The Effect Of Intracellular Acidification On The Differentiation And Drug Resistance In Leukemia Cells

Background:The chimeric BCR-ABL gene, which is created by the formation of the Philadelphia chromosome (Ph), encodes bcr-abl fusion protein. The deregulated protein tyrosine kinase activity in bcr-abl is the cause of CML and 25% ALL. Imatinib (STI571) has been developed as a specific and targeted therapy for CML. It has also shown significant activity in patients with ALL who are BCR-ABL-positive. Treatment outcome with Imatinib-based regimens has improved compared with historic controls, but most patients who do not undergo allogeneic stem cell transplantation (SCT) eventually relapse. As the major obstacle of chemotherapy, drug resistance leads to the chemoresistance and the decreased survival in leukemias. P-glycoprotein (Pgp)-mediated drug efflux has been proved to be a resistance mechanism of CML to treatment with Imatinib. The Pgp is encoded by the MDR1 gene, and could transport the lipophilia drugs out of the tumor cells, which results in the decreased therapeutic effect and the redistribution of the anticancer drugs. With the development of Pgp modulators to the third generation, the regulation of Pgp is still unsatisfactory. In recent years, it has been reported that cancer cells show a strong tendency towards an alkaline deviation of the entire homeostasis when they are resistant to therapeutic intervention.Objective:According to our previous report that Pgp-mediated multidrug resistance could be reversed by the Na+/H+exchanger 1 (NHE1) inhibitor induced intracellular acidification, we investigated here whether relapsed patients with BCR-ABL-positive CML and ALL clinically resistant to Imatinib, which is associated with Pgp overexpression, could be influenced by the regulation of NHE1.Methods: The effect of NHE1 on primary cells from newly diagnosed and relapsed patients with BCR-ABL-positive CML and ALL was analyzed. Then the molecular events linking the NHE1 and the drug resistance were further investigated.Results:High intracellular pH (pHi) and Pgp overexpression were observed in relapsed patients. After treatment with specific NHE1 inhibitor Cariporide or high K+ buffer to decrease pHi, cells from relapsed patients exhibited decreased Pgp level, enhanced Doxorubicin and Rhodaminel23 accumulation, and increased sensitivity to Imatinib treatment. Furthermore, high K+ buffer treatment induced the activation of ERK1/2, JNK and inactivation of ERK5, p38 MAPK in relapsed patients. We further used BCR-ABL-positive cell line K562/DOX with Pgp overexpression to examine the mechanism implicated in NHE1-mediated reversal of drug resistance in BCR-ABL-positive leukemia cells.Conclusion:Crosstalk of MAPK pathways mediated the reversal of Pgp-associated drug resistance in BCR-ABL-positive leukemia patients and cell lines by the inhibition of NHE1. Background:The Na+/H+ exchanger 1 (NHE1) is a ubiquitously expressed integral membrane protein that regulates intracellular pH (pHi) by removing a proton in exchange for an extracellular sodium ion. Increased pHi caused by the NHE1 has documented to provide extremely crucial signal for cell proliferation and differentiation, including myeloid differentiation which is controlled by a coordinate network of certain transcription factors. Hypoxia or mimetic hypoxia has been reported to enhance the transcriptional activity of C/EBPαby direct protein-protein interaction with hypoxia-inducible factor (HIF)-la protein, which favors acute myeloid leukemic (AML) differentiation. It is found in our preliminary experiment that hypoxia and mimetic hypoxia also induced the differentiation of K562 cells with increased pHi and NHE1 expression. However, the effects of the NHE1 on the expression of C/EBPa in K562 cells in the course of their differentiation, as well as the mechanism of hypoxia on their proliferation and differentiation, remain unknown. This study represents the first attempt to characterize the effects of hypoxia on K562 proliferation and differentiation, and furthermore, the mechanisms underlying the response in hypoxia-induced cells and the role of NHE1 in this process were analyzed.Objective:We have studied the effect of hypoxia microenvironment on K562 leukemic cells differentiation, and characterized the involvement of NHE1 in them.Methods:The K562 cells were treated with hypoxia-mimicking agent CoCl2 or under actual hypoxia culture, and the specific NHE1 inhibitor Cariporide was used to inhibit NHE1 activity. The fluorescent probe BCECF was used for pHi measurements. Gene expression was analyzed by RT-PCR. The morphological characterization was determined by Wright's staining. Signaling pathways were examined by western blotting using phosphospecific antibodies.Results:Hypoxia or mimetic hypoxia favors K562 cells differentiation with up-regulation of C/EBPa. Moreover, treatment with Cariporide under hypoxia synergistically enhanced leukemia cell differentiation. Treatment with Cariporide increased levels phosphorylated ERK5 and p38 mitogen-activated protein kinase (MAPK).Conclusions:Taken together, these results indicate the enhancement of hypoxia-induced K562 differentiation by Cariporide via MAPK signaling pathway, and suggest a possible therapeutic target of NHE1 under hypoxia microenvironment in the treatment of leukemic diseases.