Dopamine receptors in emesis: Molecular mechanisms and potential therapeutic function.

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.

Neurotrophic factor expression in expandable cell populations from brain samples in living patients with Parkinson's disease.

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.

Evidence for Bell-Shaped Dose-Response Emetic Effects of Temsirolimus and Analogs: The Broad-Spectrum Antiemetic Efficacy of a Large Dose of Temsirolimus Against Diverse Emetogens in the Least Shrew (Cryptotis parva).

Temsirolimus is a prodrug form of sirolimus (rapamycin). With its analogs (everolimus, ridaforolimus, and rapamycin), it forms a group of anticancer agents that block the activity of one of the two mammalian targets of rapamycin (mTOR) complexes, mTORC1. We investigated the emetic potential of varying doses (0, 0.5, 1, 2.5, 5, 10, 20, and 40 mg/kg, i.p.) of temsirolimus in the least shrew. Temsirolimus caused a bell-shaped and dose-dependent increase in both the mean vomit frequency and the number of shrews vomiting with maximal efficacy at 10 mg/kg (p < 0.05 and p < 0.02, respectively). Its larger doses (20 or 40 mg/kg) had no significant emetic effect. We also evaluated the emetic potential of its analogs (5, 10, and 20 mg/kg, i.p.), all of which exhibited a similar emetic profile. Our observational studies indicated that temsirolimus can reduce the shrew motor activity at 40 mg/kg, and subsequently, we examined the motor effects of its lower doses. At 10 and 20 mg/kg, it did not affect the spontaneous locomotor activity (distance moved) but attenuated the mean rearing frequency in a U-shaped manner at 10 mg/kg (p < 0.05). We then determined the broad-spectrum antiemetic potential of a 20 mg/kg (i.p.) dose of temsirolimus against diverse emetogens, including selective and nonselective agonists of 1) dopaminergic D2/3 receptors (apomorphine and quinpirole); 2) serotonergic 5-HT3 receptors [5-HT (serotonin) and 2-methyl-5-HT]; 3) cholinergic M1 receptors (pilocarpine and McN-A-343); 4) substance P neurokinin NK1 receptors (GR73632); 5) the L-type calcium (Ca2+) channel (LTCC) (FPL64176); 6) the sarcoplasmic endoplasmic reticulum Ca2+ ATPase inhibitor, thapsigargin; 7) the CB1 receptor inverse agonist/antagonist, SR141716A; and 8) the chemotherapeutic cisplatin. Temsirolimus prevented vomiting evoked by the aforementioned emetogens with varying degrees. The mechanisms underlying the pro- and antiemetic effects of temsirolimus evaluated by immunochemistry for c-fos expression demonstrated a c-fos induction in the AP and NTS, but not DMNX with the 10 mg/kg emetic dose of temsirolimus, whereas its larger antiemetic dose (20 mg/kg) had no significant effect. Our study is the first to provide preclinical evidence demonstrating the promising antiemetic potential of high doses of temsirolimus and possibly its analogs in least shrews.

Altered mitochondrial apoptotic pathway in placentas from undernourished rat gestations.

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-X(L), 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.

Altered placental development in undernourished rats: role of maternal glucocorticoids.

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.

Signal transduction pathways involved in dopamine D2 receptor-evoked emesis in the least shrew (Cryptotis parva).

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 PKCαßII 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), PKCαßII (Thr638/641), ERK1/2 (Thr202/204), and Akt (Ser473). Our results substantiate the implication of PI3K/mTOR/Akt and PI3K/PKCαßII/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.

Maternal undernutrition influences placental-fetal development.

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.

Δ9-THC and related cannabinoids suppress substance P- induced neurokinin NK1-receptor-mediated vomiting via activation of cannabinoid CB1 receptor.

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 (<10 mg/kg). We also found that the NK1 receptor antagonist netupitant can significantly suppress vomiting caused by a large emetic dose of SR141716A (20 mg/kg). In sum, Δ9-THC and related cannabinoids suppress vomiting evoked by the nonselective (SP) and selective (GR73632) neurokinin NK1 receptor agonists via stimulation of cannabinoid CB1 receptors.

AQP1 gene expression is upregulated by arginine vasopressin and cyclic AMP agonists in trophoblast cells.

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<0.05). Both SP-cAMP, a membrane-permeable and phosphodiesterase resistant cAMP, and forskolin, an adenylate cyclase stimulator significantly increased AQP1 mRNA expression in all cell lines after 2 h in a dose-dependent manner (P<0.05) with a parallel increase in protein expression. In the time course study, 5 microM of either SP-cAMP or forskolin significantly stimulated AQP1 mRNA expression after 2 h in HTR-8/SVneo cells and after 10 h in JAR and JEG-3 cells. AQP1 protein expression was highest after 20 h in both HTR-8/SVneo and JEG-3 cells (P<0.05). AVP-stimulated cAMP elevation was blocked in the presence of 9-(tetrahydro-2'-furyl) adenine (SQ22536) (100 microM), a cell-permeable adenylate cyclase inhibitor (P<0.05). These results indicate that in trophoblasts-like cells AQP1 gene expression is upregulated by both AVP and cAMP agonists. Furthermore, our data demonstrate that a cAMP-dependent pathway is responsible for the AVP effect on AQP1. Thus, modulation of AQP1 expression by maternal hormones may regulate invasion and fetal-placental-amnion water homeostasis during gestation.

Phosphaplatin Anti-tumor Effect Enhanced by Liposomes Partly via an Up-regulation of PEDF in Breast Cancer.

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.

Altered cytokine network in gestational diabetes mellitus affects maternal insulin and placental-fetal development.

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.

Cross-sectional pilot study about the liver enzymes profile in type 2 diabetic patients from an Algerian west region: Wilaya of Mostaganem.

AIMS: The magnitude of abnormal liver enzymes profile in type 2 diabetic patients is unknown in Algerian west region even though it counts among liver diseases considered as an important cause of death in type 2 diabetes. The main objective is to assess the prevalence of elevated liver enzymes levels among patients with type 2 diabetes from Algerian west region and to determine associated risk factors. MATERIALS AND METHODS: A descriptive cross sectional study was performed on 180 type 2 diabetic patients in whom anthropometric and biochemical parameters were determined. RESULTS: Twenty-five patients had abnormal elevated alanine transaminase (ALT) (13.9%) with the gender-wise prevalence being 15.9% (n=17) in women and 10.9% (n=8) in men. The prevalence of abnormal elevated aspartate transaminase (AST), gamma glutamyl transferase and alkaline phosphatase level was respectively 10% (n=18), 6.1% (n=11) and 8.9% (16). High waist circumference (OR: 5, CI: 1.04-24.04) and high blood pressure (OR: 4.86, CI: 0.94-25.12) were only associated with elevated AST. Fasting glucose >1.4g/l were associated both with elevated ALT (OR: 3.03, CI: 0.86-10.67) and AST (OR: 5.7, CI: 1.09-29.8). CONCLUSION: A relatively high prevalence of elevated liver enzymes was found in diabetic patients from west Algeria, especially in female patients.

Anti-tumor effects of pigment epithelium-derived factor (PEDF): implication for cancer therapy. A mini-review.

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.

Correlation between Gene Variants, Signaling Pathways, and Efficacy of Chemotherapy Drugs against Colon Cancers.

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.

Insights into the anti-angiogenic properties of phosphaplatins.

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.

Calcium channels, transporters and exchangers in placenta: a review.

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.

Inhibition of human trophoblast invasiveness by high glucose concentrations.

CONTEXT: Trophoblast invasion of the uterus is regulated by local microenvironmental factors. OBJECTIVE: 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. RESULTS: 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 (P < 0.01) 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 (P < 0.05). 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 (P < 0.05). In contrast, glucose transporter-3 levels were not affected by incubation in various glucose concentrations. CONCLUSIONS: 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) identification and regulation by 1,25-dihydroxyvitamin D3 in human choriocarcinoma cell line JEG-3.

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.

Absence of Activation of DNA Repair Genes and Excellent Efficacy of Phosphaplatins against Human Ovarian Cancers: Implications To Treat Resistant Cancers.

Phosphaplatins, platinum(II) and platinum(IV) complexes coordinated to a pyrophosphate moiety, exhibit excellent antitumor activities against a variety of cancers. To determine whether phosphaplatins trigger resistance to treatment by engaging DNA damage repair genes, a yeast genome-wide fitness assay was used. Treatment of yeast cells with pyrodach-2 (D2) or pyrodach-4 (D4) revealed no particular sensitivity to nucleotide excision repair, homologous recombination repair, or postreplication repair when compared with platin control compounds. Also, TNF receptor superfamily member 6 (FAS) protein was overexpressed in phosphaplatin-treated ovarian tumor cells, and platinum colocalized with FAS protein in lipid rafts. An overactivation of sphingomyelinase (ASMase) was noted in the treated cells, indicating participation of an extrinsic apoptotic mechanism due to increased ceramide release. Our results indicate that DNA is not the target of phosphaplatins and accordingly, that phosphaplatins might not cause resistance to treatment. Activation of ASMase and FAS along with the colocalization of platinum with FAS in lipid rafts support an extrinsic apoptotic signaling mechanism that is mediated by phosphaplatins.

HSP27 knockdown produces synergistic induction of apoptosis by HSP90 and kinase inhibitors in glioblastoma multiforme.

BACKGROUND/AIM: The heat-shock proteins HSP27 and HSP90 perpetuate the malignant nature of glioblastoma multiforme (GBM) and offer promise as targets for novel cancer therapeutics. The present study sought to define synergistic antitumor benefits of concurrent HSP27-knockdown and the HSP90 inhibitor, 17-N-allylamino-17-demethoxygeldanamycin (17-AAG) or, comparatively, the non-selective kinase inhibitor, staurosporine, in GBM cells. MATERIALS AND METHODS: Dose-response relations were determined for 17-AAG and staurosporine in three GBM cell lines. HSP27-targeted siRNA was administered alone or in combination with subtherapeutic concentrations of each drug and cells were evaluated for viability, proliferation and apoptosis. RESULTS: Adjuvant HSP27 knockdown with 17-AAG or staurosporine produced marked and synergistic decrease in GBM cell viability and proliferation, with robust elevation of apoptotic fractions and caspase-3 activation. CONCLUSION: HSP27 knockdown confers potent chemosensitization of GBM cells. These novel data support the development of HSP-targeting strategies and, specifically, anti-HSP27 agents for the treatment of GBM.