Combination of selol nanocapsules and magnetic hyperthermia hinders breast tumor growth in aged mice after a short-time treatment.

Short time treatment with reduced dosages of selol-loaded PLGA nanocapsules (NcSel) combined with magnetic hyperthermia (MHT) is evaluated in aged Erhlich tumor-bearing mice. Clinical, hematological, biochemical, genotoxic and histopathological parameters are assessed during 7 d treatment with NcSel and MHT, separately or combined. The time evolution of the tumor volume is successfully modeled using the logistic mathematical model. The combined therapy comprising NcSel and MHT is able to hinder primary tumor growth and a case of complete tumor remission is recorded. Moreover, no metastasis was diagnosed and the adverse effects are negligible. NcSel plus MHT may represent an effective and safe alternative to cancer control in aged patients. Future clinical trials are encouraged.

Magnetic studies of polylactic-co-glicolic acid nanocapsules loaded with selol and γ-Fe2O3 nanoparticles.

The as-prepared (MSE-NCs sample) and lyophilized (LMSE-NCs sample) polylactic-co-glicolic acid (PLGA) nanocapsules loaded with maghemite (γ-Fe2O3) nanoparticles and selol (Se-based anticancer drug) were investigated by means of dc magnetization, ac susceptibility and electron spin resonance (ESR) measurements over the temperature range of 4-300 K. The magnetic data of the as-synthesized nanocapsules containing only maghemite nanoparticles (M-NCs sample) or selol (SE-NCs sample) were also collected for comparison. The magnetic nanocapsules reveal perfect superparamagnetic (SPM) behavior only around room temperature; at temperatures lower than 200 K the SPM scaling is not observed and all samples behave as interacting superparamagnetic (ISPM) materials. The evolution from the ISPM to the SPM regime is marked by a steady decrease in the hysteretic properties of all samples, with the temperature dependence of the coercivity decreasing slower than the T1/2 behavior predicted for non-interacting SPM particles. The SPM character of the samples is also confirmed by the occurrence of a maximum in the temperature dependence of both real χ'(T) and imaginary χ''(T) components of the ac magnetic susceptibility, which shifts towards higher temperatures with increasing frequency. Moreover, upon decreasing the temperature the ESR signal shifts to lower fields and gradually broadens, following closely the predictions for the ESR of SPM particles. Additionally, an unusual giant diamagnetic response is observed at low temperatures. The ZFC magnetization is found to reverse its direction and becomes diamagnetic, whereas the FC branch remains positive. Even when compared with usual superconductors, the order of the diamagnetic susceptibility for the lyophilized sample (-10-2 emu g-1 Oe-1) is quite considerable. The nanocapsules herein reported and the presented analysis of their magnetic properties we envisage can support the engineering of magnetic nanocapsules for applications in magnetic drug delivery systems and as magnetic hyperthermia inductors in antitumor therapy.

Decoration of a Poly(methyl vinyl ether-co-maleic anhydride)-Shelled Selol Nanocapsule with Folic Acid Increases Its Activity Against Different Cancer Cell Lines In Vitro.

Due to the low therapeutic index of different chemotherapeutic drugs used for cancer treatment, the development of new anticancer drugs remains an intense field of research. A recently developed mixture of selenitetriacylglycerides, selol, was shown to be active against different cancer cells in vitro. As this compound is highly hydrophobic, it was encapsulated, in a previous study, into poly(methyl vinyl ether-co-maleic anhydride)-shelled nanocapsules in order to improve its dispersibility in aqueous media. Following this line of research, the present report aimed at enhancing the In Vitro activity of the selol nanocapsules against cancerous cells by decorating their surface with folic acid. It is known that several cancer cells overexpress folate receptors. Stable folic acid-decorated selol nanocapsules (SNP-FA) were obtained, which showed to be spherical, with a hydro-dynamic diameter of 364 nm, and zeta potential of -24 mV. In comparison to non-decorated selol nanocapsules, SNP-FA presented higher activity against 4T1, MCF-7 and HeLa cells. Moreover, the decoration of the nanocapsules did not alter their toxicity towards fibroblasts, NIH-3T3 cells. These results show that the decoration with folic acid increased the toxicity of selol nanocapsules to cancer cells. These nanocapsules, besides enabling to disperse selol in an aqueous medium, increased the toxicity of this drug In Vitro, and may be useful to treat cancer in vivo, potentially increasing the specificity of selol towards cancer cells.

Antitumor activity and systemic effects of PVM/MA-shelled selol nanocapsules in lung adenocarcinoma-bearing mice.

Selol is a semi-synthetic compound containing selenite that is effective against cancerous cells and safer for clinical applications in comparison with other inorganic forms of selenite. Recently, we have developed a formulation of poly(methyl vinyl ether-co-maleic anhydride)-shelled selol nanocapsules (SPN), which reduced the proliferative activity of lung adenocarcinoma cells and presented little deleterious effects on normal cells in in vitro studies. In this study, we report on the antitumor activity and systemic effects induced by this formulation in chemically induced lung adenocarcinoma-bearing mice. The in vivo antitumor activity of the SPN was verified by macroscopic quantification, immunohistochemistry and morphological analyses. Toxicity analyses were performed by evaluations of the kidney, liver, and spleen; analyses of hemogram and plasma levels of alanine aminotransferase, aspartate transaminase, urea, and creatinine; and DNA fragmentation and cell cycle activity of the bone marrow cells. Furthermore, we investigated the potential of the SPN formulation to cause hemolysis, activate the complement system, provoke an inflammatory response and change the conformation of the plasma proteins. Our results showed that the SPN reduced the area of the surface tumor nodules but not the total number of tumor nodules. The biochemical and hematological findings were suggestive of the low systemic toxicity of the SPN formulation. The surface properties of the selol nanocapsules point to characteristics that are consistent with the treatment of the tumors in vivo: low hemolytic activity, weak inflammatory reaction with no activation of the complement system, and mild or absent conformational changes of the plasma proteins. In conclusion, this report suggests that the SPN formulation investigated herein exhibits anti-tumoral effects against lung adenocarcinoma in vivo and is associated with low systemic toxicity and high biocompatibility.

PVM/MA-shelled selol nanocapsules promote cell cycle arrest in A549 lung adenocarcinoma cells.

BACKGROUND: Selol is an oily mixture of selenitetriacylglycerides that was obtained as a semi-synthetic compound containing selenite. Selol is effective against cancerous cells and less toxic to normal cells compared with inorganic forms of selenite. However, Selol's hydrophobicity hinders its administration in vivo. Therefore, the present study aimed to produce a formulation of Selol nanocapsules (SPN) and to test its effectiveness against pulmonary adenocarcinoma cells (A549). RESULTS: Nanocapsules were produced through an interfacial nanoprecipitation method. The polymer shell was composed of poly(methyl vinyl ether-co-maleic anhydride) (PVM/MA) copolymer. The obtained nanocapsules were monodisperse and stable. Both free Selol (S) and SPN reduced the viability of A549 cells, whereas S induced a greater reduction in non-tumor cell viability than SPN. The suppressor effect of SPN was primarily associated to the G2/M arrest of the cell cycle, as was corroborated by the down-regulations of the CCNB1 and CDC25C genes. Apoptosis and necrosis were induced by Selol in a discrete percentage of A549 cells. SPN also increased the production of reactive oxygen species, leading to oxidative cellular damage and to the overexpression of the GPX1, CYP1A1, BAX and BCL2 genes. CONCLUSIONS: This study presents a stable formulation of PVM/MA-shelled Selol nanocapsules and provides the first demonstration that Selol promotes G2/M arrest in cancerous cells.

Comparison of selected gene expression profiles in sensitive and resistant cancer cells treated with doxorubicin and Selol.

AIM OF THE STUDY: Cellular resistance is strongly correlated with the risk of failure in doxorubicin (DOX) treatment, and the knowledge of the mechanisms of resistance and its possible modulation is still very limited. MATERIAL AND METHODS: In this study, we assessed the effect of 5% Selol and DOX on the expression of genes that affect cell proliferation in the resistant KB-V1 and sensitive HeLa cell lines, using RT2 ProfilerTM PCR Array matrix "Human Cancer Drug Resistance and Metabolism" (SABiosciences). RESULTS: We showed that HeLa and KB-V1 cell lines, characterised by varying susceptibility to DOX, have different genetic profiles as regards the studied genes. KB-V1 cells show overexpression of MYC and BCL2 genes, which encode proteins with anti apoptotic properties. Selol, when used in KB-V1 cells, reduced the expression of MYC and BCL2 genes, suggested as a new therapeutic target in the treatment of cancers resistant to cytostatic drugs. CONCLUSIONS: The results suggest that Selol could be used as a modulator that enhances the cytotoxic effects of doxorubicin, particularly in cells resistant to this drug.

Are Obese Patients with Autism Spectrum Disorder More Likely to Be Selenium Deficient? Research Findings on Pre- and Post-Pubertal Children.

Selenium is involved in many metabolic pathways that are critical for life. Information concerning the metabolic effects of selenium in autism spectrum disorder (ASD) and obesity is still conflicting and incomplete. The pre- and post-pubertal selenium profiles of patients with ASD and obesity have not yet been investigated. The goal of the study was to examine selenium content before and after puberty in euthyroid children diagnosed with ASD, compared to age-matched neurotypical controls, with respect to overweight or obesity as a co-existing pathology. Serum, toenail, and 24h urine selenium levels were determined by inductively coupled plasma mass spectrometry in 287 prepubertal children (mean age 8.09 years), divided into groups: ASD with overweight/obesity (ASD+/Ob+); ASD without overweight/obesity (ASD+/Ob-); non-ASD with overweight/obesity (ASD-/Ob+); and non-ASD without overweight/obesity (ASD-/Ob-). The assessment was repeated in 258 of the children after puberty (mean age 14.26 years).The lowest serum (p < 0.001), urine (p < 0.001) and toenail (p < 0.001) selenium levels before and after puberty were observed in ASD+/Ob+ patients, and the highest in ASD-/Ob-. There were no differences in serum/toenail selenium levels between ASD+/Ob- and ASD-/Ob+ groups. The presence of ASD was associatedwith lower serum (p < 0.001) and toenail (p < 0.001) selenium in BMI-matched groups. In neurotypical patients, post-pubertal serum selenium levels were lower (p < 0.001) than pre-pubertal levels. In the multiple linear regression analyses, selenium levels showed inverse relationships with BMI (p < 0.001) and male gender (p < 0.001), irrespective of the sample type. The serum (p = 0.002) and toenail (p < 0.001) selenium levels were inversely associated with the presence of ASD. ASD, obesity/overweight, and male gender have independent impacts on selenium levels in children. Puberty may affect selenium content in neurotypical children of both genders, but not in ASD patients.

Lentinula edodes as a Source of Bioelements Released into Artificial Digestive Juices and Potential Anti-inflammatory Material.

Lentinula edodes (shiitake), an edible and medicinal mushroom, was chosen for this study with the aim of evaluating the possibility of release of bioelements into artificial digestive juices and analyzing the anti-inflammatory properties. The extracts were prepared from fruiting bodies and biomass enriched with copper (Cu), zinc (Zn), and selenium (Se). The content of bioelements was analyzed by total reflection X-ray fluorescence method. Relatively low content of elements was observed in the fruiting bodies: Cu-1.6, Zn-7.6, and Se-0.12 mg/100 g d.w. compared to mycelial cultures. The anti-inflammatory properties were evaluated in RAW 264.7 cells. Based on the levels of cyclooxygenase 2 protein, nuclear factor erythroid 2-related factor 2, and peroxisome proliferator-activated receptor γ determined using Western blot technique, it was found that the addition of bioelements enhanced the anti-inflammatory properties of mycelium. This indicates that L. edodes cultured on a suitable medium may be used as a potential component of anti-inflammatory products.

Correction to: Lentinula edodes as a Source of Bioelements Released Into Artificial Digestive Juices and Potential Anti-inflammatory Material.

The original version of this article unfortunately contained a mistake.

Preliminary study on Se-enriched Lentinula edodes mycelium as a proposal of new feed additive in selenium deficiency.

The presence of selenium in European soil is low and this causes its deficiency in livestock and, in consequence, in humans. This study aimed to obtain Lentinula (L.) edodes mycelium with the maximum content of selenium. This species was used for experiment based on its documented medicinal properties. Calves were fed with selenium-enriched L. edodes mycelium, and serum selenium concentration, average daily weight gains and selected immune parameters were estimated. The selenium-enriched mushroom was found to be safe based on cytotoxicity tests (MTT and LDH tests) and for this reason it was used for further experiments. The mean quantity of selenium in the serum of calves fed with selenium-enriched L. edodes mycelium was significantly higher than that of control calves. Additionally, the calves fed with selenium-enriched L. edodes mycelium had higher body weight gains than those of control calves. White blood cell counts and subpopulations of lymphocytes in the experimental and control calves were within the reference range. The administration of L. edodes enriched with selenium had a beneficial effect on state of health of the calves.

Selenized Plant Oil Is an Efficient Source of Selenium for Selenoprotein Biosynthesis in Human Cell Lines.

Selenium is an essential trace element which is incorporated in the form of a rare amino acid, the selenocysteine, into an important group of proteins, the selenoproteins. Among the twenty-five selenoprotein genes identified to date, several have important cellular functions in antioxidant defense, cell signaling and redox homeostasis. Many selenoproteins are regulated by the availability of selenium which mostly occurs in the form of water-soluble molecules, either organic (selenomethionine, selenocysteine, and selenoproteins) or inorganic (selenate or selenite). Recently, a mixture of selenitriglycerides, obtained by the reaction of selenite with sunflower oil at high temperature, referred to as Selol, was proposed as a novel non-toxic, highly bioavailable and active antioxidant and antineoplastic agent. Free selenite is not present in the final product since the two phases (water soluble and oil) are separated and the residual water-soluble selenite discarded. Here we compare the assimilation of selenium as Selol, selenite and selenate by various cancerous (LNCaP) or immortalized (HEK293 and PNT1A) cell lines. An approach combining analytical chemistry, molecular biology and biochemistry demonstrated that selenium from Selol was efficiently incorporated in selenoproteins in human cell lines, and thus produced the first ever evidence of the bioavailability of selenium from selenized lipids.

Selol (Se IV) modulates adhesive molecules in control and TNF-α-stimulated HMEC-1 cells.

Selol, an organic selenitetrigliceride formulation containing selenium at +4 oxidation level, has been suggested as anticancer drug. One of the causes of several diseases including cancer may be inflammation. This study aimed at determining the activity of Selol via measuring its effect on reactive oxygen species (ROS) generation, nuclear factor kappa B (NF-κB) activation, intercellular cell adhesion molecules-1 (ICAM-1), vascular cell adhesive molecule-1 (VCAM-1), and plateled-endothelial cell adhesive molecule-1 (PECAM-1) levels on control and on tumor necrosis factor-α (TNF-α)-stimulated human microvascular endothelial cells (HMEC-1). Cells were treated either with Selol 5% (4 or 8 µgSe/mL) or TNF-α (10 ng/mL) alone or with Selol concomitant with TNF-α. Selol treatment resulted in ROS generation, activation of NF-κB, downregulation of PECAM-1, VCAM-1 and slight upregulation ICAM-1 expression on the cell surface. TNF-α treatment reflected in sharp NF-κB activation, upregulation of both ICAM-1 and VCAM-1 in parallel with the downregulation of PECAM-1 expression on cell surface. Exposure to both compounds upregulated ICAM-1 and VCAM-1, downregulated PECAM-1 level on cell surface in parallel with no changes in level of NF-κB activation as compared with effects mediated by TNF-α alone. These results points to new look at Selol action since it shows a pro-inflammatory activity in parallel with effects on CAMs expression on the cell surface of human microvascular endothelial cells. However, since Selol enhances CAMs expression level when is present concomitantly with TNF-α this fact might suggest that selenium present in the condition of inflammation will make it worse.

Effects of Selol 5% supplementation on tissue antioxidant enzyme levels and peroxidation marker in healthy mice.

BACKGROUND: Selenium (Se) is an essential micronutrient for animals and humans used in the prevention or treatment of cancer. Selol is a mixture of selenitetriglycerides, containing Se(IV). It does not exhibit mutagenic activity and is less toxic than inorganic sodium selenite containing Se(IV). The antioxidant properties of the Selol were demonstrated using the blood of healthy animals. The aim of the study was to evaluate Selol as a Se supplement by determining the effect of its administration on the Se level and the antioxidant status in the tissues. METHODS: We examined the effect of long-term (28-day) Selol 5% supplementation on the activity of antioxidant enzymes, including the main selenoenzymes in healthy mice organs, such as liver, brain, lungs, and testis. Enzyme activities of the tissue homogenates and the concentration of malondialdehyde (MDA) as a biomarker of oxidative stress were measured using spectrophotometric methods. The selenium concentrations in the tissues were determined by inductively coupled plasma mass spectrometer (ICP-MS) as well. RESULTS: A significant increase in glutathione peroxidase, thioredoxin reductase, and glutathione S-transferase activity as well as the MDA concentration was observed in most of the studied tissues during the Selol 5% supplementation. CONCLUSIONS: Long-term supplementation with the new Se(IV) compound - Selol 5% significantly affects the activity of antioxidant enzymes and the redox state in healthy mice organs. In the healthy population Selol 5% seems to be a promising new antioxidant compound.

Selol nanocapsules with a poly(methyl vinyl ether-co-maleic anhydride) shell conjugated to doxorubicin for combinatorial chemotherapy against murine breast adenocarcinoma in vivo.

Nanocapsules containing selol and doxorubicin (NCS-DOX) with an oily core of selol and a shell of poly(methyl vinyl ether-co-maleic anhydride) covalently conjugated to doxorubicin were developed in a previous work. In this study, these nanocapsules showed a similar antitumour effect in comparison to the free doxorubicin (DOX) treatment, but showed no evident DOX-related cardiotoxicity, as evidenced by serum creatine kinase-MB (CK-MB) activity. The histopathological analysis showed that the free DOX treatment induced more intense morphological damage to myocardial tissues in comparison to NCS-DOX treatment. Animals treated with free DOX presented important muscle fibre degradation and animals treated with NCS-DOX, heart tissue did not present signals of muscle fibre degeneration. These results indicate that the cardiotoxicity related to DOX is reduced when this drug is carried by the NCS-DOX. Noteworthy, biodistribution analyses showed that NCS-DOX accumulated more intensely in tumours than the free DOX. Thus, this study reinforces the importance of the development of nanocapsules as drug carriers for the treatment of cancer.

Nanocapsules for the co-delivery of selol and doxorubicin to breast adenocarcinoma 4T1 cells in vitro.

Nanocapsules (NCS-DOX) with an oily core of selol and a shell of poly(methyl vinyl ether-co-maleic anhydride) covalently conjugated to doxorubicin were developed. These nanocapsules are spherical, with an average hydrodynamic diameter of about 170 nm, and with negative zeta potential. NCS-DOX effectively co-delivered the selol and the doxorubicin into 4T1 cells and changed the intracellular distribution of DOX from the nuclei to the mitochondria. Moreover, a significantly increased cytotoxicity against 4T1 cells was observed, which is suggestive of additive or synergic effect of selol and doxorubicin. In conclusion, PVM/MA nanocapsules are suitable platforms to co-deliver drugs into cancer cells.

The activity of Selol in multidrug-resistant and sensitive human leukemia cells.

Selol is a mixture of selenitetriglycerides synthesized from sunflower oil. As it contains the element selenium in its structure, it is suspected to exhibit chemopreventive and anticancer activity. In this study, the ability of Selol to inhibit cell proliferation and to induce apoptosis was investigated. Three cell lines were used: leukemia HL-60 cell line and multidrug-resistant HL-60/Dox (resistant to doxorubicin) and HL-60/Vinc (resistant to vincristine). Selol was shown to reduce the cell number as a result of treatment with increasing concentrations. For selected concentrations the evidence of apoptosis (changes in mitochondrial potential and caspase activity) was investigated, as well as changes in lysosome distribution. The study has shown that Selol overcame the cell resistance, as doxorubicin-resistant cells were more sensitive towards Selol than sensitive cells.

RP-HPLC determination of paraoxonase 3 activity in human blood serum.

The aim of the present work was to establish conditions for paraoxonase 3 (PON3) activity determination in human blood serum with simvastatin (SV) as a substrate. The activity of PON3 is considered as a good early predictor of susceptibility to premature atherosclerosis as well as of statin therapy effectiveness. The method used quantifies the SV and beta,delta-dihydroxyacid simvastatin (SVA) liberated from SV after incubation with blood serum, followed by deproteinization of the reaction mixture. Separation of SV and SVA was performed on an LC(18) column by isocratic elution with acetonitrile-K-phosphate buffer of pH 4.5 (v/v, 70:30) as a mobile phase at flow rate of 1.5 ml min(-1). Detection based on ultraviolet absorption at a wavelength of 239 nm was reliable for the simultaneous assay of SV and SVA. The applied method was sufficiently sensitive, precise and accurate for determination of low simvastatin lactone hydrolase (statinase) activity in blood serum of children (1.97-6.86 pmol min(-1) ml(-1)). The method is characterized by good linearity over the measurement range of 0.5-6 microg ml(-1) (1.194-14.3 nmol ml(-1)). Limits of detection (LOD) and quantitation (LOQ) for SV were 3.1 and 10.4 ng ml(-1), respectively. In case of SVA, LOD and LOQ were 4.7 and 14.44 ng ml(-) for a 20 microl sample, respectively. Precision and accuracy of PON3 statinase activity determination in human blood serum with SV as substrate were satisfactory and acceptable for bioanalytical methods.

Speciation of Selenium in Selenium-Enriched Sunflower Oil by High-Performance Liquid Chromatography-Inductively Coupled Plasma Mass Spectrometry/Electrospray-Orbitrap Tandem Mass Spectrometry.

The reaction of sunflower oil with selenite produces a complex mixture of selenitriglycerides with antioxidant and anticancer properties. To obtain insight into the identity and characteristics of the species formed, an analytical approach based on the combination of high-performance liquid chromatography (HPLC) with (78)Se-specific selenium detection by inductively coupled plasma mass spectrometry (ICP MS) and high-resolution (100 000), high mass accuracy (<1 ppm) molecule-specific detection by electrospray-Orbitrap MS(3) was developed. For the first time, a non-aqueous mobile phase gradient was used in reversed-phase HPLC-ICP MS for the separation of a complex mixture of selenospecies and a mathematical correction of the background signal was developed. The identical chromatographic conditions served for the sample introduction into electrospray MS. Two types of samples were analyzed: sunflower oil dissolved in isopropanol and methanol extract of the oil containing 65% selenium. HPLC-ICP MS showed 14 peaks, 11 of which could also be detected in the methanol extract. Isotopic patterns corresponding to molecules with one or two selenium atoms could be attributed by Orbitrap MS at the retention times corresponding to the HPLC-ICP MS peak apexes. Structural data for these species were acquired by MS(2) and MS(3) fragmentation of protonated or sodiated ions using high-energy collisional dissociation (HCD). A total of 11 selenium-containing triglycerol derivatives resulting from the oxidation of one or two double bonds of linoleic acid and analogous derivatives of glycerol-mixed linoleate(s)/oleinate(s) have been identified for the first time. The presence of these species was confirmed by the targeted analysis in the total oil isopropanol solution. Their identification corroborated the predicted elution order in reversed-phase chromatography: LLL (glycerol trilinoleate), LLO (glycerol dilinoleate-oleinate), LOO (glycerol linoleate-dioleinate), OOO (glycerol trioleinate), of which the extrapolation allowed for the prediction of the identity [glycerol dioleinate-stearate (OOS) and glycerol oleinate-distearate (OSS)] of the nonpolar species detected by ICP MS in the oil but not detected by electrospray MS.

A Search for the Optimum Selenium Source to Obtain Mushroom-Derived Chemopreventive Preparations.

The objective of this research was to test whether selenium-yeast (Se-yeast) is a better source of selenium than sodium selenite for accumulation in mycelia and immunoactive cell wall polysaccharides. Culture media were enriched in selenium to a concentration of 20 µg/mL. Selenium was added to the medium either in the form of sodium selenite or in form of Se-yeast (Sel-Plex; Alltech Inc., Lexington, KY). The total selenium concentrations in the mycelium biomass and in the isolated crude polysaccharides were determined using atomic absorption spectroscopy. We found that selenium accumulated more efficiently in cultures enriched with Se-yeast. A higher concentration of selenium was also found in the crude polysaccharide fractions isolated from the mycelium grown in Se-yeast-enriched media. With the use of the needle trap gas chromatography-mass spectrometry method, we found that there are significant differences in the composition of the volatile aroma and flavor compounds secreted by the mycelia cultivated in different media.

The Comparison of MTT and CVS Assays for the Assessment of Anticancer Agent Interactions.

Multiple in vitro tests are widely applied to assess the anticancer activity of new compounds, including their combinations and interactions with other drugs. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay is one of the most commonly used assays to assess the efficacy and interactions of anticancer agents. However, it can be significantly influenced by compounds that modify cell metabolism and reaction conditions. Therefore, several assays are sometimes used to screen for potential anticancer drugs. However, the majority of drug interactions are evaluated only with this single method. The aim of our studies was to verify whether the choice of an assay has an impact on determining the type of interaction and to identify the source of discrepancies. We compared the accuracy of MTT and CVS (crystal violet staining) assays in the interaction of two compounds characterized by similar anticancer activity: isothiocyanates (ITCs) and Selol. Confocal microscopy studies were carried out to assess the influence of these compounds on the reactive oxygen species (ROS) level, mitochondrial membrane potential, dead-to-live cell ratio and MTT-tetrazolium salt reduction rate. The MTT assay was less reliable than CVS. The MTT test of Selol and 2-oxoheptyl ITC, which affected the ROS level and MTT reduction rate, gave false negative (2-oxoheptyl ITC) or false positive (Selol) results. As a consequence, the MTT assay identified an antagonistic interaction between Selol and ITC, while the metabolism-independent CVS test identified an additive or synergistic interaction. In this paper, we show for the first time that the test assay may change the interpretation of the compound interaction. Therefore, the test method should be chosen with caution, considering the mechanism of action of the compound.