Abstract: Colon adenocarcinoma is one of the most common cancers worldwide, and resistance to current therapeutic modalities is a serious drawback in its treatment. Auraptene is a natural coumarin with considerable anticancer effects. The goal of this study was to introduce a novel combinatorial approach for treatment against colon adenocarcinoma cells. To do so, HT29 cells were pretreated with nontoxic auraptene and then hyperthermia was induced. Afterwards, the viability of the cells was assessed, changes induced in the cell cycle were analyzed, and the expression patterns of candidate genes were studied. Results from the MTT assay demonstrated significant (p < 0.01) decreases in cell viability when 20 g/mL auraptene was used for 72 h, heat shock was induced, and cells were allowed to recover for 24 h. Flow cytometry analysis also indicated considerable changes in the distribution of cells between the sub-G1/G1 and G/M phases of cell cycle after the combinatorial treatment. Real-time RT–PCR studies revealed significant (p < 0.01) up-regulation of P21 in the cells pretreated with auraptene after heat shock, whereas no significant change was observed in HSP27 expression. Our findings not only indicate, for the first time, that the efficacy of hyperthermia was improved by auraptene pretreatment, but also suggest that this coumarin could be used in the future to achieve more effective therapeutic outcomes

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