Urinary bladder cancer is currently the fourth most common cancer in men in Poland, with a continuously increasing incidence and mortality rate. Over the last 30 years, numerous studies have demonstrated that naturally occurring isothiocyanates possess significant chemopreventive and antitumor potential, combined with low toxicity. Their multi-targeted mode of action, including effects on mitotic division, along with their natural origin, makes them promising adjuvants for currently used oncological therapies. The metabolism of isothiocyanates via the mercapturic acid pathway leads to the formation of biologically active isothiocyanate-derived mercapturic acids, which accumulate at high concentrations in urine and urinary bladder tissue, making them particularly relevant for bladder cancer treatment.

A set of tubulin polymerization modulators (e.g., vinorelbine, colchicine, paclitaxel, combretastatin A4) was tested in vitro with structurally different mercapturic acids. Experiments included assessments of antiproliferative activity, apoptosis rate, cell cycle analysis, and glutathione levels. Antiproliferative effects were analyzed using synergy models to determine interactions. The most promising combinations were further evaluated in vivo using orthotopic cancer models established by ultrasound-guided cancer cell inoculation.

Our in vitro studies demonstrated for the first time that mercapturic acids synergistically enhance the antiproliferative activity of microtubule polymerization inhibitors (such as colchicine, vinflunine, and combretastatin A4), but not microtubule stabilizers (such as paclitaxel). This leads to reduced proliferation and increased apoptosis. We also discovered that the chemical structure of mercapturic acids influences their potency; for instance, the benzyl isothiocyanate derivative was significantly more potent than the sulforaphane derivative. Follow-up studies revealed that the observed synergistic effect resulted from an increased apoptosis rate during the G2/M phase (without substantial changes in the overall cell cycle profile), decreased glutathione and βIII-tubulin levels in combination-treated cells, and reduced tubulin polymerization in cell-free experiments. In vivo studies using orthotopic models indicated a moderate enhancement of the anticancer activity of combretastatin A4 and vinflunine by benzyl isothiocyanate-derived mercapturic acid. This effect was associated with reduced angiogenesis and tumor perfusion, without signs of increased toxicity.

These findings suggest that mercapturic acids enhance the anticancer efficacy of tubulin polymerization inhibitors (but not stabilizers), synergistically increasing mitotic catastrophe rates. This work was supported by the National Science Centre, Poland (grant no. 2017/26/D/NZ7/01152).