The Function Of Rab7b In The Mechanism Of Homoharringtonine Induced Death Of K562 Leukemic Cells And Its Role In Autophagy

Homoharringtonine is an effective antileukemia medicine developed by Chinese. The mechanism of its function are unclarified. Rab is a kind of G protein of Ras superfamily, it participate in endocytosis and exocytosis of protein transport process.In recent years,Some Rab protein such as Rab7 is find 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 or monocytic cells and is localized to lysosome-associated compartments.In our research we measured the expression of,the marker of autophagy and apoptosis pathway, trying to find the mechanism.After suppressing autophagy,we measure the modification of apoptosis.Then we transfected K562 with Rab7b and its variants and investigated their difference of apoptosis induced by HHT. We also observed the subcellar localization of Rab7b and its variants when induced by starvation and the change of localization when inhibited by varity of inhibitor of autophagy pathway.First we investigated the vitality of K562 cell when induced by HHT using MMT and the apoptosis by Annexin V/PI, it showed that apoptosis was induced by HHT,we also studied the expression of caspase3,9 and ERK1/2, Akt by Western blot, we found the expression of caspase3,9 increased, the signal pathway ERK1/2 augmented with Akt suppressed. Then,we stably transfected K562 cell with Rab7b variants Rab7b wild-type,constitutively active mutant Rab7b-Q67L and Rab7b localization deficient mutant Rab7b-ACC or Rab7b RNAi, we found by weatern blot that expression of caspase3,9 and ERK1/2 increased and Akt suppressed in overexpression of Rab7b wild-type and constitutively active mutant Rab7b-Q67L. To study the role of Rab7b in autophagy, we investigated the expression of LC3 and Rab7b in K562 cell staved by EBSS, Our result showed the expression of LC3 and Rab7b both increased in starvation situation. Rab7b colocalized with LC3 in K562 cell. When autophagy pathway was inhibited with varity of autophagy inhibitor, the localization of Rab7b to autophagosome marked with immunofluorescent marked LC3 was blocked by WM.3-MA,NEM and vinblastin, but not bafilomycin Al.it told us that Rab7b take part in the autophagosome formation process and may also the fusion of endosome and autophagosome but not the fusion between lysosome and autophagosome.Our work suggested that HHT suppressed autophagy of K562 cell to enhance apoptosis, it is caspase dependent, it is associated with suppression of Akt phosphorilation and upregulation of ERK1/2. Overexpression of Rab7b can enhanced HHT induced apoptosis of K562 cell. It may also associated with suppression of Akt phosphorilation and upregulation of ERK1/2. Rab 7b localized to autophagosome and take a role in autophagosome formation process and may also the fusion of endosome and autophagosome but not the fusion between lysosome and autophagosome.In conclusion, Our work suggested a new recognition for the mechanism of HHT treatment on leukemia cells and a new sight of the relationship between autophagy and apoptosis in leukemia chemotherapy. Also it contributed to a further recognition of autophagy pathway, and will significantly improve the research work for the prevention, diagnosis and treatment of leukemia.