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.

Cellular senescence is a permanent blockade of cell proliferation. In response to therapy-induced stress, cancer cells undergo apoptosis or premature senescence. In apoptosis-resistant cancer cells or at lower doses of anticancer drugs, therapy-induced stress leads to premature senescence. The role of this senescence in cancer treatment is discussable. First of all, the senescent cells lose the ability to proliferate, migrate, and invade. In addition, the senescent cells secrete a set of proteins (inflammatory cytokines, chemokines, growth factors) known as the senescence-associated secretory phenotype (SASP), which influences non-senescent normal cells and non-senescent cancer cells in the tumor microenvironment and triggers tumor promotion and recurrence. Recently, many studies have examined senescence induction through breast cancer therapy and potentially using this phenomenon to treat this cancer. This review summarizes the recent in vitro, in vivo, and clinical studies investigating senescence in breast cancer treatments. Senescence inductors, senolytics, as well as their action mechanism are discussed herein. Potential SASP-modulating treatment strategies are also described.

Sulforaphane (SFN), a naturally occurring chemopreventive and anticancer agent, is a nuclear factor, erythroid 2-like 2 (NFE2L2/Nrf2) inducer. Nrf2 plays a critical role in coordinating the cell defense system by initiating the transcription of cytoprotective genes, including detoxification enzymes such as NAD(P)H quinone dehydrogenase 1 (NQO1) and transport proteins such as ATP-binding cassette, subfamily C (CFTR/MRP). Recently, the essential role of Nrf2 in tumor development and progression and in the development of multidrug resistance in cancer cells has been highlighted. The aim of this study was to compare the effect of SFN on the Nrf2 system and the Nrf2-target enzymes NQO1 and MRP in human untransformed epithelial colon CRL-1790 cells and in HT-29 and Caco-2 colorectal cancer cells to elucidate the role of SFN in cancer prevention and treatment. We have demonstrated that SFN has excellent cytoprotective properties in CRL-1790 cells, as it induced Nrf2-dependent expression of MRP1 and NQO1. SFN induced Nrf2 target enzyme activity in HT-29 and Caco-2 cancer cells but regulated the Nrf2/ARE signaling pathway differently in cancer and untransformed cells.

Combination therapy is based on the beneficial effects of pharmacodynamic interaction (synergistic or additive) between combined drugs or substances. A considerable group of candidates for combined treatments are natural compounds (e.g., isothiocyanates) and their analogs, which are tested in combination with anticancer drugs. We tested the anticancer effect of the combined treatment of isothiocyanate 2-oxohexyl isothiocyanate and 5-fluorouracil in colon and prostate cancer cell lines. The type of interaction was described using the Chou-Talalay method. The cytostatic and cytotoxic activities of the most promising combined treatments were investigated. In conclusion, we showed that combined treatment with 5-fluorouracil and 2-oxohexyl isothiocyanate acted synergistically in colon cancer. This activity is dependent on the cytostatic properties of the tested compounds and leads to the intensification of their individual cytotoxic activity. The apoptotic process is considered to be the main mechanism of cytotoxicity in this combined treatment.