d-alpha-tocopheryl polyethylene glycol succinate (TPGS) induces cell cycle arrest and apoptosis selectively in Survivin-overexpressing breast cancer cells

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Abstract

d-alpha-tocopheryl polyethylene glycol succinate (TPGS) is a vitamin E derivative that has been intensively applied as a vehicle for drug delivery systems to enhance drug solubility and increase the oral bioavailability of anti-cancer drugs. Recently, it has been reported that TPGS acts as an anti-cancer agent alone or synergistically with chemotherapeutic drugs and increases the efficacy of nanoparticle formulations. In this study, we investigated the antitumor efficacy and the molecular mechanism of action of TPGS in breast cancer cell lines. Our results show that TPGS can induce G1/S cell cycle arrest and apoptosis in breast cancer cell lines (MCF-7 and MDA-MB-231) but not in “normal” (non-tumorigenic) immortalized cells (MCF-10A and MCF-12F). An investigation of the molecular mechanism of action of TPGS reveals that induction of G1/S phase cell cycle arrest is associated with upregulation of P21 and P27Kip1 proteins. Induction of apoptosis by TPGS involves the inhibition of phospho-AKT and the downregulation of the anti-apoptotic proteins Survivin and Bcl-2. Interestingly, our results also suggest that TPGS induces both caspase -dependent and -independent apoptotic signaling pathways and that this vitamin E derivative is selectively cytotoxic in breast cancer cell lines. When compared to the Survivin inhibitor YM155, TPGS was shown to be more selective for cancer cell growth inhibition. Overall our results suggest that TPGS may not only be useful as a carrier molecule for drug delivery, but may also exert intrinsic therapeutic effects suggesting that it may promote a synergistic interaction with formulated chemotherapeutic drugs.

Graphical abstract

TPGS suggested mechanism of action. CD-PCD; caspase-dependent programmed cell death, CI-PCD; caspase-independent programmed cell death.

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Introduction

Breast cancer is the most frequently diagnosed cancer and the leading cause of cancer death among women [1]. Breast cancer cells commonly have increased proliferation potency and activate potent anti-apoptotic pathways to promote their survival and drug resistance [2]. Conventional chemotherapeutic agents generally do not distinguish between tumor and cancer cells and often cause toxic side effects. Therefore, there is a need for the discovery of novel anti-cancer agents that inhibit proliferation and survival pathways selectively in cancer cells.

Emerging evidence suggests that vitamin E isoforms may be useful in the treatment of breast cancer [3], [4], [5]. Vitamin E exists in nature as a group of 8 isoforms: α-, β-, γ-, and δ- tocopherols and α-, β-, γ-, and δ- tocotrienols [reviewed in [6]]. Specifically, research in the past few years has focused on synthetic derivatives of α-tocopherol (α-TOC), such as α-tocopherol succinate (α-TOS) and α-tocopheryl ether-linked acetic acid (α-TEA). These compounds have shown enhanced pro-apoptotic potency and anti-cancer action in tumorigenic cell lines and animal models [4], [7], [8], [9]. Therefore, there is great interest for further evaluation of the most promising alpha-tocopherol synthetic derivatives using in vitro and in vivo systems.

d-alpha-tocopheryl polyethylene glycol succinate (TPGS) is an amphiphilic, water-soluble derivative of natural vitamin E which is gaining interest in the development of drug delivery systems [reviewed in [10]]. TPGS is prepared from the esterification of d-alpha-tocopheryl acid succinate (α-TOS) and PEG 1000. As such, it possesses the advantages of PEG and Vitamin E in applications of various nanocarriers for drug delivery, including the ability to extend the half-life of the drug in the plasma. The co-administration of TPGS has been shown to enhance drug solubility, inhibit P-glycoprotein mediated multi-drug resistance and increase the oral bioavailability of anti-cancer drugs [reviewed in [10]]. As an effective emulsifier, TPGS has been shown to greatly enhance the performance of nanoparticles, resulting in increased drug cellular uptake and improved in vivo pharmacokinetics [10]. Importantly, the United States’ Food and Drug Administration (FDA) as well as the European Food Safety Authority (EFSA), have already approved TPGS as a safe pharmaceutical adjuvant used in drug formulation and have estimated the safety limits for TPGS use for research purposes [11], [12].

Based on the multi-functional nature of TPGS, its displayed synergistic effectiveness with anti-cancer drugs and its potent apoptotic effects in prostate cancer cell lines [13], it is important to investigate the efficacy of this compound in breast cancer cells and to determine if its effects are cancer cell specific. It is also crucial to elucidate the underlying mechanism by which TPGS mediates its anti-cancer effects so that this compound can be combined with other chemotherapeutic agents to provide maximum anti-cancer effectiveness.

Our findings clearly demonstrate that TPGS induces G1-phase cell cycle arrest and apoptotic cell death in breast cancer cells. The ability of a carrier compound to also act as an anti-cancer agent provides new insights and a rationale for the development of effective cancer-cell-selective therapies with diminished side effects.

Section snippets

Cell cultures and reagents

MCF-7, MDA-MB-231, BT-474, SKBR3, MDA-MB-361, MCF-10A and MCF-12F cell lines were obtained from the American Type Culture Collection (ATCC) (Manassas, VA). MCF-7 and MDA-MB-231 cells were cultured in DMEM supplemented with 10% FBS and 1% antibiotic/antimycotic, BT-474 and MDA-MB-361 cells in DMEM-F12 supplemented with 10% FBS, 1% antibiotic/antimycotic and 4 mM l-glutamine, SKBR3 in McCoys media supplemented with 5% FBS and 1% antibiotic/antimycotic, MCF-10A and MCF-12F in DMEM-F12 supplemented

TPGS causes induction of cell cycle arrest and apoptosis in breast cancer cell lines

The effects of TPGS on cell viability were tested on five human breast cancer cell lines (SKBR-3, BT-474, MDA-MB-361, MCF-7 and MDA-MB-231) and two “normal” immortalized cell lines (MCF-10A and MCF-12F). TPGS caused a decrease in cell viability in all breast cancer lines tested at 24 and 48 h (Table 1). Specifically, the order of cell line sensitivity to the drug after 48 h of treatment was as follows: MCF-12F < MCF-10A < BT-474 < MDA-MB-361 < MCF-7 < MDA-MB-231 < SKBR3.

The response of the “normal”

Discussion

Vitamin E natural isoforms and synthetic derivatives are showing great promise as anti-cancer agents [6]. The FDA- and EFSA-approved use of TPGS as an adjuvant in drug formulations and delivery systems (either as coated liposomes or micellar formulation) increases the need to know exactly how it works at the molecular level.

In the present study we evaluated the molecular mechanism of action of TPGS in breast cancer cells. We show here that TPGS induces G1-phase cell cycle arrest and caspase-

Conflict of interest

The authors declare no conflict of interest.

Acknowledgments

The authors wish to thank Dr Paul Costeas, Dr Laoura Koumas and Dr Carsten Lederer for their help with flow cytometric analysis. The authors also thank Dr Chara Pitta and Christiana Savva for technical assistance. This work was supported by funding from the Department of Biological Sciences of the University of Cyprus.

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