ИСТИНА

Anti-microtubule (MT) drugs are successfully used in cancer treatment. It is usually assumed that anti-cancer effect is exerted through mitotic arrest followed by apoptosis. However, after the mitotic arrest induced by prolonged treatment with anti-MT drugs, cells display great variation of behaviors (Gascoigne, Taylor, 2008). So we addressed the question on a relationship between the mitotic arrest and cell viability. Three anti-MT drugs (nocodazole, taxol and vinorelbine) were used in a study performed on normal (3T3 fibroblasts and HaCaT keratinocytes) and cancer (A549, HT1080, U118) cells. We followed the behavior of mitotic cells in prolonged time-lapse observations using high-throughput microscopy. The threshold concentration for mitotic arrest by each drug (nocodazole, taxol, vinorelbine) was following: for 3T3 - 3nM, 3nM and 0.3-1 nM; for HaCaT - 3nM, 0.3nM, 0.1nM; for HT1080 - 30nM, 3nM, 3nM; for A549 - 1nM, 30nM, 0.3nM; and for U-118 - 3nM, 10nM, 1nM, respectively. Cells can escape death by exiting mitotic stage and going into interphase without chromosome separation. This type of behavior was prevailing in our experiments at concentrations well above the threshold level and some cells arrested in mitotic stage for 30-48 hours were able to re-enter interphase. The frequency of mitotic slippage strongly depended on the cell type and poorly correlated with drug concentration. Successful escape into interphase was rarely followed by apoptosis. To analyze overall viability under the action of anti-MT drugs, we measured cell growth by MTT test and set a threshold concentration when growth was inhibited at least two times within 72 h. For 3T3 the cell growth inhibition was achieved at the following concentrations (in order of nocodazole, taxol, vinorelbine) 300nM, 100-300nM, 100nM; for HaCaT – 100nM, 10nM, 10nM; for A549 – <100nM, 30nM-100nM, 30nM-100nM; for HT1080 – 100nM, 10nM, 3nM; for U118 – >1000nM, 30nM, 1nM. Since the growth inhibition doses were higher than those inducing mitotic arrest, we calculated a ratio between two concentrations. These ratios for each drug and tissue culture were following (in order of nocodazole, taxol, vinorelbine): 3T3 - 100, 30-100, 100-300; HaCaT - 30, 30, 3; A549 - >100, 1-3, 100-300; HT1080 - 3, 3, 1; U-118 - >300, 3, 1. It was clear that the mitotic arrest threshold dose was not equal to the effective cell growth inhibition dose, with an exception of vinorelbine for HT1080 and U118 and taxol for A549 cells. Taking into account the high ratio differences, we conclude that mitotic slippage is the major outcome for the action of MT inhibitors at minimal effective concentrations and cannot predict behavior of tumor cells.