.rhoa-rock1-erk1/2-bax Signaling Pathways In Sodium Selenite-induced Apoptosis In Leukemia Cell Regulatory Role

ObjectiveAcute promyelocytic leukaemia (APL) was considered a highly lethal form of hematological malignant disease. Although traditional therapy with all-trans retinoic acid (ATRA) has been proved to be effective in APL, relapse occurs in20%patients and the5-year disease free survival rate is only74%. In addition, ATRA treatment always evoked other side-effects such as retinoic acid syndrome or organ failure, etc. In this context, targeting mutants or superactivated molecules to elicit specific apoptosis of leukemia cells would be a rational approach to more efficacious therapy, even to gain complete remission. We have demonstrated sodium selenite potently induced apoptosis in some leukemia cells, therefore elucidation of the mechanisms involved offered a rationale for selenite used as an adjuvant in APL therapy.ContentsThe RhoA/ROCK1pathway is closely associated with the genesis and development of many human carcinomas. Constitutively-activated mutations of RhoA/ROCK1cascade are well documented in human cancers and indicate cancer progression and poor prognosis. Blockage of this pathway interferes in the proliferation and transformation of progenitor cells and can even induce cell apoptosis, therefore the RhoA/ROCK1pathway may be a potential target for cancer therapy. In some haematologic disorders that bear oncogenic KIT, FLT3, Bcr-ABL, RhoA/ROCK1is abnormally activated. A growing pile of investigations show RhoA/ROCK interacts with various signal molecules to affect apoptosis in solid tumors, therefore, analysis of RhoA/ROCK1in APL was helpful in searching for novel therapy target. In this context, effects of selenite on leukemia cells and normal monocytes were compared, the expression and activity of RhoA/ROCK1in leukemia cells were studied, the interaction between RhoA/ROCK1axis and Erk1/2were observed, and the mechanism that RhoA-ROCK1-Erk1/2-Bax cascade mediated-regulation of intrinsic apoptosis was explored.MethodsReal-Time PCR and Western blot analysis were performed to detect the alterations of protein expression and modification in mRNA levels and protein levels, respectively. Effect of certain signaling molecule on cell apoptosis was studied via chemical modification or gene expression adjustment. Immunofluorescent colocalization, Co-immunoprecipitation and GST pull-down were applied to study the protein-protein interactions. To investigate the integrity of mitochondria and cell apoptosis in situ, Annexin V/PI staining, transmission electron microscopy, mitochondrial membrane potential assay and TUNEL staining were performed. Animal models were established to study the toxicity and the antitumor properties of selenite; immunohistochemical assay was used to clarify the alterations of signaling molecules in vivo.Results1. Selenite selectively induced human leukaemia cells apoptosis. We showed that20μM sodium selenite induced mitochondria-mediated apoptosis in leukaemia NB4and Jurkat cells. However, little increase in apoptotic rates and minimal decline in cellular viability were observed in selenite-treated primary monocytes.2. RhoA/ROCK1cascade was hyper-activated in leukaemia cell lines. Immunoblot analysis revealed higher expression levels and activities (RhoA-GTP) of Rho in NB4and Jurkat cells compared with monocytes. In addition, there was no significant difference in the expression of ROCK1in leukaemia cells, but the activity of ROCK1was higher than in nonmalignant cells.3. Reactive oxygen species (ROS) are the main effector of selenite in inducing apoptosis. Selenite induced ROS production in leukaemia NB4and Jurkat cells. Hydrogen peroxide (50μM) also inhibited RhoA/ROCK2pathway and finally evoked cell apoptosis.4. RhoA/ROCK1cascade inhibition resulted in apoptosis in leukaemia cells. In leukaemia cells, the suppression of RhoA/ROCK1signaling by selenite is due to ROS production. Blockage of RhoA/ROCK1activities using inhibitors or siRNA enhanced selenite-elicited cell apoptosis; activation of RhoA/ROCK1cascade using activators or plasmid attenuated apoptosis. Furthermore, RhoA functioned in cell fate determination via ROCK1, whereas RhoA/ROCK1was not affected by selenite in primary monocytes.5. Selenite induced kinetic changes in Erk1/2activation. We observed peak Erk12activation6h after selenite treatment and a followed decrease. The acute activation of Erk1/2during the first6h is crucial for selenite-triggered apoptosis; higher activity of Erkl/2enhanced caspase activity and cell apoptosis. Nevertheless, the downregulation of Erk1/2likely had little impact on cell apoptosis. ROS was the primary inducer of Erk1/2activation. In addition, Erk1/2proteins were imported into the nucleus from the cytosol after selenite incubation.6. ROCK1served as a scaffold protein that regulates Erk1/2activity. Computational analysis revealed2conserved Erk1/2docking domains in the coiled and glycine-rich domains of ROCK1. Co-IP and GST pull-down assays suggested that ROCK1interacted with Erk2and this interaction was ROCK1activity-dependent. Moreover, RhoA enhanced the binding between ROCK1and Erk1/2.7. RhoA/ROCK1pathway and Mek1/2signaling acted in parallel to regulate Erk1/2activation and cell death. Mek1/2positively modulated Erk1/2activity, and RhoA/ROCK1pathway negatively regulated the Erk1/2signaling beyond Mek/Erk cascade. However, Mek1/2phosphorylation was not affected by RhoA and ROCK1adjustments. Inhibition of Mek1/2had no impact on inactivated-RhoA/ROCK1pathway induced Erk1/2activation and cell apoptosis.8. RhoA/ROCK1pathway affected cellular localization of Erk1/2. RhoA and ROCK1were primarily localized in cytoplasm. In untreated cells, ROCK1dominantly co-localized with Erk1/2in the cytosolic fraction and associated with RhoA through its carboxyl terminal, therefore Erk1/2was maintained in its inactive state in the cytosol. In selenite-treated cells, RhoA released ROCK1and impaired its activity, leading to Erk1/2translocate to the nucleus.9. Erk1/2regulated apoptosis via modulating Bax. Knockdown of Bax abated cell apoptosis. Activating Erk1/2promoted the expression and oligomerization of Bax, and depolarized the mitochondrion; whereas inhibiting Erk1/2downregulated the expression and oligomerization of Bax. Conversely, RhoA regulated Bax negatively.10. Selenite induces xenograft cell apoptosis in vivo. Selenite displayed minimal system toxicity to mice. In xenogragt model, selenite inhibits the growth and metastasis of malignant cells. The in situ TUNEL staining demonstrated apoptosis was induced by selenite. This result was consolidated by Western blot assay, in which cleaved caspase9and cleaved caspase3were probed. Intriguingly, RhoA-ROCK1-Erk1/2-Bax pathway was invloved in apoptosis in vivo, which was confirmed when tumor tissues were subjected to Western blot and immunohistochemistry analysis.Conclusion1. RhoA/ROCK1cascade is heperactivated in APL-and ATL-derived leukemia cells. When RhoA/ROCK1axis is inhibited, Erk1/2-Bax signaling is activated and mitochondrion mediated-apoptosis is induced. Herein RhoA/ROCK1cascade is a potential target for leukemia therapy.2. Selenite induces apoptosis specificly in leukemia cells, it may be used clinically and potentially beneficial to APL therapy.