Periodontitis promotes bacterial extracellular vesicle-induced neuroinflammation in the brain and trigeminal ganglion.

Gram-negative bacteria derived extracellular vesicles (EVs), also known as outer membrane vesicles, have attracted significant attention due to their pathogenic roles in various inflammatory diseases. We recently demonstrated that EVs secreted by the periodontopathogen Aggregatibacter actinomycetemcomitans (Aa) can cross the blood-brain barrier (BBB) and that their extracellular RNA cargo can promote the secretion of proinflammatory cytokines, such as IL-6 and TNF-α, in the brain. To gain more insight into the relationship between periodontal disease (PD) and neuroinflammatory diseases, we investigated the effect of Aa EVs in a mouse model of ligature-induced PD. When EVs were administered through intragingival injection or EV-soaked gel, proinflammatory cytokines were strongly induced in the brains of PD mice. The use of TLR (Toll-like receptor)-reporter cell lines and MyD88 knockout mice confirmed that the increased release of cytokines was triggered by Aa EVs via TLR4 and TLR8 signaling pathways and their downstream MyD88 pathway. Furthermore, the injection of EVs through the epidermis and gingiva resulted in the direct retrograde transfer of Aa EVs from axon terminals to the cell bodies of trigeminal ganglion (TG) neurons and the subsequent activation of TG neurons. We also found that the Aa EVs changed the action potential of TG neurons. These findings suggest that EVs derived from periodontopathogens such as Aa might be involved in pathogenic pathways for neuroinflammatory diseases, neuropathic pain, and other systemic inflammatory symptoms as a comorbidity of periodontitis.

Differential expression and sorting of exosomal microRNAs upon activation of the human monocyte-like cell line U937.

Extracellular vesicles such as exosomes in eukaryotes have drawn scrutiny due to their various roles in intercellular communication. Small RNAs, including microRNAs (miRNAs), are more abundant among the cargo of exosomes than other RNA types. MiRNAs loaded in secreted exosomes (or extracellular microRNAs) can be transported to recipient cells and may play a regulatory role although the miRNA loading (or sorting) mechanism in exosomes has not been clearly elucidated. Therefore, this study analyzed exosomal miRNA sequencing data from human myeloid U937 cells treated with phorbol 12-myristate 13-acetate (PMA) and compared it with data from PMA-untreated U937 cells. MiR-24 was highly expressed in the cytoplasm and exosomes of PMA-treated U937 cells. Also, miRNA pull-down and mass spectrophotometry analysis of PMA-treated U937 cells revealed that miR-24 was specifically associated with α-tubulin and hnRNP-E1 proteins. Furthermore, exosomal miR-24 was dramatically reduced when those proteins were inactivated with siRNAs, whereas cellular miR-24 showed no significant effect. We conclude that miR-24 is transported into exosomes from activated macrophages with the support of α-tubulin and hnRNP-E1.

Differential Angiogenic Potential of 3-Dimension Spheroid of HNSCC Cells in Mouse Xenograft.

The experimental animal model is still essential in the development of new anticancer drugs. We characterized mouse tumors derived from two-dimensional (2D) monolayer cells or three-dimensional (3D) spheroids to establish an in vivo model with highly standardized conditions. Primary cancer-associated fibroblasts (CAFs) were cultured from head and neck squamous cell carcinoma (HNSCC) tumor tissues and co-injected with monolayer cancer cells or spheroids into the oral mucosa of mice. Mice tumor blood vessels were stained, followed by tissue clearing and 3D Lightsheet fluorescent imaging. We compared the effect of exosomes secreted from 2D or 3D culture conditions on the angiogenesis-related genes in HNSCC cells. Our results showed that both the cells and spheroids co-injected with primary CAFs formed tumors. Interestingly, vasculature was abundantly distributed inside the spheroid-derived but not the monolayer-derived mice tumors. In addition, cisplatin injection more significantly decreased spheroid-derived but not monolayer-derived tumor size in mice. Additionally, exosomes isolated from co-culture media of FaDu spheroid and CAF upregulated angiogenesis-related genes in HNSCC cells as compared to exosomes from FaDu cell and CAF co-culture media under in vitro conditions. The mouse tumor xenograft model derived from 3D spheroids of HNSCC cells with primary CAFs is expected to produce reliable chemotherapy drug screening results given the robust angiogenesis and lack of necrosis inside tumor tissues.

Extracellular RNAs in periodontopathogenic outer membrane vesicles promote TNF-α production in human macrophages and cross the blood-brain barrier in mice.

Among the main bacteria implicated in the pathology of periodontal disease, Aggregatibacter actinomycetemcomitans (Aa) is well known for causing loss of periodontal attachment and systemic disease. Recent studies have suggested that secreted extracellular RNAs (exRNAs) from several bacteria may be important in periodontitis, although their role is unclear. Emerging evidence indicates that exRNAs circulate in nanosized bilayered and membranous extracellular vesicles (EVs) known as outer membrane vesicles (OMVs) in gram-negative bacteria. In this study, we analyzed the small RNA expression profiles in activated human macrophage-like cells (U937) infected with OMVs from Aa and investigated whether these cells can harbor exRNAs of bacterial origin that have been loaded into the host RNA-induced silencing complex, thus regulating host target transcripts. Our results provide evidence for the cytoplasmic delivery and activity of microbial EV-derived small exRNAs in host gene regulation. The production of TNF-α was promoted by exRNAs via the TLR-8 and NF-κB signaling pathways. Numerous studies have linked periodontal disease to neuroinflammatory diseases but without elucidating specific mechanisms for the connection. We show here that intracardiac injection of Aa OMVs in mice showed successful delivery to the brain after crossing the blood-brain barrier, the exRNA cargos increasing expression of TNF-α in the mouse brain. The current study indicates that host gene regulation by microRNAs originating from OMVs of the periodontal pathogen Aa is a novel mechanism for host gene regulation and that the transfer of OMV exRNAs to the brain may cause neuroinflammatory diseases like Alzheimer's.-Han, E.-C., Choi, S.-Y., Lee, Y., Park, J.-W., Hong, S.-H., Lee, H.-J. Extracellular RNAs in periodontopathogenic outer membrane vesicles promote TNF-α production in human macrophages and cross the blood-brain barrier in mice.

Inhibition of streptococcal biofilm formation by Aronia by extracellular RNA degradation.

BACKGROUND: The accumulation of oral bacterial biofilms is one of the primary etiological factors for oral diseases. Aronia melanocarpa extracts display general health benefits, including antimicrobial activities. This study evaluates the inhibitory effect of Aronia juice on oral streptococcal biofilm formation. RESULTS: Exposure to 1/10-diluted Aronia juice for 1 min significantly decreased in vitro streptococcal biofilm formation (P < 0.001). No remarkable difference was noted in streptococcal growth by Aronia under the same conditions. Interestingly, 1 week of oral rinse with diluted Aronia juice led to significantly fewer salivary streptococcal colony-forming units (CFUs) relative to oral rinsing with tap water (P < 0.05). Furthermore, Aronia exerted an extracellular RNA-degrading effect, and RNase inhibitor alleviated Aronia-dependent streptococcal biofilm inhibition. CONCLUSION: Aronia might inhibit initial biofilm formation by decomposing extracellular RNA, which plays an important role in bacterial biofilm formation. Our data suggest that oral rinsing with Aronia juice will aid in treating oral biofilm-dependent diseases easily and efficiently. © 2019 Society of Chemical Industry.

Early Growth Response 1-Dependent Downregulation of Matrix Metalloproteinase 9 and Mouse Double Minute 2 Attenuates Head and Neck Squamous Cell Carcinoma Metastasis.

BACKGROUND/AIMS: The functional relevance of early growth response-1 (EGR1) on cancer invasion remains controversial. The effect of EGR1 on the expression of MMP9, which is important for HNSCC invasion, is still disputed. There is no previous data showing the effect of EGR1 on mouse double minute 2 (MDM2), an enhancer of matrix metalloproteinase 9 (MMP9) expression. Our aim is to clarify the negative correlation between EGR1 expression and head and neck squamous cell carcinoma (HNSCC) metastasis. METHODS: EGR1 mRNA and protein expressions were compared in normal and HNSCC tissues using The Cancer Genome Atlas (TCGA) dataset analysis or immunohistochemistry (IHC), respectively. In vitro cell invasion was evaluated Matrigel invasion assay. EGR1-dependent inhibition of MDM2 transcription was assessed by promoter-luciferase assay and chromatin immunoprecipitation (ChIP). RESULTS: TCGA data showed that EGR1 mRNA levels are significantly higher in normal oral tissues as compared with HNSCC tumor tissues (adjusted P = 1.64x10-16). In addition, nonmetastatic HNSCC tissues showed significantly higher EGR1 mRNA levels as compared with metastatic tissues (adjusted P = 0.023). IHC analysis showed that primary tumor tissues expressed significantly higher levels of nuclear EGR1 compared with paired metastatic lymph node tissues (P < 0.05). EGR1 overexpression downregulated MMP9 and MDM2 protein expression. Consistent with these observations, TCGA data analysis found significantly fewer metastatic patients among a subgroup of population presenting higher EGR1 expressions with lower MMP9 and/or MDM2. CONCLUSION: Our data suggests that EGR1 prevents HNSCC metastasis through downregulation of MMP9 and MDM2. EGR1 might be a potential candidate to attenuate HNSCC metastasis.

Oxytocin inhibits head and neck squamous cell carcinoma cell migration by early growth response-1 upregulation.

The effect of oxytocin (OXT) on cancer invasion is controversial. Few studies have examined the effect of early growth response-1 (EGR1) on the invasion of head and neck squamous cell carcinoma (HNSCC). Here, we evaluated how EGR1 affects HNSCC cell migration through the molecular mechanism of OXT in exerting anti-invasion activity. Matrigel invasion and wound-healing assays were used to measure the in-vitro cell migration. The molecular mechanism of OXT was assessed by knockdown or overexpression of EGR1 in HNSCC cells. Three-dimensional (3-D) spheroids formation, followed by the image analysis for quantification was performed. OXT at 500 nmol/l increased mRNA and protein expression of E-cadherin without cytotoxicity. OXT upregulated mRNA and protein expression of EGR1 in 6 h. p53, phosphatase and tensin, and p21 expression was increased in an EGR1-dependent manner with OXT treatment. In addition, OXT significantly downregulated 3-D spheroids' formation according to spheroids' number and size. Our data showed that OXT downregulated HNSCC cell migration by EGR1 upregulation. OXT inhibited spheroids' formation of HNSCC cells under 3-D culture conditions.

Cinnamaldehydes in Cancer Chemotherapy.

Cinnamaldehyde and cinnamaldehyde-derived compounds are candidates for the development of anticancer drugs that have received extensive research attention. In this review, we summarize recent findings detailing the positive and negative aspects of cinnamaldehyde and its derivatives as potential anticancer drug candidates. Furthermore, we describe the in vivo pharmacokinetics and metabolism of cinnamaldehydes. The oxidative and antioxidative properties of cinnamaldehydes, which contribute to their potential in chemotherapy, have also been discussed. Moreover, the mechanism(s) by which cinnamaldehydes induce apoptosis in cancer cells have been explored. In addition, evidence of the regulatory effects of cinnamaldehydes on cancer cell invasion and metastasis has been described. Finally, the application of cinnamaldehydes in treating various types of cancer, including breast, prostate, and colon cancers, has been discussed in detail. The effects of cinnamaldehydes on leukemia, hepatocellular carcinoma, and oral cancer have been summarized briefly. Copyright © 2016 John Wiley & Sons, Ltd.

DJ-1 Protects Breast Cancer Cells Against 2'-Benzoyloxycinnamaldehyde-induced Oxidative Stress Independent of Nrf2.

2'-Benzoyloxycinnamaldehyde (BCA) is a promising antitumor agent. BCA effectively inhibited proliferation of MDA-MB-435 more than in MCF-7 breast cancer cells. Our recent findings showed that DJ-1 protects MCF7 cells from BCA-induced oxidative stress via its mitochondrial translocation and inhibition of the mitochondrial perturbation (Ismail et al., 2012). In this study, we addressed the question of whether Nrf2 works downstream to DJ-1 in mediating differential antiproliferation effects in MCF-7 and MDAMB-435 breast cancer cells induced by BCA treatment. BCA upregulated the expression and induced nuclear translocalization of DJ-1 and Nrf2 in only MCF-7 cells. However, in MDA-MB-435, BCA increased only Nrf2 expression without inducing DJ-1 and/or Nrf2 protein translocalization to the nucleus. Furthermore, DJ-1 knockdown decreased DJ-1 expression in both cells without affecting Nrf2 and its downstream target γ-GCS, suggesting that DJ-1-induced cell protection and works independent of Nrf2 signaling pathway.

Physical and Antibacterial Properties of Gold-Poly(ethyl methacrylate) Nanocomposites Prepared by In-Situ Polymerization.

In this paper, the physical and antimicrobial properties of gold-poly(ethyl methacrylate) nanocomposites (Au-PEMA) are evaluated. Characterization of gold nanoparticles was carried out based on UV-Vis spectroscopy and transmission electron microscopy (TEM). The specimens were characterized by attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), thermal gravimetric analysis (TGA), differential scanning calorimetry (DSC), and field emission scanning electron microscope (FE-SEM). We identified the thermal stability of Au-PEMA nanocomposites and the inhibitory effect of live bacterial attachment of Au-PEMA nanocomposites against S. mutans was also evaluated.

Akkermansia muciniphila extracellular vesicles have a protective effect against hypertension.

Akkermansia muciniphila (Am) shows a beneficial role as a probiotic in the treatment of metabolic syndrome. However, the mechanism remains to be elucidated. We tested the hypothesis that Am extracellular vesicles (AmEVs) have a protective effect against hypertension. Extracellular vesicles purified from anaerobically cultured Am were characterized by nanoparticle tracking analysis, transmission electron microscopy, and silver stain after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). AmEVs (1.0 × 1010 log particles/L) or vehicles were added into organ baths to induce vasorelaxation. In addition, AmEVs (1.0 × 108 or 1.0 × 109 particles/kg) or vehicles were injected into the tail veins of Wistar-Kyoto rats (WKYs) and spontaneously hypertensive rats (SHRs) weekly for 4 weeks. Peripheral blood mononuclear cells (PBMCs) and splenocytes isolated from both rat strains were analyzed by flow cytometry, RT-qPCR, and western blot. AmEVs affected neither vascular contraction nor endothelial relaxation in thoracic aortas. Moreover, AmEVs protected against the development of hypertension in SHRs without a serious adverse reaction. Additionally, AmEVs increased the population of T regulatory (Treg) cells and tended to reduce proinflammatory cytokines. These results indicate that AmEVs have a protective effect against hypertension without a serious adverse reaction. Therefore, it is foreseen that AmEVs may be utilized as a novel therapeutic for the treatment of hypertension.

Differential effect of cancer-associated fibroblast-derived extracellular vesicles on cisplatin resistance in oral squamous cell carcinoma via miR-876-3p.

Rationale: Platinum-based chemotherapy is commonly used for treating solid tumors, but drug resistance often limits its effectiveness. Cancer-associated fibroblast (CAF)-derived extracellular vesicle (EV), which carry various miRNAs, have been implicated in chemotherapy resistance. However, the molecular mechanism through which CAFs modulate cisplatin resistance in oral squamous cell carcinoma (OSCC) is not well understood. We employed two distinct primary CAF types with differential impacts on cancer progression: CAF-P, representing a more aggressive cancer-promoting category, and CAF-D, characterized by properties that moderately delay cancer progression. Consequently, we sought to investigate whether the two CAF types differentially affect cisplatin sensitivity and the underlying molecular mechanism. Methods: The secretion profile was examined by utilizing an antibody microarray with conditioned medium obtained from the co-culture of OSCC cells and two types of primary CAFs. The effect of CAF-dependent factors on cisplatin resistance was investigated by utilizing conditioned media (CM) and extracellular vesicle (EVs) derived from CAFs. The impacts of candidate genes were confirmed using gain- and loss-of-function analyses in spheroids and organoids, and a mouse xenograft. Lastly, we compared the expression pattern of the candidate genes in tissues from OSCC patients exhibiting different responses to cisplatin. Results: When OSCC cells were cultured with conditioned media (CM) from the two different CAF groups, cisplatin resistance increased only under CAF-P CM. OSCC cells specifically expressed insulin-like growth factor binding protein 3 (IGFBP3) after co-culture with CAF-D. Meanwhile, IGFBP3-knockdown OSCC cells acquired cisplatin resistance in CAF-D CM. IGFBP3 expression was promoted by GATA-binding protein 1 (GATA1), a transcription factor targeted by miR-876-3p, which was enriched only in CAF-P-derived EV. Treatment with CAF-P EV carrying miR-876-3p antagomir decreased cisplatin resistance compared to control miRNA-carrying CAF-P EV. On comparing the staining intensity between cisplatin-sensitive and -insensitive tissues from OSCC patients, there was a positive correlation between IGFBP3 and GATA1 expression and cisplatin sensitivity in OSCC tissues from patients. Conclusion: These results provide insights for overcoming cisplatin resistance, especially concerning EVs within the tumor microenvironment. Furthermore, it is anticipated that the expression levels of GATA1 and miR-876-3p, along with IGFBP3, could aid in the prediction of cisplatin resistance.

MicroRNA profiling in the mouse hypothalamus reveals oxytocin-regulating microRNA.

Oxytocin (Oxt), produced in the hypothalamic paraventricular and supraoptic nuclei for transport to and release from the posterior pituitary, was originally discovered through its role in lactation and parturition. Oxt also plays important roles in the central nervous system by influencing various behaviors. MicroRNAs (miRNAs), endogenous regulators of many genes, are a class of small non-coding RNAs that mediate post-transcriptional gene silencing. We performed miRNA expression profiling of the mouse hypothalamus by deep sequencing. Among the sequenced and cross-mapped small RNAs, expression of known miRNAs and unknown miRNAs candidates were analyzed. We investigated in detail one miRNA, miR-24, and found that it is a novel regulator of Oxt and controls both transcript and peptide levels of Oxt. These results provide insights into potential neurohypophysial hormone regulation mediated by miRNAs.

2-Hydroxycinnamaldehyde inhibits the epithelial-mesenchymal transition in breast cancer cells.

Since epithelial-mesenchymal transition (EMT) plays a critical role in cancer progression and in maintaining cancer stem cell properties, EMT is emerging as a therapeutic target for inhibiting the metastatic progression of cancer cells. 2'-Hydroxycinnamaldehyde (HCA) and its derivative, 2'-benzoyloxycinnamaldehyde, have recently been suggested as promising therapeutic candidates for cancer treatment. The purpose of this study is to investigate the anti-metastatic effect of HCA on breast cancer and the molecular mechanisms by which HCA regulates the transcriptional program during EMT. HCA induces epithelial reversion at nanomolar concentrations by suppressing Snail via the nuclear translocalization of GSK-3ß, which results in the transcriptional upregulation of E-cadherin. HCA also activates the transcription factor KLF17, which suppresses Id-1, indicating that HCA inhibits EMT by multiple transcriptional programs. Further, HCA treatment significantly inhibits lung metastasis in a mouse orthotopic breast cancer model. This study demonstrates the anti-metastatic effect of the non-toxic natural compound HCA through attenuation of EMT in a breast cancer model.

Identification of microRNA-size, small RNAs in Escherichia coli.

Noncoding small regulatory RNA molecules control gene expression and microRNAs provide one of the best examples in eukaryotes. However, bacterial RNAs of comparable size to eukaryotic microRNAs have received little attention. Here, we demonstrate the existence of microRNA-size, small RNAs (msRNAs) in the model bacterium Escherichia coli. We examined the small RNAs in E. coli using a deep sequencing approach, and analyzed 33.2 million small RNA clone reads after size fractionation. Bioinformatic analysis of the whole set revealed more than 400 individual msRNA species. The cellular contents of selected highly expressed msRNAs were verified by quantitative RT-PCR and northern blotting. Although, the functional significance of these RNAs is unclear, their high abundance suggests that they may play specialized roles in bacteria, analogous to miRNAs in eukaryotes.

Influence of surface characteristics on the adhesion of Candida albicans to various denture lining materials.

OBJECTIVE: This study evaluated the influence of surface characteristics of various denture lining materials on the adherence of Candida albicans. MATERIALS AND METHODS: Four different types of materials (tissue conditioners, acrylic and silicone soft liners and hard reline materials) were selected. Disk-shaped material specimens were prepared and their surface roughness values (R(a) ) measured using a profilometer. The contact angles of four reference liquids were measured on the material surfaces and surface energy parameters (total surface energy, acid and base components, degree of hydrophobicity/hydrophilicity) of the materials were calculated in accordance with acid-base theory. Specimens were incubated with C. albicans and adhering fungi quantified using the colony counting method. Data were statistically analyzed using a one-way ANOVA with Games-Howell post-hoc test (α = 0.05). Pearson correlation analysis was applied to detect correlations between surface characteristics and Candida adhesion. RESULTS: Significant differences in the surface roughness of the materials were found (p < 0.001). The acrylic soft liners were more hydrophilic than the other materials. Overall, the acrylic soft liners and tissue conditioners showed significantly greater Candida adhesion than silicone soft liners and hard reline materials (p < 0.05). The Pearson correlation analysis indicated that the base component and degree of hydrophobicity/hydrophilicity of the materials (p = 0.005/0.008), rather than the total surface energy and the surface roughness (p = 0.093/0.057), affected C. albicans adherence in a statistically significant way. CONCLUSIONS: The adhesion of C. albicans to denture lining materials can be accounted for in terms of interfacial acid-base interactions.

Up-regulation of microRNA* strands by their target transcripts.

During microRNA (miRNA) biogenesis, one strand of a 21-23 nucleotide RNA duplex is preferentially selected for entry into an RNA-induced silencing complex (RISC). The other strand, known as the miRNA* species, is typically thought to be degraded. Previous studies have provided miRNA* selection models, but it remains unclear how the dominance of one arm arises during the biogenesis of miRNA. Using miRNA sponge-like methods, we cloned four tandem target sequences (artificial target) of miR-7b* and then measured miR-7b* expression levels after transfection of the artificial target. miR-7b* levels were found to significantly increase after transfection of the artificial target. We postulate that the abundance of target transcripts drives miRNA arm selection.

Effects of Hydrogen-rich Water on Cariogenic Bacteria.

Context: Some kinds of electrolysed water have been reported to exhibit antioxidant and bactericidal activity. However, studies on the effect of electrolysed hydrogen-rich water (EHW) with a neutral pH on cariogenic bacteria are limited. Aim: This study aimed to evaluate the feasibility of using EHW as a mouthwash by examining its various effects on cariogenic bacteria. Materials and Methods: To test the bactericidal and anti-biofilm formation effects of EHW on Streptococcus mutans and Streptococcus sobrinus, bacterial growth curves, colony-forming unit (CFU) counts, and crystal violet staining of biofilms were examined after exposing the bacterial pellets to EHW or tap water as a control for one minute. In addition, the expressions of glucosyltransferase and glucan-binding proteins encoding genes were examined using real-time PCR. Results: Bacterial growth and biofilm formation were inhibited, and the number of CFUs was significantly reduced in the EHW group compared to the control group. The expression of genes encoding glucosyltransferases (gtfB, gtfC, and gtfI) and glucan-binding proteins (gbpC and dblB) were also decreased in the EHW group compared to the control. Conclusions: Exposing cariogenic bacteria to EHW at neutral pH for one minute can effectively inhibit bacterial growth and biofilm formation in vitro, suggesting that EHW is a promising mouthwash.

Phosphatase of regenerating liver-3 promotes migration and invasion by upregulating matrix metalloproteinases-7 in human colorectal cancer cells.

Phosphatase of regenerating liver (PRL)-3, a member of a subgroup of protein tyrosine phosphatases that can stimulate the degradation of the extracellular matrix, is over-expressed in metastatic colorectal cancer (CRC) relative to primary tumors. To determine whether PRL-3-induced enhancement of migration and invasion is dependent on the expression of matrix metalloproteinases (MMPs), PRL-3 was expressed in DLD-1 human CRC cells. The motility, migration and invasion characteristics of the cells were examined, and metastasis to the lung was confirmed in a nude mouse using PRL-3-overexpressing DLD-1 cells [DLD-1 (PRL-3)]. Migration and invasion of the cells were inhibited by phosphatase and farnesyltransferase inhibitors. Expression of MMPs was enhanced 3- to 10-fold in comparison to control cells, and migration and invasion were partially inhibited by small interfering RNA (siRNA) knockdown of MMP-2, -13 or -14. Importantly, siRNA knockdown of MMP-7 completely inhibited the migration and invasion of DLD-1 (PRL-3) cells, whereas overexpression of MMP-7 increased migration. The expression of MMP-7 was also downregulated by phosphatase and farnesyltransferase inhibitors. It was found that PRL-3 induced MMP-7 through oncogenic pathways including PI3K/AKT and ERK and that there is a relationship between the expression of PRL-3 and MMP-7 in human tumor cell lines. The expression of MMP-13 and -14 was very sensitive to the inhibition of farnesyltransferase; however, the migration and invasion of DLD-1 (PRL-3) cells did not strongly depend on the expression of MMP-13 or -14. These results suggest that the migration and invasion of PRL-3-expressing CRC cells depends primarily on the expression of MMP-7.