Homoharringtonine Induced Apoptosis of Leukemic Cell Line MUTZ-1 Cells Is Mediated by the Endoplasmic Reticulum Stress Pathway.

The endoplasmic reticulum (ER) is the major organelle for protein synthesis and maturation as well as the regulation of intracellular calcium (Ca²⁺) homeostasis. The accumulation of unfolded proteins in the ER lumen or depletion of Ca²⁺ from the ER lumen leads to the ER stress response known as unfold protein response. This response enhances the ability of protein folding by inducing ER resident chaperones such as glucose-regulated stress protein 78 or immunoglobulin heavy-chain binding protein (GRP78/BIP) mRNA and protects the cell from ER stress by splicing of X-box binding protein 1 (XBP-1) mRNA, and synthesis of phosphatidylcholine, the primary phospholipid of the ER membrane. However, when the ER function is severely impaired, the organelle elicits apoptotic signals. One of the components of the ER stress-mediated apoptosis pathway is C/EBP homologous protein (CHOP), also known as growth arrest- and DNA damage-inducible gene 153. Homoharringtonine (HHT), one of the alkaloids from a Chinese natural plant, Cephalotaxus, has currently been used successfully in the treatment of acute and chronic myeloid leukemias and has been shown to induce apoptosis of different types of leukemic cells in vitro. But the mechanism of how HHT causes apoptosis remains unknown. In this study, we investigated the relationship between ER stress and apoptosis of MUTZ-1 cells (cell line of MDS) treated with HHT. HHT induced growth inhibition of MUTZ-1 cells was measured by MTT method. Apoptosis was detected by FACS and observed by light and electron microscopy. Concentration of cystosolic calcium was measured with the spectrofluorometer. The mRNA expression level of CHOP, BIP and XBP1 was detected by semi-quantitative RT-PCR. We found that after exposure of MUTZ-1 to HHT (rang 0.02~0.4ug/ml) for 24 to 72 hours, the IC₅₀ and the percentage of apoptotic MUTZ-1 cells increased in a time and dose dependent manner. Typical apoptotic cells and release of Ca²⁺ from the cytosolic Ca²⁺ storage were observed. RT-PCR analysis revealed that mRNAs for ER stress-associated proapoptotic factor CHOP and an ER chaperone BiP, XBP1 are markedly increased at 4 hours after 0.05ug/ml HHT treated and reach the top level at 12hours, then decreased to the level before treatment at 24 hours. Induction of ER stress-responsive proteins (BIP and CHOP) was also observed at 12 hours. These results indicate that HHT could not only inhibit MUTZ-1 growth but also induce MUTZ-1 apoptosis and HHT-induced MUTZ-1 cell death is likely mediated by the ER stress pathway.