Mechanistic investigation of Juglone (5-hydroxy-1,4-naphthoquinone) as an anti-cancer agent in human colorectal cancer HCT116 and HT-29 cell lines.

Colorectal cancer (CRC) is one of the most prevalent causes of cancer-associated deaths globally, largely driven by non-responsiveness to drugs and metastatic progression. Natural product-derived multi-targeted anticancer agents have emerged as a promising strategy to treat or relieve various cancer conditions, including CRC. To investigate the therapeutic potential of natural bioactive compounds against colorectal cancer, we examined the effects of Juglone (5-hydroxy-1,4-naphthoquinone, [Formula: see text]), a naturally occurring constituent of the Juglandaceae family, on the human colorectal cancer cell lines HCT116 and HT-29. Flow cytometric analysis demonstrated that Juglone markedly increased early and late apoptotic cell populations, with minimal necrosis. This apoptotic effect was further confirmed by enhanced activation of caspase-3/7, -8, and - 9, indicating the involvement of both intrinsic and extrinsic apoptotic pathways. At the transcriptional level, Juglone upregulated pro-apoptotic genes (BAX, CASP3, CASP8, CASP9) while downregulating the anti-apoptotic gene BCL2, supporting a shift toward programmed cell death. Cell cycle analysis revealed significant G0/G1 phase arrest in both CRC cell lines, accompanied by suppression of CCND1, CCNB1, CDK2, and CDK4 and upregulation of CDKN1A (p21) and CDKN1B (p27). Juglone modulated key oncogenic signaling pathways by downregulating ERK1/2 and AKT1 while upregulating p38 and JNK1. Functionally, Juglone significantly suppressed cancer cell migration and invasion, indicating anti-metastatic potential. Collectively, these findings demonstrate that Juglone exerts potent anticancer effects by targeting multiple processes involved in colorectal cancer progression, including proliferation, apoptosis, cell cycle regulation, survival signaling, and metastasis. These findings demonstrated that Juglone markedly inhibited cell viability in a concentration- and time-dependent manner, exhibiting potent cytotoxic activity with IC₅₀ values in the low micromolar range after 48 h of treatment. Therefore, Juglone may represent a promising multi-targeted therapeutic candidate for colorectal cancer, warranting further in vivo evaluation.