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.

The anticancer activity of Selol involves perturbation of the redox regulation in the androgen dependent hPCa (LNCaP) cells, but not in healthy cells. After Selol treatment, intracellular Eh has increased in tumors from -223 mV to -175 mV, while in serum it has decreased (-82 mV vs -113 mV). It shows significant changes Eh in the extra- and intracellular environment. The difference decreases from 141 mV to 62 mV. The changes suggest that a tumor cell was probably directed toward apoptosis. This is exemplified in a significant decrease in cancer tumor mass by approx. 17% after the three weeks of Selol administration.

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Introduction: 5-Fluorouracil is used to treat many types of cancers such as: breast, ovarian and gastrointestinal (e.g. colon or gastric) cancer. Until recently 5-fluorouracil was investigated as a candidate for prostate cancer therapy. The antitumor activity of 5-fluorouracil as a single agent is low (approximately 30%), hence it is used mainly in polytherapy. A few years ago a new idea was observed in scientific publications to enhance effectiveness of anticancer drugs through combined treatments with naturally occurring chemopreventive agents. Use of natural compounds increases efficacy of anticancer treatment and decreases toxicity in normal cells. One of the compounds is isothiocyanate-sulforaphane. It inhibits early stages of carcinogenesis and post-initiated stages of the process. In vitro studies show that it potentiates the activity of 5-fluorouracil, oxaliplatin and doxorubicin. Additionally, our previous studies indicate that sulforaphane acts antagonistically when combined with 5-fluorouracil in normal cell lines. The aim of this study was to investigate the type of interaction between 5-fluouracil and a more cytotoxic analogue of sulforaphane-2-oxohexyl isothiocyanate-in two colon cancer and two prostate cancer cell lines. Methods: The study was performed on cancer cell lines: prostate one (LNCaP and PC-3) and colon one (Caco-2and ht-29). The cytotoxic effects of single 5-fluorouracil or isothiocyanate treatments and their combination were evaluated by the MTT assay. There was used sequential schedule. The cells were treated with isothiocyanate and left for 24 hours and then they were subsequently incubated with 5-fluorouracil for 72 hours. The types of interaction were determined through the median effect analysis as described by Chou and Talalay and only when the combination inhibited cell proliferation in more than 50%. Results: Synergic and additive types of interaction between isothiocyanate and 5-fluorouracil were observed in colon cancer cell lines. The cytotoxic effects of combination of 5-fluorouracil and 2-oxohexyl isothiocyanate were stronger than the effects of single treatments. In one prostate cell line-LNCaP-there was observed antagonism and in the second prostate cell line-PC-3there were noticed additive effects of the tested compounds. Conclusion: 2-oxohexyl isothiocyanate selectively potentiate5-fluorouracil anticancer activity in colon cancer cells lines.