Effects of pergolide mesylate on transduction efficiency of PEP-1-catalase protein

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Abstract

The low transduction efficiency of various proteins is an obstacle to their therapeutic application. However, protein transduction domains (PTDs) are well-known for a highly effective tool for exogenous protein delivery to cells. We examined the effects of pergolide mesylate (PM) on the transduction of PEP-1-catalase into HaCaT human keratinocytes and mice skin and on the anti-inflammatory activity of PEP-1-catatase against 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation using Western blot and histological analysis. PM enhanced the time- and dose-dependent transduction of PEP-1-catalase into HaCaT cells without affecting the cellular toxicity. In a mouse edema model, PEP-1-catalase inhibited the increased expressions of inflammatory mediators and cytokines such as cyclooxygenase-2, inducible nitric oxide synthase, interleukin-6 and -1β, and tumor necrosis factor-α induced by TPA. On the other hand, PM alone failed to exert any significant anti-inflammatory effects. However, the anti-inflammatory effect of co-treatment with PEP-1-catalase and PM was more potent than that of PEP-1-catalase alone. Our results indicate that PM may enhance the delivery of PTDs fusion therapeutic proteins to target cells and tissues and has potential to increase their therapeutic effects of such drugs against various diseases.

Research highlights

► We studied effects of pergolide mesylate (PM) on in vitro and in vivo transduction of PEP-1-catalase. ► PEP-1-catatase inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation. ► PM enhanced the transduction of PEP-1-catalase into HaCaT cells and skin tissue. ► PM increased anti-inflammatory activity of PEP-1-catalase. ► PM stimulated therapeutic action of anti-oxidant enzyme catalase in oxidative-related diseases.

Introduction

Reactive oxygen species (ROS) such as hydroxyl radicals, superoxide anion and hydrogen peroxide, are produced from various normal cellular processes and lead to cell damage, cell death and consequently, several diseases including inflammation, cancer, allergy, and ischemia [1], [2], [3]. Anti-oxidant enzymes, like superoxide dismutase (SOD), catalase, and glutathione peroxidase play important roles in decreasing overproduced ROS, maintaining a balance of the redox state and protecting cells against numerous sources of oxidative damage [4], [5], [6], [7]. This suggests that these anti-oxidant enzymes may be useful as therapeutic molecules. However, delivering several proteins with therapeutic potential into cells is difficult due to their size and biochemical properties, making it problematic to utilize such proteins as therapeutic drugs [8].

Many researchers have reported that therapeutic proteins fused with protein transduction domains (PTD) such as Tat and PEP-1 can be efficiently delivered into cells [9], [10], [11], [12], [13], [14], [15], [16]. Also, we constructed the expression vectors of PEP-1-catalase, PEP-1-rpS3, and Tat-SOD fusion proteins. PEP-1-catalase fusion protein was reported to be rapidly transduced into astrocytes and gerbil brain and protected neuronal cells against ischemic damage [17]. The transduction of Tat-SOD into pancreatic beta cells improved the diabetic status of mice in a diabetic mouse model [18]. In addition, transduced PEP-1-rpS3 prevented TPA-induced skin inflammation in a mice model by inhibiting the expression of pro-inflammatory mediator and cytokines such as cyclooxygenase-2 (COX-2), interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) [19].

Dopamine receptor agonists are divided into ergolines and non-ergolines. Ergolines are derived from ergot alkaloids and include bromocriptine, pergolide, lisuride and cabergoline. They bind to dopaminergic D1 receptors, adrenergic and 5-hydroxytryptamine receptors as well as dopaminergic D2 receptors. Pergolide mesylate (PM) is a synthetic ergoline derivative and is used in the treatment of Parkinson’s disease (PD) [20]. Also, some studies have reported that pergolide has free radical scavenging activity and an inhibitory activity on phospholipid peroxidation in rat brain and induces Cu,Zn-SOD activity in rat striatum [21], [22]. It exerts its neuroprotective activity by interfering with NF-κB translocation in neuronal cell line exposed to H2O2[23], [24], [25]. However, the effect of PM on the cellular membrane conformation and its anti-inflammatory effect on skin cells and tissue are still unknown.

Therefore, in this study, we investigated whether PM could enhance the transduction efficiency and thereby the anti-inflammatory effects of PEP-1-catalase. Subsequently we found that PM enhanced the transduction efficiency of PEP-1-catalase in vitro and in vivo, and that in turn increased the efficiency with which it inhibited the expression levels of anti-inflammatory mediators and cytokines.

Section snippets

Materials

PM was purchased from Sigma–Aldrich (St. Louis, MO, USA). Primary antibodies against COX-2, iNOS, IL-6, IL-1β and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All other chemicals and reagents were obtained from Sigma–Aldrich (MO, USA) unless otherwise stated.

Cell culture and MTT assay

HaCaT human keratinocytes were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% heat-inactivated fetal bovine serum and antibiotics (100 μg/ml streptomycin and 100 U/ml penicillin) and kept at 37

Effect of PM on in vitro and in vivo transduction efficiency of PEP-1-catalase

Protein transduction domains (PTDs) such as Tat and PEP-1 are able to deliver exogenous macromolecules to cells and animals [9], [15], [16], [28]. Several studies have suggested that PTD fusion therapeutic molecules may be useful drugs against various disorders [29], [30], [31], [32]. Anti-oxidant enzymes, such as superoxide dismutase (SOD), catalase and glutathione peroxidase, are known to maintain the cellular redox balance and work as a cellular defenses mechanism. Earlier we reported that

Acknowledgments

This work was supported by the Priority Research Centers Program grant (2009-0093812) and by the Regional Research Universities Program/Medical & Bio-material Research Center grant through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology.

References (40)

1

These authors equally contributed to this work.

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