Synthesis and evaluation of KR-12, an LL-37 fragment, and its short-chain fatty acid derivatives: selective cytotoxicity in colorectal cancer cells and anti-tumor efficacy in an azoxymethane/DSS-induced colitis-associated cancer mouse model.

[BACKGROUND] Colorectal cancer (CRC) remains a major global health challenge, with increasing incidence, particularly among individuals under 50 years of age. Cathelicidin LL-37, a multifunctional antimicrobial peptide, has shown promise in cancer treatment, particularly for its anti-inflammatory effects.

[METHODS] Using in vitro and in vivo models, we investigated the anticancer potential of KR-12 amide, the shortest active fragment of LL-37, and its short-chain fatty acid (SCFA)-modified derivatives (acetyl-, propionyl-, and butyryl-KR-12-NH). Peptides were synthesized by Fmoc solid-phase synthesis and purified by RP-HPLC. Their cytotoxicity was assessed in colon cancer HT-29 and normal colon epithelial CCD 841 CoN cell lines using MTT viability assays. In vivo efficacy was evaluated in a mouse (male Balb/C mice) azoxymethane/dextran sodium sulfate (AOM/DSS) model of colitis-associated colorectal cancer (CACRC). Tumor burden was quantified by macroscopic and histological scoring, while inflammation was assessed through myeloperoxidase activity, ELISA-based cytokine profiling (IL-1β, IL-6, TNF-α), and microscopic evaluation of colon architecture.

[RESULTS] For all tested compounds, except KR-12-NH modified with butyric acid, the concentrations needed for 50% growth inhibition were lower for colon cancer cell line HT-29 than for healthy colon epithelial cells CCD 841 CoN. The IC₅₀ values for KR-12 amide and propionyl-KR-12-NH against HT-29 cells were 236.7 µM and 309.0 µM, respectively, compared with 347.3 µM and 422.1 µM for CCD 841 CoN cells. In the AOM/DSS-induced murine model, rectal administration of KR-12-NH and propionyl-KR-12-NH significantly reduced total tumor number compared with AOM/DSS-only animals (p = 0.02 and p = 0.03, respectively), accompanied by lower macroscopic (both p < 0.001) and microscopic disease scores (p = 0.005 and p = 0.01). Both compounds also significantly decreased proinflammatory cytokines: rectal KR-12-NH lowered IL-6 levels (p = 0.05), while rectal propionyl-KR-12-NH reduced IL-6 (p = 0.02) and TNF-α (p = 0.01).

[CONCLUSIONS] These findings provide a foundation for further investigation of cathelicidin derivatives in colorectal cancer therapy.