The expression of claudins (CLDNs), major components of tight junctions (TJs), is abnormal in various solid tumors. CLDN14 is highly expressed in human colorectal cancer (CRC) tissues and confers chemoresistance. CLDN14 may become a novel therapeutic target for CRC, but CLDN14‐targeting drugs have not been developed. Here, we searched for a CLDN14‐targeting peptide, which can suppress CLDN14 expression and chemoresistance using human CRC‐derived DLD‐1 and LoVo cells. Among some short peptides which mimic the second extracellular loop structure of CLDN14, PSGMK most strongly suppressed the protein expression of CLDN14. The mRNA expression of other endogenous TJ components was unchanged by PSGMK. The PSGMK‐induced reduction of CLDN14 protein was inhibited by chloroquine, a lysosome inhibitor, and monodansylcadaverine, a clathrin‐dependent endocytosis inhibitor, indicating that PSGMK may enhance endocytosis and lysosomal degradation of CLDN14. In a three‐dimensional culture model, the oxidative stress was significantly reduced by PSGMK, whereas hypoxia stress was not. Furthermore, the expression levels of nuclear factor erythroid 2‐related factor 2, an oxidative stress response factor, and its target genes were decreased by PSGMK. These results suggest that PSGMK relieves stress conditions in spheroids. The cell viability of spheroids was decreased by anticancer drugs such as doxorubicin and oxaliplatin, which was exaggerated by the cotreatment with PSGMK. Our data indicate that CLDN14‐targeting peptide, PSGMK has an anti‐chemoresistance effect in CRC cells.
