SyB-0702

Heat shock protein 32 (Hsp32), also known as heme oxygenase 1 (HO-1), has recently been identified as a potential target in various hematologic malignancies. We provide evidence that Hsp32 is constitutively expressed in primary leukemic cells in patients with acute myeloid leukemia (AML) and in various AML cell lines (HL60, U937, KG1). Expression of Hsp32 mRNA was demonstrable by qPCR, and expression of the Hsp32 protein by immunocytochemistry and Western blotting. The stem cell-enriched CD34+/CD38+ and CD34+/CD38- fractions of AML cells were found to express Hsp32 mRNA in excess over normal CD34+ progenitor cells. Two Hsp32-targeting drugs, pegylated zinc-protoporphyrin (PEG-ZnPP) and styrene-maleic-acid-copolymer-micelle-encapsulated ZnPP (SMAZnPP), were found to inhibit cytokine-dependent and spontaneous proliferation in all 3 AML cell lines as well as in primary AML cells. Growth inhibitory effects of SMA-ZnPP and PEG-ZnPP were dose-dependent with IC50 values ranging between 1 and 20 μM, and were accompanied by apoptosis as evidenced by light- and electron microscopy, Tunel assay, and caspase-3 activation. Finally, we were able to demonstrate that SMA-ZnPP inhibits cytokine-dependent proliferation of CD34+/CD38+ and CD34+/CD38- AML progenitor cells in vitro in all patients as well as leukemiainitiation of AML stem cells in NOD-SCID IL-2Rγ(-/-) (NSG) mice in vivo. Together, our data suggest that Hsp32 plays an important role as a survival factor in leukemic stem cells and as a potential new target in AML.

SMA-ZnPP and PEG-ZnPP are micellar drugs, encapsulating zinc protoporphyrin IX (ZnPP) with styrene maleic acid copolymer (SMA) and covalent conjugate of ZnPP with polyethylene glycol (PEG) respectively. Their intracellular uptake rate and subcellular localization were investigated. We found SMA-ZnPP showed higher and more efficient (about 2.5 times) intracellular uptake rate than PEG-ZnPP, although both SMA-ZnPP and PEG-ZnPP micelles were localized at endoplasmic reticulum (ER) and inhibited the target enzyme heme oxygenase 1 (HO-1) similarly. Both micellar ZnPP were taken up into the tumor cells by endocytosis. Furthermore SMA-ZnPP and PEG-ZnPP were examined for their drug releasing mechanisms. Liberation of ZnPP from the SMA micelle appears to depend on cellular amphiphilic components such as lecithin, while that for PEG-ZnPP depends on hydrolytic cleavage. These results indicate that these micelle formulations make water insoluble ZnPP to water soluble practical anticancer agents.