Rab7b suppresses autophagy and induces apoptosis of K562 leukemic cells treated with homoharringtonine

Abstract 4884

Homoharringtonine is an effective anti-leukemia medicine developed by Chinese. It has been found to induce differentiation and apoptosis of leukemia cells. However, the mechanism of its anti-leukemia function has not been fully understood. Rab is a kind of G protein of Ras superfamily and participates in endocytosis and exocytosis of protein transport process. In recent studies, some Rab proteins such as Rab7 are found to be associated with cellular autophagy and apoptosis. We previously identified a new small GTPase homologous to Rab7, named Rab7b, which is selectively expressed in promyeloid and monocytic cells and is localized to lysosome-associated compartments.

To investigate the roles of Rab7b in acute myeloid leukemia, we used leukemia cell line K562 as a model in the present study. After treatment of K562 cells with various doses of HHT, cell viability and apoptosis were measured by MTT assay and Annexin V/PI staining respectively. Protein expression of LC3, a marker of autophagy, and caspase3, 9, ERK1/2,Akt were determined by Western blot analysis. Using stable gene transfection, several Rab7b variants, including Rab7b wild-type, active mutant Rab7b-Q67L and localization-deficient mutant Rab7b-ΔCC as well as Rab7b RNAi were transfected in to K562 cells and their roles in regulation of apoptosis in K562 leukemia cells induced by HHT were further evaluated.

Our data showed that the viability of the K562 cells was greatly reduced by HHT treatment in a dose- and time- dependant manner. Treatment of the K562 with HHT significantly increases apoptosis in the cells as measured by Annexin V/PI staining. Using Western blot analysis, we further determined that the expression of caspase3, 9 was increased, and ERK1/2 augmented with Akt was suppressed in the cells treated with HHT. After suppressing autophagy with 3-MA, apoptosis was enhanced in the K562 cells treated with HHT. (p<0.05). By constitutively expression of Rab7B and variants in K562 cells, we found that the rate of apoptotic cells are much higher in the K562 cells transfected with Rab7b wild-type and Rab7b-Q67L variants, along with increased expression of caspase3, 9, ERK1/2 and decreased expression of Akt in the transfectants with Rab7b wild-type and active mutant Rab7b-Q67L.

Our study suggests that HHT is able to suppress autophagy and enhance apoptosis in K562 leukemia cells in a caspase-dependent way, which is associated with suppression of Akt phosphorylation and upregulation of ERK1/2. Over-expression of Rab7b can enhance HHT induced apoptosis in K562 cells, which may also be associated with suppression of Akt phosphorylation and upregulation of ERK1/2. Taken together, our study elucidates a new recognition for the mechanism of HHT in anti-leukemia therapy and provides a new insight into understanding the relationship between autophagy and apoptosis in leukemia cells induced chemotherapy.