Impact of calcium ion on cytotoxic effect of the boroxine derivative, K₂[B₃O₃F₄OH]

Abstract

The effect of Ca²⁺ ions on the cytotoxic ability of boron heterocyclic compound dipotassium-trioxohydroxytetrafluorotriborate (K₂[B₃O₃F₄OH]), on in vitro tumor cells (mammary adenocarcinoma 4T1, melanoma B16F10 and squamous cell carcinoma SCCVII) and non-tumoral fibroblast cells (mouse dermal L929 and hamster lung V79) was examined. At small concentrations of Ca²⁺ ions (0.42 mM), K₂[B₃O₃F₄OH] (3.85 mM) has a very strong cytotoxic effect on all cancer cells tested (89.1, 85.6 and 84.6%) and significantly less effect on normal cells (19.5 and 24.2%), respectively. Applying larger concentrations of Ca²⁺ ions (9.42–72.42 mM), at the same concentration of K₂[B₃O₃F₄OH], no significant cytotoxic effect was detected on cancer cells and normal cells investigated. The selective ability of K₂[B₃O₃F₄OH], in the medium with a low concentration of Ca²⁺ ions has a strong cytotoxic effect on cancer cells and very weak effect in normal cells, opens up the possibility of its application in antitumor therapy.

Keywords:

• Anticancer activity
• boroxine
• calcium ion
• drug research

Introduction

Boroxines are the dehydration products of organoboronic acids and their derivatives such as trimethylboroxine and triphenylboroxine1,2. Due to their unique electronic structures, these compounds are currently under investigation as possible enzyme inhibitors and therapeutics. In the development of boronic acid-based enzyme inhibitors as potential antitumor drugs, as target specificity within a wide family of these compounds, is the possibility to avoid different side effects during their application. The recent studies3–7 investigated the kinetic parameters and inhibition mechanisms of halogenated boroxine dipotassium-trioxohydroxytetrafluorotriborate (K₂[B₃O₃F₄OH]) and some other boron-containing anions on enzymes catalase and human carbonic anhydrases. It was shown that its mM concentration could significantly reduce catalase activity and that K₂[B₃O₃F₄OH] is a potent inhibitor of some human carbonic anhydrases with a KI ranging from of 8.0 to 93 μM. In one study6, it was hypothesized that the local application of K₂[B₃O₃F₄OH]-containing cream or by its intra-tumor injection at level of mM concentration could significantly reduce catalase activity and increase the concentration of H₂O₂ and accordingly produce beneficial effects in tumor tissue alone. In other study7, it was proposed that K₂[B₃O₃F₄OH] binds to the Zn(II) from active site of enzyme carbonic anhydrase, coordinating to the metal ion monodentately through its Boron-OH functionality.

K₂(B₃O₃F₄OH) is explored as useful in treatment of benign and malignant skin changes, such as nevus or skin cancer8,9. First published effects of its bioactivity, revealed a potential for inhibition of lymphocytes proliferation and cell growth of basal cell carcinoma culture as well as certain clastogenic potential10. Recently it has been confirmed that selected flavonoids may inhibit damages of genetic material in human lymphocytes induced by K₂(B₃O₃F₄OH)11. Previous findings of antitumor activity of K₂(B₃O₃F₄OH) in vitro and in vivo on 4T1 mammary carcinoma, B16F10 melanoma and squamous cell carcinoma SCVII revealed inhibitory effects on cell proliferation in concentrations of 1 mM while concentrations of less than 0.1 mM do not significantly affect cell growth in vitro. K₂(B₃O₃F₄OH) slows the growth of three tested tumors in vivo compared to control and regardless of the route of administration (intraperitoneally, intratumor, per oral or as an ointment)12.

Ca²⁺ ions play an important role for cell signaling. Physiological roles for calcium signaling include muscle contraction, neuronal transmission as in an excitatory synapse, cellular mobility (including the movement of flagella and cilia), fertilisation, as well as cell growth or proliferation. Cancer cell proliferation and apoptosis depend on the intracellular Ca²⁺ ions concentration, and the expression of numerous ion channels with the ability to control intracellular Ca²⁺ ions concentrations has been correlated with cancer13. Specific signals can trigger a sudden increase in the cytoplasmic Ca²⁺ ion levels up to 0.5–1.0 mM by opening channels and release of Ca²⁺ ions from the endoplasmic reticulum which lead to Ca²⁺ ion entries from outside the cell. Many of Ca²⁺ ions mediated events occur when the released Ca²⁺ ions bind to calmoduline and activate the regulatory Ca²⁺ ions-calmodulin-dependent protein kinases or other effector proteins. Since exogenous and endogenous factors can impact this cell signaling process, it is interesting also to investigate the impact of Ca^{2 + i} ions on possible cytotoxic or proliferation effect of some drug14,15. In a recent article16, the authors concluded that inhibition of low-level endoplasmic reticulum-to-mitochondria Ca²⁺ ion transfer is toxic, specifically to cancer cells. An unexpected dependency on this transfer to mitochondria exists for a viability of tumorigenic cells, suggesting that mitochondrial Ca²⁺ ion addiction is a novel feature of cancer cells. These studies suggest the existence of completely unexpected new targets for which drugs could be developed to kill cancer cells specifically by targeting Ca²⁺ ion release from the endoplasmic reticulum and Ca²⁺ ion uptake by mitochondria. A major challenge now is to discover drugs that can do this in new cancer therapies.

According to above finding, the objective of our study was to investigate the impact of Ca²⁺ ions on cytotoxic effect of K₂[B₃O₃F₄OH]. For this purpose, tumor cells (mammary adenocarcinoma 4T1, melanoma B16F10 and squamous cell carcinoma SCCVII) and non-tumoral fibroblast cells (mouse dermal L929 and hamster lung V79) were used.

Materials and methods

Chemicals

Dipotassium-trioxohydroxytetrafluorotriborate (K₂[B₃O₃F₄OH]) was prepared as reported in the literature17. All other compounds used in this study were commercially available, highest purity reagents, from Sigma-Aldrich (Buchs, Switzerland).

Cell lines

Mouse mammary adenocarcinoma 4T1 and mouse melanoma B16F10 cell lines were purchased from American Type Culture Collection (ATCC, Manassas, VA), and mouse squamous cell carcinoma SCCVII cell line was obtained from BC Cancer Research Centre (Vancouver, Canada). Non-tumoral cell lines, mouse dermal fibroblasts L929 and the Chinese hamster lung fibroblasts V79 were purchased from American Type Culture Collection (ATCC, Manassas, VA). Cell were grown in a RPMI 1640 medium (Sigma-Aldrich, Buchs, Switzerland) supplemented with 10% FCS (Sigma-Aldrich, Buchs, Switzerland), in a humidified atmosphere of 5% CO₂ in air and at temperature 37 °C.

Determination of cytotoxic activity in vitro

Experiments were carried out in microtiter plates with 96 wells and 1 × 10⁴ cells/250 μl of medium was applied in each well. After 24 h, when the cells reached confluence, the old cultured medium was replaced with a fresh one and K₂[B₃O₃F₄OH] was added to the cultures to the final concentrations of 3.85 mM with the addition of different concentrations of Ca²⁺ ions in corresponding cells with a final concentration: (A) 0.42 mM (Ca²⁺ ions from RPMI 1640 medium), (B) 9.42 mM, (C) 18.42 mM, (D) 36.42 mM and (E) 72.42 mM. Control cells (Control 1) were incubated in RPMI medium without addition of K₂[B₃O₃F₄OH] and Ca²⁺ ions. Control cells (Control 2) were incubated in RPMI medium without addition of K₂[B₃O₃F₄OH] in the presence of different concentrations of Ca²⁺ ions 9.42–72.42 mM. The cells were incubated for the next 24 h and cytotoxicity test with crystal violet was performed to measure cell growth inhibition rate. In short, cells were fixed by the addition of a 3% solution of formalin for 15 min, washed with deionized water and dried in air. After that, cells were stained with 0.1% crystal violet for 20 min, then extensively washed with deionized water and left to dry overnight. The dye was extracted from the cells using a 10% solution of acetic acid and then absorbance was measured at 590 nm using a microplate reader. The absorbance at 590 nm is proportional to the number of surviving cells. Each experiment was done in quadruplicate. Inhibition of cell growth I (%) relative to controls was calculated according to the formula: I = (C−T)/C × 100, where T denotes the mean absorbance of treated cells, and C indicates the mean absorbance of untreated (Control 1) cells. Corresponding inhibition values are presented in figures and Table 1.

Table 1. Impact of calcium ion on cytotoxic effect of 3.85 mM K₂[B₃O₃F₄OH] on different tumoral and non-tumoral cell lines.

	% of inhibition
[Ca^2+] (mM)	SCVII^a	B16F10^b	4T1^c	L929^d	V79^e
0.42*	84.6	85.6	89.1	19.5	24.2
9.42	30.7	40.0	23.9	2.6	3.2
18.42	6.1	13.6	13.0	3.9	0.0
36.42	0.0	7.4	13.0	2.6	3.2
72.42	3.1	3.1	0.0	15.6	48.3

*RPMI 1640 medium containing 0.42 mM calcium ions.

Estimated value of IC₅₀ is at:

^a [Ca²⁺] = 6 mM.

^b [Ca²⁺] = 7 mM.

^c [Ca²⁺] = 6 mM.

^d The IC₅₀ value is not reached.

^e The IC₅₀ value is not reached.

Statistical analysis

The obtained results were expressed as average ± standard deviation (SD). To evaluate differences between the groups, a one-way ANOVA followed by LSD post hoc test of multiple comparisons was used. Statistical analyses were performed using the Statistica software package (StatSoft, Tulsa, OK). Significant level was set at p < 0.05.

Results

Cytotoxic activity in cancer cell lines

The effect of Ca²⁺ ions on cytotoxic activity of K₂[B₃O₃F₄OH] was evaluated on melanoma B16F10 (Figure 1), squamous cell carcinoma SCCVII (Figure 2) and mouse mammary adenocarcinoma 4T1 (Figure 3) tumor cell lines. Ca²⁺ ion concentrations were varied from 0.42 to 72.42 mM, while the concentration of K₂[B₃O₃F₄OH] was constant, 3.85 mM. In the RPMI 1640 medium containing 0.42 mM Ca²⁺ ions, K₂[B₃O₃F₄OH] significantly reduced the number of surviving cells in all the tested cancer cell lines compared to the control group (columns A compared to columns Control 1 in Figures 1–3). Inhibition of cell growth (I) was 85.6%, 84.6%, and 89.1%, respectively. Thereafter, the impact of different higher concentrations of Ca²⁺ ions (9.42–72.42 mM) on cytotoxic effect of K₂[B₃O₃F₄OH] was evaluated (clustered columns B, C, D and E in Figure 1–3). Generally, in all the tested cancer cell lines, Ca²⁺ ions reduced cytotoxic activities of K₂[B₃O₃F₄OH] almost to zero, depending on Ca²⁺ ion concentrations. The results demonstrated that Ca²⁺ ions at tested concentrations, without K₂[B₃O₃F₄OH], did not influence significantly on the number of surviving cells (columns Control 2 in Figure 1–3).

Figure 1. Cytotoxic effect of K₂[B₃O₃F₄OH] on mouse melanoma cell line B16F10. (Control 1) Cells without the addition of K₂[B₃O₃F₄OH] and in the absence of Ca²⁺ ions; (Control 2) cells without the addition of K₂[B₃O₃F₄OH] and in the presence of different concentrations of Ca²⁺ ions 9.42–72.42 mM (average value); (A–E) cells after addition of 3.85 mM K₂[B₃O₃F₄OH] and in the presence of Ca²⁺ ions: (A) 0.42 mM, (B) 9.42 mM, (C) 18.42 mM, (D) 36.42 mM and (E) 72.42 mM.

Figure 2. Cytotoxic effect of K₂[B₃O₃F₄OH] on mouse squamous carcinoma cell line SCCVII. (Control 1) Cells without the addition of K₂[B₃O₃F₄OH] and in the absence of Ca²⁺ ions; (Control 2) cells without the addition of K₂[B₃O₃F₄OH] and in the presence of different concentrations of Ca²⁺ ions 9.42–72.42 mM (average value); (A–E) cells after the addition of 3.85 mM K₂[B₃O₃F₄OH] and in the presence of Ca²⁺ ions: (A) 0.42 mM, (B) 9.42 mM, (C) 18.42 mM, (D) 36.42 mM and (E) 72.42 mM.

Figure 3. Cytotoxic effect of K₂[B₃O₃F₄OH] on mammary adenocarcinoma 4T1 cell line. (Control 1) Cells without the addition of K₂[B₃O₃F₄OH] and in the absence of Ca²⁺ ions; (Control 2) cells without the addition of K₂[B₃O₃F₄OH] and in the presence of different concentrations of Ca²⁺ ions 9.42–72.42 mM (average value); (A–E) cells after the addition of 3.85 mM K₂[B₃O₃F₄OH] and in the presence of Ca²⁺ ions: (A) 0.42 mM, (B) 9.42 mM, (C) 18.42 mM, (D) 36.42 mM and (E) 72.42 mM.

Cytotoxic activity in non-tumor cell lines

The effect of Ca²⁺ ions on cytotoxic activity of K₂[B₃O₃F₄OH] was evaluated on mouse dermal fibroblasts (Figure 4), and on hamster lung fibroblast V79 (Figure 5) cell lines. In the presence of Ca²⁺ ions (0.42 mM) from RPMI 1640 medium, K₂[B₃O₃F₄OH] (3.85 mM) had a low cytotoxic effect on mouse dermal fibroblast L929 cell line (I = 19.5%). At different higher concentrations of Ca²⁺ ions (9.42–72.42 mM), there is no impact on its activity (clustered columns B–E in Figure 4). In case of hamster lung fibroblast V79 cell line, in the presence of Ca²⁺ ions of the concentration 0.42 mM, K₂[B₃O₃F₄OH] (3.85 mM) has a weak cytotoxic activity (I = 24.2%), which is almost suspended at different higher concentrations of Ca²⁺ ions (9.42–72.42 mM) (clustered columns B–E in Figure 5).

Figure 4. Cytotoxic effect of K₂[B₃O₃F₄OH] on mouse dermal fibroblast L929 cell line. (Control 1) Cells without the addition of K₂[B₃O₃F₄OH] and in the absence of Ca²⁺ ions; (Control 2) cells without the addition of K₂[B₃O₃F₄OH] and in the presence of different concentrations of Ca²⁺ ions 9.42–72.42 mM (average value); (A–E) cells after the addition of 3.85 mM K₂[B₃O₃F₄OH] and in the presence of Ca²⁺ ions: (A) 0.42 mM, (B) 9.42 mM, (C) 18.42 mM, (D) 36.42 mM and (E) 72.42 mM.

Figure 5. Cytotoxic effect of K₂[B₃O₃F₄OH] on hamster lung fibroblast V79 cell line. (Control 1) Cells without the addition of K₂[B₃O₃F₄OH] and in the absence of Ca²⁺ ions; (Control 2) cells without the addition of K₂[B₃O₃F₄OH] and in the presence of different concentrations of Ca²⁺ ions 9.42–72.42 mM (average value); (A–E) cells after the addition 3.85 mM of K₂[B₃O₃F₄OH] and in the presence of Ca²⁺ ions: (A) 0.42 mM, (B) 9.42 mM, (C) 18.42 mM, (D) 36.42 mM and (E) 72.42 mM.

Discussion and conclusions

Boron heterocyclic compound K₂[B₃O₃F₄OH] is an anhydride of boronic acid. It is one of the substances whose structural basis is boroxine ring. Research of bioactive potential of this molecule has been the subject of multiple studies. The well-known ability of this substance is to significantly affect the proliferation and cytotoxicity of various cancer cells, but the understanding of these effects is insufficient. Recently published results of in vitro and in vivo investigation12 on the 4T1 breast adenocarcinoma, B16F10 melanoma, and squamous cell carcinoma SCCVII, undoubtedly showed that K₂[B₃O₃F₄OH] can affect the growth of cancer cells in an unusual way. Cell proliferation is dependent on the concentration of K₂[B₃O₃F₄OH] so that the concentration of 0.1 mM and less does not affect the growth of cells, or concentration of 1 mM or more, substantially slows their growth. Under in vivo conditions, K₂[B₃O₃F₄OH] slowed the growth of all the three tested cancer cells. There is almost no difference in the antitumor effect when K2 [B3O3F4OH] has administered intraperitoneally, intratumorally, orally, or as a cream.

So far it is not known why K₂[B₃O₃F₄OH] has anticancer activity in several types of cancer cells and why the anticancer intensity does not change in proportion to its concentration. It is not known how this molecule finds the cancer cells among a large number of normal cells. We assume that these small inorganic molecules, in some unknown way, recognize cancer cells or cancer cells have a common feature that is attractive for molecules K₂[B₃O₃F₄OH]. In order to find the answers to these questions, we decided to investigate the influence of Ca²⁺ ions on its anticancer ability. It is known that Ca²⁺ ions regulate many cellular functions, including cell metabolism. The recent research reveals an extremely important role of Ca²⁺ ions and its concentration in stopping the growth of a cancer cell. Cardenas et al.16 have indicated that the mitochondria in cancer cells depends on the concentration of Ca²⁺ ions and claims that it is a general feature of the cancer cells. They showed that the inhibition of the transfer of Ca²⁺ ions in mitochondria is toxic only to cancer cells, but not to normal cells. Zhao et al.18 have proposed a treatment of cancer by the process of calcification of cancer cells. The procedure is based on the fact that many types of cancer cells have increased expression of folate receptors. This means that these receptors can selectively take folate molecules which with a carboxylic group can specifically bind Ca²⁺ ions. This leads to an increase in the concentration of Ca²⁺ ions and calcification in cancer cells. Finally, the resulting mineral compressed (encapsulated) tumor cells and it can induce their death. Both of these studies point out that Ca²⁺ ion and its concentration significantly affects the anticancer processes. This was the impetus for this research which aims evaluation of how Ca²⁺ ions in different concentrations can affect the cytotoxic ability of K₂[B₃O₃F₄OH] which stops the growth of several cancer and normal cells. These effects were examined in vitro for cancer cells (mammary adenocarcinoma 4T1, B16F10 melanoma and squamous cell carcinoma SCCVII) and normal fibroblasts (L929 mouse dermal and hamster lung V79). As described in Results section, Ca²⁺ ion concentrations varied from 0.42 to 72.42 mM, while the concentration of K₂[B₃O₃F₄OH] was constant, 3.85 mM. An amount of 0.42 mM of Ca²⁺ ions is present in the formulation of the RPMI 1640 medium.

The results of this research are:

1. In the medium of the low concentration of Ca²⁺ ions (0.42 mM), K₂[B₃O₃F₄OH] (3.85 mM) had a very strong cytotoxic effect on all tested cancer cells (89.1, 85.6 and 84.6%).

2. In the medium with the same concentration of Ca²⁺ ions (0.42 mM), K₂[B₃O₃F₄OH] (3.85 mM) had a significantly lower effect on normal cells (19.5% and 24.2% respectively).

3. In the medium with higher concentrations of Ca²⁺ ions (9.42–72.42 mM), with corresponding concentration of K₂[B₃O₃F₄OH], no significant cytotoxic effect on cancer cells and normal cells was detected.

These results indicate that the significant selective ability of K₂[B₃O₃F₄OH] exists only in the medium of a small concentration of Ca²⁺ ions, under the same conditions, at the same time it has a very strong cytotoxic effect on different types of cancer cells, a significantly weaker effect on normal cells. Based on these results, it can be assumed that K₂[B₃O₃F₄OH] has the ability to affect the anticancer processes in which Ca²⁺ ions and their concentrations play an important role. According to a study by Cardenas et al.16, in future investigations, it would be very interesting to investigate the impact of K₂[B₃O₃F₄OH] to inositol 1,4,5-trisphosphate receptors (InsP₃Rs) in Ca²⁺ ion channels and its consequences for inhibition of Ca²⁺ ion transfers from endoplasmic reticulum to mitochondria. This is supported by the fact that K₂[B₃O₃F₄OH] finds cancer cells throughout the body, affecting the proliferation and cytotoxicity of various types of cancer cells and that there is a discontinuity effect on cancer cells depending on its concentration12. The selective ability of K₂[B₃O₃F₄OH] that, in the medium with a low concentration of Ca²⁺ ions has a strong cytotoxic effect on cancer cells and very weak effect in normal cells, opens up the possibility of its application in antitumor therapy.

Declaration of interest

This work has been fully supported by the Croatian Science Foundation under the project number (IP-2014-09-6897). The authors report no conflict of interest.