Elsevier

Biochemical Pharmacology

Volume 90, Issue 3, 1 August 2014, Pages 212-225
Biochemical Pharmacology

CXCL12/CXCR4 axis confers adriamycin resistance to human chronic myelogenous leukemia and oroxylin A improves the sensitivity of K562/ADM cells

https://doi.org/10.1016/j.bcp.2014.05.007Get rights and content

Abstract

This study was aimed at investigating the reversal effect of oroxylin A, a naturally bioactive monoflavonoid separated and purified from Scutellaria baicalensis Georgi, in human chronic myeloid leukemia (CML) and the underlying mechanisms. The results showed that CXCL12 could enhance the resistance of K562 cells to adriamycin (ADM) by increasing the expression of CXCR4, up-regulating the downstream PI3K/Akt pathway, and promoting translocation of NF-κB dimers into nucleus and subsequently decreasing the expression of apoptosis-related proteins in K562 cells. And we found that ADM resistance was partially reversed by CXCR4 siRNA transfection. Moreover, the sensitivity enhancement of oroxylin A was demonstrated by decreasing the expression of CXCR4 at both protein and mRNA levels, via PI3K/Akt/NF-κB pathway and triggering the apoptosis pathway in vitro. In addition, the in vivo study showed that oroxylin A increased apoptosis of leukemic cells with low systemic toxicity, and the mechanism was the same as in vitro study. In conclusion, all these results showed that oroxylin A improved the sensitivity of K562/ADM cells by increasing apoptosis in leukemic cells and decreasing the expression of CXCR4 and PI3K/Akt/NF-κB pathway, and probably served as a most promising agent for CML treatment.

Introduction

Chronic myeloid leukemia (CML) is a hematological disease that represents 20% of all adult leukemia [1]. It is characterized by accumulation and proliferation of myeloid cells and the precursors, and it is associated with the BCR-ABL fusion gene lesion [2]. BCR-ABL specific tyrosine kinase inhibitors (TKIs), such as Imatinib, Nilotinib and Dasatinib, have improved the 10-years survival time in 80% CML patients [1]. Due to CML genomic instability and activation of additional mechanisms, it starts with a chronic phase and progresses to blast crisis, becoming increasingly resistant to chemotherapeutics [3]. Currently in clinical trials, several novel drugs have been designed to target biological features of CML cells, not only to improve the therapeutic effect but also to overcome drug resistance of CML cell lines.

CXCR4, a seven-transmembrane G-protein-coupled chemokine receptor, is expressed in hematopoietic and epithelial cancer cells [4]. CXCR4 is reported to be an essential factor for survival and drug resistance in leukemia. CXCL12 (SDF-1), a ligand for CXCR4, is a soluble pro-survival factor produced by neighboring stromal cells and actively potentiates CXCR4 expression. The binding of CXCL12 to CXCR4 induces several intracellular signals related to chemotaxis, homing, cell survival and proliferation [5]. Marrow stromal cells (MSC), the primary component of bone marrow microenvironment, could constitutively secret CXCL12 [6]. Accumulating evidence suggests that leukemia cells are protected by MSCs [7]. Indirect communication through CXCL12 and direct contact between leukemia cells and MSCs have been demonstrated to be essential for CML cell survival and resistance [8]. Activation of CXCR4 induces CML cell homing to the bone marrow microenvironment, where CXCL12 exists protecting CML cells from chemotherapeutics-induced apoptosis [9]. For these reasons, the CXCL12/CXCR4 axis has been regarded as a potential target for new therapeutic strategies. The adhesive interactions between leukemia with stromal cells that confer survival and drug resistance signals could be disrupted by the CXCR4 antagonists. Owing to the blockade of the CXCL12/CXCR4 axis the leukemia cells were changed from the marrow, more accessible to chemotherapeutics [9].

There is evidence that targeting the leukemia microenvironment and inhibition of CXCR4 can overcome resistance to kinase inhibitors and chemotherapy in acute myelocytic leukemia (AML) cells [10]. Activation of CXCL12/CXCR4 pathway has been reported to regulate cell migration, survival and proliferation through G proteins, PI3K/Akt and extracellular signal-regulated kinase signals [11]. When CXCR4 is activated, PI3K as one of downstream effectors of CXCR4-mediated signaling is also activated, which confer cancer cells drug resistance through the PI3K/Akt pathway [12]. In recent years, some novel therapeutic interventions targeting the microenvironment/leukemia interaction focusing on SDF1/CXCR4, ILK/PI3K/Akt were proposed [13]. For example, CXCR4 antagonists such as plerixafor (AMD3100) and T140 can disrupt tumor cells/stromal interactions and block the microenvironment protection of leukemic cells from chemotherapeutics-induced apoptosis [14]. Isoform-selective PI3K inhibitors could inhibit CXCR4 signaling and overcome stromal cell-mediated drug resistance and survival in chronic lymphocytic leukemia [15]. Totally, CXCR4 antagonists can mobilize leukemia cells from their protective marrow microenvironment, and then the mobilized leukemia cells would become more accessible to chemotherapeutics, even reverse drug resistant.

Oroxylin A (C16H12O5) is one of bioactive flavonoids separated and purified from Scutellaria baicalensis Georgi. It has been widely used for anti-inflammation, anticancer, antiviral and antibacterial infections [16]. Previous studies have shown that oroxylin A was effective in reversing multi-drug resistance. As an active flavonoid, combination oroxylin A with 5-fluorouracil (5-FU) could increase human hepatoma cell sensitivity to 5-FU in vivo [17] and in vitro [18]. It has recently been reported that oroxylin A could reverse multi-drug resistance in MCF7/ADR cells by arresting cells at G2/M checkpoint [19]. Furthermore, it reversed CAM-DR of HepG2 cells through inhibiting integrin β1 and its related PI3K/Akt signaling pathway [20]. Meanwhile, oroxylin A could reverse multi-drug resistance in human hepatoma BEL7402/5-FU cells via downregulation of P-glycoprotein expression by inhibiting NF-κB signaling pathway [21].

In this article, we investigated whether oroxylin A reverse the drug resistance by inhibiting CXCL12/CXCR4 axis and its related PI3K/Akt/NF-κB pathway. Besides, we examined the antitumor effect BALB/c mice model bearing K562/ADM tumor.

Section snippets

Reagents and antibodies

Oroxylin A (purity >99%) was prepared at China Pharmaceutical University (Nanjing, China). The compound was dissolved in dimethyl sulfoxide (DMSO) (Sigma–Aldrich, St. Louis, USA) to a concentration of 100 mM and stored at −20 °C. The culture medium was RPMI-1640 (Gibco, Invitrogen Corporation, Carlsbad, CA). Dye DAPI (Invitrogen, USA) was used to detected apoptosis cells. The final DMSO concentration did not exceed 0.1% throughout the study. Adriamycin and AMD3100 were purchased from Sigma–Aldrich

CXCR4 mediates drug resistance in K562/ADM cells

Doubling time (DT) has emerged as an important surrogate marker of disease progression and survival in vivo and vitro [29]. The doubling time of K562 cells was 40.65 h, but those of K562/ADM cells were 39.05 h respectively (P > 0.05; Fig. 1A). There was no significant difference between doubling times of K562 and K562/ADM cells.

Resistance index (RI) was important for assessing the resistance of MDR cells to various anticancer drugs [30]. As shown in Fig. 1B, the IC50 of Adriamycin (ADM) in K562/ADM

Discussion

The emergence of multidrug resistance (MDR) in leukemia is a major obstacle to successful cancer chemotherapy [42]. Among the molecular events leading to cross resistance to multiple drugs in CML cells, higher level of CXCR4 seems to be very important [43], and our study is consistent with above description: CXCR4 expression in both protein and mRNA level is higher in drug-resistant K562/ADM cells compared with sensitive (K562) cells, which suggested that the resistance in K562/ADM cells occur

Conflict of interest

None declared.

Acknowledgments

This work was supported by the Project Program of State Key Laboratory of Natural Medicines, China Pharmaceutical University (No. JKGZ201101, and SKLNMZZJQ201302), Fundamental Research Funds for the Central Universities (No.JKP2013010), the National Natural Science Foundation of China (No. 81001452, 91029744 and 81173086), the National Science & Technology Major Project (No. 2012ZX09103101-050 and 2012ZX09304-001) and Program for Changjiang Scholars and Innovative Research Team in University

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