PRL-3 down-regulates PTEN expression and signals through PI3K to promote epithelial-mesenchymal transition.

PRL-3 is a metastasis-associated phosphatase. We and others have shown that its overexpression increases cell motility and invasiveness. These phenotypic changes are reminiscent of the epithelial-mesenchymal transition (EMT) that occurs during embryonic development and oncogenesis. The EMT is a complex process that converts epithelia into migratory mesenchymal cells. We here attempt to unravel the underlying mechanistic basis of these phenomena. HeLa cells transiently expressing EGFP-PRL-3 (HeLa-PRL-3) exhibit reduced levels of paxillin. Similarly, Chinese hamster ovary cells stably expressing myc-PRL-3 (CHO-PRL-3) also show marked reductions in paxillin, phosphorylated paxillin-Tyr(31), and vinculin at focal adhesion complexes and notable reductions in the levels of RhoA-GTP, Rac1-GTP, and filamentous-actin filaments. DLD-1 human colorectal cancer cells engineered to express EGFP-PRL-3 (DLD-1-PRL-3) underwent changes consistent with EMT. In these cells, PRL-3 activates Akt and inactivates glycogen synthase kinase-3beta as assessed by phosphospecific antibodies. PRL-3 up-regulates mesenchymal markers fibronectin and Snail and down-regulates epithelial markers E-cadherin, gamma-catenin (plakoglobin), and integrin beta(3), which are major effectors in the EMT pathway. The changes in these EMT characteristics brought about by PRL-3 can be abrogated by the phosphoinositide 3-kinase (PI3K) inhibitor LY294002, implying that PRL-3 acts upstream of PI3K and could play an initiating role to trigger the EMT switch during cancer metastasis. In addition, PRL-3 can down-regulate phosphatase and tensin homologue deleted on chromosome 10, which is an important antagonist of PI3K, further reinforcing PI3K/Akt function in PRL-3-triggered EMT. Catalytically inactive PRL-3 (C104S) was impaired in the above PRL-3-mediated events, indicating that these properties require phosphatase activity. Targeting PRL-3 may thus be a useful strategy to impede cancer cell invasion and metastasis.

Antibiotics Used in Regenerative Endodontics Modify Immune Response of Macrophages to Bacterial Infection.

INTRODUCTION: Ciprofloxacin, amoxicillin, and metronidazole are antibiotics used in regenerative endodontic therapy (RET). Although their antimicrobial properties are well-documented, there is a lack of information on the effects of these antibiotics on the immune response by host macrophages and periapical healing. Thus, this study had 2 objectives: (1) to determine the immune response of macrophages to bacterial infection in response to the combination of ciprofloxacin or amoxicillin and metronidazole and (2) using conditioned media produced by these macrophages to simulate the periapical microenvironment, to determine the impact on the expression of extracellular matrix (ECM) components by periodontal fibroblasts. METHODS: Macrophages were treated with ciprofloxacin and metronidazole or amoxicillin and metronidazole at 10-1000 µg/mL. The treated macrophages were exposed to lipopolysaccharide, and the pro- and anti-inflammatory cytokines produced were quantified with enzyme-linked immunosorbent assay. Periodontal fibroblasts were treated with conditioned media from these treated macrophages, and the expression of ECM genes was determined by quantitative polymerase chain reaction. RESULTS: Lipopolysaccharides elicited the production of proinflammatory cytokines interleukin 1 beta and tumor necrosis factor alpha by macrophages, but this was suppressed by ciprofloxacin and metronidazole. Moreover, only conditioned media from macrophages treated with ciprofloxacin and metronidazole rescued microbial-induced down-regulation of ECM genes by periodontal fibroblasts. Specifically, ciprofloxacin was the antibiotic responsible for these observations. In contrast, these effects were not observed with amoxicillin and metronidazole. CONCLUSIONS: Apart from disinfection of the root canal system, the combination of ciprofloxacin and metronidazole also exerts an immunomodulatory effect, which may aid in periapical healing.

Cysteamine Enhances Biofilm Eradication Efficacy of Calcium Hydroxide.

INTRODUCTION: Calcium hydroxide (Ca[OH]2) is a widely used interappointment dressing, but its antibacterial property is compromised by dentin. Hence, the addition of chlorhexidine (CHX) with Ca(OH)2 has been proposed. However, the antimicrobial efficacy of this mixture compared with Ca(OH)2 alone is currently still debatable. Cysteamine is a mucolytic agent used to reduce the viscosity of mucus through the disruption of proteins, which are also important components of the extracellular matrix of biofilms. The aims of this study were to determine the efficacy of cysteamine alone and in combination with Ca(OH)2 to eradicate Enterococcus faecalis biofilm compared with CHX with Ca(OH)2, and to determine if this effect is affected by dentin. METHODS: The biofilm eradication efficacies of Ca(OH)2 alone and with cysteamine were determined using 7-day E. faecalis biofilm cultured on dentin discs and compared with Ca(OH)2 with 2% CHX. The effects of dentin on the efficacies of Ca(OH)2 alone and with either cysteamine or CHX were examined. RESULTS: Cysteamine alone completely abolished E. faecalis biofilm at 200 mg/mL. The combination of Ca(OH)2 with either cysteamine at 10 mg/mL or 2% CHX completely obliterated E. faecalis biofilm. Cysteamine with Ca(OH)2 completely eradicated E. faecalis biofilm despite preincubation with dentin, whereas CHX with Ca(OH)2 was less effective. CONCLUSIONS: Cysteamine effectively eliminated E. faecalis biofilm and showed synergistic effects in combination with Ca(OH)2, which were unaffected by dentin. Hence, our findings support the use of cysteamine as a potential adjunct to Ca(OH)2 as an interappointment dressing.

Rapid method for the detection of root canal bacteria in endodontic therapy.

INTRODUCTION: Complete eradication of microorganisms is essential for successful root canal therapy. However, current methods to evaluate persistent bacteria after therapy are not widely practiced. Adenosine triphosphate (ATP) is an indicator of viable cells. The bioluminescence-based ATP assay is easy to perform, and results can be obtained in a clinically relevant time frame of 5 minutes. The aims of this study were to evaluate the sensitivity of the ATP detection method and the specificity of this assay for viable cells and to compare the ATP and culture methods from root canal samples of patients undergoing endodontic treatment. METHODS: The sensitivity of the ATP assay was determined using bacterial species commonly isolated from root canals. Bacteria were treated with sodium hypochlorite; after which, culture plating and the ATP assay were performed. Forty-three root canal samples before (S1) and after (S2) instrumentation and 36 samples after the removal of calcium hydroxide dressing (S3) were collected from patients undergoing root canal treatment and subjected to ATP assay and anaerobic culture. RESULTS: The sensitivity of the ATP assay was determined to be between 10 and 100 bacterial cells. This method of detection also correlated well with the presence of viable bacteria. The ATP readings obtained allowed clear segregation of anaerobic culture-positive and -negative samples obtained from infected root canals of patients. CONCLUSIONS: The ATP detection method can be used as a rapid tool to determine the presence of viable bacteria during root canal therapy. This method may be potentially useful as an adjunct to root canal treatment.

Matrix Metalloproteinase Inhibitor as an Antimicrobial Agent to Eradicate Enterococcus faecalis Biofilm.

INTRODUCTION: Successful endodontic treatment outcomes require new strategies for the complete eradication of microbial biofilms in the root canal system. Matrix metalloproteinases (MMPs) are essential enzymes in microbial cell growth and homeostasis, and they require transition metal ion cofactors to function. Targeting MMP activity also preserves dentin collagen integrity. In this study, 1,10-phenanthroline-5,6-dione (Phendione), a metal chelator, was tested as a potentially novel antimicrobial agent against Enterococcus faecalis and inhibitor of human MMP in the root canal. METHODS: Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of Phendione on E. faecalis were determined. The antimicrobial properties of Phendione in the presence of dentin powder and various transition metal ions were examined. The ability of Phendione to inhibit human MMP-2 was subsequently tested. The efficacy of Phendione against E. faecalis biofilm was determined by exposure of 7-day-old E. faecalis biofilms to Phendione. RESULTS: The MIC and MBC of Phendione were 2.0 µg/mL and 16 µg/mL, respectively, whereas 64 µg/mL was required to kill E. faecalis biofilm. Phendione completely eradicated E. faecalis despite dentin preincubation. The presence of Zn(2+), and to a lesser extent Fe(2+), abrogated the antimicrobial effect of Phendione. In addition, Phendione at MIC and MBC significantly inhibited human MMP-2 activity. CONCLUSIONS: Phendione effectively eradicated E. faecalis biofilms and significantly inhibited human MMP-2 through its ability to chelate metal ions. The antibacterial property of Phendione was preserved in the presence of dentin. Phendione can potentially be applied in endodontic treatment as both an antimicrobial agent and MMP inhibitor.

Isolation of alkaline-tolerant bacteria from primary infected root canals.

INTRODUCTION: Alkaline-tolerant bacteria in primary infected root canals could have enhanced survival capacity against antimicrobials commonly used in root canal treatment. The aims of this study were to isolate and characterize alkaline-tolerant bacteria before endodontic treatment (S1), after chemomechanical root canal preparation (S2), and after calcium hydroxide dressing (S3). METHODS: Bacteriologic samples were obtained from 43 primary infected root canals. Samples were inoculated into culture media at a pH of 9 and incubated anaerobically. The identities of bacterial isolates were determined by 16S ribosomal RNA sequencing. RESULTS: All S1 samples were culture positive, with 70% harboring bacteria tolerating a pH of 9. Gram-positive bacteria Pseudoramibacter alactolyticus and Streptococcus spp were the most frequently isolated strains with a prevalence of 54%. Of 13 culture-positive S2 samples, 8 isolates tolerated a pH of 9, namely Streptococcus sanguinis, Enterococcus faecalis, Enterobacter cancerogenus, Streptococcus oralis, and Fusobacterium nucleatum. Seven of these 8 isolates (88%) were correspondingly isolated at S1. All 3 culture-positive S3 samples tolerated a pH of 9, namely S. sanguinis and E. faecalis, which were also isolated in the corresponding S1 and S2 samples. CONCLUSIONS: We showed that the presence of alkaline-tolerant Streptococcus and Enterococcus spp in primary infected root canals could lead to their persistence during and after root canal treatment and could pose a challenge to current treatment efficacy.

N-acetylcysteine inhibits growth and eradicates biofilm of Enterococcus faecalis.

INTRODUCTION: The aims of this study were to evaluate the antibacterial and biofilm eradication efficacies of N-acetylcysteine (NAC) on Enterococcus faecalis. METHODS: The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of NAC on E. faecalis were determined. In addition, the ability of dentin powder to neutralize the antibacterial activity of NAC was examined. Calcium hydroxide, a commonly used intracanal medicament, was included as a comparison. The efficacy of NAC on E. faecalis biofilms was tested by exposure of 21-day old E. faecalis biofilms to NAC. RESULTS: NAC was most bactericidal at pH 11 with MIC and MBC of 1.56 mg/mL and 12.5 mg/mL, respectively. Although preincubation of calcium hydroxide with dentin powder abolished its antibacterial effects, NAC completely killed E. faecalis regardless of dentin powder preincubation. In addition, prolonged incubation of NAC with dentin powder (up to 3 weeks) did not significantly reduce its antibacterial activity on E. faecalis. Furthermore, NAC also effectively eradicated E. faecalis biofilms. CONCLUSIONS: NAC was bactericidal against both the planktonic and biofilm forms of E. faecalis. This antibacterial property of NAC was unaffected by the presence of dentin.

PRL-3 initiates tumor angiogenesis by recruiting endothelial cells in vitro and in vivo.

We show here that PRL-3 protein is expressed in fetal heart, developing blood vessels, and pre-erythrocytes but not in their mature counterparts. These observations imply that PRL-3 may be involved in the early development of the circulatory system. Because PRL-3 mRNA had been reported to be consistently elevated in metastatic samples derived from colorectal cancers, we attempted to investigate if PRL-3 might be involved in tumor angiogenesis and if PRL-3-expressing cells could cross-talk to human umbilical vascular endothelial cells (HUVEC) by using an in vitro coculture system. HUVECs were grown with fibroblasts, which were later overlaid with PRL-3-expressing cells. We observed that both PRL-3-expressing Chinese hamster ovary (CHO) cells and PRL-3-expressing DLD-1 human colon cancer cells could redirect the migration of HUVECs toward them; in addition, PRL-3-expressing DLD-1 cells could enhance HUVEC vascular formation. In vivo injection of PRL-3-expressing CHO cells into nude mice to form local tumors resulted in the recruitment of host endothelial cells into the tumors and initiation of angiogenesis. We further showed that PRL-3-expressing cells reduced interleukin-4 (IL-4) expression levels and thus attenuated IL-4 inhibitory effects on the HUVEC vasculature. Our findings provide direct evidence that PRL-3 may be involved in triggering angiogenesis and establishing microvasculature and it may serve as an attractive therapeutic target with respect to both angiogenesis and cancer metastasis.