Effect of Etidronate and Ibandronate on Cytosolic Ca 2 + in HT 29 and Parasite Cell Line from Echinococcus Granulosus sensu lato

Abstract: Background: The bisphosphonates are synthetic analogs of pyrophosphate in which two phosphates are connected through carbon instead of oxygen. They are approved compounds for the treatment of hypercalcemia, bone diseases and they have been proposed to treat infectious diseases. Bisphosphonates’ main mechanisms of action are on calcium metabolism, inhibition of protein prenylation and on ATP synthesis. In a previous work, the antiparasitic activity of bisphosphonates on a cell line from Echinococcus granulosus, sensu lato protoscoleces, 30 μM etidronate and ibandronate have antiproliferative activity after 72 h of incubation, decreasing intracellular ATP and only etidronate increased intracellular total calcium concentration.


INTRODUCTION
The bisphosphonates (BF) are synthetic analogs of pyrophosphate in which two phosphates are connected through carbon instead of oxygen.The BF binds to bone by the chemical group P-C-P while the NH 3 -and lateral chains confer them with other pharmacological properties.They are approved compounds for the treatment of hypercalcemia, bone diseases and osseous cancer metastasis acting on pH regulation, hydroxyapatite stability [1] and Ca 2+ metabolism [2,3].
The BF have been proposed to treat infectious diseases [4,5].In our laboratory, we are studying a parasitic worldwide distributed diseases caused by Echinococcus granulosus, sensu lato (E.granulosus,s.l), an etiological agent of Cystic Echinococcosis (CE).The E. granulosus, s.l development requires two hosts: the embryonic eggs originated by the worm stage in the dog intestine released with the feces, preserved in humid environments to find the intermediary ungulate host (ovine, bovine, camelid and porcine) or humans, who ingested the eggs.The embryos invade the hosts' organs by passing through the intestinal barrier; there they develop the larvae disease, CE.The metacestode, larvae, is located mainly in the liver and the lungs and could primarily be implanted or disseminated to other organs by spreading embryonic corps, protoscoleces (pe).Metacestode develops a cyst, external laminal membrane is rich in calcium and, inside the hydatid fluid (HF) are high-calcified isolate cells.Membrane calcification is associated with parasite viability lost [6] affecting membrane permeability [7].
The pharmacological effect of five BF was studied in our laboratory on an in-vitro parasite model constituted by a cell line, EGPE, isolated from bovine's pe, genotype G1 by Cytochrome c oxidase, subunit 1 (DCO1) sequence [8].EGPE forms cystic colonies in agarose and BF decreased colonies development and ATP synthesis.Nevertheless, BF effect has not always correlated with the total intracellular calcium increased.Cells treated with 30 µM etidronate (HEDP) showed total intracellular calcium increased, but cells treated with ibandronate (IB) did not, they were measured with the colorimetric method [9].
In order to investigate if the antiproliferative effect is dependent on BF role on cytoplasmic ionic calcium ([Ca  2+ ] c ) we studied the effect of HEDP and the IB on ] c ) could be related to the pharmacological effects on cell viability.

2+
] c and Incubation Conditions

Salts Solutions
Calcium salts, CaCl 2 (Anedra) and CaGluc 2 (Sigma), prepared in deionized water and used at final concentration of 1.5 mM Ca 2+ .The BF, HEDP and IB, were prepared in HBSS, pH 7.5 and were used at a final concentration of 30 µM, the concentration had an antiproliferative effect on EGPE cells [9].

Cytoplasmic Ca 2+ Measurement
It was performed in spectrofluorometer Glomax multifunction (Promega) heated at 25 °C.Fluorescence (Fl) was measured in extinction and emission (Ex/ Em) wavelengths, 495-516 nm, every 1.5 min.The minimum fluorescence (Fl min ) or baseline, were values obtained from cell suspension (90 µl) with HBSS (10 µl) in the first 5 min.Then, any calcium salt solution was added to each well at final concentration of 1.5mM (10 µl).When fluorescence values reached the plateau, after 15-20 min, 9 µM ionomycin (io) was added (1 µl) to obtain the maximum value of fluorescence (Fl max ).The total time of fluorescence recorded was 30 min and the final volume/ well was 111 µl.Measures were performed by triplicate in 3 independent experiments (Figs.1a and 1b).

Studies of Bisphosphonates Effect
BF were added in sample wells and then the calcium salts were added.The values of [Ca 2+ ] c were recorded and compared to those obtained with calcium salts without BF but, with HBSS instead, controls.

Net [Ca 2+ ] c in HT29 and EGPE cells
It was calculated to compare nM [Ca 2+ ] c between cell lines, proteins were measured by Bradford method and the values of nM [Ca 2+ ] c / µg prot were calculated in cells defied with CaGluc 2 .

Calcium Concentration and Statistics
To calculate the [Ca 2+ ] c the formula (Gee KR, 2000) was used where [Ca 2+ ] c = Kd * (Fl-Fl min )/ (Fl max -Fl).Where Fl min was the fluorescence value obtained before calcium salts addition in the same sample (first 5 min) and Fl max was the maximum fluorescence value obtained after io addition (5 to 10 minutes) and Fl was the fluorescence value obtained in the plateau after calcium salts addition and before io (15-20 min) (Fig. 1).For the BF effect, statistic differences were calculated using contrasting samples, cells treated with BF with controls were incubated with calcium salts, cells without BF performed at the same time.The χ 2 test and "student's t" test were used to evaluate differences on BF effect on cell growth and differences on nM Ca 2+ / cell or nM Ca 2+ / µg proteins.Results are expressed in mean ± standard deviation (SD).

The Calcium Salts Drive Cytoplasmic Ionic Calcium was Related with Polarized Cell Condition
HT29 and EGPE labeled cells were incubated in hyperpolarized cell condition and after 1.50 mM CaCl 2 or CaGluc 2 addition, both cell lines showed that Cl -, compared with Gluc -, significantly favored the increase of [Ca 2+ ] c ; p< 0.05, "student`s t" test, (Figs.3a and 3b).
The difference on [Ca  2+ ] c related to Cl -compared with Gluc -, calcium salt, in both cell lines, was lost in depolarized cell condition (Figs.3c and 3d).However, in HT29 cells, p< 0.05 less [Ca  2+ ] c was accounted in depolarized cells condition compared with hyperpolarized status, whatever calcium salt used, Cl -or Gluc -.Instead in EGPE cells, this difference was only found with Cl -.

Effect of Bisphosphonates on [Ca 2+ ] c Marked more
Differences between HT29 and EGPE Cell Lines.
Differences in [Ca  2+ ] c , between both cell lines were more evident with the addition of BF, always compared samples with controls, incubated cells in the same conditions adding calcium salts.The addition of 30 µM HEDP on hyperpolarized cells condition, in EGPE, cells decreased [Ca 2+ ] c only with CaCl 2 (Fig. 3a) and, on HT29 cells [Ca 2+ ] c increased, independent of calcium salt added (Fig. 3b).On the contrary, the results obtained in depolarized cells condition showed that HEDP decreased significantly [Ca  2+ ] c in both cell lines, but on EGPE this effect was only observed with CaGluc 2 (Figs.3c and 3d).

DISCUSSION
In a previous work, the BF effect on parasite's EGPE cell line from E. granulosus G1 was studied.Bisphosphonates decreased cell proliferation and only the HEDP increased the total intracellular calcium concentration [9].In this work, the HEDP and IB have a direct effect on [Ca  2+ ] c in two cell lines, EGPE and mammalian HT29 cells, in the whole cell by the spectrofluorometric method.The concentration of BF used was the same of its antiproliferative effect in vitro on EGPE cells.In this work we observed a selective decreased [Ca  2+ ] c only on parasite cell line, in hyperpolarized condition, disclosing an associated antiproliferative mechanism.
A wide range of mechanisms regulates [Ca 2+ ] c , proteins and inorganic compounds chelate the calcium ions.These mech-anisms are critical, changes in the cytosolic Ca 2+ concentration regulates the most of enzymes and pathways involved in normal cell physiology [10,11].The sources of ionic calcium are extracellular or intracellular.The intracellular calcium is released through the endoplasmic reticulum (ER), involving the inositol triphosphate (IP3) and its receptors [12].The Ca 2+ binds oxygen-bearing proteins, altering its conformation and by this mechanism distinguishes the main cation concentration, K + or Na + , depolarized or hyperpolarized cells [13].The Cl - facilitated the increase of [Ca 2+ ] c on both cell lines in [Na + ] o , as it has been described in endothelial cells [14] and secretory cells [15].Ca 2+ -activated Cl -channels could explain the preferred calcium conductance by Cl -over Gluc -, probably by Na + / Ca 2+ pump activation in plasmatic membrane.The higher regulation, involving Na + influx, probably in response to an increase of osmolality produced by Gluc -in the extracellular space.Results obtained by Ibarra & Reisin, 1994 [16] had observed in pe more depolarization to K + than Na + , and 50% of Na + current was blocked by amiloride, which decreased of total intracellular calcium on EGPE cells [9].The HF has the same concentrations of sodium, chloride, bicarbonate, and higher potassium and calcium and lower concentration of phosphate than plasma [17].Nevertheless, potassium, magnesium, calcium, and bicarbonate were higher in pe than in HF, and chloride was not detected in pe [18], but in isolated pe membrane have described small permeability to Cl - [19].The adaptive ionic balance of the pe to HF could explain the differences, between cell lines, of [Ca  2+ ] c regulation related with [Na + ] o and [K + ] o .Other difference between both cell lines is the tolerance of EGPE cells to the ionic calcium concentration/ µg proteins, compared with HT29 cells.
For the BF, a pleiotropic action mechanism has been proposed.BF are highly ionized at physiological pH and the affinity for hydroxyapatite is favored in the HEDP over the IB [20].On soft tissues, HEDP decreases dystrophic calcification compensating PPi deficit [21].HEDP regulates the extracellular and intracellular [Ca  2+ ], reduces ROS generation and cell apoptosis in glutamate injured PC12 cells [22].For amino-BF, as IB, the pharmacological effect is related to cholesterol metabolism on protein prenylation [5,23].HEDP, IB and others BF need 72 h to display the antiproliferative effect [9,24], pointing to metabolic action mechanisms, in which Ca 2+ could be involved.And, in calcification of Stylophorum's ectoderm, was described as participation of voltage-dependent calcium channel (Cav1), related with calcium L-type family [25] and BF inhibit the Ca 2+ L-channel inhibitors [2].

ETHICS APPROVAL AND CONSENT TO PARTI-CIPATE
Not applicable.

HUMAN AND ANIMAL RIGHTS
No animals/humans were used for studies that are the basis of this research.
Differences in the BF effect on cell lines were evident in hyperpolarization.On HT29 cells we observed the synergic effect of HEDP, Na Moreover, results obtained with EGPE cells and HEDP agrees with our previous findings [9], suggesting the effect of HEDP on Ca 2+ stabilization, firstly localized in plasmatic cell membrane.

CONCLUSION
The HEDP was the BF, which presented more selectivity in its effect respect to cell line, EGPE decreased [Ca  2+ ] c incubated in hyperpolarized condition, while in the same conditions [Ca  2+ ] c increased by the effect of HEDP on HT29 cell line.IB disclosed discrete differences between the studied cell lines, may be due mainly by its effect on intracellular membranes.BF-Cl -transport and cells response to osmolality compensated by Na + transport require further investigation This work was supported by a grant from Fundación Iberoamericana de Estudios Superiores (FIES), Buenos Aires, Argentina.Lilian Andrea Granada Herrera is a student of the Maestría en Investigación Clínica Farmacológica, Universidad Abierta Intreramericana, Argentina.Mariana Ferrulli is a student of Biotechnology, Universidad Nacional de Quilmes, Argentina and she obtained a fellowship from FIES to perform this work in CAECIHS, UAI.Patent pending "Linea celular de protoecolices de Echinococcus granulosus SPP.Procedimiento para la obtener estructuras quísticas y método para evaluar la actividad de drogas antiparasitarias".Instituto Nacional de la Propiedad Industrial (INPI) Argentina P-090102320, Inventor Alicia G Fuchs, owner FIES.
Authors: Mariana Ferrulli, Fernando Gabriel Perez Rojo and Lilian Andrea Granada Herrera performed the research.Andrea Maglioco collected data and Emilio AJ Roldán contributed important reagents.Alicia G Fuchs designed the research, analyzed data and wrote the manuscript.

Fig. ( 1
Fig. (1).Calibration of fluorescence measurement in EGPE (a) and HT29 (b) cell lines.Labelled cells with Fluo-4,AM were incubated in medium 149 mM [Na + ] o and 2.22 mM [K + ] o , as described in material and methods.Control are labelled cells (Fluo-4,AM) with the addition of HBSS (--) instead calcium salt.Aliquots of 0.5 mM CaGluc 2 was added, in samples (----), as indicated in the figures, thin arrows, three times.When samples reach a plateau 9 µM ionomicin was added, in sample and control, gross arrow.Fluorescence values were obtained and expressed as Flourescence= [(Fl-Fl min )/ Fl min ].The Fl min is the last value of fluorescence obtained before calcium salt addition and Fl corresponds to the values obtained in each point of measure.Samples and controls run in duplicate and showed mean ± SD.

Fig. ( 2 )
Fig. (2).HT29 Cells Growth, Arrow indicate the beginning of bisphosphonates (BF) treatment, control, without BF cells , cells incubated with 30 µM HEDP or IB .Every point is the mean ± SD of a triplicate sample.Only the viable cells were graphed.