Cancer Letters

Cancer Letters

Volume 375, Issue 2, 1 June 2016, Pages 199-208
Cancer Letters

Original Articles
Novel anticancer compound [trifluoromethyl-substituted pyrazole N-nucleoside] inhibits FLT3 activity to induce differentiation in acute myeloid leukemia cells

https://doi.org/10.1016/j.canlet.2016.02.028Get rights and content

Highlights

  • G-11 is a novel trifluoromethyl-substituted pyrazole N-nucleoside showing strong and selective antiproliferative activities against various hematological and solid tumor cell lines.

  • G-11 induces cellular differentiation of the promyelocytic leukemia HL-60 cells to monocyte/macrophage and granulocyte.

  • G-11 inhibits the induced activation of the oncogenic kinase FLT3 (FMS-like tyrosine kinase 3, CD135), and thereby promotes differentiation of HL-60 cells.

  • In addition to the wild-type FLT3, G-11 also inhibits the constitutively active mutant type of FLT3 containing internal tandem repeats mutations (ITD).

  • Molecular Modeling studies show that G-11 docks within the ATP binding pocket of FLT3 in a comparable manner to the anticancer drug quizartinib, thus providing additional evidence supporting the development of G-11 as an anticancer agent for relapse free therapeutic strategies of AML patients.

Abstract

Anticancer properties of chemically synthesized compounds have continuously been optimized for better efficacy and selectivity. Derivatives of heterocyclic compounds are well known to have selective antiproliferative effect against many types of cancer. In this study, we investigated the ability of an indigenously synthesized anticancer molecule, G-11 [1-(2”,3”,4”,6”-Tetra-O-acetyl-β-D-glucopyranosyl)-4-(3'-trifluoromethylphenylhydrazono)-3-trifluoromethyl-1,4-dihydropyrazol-5-one], to cause selective cytotoxicity and induce differentiation in the acute myeloid leukemia HL-60 cells. G-11 was able to exert cytotoxic effect on hematological (Jurkat, U937, K562, HL-60, CCRF-SB) and solid tumor (MCF-7, HepG2, HeLa, Caco-2) cell lines, with IC50 values significantly lower than noncancerous cells (HEK-293, BJ and Vero) and normal peripheral blood mononuclear cells. G-11 induced differentiation of HL-60 cells to granulocytes and monocytes/macrophages by inhibiting the activation of FLT3 (CD135 tyrosine kinase). ITD-FLT3 mutation found in many acute myeloid leukemia patients could also be targeted by G-11 as exhibited by its inhibitory effect on MOLM-13 and MV4-11 cell lines. Molecular docking studies suggest the involvement of Leu616, Asp698, Cys694 and Cys828 residues in binding of G-11 to FLT3. The ability of G-11 to cause selective cytotoxicity and induce differentiation in cancer cells could be clinically relevant for therapeutic gains.

Introduction

Chemotherapy is one of the most significant treatment modalities in cancer management. It has been realized that the principal obstacle to the clinical efficacy of chemotherapy is due to possible toxicity to normal tissues of the body and development of drug resistance [1], [2], [3], [4], [5]. To overcome these obstacles, the design and discovery of non-traditional, efficient and safe chemical agents is the prime objective of contemporary medicinal chemistry. Over the past two decades, heterocyclic compounds containing pyrazole have received considerable attention owing to their diverse chemotherapeutic potential, including versatile antineoplastic activities. Nucleoside analogs are a pharmacologically diverse family, which includes cytotoxic compounds, antiviral agents, and immunosuppressive molecules. The anticancer nucleosides include several analogs of physiological pyrimidine and purine nucleosides and nucleobases [6]. Initially, nucleobase analogues, such as fluorinated pyrimidines, were investigated as antimetabolite chemotherapeutic agents on cancer cells. Later, pyrimidine analogs-Ara-C and Gemcitabine were used in cancer therapy [7]. Among the currently available pyrimidine analogs, cytarabine is extensively used in the treatment of acute leukemia; gemcitabine has activity in various solid tumors and some hematological malignant diseases; and the fluoropyrimidines-fluorouracil and capecitabine have shown activity in colorectal and breast cancers [8], [9]. Success with pyrimidine nucleoside analogs in cancer therapy led to the discovery of purine nucleoside analogs. For more than six decades, 6-mercaptopurine and 6-thioguanine have been used as inhibitors of nucleic acid metabolism in acute lymphoblastic leukemia [10], [11]. Currently, the purine nucleoside analogs fludarabine, cladribine, and pentostatin are used for treating hematological malignancies [11], [12]. Pyrazole derived molecules have been used as anti-leukemic [13], [14], [15] antitumor [16], [17], [18], [19] and anti-proliferative agents [20], [21].

A hallmark of cancer is disruption of differentiation within cancer cells. Internal tandem duplication (ITD) mutations of the FLT3 (FMS-like tyrosine kinase 3, CD135) occur commonly in acute myeloid leukemia (AML) and are associated with poor survival. Inhibition of FLT3 in ITD mutant cell line caused terminal myeloid differentiation in vivo encouraging efforts to develop FLT3 inhibitors [22]. Studying the effect of chemotherapeutic agents on cell differentiation is significant in terms of understanding the development of drug resistance over the course of chemotherapy. There have been reports about cell differentiation caused by purine analogs in HL-60 cells [23] and small cell lung carcinoma (SCLC) [24]. Developing chemotherapeutic agents which effectively lead to differentiated cells and stop proliferation will be vital in developing resistance free therapy for cancer. Differentiation of immune cells would add another dimension of the effect of these chemotherapeutic agents acting via changes in immune response.

Molecular docking allows prediction of ligand binding poses (position, orientation and conformation) and estimation of ligand binding affinities within certain targeted receptor(s). These are essential prerequisites towards designing and subsequent optimization of novel bioactive compounds including new anticancer agents [25]. Docking involves fitting virtual ligands, usually derived from large virtual libraries, into targeted binding sites employing computer algorithms. Docking algorithms rely on force fields to calculate attractive and repulsive interactions within virtual ligand-protein complexes [26].

We recently reported anticancer activity of several new trifluoromethyl-substituted pyrazole N-nucleosides and their corresponding glucoside derivatives [27]. One of the potential molecules named G-11 [1-(2”,3”,4”,6”-Tetra-O-acetyl-β-D-glucopyranosyl)-4-(3'-trifluoromethylphenylhydrazono)-3-trifluoromethyl-1,4-dihydropyrazol-5-one] (Fig. 1A) has been successfully tested to cause apoptosis mediated cell death in HL-60 cells (manuscript in communication). In this report, we provide molecular evidence for G-11 induced cell differentiation and cytotoxicity. Its mechanism of action involves FLT3 inactivation that could lead to differentiation. Commonly found mutant FLT3 in AML patients can be targeted with significant efficacy as suggested by the effect of G-11 on representative cell lines. Molecular docking studies provide detailed insights about the binding of G-11 to its possible target molecule – FLT3, in a way that is similar to the latest promising FLT3 inhibitor – quizartinib. Our findings thus suggest G-11 as a novel anticancer molecule that has potential for selectively targeting hematological and epithelial malignancies. The ability of G-11 to induce differentiation in HL-60 cells makes it a promising candidate to develop relapse free therapeutic regimen for AML and probably other types of cancer.

Section snippets

Reagents and antibodies

Synthesis and characterization of G-11 [1-(2”,3”,4”,6”-Tetra-O-acetyl-β-D-glucopyranosyl)-4-(3'-trifluoromethylphenyl hydrazono)-3-trifluoromethyl-1,4-dihydropyrazol-5-one] was previously described [27]. NBT (Nitroblue-tetrazolium), TPA [1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3), 12-O-tetradecanoylphorbol-13-acetate], etoposide, DMSO (dimethyl sulfoxide), components of lysis buffers, protease inhibitors (PMSF, pepstatin A, leupeptin, and aprotinin), phosphatase inhibitor cocktail-2, denaturing

G-11 is more cytotoxic to cancer cell lines than normal cells

We analyzed the effect of G-11 on cell proliferation in nine different human cancer and three mammalian non-cancerous cell lines. Five human blood-derived cancer cell lines; Jurkat [acute T cell leukemia], K562 [chronic myelogenous leukemia], U937 [B-cell lymphoma (histiocytic lymphoma)], HL60 [promyelocytic leukemia] and CCRF-SB [acute B-lymphoblastic leukemia] and four solid tumor-derived cell lines; MCF-7 [breast adenocarcinoma], HepG2 [hepatocellular carcinoma], HeLa [cervical

Discussion

We have earlier demonstrated the potential capacity of an indigenously synthesized heterocyclic compound ‘G-11’ to act as anticancer agent [27] and studied its mechanism of causing cell death (manuscript in communication). In this report (i) we establish the selective anticancer property of G-11 in different types of cancer cells by studying its cytotoxic effect, (ii) implicate FLT3 as mediator of cell differentiation caused by G-11, and (iii) suggest molecular details of G-11 binding to FLT3

Funding

This project is supported by a research grant from King Abdulaziz City for Science and Technology (KACST, Riyadh, Saudi Arabia; Grant number- AT-34-136) for Saleh AM. Publication cost is supported by King Abdullah International Medical Research Center (KAIMRC) in Riyadh, Saudi Arabia.

Authors' contributions

AMS obtained the funds, designed the research project, supervised all experiments and drafted the manuscript; MOT and RAT performed the docking study; MAA planned and edited the manuscript, and generated the scheme for FLT3 signaling; MAQ and SAR characterized G-11 molecule. All authors reviewed the manuscript.

Conflict of interest

The authors declare that they have no competing interests.

Acknowledgments

We are grateful to Dr. Ibrahim M Abdou from United Arab Emirates University for providing the G-11 compound.

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