Vanadium compounds affect growth and morphology of human rhabdomyosarcoma cell line

Introduction

Vanadium (atomic number 23) is a period 4, VB group element, which appears in compounds at different stages of oxidation (-III, -I, 0 and from +I to +V). According to Aureliano and Gandara 2005 [1] and Soares et al. 2006 [2], inside various types of cells, 98% of vanadium appears as vanadyl ions [V(IV)], whereas vanadate forms not only monomers but also dimers, tetramers, pentamers and decamers that interact with proteins. The majority of mammalian tissues contain approximately 20 nM of vanadium; the tissue deposit decreases in the following descending order: bone > liver = kidney = spleen > blood > muscle > brain. In living organisms, vanadium affects the growth, development and differentiation of some species, and as it has been known since 1899, it acts as an insulin-mimetic, antidiabetic compound, while since the second half of the 20th century, vanadium has been acknowledged as a “drug”, affecting such mitogenic cell responses as apoptosis, proliferation and neoplastic transformation. Vanadium compounds are capable of operating as pro- or antineoplastic agents, i.e. of inducing growth or inhibiting proliferation and survival of neoplastic cells. They act on both animal and human cells, affecting not only neoplastic, cancer cells but also normal cells. The results of vanadium activity have been reported as those of anti-tumour drugs by many investigators (Ban et al. 2000 [3], Evangelou et al. 1997 [4], Molinuevo et al. 2004 [5], Scrivens et al. 2003 [6], Zhang et al. 2001 [7], Wozniak and Blasiak 2004 [8]), similarly as in our preliminary experiments (Holko et al. 2008 [9], Kordowiak et al. 2007 [10] Klein et al. 2008 [11]); however, some authors have not confirmed this effect and suggest a contrary action of this element (Sakai 1997 [12], Ding et al. 1999 [13], Rodriguez-Mercado et al. 2003 [14], Shi et al. 2004 [15], Zhang et al. 2002 [16]).

This paper presents a comparison between the influence of two inorganic salts, orthovanadate and metavanadate [V(V)] and BMOV(bis (maltolato)oxovanadium), an organic vanadyl derivative, and vanadium sulphate [V(IV)] on growth, survival and morphology of the human cancer RMS cell line.

Material and methods

Reagents



Sodium orthovanadate (Na3VO4), DMEM, F12, glucose, L-glutamine, trypsin, tylosine, EDTA, albumin, penicillin, streptomycin, N-hexamethylpararosaniline (crystal violet = CV) or bromide 3-(4,5-dime­thylthiazol-2)-2,5-diphenyltetrazolium (MTT), glutaraldehyde 8% or 25% were obtained from Sigma Chemical Company St Louis USA, vanadyl sulphate (VOSO4) hydrate comes from Aldrich Chem. Comp Inc, sodium metavanadate (NaVO3) comes from Fluka, bovine serum and physiological buffered saline (PBS) came from WSS Lublin Poland: fetal bovine serum (FBS) was obtained from Biowest, South American Origin. For electron microscopy, Spurr epoxy resin from Pellco Co. and formaldehyde and osmium tetroxide were purchased from Polysciences Inc., test tubes 15 and 50 ml, Eppendorf tubes 1.5 ml, Falcon bottles of areas 25 and 75 cm, 96-well plates, 10 cm plates, sterilized filters with pore size 0.22 m were obtained from Technoplastic Products AG, Switzerland. All other reagent at analytical grade came from POChem Gliwice, Poland. Bis(maltolato)oxovanadium (BMOV) was synthesized and characterized in Chemical Faculty UJ.

Cell culture

The human cancer rhabdomyosarcoma (RMS) cell line was obtained from the Institute of Immunology and Experimental Therapy, Wroclaw, Poland.

Cell proliferation assays

This investigation was conducted employing two staining methods: N-hexamethylpararosaniline (crystal violet = CV) or bromide 3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium (MTT). The cells were seeded on 24-well plates at a density of 12×103 per well in 0.8 ml DMEM. After 24 h the medium was replaced by serum-free medium and the cells were exposed to from 0 (control without vanadium) to 60 M of the four vanadium salts. Within 48 h of incubation with two inorganic salts, Na3VO4, NaVO3 [V(V) as anions], or BMOV (an organic vanadyl derivative, i.e. bis(maltolato)oxovanadium), VOSO4 where [V(IV) as cations] from 0 (control without vanadium) up to 60 M in serum-free medium, the mean values of IC50 of these two tests for RMS were calculated. This paper presents a comparison between the influence of these four vanadium compounds on growth, survival and morphology of the human RMS cell line. The methods of Gillies et al. 1986 [17] and Mosmann 1983 [18] were used to determine proliferation or survival of these cells. Each experiment was repeated at least seven times in threefold irrespectived tests.

Statistical analysis

The results are expressed as mean ± standard error (SEM). Differences between the vanadium-treated cells and control cells were evaluated statistically using Wilcoxon’s matched pair test according to Statsoft Statistica program (Motulsky 1996 [19] Statsoft Statistica manuals [20]). P values lower than 0.05 were considered significant. CV and MTT results were used to obtain a hypothetical dose-dependent curve. The statistical analysis of the obtained dose-dependent curves was performed by MANOVA according to the Statsoft Statistica software.

Electron microscopic examination

After passaging, RMS cells were plated (in the amount of 5×106/plate) on plates 10 cm in diameter and 15 ml of DMEM with 5% FBS and 2 mM glutamine and 0.45% glucose, penicillin (100 units/ml) and streptomycin (100 g/ml) was added to each plate. After 24 h the medium was replaced by standard defined serum-free medium DMEM/F12 (1 : 1) with the investigated concentration of four vanadium salts (see above). In the case of control cells, the medium consisted solely of DMEM/F12 without the addition of vanadium compounds. After 48 h at 37°C, 14 ml of the liquid was discarded from each plate, the cells were scraped away from the plate and placed in 1.5 ml Eppendorf tubes with approximately 1 ml of the liquid. The tubes were centrifuged in an MPW-360 centrifuge at 1000 rev/min for 1 min. After discarding the supernatant, the cells in the form of pellets were underlayered with 1.5 ml of 4% glutaraldehyde at 4°C and allowed to remain at the same temperature for approximately 60 min. Subsequently, the samples were transferred to the Chair of Pathomorphology, Collegium Medicum Jagiellonian University. After centrifuging, all the samples were washed in buffer. Subsequently, the samples were postfixed in 1% osmium tetroxide. After dehydration in graded concentrations of ethyl alcohol and propylene oxide, the tissue was embedded in Spurr medium. The samples were sectioned with an ultramicrotome Reichert Ultracut S using a diamond knife. Semi-thin sections were stained with methylene blue and ultra-thin sections with 8% uranyl acetate dissolved in 50% methanol and then in lead citrate according to Venable and Coggeshal 1965 [21]. All studies were performed under the electron microscope Zeiss EM 900 operating at 80 kV.

Results

The effect of vanadium cations (i.e. BMOV and VOSO4) or anions (Na3VO4 and NaVO3) on autocrine growth of rhabdomyosarcoma (RMS) human childhood malignancies is shown in Fig 1. The figure summarizes the mean values ± SD obtained by two different methods: CV (crystal violet) and MTT. Statistically significant values as compared with the control within 20-40 M vanadium ion concentration were obtained. Lower than 20 M vanadium ion concentration (excluding NaVO3 which obtained the value at 10 M) were not statistically significant; greater than 40 M, almost all cells demonstrate destruction or death and are not shown. Table I showed comparison of the influence on RMS cell line growth inhibition by four investigated vanadium compounds within the investigated concentration.

Table II presents the mean values of IC50 from the CV and MTT study. The last result was important, because within these concentrations the cells were prepared for morphological investigations. Results of the study are shown in Figs. 2-6. Ultrastructurally, RMS cells did not show the presence of contractile filaments, either thick or thin. In the whole investigated group the time of cells’ incubation was 48 h and cells were observed under primary magnification of EM 4500×. Control (without vanadium compounds) surviving cells demonstrated a large number of small vacuoles and small cytoplasmic fissures. In groups submitted to action of both V(V) compounds there were encountered smooth cell membranes often lacking cytoplasmic processes or microvilli. Scarce large vacuoles at times were filled with biological material. In cells submitted to NaVO3 influence, large fissures in the perinuclear zone and smaller cytoplasmic ones were observed. Cells subjected to vanadium derivatives V(IV) had less or more plicated cell membranes often with preserved microvilli. Large vacuoles (often digestive) appeared in cells submitted to 45 M BMOV action; after influence of c. 30 M VOSO4 the cells demonstrated fairly small cytoplasmic vacuoles. A predominantly characteristic observation in RMS cells was chiefly pleomorphic nuclei with a visible amount of heterochromatin and large nucleoli. In all investigated groups a large number of markedly damaged or dead cells were noted.

Discussion

Vanadium compounds in low concentrations (c. ng/g of food) act as one of the essential microelements for some species from algae to vertebrates, such as chicken or rat, but not for humans, who daily receive the element with food and fluid intake. The mean vanadium concentration in blood, ranging from 0.032 to 0.095 ng/ml in the general population, reaches a much higher level (even 1000-fold) in people exposed to high vanadium concentration levels in the air, e.g. industrial workers [22]. The molecular mechanism of intracellular vanadium action is not yet fully understood. While vanadium’s influence as an insulin-mimetic agent acting towards normalization of metabolism of lipids and carbohydrates is relatively well known [23], intensive investigations of this microelement’s effect on proliferation and viability of cells (including cancer cell lines) are in progress. Vanadium derivatives as phosphate analogues inhibited protein tyrosine phosphatases and activated kinases [2, 19, 25, 26], influenced gene expression, DNA repair mechanisms, cell growth, apoptosis, and acted as pro-cancerogenic [e.g. 24-26] or anti-cancerogenic “drugs” [15, 27, 28].

In the anti-tumour activity of vanadium, two activity levels may be distinguished. The former consists in a cytoprotective effect exerted upon experimentally induced cancers in animal models. Administration of vanadium derivatives results in a decreased rate of proliferation and/or mass and number of newly developing tumours. Chakraborty et al. [29-32] believe that anti-tumour vanadium activity consists in induction and/or stabilization of liver enzymes, as well as inhibition of activity of -glutamyl transpeptidase and placental glutathione S-transferase. The other mechanism involves decreasing the proliferation activity of specific tumour cell lines. The latter stage is subject to extensive studies. Having entered the cell, vanadium compounds are believed to affect nuclear DNA, resulting in formation of cross-links between DNA strands, which hinders or even completely prevents DNA replication, thus sending the cells on the path to apoptosis. In the opinion of Young and Wang [28], the vanadium-selenium complex exhibits multidirectional activity, causing inhibition of S189 and H22 life cycle in S and G2/M phase; the mechanism underlying the process depends on Ca2+ and Mg2+ accumulation in the cell, an increase in the amount of free radicals, a decrease of pH in mitochondrial membranes and a decreased potential of these membranes. Barrio et al. [33] demonstrated antiproliferative abilities of the vanadium-trehalose complex, which depended on the concentration employed (UMR106 cell line) and appeared at 50-100 M. ERK kinase stimulation in the cells was also noted, with the activity dependent upon the concentration of the compound and time of exposure. Molinuevo et al. [27] observed vanadium (IV) complex-evoked inhibition of adhesion, migration and colony forming in the osteosarcoma UMR cell line.

Our previous investigations showed the effect of vanadium cation (as vanadyl sulphate) as well as anion (orthovanadate) on autocrine growth and viability of the rat hepatoma cell line H35-19 [10], similarly as on some human carcinoma cell lines (lung, kidney and prostate) [9, 11]. It has seemed interesting to study in particular the effect of four vanadium compounds (as two cations, VOSO4 and BMOV [V(IV)], and two anions, Na3VO4 and NaVO3 [V(V)]) on cell growth and viability, and to carry out a parallel investigation of the morphology of rhabdomyosarcoma, a human cancer cell line. Rhabdomyosarcoma (RMS) arises mainly in the head and neck region, genitourinary tract and extremities; it is the most common childhood malignancy. Permanent activation of signal transduction pathways promotes RMS growth.

Within the cell, VO2+ cation was a dominant form; when bound to an organic ligand (BMOV), it was more absorbable and less toxic than VOSO4 [34]. Both [V(IV)] compounds at concentrations of approximately 10 M resulted in a statistically significant decrease in proliferation rate of the investigated cells; when the concentration increased above 20 M, they depressed respiratory activity in RMS. The half maximal inhibitory concentration (IC50) of VOSO4 metavanadate was lower by 1/3 (28 M) as compared to BMOV (47 M). Orthovanadate showed an intermediate value (40 M). The compounds exerted a dose and incubation time-dependent cytotoxic effect on the investigated cell line; the onset of proapoptotic activity was noted after 24-48 hours of incubation, while longer incubation periods resulted in predominance of a necrotic effect.

Electron microscopic study confirmed a relatively high number of dead cells. Four vanadium derivatives resulted in ultrastructural changes in RMS cells. In the case of V(V), the cell membranes were smoothened out, with obliteration of the cytoplasmic processes and microvilli, while the vacuoles were large; following exposure to NaVO3, large fissures were seen around the nucleus and smaller ones in the cytoplasm. As the effect of vanadium V(IV) derivatives, the cell membranes were plicated to a greater or lesser degree (similarly as in the control cells) and the microvilli were preserved. Large, scant and often digestive vacuoles were observed in the cells subjected to the effect of c. 50 M BMOV, while following the application of c. 30 M VOSO4, small cytoplasmic vacuoles were noted, similarly as in the controls. Ultrastructurally, RMS cells did not show the presence of contractile filaments, either thick or thin. As it follows from the literature, single thin filaments observed at times in early-stage rhabdomyoblasts are not present in RMS cells. Also characteristic arrangements of single thick filaments surrounded by thin filaments are almost never encountered in poorly differentiated rhabdomyoblasts, and even less so in RMS [35].

Similarly as in our investigations, embryonal RMS often manifests cells the morphology of which is difficult to fully diagnose. In such situations, establishing the diagnosis is facilitated by clinical data, as well as by histopathology results. In the case of cultured cells, we are sure of both the origin and the cell line. The presence of cell dedifferentiation (especially in consecutive passages) is a well-known phenomenon. A predominantly characteristic observation in RMS cells was chiefly pleomorphic nuclei with a visible amount of heterochromatin and large nucleoli. Similarly as in other tumour cell lines, cells in various differentiation stages were also noted. In the present investigations, no significant difference was observed between particular experimental groups, while the cells originating from these groups were markedly more damaged, most likely in consequence of vanadium activity. In RMS cells, practically no cytoskeleton elements were noted in either group, control or experimental, in contrast to the results of similar investigations carried out in human lung cancer cells of the A549 line, where cytoskeletal elements were seen as bundles or single intermediate filaments or microtubules (paper being prepared for publication).

A comparison of the present results with the results obtained previously by our investigative team demonstrated the highest sensitivity to vanadates and vanadyl sulphate (IC50 up to 5 M) of rat hepatoma cells (H35-19) as compared to the four investigated lines of human cells (A549 lung, DU145 prostate, HTB kidney and RMS). The highest resistance to the effect of Na3VO4 was seen in RMS cells (approx. 40 M) and HTB cells (20 M), while the A549 and DU145 lines reached IC50 already at several micromoles (5-10 M). Under the effect of VOSO4, both the cell lines achieved IC50 at a concentration value that was 50% lower as compared to RMS (15 M versus 30 M). The lowest effect was exerted on RMS cells by BMOV, since the concentration that needed to be employed reached up to 50 M. Analysing the resultant values, one may state that rat hepatoma cells are more sensitive to the effect of inorganic vanadium salts in comparison to the four investigated lines of human cancer cells. The highest resistance to the effect of these compounds was seen in the HTB and RMS cell lines.

In the investigated RMS line, a similar effect of approximately 50% growth inhibition was evoked by metavanadate and vanadyl sulphate, with IC50 occurring at < 30 M. The weakest effect was exerted by BMOV at c. 50 M, while orthovanadate showed an intermediate effect at c. 40 M. These findings are generally in agreement with data reported by other authors investigating the properties of this microelement. In the case of NaVO3, one should take into consideration the highest toxicity of this derivative, while BMOV showed the mildest effect both in vivo and in vitro [36].

Acknowledgements

The authors are grateful to Maria Kawecka MSc and Alicja Strzelbicka MSc for their excellent technical help during experiments.

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Address for correspondence

Wojciech Dąbroś PhD

Chair of Pathomorphology

Faculty of Medicine

Collegium Medicum Jagiellonian University

ul. Grzegórzecka 16

31-531 Kraków, Poland

e-mail: mpdabros@cyf-kr.edu.pl