Drug Deliv. and Transl. Res. DOI 10.1007/s13346-015-0230-6
SHORT COMMUNICATION
TAT cell-penetrating peptide modulates inflammatory response and apoptosis in human lung epithelial cells Hyunhee Kim 1,2 & Serisha Moodley 1,3 & Mingyao Liu 1,2,3,4
# Controlled Release Society 2015
Abstract Cell-penetrating peptides (CPPs) are commonly used as delivery vehicles for the introduction of a variety of macromolecules into cells. Trans-activator of transcription (TAT) is the most commonly used CPP and, as a delivery vehicle, is assumed to be biologically inert. In this study, we pretreated human lung epithelial cells with TAT prior to stimulation with phorbol 12,13-dibutyrate (PDBu), a protein kinase C (PKC) activator. Surprisingly, TAT alone inhibited the production of multiple cytokines induced by PKC activation. Furthermore, PKC activation-induced IκBα degradation was partially reduced by TAT. Moreover, TAT treatment alone induced apoptosis in a dose-dependent manner, influenced expression of several B cell lymphoma 2 (Bcl-2) family members and increased caspase 3 cleavage at a high dose. These findings suggest that TAT as a delivery vehicle should be used cautiously, as it may affect the inflammatory response, as well as signals related to apoptosis.
Keywords TAT cell-penetrating peptide . Apoptosis . Inflammation * Mingyao Liu [email protected] 1
Latner Thoracic Surgery Research Laboratories, Toronto General Research Institute University Health Network, Toronto, Ontario, Canada
2
Department of Physiology, University of Toronto, Toronto, Ontario, Canada
3
Institute of Medical Science, University of Toronto, Toronto, Ontario, Canada
4
Department of Surgery, Faculty of Medicine, University of Toronto, 101 College Street, Room TMDT 2-814, Toronto, Ontario M5G 1L7, Canada
Since its discovery in 1988, as a part of HIV-1 virus regulatory protein, trans-activator of transcription (TAT) peptide is known to contain a protein transduction domain and, thus, is considered to be a cell-penetrating peptide (CPP) that is a useful tool for the intracellular delivery of macromolecules [1]. The arginine-rich transduction domain, YGRK KRRQRRR, allows TAT-conjugated molecules to enter cells via micropinocytosis and other transfer mechanisms [2]. So far, TAT CPP has been used to introduce a variety of molecules, such as peptides, proteins, siRNAs, antisense oligonucleotides, nanoparticles, polymers, liposomes, as well as therapeutic and diagnostic reagents [3]. Clinical trials using TAT CPP-conjugated peptides have been conducted to test transdermal delivery and treatment of skin disease, and a TATprotein kinase C (PKC) inhibitor clinical trial is being developed for the treatment of acute myocardial infarction [2]. Thus, it is important to determine whether TAT CPP alone influences cellular homeostasis. In this study, we investigated the inflammatory response and cell survival of lung epithelial cells after treatment with the drug delivery vehicle, TAT CPP. PKC activation is one of the important signal transduction pathways that are activated during acute lung injury [4]. We treated human lung epithelial BEAS-2B cells with a PKC activator (phorbol 12,13-dibutyrate (PDBu); 500 nM for 4 h), collected the culture medium, and measured cytokines via cytometric beads array (CBA). The production of multiple cytokines was increased significantly [5]. Surprisingly, pretreatment of PDBu stimulated BEAS-2B cells with TAT CPP alone (1 μM for 1 h), inhibited PKC activation-induced increase of 13 cytokines: fractalkine, G-CSF, GRO1-α, GMCSF, IL-6, IL-7, IL-12(p70), MIP-1α, TNF-α, TGF-α, TNF-β, VEGF, and IFNγ (Fig. 1a). Results of IL-6 and eotaxin are shown as examples of cytokines that are inhibited or not affected by TAT CPP, respectively (Fig. 1b). This nonspecific effect may interfere the interpretation of results. For
Drug Deliv. and Transl. Res.
A Cytokines increased by PDBu (fold of increase)
Not affected by PDBu and TAT EGF IL-1a FGF-2 IL-3 IL-17 MCP-3
Inhibited by TAT PDBu
Fractalkine G-CSF GM-CSF GRO1-α IL-6 IL-7 IL-12 (p70) MIP-1α TGF-α TNF-α TNF-β VEGF IFNγ
2.6 76.4 25.9 12.8 7.9 3.1 3.6 1.5 6.3 50.1 3.1 2.5 5.0
No effected by TAT PDBu plus TAT
1.5 30 17.9 3.1 2.1 0.6 1.9 1.1 4.7 37.2 2.3 1.5 1.4
PDBu
Eotaxin IL-8 IP-10 IFNa2 MIP-1β PDGF-αα
1.8 14.9 1.1 1.9 1.4 1.7
PDBu plus TAT
1.7 17.6 1.1 1.7 1.1 1.8
B
D
TAT
PDBu Control
C
DMSO
Fig. 1 The inhibitory effects of TAT peptide on PDBu-induced cytokine production and signal transduction. a Multiplex cytometric beads assay demonstrated that TAT peptide pretreatment (1 μM for 1 h) inhibited 13 cytokines induced by 500 nM PDBu (a PKC activator) in BEAS-2B cells (n=4; p
SHORT COMMUNICATION
TAT cell-penetrating peptide modulates inflammatory response and apoptosis in human lung epithelial cells Hyunhee Kim 1,2 & Serisha Moodley 1,3 & Mingyao Liu 1,2,3,4
# Controlled Release Society 2015
Abstract Cell-penetrating peptides (CPPs) are commonly used as delivery vehicles for the introduction of a variety of macromolecules into cells. Trans-activator of transcription (TAT) is the most commonly used CPP and, as a delivery vehicle, is assumed to be biologically inert. In this study, we pretreated human lung epithelial cells with TAT prior to stimulation with phorbol 12,13-dibutyrate (PDBu), a protein kinase C (PKC) activator. Surprisingly, TAT alone inhibited the production of multiple cytokines induced by PKC activation. Furthermore, PKC activation-induced IκBα degradation was partially reduced by TAT. Moreover, TAT treatment alone induced apoptosis in a dose-dependent manner, influenced expression of several B cell lymphoma 2 (Bcl-2) family members and increased caspase 3 cleavage at a high dose. These findings suggest that TAT as a delivery vehicle should be used cautiously, as it may affect the inflammatory response, as well as signals related to apoptosis.
Keywords TAT cell-penetrating peptide . Apoptosis . Inflammation * Mingyao Liu [email protected] 1
Latner Thoracic Surgery Research Laboratories, Toronto General Research Institute University Health Network, Toronto, Ontario, Canada
2
Department of Physiology, University of Toronto, Toronto, Ontario, Canada
3
Institute of Medical Science, University of Toronto, Toronto, Ontario, Canada
4
Department of Surgery, Faculty of Medicine, University of Toronto, 101 College Street, Room TMDT 2-814, Toronto, Ontario M5G 1L7, Canada
Since its discovery in 1988, as a part of HIV-1 virus regulatory protein, trans-activator of transcription (TAT) peptide is known to contain a protein transduction domain and, thus, is considered to be a cell-penetrating peptide (CPP) that is a useful tool for the intracellular delivery of macromolecules [1]. The arginine-rich transduction domain, YGRK KRRQRRR, allows TAT-conjugated molecules to enter cells via micropinocytosis and other transfer mechanisms [2]. So far, TAT CPP has been used to introduce a variety of molecules, such as peptides, proteins, siRNAs, antisense oligonucleotides, nanoparticles, polymers, liposomes, as well as therapeutic and diagnostic reagents [3]. Clinical trials using TAT CPP-conjugated peptides have been conducted to test transdermal delivery and treatment of skin disease, and a TATprotein kinase C (PKC) inhibitor clinical trial is being developed for the treatment of acute myocardial infarction [2]. Thus, it is important to determine whether TAT CPP alone influences cellular homeostasis. In this study, we investigated the inflammatory response and cell survival of lung epithelial cells after treatment with the drug delivery vehicle, TAT CPP. PKC activation is one of the important signal transduction pathways that are activated during acute lung injury [4]. We treated human lung epithelial BEAS-2B cells with a PKC activator (phorbol 12,13-dibutyrate (PDBu); 500 nM for 4 h), collected the culture medium, and measured cytokines via cytometric beads array (CBA). The production of multiple cytokines was increased significantly [5]. Surprisingly, pretreatment of PDBu stimulated BEAS-2B cells with TAT CPP alone (1 μM for 1 h), inhibited PKC activation-induced increase of 13 cytokines: fractalkine, G-CSF, GRO1-α, GMCSF, IL-6, IL-7, IL-12(p70), MIP-1α, TNF-α, TGF-α, TNF-β, VEGF, and IFNγ (Fig. 1a). Results of IL-6 and eotaxin are shown as examples of cytokines that are inhibited or not affected by TAT CPP, respectively (Fig. 1b). This nonspecific effect may interfere the interpretation of results. For
Drug Deliv. and Transl. Res.
A Cytokines increased by PDBu (fold of increase)
Not affected by PDBu and TAT EGF IL-1a FGF-2 IL-3 IL-17 MCP-3
Inhibited by TAT PDBu
Fractalkine G-CSF GM-CSF GRO1-α IL-6 IL-7 IL-12 (p70) MIP-1α TGF-α TNF-α TNF-β VEGF IFNγ
2.6 76.4 25.9 12.8 7.9 3.1 3.6 1.5 6.3 50.1 3.1 2.5 5.0
No effected by TAT PDBu plus TAT
1.5 30 17.9 3.1 2.1 0.6 1.9 1.1 4.7 37.2 2.3 1.5 1.4
PDBu
Eotaxin IL-8 IP-10 IFNa2 MIP-1β PDGF-αα
1.8 14.9 1.1 1.9 1.4 1.7
PDBu plus TAT
1.7 17.6 1.1 1.7 1.1 1.8
B
D
TAT
PDBu Control
C
DMSO
Fig. 1 The inhibitory effects of TAT peptide on PDBu-induced cytokine production and signal transduction. a Multiplex cytometric beads assay demonstrated that TAT peptide pretreatment (1 μM for 1 h) inhibited 13 cytokines induced by 500 nM PDBu (a PKC activator) in BEAS-2B cells (n=4; p
