Biological Effects Of CIAPIN1in Tumors

Background:CML (chronic myeloid leukemia) is a kind of common hematologic malignancies and is characterized by the formation of the Ph (Philadelphia) chromosome and bcr-abl fusion gene. CIAPIN1(cytokine-induced apoptosis inhibitor1) is a newly identified antiapoptotic molecule which does not show any homology to known apoptosis regulatory molecules such as Bcl-2family, caspase family. CIAPIN1is proven to be a downstream effector of the receptor tyrosine-Ras signaling pathway. The expression of CIAPIN1is closely related to the development of tumor. However, its potential role in regulating myeloid differentiation remains unclear.Objective:Our study was aimed to investigate the biological effect CIAPIN1on the granulocytic differentiation of K562cells and the molecular mechanisms involved in the process.Methods:The RNA interference technique was conducted to silence the expression of CIAPIN1in K562cells. The interference efficiency was examined by real-time quantitative PCR, western blotting and immunofluorescence assay. The effect of CIAPIN1silencing on granulocytic differentiation of K562cells was confirmed by morphological and ultramicrostructural changes observation and myeloid-associated genes detection. The changes of NHE1expression in CIAPIN1-silenced K562cells were examined by real-time quantitative PCR and western blotting. The effect of NHE1changes on granulocytic differentiation of CIAPIN1-silenced K562cells was detected and the regulating correlation between CIAPIN1and NHE1was elucidated. The changes of ERK1/2phosphorylation were detected by western blotting after NHE1expression was changed in CIAPIN1-silenced K562cells. The potential role of ERK1/2signaling pathway in granulocytic differentiation of K562cells was examined by inhibition of ERK1/2after NHE1inhibition in CIAPIN1-silenced K562cells.Results:The expression of CIAPIN1in K562cells was depleted when transfection with CIAPIN1shRNA plasmids. We found depletion of CIAPIN1by shRNA led to granulocytic differentiation of K562cells. Meanwhile, the decrease of NHE1and up-regulation of phosphorylated ERK1/2were observed after CIAPIN1depletion. Interestingly, targeted inhibition of NHE1further promoted the differentiation of K562cells with CIAPIN1silencing. Accordingly, ectopic expression of NHE1reversed this phenotype. Furthermore, ERK1/2inhibition with the chemical inhibitor, PD98059, abolished CIAPIN1silencing-induced granulocytic differentiation of K562cells after NHE1inhibition.Conclusion:Taken together, our results demonstrated that CIAPIN1knock-down induced K562CML cells to differentiate toward more mature granulocytic stage. Moreover, in our study we first found NHE1was the target of CIAPIN1to regulate the differentiation of K562cells. The activation of ERK1/2signaling pathway was essential in CIAPIN1depletion-induced differentiation of K562cells. Our findings implied CIAPIN1and NHE1could be new targets in developing therapeutic strategies against leukemia. Background:CML (chronic myeloid leukemia) is a myeloproliferative disorder of pluripotent hematopoietic stem cells and is characterized by the formation of the Ph (Philadelphia) chromosome and bcr-abl fusion gene. The tyrosine kinase inhibitor Imatinib is a specific targeted molecular drug for the treatment of Ph chromosome positive CML, particularly in chronic phrase. Although it is considered as one of the most effective drugs and the first-line treatment for CML, Imatinib resistance seems unavoidable and occurs frequently during its clinical application. In addition to the bcr-abl duplication or mutation, abnormal expression of some other gene or alternative signaling pathway activation may also contribute to the resistance to Imatinib. CIAPIN1(cytokine induced apoptosis inhibitor1) is a newly identified antiapoptotic molecule which does not show any homology to known apoptosis regulatory molecules such as Bcl-2family, caspase family. CIAPIN1is proven to be a downstream effector of the receptor tyrosine-Ras signaling pathway. The expression of CIAPIN1is closely related to the development of tumor. However, studies on the role of CIAPIN1in CML are still unavailable. As Imatinib has emerged as the leading compound to treat CML and selectively induces growth arrest and apoptosis of bcr-abl positive leukemia cells, we then address the question of whether CIAPIN1knock-down could sensitize K562cells to Imatinib treatment.Objective:To investigate the biological effects of CIAPIN1on the sensitivity of K562chronic myeloid leukemia cells to Imatinib and the related mechanisms.Methods:1. Bone marrow samples were obtained from14adult-ALL (acute lymphoblastic leukemia) patients,20AML (acute myeloid leukemia) patients,37CML patients and16CLL (chronic lymphocytic leukemia) patients in our hospital from August2011to August2012. Bone marrow samples of8healthy volunteers were used as control. Bone marrow mononuclear cells were prepared by Ficoll-Hypaque density gradient centrifugation. The expression level of CIAPIN1in primary bone marrow cells was assayed by real-time quantitative PCR.2. CIAPIN1shRNA plasmids were constructed, transfected into K562cells and the stable clones were selected with G418(termed as interference group). K562cells transfected with CIAPINl unrelated sequence plasmid were used as control (termed as control group). MTT assay was used to detected cell viability. Cell colony forming experiment was used to detect colony formation ability. Flow cytometry was used to detect cell cycle. Hoechst33258staining and Annexin V-APC/PI apoptosis analysis were used to detect apoptosis. BALB/C-nu/nu mice were conducted to detect tumorigenicity.3. Both the interference group and the control group were treated with Imatinib. MTT assay was used to detected cell viability. Cell colony forming experiment was used to detect colony formation ability. Flow cytometry was used to detect cell cycle. Hoechst33258staining and Annexin V-APC/PI apoptosis analysis were used to detect apoptosis.4. The western blotting technique was used to detect Bcl-2, caspase, Pgp and NF-κB signaling pathway relative proteins. Nuclear localization of NF-κB p65protein was determined by immunofluorescence assay.Results:1. The mRNA expression level of CIAPIN1was statistically higher in CML patients preliminarily diagnosed and K562CML cell line compared with control (P<0.05).2. CIAPIN1knock-down inhibited proliferation of K562cells both in vitro and in vivo.3. CIAPIN1knock-down inhibited proliferation of K562cells with Imatinib treatment; CIAPIN1knock-down promoted Imatinib-induced apoptosis of K562cells.4. In K562cells with Imatinib treatment, CIAPIN1knock-down not only up-regulated Bid, Bim and cleaved PARP but also activated caspase cascade. By contrast, CIAPIN1knock-down repressed expressions of Bcl-xl, Bcl-2and Mcl-1. The positive correlation occurred between CIAPIN1and Pgp expressions. CIAPIN1knock-down decreased NF-κB signaling pathway activity of K562cells. NF-κB inhibitor Bayl1-7082could further block NF-κB activity in K562cells with CIAPIN1knock-down. In addition, NF-κB inhibitor Bayl1-7082could inhibit growth and promote apoptosis of K562cells with CIAPIN1depletion.Conclusion:CIAPIN1knock-down sensitized K562CML cells to Imatinib treatment. The promotion effect of CIAPIN1knock-down on Imatinb-induced apoptosis could be partly mediated by regulation of Bcl-2family, caspase family and Pgp expression. Inhibition of NF-κB signaling pathway activity was essential in CIAPIN1depletion-induced sensitivity of K562cells to Imatinib. Background:Breast cancer is one of the most common malignant tumors in women and distant metastasis is the leading cause of treatment failure and death in patients with breast cancer. Consequently, the elucidation of the mechanisms governing breast cancer metastasis is pressing and remains a major challenge. CIAPIN1(cytokine-induced apoptosis inhibitor1) is a newly identified antiapoptotic molecule and has been proved to be closely correlated with many malignant tumors. NHE1(Na+/H+exchanger1), belonging to Na+/H+exchangers family and expresses ubiquitously in mammal cells, is an integral membrane protein and higher expresses in tumor cells. The primary regulator of pHi and pHe is known to be NHE1, which can catalyze the extrusion of intracellular proton ions in exchange for extracellular sodium ions. Thus, NHE1activation may induce extracellular acidification and intracellular alkalinization and this acid-base microenvironment homeostasis is thought to be critical to activation of extracellular proteases and degradation of the ECM resulting in motility and invasion of the tumor cell. A substantial body of evidence indicates that NHE1involves in motility and invasion of many solid tumors including breast cancer. However, the exact mechanism underlying this process is still poorly understood.Objective:To investigate the biological role of CIAPIN1in migration and invasion of MDA-MB-231cells; to elucidate the direct regulation model between CIAPIN1and NHE1; to explore the signaling pathway involved.Methods:1. The expression of CIAPIN1in two breast cancer cell lines MDA-MB-231and MCF-7was detected by real-time quantitative PCR and western blotting;2. The lentiviral expression vector of CIAPIN1was constructed and infected MDA-MB-231cells. The migration and invasion was detected by wound-healing and transwell assays;3. The expression of NHE1in CIAPIN1over-expressed MDA-MB-231cells was detected by real-time quantitative PCR and western blotting. The migration and invasion of MDA-MB-231cells with CIAPIN1over-expression after NHE1inhibition was detected by wound-healing and transwell assays;4. The expression of phosphorylated ERK1/2in CIAPIN1over-expressed MDA-MB-231cells after NHE1inhibition was detected by western blotting. The migration and invasion of MDA-MB-231cells with CIAPIN1over-expression after phosphorylated ERK1/2inhibition was detected by wound-healing and transwell assays;5. The expressions of MMP2and MMP9in MDA-MB-231cells with indicated treatments were detected by western blotting.Results:1. The expression of CIAPIN1was lower in MDA-MB-231cells than that in MCF-7cells;2. The migration and invasion of MDA-MB-231cells was inhibited after CIAPIN1over-expression. The expressions of MMP2and MMP9were down-regulated in CIAPIN1over-expressed MDA-MB-231cells;3. The decreased expressions of NHE1and phosphorylated ERK1/2were observed after CIAPIN1over-expression;4. Taregeted inbition of NHE1in CIAPIN1over-expressed MDA-MB-231cells further inhibited cells’migration and invasion. The expressions of MMP2and MMP9were further down-regulated;5. The decreased expression of phosphorylated ERK1/2was observed in CIAPIN1over-expressed MDA-MB-231cells after NHE1inhibition. The migration and invasion of MDA-MB-231cells was inhibited at the greatest extent by targeted inhibition of phosphorylated ERK1/2and NHE1together. The expressions of MMP2and MMP9were also inhibited at the greatest extent.Conclusion:Taken together, our results demonstrated that CIAPIN1over-expression inhibited the migration and invasion of MDA-MB-231cells. Moreover, we found CIAPIN1taregeted NHE1to regulate metastasis of MDA-MB-231cells. The inhibition of ERK1/2signaling pathway was essential in CIAPIN1-mediated metastasis of MDA-MB-231cells. Our findings implied CIAPIN1and NHE1could be new targets in developing therapeutic strategies against breast cancer.