<p>Here, we report that long-term exposure of PANC-1 human pancreatic cancer cells to indirubin derivatives (Epox/5-Br-Ind) resulted in increased sensitivity to anticancer drugs such as 5-fluorouracil (5-FU) and gemcitabine (Gem). Epox/5-Br-Ind is an indirubin derivative bearing an oxirane moiety that is known to potently and continuously inhibit the proliferation of HepG2 liver cancer cells. We evaluated the effects of Epox/5-Br-Ind on PANC-1 proliferation from the perspectives of acute and chronic toxicity: acute toxicity was assessed using resazurin-based assays, while chronic toxicity was evaluated by comparing cumulative population doubling levels (CPDLs). Our results showed that Epox/5-Br-Ind compounds exhibited acute toxicity (IC₅₀ value = 1.8 μM) against PANC-1, but the concentration required to arrest cell proliferation over long-term exposure was only 0.1 μM. Flow cytometric analyses further revealed that chronic exposure to 0.1 μM Epox/5-Br-Ind induced a pronounced accumulation of cells in the S/G2/M phases and a clear increase in Annexin V–positive populations, indicating that sustained growth inhibition was accompanied by cell-cycle arrest and apoptosis induction. Results of docking simulations suggested that Epox/5-Br-Ind, like other indirubin derivatives, may inhibit the activity of common cyclin-dependent kinases (CDKs). Further analysis of gene expression profiles revealed time-dependent upregulation of Caspase-3 and -7 following exposure to Epox/5-Br-Ind, thereby paralleling CDK inhibition. However, we also observed concurrent upregulation of CDK2, suggesting a compensatory response to CDK4/6 inhibition. PANC-1 cells exposed to 0.1 μM Epox/5-Br-Ind for a prolonged period showed IC₅₀ values of 1.2 μM and 2.0 μM for 5-FU and Gem, respectively; these values were approximately 1/10 of the control group. Overall, our findings suggest that Epox/5-Br-Ind is a promising chemosensitizer and therapeutic candidate for combination treatment strategies to treat pancreatic cancer.</p><p></p>