Suppressed Wound Healing Research Articles

E-cigarette flavoring chemicals and vehicles adversely impact the functions of pigmented human retinal ARPE-19 cells

Electronic cigarettes (ECs) have been shown to adversely impact the human eye's retinal pigment epithelium (RPE). Flavored e-liquids induced cytotoxicity in unpigmented human ARPE-19 cells independent of nicotine's presence in my previous study. In the current study, human ARPE-19 cells pigmented by sepia melanin were employed to examine the effects of four flavoring chemicals, vanillin, menthol, furanone, and cinnamaldehyde, and EC vehicles propylene glycol (PG)/vegetable glycerin (VG) ratios (0:100, 80:20, 100:0% v/v), on metabolic activity, membrane integrity, oxidative stress, and wound healing capacity of these cells. Results demonstrate that cinnamaldehyde was the most cytotoxic flavoring, and all vehicles showed marked cytotoxicity at the highest concentration of 10%. All four flavorings elicited a significant production of reactive oxygen species (ROS), while the three vehicles did not impact ROS levels. Vanillin significantly (p < 0.05) suppressed wound healing, while furanone and cinnamaldehyde had no effects, although menthol promoted wound healing at the lowest concentration. Moreover, the vehicles with two ratios of 0:100 PG/VG and 80:20 PG/VG suppressed wound healing. Together, these results suggest that vanillin and VG-containing vehicles exert the greatest adverse effects on ARPE-19 cells. These findings underscore the potential harm that exposure to ECs can cause to the human retina.

Restoration of Radiosensitivity by Soya Isoflavone Genistein is Accomplished by Facilitating DNA Damage Response in Radioresistant Cervical Cancer in vitro

ABSTRACT Context: Enhancing radiotherapeutic efficacy in tumor cells and sparing the normal tissues are major clinical concerns for the betterment of cancer therapy. Genistein (GEN) being a radiosensitizer ameliorates the effectiveness of radiation-induced cell killing by inducing DNA damage. This molecule is accountable for minimizing radiation-related toxicity and protecting healthy cells. However, the explicit mechanism of action of such molecules needs exploration. Aims: The objective of this study is to investigate the mechanistic action of GEN in cervical cancer cell radiosensitization. Settings and Design: Cervical squamous carcinoma cell SiHa and a radioresistant subline SiHa/RR (developed and isolated from SiHa) were taken for this study. The experiments were performed by pretreating the cells with IC30 dose of GEN, followed by acute irradiation to detect the impact of GEN in imparting radiosensitivity. Subjects and Methods: Optimal dose selection of GEN was performed by MTT assay, and radiosensitizing potency was determined by pretreating the cells with IC30 dose of GEN, followed by challenging with acute incremental doses of radiation. Mechanistic parameters were checked by clonogenic assay, cell cycle analysis, DNA damage estimation, apoptosis, and wound healing-sphere-forming assay. Statistical Analysis Used: Statistical analysis was performed in GraphPad software by performing the Student’s t-test. Results: Results depicted decreased numbers of colonies, increased frequency of DNA damage and apoptotic cells, and suppressed wound healing ability along with restrained sphere-forming ability upon the intervention of cells with GEN before radiation exposure. Such observations implied that GEN pretreatment renders improved radiosensitivity in cervical cancer by increased DNA damage-mediated G2/M arrest with subsequent apoptosis. Conclusions: GEN by inducing DNA damage stimulates radiation-induced cell killing in vitro.

Growth and Migration Blocking Effect of Nanaomycin K, a Compound Produced by Streptomyces sp., on Prostate Cancer Cell Lines In Vitro and In Vivo.

Since castration-resistant prostate cancer (CRPC) acquires resistance to molecularly targeted drugs, discovering a class of drugs with different mechanisms of action is needed for more efficient treatment. In this study, we investigated the anti-tumor effects of nanaomycin K, derived from "Streptomyces rosa subsp. notoensis" OS-3966. The cell lines used were LNCaP (non-CRPC), PC-3 (CRPC), and TRAMP-C2 (CRPC). Experiments included cell proliferation analysis, wound healing analysis, and Western blotting. In addition, nanaomycin K was administered intratumorally to TRAMP-C2 carcinoma-bearing mice to assess effects on tumor growth. Furthermore, immuno-histochemistry staining was performed on excised tissues. Nanaomycin K suppressed cell proliferation in all cell lines (p < 0.001) and suppressed wound healing in TRAMP-C2 (p = 0.008). Nanaomycin K suppressed or showed a tendency to suppress the expression of N-cadherin, Vimentin, Slug, and Ras in all cell lines, and suppressed the phosphorylation of p38, SAPK/JNK, and Erk1/2 in LNCaP and TRAMP-C2. In vivo, nanaomycin K safely inhibited tumor growth (p = 0.001). In addition, suppression of phospho-Erk1/2 and increased expression of E-cadherin and cleaved-Caspase3 were observed in excised tumors. Nanaomycin K inhibits tumor growth and suppresses migration by inhibiting epithelial-mesenchymal transition in prostate cancer. Its mechanism of action is related to the inhibition of phosphorylation of the MAPK signaling pathway.

Is Green Tea All It Is Brewed Up to Be? Our Results Suggest It May Not Be

Abstract Inflammation underlies multiple disorders, including cardiovascular disease, diabetes mellitus, and cancer. Naturally occurring compounds are being investigated for potential medicinal benefit in the treatment of multiple disorders. The published literature indicates that epigallocatechin gallate (EGCG), derived from green tea leaves, has antioxidant, anti-inflammatory, anti-cancer, and antimicrobial properties. Through a series of cell-culture-based assays, EGCG was determined to be strongly proinflammatory at 10 µM and cytotoxic at 50 µM, concentrations utilized in multiple published reports, in both vascular endothelial cells and monocyte-derived macrophages. Furthermore, EGCG suppressed wound healing in vascular endothelial cells at both 10 and 50 µM. A 1-µM EGCG treatment appeared to have no effect on vascular endothelial cells or monocyte-derived macrophages when carried out for less than 3 days. Concentrations of 1, 10, and 50 µM EGCG were assessed for the ability to promote the biocompatibility of polymers using a THP-1 cell adhesion assay and a Chandler Loop ex vivo model of blood perfusion using whole equine blood. No significant changes in the number of adhered cells were seen in either assay, suggesting that EGCG does not promote the biocompatibility of commonly used polymers in medical devices. Taken together, our results indicate that EGCG is proinflammatory at the concentrations assessed and not suitable as a therapy to promote biocompatibility.

Ceramide kinase regulates acute wound healing by suppressing 5-oxo-ETE biosynthesis and signaling via its receptor OXER1

The sphingolipid, ceramide-1-phosphate (C1P), has been shown to promote the inflammatory phase and inhibit the proliferation and remodeling stages of wound repair via direct interaction with group IVA cytosolic phospholipase A2, a regulator of eicosanoid biosynthesis that fine-tunes the behaviors of various cell types during wound healing. However, the anabolic enzyme responsible for the production of C1P that suppresses wound healing as well as bioactive eicosanoids and target receptors that drive enhanced wound remodeling have not been characterized. Herein, we determined that decreasing C1P activity via inhibitors or genetic ablation of the anabolic enzyme ceramide kinase (CERK) significantly enhanced wound healing phenotypes. Importantly, postwounding inhibition of CERK enhanced the closure rate of acute wounds, improved the quality of healing, and increased fibroblast migration via a “class switch” in the eicosanoid profile. This switch reduced pro-inflammatory prostaglandins (e.g., prostaglandin E2) and increased levels of 5-hydroxyeicosatetraenoic acid and the downstream metabolite 5-oxo-eicosatetraenoic acid (5-oxo-ETE). Moreover, dermal fibroblasts from mice with genetically ablated CERK showed enhanced wound healing markers, while blockage of the murine 5-oxo-ETE receptor (oxoeicosanoid receptor 1) inhibited the enhanced migration phenotype of these cell models. Together, these studies reinforce the vital roles eicosanoids play in the wound healing process and demonstrate a novel role for CERK-derived C1P as a negative regulator of 5-oxo-ETE biosynthesis and the activation of oxoeicosanoid receptor 1 in wound healing. These findings provide foundational preclinical results for the use of CERK inhibitors to shift the balance from inflammation to resolution and increase the wound healing rate.

Exhausting hsa_circ_0072088 restrains proliferation, motility and angiogenesis of breast carcinoma cells through regulating miR-1236-3p and RRM2 in a ceRNA pathway

BackgroundHsa_circ_0072088 (circ_0072088) is a novel and informative circular RNA in several human cancers, including breast carcinoma (BRCA). Herewith, we aimed at investigating the role and mechanism of circ_0072088 in BRCA cells. MethodsExpression of circ_0072088, microRNA (miR)-1236-3p, ribonucleotide reductase subunit M2 (RRM2), and epithelial-to-mesenchymal transition (EMT)-related markers E-cadherin, N-cadherin and Vimentin was detected by quantitative PCR and/or western blotting. Cell proliferation was measured by assays using MTS, EdU, colony formation, immunohistochemistry and nude mice tumorigenicity. Migration and/or invasion and angiogenesis were determined by scratch wound assay, transwell assay, and Capillary-like tube formation assay. Intermolecular interdependence was determined by bioinformatics algorithms, dual-luciferase reporter assay, RNA pull-down assay and Pearson's correlation coefficient analysis. ResultsCirc_0072088 was upregulated in BRCA patients’ tumors, and its high expression was associated with clinical metastasis and unfavorable disease-free survival. Interfering circ_0072088 could weaken cell viability, colony formation, EdU incorporation, and tumor growth of BRCA cells; moreover, circ_0072088 interference suppressed wound healing, migration/invasion and EMT process of BRCA cells, and tube formation of endothelial cells. Strikingly, either miR-1236-3p inhibitor or RRM2 overexpression could significantly abate in vitro roles of circ_0072088 blockage. Allied with that, circ_0072088 was a sponge of miR-1236-3p in regulating expression of miR-1236-3p-specific RRM2. ConclusionInhibiting circ_0072088 might treat BRCA via miR-1236-3p/RRM2 axis, suggesting circPHKA2 as a novel molecular inhibitor of RRM2, a Food and Drug Administration (FDA)-approved drug target.

Identification of novel key genes associated with the metastasis of prostate cancer based on bioinformatics prediction and validation

BackgroundMetastatic prostate cancer (PCa) is a lethal tumor. However, the molecular mechanisms underlying PCa progression have not been fully elucidated.MethodsTranscriptome expression profiling and clinical information on primary and metastatic PCa samples were obtained from TCGA. R software was used to screen the DEGs, and LASSO logistical regression method was utilized to identify the pivotal PCa metastasis-related DEGs. The transcriptional expression levels of the key genes were analyzed using the UALCAN database, and the corresponding protein expression were validated by Immunohistochemistry (IHC). Survival analysis of the key genes was performed using the GEPIA database. Wound healing assay and Transwell assay were conducted to determine whether knockdown of the key genes influence the migration and invasion abilities of PCa cells (22Rv1 and PC3). GSEA was performed to predict key genes-mediated signaling pathways for the development of PCa. Western blotting was used to evaluate the expression changes of E-cadherin, Twist1, and Vimentin in PCa cells with the key genes silencing. An in vivo mouse metastatic model for PCa was also generated to verify the important role of ISG15 and CST2 in PCa metastasis.ResultsA comparison between primary and metastatic PCa tissues was conducted, and 19 DEGs were screened. Among these, three key genes were identified that might be closely associated with PCa progression according to the LASSO logistical analysis, namely ISG15, DNAH8, and CST2. Further functional experiments revealed that knockdown of ISG15 and CST2 suppressed wound healing, migration, and invasion of PCa cells. To explore the molecular mechanism of ISG15 and CST2 in the development of PCa, GSEA was performed, and it was found that both genes play crucial roles in cell adhesion molecules, extracellular matrix-receptor interaction, and focal adhesion. Western blotting results exhibited that inhibiting ISG15 and CST2 led to increase the expression of E-cadherin and decrease the expression of Twist1 and Vimentin. Additionally, the metastatic in vivo study demonstrated that both PC3 and 22Rv1 cells expressing with luciferase-shISG15 and luciferase-shCST2 had significantly lower detectable bioluminescence than that in the control PCa cells.ConclusionISG15 and CST2 may participate in PCa metastasis by regulating the epithelial-mesenchymal transition (EMT) signaling pathway. These findings may help to better understand the pathogenetic mechanisms governing PCa and provide promising therapeutic targets for metastatic PCa therapy.

Puerarin Improves Dexamethasone-Impaired Wound Healing In Vitro and In Vivo by Enhancing Keratinocyte Proliferation and Migration

The delayed and impaired wound healing caused by dexamethasone (DEX) is commonly reported. Puerarin, the major isoflavone found in Pueraria montana var. lobata (Willd.) Sanjappa &amp; Pradeep promoted the wound healing process in diabetic rats. However, the effects and underlying mechanisms of puerarin on DEX-impaired wound healing have not been investigated. This study examined the potential uses of puerarin in upregulating keratinocyte proliferation and migration in dexamethasone (DEX)-suppressed wound healing model. The effects of puerarin on wound healing in vivo were investigated by taking full-thickness 5 mm punch biopsies from the dorsal skin of BALB/c mice and then treating them topically with 0.1% DEX. For the in vitro study, DEX-treated HaCaT cells were used to examine the effects of puerarin on DEX-induced keratinocyte proliferation and migration and the mechanisms of its action. Puerarin, when applied topically, accelerated the wound closure rate, increased the density of the capillaries, and upregulated the level of collagen fibers and TGF-β in the wound sites compared to the DEX-treated mice. Puerarin promoted the proliferation and migration of keratinocytes by activating the ERK and Akt signaling pathways in DEX-treated HaCaT cells. In conclusion, puerarin could be effective in reversing delayed and disrupted wound healing associated with DEX treatments.

GDF15 knockdown suppresses cervical cancer cell migration invitro through the TGF-β/Smad2/3/Snail1 pathway.

Growth differentiation factor 15 (GDF15), a member of the transforming growth factor β (TGF-β) superfamily, is a prognostic biomarker of cervical cancer. In addition, GDF15 has been reported to enhance the migration of colorectal cancer cells and liver cancer stem-like cells. However, the mechanism by which GDF15 promotes cervical cancer cell migration is not completely understood. Here, we report that GDF15 expression is enhanced in cervical cancer tissues, as well as in cultured cervical cancer cells. ShGDF15 transfection markedly inhibited expression of Vimentin, N-cadherin and Snail1, and resulted in up-regulation of E-cadherin expression in HT-3 and HeLa cells. Moreover, knockdown of GDF15 suppressed wound healing rate and reduced the number of invasive cells. Furthermore, knockdown of GDF15 significantly suppressed the expression of phosphorylated Smad2 and Smad3. The addition of TGF-β1 partially abolished the inhibitory effects of GDF15 knockdown on the migration and invasion of cervical cancer cells. In summary, we report here that GDF15 knockdown inhibits migration and invasion of cervical cancer cells invitro through the TGF-β/Smad2/3/Snail1 pathway.

Enhanced photocatalytic and photothermal properties of ecofriendly metal-organic framework heterojunction for rapid sterilization

Injured tissues are susceptible to infections that suppress wound healing. Rapid and safe sterilization is therefore urgent for bacteria-infected wounds, especially under harsh conditions without antibiotic availability. In this work, we developed a photoresponsive metal–organic framework (MOF) heterojunction that responds to 660 nm light irradiation. It is composed of two kinds of MOFs (Prussian blue [PB] and PCN-224), which exhibited an enhanced photocatalytic performance due to the accelerated charge transfer of the heterojunction and the fast separation of photogenerated electron-hole pairs between PB and PCN-224. The combination of the enhanced photocatalytic performance and the intrinsic photothermal effect of PB endowed the MOFs heterojunction with highly effective sterilizing rates of 99.84% and 99.3% against Staphylococcus aureus and its biofilm after 660 nm light irradiation for 15 min, respectively. The iron and zirconium ions released from PB-PCN-224 composites were biocompatible and their cytotoxicity was negligible. More importantly, in vivo experiments showed that PB-PCN-224 can expedite wound healing.

An overview of superficial radiation of surgical keloidectomy sites to reduce keloid recurrences

AbstractBackgroundKeloids adversely impact medical, cosmetic and psychological health and are detrimental to the quality of life of those affected. The clinical challenge following surgical excision of keloids is the high, 71%, recurrence rate. Based on its ability to suppress wound healing, judicious use of low‐dose, fractionated superficial radiation therapy (SRT) has been successful in reducing post‐keloidectomy keloid recurrences.ObjectiveThis overview focusses on the reported safety and efficacies of differing dosing and fractionation regimens of SRT of keloidectomy surgical sites to reduce keloid.MethodsA literature review regarding superficial radiation of surgical keloidectomy sites to reduce keloid recurrences.ResultsPostsurgical treatment of keloid excision suture lines with several fractions of SRT reduces keloid recurrence rates. An effective and safe treatment protocol is a biologically effective dose of 3000 cGy (BED 30) with three fractions of 600 cGy on postoperative days 1, 2, and 3. Fractionation of the SRT dose reduces the risk of hyperpigmentation and other adverse events.ConclusionJudicious use of low‐dose, fractionated superficial radiation therapy safely and effectively reduces the rate of post‐keloidectomy keloid recurrences.

Different nitrogen sources speed recovery from corallivory and uniquely alter the microbiome of a reef-building coral.

Corals are in decline worldwide due to local anthropogenic stressors, such as nutrient loading, and global stressors, such as ocean warming. Anthropogenic nutrient loading, which is often rich in nitrate, inhibits coral growth and worsens corals’ response to warming while natural sources of nitrogen, such as ammonium from fish excretion, promotes coral growth. Although the effects of nutrient loading and ocean warming have been well-studied, it remains unclear how these factors may interact with biotic processes, such as corallivory, to alter coral health and the coral microbiome. This study examined how nitrate vs. ammonium enrichment altered the effects of increased seawater temperature and simulated parrotfish corallivory on the health of Pocillopora meandrina and its microbial community. We tested the effects of nitrogen source on the response to corallivory under contrasting temperatures (control: 26 °C, warming: 29 °C) in a factorial mesocosm experiment in Moorea, French Polynesia. Corals were able to maintain growth rates despite simultaneous stressors. Seawater warming suppressed wound healing rates by nearly 66%. However, both ammonium and nitrate enrichment counteracted the effect of higher temperatures on would healing rates. Elevated seawater temperature and ammonium enrichment independently increased Symbiodiniaceae densities relative to controls, yet there was no effect of nitrate enrichment on algal symbiont densities. Microbiome variability increased with the addition of nitrate or ammonium. Moreover, microbial indicator analysis showed that Desulfovibrionaceae Operational taxonomic units (OTUs) are indicators of exclusively temperature stress while Rhodobacteraceae and Saprospiraceae OTUs were indicators of high temperature, wounding, and nitrogen enrichment. Overall, our results suggest that nitrogen source may not alter the response of the coral host to simultaneous stressors, but that the associated microbial community may be distinct depending on the source of enrichment.

Varroa destructor parasitism has a greater effect on proteome changes than the deformed wing virus and activates TGF-\u03b2 signaling pathways

Honeybee workers undergo metamorphosis in capped cells for approximately 13 days before adult emergence. During the same period, Varroa mites prick the defenseless host many times. We sought to identify proteome differences between emerging Varroa-parasitized and parasite-free honeybees showing the presence or absence of clinical signs of deformed wing virus (DWV) in the capped cells. A label-free proteomic analysis utilizing nanoLC coupled with an Orbitrap Fusion Tribrid mass spectrometer provided a quantitative comparison of 2316 protein hits. Redundancy analysis (RDA) showed that the combination of Varroa parasitism and DWV clinical signs caused proteome changes that occurred in the same direction as those of Varroa alone and were approximately two-fold higher. Furthermore, proteome changes associated with DWV signs alone were positioned above Varroa in the RDA. Multiple markers indicate that Varroa activates TGF-β-induced pathways to suppress wound healing and the immune response and that the collective action of stressors intensifies these effects. Furthermore, we indicate JAK/STAT hyperactivation, p53-BCL-6 feedback loop disruption, Wnt pathway activation, Wnt/Hippo crosstalk disruption, and NF-κB and JAK/STAT signaling conflict in the Varroa–honeybee–DWV interaction. These results illustrate the higher effect of Varroa than of DWV at the time of emergence. Markers for future research are provided.

Post-traumatic pulmonary infection: the innate immune response protects the lung at the expense of the healing cutaneous wound

Abstract Hospitalized trauma patients have an increased risk of pulmonary infection. The acute response to injury, which relies on the coordinated activity of recruited leukocytes, may affect the early, innate immune-mediated control of lung infection. We hypothesized that a cutaneous wound would inhibit the ability of innate leukocytes to respond to a subsequent pulmonary bacterial infection. To address this, we developed a murine model of post-injury pneumonia. Mice were wounded by the dorsal subcutaneous implantation of polyvinyl alcohol sponges, which models the acute stages of wound repair, and a cohort was infected intranasally with the opportunistic bacterium Klebsiella oxytoca. For comparison, a control group was only infected. Wounding did not alter the early control of lung bacterial infection, the infiltration of innate leukocytes into the infected lung, or cytokine and chemokine levels in the bronchoalveolar lavage fluid. In contrast, pulmonary infection suppressed wound healing, caused decreased monocyte and neutrophil trafficking and lower cytokine and chemokine concentrations in the wound. Exogenous delivery of CCL2 and CXCL1 to the wound increased the number of neutrophils and accelerated healing. However, this led to increased pulmonary bacterial burden, highlighting the balance of cellular responses required to protect the lung. These data suggest that the innate immune system prioritizes the protection of one site above the other when faced with competing inflammatory insults. Here, early control of lung infection took priority over the healing wound. This work aims to elucidate mechanisms by which the innate immune system responds to multiple insults, which may be broadly applicable to inflammatory conditions.

Effects of Vasopressin and Norepinephrine on Wound Healing of Vascular Smooth Muscle and Endothelium

ObjectivesHemorrhagic shock as a result of trauma or surgical bleeding is frequently temporized with administration of vasopressors in order to augment systemic vascular resistance while resuscitation measures are employed. This mimics the treatment strategy of other forms of shock where systemic vascular resistance is low and vasopressors are heavily relied upon to resuscitate the patient. Blood vessels respond to direct exposure to vasopressin and norepinephrine via receptors on smooth muscle and endothelial cells and ultimately result in vasoconstriction which contributes to a low flow state and increases shear stress within vessels. The effect of vasopressors on vascular cells with regard to direct cellular injury and repair has not been well‐studied. The objective of this study is to examine the in vitro effect of vasopressin and norepinephrine on the rate of healing of vascular cells after wounding.MethodsWounding and measurement of healing rate of cells were performed using Electric Cell‐substrate Impedance Sensing (ECIS) technology. A specified area of a monolayer of cells was wounded via high electrical charge, and healing of the cell layer was assessed via measurement of resistance (ohms) over time. Pig vascular smooth muscle cells (SMCs) and pig vascular endothelial cells (ECs) were seeded in 96‐well plates. Immediately after wounding, cells were treated with either lysine‐vasopressin (LVP; 0, 0.001, 0.01, 0.1, 1, 10, 100, 1000 ηM; n=6/group), or norepinephrine (NE; 0, 0.001, 0.01, 0.1, 1, 10, 100, 1000 μM; n=6/group). Data were recorded continuously for another 18 hours. Statistical comparisons were made using two‐way ANOVA and Tukey HSD post hoc analysis(alpha level = 0.05).ResultsWhen SMCs were injured, NE treatment significantly suppressed healing rate over the course of 10 hours (Time × Dose, F(133,665) = 1.955, p&lt;0.0001), whereas LVP had minimal effect on healing of SMCs (F(133,665) = 0.6346, p=0.999). When ECs were injured, both NE (F(133,665) = 3.716, p&lt;0.0001), and LVP (F(133,665) = 3.058, p&lt;0.0001) significantly suppressed rate of healing.ConclusionsNE significantly suppressed the wound healing response of both SMCs and ECs, whereas LVP suppressed wound healing of ECs only. This strongly suggests that the use of different vasopressors during treatment and resuscitation can elicit unique effects on vascular healing in the body. We plan to further explore the physiological effects of vasopressors on post‐injury healing of blood vessels to aid in optimizing resuscitation strategies that reduce vascular injury and end organ damage in the period after recovery from trauma and shock.Support or Funding InformationThis project was funded by the U.S. Army Medical Command. The views expressed in this abstract are those of the authors and do not reflect the official policy or position of the Department of the Army, Department of Defense, or the U.S. Government.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Lack of plakoglobin impairs integrity and wound healing in corneal epithelium in mice

We generated cornea-specific plakoglobin (Jup; junctional plakoglobin) knockout mice in order to investigate the function of plakoglobin on the maintenance of the homeostasis of corneal epithelium in mice. Cornea epithelium-specific conditional knockouts (JupCEΔ/CEΔ) (cKO) were obtained by breeding keratin12-Cre (Krt12-Cre) mice to Jup-floxed (Jupf/f) mice. Light and transmission electron microscopic and immunohistochemical analyses were carried out to determine consequence of the loss of plakoglobin on maintaining corneal epithelium integrity under mechanical stress, e.g., brushing and wound healing. Immunohistochemistry analysis demonstrated that, although Jup ablation did not affect BrdU incorporation, basal cell-like cells labeled for keratin 14 were ectopically present in the supra-basal layer in mutant corneal epithelium, suggestive of altered cell differentiation. Plakoglobin-deficient epithelium exhibits increased fragility against mechanical intervention when compared to wild-type controls under identical treatment. Closure of an epithelial defect was significantly delayed in JupCEΔ/CEΔ epithelium. Our findings indicate that the lack of plakoglobin significantly affects corneal epithelium differentiation, as well as its structural integrity. Plakoglobin is essential to the maintenance of the structure of the corneal epithelium and its wound healing.Homeostasis of the corneal epithelium is critical in exerting a barrier function to noxious external stimuli. This study shows that that plakoglobin, a member of the catenin protein family, is essential to the maintenance of homeostasis of the corneal epithelium in mice. Corneal epithelium-specific deletion of the plakoglobin gene impairs tissue integrity against mechanical intervention and also suppresses wound healing without affecting cell proliferation.

Abstract 814: Aurora kinase: A target modulating invasiveness of lung adenocarcinoma

Abstract Introduction: Lung cancer is the major cause of cancer-related deaths in United States and lung adenocarcinoma (LUAD) is its most common subtype exhibiting highly variable molecular signature. Identification of patients at high risk of developing invasive tumor is important for their better management. Experimental procedure: We obtained 53 early-stage LUAD patient's tumor samples, histopathologically classified into 32 indolent (adenocarcinoma in situ, minimally invasive and lepidic predominant adenocarcinoma) and 21 invasive (acinar predominant, papillary predominant, micropapillary predominant and solid predominant adenocarcinoma) subtypes and profiled their transcriptomes by RNA sequencing. We clustered these samples in an unsupervised manner followed by additional supervised clustering into 2 groups. Using our signature genes, we categorized 51 lung adenocarcinoma cell lines from Cancer Cell Line Encyclopedia database based on their relative invasiveness scores derived from elastic network trained in our dataset. We chose NCI-H1792, NCI-H1373, SK-LU-1 and NCI-H2009 representing high while HCC1833, Calu3 and NCI-H3255 representing low invasive signature for our in vitro functional assays. Summary: Our clustering significantly overlapped with the groups classified by histology and we defined 21 tumors as “invasive” and 32 as “non-invasive” early stage LUAD. We identified 1,322 differentially expressed genes between two groups with 526 upregulated and 796 downregulated genes in invasive tumors. Among the enriched signature genes, AURKA and AURKB were significantly upregulated in the “invasive” group. We used two potent aurora kinase inhibitors AMG 900 and PF-03814735 to determine the effect of aurora kinase inhibition on invasiveness of LUAD cells. AMG 900 and PF-03814735 significantly inhibited Aurora kinase (Aur-) A and B activity, but did not affect cell viability (IC50 ≥2μM) of all cell lines regardless of their invasiveness score. Of note, both drugs significantly suppressed wound healing, migration and invasion of all four “invasive” cell lines at much lower 100nM. Moreover, the cells with low invasive signature used in the study showed little migration and invasion thus confirming the validity of our elastic network analysis for their relative invasiveness. Conclusion: Here, we have identified a robust gene signature that distinguishes invasive to indolent non-invasive early-stage LUADs, which may lead to development of assays on clinical specimens such as biopsy to identify patients at high risk of developing invasive LUAD at an early stage. This gene signature also suggests Aur-A and B promote invasiveness in early stage LUAD. Our in vitro functional data suggest that targeting Aur-A and B could be a promising approach for management of early stage LUAD patients who are more likely to develop an invasive tumor. Citation Format: Abhilasha Sinha, Seungyeul Yoo, Hideo Watanabe, Jun Zhu, Charles A. Powell. Aurora kinase: A target modulating invasiveness of lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 814.

Platelet-Rich Plasma May Offer a New Hope in Suppressed Wound Healing When Compared to Mesenchymal Stem Cells.

Background: The present study investigated the effectiveness of platelet-rich plasma (PRP) and mesenchymal stem cells (MSCs) in wound healing suppressed by corticosteroid in rats. Methods: Forty rats were separated into four groups. To disrupt the wound-healing processes, intraperitoneal single dose 10 mg/kg methylprednisolone was administered to all rats with the exception of Sham-S group. Then, full-thickness incision was performed to the abdominal skin of all animals, and PRP or MSCs were applied to the incision line except the Sham-S and Sham-M group animals. Ten days later, all animals were sacrificed to investigate: tissue collagenization, inflammation, and re-epithelialization grades histopathologically; and tissue hydroxyproline (HP), interleukin-1β (IL-1β), tumor necrosis factor-α levels biochemically. Results: Collagenization (p = 0.003) and inflammation grade (p = 0.002) values were higher in PR group. Tissue HP level value was found to be high in MC group (p < 0.001). Tissue IL-1β level value of Sham-M group was lower than those of other groups (p < 0.001). Conclusions: This preliminary study revealed that PRP could improve the histopathological grades in wound healing which was suppressed by corticosteroid in rats, while MSCs could show their therapeutic effects via biochemical route. These positive effects were more salient in PR group.

D Rhamnose β-hederin inhibits migration and invasion of human breast cancer cell line MDA-MB-231

Many natural products have been shown to have inhibitory effects on the metastatic process of various cancers including breast cancer. An active triterpenoid saponin D Rhamnose β-hederin (DRβ-H) from Clematis ganpiniana, has been known induce the apoptosis of breast cancer cells, but the effect of DRβ-H on the metastasis of breast cancer cells is largely unknown. In this study, we demonstrated that a non-cytotoxic concentration of DRβ-H markedly suppressed wound healing migration, migration through the chamber and invasion through the matrigel. In addition, DRβ-H regulated expression of RNPC1, E-cadherin proteins of MDA-MB-231 cells. Furthermore, RNPC1 knockdown decreased the DRβ-H-induced up-regulation of RNPC1 and E-Cadherin in MDA-MB-231 cells. RNPC1 knockdown reduced the anti-metastasis activities of DRβ-H, meaning that the up-rugulation of RNPC1 by DRβ-H is essential for its anti-metastatis activities. These results suggest that DRβ-H might be a potential therapeutic candidate for the treatment of breast cancer metastasis.

Structure Identification and In Vitro Anticancer Activity of Lathyrol-3-phenylacetate-5,15-diacetate.

Natural products from the genus Euphorbia show attention-attracting activities, such as anticancer activity. In this article, classical isolation and structure identification were used in a study on Caper Euphorbia Seed. Subsequently, MTT and wound healing assays, flow cytometry, western blotting, Hoechst 33258 staining and fluorescence microscopy examination were applied to investigate the anticancer activity of the obtained compounds. In a result, lathyrol-3-phenyl- acetate-5,15-diacetate (deoxy Euphorbia factor L1, DEFL1) was isolated from Caper Euphorbia Seed. Moreover, the NMR signals were totally assigned. DEFL1 showed potent inhibition against lung cancer A549 cells, with an IC50 value of 17.51 ± 0.85 μM. Furthermore, DEFL1 suppressed wound healing of A549 cells in a concentration-dependent manner. Mechanically, DEFL1 induced apoptosis, with involvement of an increase of reactive oxygen species (ROS), decrease of mitochondrial membrane potential (ΔΨm), release of cytochrome c, activity raise of caspase-9 and 3. Characteristic features of apoptosis were observed by fluorescence microscopy. In summary, DEFL1 inhibited growth and induced apoptosis in lung cancer A549 cells via a mitochondrial pathway.