Elsevier

Life Sciences

Volume 183, 15 August 2017, Pages 60-68
Life Sciences

Pentoxifylline induces apoptosis of HepG2 cells by reducing reactive oxygen species production and activating the MAPK signaling

https://doi.org/10.1016/j.lfs.2017.05.029Get rights and content

Abstract

Aims

Pentoxifylline (PTX) is a methylxanthine derivative and has potent anti-tumor activity. This study aimed at investigating the anti-HCC effects of PTX and associated molecular mechanisms.

Main methods

The effects of varying doses of PTX on viability, cell cycle and apoptosis of HepG2 cells were determined by MTT and flow cytometry, respectively. The effects of PTX on the production of reactive oxygen species (ROS), expression of pro- and anti-apoptotic regulators and activation of the MAPK signaling in HepG2 cells were analyzed by flow cytometry and Western blot assays. The effects of PTX on the growth of implanted HepG2 cells and their apoptosis in mice were examined.

Key findings

Our results indicated that PTX inhibited proliferation of HepG2 cells and induced HepG2 cell cycle arrest at G0/G1 phase and apoptosis in a dose- and time-dependent manner. Treatment with PTX reduced levels of ROS and Bcl-XL expression, but increased caspase 3 and caspase 9 expression and JNK and ERK1/2 phosphorylation in HepG2 cells. Pre-treatment with n-acetyl-l-cysteine (NAC), a ROS scavenger, enhanced PTX-mediated cell cycle arrest, apoptosis and the JNK and ERK MAPK activation, while pre-treatment with SP600125 or PD98509 attenuated PTX-mediated effects in HepG2 cells. Treatment with PTX inhibited the growth of implanted HCC and promoted HCC apoptosis in mice.

Significance

Our data demonstrate that PTX inhibits proliferation of HepG2 cells and induces HepG2 cell apoptosis by attenuating ROS production and enhancing the MAPK activation in HepG2 cells.

Introduction

Hepatocellular carcinoma (HCC) is a common cancer in the world with an increasing incidence and is associated with high morbidity and mortality [1]. Currently, therapeutic strategies for HCC include tumor resection, pre-operative portal vein embolization, percutaneous radiofrequency, cryoablation, alcohol or microwave ablation, arterially directed therapies, external-beam radiation therapy (EBRT), local chemotherapy, liver transplantation, targeted therapies and immunotherapy. However, the therapeutic efficacy of these strategies is limited. It is notable that some traditional Chinese medicines have potent anti-tumor effects [2], [3]. Discovery and validation of new therapeutic medicines is of significance in management of patients with HCC [4], [5].

Pentoxifylline (1-[5-oxohexyl]-3,7-dimethyl-xanthine], oxpentifylline, PTX) is a derivative of methylxanthine (Fig. 1A). Functionally, PTX can inhibit the activity of phosphodiesterase, increase intracellular cAMP concentrations, and has been previously used to treat peripheral vascular disease and intermittent claudication [6]. Previous studies have shown that treatment with PTX at a therapeutic dose protects from malathion-induced oxidative damage in rat liver and promotes the regeneration of hepatocytes in animal models [7], [8]. Furthermore, PTX has inhibits TNF-α production in macrophages and improves graft survival of orthotopic liver transplantation in rats [9]. In addition, PTX prevents the development of a hyperdynamic circulatory state and hepatopulmonary syndrome in cirrhotic rats [10] and inhibits stellate cell proliferation [11]. Moreover, PTX can inhibit the proliferation of various types of cancer cells and enhance the sensitivity of tumor cells to chemotherapeutic reagents and radiotherapy [12], [13]. PTX has potent anti-oxidant and anti-inflammatory activity, and has been shown to inhibit cell adhesion [14], [15]. However, little is known on the molecular mechanisms underlying the action of PTX in inhibiting HCC proliferation and inducing HCC apoptosis.

Therapeutic strategies to induce tumor cell apoptosis have been thought to be one of the effectively therapeutic methods to treat cancer [16]. Apoptosis is a process regulated by a series of enzymes and genes under physiological or pathological condition [17]. Apoptosis is mediated by caspase cascade and regulated by the MAPK signaling [18], [19]. Indeed, several therapeutic reagents directly target the MAPK signaling [20]. In addition, while high levels of ROS are toxic to HCC, through inducing HCC cell apoptosis, low to moderate levels of ROS may promote the growth of HCC [15], [21], [22]. Previous studies have shown that PTX can attenuate ROS production [23], [24]. We hypothesize that PTX may enhance the MAPK signaling and inhibit ROS production to promote apoptosis of HCC cells.

In this study, we employed HCC HepG2 cell line to determine the therapeutic effect of different doses of PTX and their molecular mechanisms in vitro and in vivo.

Section snippets

Reagents

Pentoxifylline (PTX), DCFH-DA (2′,7′-dichlorodihydrofluorescein diacetate) and NAC (N-acetyl-l-cysteine) were purchased from Sigma-Aldrich (St. Louis, USA). SP600125 and PD98059 were purchased from Beyotime (Nanjing, Jiangsu, China). Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit was purchased from BD Biosciences (San Jose, USA). MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was obtained from Biomart (Wuhan, Hubei, China). Primary antibodies against

PTX inhibits proliferation of HepG2 cells

To determine the potential toxicity of PTX, HepG2 cells were treated with vehicle or varying concentrations of PTX at 1, 2, 4, 8 or 16 mM for 24, 48 or 72 h in FSB-free medium (SFM) or complete medium. Proliferation of individual groups of cells was determined by MTT and the inhibition rates of different concentrations of PTX on proliferation of HepG2 cells were calculated in Fig. 1B. Treatment with PTX in SFM inhibited proliferation of HepG2 cells in a dose- and time-dependent manner. The IC50

Discussion

PTX is an alkaloid, which derived theobromine from coffee beans, joined the ethyl ketone in [6]. PTX has anti-inflammatory, anti-fibrotic and anti-tumor activity and has been used for the treatment of hemodynamic disorders, peripheral vascular disease and a variety of other conditions [30], [31]. Previous studies have shown that treatment with PTX benefits patients with NAFLD [32], [33]. PTX treatment decreased hepatic contents of serum triglyceride, total cholesterol, free fatty acids, and

Conclusions

In summary, our data demonstrated that PTX had potent cytotoxicity against HepG2 cells in vitro and in vivo by inducing HepG2 cell cycle arrest at G0/G1 phase and apoptosis. Furthermore, the mechanisms underlying the therapeutic effects of PTX were associated with a reduction in ROS generation and activation of JNK and ERK signaling in HepG2 cells. Therefore, PTX may be a valuable therapy for the future treatment of HCC.

Abbreviations

    PTX

    pentoxifylline

    SFM

    free medium

    FBS

    fetal bovine serum

    IC50

    50% inhibitory concentration

    ROS

    reactive oxygen species

    MAPK

    mitogen-activated protein kinases

    ERK1/2

    extracellular stress-related kinase-1/2

    p-ERK1/2

    phospho-ERK1/2, phospho-extracellular stress-related kinase-1/2

    JNK

    c-Jun N-terminal kinase

    p-JNK

    phospho-JNK, phospho-c-Jun N-terminal kinase

    P38

    p38MAPK, p38 mitogen-activated protein kinase

    p-P38

    p-p38MAPK, phospho- p38 mitogen-activated protein kinase

    PD

    PD98059, ERK specific inhibitor

    SP

    SP600125, JNK

Conflict of interest statement

None.

Author's contribution

Yan Wang and Lei Dong conceived and designed the experiments; Yan Wang, Jing Li and Boxin Shang performed the experiments; Yan Wang analyzed the data and wrote the paper; Lei Dong and Miaosha Luo contributed reagents/materials/analysis tools.

Acknowledgements

This study was supported by ‘Twelfth Five-Year’ National Science and Technology Support Program (No. 2012BAJ18B03-03).

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