Mechanistic Profiling of Phenethyl Isothiocyanate in CCRF-CEM Acute Lymphoblastic Leukemia Cells

Background/Objectives: Phenethyl isothiocyanate (PEITC), a bioactive compound found in cruciferous vegetables, exhibits potent anti-cancer activity, but its molecular mechanisms in acute lymphoblastic leukemia (ALL) remain poorly defined. This study aimed to investigate the anti-leukemic effects of PEITC. Methods: PEITC cytotoxicity was assessed in human cancer cell lines using resazurin reduction assays. Microarray and Ingenuity Pathway Analysis (IPA) were used to investigate altered molecular signaling pathways. Functional assays were performed to assess DNA damage, cell-cycle progression, apoptosis, autophagy, mitochondrial membrane potential, microtubule organization and endothelial tube formation. Western blotting was performed to examine pathway-related proteins, and preliminary in vivo antitumor activity was evaluated using a CCRF-CEM zebrafish xenograft model. Results: PEITC exerted potent cytotoxicity against CCRF-CEM cells (IC₅₀ = 1.76 ± 0.07 µM). Microarray and IPA analyses predicted inhibition of PI3K/AKT/mTOR signaling, which was further supported by Western blot analysis. Functional assays demonstrated that PEITC induced DNA damage, cell cycle perturbation, mitochondrial dysfunction, and apoptotic and autophagic responses. PEITC also disrupted microtubule organization in GFP-tagged α-tubulin-expressing U2OS cells. Furthermore, PEITC suppressed endothelial tube formation in EA.hy926 cell lines and exhibited preliminary in vivo antitumor activity in a zebrafish xenograft model (n = 10). Conclusions: These findings provide mechanistic insights into the anti-leukemic activity of PEITC and demonstrate its effects on multiple cellular processes associated with leukemia cell survival and proliferation, supporting further investigation of its therapeutic potential in ALL.