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Further mechanistic studies revealed that penfluridol influenced cell apoptosis and glycolysis in CRC cells by down-regulating hexokinase-2 (HK-2). The pro-apoptotic effect and glycolytic inhibition-induced by penfluridol were effectively reversed by HK-2 overexpression. Consistent with in vitro results, penfluridol could also suppress tumor growth and trigger apoptosis in vivo. Conclusion Penfluridol triggers mitochondrial-mediated apoptosis and induces glycolysis inhibition via modulating HK-2 in CRC and provides a theoretical basis t