APE1 Promotes Pancreatic Cancer Proliferation through GFRα1/Src/ERK Axis-Cascade Signaling in Response to GDNF

Pancreatic cancer is the worst exocrine gastrointestinal cancer leading to the highest mortality. Recent studies reported that aberrant expression of apurinic/apyrimidinic endodeoxyribonuclease 1 (APE1) is involved in uncontrolled cell growth. However, the molecular mechanism of APE1 biological role remains unrevealed in pancreatic cancer progression. Here, we demonstrate that APE1 accelerates pancreatic cancer cell proliferation through glial cell line-derived neurotrophic factor (GDNF)/glial factor receptor α1 (GFRα1)/Src/ERK axis-cascade signaling. The proliferation of endogenous APE1 expressed-MIA PaCa-2, a human pancreatic carcinoma cell line, was increased by treatment with GDNF, a ligand of GFRα1. Either of downregulated APE1 or GFRα1 expression using small interference RNA (siRNA) inhibited GDNF-induced cancer cell proliferation. The MEK-1 inhibitor PD98059 decreased GDNF-induced MIA PaCa-2 cell proliferation. Src inactivation by either its siRNA or Src inhibitor decreased ERK-phosphorylation in response to GDNF in MIA PaCa-2 cells. Overexpression of GFRα1 in APE1-deficient MIA PaCa-2 cells activated the phosphorylation of Src and ERK. The expression of both APE1 and GFRα1 was gradually increased as progressing pancreatic cancer grades. Our results highlight a critical role for APE1 in GDNF-induced pancreatic cancer cell proliferation through APE1/GFRα1/Src/ERK axis-cascade signaling and provide evidence for future potential therapeutic drug targets for the treatment of pancreatic cancer

Colonization and Infection of the Skin by S. aureus: Immune System Evasion and the Response to Cationic Antimicrobial Peptides

Staphylococcus aureus (S. aureus) is a widespread cutaneous pathogen responsible for the great majority of bacterial skin infections in humans. The incidence of skin infections by S. aureus reflects in part the competition between host cutaneous immune defenses and S. aureus virulence factors. As part of the innate immune system in the skin, cationic antimicrobial peptides (CAMPs) such as the β-defensins and cathelicidin contribute to host cutaneous defense, which prevents harmful microorganisms, like S. aureus, from crossing epithelial barriers. Conversely, S. aureus utilizes evasive mechanisms against host defenses to promote its colonization and infection of the skin. In this review, we focus on host-pathogen interactions during colonization and infection of the skin by S. aureus and methicillin-resistant Staphylococcus aureus (MRSA). We will discuss the peptides (defensins, cathelicidins, RNase7, dermcidin) and other mediators (toll-like receptor, IL-1 and IL-17) that comprise the host defense against S. aureus skin infection, as well as the various mechanisms by which S. aureus evades host defenses. It is anticipated that greater understanding of these mechanisms will enable development of more sustainable antimicrobial compounds and new therapeutic approaches to the treatment of S. aureus skin infection and colonization

Colonization and Infection of the Skin by S. aureus: Immune System Evasion and the Response to Cationic Antimicrobial Peptides

Staphylococcus aureus (S. aureus) is a widespread cutaneous pathogen responsible for the great majority of bacterial skin infections in humans. The incidence of skin infections by S. aureus reflects in part the competition between host cutaneous immune defenses and S. aureus virulence factors. As part of the innate immune system in the skin, cationic antimicrobial peptides (CAMPs) such as the β-defensins and cathelicidin contribute to host cutaneous defense, which prevents harmful microorganisms, like S. aureus, from crossing epithelial barriers. Conversely, S. aureus utilizes evasive mechanisms against host defenses to promote its colonization and infection of the skin. In this review, we focus on host-pathogen interactions during colonization and infection of the skin by S. aureus and methicillin-resistant Staphylococcus aureus (MRSA). We will discuss the peptides (defensins, cathelicidins, RNase7, dermcidin) and other mediators (toll-like receptor, IL-1 and IL-17) that comprise the host defense against S. aureus skin infection, as well as the various mechanisms by which S. aureus evades host defenses. It is anticipated that greater understanding of these mechanisms will enable development of more sustainable antimicrobial compounds and new therapeutic approaches to the treatment of S. aureus skin infection and colonization

Bacterial Outer Membrane Vesicles Promote Lung Inflammatory Responses and Macrophage Activation via Multi-Signaling Pathways

Emerging evidence suggests that Gram-negative bacteria release bacterial outer membrane vesicles (OMVs) and that these play an important role in the pathogenesis of bacterial infection-mediated inflammatory responses and organ damage. Despite the fact that scattered reports have shown that OMVs released from Gram-negative bacteria may function via the TLR2/4-signaling pathway or induce pyroptosis in macrophages, our study reveals a more complex role of OMVs in the development of inflammatory lung responses and macrophage pro-inflammatory activation. We first confirmed that various types of Gram-negative bacteria release similar OMVs which prompt pro-inflammatory activation in both bone marrow-derived macrophages and lung alveolar macrophages. We further demonstrated that mice treated with OMVs via intratracheal instillation developed significant inflammatory lung responses. Using mouse inflammation and autoimmune arrays, we identified multiple altered cytokine/chemokines in both bone marrow-derived macrophages and alveolar macrophages, suggesting that OMVs have a broader spectrum of function compared to LPS. Using TLR4 knock-out cells, we found that OMVs exert more robust effects on activating macrophages compared to LPS. We next examined multiple signaling pathways, including not only cell surface antigens, but also intracellular receptors. Our results confirmed that bacterial OMVs trigger both surface protein-mediated signaling and intracellular signaling pathways, such as the S100-A8 protein-mediated pathway. In summary, our studies confirm that bacterial OMVs strongly induced macrophage pro-inflammatory activation and inflammatory lung responses via multi-signaling pathways. Bacterial OMVs should be viewed as a repertoire of pathogen-associated molecular patterns (PAMPs), exerting more robust effects than Gram-negative bacteria-derived LPS

P5 inhibits <i>P</i>. <i>acnes</i>-induced intracellular Ca²⁺ mobilization in HK cells.

<p>Fura-2-loaded HKs on glass coverslips were treated with <i>P</i>. <i>acnes</i> (1 x 10⁸ CFU/150 μl) in the presence or absence of 0.8 μM P5 or P4. Uninfected HKs treated with P5 and P4 served as negative controls. The intracellular free Ca²⁺ concentration was determined by measuring the ratio of fura-2 fluorescence at 510 nm elicited by excitation at 340 and 380 nm. The peaks in this figure represent the simultaneous intracellular Ca²⁺ responses of different cells to <i>P</i>. <i>acnes</i> or the indicated AMP.</p

Suppression of <i>Propionibacterium acnes</i> Infection and the Associated Inflammatory Response by the Antimicrobial Peptide P5 in Mice

<div><p>The cutaneous inflammation associated with acne vulgaris is caused by the anaerobic bacterium <i>Propionibacterium acnes</i> through activation of the innate immune system in the skin. Current standard treatments for acne have limitations that include adverse effects and poor efficacy in many patients, making development of a more effective therapy highly desirable. In the present study, we demonstrate the protective effects of a novel customized α-helical cationic peptide, P5, against <i>P</i>. <i>acnes</i>-induced inflammatory responses <i>in vitro</i> and <i>in vivo</i>. Application of P5 significantly reduced expression of two inflammatory cytokines IL-8 and TNF-α in <i>P</i>. <i>acnes</i>-treated primary human keratinocytes, where P5 appeared to act in part by binding to bacterial lipoteichoic acid, thereby suppressing TLR2-to-NF-κB signaling. In addition, in a mouse model of acne vulgaris, P5 exerted both anti-inflammatory and antimicrobial effects against <i>P</i>. <i>acnes</i>, but exerted no cytotoxic effects against skin cells. These results demonstrate that P5, and perhaps other cationic antimicrobial peptides, offer the unique ability to reduce numbers <i>P</i>. <i>acnes</i> cells in the skin and to inhibit the inflammation they trigger. This suggests these peptides could potentially be used to effectively treat acne without adversely affecting the skin.</p></div

<i>P</i>. <i>acnes</i>-induced secretion of IL-8 and TNF-α from HKs was inhibited by P5 treatment.

<p>IL-8 (A) and TNF-α (B) were measured in culture supernatants after incubating <i>P</i>. <i>acnes</i>-infected HK cells for 24 h in the presence or absence of 1.6 μM P5, P4 or CA-MA. The data shown are representative of triplicate experiments. All values are expressed as mean ± SD. *p< 0.001.</p

Effects of intradermal injection of P5 on <i>P</i>. <i>acnes</i> cell growth and <i>P</i>. <i>acnes</i>-induced inflammation in ICR mice.

<p>(A) Inflammation-associated erythema was visualized 24 h after injection of live <i>P</i>. <i>acnes</i> (1x10⁸ CFU/20 μl in PBS), <i>P</i>. <i>acnes</i> plus P5 (1.6 μM) or P5 alone into the ears of ICR mice. (B) Percent differences (right <i>vs</i>. left (control) ear) in ear edema compared among treatment groups every 24 h for 96 h. (C) Total number of <i>P</i>. <i>acnes</i> (CFUs) recovered from the ears of mice in the indicated treatment groups. All values represent mean ± SD of three individual experiments (*<i>P</i><0.001). Untreated, uninfected ears served as a negative control.</p

Morphological perturbations and blebs induced in <i>P</i>. <i>acnes</i> by P5.

<p>Shown are <i>P</i>. <i>acnes</i> cells after incubation for 20 min in the absence (A) and presence of CA-MA (B), P5 (C) and P4 (D) at a concentration of 1/2 MBC. Arrows point to morphological perturbations and blebs, which were clearly visible following treatment with P5.</p

P5 inhibits expression of IL-8 and TNF-α mRNA induced by <i>P</i>. <i>acnes</i> infection in HKs.

<p>Total RNA was collected from <i>P</i>. <i>acnes</i>-infected HKs with and without P5 treatment. Expression of IL-8 (A) and TNF-α (B) mRNA was measured using quantitative RT-PCR with human IL-8- and TNF-α-specific primers. The relative level of each mRNA was normalized to the expression of 18S rRNA. All data were compared to the untreated control values. The data shown are representative of triplicate experiments. All values are expressed as the mean ± SD. *<i>p</i><0.001.</p