Identification Of Target Protein For Mechanistic Studies Of Anti-leukemia Activity Of Homoharringtonine

BackgroudLeukemia is a common malignant neoplasm of hematopoietic system. However, its pathogenesis and molecular mechanism of development is still not clear. In recent years, combination chemotherapy is one of most frequently means used to treat leukemia in clinical practice.Homoharringtonine (HHT) is a natural alkaloid that extracted from Cephalotaxus fortunei. Its antitumor activity was first reported by Chinese investigators in the 1970s. It has been proved that this agent used to be considered the most effective treatment for acute myelogenous leukemia. In addition, a series of clinical trials confirmed the therapeutic value of HHT for patients with chronic myeloid Leukemia (CML) after failure on imatinib therapy, suggesting homoharringtonine could be used as salvage therapy for patients who have imatinib-resistant CML. So far, little is known about the molecular mechanism of its action. Preclinical studies demonstrate that HHT exerts its antitumor activity via inhibition of protein synthesis and induction of cell apoptosis. However, these studies could not answer the question that what is the direct target protein of HHT.In order to further determine the specific mechanism of the drug, we identified the direct target proteins of HHT by preparing the affinity column in this study. We also established human leukemia cells stably expressing high levels of the target protein and employed a series of technologies, including gene-chip array, real-time PCR and immune, to explore possible mechanisms of HHT in leukemia.Materials and Methods1. Cell culture:(1) Human leukemia cell lines:acute monocytic leukemia cell lines, THP1 and U937; acute myelogenous leukemia cell line, Kasumi-1; chronic myelogenous leukemia cell line, K562;(2) Plat-GP cells:a 293T-derived murine-leukemia-virus-based packaging cell line.2. Cell proliferation assay with MTS assay.3. Cell cycles assay of leukemia cell line with Flow cytometry.4. Apoptosis assay of leukemia cell line with Flow cytometry and PI/Annexin-V assay.5. Prepare the affinity column of HHT with Streptavidin agarose resins and biotin and identify by LC-MS mass.6. Protein elution and identification:wash the affinity column with PBS, NaCl and HHT solutions respectively; dialyze and dry the target proteins; stain the protein bands on SDS-PAGE; excise and digest the protein bands; identify the target proteins through protein Mass spectrum.7. Establishment of cells stably over-expressing the target protein:cells were transduced by infection with retrovirus-containing supernatants of transfected Plat-GP cells and selected with puromycin in maintenance medium.8. Gene expression assay in leukemia cell line stably over-expressing the target protein with Affymetrix GeneChip(?) Human Genome U133 Plus 2.0 Array.9. Assay of mRNA expression in leukemia cells after treatment with HHT by real-time PCR.10. Protein expression assay in leukemia cells after treatment with HHT by Western blot.Results1. Homoharringtonine inhibited significantly the proliferation of acute myelogenous leukemia cell lines, THP1, U937 and Kasumi-1, and chronic myelogenous leukemia cell line, K562. The values of IC50 were 22.37 ng/ml,11.09 ng/ml,14.93 ng/ml and 37.38 ng/ml at 48 hours respectively.2. Homoharringtonine induced cell apoptosis. The percentage of apoptosis cells after treatment with HHT increased obviously.3. Actin-binding protein, Non muscle myosin heavy chain IIA, is the direct target protein of HHT by preparing HHT affinity column. HHT also could up-regulate the protein.4. Results of microarray showed that over-expressing NMHC IIA could influence expression levels of signal molecules in cell cycle and MAPK pathways in leukemia cells.5. Western Blot and RT-PCR confirmed the results of microarray. Either treatment with HHT or high expression of NMHC IIA could up-regulate the expression level of proteins in cell-cycle and MAPK pathways, such as c-jun and JNK.Conclusion1. Homoharringtonine could inhibit the proliferation of leukemia cell lines and induce apoptosis of these cells.2. Homoharringtonine directly binds and up-regulates the actin-binding protein, non muscle myosin heavy chain IIA.3. Homoharringtonine affects expression levels of cell cycle-related proteins via up-regulation of NMHC IIA, promoting Glâ†'S cell phage transiton.4. Activation and up-regulation of JNK/c-jun signaling pathway via up-regulating NMHC IIA might be one of mechanisms for Homoharringtonine-induced apoptosis.