight with increased plasma leptin. To address this paradox, we investigated the effects of FR during pregnancy on growth and leptin levels in maternal, placental, and fetal sites. From embryonic day E10, control pregnant rats received ad libitum (AdLib) food, whereas study rats were 50% FR. At gestational ages, E16 and E20, the alterations in maternal body composition, retroperitoneal versus subcutaneous adipose leptin expression, and plasma leptin levels were studied. Furthermore, these changes were related to non-pregnant (NP) status and placental/fetal growth and leptin levels. As compared to NP, both FR and AdLib dams showed a progressive increase in body and lean body mass. However, total body fat was reduced in FR dams but remained unchanged in AdLib dams. Furthermore, plasma leptin levels in FR dams were markedly increased at E20 unlike AdLib dams, which showed moderate increments at E16 and E20. Additionally, FR dams showed significantly decreased leptin expression in subcutaneous and notably unaltered levels in retroperitoneal adipose tissue, suggesting an alternate source of elevated maternal plasma leptin. More importantly, the FR dams had reduced placental weights with paradoxical increased leptin expression at both gestations. Thus, increased plasma leptin levels at E20 in maternal FR pregnancies may be explained, in part, by upregulation of placental leptin. Despite maternal and placental hyperleptinemia during FR pregnancies, the growth-restricted FR fetus had reduced leptin levels. These findings have important implications for pregnancy outcome and fetal growth.

Aquaporins (AQPs) are water channels that regulate water flow in many tissues. As AQP1 is a candidate to regulate placental fluid exchange, we sought to investigate the effect of arginine vasopressin (AVP) and cAMP agonists on AQP1 gene expression in first trimester-derived extravillous cytotrophoblasts (HTR-8/Svneo) and two highly proliferative carcinoma trophoblast-like cell lines but with a number of functional features of the syncytiotrophoblast namely; JAR and JEG-3 cells. Our data demonstrated that AVP (0.1 nM) significantly increased the expression of AQP1 mRNA at 10 h in HTR-8/SVneo and JEG-3 cells (P

Damage of the placenta resulting from ischemia-reperfusion is important to the pathophysiology of preeclampsia. Here we investigated whether low concentrations of glyceryl trinitrate (GTN), a nitric oxide mimetic with anti-apoptotic properties, inhibit hypoxia/reoxygenation-induced apoptosis in the syncytiotrophoblast of chorionic villous explants from human placentas. Compared with villi analyzed immediately after delivery or maintained under normoxic conditions, villi exposed to a 6-hour cycle of hypoxia/reoxygenation exhibited greater numbers of syncytiotrophoblasts with terminal dUTP nick-end labeling (TUNEL)-positive nuclei in the syncytiotrophoblast. This increased number of TUNEL-positive nuclei was paralleled by higher levels of 4-hydroxynonenal (marker of lipid peroxidation), nitrotyrosine residues, and active caspase-3 and polyADP-ribose polymerase expression. Morphological analysis of explants exposed to hypoxia/reoxygenation revealed apoptotic and aponecrotic features similar to those of chorionic villi from preeclamptic pregnancies. Treatment with GTN during the hy-poxia/reoxygenation cycle blocked the increases in the number of TUNEL-positive nuclei and in the levels of 4-hydroxynonenal, nitrotyrosine, and active caspase-3. Incubation with GTN also attenuated the hypoxia/reoxygenation-induced polyADP-ribose polymerase expression and the apoptotic and aponecrotic morphological alterations. These results suggest that small concentrations of nitric oxide protect chorionic villi from hypoxia/reoxygenation-induced damage and provide a rationale for the use of low doses of nitric oxide mimetics in the treatment and/or prevention of preeclampsia.

Platinum-based anticancer drugs, including cisplatin and carboplatin, have been cornerstones in the treatment of solid tumors. We report here that these DNA-damaging agents, particularly cisplatin, induce apoptosis through plasma membrane disruption, triggering FAS death receptor via mitochondrial (intrinsic) pathways. Our objectives were to: quantify the composition of membrane metabolites; and determine the potential involvement of acid sphingomyelinase (ASMase) in the FAS-mediated apoptosis in ovarian cancer after cisplatin treatment. The resulting analysis revealed enhanced apoptosis as measured by: increased phosphocholine, and glycerophosphocholine; elevated cellular energetics; and phosphocreatine and nucleoside triphosphate concentrations. The plasma membrane alterations were accompanied by increased ASMase activity, leading to the upregulation of FAS, FASL and related pro-apoptotic BAX and PUMA genes. Moreover FAS, FASL, BAX, PUMA, CASPASE-3 and -9 proteins were upregulated. Our findings implicate ASMase activity and the intrinsic pathways in cisplatin-mediated membrane demise, and contribute to our understanding of the mechanisms by which ovarian tumors may become resistant to cisplatin.

Pre-eclampsia, a hypertensive disorder of pregnancy, affects 5 to 7% of pregnancies. Oxidative stress-induced placental injury and subsequent release of placental debris into the maternal circulation are key pathogenic events in the progression of pre-eclampsia. Women who smoke cigarettes throughout pregnancy are 33% less likely to develop this disorder than nonsmoking women. We postulated that elevated carbon monoxide concentrations in serum of smoking women inhibits apoptosis and debris shedding of trophoblast cells exposed to ischemia-reperfusion injury because carbon monoxide has cytoprotective effects on endothelial and smooth muscle cells in culture. This may be responsible for the reduced risk of pre-eclampsia in smoking women. To assess the cytoprotective properties of carbon monoxide within placental tissue, carbon monoxide treatments were administered to in vitro hypoxia/reoxygenation-insulted villous explants cultured from term human placenta. Induction of apoptosis was assessed using molecular and morphological approaches. Placental villous explants treated with carbon monoxide demonstrated 60% less hypoxia/reoxygenation-induced apoptosis in the differentiated syncytiotrophoblast layer compared with untreated explants undergoing a similar insult. In addition, retention of intact syncytial membranes was observed in carbon monoxide-treated explants. These observations indicate that carbon monoxide has potent antiapoptotic properties within human placenta and may hold therapeutic potential in the treatment of pre-eclampsia.

Glucose deprivation has been shown to increase the invasive and metastatic potential of tumour cells. In the present study, we determined whether the enhanced tumour cell invasiveness resulting from glucose deprivation is linked to increased activity of enzymes required for extracellular matrix degradation. Results of in vitro invasion assays revealed that the invasiveness of human MDA-MB-231 and MCF-7 breast carcinoma cells and MCF-10A1 normal breast cells was, respectively, 3.9-, 2.9-, and 2.1-fold higher when they were incubated under glucose-deprivation (0.2 mM glucose) than when incubated under physiological blood glucose levels (5 mM). This effect of glucose deprivation on invasion correlated with increased urokinase plasminogen activator (uPA) and plasmin activity. Glucose deprivation did not increase the levels of gelatinase and plasminogen activator inhibitor-1 secretion, or the expression of cell-associated uPA receptor. To determine whether the increased invasiveness resulting from glucose deprivation is causally linked to increased uPA activity, invasion assays were conducted using MDA-MB-231 cells incubated in 0.2 mM or 5 mM glucose in the presence of a neutralising anti-uPA antibody. Results revealed that the anti-uPA antibody significantly inhibited invasion in a dose-dependent manner and to a much greater extent in cells incubated in 0.2 mM glucose than in cells incubated in 5 mM glucose. These results suggest that low glucose levels in malignant cancers increase tumour cell invasiveness by stimulating uPA and plasmin activity.

Trophoblast invasion of the uterus is regulated by local microenvironmental factors.Because certain conditions may affect uterine glucose levels during placentation, the aim of this study was to determine the effect of glucose concentration on trophoblast invasion.Compared with incubation in 0.2 and 2.5 mm glucose, a 24-h incubation in increasing glucose concentrations (5 and 10 mm) resulted in up to a 62% inhibition (P0.01) of the in vitro invasiveness of immortalized HTR-8/SVneo trophoblasts. This decreased invasiveness in 5 and 10 mm glucose was paralleled by inhibition of a plasminogen activator (PA) activity corresponding to active urokinase-type PA (uPA). Inhibition of pro-uPA binding to the uPA receptor decreased the invasiveness of cells incubated in 0.2 and 2.5 mm glucose to levels observed in cells incubated in higher glucose concentrations (P0.05). Gelatin zymography and Western blot analysis revealed that the levels of matrix metalloproteinase-2 and -9, PA inhibitor-1, and uPA receptor were unaffected by glucose. Glucose transporter-1 levels were 26 and 34% higher in cells cultured in 2.5 and 0.2 mm glucose, respectively, vs. 5 or 10 mm glucose (P0.05). In contrast, glucose transporter-3 levels were not affected by incubation in various glucose concentrations.These findings indicate that high glucose concentrations inhibit the invasiveness of HTR-8/SVneo cells by preventing uPA activation. Therefore, through its effects on uPA activity, glucose may be an important regulator of trophoblast invasiveness during implantation and placentation.

Calbindin-D28k (CaBP28k) is a cytosolic calcium (Ca2+)-binding protein expressed in tissues such as intestine, kidneys and placenta. This protein is thought to be involved in Ca2+ homeostasis. While it is well known that CaBP28k is influenced by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] in the intestine and kidneys, nothing is known regarding the regulation of this protein in trophoblasts of human placenta. We used JEG-3 syncytiotrophoblast-like carcinoma cell line to study the regulation of CaBP28k in correlation with 1,25(OH)2D3 receptor (VDR) following 1,25(OH)2D3 treatments. Our data demonstrated for the first time that both CaBP28k mRNA and protein were highly induced by the addition of 1,25(OH)2D3 in dose-dependent manner. Moreover, the increase and subsequent decrease in the expression of CaBP28k and VDR mRNAs indicates the transient nature of the changes in gene expression in response to 1,25(OH)2D3. This is in contrast with the temporal pattern of increasing protein for CaBP28k and VDR. We also showed that new RNA and protein syntheses are required for 1,25(OH)2D3-induced upregulation of CaBP28k. Furthermore, a 25-carboxylic ester analogue of 1,25(OH)2D3, ZK159222, used as an antagonist of 1,25(OH)2D3 signaling confirmed that indeed 1,25(OH)2D3 was implicated in the induction of CaBP28k. These novel findings are a contribution to the processes that drive CaBP28k expression regulation in human placenta.

During pregnancy, the calcium (Ca(2+)) transport machinery of the placenta is solely responsible for the nutrient supply to the developing fetus, where active Ca(2+) transport occurs from the mother to the fetus. As part of a larger study to determine the role of Ca(2+) in placental transport in vivo, we questioned whether calbindin-D9k (CaBP9k), which is mainly expressed in duodenum, uterus, and placenta of several mammals, is present in cytotrophoblast cells and syncytiotrophoblasts of human term placenta. We were interested in this protein because of its potential importance in serving as an indicator of Ca(2+) availability and utilization in the placenta. Here, we demonstrated that CaBP9k transcript is present in both cell types, with a lower expression in cytotrophoblast cells as compared to syncytiotrophoblasts. Moreover, we showed by immunochemistry that CaBP9k protein was present in cytotrophoblast and syncytiotrophoblast placental tissue sections as well as in cultured cells. The occurrence of CaBP9k protein in trophoblast cells was further confirmed by Western blot analysis. Thus, these results indicate for the first time that CaBP9k is unequivocally expressed by trophoblast cells from human term placenta.

Calbindin-D28k (CaBP28k) belongs to a large class of eucaryotic proteins that bind calcium (Ca2+) to a specific helix-loop-helix structure. To date, this protein was mainly linked to brain, kidneys, and pancreas. Here, we demonstrate for the first time the existence of CaBP8k in the human placental trophoblasts of the human term placenta. Placental Ca2+ transfer from maternal to fetus is crucial for fetal development, although the biochemical mechanisms responsible for this process are largely unknown. In the current study, we have investigated the 45Ca2+ uptake by human trophoblast cells in correlation with the expression CaBP28k. The expression of CaBP28k was determined by Northern blot analysis, reverse transcriptase polymerase chain reaction (RT-PCR), immunochemistry, and Western blot analysis. Indeed, Northern blot analysis revealed the presence of a CaBP28k transcript in syncytiotrophoblasts, cytotrophoblast cells, and HEK-293 cells. This was further confirmed by RT-PCR analysis followed by...

Gestational diabetic mellitus (GDM) pregnancies have an increased risk of macrosomic infants and large placental mass, though the mechanisms explaining each of these is uncertain. We sought to evaluate the contribution of apoptosis to placental size and the expression of glucose transporters (SLC2A) in GDM pregnancies. Maternal age and pre-pregnancy body weight were documented. Newborn weights were recorded after delivery. Placentas 37–40-week gestation from control patients (no pregnancy complication) (n = 5), or with GDM (n = 5) were weighed immediately after delivery. Villous samples (4 mm diameter) were collected and divided into specimens; one was fixed in 4% paraformaldehyde for immunostaining using terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling (TUNEL) and activated caspase-3. The other specimen was snap frozen in liquid nitrogen and stored at −80°C for active caspase-3, poly(ADP-ribose) polymerase (PARP), SLC2A1 and SLC2A3 gene expression analysis. Our results showed that maternal age and pre-pregnancy body weight were significantly higher in the GDM group when compared with those from the controls (P < 0.05). The mean neonatal birth weight and placenta weight were significantly higher in the GDM group compared with that from the controls (P < 0.05). The apoptotic index of placentas (0.05 ± 0.01 v. 0.17 ± 0.04, P < 0.04), active caspase-3 polypeptide fragments and PARP protein were significantly decreased in GDM placentas as compared with controls. Further, the level of placental SLC2A1 protein expression was ∼3-fold higher in GDM placentas. Our results suggest that reduced apoptosis in GDM placentas may contribute to increased placental tissue, which together with enhanced SLC2A1 expression, could play a role in fetal macrosomia.

The purpose of this study is to correlate genetic mutations, amino-acid variants, signaling pathways with platiunum based drug activity to shed light on personalized treatment of cancer. Platinum anticancer drugs such as cisplatin, carboplatin, and oxalipaltin are widely used to treat a variety of cancers. Although DNA is the molecular target for these platinum therapies, their efficacies, toxicities, and resistance mechanisms vary widely among various categories and sub-categories of cancers. To comprehend this great variability, an integrated analysis was performed to determine the impact of somatic mutations on protein functions, signaling pathways, and drug activity (sensitivity or resistance) among the US National Cancer Institute (NCI) 60 human tumor cell lines based on Z-scores to predict a priori treatment outcome using CellMiner (http://discover.nci.nih.gov/cellminer). Specifically, somatic mutations of significantly mutated genes from the cancer genome atlas (TCGA) were analyzed along with the driver genes (oncogenes and tumor suppressor genes) from catalogue of somatic mutations in cancer (COSMIC) for total 188 genes that belong to more than 20 different signaling pathways. The functional impact of individual amino-acid variant for each gene and its correlation with the activity of carboplatin, cisplatin and oxaliplatin for each cancer cell line were explored. Particular attention was given to colon cancer for which nearly 40% of tumors are known to have mutated KRAS (Kirsten rat sarcoma viral oncogene homolog) gene. Resulting analysis revealed that colon cancer cell lines with KRAS mutations for codon 12 (G12V-mutant from SW620 cell line) and codon 13 (G13D-mutant from HCT116 and HCT15 cell lines) correlated with the sensitivity to oxaliplatin. Conversely, oxaliplatin resistant HCC2998 colon cancer cell line did not show any correlation with mutated KRAS for codon 146 (A146T-mutant). Notably, all the colon cancer cell lines were resistant to both carboplatin and cisplatin with no correlation to the KRAS mutants. Based on our integrated analysis we further predicted gene networks related to oxaliplatin activity for colon cancer. The network includes the epidermal growth factor (EGFR) signaling pathway that involves PIK3CA, PIK3CG and MTOR from the phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) (PIK3/mTOR) pathway, and JAK3 from Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway in addition to KRAS from Mitogen-activated protein kinases (MAPK) pathway. We conclude that instead of considering all mutations of a gene in the same way to assess their clinical significance, it may be beneficial to categorize them into different classes based on their functional impact and efficacies towards the anti-cancer drugs for personalized treatment. Similar analytical approach is being extended to non-small cell lung and ovarian cancers where platinum therapies are widely used. S. Tripathi gratefully acknowledges the support through a training fellowship from a grant by the Cancer Prevention Research Institute of Texas (Grant No. RP140113 to R.N.Bose). Citation Format: Swarnendu Tripathi, Louiza Belkacemi, Margaret S. Cheung, Rathindra N. Bose. Correlation between oncogenic mutations, signaling pathways, and efficacy of platinum-based drugs against colorectal cancers. [abstract]. In: Proceedings of the AACR Special Conference on Computational and Systems Biology of Cancer; Feb 8-11 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 2):Abstract nr B1-02.

Aim Increased placental growth secondary to reduced apoptosis may contribute to the development of macrosomia in GDM pregnancies. We hypothesize that reduced apoptosis in GDM placentas is caused by dysregulation of apoptosis related genes from death receptors or mitochondrial pathway or both to enhance placental growth in GDM pregnancies. Methods Newborn and placental weights from women with no pregnancy complications (controls; N=5), or with GDM (N=5) were recorded. Placental villi from both groups were either fixed for TUNEL assay, or snap frozen for gene expression analysis by apoptosis PCR microarrays and qPCR. Results Maternal, placental and newborn weights were significantly higher in the GDM group vs. Controls. Apoptotic index of placentas from the GDM group was markedly lower than the Controls. At a significant threshold of 1.5, seven genes (BCL10, BIRC6, BIRC7, CASP5, CASP8P2, CFLAR, and FAS) were down regulated, and 13 genes (BCL2, BCL2L1, BCL2L11, CASP4, DAPK1, IκBκE, MCL1, NFκBIZ, NOD1, PEA15, TNF, TNFRSF25, and XIAP) were unregulated in the GDM placentas. qPCR confirmed the consistency of the PCR microarray. Using Western blotting we found significantly decreased placental pro-apoptotic FAS receptor and FAS ligand (FASL), and increased mitochondrial anti-apoptotic BCL2 post GDM insult. Notably, caspase-3, which plays a central role in the execution-phase of apoptosis, and its substrate poly (ADP-ribose) polymerase (PARP) were significantly down regulated in GDM placentas, as compared to non-diabetic Control placentas. Conclusion Maternal GDM results in heavier placentas with aberrant placental apoptotic and inflammatory gene expression that may account, at least partially, for macrosomia in newborns.

Cell-based therapies offer promise for patients with Parkinson's disease (PD); however, durable and effective transplantation substrates need to be defined. This study characterized the feasibility and growth properties of primary cultures established from small-volume brain biopsies taken during deep brain stimulation (DBS) surgery in patients with PD. The lineage and expression of neurotrophic factors with known beneficial actions in PD-affected brain circuitry were also evaluated. Nineteen patients with PD undergoing DBS surgery consented to brain biopsies prior to electrode implantation. Cultures from these samples exhibited exponential and plateau phases of growth and were readily expanded throughout multiple passages. There was robust expression of progenitor markers and the unexpected colocalization of neural and mesenchymal proteins. The oligodendrocyte transcription factor, Olig1, and the myelin-specific sphingolipid, galactocerebroside, were coexpressed with each of glial-derived neurotrophic factor, brain-derived neurotrophic factor, and cerebral dopamine neurotrophic factor. Fluorescence-activated cell sorting demonstrated homogeneous expression of both nestin and Olig1 throughout the expanded cultures. Cells remained viable after a year in cryostorage. These findings confirm the feasibility of small brain biopsies as an expandable source of autologous cell substrate in living patients and demonstrate the complex phenotype of these cells, with implications for therapeutic application in PD and other neurological diseases.

Pigment Epithelial Derived Factor (PEDF) is a secreted glycoprotein that exhibits several biological activities, most notably as a potent anti-angiogenic agent as well as a neurotrophic factor. As such, this molecule has potential application as therapeutics for cancer and a variety of neurological diseases. However, prior art methods directed towards enhancing and stabilizing PEDF expression in vivo have met with little success. Therefore, methods that could lead to the stable expression of PEDF in vivo are still needed. We have recently shown that PEDF protein is overexpressed in tumor cells treated with platinum based drugs; phosphaplatins that are platinum-(II) and platinum-(IV) complexes coordinated to a pyrophosphate moiety. The objective of this study is to extend our previous findings by measuring PEDF expression in both malignant cells and normal neuronal tissues after treating them with these compounds. We also investigated the impact of PEDF upregulation on important biological processes such as apoptosis, proliferation and invasion of tumor cells. Our data showed that there was a significant upregulation (over 4-fold) of PEDF in brain of mice treated with phosphoplatins when compared to the control animals. The compounds were found to stimulate PEDF expression in cultured neuronal cells when assessed in vitro (P Citation Format: Louiza Belkacemi, Armando Rivera, Lu Yang, Rathindra N. Bose, Shaun X. Zhang. Phosphaplatins are potent inducers of pigment epithelial- derived factor (PEDF). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3806.

Efficacies, toxicities, and resistance mechanisms of chemotherapy drugs, such as oxaliplatin and 5-fluorouracil (5-FU), vary widely among various categories and subcategories of colon cancers. By understanding the differences in the drug efficacy and resistance at the level of protein–protein networks, we identified the correlation between the drug activity of oxaliplatin/5-FU and gene variations from the US National Cancer Institute-60 human cancer cell lines. The activity of either of these drugs is correlated with specific amino acid variant(s) of KRAS and other genes from the signaling pathways of colon cancer progression. We also discovered that the activity of a non-DNA-binding novel platinum drug, phosphaplatin, is comparable with oxaliplatin and 5-FU when it was tested against colon cancer cell lines. Our strategy that combines the knowledge from pharmacogenomics across cell lines with the molecular information from specific cancer cells is beneficial for predicting the outcome of a possible combination therapy for personalized treatment.

Julie L Boles,1 Michael G Ross,1 Ron Beloosesky,2 Mina Desai,1 Louiza Belkacemi11Department of Obstetrics and Gynecology, Harbor-UCLA Medical Center, Los Angeles Biomedical Research Institute at Harbor-UCLA, David Geffen School of Medicine at UCLA, University of California, Los Angeles, Torrance, CA, USA; 2Department of Obstetrics and Gynecology, Rambam Medical Center, Haifa, IsraelBackground: Cerebral palsy is a nonprogressive motor impairment syndrome that has no effective cure. The etiology of most cases of cerebral palsy remains unknown; however, recent epidemiologic data have demonstrated an association between fetal neurologic injury and infection/inflammation. Maternal infection/inflammation may be associated with the induction of placental cytokines that could result in increased fetal proinflammatory cytokine exposure, and development of neonatal neurologic injury. Therefore, we sought to explore the mechanism by which maternal infection may produce a placental inflammatory response. We specifically examined rat placental cytokine production and activation of the Toll-like receptor 4 (TLR4) pathway in response to lipopolysaccharide exposure at preterm and near-term gestational ages.Methods: Preterm (e16) or near-term (e20) placental explants from pregnant rats were treated with 0, 1, or 10 µg/mL lipopolysaccharide. Explant integrity was assessed by lactate dehydrogenase assay. Interleukin-6 and tumor necrosis alpha levels were determined using enzyme-linked immunosorbent assay kits. TLR4 and phosphorylated nuclear factor kappa light chain enhancer of activated B cells (NFκB) protein expression levels were determined by Western blot analysis.Results: At both e16 and e20, lactate dehydrogenase levels were unchanged by treatment with lipopolysaccharide. After exposure to lipopolysaccharide, the release of interleukin-6 and tumor necrosis alpha from e16 placental explants increased by 4-fold and 8–9-fold, respectively (P < 0.05 versus vehicle). Conversely, interleukin-6 release from e20 explants was not significantly different compared with vehicle, and tumor necrosis alpha release was only 2-fold higher (P < 0.05 versus vehicle) following exposure to lipopolysaccharide. Phosphorylated NFκB protein expression was significantly increased in the nuclear fraction from placental explants exposed to lipopolysaccharide at both e16 and e20, although TLR4 protein expression was unaffected.Conclusion: Lipopolysaccharide induces higher interleukin-6 and tumor necrosis alpha expression at e16 versus e20, suggesting that preterm placentas may have a greater placental cytokine response to lipopolysaccharide infection. Furthermore, increased phosphorylated NFκB indicates that placental cytokine induction may occur by activation of the TLR4 pathway.Keywords: cytokines, lipopolysaccharide, NFκB, placenta, rat pregnancy

Maternal undernutrition (MUN) during pregnancy results in intrauterine growth-restricted (IUGR) fetuses and small placentas. Although reduced fetal nutrient supply has been presumed to be etiologic in IUGR, MUN-induced placental dysfunction may occur prior to detectable fetal growth restriction. Placental growth impairment may result from apoptosis signaled by mitochondria in response to reduced energy substrate. Therefore, we sought to determine the presence of mitochondrial-induced apoptosis under MUN and ad libitum diet (AdLib) pregnancies. Pregnant rats were fed an AdLib or a 50% MUN diet from embryonic day 10 (E10) to E20. At E20, fetuses and placentas from proximal- and mid-horns (extremes of nutrient/oxygen supply) were collected. Right-horn placentas were used to quantify apoptosis. Corresponding left-horn placentas were separated into basal (hormone production) and labyrinth (feto-maternal exchange) zones, and protein expression of the mitochondrial pathway was determined. Our results show that the MUN placentas had significantly increased apoptosis, with lower expression of cytosolic and mitochondrial anti-apoptotic Bcl2 and Bcl-XL, and significantly higher expression of pro-apoptotic Bax and Bak especially in the labyrinth zone. This was paralleled by higher coimmunostaining with the mitochondrial marker manganese superoxide dismutase (MnSOD), indicating transition of pro-apoptotic factors to the mitochondrial membrane. Also, cytosolic cytochrome c and activated caspases-9 and -3 were significantly higher in all MUN. Conversely, peroxisome proliferator-activator receptor-γ (PPARγ), a member of the nuclear receptor family with anti-apoptotic properties, was significantly downregulated in both zones and horns. Our results suggest that MUN during rat pregnancy enhances mitochondria-dependent apoptosis in the placenta, probably due to the downregulation of PPARγ expression.

To investigate the effect of maternal undernutrition (MUN) during pregnancy on fetal and placental weight, amniotic fluid (AF) volume, AF osmolality and ion concentrations at gestational ages E16 and E20. We also quantified protein expression of water channels (aquaporins; AQPs).Pregnant rat dams were fed an ad libitum diet (AdLib; n = 6) or were 50% MUN (n = 6) beginning at E10 of gestation. At E16 and E20, we assessed the effect of MUN on fetal and placental weights, AF volume and osmolality, and placental expression of AQP1, 8 and 9. We focused on two uterine positions (proximal and mid-horns) with the extremes of nutrient/oxygen supply. We also separately studied the basal zone (hormone production) and the labyrinth zone (feto-maternal exchange).We showed that at E16, MUN fetal, and placental weights were unchanged and that, similarly, MUN AF volume, osmolality were comparable to AdLib. At E20, however, MUN fetal and placental zonal weights were significantly decreased. Inversely, due to MUN, maternal and fetal plasma osmolality and Na+ concentrations were significantly increased. Further, MUN AF volume was significantly reduced, while AF osmolality and Na+ concentration were increased at E20.Placental basal zone showed variable changes in AQP expression unrelated to position in the uterus or the gestational age (and thus severity of the fetal/placental growth restriction). In the labyrinth zone, MUN placental AQP1 was significantly decreased, whereas AQP8 and 9 expressions were significantly increased at E16 and E20. Dysregulation of AQPs' expression prior to the occurrence of oligohydramnios may represent a compensatory mechanism under conditions of early MUN.

Quantification of metabolic changes by magnetic resonance and mechanism of cell death in ovarian cancer Louiza Belkacemia*, Leila Maurmannb and Rathindra N Bosea aDepartment of Biology and Biochemistry, University of Houston, Houston, TX 77204 bDepartment of Chemistry, Kansas State University, Manhattan, KS 6650 Ovarian cancer is the most frequent cause of death from gynecologic malignancy. Presently most ovarian cancers are diagnosed when the disease has progressed to stage III or IV. Therefore there is an urgent need for the identification of new biomarkers for early diagnosis and drug efficacy. Changes in metabolite concentrations have been associated with tumor regression or progression. Nuclear magnetic resonance (NMR) has been used to identify molecular markers during cell death (apoptosis), especially the onset of membrane disintegration. Cisplatin is the most commonly used drug in chemotherapy of ovarian cancer. To date, most research has been focused on the cisplatin-DNA reaction. Thus it is essential to evaluate the effect of cisplatin on binding to other biomolecules in the cell. Chinese hamster ovarian (CHO) cells, which, like resistant ovarian cancer cells, require high cisplatin concentrations to induce cell death, were treated with 50 μM cisplatin at different time intervals. Metabolite concentrations were determined by one and two dimensional 31P and 1H NMR. Moreover, the A2780 cisplatin-sensitive cell line and its resistant variant were evaluated for the mechanism of cell death. Analysis revealed that enhanced apoptosis was associated with significantly increased phosphocholine and glycerophosphocholine metabolites and marked elevation of cellular phosphocreatine and nucleoside triphosphate concentrations at 6 hrs and decreased thereafter. Plasma membrane alterations were paralleled by an upregulation of expression of Fas and related proapoptotic Bax and PUMA genes, and Fas and caspase-3 and -9 proteins. These findings suggest that NMR may be used as a tool to monitor tumors and drug efficacy in humans using for instance tissue biopsies. Further, NMR measurements of cell extracts may provide significant insight on pathways to apoptosis. Citation Format: Louiza Belkacemi. Quantification of metabolic changes by magnetic resonance and mechanism of cell death in ovarian cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1832. doi:10.1158/1538-7445.AM2015-1832

Calcium (Ca2+) entry in cells is crucial for development and physiology of virtually all cell types. It acts as an intracellular (second) messenger to regulate a diverse array of cellular functions, from cell division and differentiation to cell death. Among candidates for Ca2+ entry in cells are-voltage-dependant Ca2+ channels (VDCCs), transient receptor potential (TRP)-related Ca2+ channels and store-operated Ca2+ (SOC) channels. Plasma membrane Ca2+-ATPases (PMCA) and Na+/Ca2+ exchanger (NCX) are mainly responsible for Ca2+ extrusion. These different Ca2+channels/transporters and exchangers exhibit specific distribution and physiological properties. During pregnancy, the syncytiotrophoblast layer of the human placenta transfers as much as 30 g of Ca2+ from the mother to the fetus, especially in late gestation where Ca2+ transport through different channels must increase in response to the demands of accelerating bone mineralization of the fetus. The identification and characterization of the different Ca2+ channels/transporters and exchangers on the brush-border membrane (BBM) facing the maternal circulation, and the basal plasma membrane (BPM) facing the fetal circulation; placental membrane of the syncytiotrophoblasts have been the focus of numerous studies. This review discusses current views in this field regarding localization and functions during transcellular Ca2+ entry and extrusion from cells particularly in the placenta.

Adenylyl cyclase (AC) activity is ubiquitous in mammalian cells, and various forms of this enzyme exist that widely differ with regard to tissue distribution, abundance, and modes of regulation. Human placenta is made, among others, of cytotrophoblast cells and syncytiotrophoblasts. This latter is a polynucleate structure that originates from the differentiation of proliferative mononucleated cytotrophoblast cells, the placental stem cell, into syncytiotrophoblasts. In vitro, this differentiation process is associated with a concomitant increase in cellular levels of cAMP and enhanced expression of genes representative of syncytiotrophoblasts endocrine activity. Thus, in this study we evaluated the differential distribution of AC isoforms in cytotrophoblast cells and syncytiotrophoblasts by reverse transcription-polymerase chain reaction (RT–PCR) using total RNA or purified mRNA. Our results demonstrate that all membrane and soluble AC mRNA isoforms are present in both cell types. Interestingly in syncytiotrophoblasts, AC4 and AC8 mRNA are highly expressed, while AC1, AC2 mRNA are less abundant when compared to cytotrophoblast cells. Additionally, the soluble AC is expressed in both trophoblast cells, but its expression is greatly reduced in differentiated cells, syncytiotrophoblasts. The presence of these AC proteins in both cells was confirmed by Western blotting. Taken together, these data help us to characterize the different AC isoforms in human cytotrophoblast cells and syncytiotrophoblasts, and demonstrate that the AC isoforms expression seems to be mainly modulated in groups 1 and 2. Moreover, the important decrease of the soluble AC isoform in syncytiotrophoblasts as compared to cytotrophoblast cells could suggest an important role of this AC in the extravillous trophoblast formation.

During gestation, transport by the placenta is solely responsible for nutrient supply to the developing fetus. In this context, calcium (Ca2+) transport machinery of the placenta thus represents the primary tissue site for regulating fetal Ca2+ homeostasis. In humans, the transplacental movements of Ca2+ increase dramatically during the last trimester of gestation, when fetal skeletal mineralization is at its highest. However, little is known about the exact mechanism of transport. Evidence suggests that some developmentally expressed cytosolic Ca2+-binding proteins (CaBPs) have an important role in regulating or shuttling cytosolic Ca2+ since they are endowed with a high affinity for Ca2+ (∼106 M −1). CaBPs belong to a large family of eukaryotic proteins containing a specific helix-loop-helix structure, referred to as the EF-hand motif, which counts more than 200 members. Several of these CaBPs were identified in the placenta: CaBP9k, CaBP28k, CaBP57k, oncomodulin, S-100P, S-100α, and S-100β. This review discusses the current views in this field to guide future investigations into the localization and functions of CaBPs during Ca2+ intracellular homeostasis in the placenta.

Pigment epithelium-derived factor (PEDF) is a secreted glycoprotein and a non-inhibitory member of the serine protease inhibitor (serpin) family. It is widely expressed in human fetal and adult tissues but its expression decreases with age and in malignant tissues. The main anti-cancer activities of PEDF derive from its dual effects, either indirectly on the tumor microenvironment (indirect antitumor action) or directly on the tumor itself (direct antitumor influence). The indirect antitumor activities of PEDF were uncovered from the early findings that it stimulates retinoblastoma cell differentiation and that additionally it possesses anti-angiogenic, anti-tumorigenic and anti-metastatic properties. The mechanisms of its direct antitumor effect, however, have not been fully elucidated. This review highlights recent progress in our understanding of the multifunctional activities of PEDF and, in particular, its anti-cancer signaling mechanisms. Additionally, we discuss the possibility of using novel phosphaplatin compounds that can upregulate PEDF expression as a chemotherapy for cancer treatment.

Maternal undernutrition (MUN) during pregnancy may lead to fetal intrauterine growth restriction (IUGR), which itself predisposes to adult risk of obesity, hypertension, and diabetes. IUGR may stem from insufficient maternal nutrient supply or reduced placental nutrient transfer. In addition, a critical role for maternal stress-induced glucocorticoids (GCs) has been suggested to contribute to both IUGR and the ensuing risk of adult metabolic syndrome. While GC-induced fetal organ defects have been examined, there have been few studies on placental responses to MUN-induced maternal stress. Therefore, we hypothesize that 50% MUN associates with increased maternal GC levels and decreased placental HSD11B. This in turn leads to decreased placental and fetal growth, hence the need to investigate nutrient transporters. We measured maternal serum levels of corticosterone, and the placental basal and labyrinth zone expression of glucocorticoid receptor (NR3C1), 11-hydroxysteroid dehydrogenase B 1 (HSD11B-1) predominantly activates cortisone to cortisol and 11-dehydrocorticosterone (11-DHC) to corticosterone, although can sometimes drive the opposing (inactivating reaction), and HSD11B-2 (only inactivates and converts corticosterone to 11-DHC in rodents) in control and MUN rats at embryonic day 20 (E20). Moreover, we evaluated the expression of nutrient transporters for glucose (SLC2A1, SLC2A3) and amino acids (SLC38A1, 2, and 4). Our results show that MUN dams displayed significantly increased plasma corticosterone levels compared to control dams. Further, a reduction in fetal and placental weights was observed in both the mid-horn and proximal-horn positions. Notably, the placental labyrinth zone, the site of feto-maternal exchange, showed decreased expression of HSD11B1-2 in both horns, and increased HSD11B-1 in proximal-horn placentas, but no change in NR3C1. The reduced placental GCs catabolic capacity was accompanied by downregulation of SLC2A3, SLC38A1, and SLC38A2 expression, and by increased SLC38A4 expression, in labyrinth zones from the mid- and proximal-horns. In marked contrast to the labyrinth zone, the basal zone, which is the site of hormone production, did not show significant changes in any of these enzymes or transporters. These results suggest that dysregulation of the labyrinth zone GC "barrier", and more importantly decreased nutrient supply resulting from downregulation of some of the amino acid system A transporters, may contribute to suboptimal fetal growth under MUN.