Guoqiang Chang

NHE1 mediates MDA-MB-231 cells invasion through the regulation of MT1-MMP

Na⁺/H⁺ exchanger 1 (NHE1), an important regulator of intracellular pH (pH(i)) and extracellular pH (pH(e)), has been shown to play a key role in breast cancer metastasis. However, the exact mechanism by which NHE1 mediates breast cancer metastasis is not yet well known. We showed here that inhibition of NHE1 activity, with specific inhibitor Cariporide, could suppress MDA-MB-231 cells invasion as well as the activity and expression of MT1-MMP. Overexpression of MT1-MMP resulted in a distinguished increase in MDA-MB-231 cells invasiveness, but treatment with Cariporide reversed the MT1-MMP-mediated enhanced invasiveness. To explore the role of MAPK signaling pathways in NHE1-mediated breast cancer metastasis, we compared the difference of constitutively phosphorylated ERK1/2, p38 MAPK and JNK in non-invasive MCF-7 cells and invasive MDA-MB-231 cells. Interestingly, we found that the phosphorylation levels of ERK1/2 and p38 MAPK in MDA-MB-231 cells were higher than in MCF-7 cells, but both MCF-7 cells and MDA-MB-231 cells expressed similar constitutively phosphorylated JNK. Treating MDA-MB-231 cells with Cariporide led to decreased phosphorylation level of both p38 MAPK and ERK1/2 in a time-dependent manner, but JNK activity was not influenced. Supplementation with MAPK inhibitor (MEK inhibitor PD98059, p38 MAPK inhibitor SB203580 and JNK inhibitor SP600125) or Cariporide all exhibited significant depression of MDA-MB-231 cells invasion and MT1-MMP expression. Furthermore, we co-treated MDA-MB-231 cells with MAPK inhibitor and Cariporide. The result showed that Cariporide synergistically suppressed invasion and MT1-MMP expression with MEK inhibitor and p38 MAPK inhibitor, but not be synergistic with the JNK inhibitor. These findings suggest that NHE1 mediates MDA-MB-231 cells invasion partly through regulating MT1-MMP in ERK1/2 and p38 MAPK signaling pathways dependent manner.

Inhibition of K562 leukemia angiogenesis and growth by selective Na+/H+ exchanger inhibitor cariporide through down-regulation of pro-angiogenesis factor VEGF.

To investigate the effect of inhibition of Na(+)/H(+) exchanger isoform1 (NHE1) on K562 leukemia-driven angiogenesis, the selective NHE1 inhibitor cariporide was used. Cariporide treatment of K562 resulted in a decrease in pHi and down-regulation of VEGF secretion. The proliferation, migration and in vitro tube formation of human umbilical vein endothelial cells was decreased in cariporide treated K562 condition medium (CM) while VEGF supplement could partially restore the inhibitory effect. Subcutaneous injection of nude mice with cariporide inhibited K562 tumor growth with a reduction of the density of microvessels compared to the control group.

CD44 targets Wnt/β-catenin pathway to mediate the proliferation of K562 cells.

BackgroundChronic myeloid leukemia is a clonal myeloproliferative disorder disease in which BCR/ABL plays an important role as an oncoprotein and molecular target. Despite the success of targeted therapy using tyrosine kinase inhibitors, CML remains largely incurable, most likely due to the treatment resistance after firstly chemical therapy. So know well the unique molecular pathway of CML is very important.MethodsThe expressions of CD44 in different leukemia patients and cell lines were detected by real-time PCR and western blotting. The effects of CD44 on proliferation of K562 cells were determined using the MTT and colony formation assays, and even in a nude mouse transplantation model. Then, the cell cycle changes were detected by flow cytometric analysis and the early apoptosis of cells was detected by the annexin V/propidium iodide double-staining assay. The expressions of the cycles and apoptosis-related proteins p21, Cyclin D1 and Bcl-2 were analyzed by western blot and real-time PCR assay. Finally, the decreased nuclear accumulation of β-catenin was detected by western blotting and immunefluorescence.ResultsFirstly, we showed that CD44 expression was increased in several kinds of leukemia patients and K562 cells. By contrast, the down-regulation of CD44 resulted in decreased proliferation with a G0/G1 arrest of cell cycle in K562 cells according to the MTT assay and the flow cytometric analysis. And no significant induction of both the early and late phases of apoptosis was shown by the annexin V-FITC and PI staining. During this process, p21 and cyclin D1 are the major causes for cell cycle arrest. In addition, we found CD44 down-regulation decreased the expression of β-catenin and increased the expression of phosphorylated β-catenin. The instability of Wnt/β-catenin pathway induced by increased expression of p-β-catenin resulted in a decreased nuclear accumulation in CD44 silenced K562 cells. In the nude mouse transplantation model, we also found the same results.ConclusionsThese results show that K562 cells depend to a greater extent on CD44 for proliferation, and CD44 down-regulation may induce a cell cycle arrest through Wnt/β-catenin pathway. CD44 blockade may be beneficial in therapy of CML.

Na+/H+ exchanger 1 inhibition contributes to K562 leukaemic cell differentiation.

The effect of hypoxia on the differentiation of chronic myeloid leukaemic K562 cells were studied, as was the role of the NHE1 (Na+/H+ exchanger 1). Hypoxia induced differentiation of K562 cells as seen by modifications in their morphological features, up‐regulation of C/EBPα (CCAAT/enhancer‐binding protein α), and marked IL‐8 (interleukin‐8) release. Inhibition of NHE1 under hypoxia additionally enhanced the level of C/EBPα and further promoted leukaemic cells differentiation. Pharmacological inhibition of p38 MAPK (mitogen‐activated protein kinase) also significantly suppressed C/EBPα expression under hypoxia conditions after NHE1 inhibition. These results indicate the enhancement of hypoxia‐induced K562 differentiation by NHE1 inhibition, which may be due to up‐regulation of C/EBPα via p38 MAPK signalling pathway, which suggests a possible therapeutic target of NHE1 under hypoxia microenvironment in the treatment of leukaemic diseases.

Nuclear accumulation of Calcineurin B Homologous Protein 2 (CHP2) results in enhanced proliferation of tumor cells

The interaction between calcineurin B homologous protein 2 (CHP2) and Na+/H+ exchanger 1 (NHE1), two membrane proteins, is essential for protecting cells from serum deprivation–induced death. Although four putative EF‐hands in CHP2 had been predicted for years, Ca2+‐binding activities of these motifs have not been tested yet, their role in this process remain poorly understood. To identify Ca2+‐binding motifs required for the stable formation of CHP2/NHE1 complexes, we developed a mutagenesis‐based assay in PS120 cells. We found that 45Ca2+ bond to two EF‐hand motifs (EF3 and 4) of CHP2 proteins with high affinity. Complex formation between CHP2 and the CHP2 binding domain of NHE1 resulted in a marked increase in the Ca2+‐binding affinity of CHP2. Co‐immunoprecipitation and distribution of GFP‐tagged CHP2‐EF3m/4m also indicated that Ca2+ affected the membrane location of CHP2 to interact with NHE1. The C‐terminal region of CHP2 contains a nuclear export sequence (NES). When the six leucines of NES were mutated to alanines, the resulting CHP2 protein was predominantly localized to the nucleus. Furthermore, mutation of the NES resulted in enhanced proliferation and oncogenic potential of HeLa cells. Together, these results show that calcium and NES control the subcellular distribution of CHP2 and then distinctively regulate cell proliferation.

Down-regulation of the P-glycoprotein relevant for multidrug resistance by intracellular acidification through the crosstalk of MAPK signaling pathways

In our previous study, we have found that the tumor multidrug resistance mediated by P-glycoprotein could be reversed by sustained intracellular acidification through down-regulating the multidrug resistance gene 1 mRNA and P-glycoprotein expression. However, the molecular events linking the intracellular acidification and the regulation of P-glycoprotein remain unclear. In the present study, the molecular pathways involved in the regulation of P-glycoprotein expression by the intracellular acidification were investigated. We found that the P-glycoprotein expression was down-regulated by the intracellular acidification through inhibition of p38 mitogen-activated protein kinase (MAPK) and the activation of c-Jun N-terminal kinase (JNK) in the resisitant K562/DOX cells. In the sensitive K562 and HL60 cell lines, the changes of the p38 MAPK expression after the acidification are not as obvious as that of K562/DOX cells, but the activation of extracellular signal-regulated kinase (ERK) is also observed, which indicates that the down-regulation of p38 MAPK by the intracellular acidification might be the resistant cell line specific. Blockade of ERK and JNK signaling by the inhibitors or RNA interference increased p38MAPK activities suggesting that cross-talk within MAPKs is also important for this response. Our study provides the first direct evidence that the reversal of P-glycoprotein-mediated multidrug resistance by intracellular acidification is mediated by the crosstalk of MAPK signaling pathways.

Decreased Intracellular pH Induced by Cariporide Differentially Contributes to Human Umbilical Cord-Derived Mesenchymal Stem Cells Differentiation

Background/Aims: Na+/H+ exchanger 1 (NHE1) is an important regulator of intracellular pH (pHi). High pHi is required for cell proliferation and differentiation. Our previous study has proven that the pHi of mesenchymal stem cells is higher than that of normal differentiated cells and similar to tumor cells. NHE1 is highly expressed in both mesenchymal stem cells and tumor cells. Targeted inhibition of NHE1 could induce differentiation of K562 leukemia cells. In the present paper we explored whether inhibition of NHE1 could induce differentiation of mesenchymal stem cells. Methods: MSCs were obtained from human umbilical cord and both the surface phenotype and functional characteristics were analyzed. Selective NHE1 inhibitor cariporide was used to treat human umbilical cord-derived mesenchymal stem cells (hUC-MSCs). The pHi and the differentiation of hUC-MSCs were compared upon cariporide treatment. The putative signaling pathway involved was also explored. Results: The pHi of hUC-MSCs was decreased upon cariporide treatment. Cariporide up-regulated the osteogenic differentiation of hUC-MSCs while the adipogenic differentiation was not affected. For osteogenic differentiation, β-catenin expression was up-regulated upon cariporide treatment. Conclusion: Decreased pHi induced by cariporide differentially contributes to hUC-MSCs differentiation.

C/EBP ζ targets to neutrophil gelatinase-associated lipocalin (NGAL) as a repressor for metastasis of MDA-MB-231 cells.

Breast cancer is a leading cause of morbidity in women worldwide. neutrophil gelatinase-associated lipocalin (NGAL), a useful biomarker of ER negative (ER(-)) breast cancer, promotes local tumor invasion and lymph node metastasis. We first identified the distinctive expression of NGAL in two breast cancer cell lines MCF7 and MDA-MB-231 cells, and then confirmed NGAL as a critical inducer of metastasis. Finally, the transcriptional factor CCAAT enhancer-binding proteins ζ (C/EBP ζ) was overexpressed in MDA-MB-231 cells. Consistent with the effect of NGAL knockdown, C/EBP ζ overexpression caused the significant changes that could prevent cell metastasis. C/EBP ζ overexpression induced a strong decrease in NGAL and matrix metalloproteinases (MMPs) expressions as determined by quantitative real time PCR and Western blotting. To identify the potential role of C/EBP ζ on regulating of NGAL in breast cancer, we established the dual-luciferase reporter assay for NGAL in MDA-MB-231 cells cotransfected with C/EBP ζ. Promoter reporter assays determined that C/EBP ζ directly repressed the human NGAL gene promoter activity by inhibiting the NGAL transcription. Taken together, this work identified that the C/EBP ζ overexpression downregulated NGAL to inhibit migration and invasion of breast cancer, which could be used as a novel strategy for breast cancer therapy.

CUEDC2 sensitizes chronic myeloid leukemic cells to imatinib treatment

CUEDC2, a newly reported protein, has been found to be ubiquitously expressed in human tissues and repress NF-κB activity. To study the role of CUEDC2 in chronic myeloid leukemia (CML), we explored the function of CUEDC2 in CML cells through using the CML cell line K562 and its imatinib resistant cells K562/G01. K562 cells expressed a relatively higher level of CUEDC2 compared to K562/G01 cells. Knockdown of CUEDC2 in K562 cells resulted in decreased cell apoptosis after imatinib treatment; when CUEDC2 was overexpressed in K562/G01 cells, imatinib induced more cell apoptosis. By analyzing the activity of NF-κB, the results indicated a negative association between the expression of CUEDC2 and NF-κB signaling pathway in these CML cells. Our data suggested that the expression level of CUEDC2 has an inverse correlation with imatinib resistance and activity of NF-κB signaling pathway in CML cells, CUEDC2 could regulate imatinib sensitivity in CML cells at least partially through NF-κB signaling pathway.

Cariporide sensitizes leukemic cells to tumor necrosis factor related apoptosis-inducing ligand by up-regulation of death receptor 5 via endoplasmic reticulum stress–CCAAT/enhancer binding protein homologous protein dependent mechanism

Abstract CCAAT/enhancer binding protein homologous protein (CHOP) expression increases when Na+–H+ exchanger 1 (NHE1) is inhibited. Endoplasmic reticulum (ER) stress has been shown to trigger tumor cell death through CHOP. We therefore hypothesized that NHE1 activity correlates with ER stress and confers pharmaceutical potential to NHE1 inhibitor as an anti-tumor agent. The present study showed that treatment with the NHE1 inhibitor cariporide led to ER stress-induced up-regulation of the death receptor 5 (DR5) which is mediated by CHOP at the transcriptional level. We also determined that ER stress-induced Janus kinase (JNK) activation was responsible for the modulation of CHOP. Combining cariporide with tumor necrosis factor related apoptosis-inducing ligand (TRAIL) led to a significantly enhanced level of apoptosis that was abrogated by siRNA silencing of CHOP. This study provides a potential mechanistic rationale for the use of NHE1 inhibitor in combination with DR5 agonists to induce apoptosis in leukemia.