Pentoxifylline induces apoptosis of HepG2 cells by reducing reactive oxygen species production and activating the MAPK signaling☆
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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The experimental protocol was approved by the Animal Care and Research Committee of Xian Jiaotong University.