Nemorosone blocks proliferation and induces apoptosis in leukemia cells.
OBJECTIVE
This work is aimed at characterizing nemorosone, isolated from Clusia rosea, as a potential antileukemic agent. In addition, we analyzed its influence on hematopoiesis in a mouse model.


METHODS
The isolation of nemorosone was carried out employing the RP-HPLC (reversed phase high-performance liquid chromatography) technique. Cytotoxicity was assessed in human leukemia cell lines including parental and chemotherapy-refractory sublines based on the MTT compound. Its effects on the cell cycle were analyzed using FACS (fluorescence-activated cell sorting) and Western blot techniques. Studies on the drug-induced early apoptotic process were carried out by means of fluorescence microscopy. Major signal transducers and the enzymatic inhibition of immunoprecipitated Akt/PKB were detected by Western blot. Hematopoiesis was analyzed in NMRI nu/nu mice after chronic nemorosone treatment, measuring hematological parameters by conventional laboratory techniques.


RESULTS
Nemorosone proved cytotoxic in both parental and chemoresistant leukemia cell lines with IC50 values between 2.10 and 3.10 mg/ml. No cross-resistances could be detected. Cell cycle studies showed apoptosis induction accompanied by an increase in the G0/G1 population in both cell lines studied, whereas a significant decrease in the S-phase was found in Jurkat cells. Nemorosone induced a down-regulation of cyclins A, B1, D1, and E as well as a dephosphorylation of cdc2. Major signal transduction elements such as ERK1/2 and p38 MAPK, as well as important oncoproteins such as c-Myb and BCR/ABL were also found down-regulated. The enzymatic activity of immunoprecipitated Akt/PKB was substantially inhibited in vitro. Moreover, subchronic nemorosone treatment induced reversible monocytosis and thrombocytosis in the mouse model examined.


CONCLUSIONS
Here, we demonstrate for the first time that nemorosone exerts cytotoxicity in leukemia cells, partly by targeting the Akt/PKB signal transducer, affecting protein levels and cell cycle progression. Finally, in vivo studies suggest that nemorosone significantly affects hematopoiesis in mice.
Díaz-Carballo
D
D
Abteilung für Klinische Pharmakologie, Ruhr-Universität Bochum, Germany. david.diaz-carballo@rub.de
Malak
S
S
Freistühler
M
M
Elmaagacli
A
A
Bardenheuer
W
W
Reusch
H P
HP
D000818:Animals; D000972:Antineoplastic Agents, Phytogenic / Q000008:administration & dosage / Q000302:isolation & purification / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D001577:Benzophenones / Q000008:administration & dosage / Q000302:isolation & purification / Q000494:pharmacology*; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D031251:Clusia / Q000737:chemistry*; D016503:Drug Delivery Systems; D019008:Drug Resistance, Neoplasm; D005260:Female; D006410:Hematopoiesis / Q000187:drug effects; D006801:Humans; D020128:Inhibitory Concentration 50; D007938:Leukemia / Q000188:drug therapy* / Q000473:pathology; D051379:Mice; D008819:Mice, Nude; D051057:Proto-Oncogene Proteins c-akt / Q000187:drug effects / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects
D000972:Antineoplastic Agents, Phytogenic; D001577:Benzophenones; C437463:nemorosone; D051057:Proto-Oncogene Proteins c-akt