Modulating claudin-2 with CRISPR-Cas9 to improve photodynamic therapy outcomes in colorectal cancer

Claudin-2 (CLDN2) is a tight junction protein that is overexpressed in colorectal cancer (CRC) and is associated with chemoresistance. Photodynamic therapy (PDT) is an emerging treatment that utilizes a photosensitizer (in this case, chlorin e6 [Ce6]) and light to generate cytotoxic reactive oxygen species (ROS). This paper investigated the influence of the combination of Ce6-PDT and CRISPR-Cas9-mediated CLDN2 knockout (KO) on the relative metabolic activity of the CRC cell line. CRISPR-Cas9 was used to produce HCT116 cells with CLDN2 KO. Ce6 was placed on the cells, and the red laser (659 nm, 6 J/cm²) was used to illuminate the cells. The relative metabolic activity, migration, Apoptosis, cell cycle, and ROS generation, gene expression, protein expression were measured by MTT assay, wound healing assay, flow cytometry, DCFH-DA method, RT-PCR, western blot and bioinformatics, respectively. PDT significantly decreased the relative metabolic activity and/or migration, more in CLDN2KO cells (p < 0.0001) than in the WT. The CLDN2KO cells had a high level of Apoptosis (46.56 ± 2.05%), compared to the WT (26.03 ± 6.72%), the p = 0.0072. The production of ROS was also increased to 779.51 % in CLDN2KO cells, which is higher than the production in WT cells at 767.10 %. Upregulation of P53 and BAX following PDT was greatly enhanced, and BCL2 expression was significantly reduced as compared to wild-type groups. Coexistence of Ce6-PDT with CLDN2 KO enhances the Apoptosis of the CRC cell line. The Western blot results showed a decrease in ZO-1 and Occludin proteins after Ce6-PDT. Bioinformatics analysis demonstrated that increased CLDN2 expression in CRC, associated with multiple genes and implicated in various cellular pathways. Although the initial results are encouraging regarding the possibility of synergy, further studies are needed to determine its effectiveness and safety in clinical practice. The results of this research suggest the possibility of a therapeutic approach aimed at enhancing the effectiveness of CRC treatment through genetic regulation with the aid of PDT.