Homoharringtonine affects the JAK2-STAT5 signal pathway through alteration of protein tyrosine kinase phosphorylation in acute myeloid leukemia cells.
OBJECTIVE
Homoharringtonine (HHT) was efficient in therapying patients with acute myeloid leukemia (AML) in China, but little is known about the mechanism of its action. As the abnormal activation of JAK2 associated pathway is important to AML, we try to explore the effect of HHT on JAK2-STAT pathway in AML cells, thus supplying theoretical basis for wider use of HHT.


METHODS
The cell viability was tested by MTT. Apoptosis was tested by flow cytometry. RT-PCR was used to measure the expression of JAK2, STAT5 and the effect gene Bcl-xL. The signal proteins such as p-JAK2, p-STAT5, p-AKT, p-ERK activated by abnormal activated JAK2 were tested by Western blotting.


RESULTS
HHT obviously inhibited the viability of primary AML cells and AML cell lines HEL, K562 and HL-60 cells, AnnexinV-PI double staining confirmed early apoptosis in a dose-dependent manner. In immunoblotting analysis, when AML cells were affected by HHT for 6 h (much ahead of the time when apoptosis could be induced). The expressions of p-JAK2, p-STAT5, and p-AKT were down-regulated, while the total JAK2, STAT5 and AKT protein levels were stable. There were no changes in p-ERK and BcL-xL proteins. When it prolonged to 24 h, Bcl-xL decreased obviously. Similar results were obtained by using JAK2 specific inhibitor AG490.


CONCLUSIONS
HHT possibly acts as a broad-spectrum PTK inhibitor and inhibits the phosphorylation of the signal proteins caused by oncogenic proteins such as JAK2V617F, BCR/ABL, thus blocking the survival and proliferative signal pathway of malignant cells.
Tong
Hongyan
H
Department of Hematology, First Affiliated Hospital, College of Medicine, Zhejiang University, Zhejiang, China. hongyantong@yahoo.com.cn
Ren
Yanling
Y
Zhang
Fengjuan
F
Jin
Jie
J
D019943:Amino Acid Substitution; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology* / Q000627:therapeutic use; D017209:Apoptosis / Q000187:drug effects; D002470:Cell Survival; D004305:Dose-Response Relationship, Drug; D004354:Drug Screening Assays, Antitumor; D016044:Fusion Proteins, bcr-abl; D018922:HL-60 Cells; D006248:Harringtonines / Q000494:pharmacology* / Q000627:therapeutic use; D000077863:Homoharringtonine; D006801:Humans; D053614:Janus Kinase 2 / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D020014:K562 Cells; D015470:Leukemia, Myeloid, Acute / Q000188:drug therapy* / Q000201:enzymology / Q000235:genetics; D020125:Mutation, Missense; D051058:Oncogene Protein v-akt / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology* / Q000627:therapeutic use; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D050799:STAT5 Transcription Factor / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects*; D020032:Tyrphostins / Q000494:pharmacology; D051020:bcl-X Protein / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism
D000972:Antineoplastic Agents, Phytogenic; C494811:BCL2L1 protein, human; D006248:Harringtonines; D047428:Protein Kinase Inhibitors; D050799:STAT5 Transcription Factor; D020032:Tyrphostins; C095512:alpha-cyano-(3,4-dihydroxy)-N-benzylcinnamide; D051020:bcl-X Protein; D000077863:Homoharringtonine; D011505:Protein-Tyrosine Kinases; D016044:Fusion Proteins, bcr-abl; C507924:JAK2 protein, human; D053614:Janus Kinase 2; D051058:Oncogene Protein v-akt