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Background: 5-Fluorouracil (5-FU) has been used as a standard chemotherapeutic agent for colorectal cancer (CRC). However, chemo-resistant cancer cells can survive under chemotherapeutic regimens, resulting in cancer recurrence. Thus, a novel approach is required to overcome chemo-resistance in CRC. Objective: Recently, oleuropein (OLP), an extract derived from olive leaves, has shown anticancer effects and induces apoptosis in cancer cells; however, the role of OLP in 5-FU-resistant cells remains to be elucidated. Therefore, we have assessed the anticancer effect of OLP on tumor spheres and 5-FU-resistant cells, but also synergistic effect in combination of OLP and 5-FU. Results: Our findings revealed that OLP had a marked suppressive effect on tumor sphere formation capacity. Annexin V-PI staining data showed that OLP-induced apoptosis occurred in a dose-dependent manner. Mitochondrial fragmentation and mitochondrial superoxide production were observed following treatment with OLP. OLP treatment drastically suppressed the metastatic potential, including cell migration ability and anchorage-independent growth capability. In addition, combination treatment with 5-FU and OLP revealed a synergistic effect on cell viability in DLD-1 cells and 5-FU-resistant cells. Conclusion: These findings showed the suppressive effects of OLP against the colorectal tumor spheres and 5-FU-resistant cells. In addition, these results offer the new insights into novel therapeutic strategies for combining 5-FU and OLP in patients with chemo-resistant CRC. [ABSTRACT FROM AUTHOR]