Metabolic regulation of calcium pumps in pancreatic cancer: role of phosphofructokinase-fructose-bisphosphatase-3 (PFKFB3).

BACKGROUND: High glycolytic rate is a hallmark of cancer (Warburg effect). Glycolytic ATP is required for fuelling plasma membrane calcium ATPases (PMCAs), responsible for extrusion of cytosolic calcium, in pancreatic ductal adenocarcinoma (PDAC). Phosphofructokinase-fructose-bisphosphatase-3 (PFKFB3) is a glycolytic driver that activates key rate-limiting enzyme Phosphofructokinase-1; we investigated whether PFKFB3 is required for PMCA function in PDAC cells. METHODS: PDAC cell-lines, MIA PaCa-2, BxPC-3, PANC1 and non-cancerous human pancreatic stellate cells (HPSCs) were used. Cell growth, death and metabolism were assessed using sulforhodamine-B/tetrazolium-based assays, poly-ADP-ribose-polymerase (PARP1) cleavage and seahorse XF analysis, respectively. ATP was measured using a luciferase-based assay, membrane proteins were isolated using a kit and intracellular calcium concentration and PMCA activity were measured using Fura-2 fluorescence imaging. RESULTS: PFKFB3 was highly expressed in PDAC cells but not HPSCs. In MIA PaCa-2, a pool of PFKFB3 was identified at the plasma membrane. PFKFB3 inhibitor, PFK15, caused reduced cell growth and PMCA activity, leading to calcium overload and apoptosis in PDAC cells. PFK15 reduced glycolysis but had no effect on steady-state ATP concentration in MIA PaCa-2. CONCLUSIONS: PFKFB3 is important for maintaining PMCA function in PDAC, independently of cytosolic ATP levels and may be involved in providing a localised ATP supply at the plasma membrane.

Targeting tumour energy metabolism potentiates the cytotoxicity of 5-aminolevulinic acid photodynamic therapy.

BACKGROUND: Cancerous cells usually exhibit increased aerobic glycolysis, compared with normal tissue (the Warburg effect), making this pathway an attractive therapeutic target. METHODS: Cell viability, cell number, clonogenic assay, reactive oxygen (ROS), ATP, and apoptosis were assayed in MCF-7 tumour cells and corresponding primary human mammary epithelial cells (HMEC). RESULTS: Combining the glycolysis inhibitors 2-deoxyglucose (2DG; 180 mM) or lonidamine (300 µM) with 10 J cm(-2) 5-aminolevulinic acid (ALA) photodynamic therapy (PDT) increases MCF-7 cytotoxicity (by 3.5-fold to 70% death after 24 h, and by 10-fold in 9-day clonogenic assays). However, glycolysis inhibition only slightly increases HMEC PDT cytotoxicity (between two-fold and three-fold to a maximum of 9% death after 24 h). The potentiation of PDT cytotoxicity only occurred if the glycolysis inhibitors were added after ALA incubation, as they inhibited intracellular accumulation of photosensitiser if coincubated with ALA. CONCLUSION: As 2DG and lonidamine are already used as cancer chemotherapeutic agents, our results are directly translatable to combination therapies with existing topical PDT.

Modulation of Ca2+ oscillations by phosphorylation of Ins(1,4,5)P3 receptors.

Activation of InsP(3)Rs (InsP(3) receptors) represents the major mechanism underlying intracellular calcium release in non-excitable cells such as hepatocytes and exocrine cells from the pancreas and salivary glands. Modulation of calcium release through InsP(3)Rs is therefore a major route whereby the temporal and spatial characteristics of calcium waves and oscillations can potentially be 'shaped'. In this study, the functional consequences of phosphoregulation of InsP(3)Rs were investigated. Pancreatic and parotid acinar cells express all three types of InsP(3)R in differing abundance, and all are potential substrates for phosphoregulation. PKA (protein kinase A)-mediated phosphorylation of InsP(3)Rs in pancreatic acinar cells resulted in slowed kinetics of calcium release following photo-release of InsP(3). In contrast, activation of PKA in parotid cells resulted in a marked potentiation of calcium release. In pancreatic acinar cells the predominant InsP(3)R isoform phosphorylated was the type 3 receptor, while the type 2 receptor was markedly phosphorylated in parotid acinar cells. In order to further decipher the effects of phosphorylation on individual InsP(3)R subtypes, DT-40 cell lines expressing homotetramers of a single isoform of InsP(3)R were utilized. These data demonstrate that phosphoregulation of InsP(3)Rs results in subtype-specific effects and may play a role in the specificity of calcium signals by 'shaping' the spatio-temporal profile of the response.

A model of calcium waves in pancreatic and parotid acinar cells.

We construct a mathematical model of Ca(2+) wave propagation in pancreatic and parotid acinar cells. Ca(2+) release is via inositol trisphosphate receptors and ryanodine receptors that are distributed heterogeneously through the cell. The apical and basal regions are separated by a region containing the mitochondria. In response to a whole-cell, homogeneous application of inositol trisphosphate (IP(3)), the model predicts that 1), at lower concentrations of IP(3), the intracellular waves in pancreatic cells begin in the apical region and are actively propagated across the basal region by Ca(2+) release through ryanodine receptors; 2), at higher [IP(3)], the waves in pancreatic and parotid cells are not true waves but rather apparent waves, formed as the result of sequential activation of inositol trisphosphate receptors in the apical and basal regions; 3), the differences in wave propagation in pancreatic and parotid cells can be explained in part by differences in inositol trisphosphate receptor density; 4), in pancreatic cells, increased Ca(2+) uptake by the mitochondria is capable of restricting Ca(2+) responses to the apical region, but that this happens only for a relatively narrow range of [IP(3)]; and 5), at higher [IP(3)], the apical and basal regions of the cell act as coupled Ca(2+) oscillators, with the basal region partially entrained to the apical region.

Experimental assessment of lanthanide ion donor preference: spectroscopic and theoretical dissection of static charge and dynamic polarisation contributions to axial ligation in a C4-symmetric chiral europium complex.

Measurements of the equilibrium constants for ligand exchange (MeCN, 295 K) involving the axial donor in a C4-symmetric, mono-capped, square antiprismatic cationic Eu complex, supported by calculations based on an electrostatic perturbation model, have been interpreted in terms of a predominant ligand polarisation interaction defined by observation of the hypersensitive delta J = 2 normalised emission intensity, in association with measurements correlating delta J = 1 band splitting and 1H NMR dipolar shifts that vindicate Bleaney's theory of magnetic anisotropy.

Pharmacological evaluation of the role of cytochrome P450 in intracellular calcium signalling in rat pancreatic acinar cells.

We have investigated whether the cytochrome P450 system is involved in Ca(2+) signalling in rat pancreatic acinar cells. Intracellular free [Ca(2+)] ([Ca(2+)](i)) was measured in collagenase-isolated cells using fura-2 microspectrofluorimetry and imaging. The imidazole P450 inhibitor ketoconazole (5 - 50 microM) inhibited [Ca(2+)](i) oscillations induced by cholecystokinin octapeptide (CCK). However, ketoconazole also raised baseline [Ca(2+)](i) when applied in the absence of CCK. These effects were mimicked by 5 - 50 microM SKF96365, an imidazole widely used as an inhibitor of Ca(2+) entry. The non-imidazole P450 inhibitor proadifen (SKF525A) inhibited CCK-induced [Ca(2+)](i) oscillations at a concentration of 10 - 50 microM. Proadifen alone caused intracellular Ca(2+) release at 25 or 50 microM, but not at 10 microM. Octadecynoic acid and 1-aminobenzotriazole, structurally-unrelated non-imidazole P450 inhibitors, did not alter baseline [Ca(2+)](i) or CCK-evoked oscillations. We compared cumulative CCK dose-response relationship in control cells and in cells where P450 had been induced by prior injection of animals with beta-naphthoflavone. Only minor differences were apparent, with induced cells showing some decrease in responsiveness at moderate and higher concentration of CCK (30 pM - 3 nM). Direct assessment of depletion-activated Ca(2+) entry showed no clear differences between control and induced cells. In conclusion, we could find no compelling evidence for a role of P450 in controlling Ca(2+) signalling generally, or Ca(2+) entry in particular, in pancreatic acinar cells. Induction of P450 is therefore probably toxic to acinar cells via a Ca(2+)-independent mechanism.

Molecular and functional identification of a Ca2+ (polyvalent cation)-sensing receptor in rat pancreas.

The balance between the concentrations of free ionized Ca2+ and bicarbonate in pancreatic juice is of critical importance in preventing the formation of calcium carbonate stones. How the pancreas regulates the ionic composition and the level of Ca2+ saturation in an alkaline environment such as the pancreatic juice is not known. Because of the tight cause-effect relationship between Ca2+ concentration and lithogenicity, and because hypercalcemia is proposed as an etiologic factor for several pancreatic diseases, we have investigated whether pancreatic tissues express a Ca2+-sensing receptor (CaR) similar to that recently identified in parathyroid tissue. Using reverse transcriptase-polymerase chain reaction and immunofluorescence microscopy, we demonstrate the presence of a CaR-like molecule in rat pancreatic acinar cells, pancreatic ducts, and islets of Langerhans. Functional studies, in which intracellular free Ca2+ concentration was measured in isolated acinar cells and interlobular ducts, show that both cell types are responsive to the CaR agonist gadolinium (Gd3+) and to changes in extracellular Ca2+ concentration. We also assessed the effects of CaR stimulation on physiological HCO3- secretion from ducts by making measurements of intracellular pH. Luminal Gd3+ is a potent stimulus for HCO3- secretion, being equally as effective as raising intracellular cAMP with forskolin. These results suggest that the CaR in the exocrine pancreas monitors the Ca2+ concentration in the pancreatic juice, and might therefore be involved in regulating the level of Ca2+ in the lumen, both under basal conditions and during hormonal stimulation. The failure of this mechanism might lead to pancreatic stone formation and even to pancreatitis.

Analysis of calcium oscillations in pancreatic acinar cells.

Since they were first reported in hepatocytes in 1986, agonist-evoked oscillations in intracellular free calcium ([Ca2+]i) have been described in numerous cell types, including pancreatic exocrine and endocrine cells. Pancreatic acinar cells show both 'sinusoidal' [Ca2+]i oscillations (when stimulated with muscarinic agonists) and so-called 'baseline [Ca2+]i spiking' (when stimulated with cholecystokinin, bombesin or analogues of either peptide). These agonist-evoked [Ca2+]i oscillations present novel problems to the investigator, particularly since they show considerable cell-to-cell variability. In this review, we discuss the various approaches available for analysing [Ca2+]i oscillations, particularly with respect to assessing whether drugs or other experimental interventions alter the oscillation pattern.

Persistence of Schistosoma haematobium infection despite multiple courses of therapy with praziquantel.

A 7-year-old boy, who had returned to the United States in June 1991 after a 3-year stay in Malawi, was evaluated in October 1991 because of hematuria. He was excreting Schistosoma haematobium eggs and was treated with praziquantel (PZQ; approximately 40 mg/kg). He may have spit up < or = 30% of this dose, and a concomitant Giardia lamblia infection might have caused malabsorption of PZQ. Because of persistent excretion of viable eggs, he was retreated with PZQ in January and May 1992. Egg excretion was first quantified 2 months following his second course of PZQ; at that time it was 35 eggs per 10 mL of urine. He excreted viable eggs at least as late as October 1992, 5 months after his third PZQ course. Experimental administration of chemotherapy to hamsters infected with the S. haematobium strain demonstrated that it was susceptible to PZQ. Repeated courses of therapy with PZQ may be necessary to cure S. haematobium infection, and both parasite and host factors should be considered if infection persists.

Response of drug resistant isolates of Schistosoma mansoni to antischistosomal agents.

The susceptibility of four isolates of Schistosoma mansoni (BH, MAP, MPR-1 and K) to four multiple doses of anti-schistosomal agents (hycanthone, niridazole, oxamniquine, and praziquantel) were evaluated in infected female Swiss albino mice. These schistosomal isolates had been maintained in the laboratory without further drug pressure for 20 to 30 generations. Multiple dosage regimens were used for each drug against each isolate of S. mansoni to generate ED50 (effective dose 50%) values. Results demonstrated that the K isolate is resistant to niridazole, the MPR-1 isolate to oxamniquine, and the MAP isolate to both hycanthone and oxamniquine. The BH isolate was susceptible to all drugs and was used as the reference isolate. All isolates were susceptible to parziquantel. The significance of the difference in response of the MPR-1 and MAP isolates is discussed. These results confirm the resistance of these isolates of S. mansoni to three schistosomicides and demonstrate that the resistance of these isolates are stable over long periods of time without exposure to drugs.

Efficacy of Niclosamide as a potential topical antipenetrant (TAP) against cercariae of Schistosoma mansoni in monkeys.

A 1% (W/V) formulation of Niclosamide (2', 5-Dichloro-4-nitrosalicylanilide) (TAP) was tested on Cebus apella monkeys as a topical prophylactic against schistosomiasis mansoni. Two experiments were conducted using the same formulation. In the first experiment, the TAP provided complete protection against schistosomiasis for 3 days. Of the 4 monkeys treated with TAP 7 days before exposure to Schistosoma mansoni cercariae, 2 were completely protected. The remaining 2 monkeys of the 7 day treatment group had a 78% or greater reduction in adult worm burdens when compared to the placebo treated monkeys. The second experiment was designed to determine the time between day 3 and 7 when the TAP no longer provided complete protection. However, all of the TAP treated monkeys in this experiment were completely protected, even the monkeys treated 7 days earlier. In both experiments, all monkeys used as infection controls and those receiving only the placebo became infected and showed typical experimental schistosomiasis. These results demonstrate that the TAP could provide fast acting, short-term protection to people who must enter cercariae infested water.

Giardia lamblia: phospholipid analysis of human isolates.

Thin layer chromatograms for phospholipids obtained from 11 human Giardia lamblia isolates and their culture media have shown that phosphatidylcholine and sphingomyelin are the predominant phospholipid classes in all samples. A decrease in the relative percentage of the different classes, especially of phosphatidylcholine, was noticed in the medium after Giardia growth. Fatty acid analysis of the parasite phosphatidylcholine demonstrated that while oleate and palmitate were the major fatty acids in most isolates, arachidonate predominated in two of those studied. Some isolates contained small amounts of myristate, which was not present in the phosphatidylcholine of the culture medium. Moreover, stearate and linoleate predominated in phosphatidylcholine obtained from both media types. The saturated/unsaturated fatty acid ratio also varied for the different isolates. These results appear to suggest heterogeneity in the metabolic activity and utilization of lipid molecules between Giardia isolates.

Efficacy of alternating therapy with oxamniquine and praziquantel to treat Schistosoma mansoni in children following failure of first treatment.

Two hundred children infected with Schistosoma mansoni were treated with either 20 mg/kg oxamniquine or 60 mg/kg praziquantel. Cure rates (about 85%) were similar as was the percentage reduction (80%) in egg counts in uncured children. Treatment with the alternative drug of children not cured with the first treatment resulted in negative stools in 11 of 12 cases examined one month after the second round of therapy. In order to minimize the risk of the development of drug resistance, our data suggest that infected patients be treated with one drug, and therapeutic failures with another. Evidence from experiments in mice with isolates obtained after failures of one treatment in children suggests that therapeutic failure does not necessarily indicate the presence of drug-resistant schistosomes. The value of using mice to assess drug resistance in schistosomes is questioned.

Inhibition of in vitro RNA synthesis by hycanthone, oxamniquine and praziquantel.

The schistosomicides, hycanthone, oxamniquine and praziquantel, were found to inhibit the in vitro RNA synthesis using isolated hamster liver nuclei. Preincubation of the nuclei with these drugs revealed that the inhibitory effect of oxamniquine was irreversible and progressed with time, whereas that of hycanthone and praziquantel was reversible. On the other hand, hycanthone and praziquantel have a high affinity for DNA but oxamniquine does not. The data indicate that the mechanism of inhibition by oxamniquine is different from that of hycanthone and praziquantel.

Conventional Giemsa-stained and C-banded chromosomes of seven strains of Schistosoma mansoni.

Karyotypes stained with conventional Giemsa and with a C-banding method were compared among 7 strains of Schistosoma mansoni: 2 from Puerto Rico and 1 each from St. Lucia, Brazil, Venezuela, Egypt, and Kenya. A few differences were noted in relative lengths and centromeric indexes, but overall karyotypes of all strains were similar, with 2n = 16. The W chromosome of the female of all strains had a relatively large heterochromatic block, distinguishing the female from the male karyotype.

Rate of action of schistosomicides in mice infected with Schistosoma mansoni.

Mice infected with adult Schistosoma mansoni were dosed with a single oral dose of 125 or 250 mg/kg oltipraz, 50 or 100 mg/kg oxamniquine, or 200 or 400 mg/kg praziquantel. The mortality rate of worms and oogram changes were determined between 1 and 16 weeks after dosing. The time required between dosing and postmortem to obtain maximum effectiveness was 1 week for praziquantel, 2 weeks for oxamniquine and 8 weeks for oltipraz. Changes in oograms persisted throughout most of the experiment, although relapse has been observed at the 4th week on.

The detection of IgE by radio allergosorbent technique (R.A.S.T.) and ELISA in Egyptian cases of schistosomiasis.

Studies were carried out on the radioallergosorbent test (R.A.S.T.) in comparison with the Elisa for the diagnosis of schistosomiasis. Using sera from known S. mansoni, S. haematobium and both S. mansoni and S. haematobium cases, R.A.S.T. showed high sensitivity; 72%, 55% and 75% respectively, in the detection and quantification of specific IgE antibodies. While in case of ELISA only 20% of patients were positive. With control sera, both tests showed 100% negative. It is concluded that R.A.S.T. is more specific and superior than ELISA for IgE detection in schistosomiasis.

New trends in the sero-diagnosis of schistosomiasis in Egypt.

A comparison has been made of a newly modified diffusion-in-gel enzyme-linked immunosorbent technique (DIG-ELISA) and an established, standardized ELISA, using sera as well as whole blood adsorbed on filter paper discs. With known human cases of S. mansoni and/or S. haematobium, both tests showed great sensitivity in the diagnosis of schistosomiasis; 93%-95% positive for ELISA and 92%-93% positive by DIG-ELISA on using egg antigens, while 92% positive for ELISA and 91% positive by DIG-ELISA when worm antigens were used. DIG-ELISA is characterised by its technical simplicity, high sensitivity which make it an excellent screening serological technique especially in endemic areas. Moreover, the use of whole blood on filter paper discs (paper disc DIG-ELISA), rather than serum from venipuncture should add more advantages for its field application.

The molluscicidal activity of Phytolacca dodecandra. I. Location of the activating esterase.

A number of methods have been used to extract molluscicidal saponins from the dried berries of Phytolacca dodecandra. The potency of the extract has been determined to depend on the release of an enzyme found only in the seed and breaking the seed is critical to the extraction process. The enzyme is inactivated by heat of alcohol. The highest potency extract is made from a cold water extraction of finely ground dried berries.