Cancer Letters

Cancer Letters

Volume 433, 1 October 2018, Pages 117-130
Cancer Letters

Original Articles
Novel combined Ato-C treatment synergistically suppresses proliferation of Bcr-Abl-positive leukemic cells in vitro and in vivo

https://doi.org/10.1016/j.canlet.2018.06.027Get rights and content

Highlights

  • ATO acts synergistically with CDDP to inhibit growth of TKI-resistant CML cells.

  • Ato-C (ATO/CDDP) substantially induces apoptosis of Ph + CML cells.

  • Ato-C significantly downregulates expression of MYC oncogenic signature.

  • Ato-C decreases phosphorylation level of FOXO transcriptional factor.

  • Ato-C significantly reduces tumor growth compared with either monotherapy in vivo.

Abstract

Chronic myelogenous leukemia (CML) accounts for 15–20% of all leukemias affecting adults. Despite recent advances in the development of specific Bcr-Abl tyrosine kinase inhibitors (TKIs), some CML patients suffer from relapse due to TKI resistance. Here, we assessed the efficacy of a novel combinatorial arsenic trioxide (ATO) and cisplatin (CDDP) treatment (Ato-C) in human Bcr-Abl-positive leukemic cells. Combination index analyses revealed that a synergistic interaction of ATO and CDDP elicits a wide range of effects in K562, KU-812, MEG-A2, and KCL-22 cells. Notably, Ato-C synergistically enhanced apoptosis and decreased the survival of both acquired TKI-resistant CML cells and the cells expressing mutant Bcr-AblT315I. In addition, Ato-C dramatically decreased the phosphorylation level of forkhead transcription factor FOXO1/3a and STAT5 as well as c-Myc protein level. Interestingly, results of gene set enrichment analysis showed that Ato-C significantly downregulates the expression of MYC- and/or E2F1-target genes. Furthermore, Ato-C significantly suppressed the proliferation of MEG-A2-derived tumor when compared with that following monotherapy in vivo. Collectively, these results suggest that combined Ato-C treatment could be a promising alternative to the current therapeutic regime in CML.

Introduction

Currently, chronic myelogenous leukemia (CML) accounts for 15–20% of all leukemias affecting adults [1]. Recent therapeutics for CML were developed by targeting the Bcr-Abl fusion protein generated from a Philadelphia (Ph) chromosome with reciprocal translocation of chromosomes 9 and 22 [[2], [3], [4], [5]]. Bcr-Abl tyrosine kinase inhibitors (TKIs) dramatically improved the survival rate of patients with CML; novel TKIs called second and/or third generation have been clinically developed for the treatment of newly developed and relapsed CML [1,[6], [7], [8], [9], [10]]. Despite the advances in CML therapeutics, it has been reported that prolonged TKI therapy can produce a sub-population of CML cells with a Bcr-Abl gene mutation that leads to resistance to TKI therapy, which results in a poorer prognosis [1,[11], [12], [13], [14], [15], [16]]. Eventually, 10–15% of patients remain resistant to TKIs and at risk of disease progression [17,18]. Although hematopoietic stem cell transplantation is one of the best alternative therapies for CML, there are difficulties in finding the HLA-matched donors as well as age limitations [18,19]. Therefore, novel therapeutic agents and/or strategies are required to improve both the disease outcome and the quality of life for patients with CML.

Combination therapies have been developed to improve response rates and to prevent drug resistance or relapse [20]. Arsenic trioxide (ATO) has attracted worldwide interest in the field of oncology because of its substantial anti-cancer activity in patients with acute promyelocytic leukemia (APL) [21,22]. Currently, all-trans retinoic acid (ATRA) combined with ATO therapy is considered as one of the standard induction therapy regimens for patients newly diagnosed with APL [23]. In CML, arsenic combined with imatinib and/or its derivatives exerts promising anti-cancer activity by enhancing apoptosis [24,25]. Interestingly, Zhang et al. and we independently reported that ATO and cisplatin (CDDP) synergistically increase their cytotoxic effects in solid cancer cell lines [26,27]. Therefore, accumulating evidence implicates that ATO could be an important component of combined treatment for cancer. Here, we examined the efficacy of combined ATO-CDDP (Ato-C) treatment in human Bcr-Abl-positive (Ph+) CML cells both in vitro and in vivo. We also examined the efficacy of Ato-C in the acquired imatinib-resistant Ph+ CML cells as well as cells expressing mutant Bcr-AblT315I. Moreover, we identified a molecular mechanism underlying the anti-tumor effects induced by Ato-C.

Section snippets

Cell culture and reagents

Human CML cell lines KU-812, KCL-22, MEG-A2, K562, and a murine pro-B cell line Ba/F3 were obtained from the Japanese Collection of Research Bioresource Cell Bank. HL-60, NB4, U266, and THP-1 cells were kindly provided by Hiroshi Miwa (Department of Hematology, Mie University, Mie, Japan). The cells expressing native Bcr-Abl (p210) or mutant Bcr-Abl (p210T315I) were generated by nucleofection of parental cells with the retroviral vector Mig210 or Mig210/Thr315Ile, respectively. Mig210 was a

ATO acts synergistically with CDDP to inhibit growth of Ph+ CML cell lines

In the past, both ATO and CDDP monotherapies have been clinically shown to exhibit anti-cancer activity. To assess their synergistic effects on Ph+ CML cells, we first tested the combined effect of Ato-C on the proliferation of K562, KU-812, KCL-22, and MEG-A2 cells. The combined treatment of Ato-C significantly suppressed the proliferation of all the Ph+ CML cells as compared to ATO or CDDP alone (Fig. 1A, left bar graph; Supplementary Fig. S1A), while the combined treatment partially

Discussion

In the past decade, the molecular-targeted drugs TKIs have been used as a first-line treatment for CML, but patients with relapsed CML are often suffering from TKI-resistance. To improve clinical outcomes, a novel alternative therapy is certainly warranted for patients with relapsed or refractory CML. The present study is the first to report that a novel combined Ato-C treatment synergistically inhibited growth and induced apoptosis of both parental and acquired TKI-resistant Ph+ CML cell

Ethical standards

This research complies with the ethical guidelines of the Japanese Ministry of Health, Labour, and Welfare.

Conflicts of interest

The authors declare that they have no competing interests.

Acknowledgments

We thank Dr. Masanao Okumura for animal care assistance. This work was partly supported by Grants-in-Aid for Scientific Research (KAKENHI) from the Japan Society for the Promotion of Science [15K19561 to A.O.], and the Research Grant from THE Hori Sciences and Arts Foundation [to A.O.], YOKOYAMA Foundation for Clinical Pharmacology [to A.O.] and Nagao Memorial Fund [to A.O.].

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