Cancer Letters

Cancer Letters

Volume 276, Issue 1, 8 April 2009, Pages 95-101
Cancer Letters

System L amino acid transporter inhibitor enhances anti-tumor activity of cisplatin in a head and neck squamous cell carcinoma cell line

https://doi.org/10.1016/j.canlet.2008.10.035Get rights and content

Abstract

LAT1, a subunit of heterodimeric system L transporter responsible for transporting neutral amino acids into cells, has been investigated in several cancers because of its onco-fetal nature. Based on the studies of its functional inhibition, LAT1 has been proposed to be a new molecular target of a cancer therapy. We have shown here that human head and neck cancer cell line, Hep-2, expresses both LAT1 and 4F2hc, another subunit of system L transporter. An inhibitor of system L, 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH), inhibited leucine uptake by the cells. BCH administration or restriction of essential amino acid leucine decreased viability of Hep-2 cells. Co-administration of cisplatin with BCH reduced the viability of the cells more than either agent alone. When BCH treatment preceded cisplatin administration, reduction in Hep-2 cell viability was additive. In contrast, when BCH was given after cisplatin treatment, synergistic effect in decreasing the number of viable cells was obtained. BCH treatment decreased the phosphorylation of mTOR, p70S6K and 4EBP1, suggesting that BCH enhanced anti-tumor action of cisplatin by inhibiting mTOR pathway. This potentiation may be used to reduce cisplatin exposure to alleviate many unwanted toxicity of the drug.

Introduction

Recent studies have revealed that amino acids, especially leucine, are signaling molecules which regulate cellular metabolism, gene expression, cell growth and cell survival [1], [2], [3]. The mechanisms include actions on signaling pathways involving the activation of mammalian target of rapamycin (mTOR), which integrates nutritional and growth factor signals [4], [5]. The amino acids that stimulate mTOR signaling pathway are transported into cells through plasma membrane system L amino acid transporters [6]. System L transports bulky neutral amino acids in a Na+-independent manner [6], [7]. At this moment, four isoforms of system L transporters have been identified: LAT1, LAT2, LAT3 and LAT4 [8], [9], [10], [11]. LAT1 and LAT2 are subunits of heterodimeric proteins composed of a light chain (LAT1 or LAT2) and a heavy chain (4F2hc) [12], [13]. Among them, LAT1 has been shown to be highly expressed in various tumors [14], [15], [16], [17], [18], [19], [20], [21].

Head and neck cancers occur more than 500,000 cases/year world wide [22]. The mainstay in the treatment of head and neck cancers is surgery. However, radical resections of the tumors often lead to severe functional impairments in the region. In order to preserve voice and swallowing functions, it would be desirable to minimize surgical resections by adding non-surgical therapies to treatment regimens. Among such non-surgical therapies, cisplatin-based chemotherapy is often used in combination with surgery [23], [24], [25]. However, cisplatin toxicity including renal tubular damage, bone marrow suppression and ototoxicity is a major obstacle given that many patients with head and neck cancers are old.

Previous studies suggested that the inhibition of mTOR by rapamycin administration enhances the anti-tumor effect of cisplatin in cancer chemotherapy [26], [27]. The inhibition of system L amino acid transporter is expected to cause the similar effect on cisplatin action because the transporter, which allows amino acids to enter the cells, is the upstream of mTOR in amino acid signaling [2]. In this study, to explore potential means to reduce cisplatin dose without compromising its anti-cancer activity, we examined whether an inhibitor of system L transporter LAT1, 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH), sensitizes head and neck cancer cell line to cisplatin.

Section snippets

Reagents

Cisplatin and dithiothreitol (DTT) were purchased from Wako. All the other reagents were purchased from Sigma unless otherwise indicated.

Cell culture

Hep-2 cells derived from human laryngeal squamous cell carcinoma [28] (American Type Culture Collection) were cultured in Eagle’s Minimum essential medium (MEM) with 10% fetal bovine serum (FBS, Invitrogen), 2.2 g/l, sodium bicarbonate, 100 U/ml penicillin and 100 μg/ml streptomycin. The cells were maintained in a 37 °C humidified incubator with 5% CO2. In the

Hep-2 cells expressed LAT1 and 4F2hc

It has been shown that LAT1 is expressed in fetal tissues and in various cancers. We examined the presence of LAT1 and 4F2hc in Hep-2 cells by western blot analysis. As shown in Fig. 1A, the antibodies against LAT1 and 4F2hc both recognized the 125-kDa-protein in the non-reducing condition (DTT−), while a 37-kDa-protein for LAT1 and an 85-kDa-protein for 4F2hc were detected in the reducing condition (DTT+), suggesting that Hep-2 cell expresses both LAT1 and 4F2hc and that they form a

Discussion

We have shown that a human head and neck cancer cell line, Hep-2, expresses LAT1 and 4F2hc and that the transporter formed as a heterodimer of these molecules plays an important role in tumor cell viability through amino acid transport function. In agreement with other reports on various LAT1 positive tumors or tumor cell lines, targeting system L transporter by relatively specific inhibitor BCH resulted in decreased tumor cell viability [17], [18]. Furthermore, for the first time to our

Conflicts of interest

None declared.

Acknowledgements

This work was support in part by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan, the Japan Society for the Promotion of Sciences and Setsuro Fujii Memorial the Osaka Fundation for Promotion of Fundamental Medical Research.

References (43)

  • I. Beuvink et al.

    The mTOR inhibitor RAD001 sensitizes tumor cells to DNA-damaged induced apoptosis through inhibition of p21 translation

    Cell

    (2005)
  • J.H. Yoon et al.

    Expression and functional characterization of the system L amino acid transporter in KB human oral epidermoid carcinoma cells

    Cancer Lett.

    (2004)
  • N. Hay et al.

    Upstream and downstream of mTOR

    Genes Dev.

    (2004)
  • Y. Ishizuka et al.

    Leucine induces phosphorylation and activation of p70S6K in cortical neurons via the system L amino acid transporter

    J. Neurochem.

    (2008)
  • E. Petroulakis et al.

    mTOR signaling: implications for cancer and anticancer therapy

    Br. J. Cancer

    (2006)
  • R.J. Shaw et al.

    Ras, PI(3)K and mTOR signalling controls tumour cell growth

    Nature

    (2006)
  • H.N. Christensen

    Role of amino acid transport and countertransport in nutrition and metabolism

    Physiol. Rev.

    (1990)
  • D.L. Oxender et al.

    Evidence for two types of mediation of neutral and amino-acid transport in Ehrlich cells

    Nature

    (1963)
  • Y. Kanai et al.

    Heterodimeric amino acid transporters: molecular biology and pathological and pharmacological relevance

    Curr. Drug Metab.

    (2001)
  • F. Verrey et al.

    CATs and HATs: the SLC7 family of amino acid transporters

    Pflugers Arch.

    (2004)
  • D.K. Kim et al.

    Characterization of the system L amino acid transporter in T24 human bladder carcinoma cells

    Biochim. Biophys. Acta

    (2002)
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