Comparison of the Effects of Sodium Orthovanadate and Sodium Metavanadate on the Growth and Survival of DU-145 Prostate Cancer Cells

Abstract

Prostate cancer is one of the most prevalent malignancies in developed countries. Although new anticancer agents are being evaluated in models of this cancer, there is a scarcity of studies on the effects of sodium metavanadate (NaVO₃). Therefore, the aim of this study was to investigate the effects of sodium metavanadate on the growth, viability, and survival of prostate cancer cells and to compare these results with those for sodium orthovanadate (Na₃VO₄). The experiments were conducted on DU-145 cells incubated with either sodium metavanadate or sodium orthovanadate. The impact of these vanadium compounds on cell growth was assessed using the MTT assay and crystal violet staining. Subsequently, the effects of sodium metavanadate and sodium orthovanadate on cell survival and the cell cycle were examined using propidium iodide and Hoechst 33342 staining, followed by analysis with fluorescence microscopy and flow cytometry. Both sodium metavanadate and sodium orthovanadate inhibited the growth of DU-145 cells, with IC₅₀ values of 12.74 ± 5.38 µM and 8.94 ± 2.90 µM, respectively. This inhibition was attributed to apoptosis and necrosis, as well as cell cycle arrest at the G₁/S checkpoint. The findings suggest that sodium metavanadate and sodium orthovanadate are potential anticancer agents against prostate cancer, effective at low concentrations.

Introduction

Vanadium forms a large group of chemical compounds [1]. Among these, the inorganic vanadium compounds, such as vanadyl sulfate and sodium orthovanadate, are the most studied and significant for medical applications [2]. Additionally, vanadium forms organic derivatives, which involve inorganic vanadium complexes with various organic substances [3, 4]. These include vanadyl(IV) complexes with 1,10-phenanthroline and polypyridyl [5], flavonoids [6], and already approved drugs such as zoledronate, alendronate, cetirizine, and ibuprofen [7].

It is estimated that only about 40% of vanadium kompaund can be absorbed from the gastrointestinal tract [8]. Following their distribution throughout the body via the bloodstream, vanadium compounds accumulate mainly in the bones and kidneys [9], and to a lesser extent in the liver [9], which indicates the target organs affected by vanadium compounds.

Vanadium compounds are known inhibitors of protein tyrosine phosphatases (PTP) [10, 11]. This inhibition is due to their spatial structure and interaction with the cysteine residue in the catalytic center of PTP. The reduction in PTP activity leads to increased phosphorylation of tyrosine residues in cells exposed to vanadium compounds [3, 12]. This enhances signal transmission or facilitates signal transduction through various intracellular signaling pathways, including increased activity of the insulin receptor and the insulin-like growth factor-I receptor (IGF-IR) [3]. Consequently, vanadium compounds possess insulin-mimetic properties, reducing blood glucose levels and being investigated as potential antidiabetic drugs [13–17].

Vanadium compounds also exhibit anticancer properties by inhibiting the proliferation and growth of various cancer types [18–21]. This antiproliferative effect is mediated by various mechanisms due to the nonspecific action of vanadium compounds. They affect cell cycle phosphatases, particularly cell division control protein 25 homolog (Cdc25)A [22] and Cdc25C [23], and may also inhibit the cell cycle through the activation of extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) [24]. Furthermore, vanadium compounds induce apoptosis in cancer cells, associated with their effects on mitochondria [25, 26] and the p53 protein [27]. Animal studies also demonstrate the anticancer properties of vanadium compounds [18, 21].

However, there is a scarcity of detailed studies on the effects of vanadium compounds on prostate cancer, one of the most common cancers among men in developed countries [28]. It is estimated that over 1.25 million new cases are diagnosed worldwide annually, with nearly 360,000 deaths, placing prostate cancer third among cancers in terms of incidence and mortality, following lung and breast cancers. Consequently, new anticancer drugs are frequently tested on models of this cancer.

The aim of this study is to analyze the anticancer activity of two inorganic vanadium compounds, sodium metavanadate (NaVO₃) and sodium orthovanadate (Na₃VO₄), in the DU-145 prostate cancer cell model, one of the most frequently studied prostate cancer models. The specific research objectives include:

• evaluating the effect of selected vanadium compounds on DU-145 prostate cancer cell growth, including precise determination of IC₅₀ (50% inhibition of control growth),

• assessing their effect on DU-145 prostate cancer cell viability, including pro-apoptotic and necrotic effects on cancer cells,

• determining the precise impact of the selected vanadium compounds on DU-145 prostate cancer cell proliferation, with particular emphasis on their effect on the cell cycle of these cells.

Materials and Methods

Cell Culture

Experiments were conducted using DU-145 cells, which are prostate cancer cells isolated from a brain metastasis [29]. The cells were purchased from American Type Culture Collection (ATCC), Manassas, Virginia (USA). These cells are widely used as a research model for prostate cancer in studies of anticancer agents [30, 31]. Cells were cultured in MEM Eagle medium with L-glutamine (CytoGen – Polska Sp. z o.o.), 1 mM sodium pyruvate (CytoGen – Polska Sp. z o.o.), essential amino acids (CytoGen – Polska Sp. z o.o.), and antibiotics (penicillin 100 µg/ml and streptomycin 100 J/ml) (PAA Laboratories GmbH), supplemented with 10% fetal bovine serum (FBS) (PAA Laboratories GmbH). Cultures were maintained at 37 °C in a humidified atmosphere with 5% CO₂. DU-145 cells were passaged twice weekly using 0.05% trypsin in 0.9% NaCl with 0.02% EDTA (Sigma-Aldrich). For growth and viability assays, DMEM/Ham’s F12 medium with L-glutamine (CytoGen – Polska Sp. z o.o.), albumin (0.5 mg/ml), transferrin (5 µg/ml), sodium selenite (2 ng/ml) (Sigma-Aldrich), and antibiotics (penicillin 100 µg/ml and streptomycin 100 J/ml) (PAA Laboratories GmbH) was used.

Analysis of the Effects of Vanadium Compounds on DU-145 Cell Growth

The effects of sodium orthovanadate and sodium metavanadate on DU-145 cell growth were analyzed using the 3-(4,5-dimethylthiazol-2-yl)−2,5-diphenyl tetrazolium bromide (MTT) assay [32, 33] and crystal violet (CV) staining [34]. DU-145 cells were seeded in 96-well plates (FALCON) at a density of 5000 or 3000 cells (for 120-h incubation) per well. After 24 h of incubation, the medium was replaced with DMEM/Ham’s F12. Following another 24 h, MTT or CV staining was performed in 12 wells to estimate the initial cell count. In the remaining wells, the medium was replaced, and fresh sodium orthovanadate. CAS Number: 13721–39-6 (Sigma-Aldrich) or sodium metavanadate, CAS Number: 13718–26-8 (Fluka) was added at concentrations of 2 µM, 5 µM, 10 µM, and 25 µM. Control wells contained cells in medium without vanadium compounds. Vanadium compounds were prepared as 2 mM solutions in PBS. Fresh solutions were prepared before each experiment involving vanadium compounds.

For the MTT assay, after 48, 72, and 120 h of incubation with vanadium compounds, 0.1 mg MTT (Sigma-Aldrich) in PBS was added to the medium. Cells were incubated for 2 h, after which the medium was removed and isopropanol (Avantor Performance Materials Poland S.A.) was added. After shaking the plates, absorbance was measured at 570 nm. Six measurements were taken for each concentration and incubation time. The experiment was repeated three times.

For CV staining, cells were fixed with methanol. After removing methanol, a CV solution (0.5% crystal violet (Sigma-Aldrich) with 20% methanol (Avantor Performance Materials Poland S.A.)) was added. After incubation with CV, cells were rinsed with running water, and a decolorizing solution (0.1 mol citric acid buffer (Sigma-Aldrich) in 50% methanol (Avantor Performance Materials Poland S.A.)) was added. After shaking the plates, absorbance was measured at 540 nm. The readings were performed using a Spectra FLUOR Plus microplate reader (Tecan).

Six measurements were taken for each concentration and incubation time. The experiment was repeated three times.

The percentage inhibition of DU-145 cell growth was calculated using the formula:

$$\left[1,-,\frac{A_i-A_0}{A_C-A_0}\right]\ast 100$$

where:

A_i is the estimated cell count at a given concentration of vanadium compound and incubation time,

A_c is the control cell count after the specified incubation time,

A₀ is the initial cell count [35].

The IC₅₀ (50% inhibition of control growth) was calculated using GraphPad Prism 5.04 Trial (GraphPad Software) with the "Dose–response curves – Inhibition: log(inhibitor) vs. normalized response – Variable slope" model using the regression curve described by the equation:

$$G_i=\frac{100}{{1+100}^{\left(L,o,g,I,C_{50},-,S_{\mathit{MLog}}\right)H_s}}$$

where:

G_i is the mean cell count relative to the control,

IC₅₀ is the concentration causing 50% inhibition of control growth,

S_MLog is the logarithm of the molar concentration of the vanadium compound,

H_s is the hill slope.

Analysis of the Effects of Vanadium Compounds on DU-145 Cell Viability

The effects of sodium orthovanadate and sodium metavanadate on DU-145 cell viability were analyzed using propidium iodide (PI) (Sigma-Aldrich) and Hoechst 33,342 (Sigma-Aldrich) staining [23]. Cells were seeded in 24-well plates (Renner GmbH) at a density of 20,000 cells per well. After 24 h of incubation, the medium was replaced with DMEM/Ham’s F12. Following another 24 h, the medium was replaced with fresh sodium metavanadate (11 µM and 22 µM) or sodium orthovanadate (8 µM and 16 µM). The first concentration corresponds to the calculated IC₅₀ from the MTT and CV growth assays after 48 h of incubation with the vanadium compounds.

The second concentration is twice the calculated IC₅₀. Positive controls included taxol (Sigma-Aldrich) at concentrations of 20 nM and 50 nM [36]. Cells incubated without vanadium compounds or taxol served as controls. DU-145 cells were incubated for 48 h with the test substances. After this period, Hoechst 33,342 (5 µg/ml) and PI (1 µg/ml) were added. Stained cells were then photographed using a D5000 digital camera (Nikon) mounted on a fluorescence microscope (Olympus IX50). The captured images were used to count viable, apoptotic, and necrotic cells.

The experiment was repeated three times.

Analysis of the Effects of Vanadium Compounds on the Cell Cycle of DU-145 Cells

To analyze the effects of sodium orthovanadate and sodium metavanadate on the cell cycle of DU-145 cells, PI staining and flow cytometry were utilized [37]. DU-145 cells were seeded at a density of 250,000 cells per 60 mm Petri dish. After 24 h of incubation, the medium was replaced with DMEM/Ham’s F12. Following another 24 h, the medium was replaced with fresh sodium metavanadate (11 µM) or sodium orthovanadate (8 µM), concentrations corresponding to the IC₅₀ values calculated for 48-h incubation with each vanadium compound. Control cells were incubated without the vanadium compounds. After 48 h of incubation, cells were trypsinized and resuspended in PBS with 2% FBS. Cells were then fixed with ice-cold 70% ethanol. After removing the ethanol, cells were resuspended in a staining solution (0.1% Triton-X-100 (Sigma-Aldrich), 20 µg/ml PI (Sigma-Aldrich) 0.2 µg/ml RNAse (Sigma-Aldrich) in PBS (Biomed) with 2% FBS (PAA Laboratories GmbH)). After 30 min of incubation, stained cells were analyzed using the flow cytometer LSR II (Becton Dickinson). A total of 20,000 cells were counted.

Data were analyzed using FCS Express 4 Plus (De Novo Software) software.

Statistical Analysis

Means and standard deviations were calculated for each experimental parameter. Statistical significance of each value relative to the control was assessed using Wilcoxon’s matched pair test with the Statsoft Statistica program. P values less than 0.05 were considered significant.

Results

Sodium Orthovanadate and Sodium Metavanadate Inhibit DU-145 Cell Growth at Micromolar Concentrations

Sodium orthovanadate and sodium metavanadate significantly inhibit the growth of DU-145 cells at micromolar concentrations during several days of incubation. Sodium orthovanadate exhibits a stronger effect on DU-145 cells, with an IC₅₀ of 8.94 ± 2.90 µM (Fig. 1; Table 1). In contrast, the mean IC₅₀ for all experiments conducted with sodium metavanadate was 12.74 ± 5.38 µM.

Fig. 1.

Sodium orthovanadate and sodium metavanadate inhibit the growth of DU-145 cells. DU-145 cells were cultured in a medium supplemented with sodium orthovanadate or sodium metavanadate for 48 h (A, B), 72 h (C, D), or 120 h (E, F). Cell counts were estimated using CV staining (A, C, E) or MTT assay (B, D, F). * – p < 0.05; ** – p < 0.01; *** – p < 0.001 compared to control

Table 1.

IC₅₀ values for each incubation time and each tested vanadium compound [µM]

	CV staining		MTT assay
Incubation time	IC50	Confidence interval greater than 95%	IC50	Confidence interval greater than 95%	Mean
NaVO3
48 h	7.88	5.98–10.39	14.53	13.34–15.83	11.21
72 h	21.90	13.14–36.5	13.48	9.89–18.39	17.69
120 h	11.57	9.94–13.48	7.07	6.15–8.13	9.32
Mean	13.78±7.27		11.69±4.04		12.74±5.38
Na3VO4
48 h	6.41	3.76–10.95	8.35	6.68–10.45	7.38
72 h	10.93	8.18–14.59	13.62	11.16–16.63	12.28
120 h	8.47	6.63–10.81	5.89	3.95–8.77	7.18
Mean	8.30±2.26		9.29±3.95		8.94±2.90

Sodium Orthovanadate and Sodium Metavanadate Induce Apoptosis and Necrosis in DU-145 Cells

After 48 h of incubation, sodium orthovanadate and sodium metavanadate induced apoptosis and necrosis in DU-145 cells (Fig. 2). The percentage of apoptotic cells at each tested concentration of the vanadium compounds ranged from 16 to 20%. In contrast, the extent of necrosis varied with the concentration of the vanadium compounds. At the IC₅₀ concentration, these compounds caused necrosis in 15% of DU-145 cells. Doubling the concentration increased the percentage of necrotic cells to 26–27%. Taxol, used as a control, induced apoptosis in 40–50% of DU-145 cells at the tested concentrations.

Fig. 2.

Sodium orthovanadate and sodium metavanadate induce apoptosis and necrosis in DU-145 cells. DU-145 cells were cultured in a medium supplemented with sodium orthovanadate, sodium metavanadate, or taxol for 48 h. The number of apoptotic and necrotic cells was assessed using PI and Hoechst 33,342 staining. * – p < 0.05; ** – p < 0.01; *** – p < 0.001, **** – p < 0.0001 compared to control

Vanadium Compounds Inhibit the Cell Cycle of DU-145 Ccells at the G₁/S Checkpoint

A 48-h incubation with sodium metavanadate (11 µM) or sodium orthovanadate (8 µM) increased the number of DU-145 cells in the G₁ phase and decreased the number in the S phase of the cell cycle. This suggests that both vanadium compounds inhibit the cell cycle at the G₁/S checkpoint (Table 2).

Table 2.

Effect of the tested vanadium compounds on the cell cycle of DU-145 cells

Cell cycle phase	G1 [%]	S [%]	G2 [%]
Control	62.31 ± 4.73	23.12 ± 1.70	14.57 ± 1.09
Sodium metavanadate	69.50 ± 5.20 *	16.21 ± 1.22 *	14.29 ± 1.07
Sodium orthovanadate	68.71 ± 5.22 *	16.89 ± 1.27 *	14.40 ± 1.08

* – p < 0.05 compared to control

Discussion

Vanadium Compounds Inhibit the Growth of DU-145 Cells

In this study, sodium metavanadate and sodium orthovanadate reduced the growth of DU-145 cells at micromolar concentrations. The calculated mean IC₅₀ values for sodium metavanadate and sodium orthovanadate were 12.74 ± 5.38 µM and 8.94 ± 2.90 µM, respectively. These results align with existing literature on the effects of sodium orthovanadate on various cell lines, including HL60 cells (acute leukemia) [38], HepG2, SK-Hep-1, and Hep3B cells (hepatocellular carcinoma) [18], HTB44 cells (kidney carcinoma) [35], A549 cells (non-small cell lung carcinoma) [35], and Cal27 cells (oral squamous cell carcinoma) [19].

However, the IC₅₀ value for a given vanadium compound varies depending on the cell line. For instance, in 8505 C cells (anaplastic thyroid carcinoma) [21], H35-19 cells (rat hepatoma) [39], SH-SY5Y (neuroblastoma) [40], 4T1 (murine breast cancer) [41], and A375 (melanoma) [42], the IC₅₀ is around or below 5 µM. In contrast, for DBTRG.05MG cells (human glioblastoma multiforme) [43], the IC₅₀ is higher than in DU-145 cells, at 22 µM after 72 h of incubation. In contrast, ammonium metavanadate inhibits the growth of UACC-62 (melanoma) with an IC₅₀ of 25 µM after 48 h [44].

Some cell lines, such as TPC-1 cells (papillary thyroid carcinoma) [45], exhibit even higher IC₅₀ values, around 50 µM, with lower concentrations of sodium orthovanadate (< 10 µM) promoting cell growth (Table 3).

Table 3.

Comparison of the obtained viability test results with other research studies

Vanadium compound	Cancer model	IC50 [µM]	Source
Metavanadate
Ammonium metavanadate	UACC-62 (melanoma)	25.0 (48 h)	[44]
Sodium metavanadate	DU-145 (prostate cancer)	11.21 (48 h)	our results
Sodium metavanadate	A375 (melanoma)	4.7 (72 h)	[42]
Sodium metavanadate	8505 C (anaplastic thyroid carcinoma)	2.50 (48 h)	[21]
Sodium metavanadate	4T1 (murine breast cancer)	1.92 (48 h)	[41]
Orthovanadate
Sodium orthovanadate	TPC-1 (papillary thyroid carcinoma)	50 (24 h)	[45]
Sodium orthovanadate	HepG2, Hep3B and SK-Hep-1 (hepatocellular carcinoma)	30 (48 h)	[18]
Sodium orthovanadate	DBTRG.05MG (glioma)s	25 (72 h)	[43]
Sodium orthovanadate	Cal27 (oral squamous cell carcinoma)	25 (72 h)	[19]
Sodium orthovanadate	A549 (lung adenocarcinoma)	9.57 (72 h)	[35]
Sodium orthovanadate	DU-145 (prostate cancer)	7.38 (48 h)	our results
Sodium orthovanadate	H35-19 (rat hepatoma)	5.0 (48 h)	[39]
Sodium orthovanadate	SH-SY5Y (neuroblastoma)	5.0 (48 h)	[40]
Sodium orthovanadate	8505 C (anaplastic thyroid carcinoma)	3.55 (48 h)	[21]

The tested vanadium compounds affect cell viability, influencing both the number of DU-145 cells and their proliferation rate. It should be emphasized, however, that these compounds also interfere with the parameters measured by the MTT and CV assays. Vanadium compounds impair mitochondrial function, leading to a decrease in mitochondrial membrane potential [25, 26, 46]. Since the MTT assay relies on the metabolic conversion of MTT into formazan by viable cells [32, 33], and this process depends in part on functional mitochondria [47], reduced mitochondrial activity leads to a weaker MTT signal. In addition, vanadium compounds impair cell adhesion and weaken cell–cell junctions [48]. This has a direct impact on the CV assay, which measures total cell mass through crystal violet staining. During the rinsing step, weakly attached cells – including viable but poorly adherent ones – are washed away, resulting in lower dye retention and reduced absorbance values [34]. Consequently, the effects of vanadium compounds on mitochondria and adhesion alter the readouts of both MTT and CV assays and thus affect the calculated IC₅₀ values.

Inhibition of DU-145 Cell Growth by Sodium Metavanadate and Sodium Orthovanadate is due to the Induction of Apoptosis and Necrosis

Sodium metavanadate and sodium orthovanadate induced apoptosis and necrosis in DU-145 cells at IC₅₀ concentrations and above. This is consistent with literature showing that sodium orthovanadate induces apoptosis in various cell lines, including HL60 cells [38], 8505 C cells [21], SH-SY5Y cells [42], and Cal27 cells [19]. Studies on A549 cells also demonstrated that sodium metavanadate induces apoptosis [20]. Different in vitro models exhibit varying susceptibility to vanadium compounds. For instance, sodium orthovanadate at 4 µM induces apoptosis in 40% of 8505 C cells [21], whereas in DU-145 cells, 8 µM sodium orthovanadate induces apoptosis in only 20% of cells.

The pro-apoptotic effect of vanadium compounds is associated with their action on mitochondria [25, 26], leading to reduced mitochondrial membrane potential and apoptosis induction [18, 21]. Vanadium compounds also induce apoptosis through p53 activation [27]. However, many cancers harbor TP53 gene mutations, rendering p53 nonfunctional [49].

Sodium metavanadate and sodium orthovanadate also induce necrosis in DU-145 cells, likely due to the generation of reactive oxygen species (ROS), which cause cellular damage, including lipid peroxidation [50–52]. Vanadium in the + 5 oxidation state is reduced intracellularly to the vanadyl ion (+ 4 oxidation state), which reacts with hydrogen peroxide (H₂O₂), leading to oxidative damage [53, 54].

Vanadium compounds also induce oxidative stress through Fenton-type reactions and by acting on mitochondria [55]. The pro-oxidative properties may be partly attributed to decavanadate. Vanadium compounds accumulate in mitochondria in the form of polyoxovanadates, particularly decavanadate, thereby damaging these organelles [56, 57]. As a consequence, mitochondrial activity and ROS generation are reduced.

Inhibition of DU-145 Cell Growth by Sodium Metavanadate and Sodium Orthovanadate is due to Cell Cycle Arrest

Sodium metavanadate and sodium orthovanadate reduced the growth of DU-145 cells by inhibiting cell proliferation. Both compounds caused cell cycle arrest at the G₁/S checkpoint, increasing the number of cells in the G₁ phase. These findings are consistent with studies on HepG2 cells [24, 58], where sodium orthovanadate exhibited similar effects on the cell cycle, dependent on ERK MAPK activation. However, studies on HL60 cells [38], 8505 C cells [21], and SH-SY5Y cells [42] showed that sodium orthovanadate causes G2/M cell cycle arrest, indicating that the effects of sodium orthovanadate vary with the experimental model.

Conclusions: Sodium metavanadate and sodium orthovanadate exhibit anticancer properties at concentrations slightly higher than those used in experimental antidiabetic therapy using vanadyl sulfate

In this study, it was demonstrated that vanadates inhibit the growth of DU-145 prostate cancer cells at micromolar concentrations. Sodium orthovanadate inhibited growth with an IC₅₀ of 8.94 ± 2.90 µM, while sodium metavanadate showed an IC₅₀ of 12.74 ± 5.38 µM. This effect results from the induction of apoptosis and necrosis in DU-145 cells by the tested vanadium compounds. Their activity is also associated with cell cycle arrest at the G1/S checkpoint.

The calculated IC₅₀ values for sodium metavanadate and sodium orthovanadate were only twice as high as the concentrations used for vanadyl sulfate in experimental antidiabetic therapy [17, 59]. At a concentration of 5 µM, vanadyl sulfate showed no significant toxicity in patients, even after 2.5 years of therapy [17]. Oral administration of vanadyl sulfate only caused mild diarrhea at the beginning of the experimental therapy. However, the therapeutic window for vanadium compounds is very narrow. At blood concentrations around 50 µM, vanadium compounds exhibit strong toxicity [60] and, therefore, assessing the susceptibility of cancer cells to a given vanadium compound is essential in planning anticancer therapy.

However, vanadates exhibit varying toxicity across different cell lines. In TPC-1 (papillary thyroid carcinoma) cells, the IC₅₀ is 50 µM [45], which represents a toxic concentration for humans [60]. In contrast, A549 (lung adenocarcinoma) [35], A375 (melanoma) [42], H35-19 (rat hepatoma) [39], and SH-SY5Y (neuroblastoma) [40] cells show high sensitivity to the tested vanadium compounds, with IC₅₀ values ranging from 5 to 10 µM, similar to the IC₅₀ values obtained for the DU-145 line. For this reason, this concentration range should be considered in the planning of future anticancer therapies using vanadates.