Enhancement of cytotoxicity of natural product drugs against multidrug resistant variant cell lines of human head and neck squamous cell carcinoma and breast carcinoma by tesmilifene

doi:10.1016/j.canlet.2008.09.021

Cancer Letters

Volume 274, Issue 2, 18 February 2009, Pages 279-289

https://doi.org/10.1016/j.canlet.2008.09.021 Get rights and content

Abstract

N,N-diethyl-2-[4-(phenylmethyl)phenoxyl]ethanamine (tesmilifene), a tamoxifen derivative with antihistamine activity, greatly enhanced the survival of doxorubicin-treated, advanced stage breast cancer patients in a phase III trial. However, the molecular basis of tesmilifene action is not firmly established. The effects of tesmilifene on activity of several anticancer drugs was investigated using human head and neck squamous cell carcinoma (HNSCC) and breast carcinoma cell lines as a model system. Multidrug resistant (MDR) variants of an HNSCC cell line, HN-5a/V15e, and a breast carcinoma cell line, MCF-7/V25a, both highly overexpressed mdr1 (ABCB1) mRNA and the proteins P-glycoprotein and glutathione transferase-pi. Drug sensitivities were measured by a vital stain after 4 days of continuous exposure to anticancer drug in the absence and presence of tesmilifene at a concentration that alone had no antiproliferative effect. Tesmilifene had minimal effect on drug cytotoxicity against the parental cell lines. However, the same tesmilifene treatment enhanced cytotoxicity of docetaxel, paclitaxel, epirubicin, doxorubicin, and vinorelbine against both MDR cell lines by up to 50%. Flow cytometric measurement of annexin V/propidium iodide staining demonstrated that tesmilifene increased the killing of HN-5a/V15e cells caused by docetaxel after 24 and 48 h exposure. Tesmilifene increased accumulation of radiolabelled vincristine in HN-5a/V15e cells, over 4 h, by up to 100%. The results suggest that tesmilifene might be effective in the treatment of tumors that are resistant to natural product drugs. The mechanism of enhancement appears to be related to expression of an ABC pump-dependent, MDR phenotype.

Introduction

The recurrence of patient tumors due to propagation of cryptic colonies of drug-resistant cells remains a formidable obstacle to the success of anticancer chemotherapy. Although much work has been undertaken to develop modulators of resistance, their use clinically has been limited by a lack of selectivity of enhancement of drug toxicity specifically to cancer cells. However, several compounds have shown promise as selective enhancers of antitumor effect. The antihistamine l-histidinol, when administered to tumor-bearing mice, increased the antitumor effect of antimetabolites, alkylating agents, and anthracyclines, while at the same time protecting bone marrow cells from toxicity [1]. Another compound with antihistamine activity as well as binding activity to anti-estrogen receptors is the tamoxifen derivative tesmilifene {N,N-diethyl-2-[4-(phenylmethyl)phenoxy]ethanamine; DPPE} [2]. Tesmilifene has been shown to enhance the cytotoxicity of doxorubicin against tumors in mice, and, like l-histidinol, it protected bone marrow progenitor cells [3]. Tesmilifene is beneficial in the clinical treatment of breast cancer when used in combination with doxorubicin [4] and possibly also with mitoxantrone against prostate cancer [5]. However, information is limited about the tumor types and drug classes with which tesmilifene could interact to increase drug efficacy as well as the mechanism by which this occurs.

A preliminary report suggested that tesmilifene acted as a classical P-glycoprotein (Pgp) inhibitor, enhancing drug accumulation in Pgp-overexpressing cells [6]. However, these experiments were conducted with concentrations of tesmilifene that are much higher than those achievable clinically, and did not necessarily represent the mechanism responsible for the clinical antitumor activity of tesmilifene. Therefore, it was necessary to demonstrate whether tesmilifene, at clinically achievable concentrations, could enhance drug cytotoxicity against a cell culture model expressing a moderate, clinically relevant level of Pgp-dependent multidrug resistance. This model could then be investigated to gain some insight as to how tesmilifene enhanced cytotoxicity under these conditions.

Preliminary work from this laboratory demonstrated that tesmilifene, although not cytotoxic itself at clinically achievable concentrations (up to 7.5 μM), was able to enhance docetaxel cytotoxicity against a multidrug resistant (MDR) variant of a head and neck squamous cell carcinoma (HNSCC) cell line (HN-5a/V15e) [7]. The data suggested that enhancement of drug cytotoxicity by tesmilifene was a function of whether a cell line expressed Pgp, and whether the drug with which it was being used was a substrate for Pgp. This study has been expanded to determine whether this phenomenon occurs with other cell lines and other chemotherapy agents, and to better understand the mechanism responsible.

It was undertaken to test tesmilifene with a series of natural product drugs against both the HNSCC MDR variant, and against an MDR variant derived from the breast carcinoma cell line MCF-7. The results of these studies suggest that tesmilifene enhances natural product drug cytotoxicity by, at least in part, enhancing cellular drug accumulation, potentially through an inhibitory effect on Pgp-dependent drug efflux. Tesmilifene may be an important addition to the treatment of drug-refractory tumors and of occult colonies of drug-resistant cells in primary tumors.

Section snippets

Materials

Cell culture medium and fetal bovine serum were purchased from Invitrogen, Inc. (Burlington, Ont., Canada). Cell culture plasticware was obtained from Invitrogen, Fisher Scientific (Unionville, Ontario), and VWR Canlab (Mississauga, Ontario). Tesmilifene was provided in solution by YM Biosciences, Inc. Other drug stock solutions were purchased from the London Regional Cancer Program pharmacy.

Cell lines

The human HNSCC cell line HN-5a and its vincristine-resistant variant line, as well as the

Characterization of MDR cell lines

It had previously been demonstrated in this laboratory, but reported only in abstract form, that tesmilifene was able to enhance cytotoxicity of docetaxel against a vincristine-resistant variant of the HNSCC cell line HN-5a. It was therefore important to confirm this activity in a second drug-resistance cell culture model, to determine what types of anticancer drugs could be affected by this activity, and to determine a mechanism by which this occurred. To this end, it was therefore necessary

Discussion

The clinical use of tesmilifene as an adjunct to anticancer chemotherapy evolved from the original observation that l-histidinol enhanced cytotoxicity of anticancer drugs against tumor cells, while at the same time protecting normal cells, both in vitro and in vivo [1], [3]. The antihistamine tesmilifene, which is similar in structure to tamoxifen, possesses anti-estrogenic activity [3]. In mice, tesmilifene protected bone marrow cells from the cytotoxic effects of doxorubicin, and enhanced the

Conflict of interest statement

Dr. Ferguson and Mrs. Brisson were paid from an operating grant provided by YM Biosciences, Inc. Dr. Vincent is a consultant for YM Biosciences, Inc.

Funding

This study was funded in part by YM Biosciences, Inc., and by the London Regional Cancer Program, London Health Sciences Centre, London, Ontario, Canada. The decision to submit this manuscript for publication was approved by YM Biosciences, Inc., under the terms of a Material Transfer Agreement, by which tesmilifene was provided to the investigators at no charge. YM Biosciences, Inc., were not involved in planning of experiments, in collection, analysis or interpretation of data, or in the

Acknowledgements

The authors thank Carolina Phay for excellent assistance with flow cytometry. This work was supported by YM Biosciences, Inc., and the London Regional Cancer Program - London Health Sciences Centre.

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Morphology studies were performed on a Hitachi S-3400N (Ottawa, ON, Canada) with a current of 99 μA and a voltage of 20–30 kV. The in vitro cytotoxicity studies were performed using the human head and neck squamous cell carcinoma (HNSCC) cell line HN-5a [26]. The HN-5a cells were grown in α-MEM medium supplemented with 10% fetal bovine serum and antibiotics (growth medium).
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^☆: These results have been reported previously, in part, in abstract form: Proc. Am. Assoc. Cancer Res., 48: 546, 2007.

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Enhancement of cytotoxicity of natural product drugs against multidrug resistant variant cell lines of human head and neck squamous cell carcinoma and breast carcinoma by tesmilifene☆

Abstract

Introduction

Section snippets

Materials

Cell lines

Characterization of MDR cell lines

Discussion

Conflict of interest statement

Funding

Acknowledgements

J. Urol.

J. Nutr.

J. Gastroint. Surg.

Exp. Mol. Pathol.

Med. Hypotheses

FEBS Lett.

J. Biol. Chem.

A novel approach for improving the efficacy of experimental cancer chemotherapy using combinations of anticancer drugs and l-histidinol

Anticancer Res.

Results of a clinical trial in humans with refractory cancer of the intracellular histamine antagonist, N,N-diethyl-2-[4-(phenylmethyl)phenoxy]ethanamine-HCl, in combination with various single antineoplastic agents

J. Clin. Oncol.

Increased therapeutic index of antineoplastic drugs in combination with intracellular histamine antagonists

J. Natl. Cancer Inst.

Phase III study of N,N-diethyl-2-[4-(phenylmethyl)phenoxy]ethanamine (BMS-217380-01) combined with doxorubicin alone in metastatic/recurrent breast cancer, National Cancer Institute of Canada Clinical Trials Group Study MA.19

J. Clin. Oncol.