CA4P decreases expression of VCAM-1 in vivo and on HUVECs, and reduces AML adhesion and survival. (A) CA4P decreases VCAM-1 expression in vivo. NOD-SCID mice with subcutaneous HL60 AML tumors were treated with CA4P or PBS (untreated control). Immunofluorescence for VCAM-1 (red staining) shows significantly decreased VCAM-1 expression in CA4P-treated tumors. (B) CA4P prevents up-regulation of VCAM-1 in HUVECs. VCAM-1 expression in IL-1–activated HUVECs, with or without addition of CA4P, was measured by flow cytometry. (C,D) CA4P reduces leukemic cell adhesion to HUVECs. Adhesion of GFP⁺ U937 (C) and HL60 (D) leukemic cells to IL-1–activated HUVECs treated with various concentrations of CA4P was measured. The number of adherent cells is expressed as a percentage of total leukemic cells and representative of 3 independent experiments performed in triplicate plus or minus SEM (*P < .05 compared with CA4P-untreated control). (E,F) Leukemic cells adherent to HUVECs are more resistant to CA4P. Survival of GFP⁺ U937 (E) and HL60 (F) leukemic cells cocultured with HUVECs is expressed as a percentage of total cells. Compared are adherent (ie, attached to IL-1β–activated HUVECs) versus nonadherent leukemic cells (ie, cultured with nonactivated HUVECs). Results are representative of 3 independent experiments performed in triplicate plus or minus SEM (*P < .05). (G) Proposed mechanisms for antileukemic activity of CA4P. Right panel: CA4P modulates leukemia-vascular stroma interactions. Leukemic cells adhere to vascular stromal cells expressing VCAM-1, creating a chemoprotected niche. CA4P down-regulates VCAM-1 on vascular stromal cells, leading to decreased adhesion of leukemic cells and increased chemosensitivity. Left panel: CA4P causes leukemic cell death by targeting mitochondria. CA4P causes cell death by disrupting mitochondrial function, leading to accumulation of ROS, which in turn enhance mitochondrial damage and cause cellular damage via free radicals. Proapoptotic mitochondrial membrane proteins are released from the damaged mitochondria, contributing to cell death.