ABSTRACT
In pancreatic cancer (PC) as intractable solid cancer, current research is focused mainly on targeted immunotherapies such as antibodies and immune cell modulators. To identify promising immune-oncological agents, animal models that recapitulate the essential features of human immune status are essential. To this end, we constructed an orthotopic xenograft model using CD34+ human hematopoietic stem cell-based humanized NOD scid gamma mouse (NSG) mice injected with luciferase-expressing PC cell lines AsPC1 and BxPC3. The growth of orthotopic tumors was monitored using noninvasive multimodal imaging, while the subtype profiles of human immune cells in blood and tumor tissues were determined by flow cytometry and immunohistopathology. In addition, the correlations of blood and tumor-infiltrating immune cell count with tumor extracellular matrix density were calculated using Spearman's test. Tumor-derived cell lines and tumor organoids with continuous passage capacity in vitro were isolated from orthotopic tumors. It was further confirmed that these tumor-derived cells and organoids have reduced PD-L1 expression and are suitable for testing the efficacy of specific targeted immunotherapeutic agents. These animal and culture models could facilitate the development and validation of immunotherapeutic agents for intractable solid cancers including PC.
Subject(s)
Pancreatic Neoplasms , Humans , Animals , Mice , Xenograft Model Antitumor Assays , Antigens, CD34 , Organoids , Mice, SCID , Disease Models, Animal , Mice, Inbred NOD , Pancreatic NeoplasmsABSTRACT
Studies have been actively conducted to ensure that gadolinium-based contrast agents for magnetic resonance imaging (MRI) are accompanied by various biological functions. A new example is the anti-inflammatory theragnostic MRI agent to target inflammatory mediators for imaging diagnosis and to treat inflammatory diseases simultaneously. We designed, synthesized, and characterized a Gd complex of 1,4,7-tris(carboxymethylaza) cyclododecane-10-azaacetylamide (DO3A) conjugated with a nonsteroidal anti-inflammatory drug (NSAID) that exerts the innate therapeutic effect of NSAIDs and is also applicable in MRI diagnostics. Gd-DO3A-fen (0.1 mmol/kg) was intravenously injected into the turpentine oil-induced mouse model, with Gd-DO3A-BT as a control group. In the in vivo MRI experiment, the contrast-to-noise ratio (CNR) was higher and persisted longer than that with Gd-DO3A-BT; specifically, the CNR difference was almost five times at 2 h after injection. Gd-DO3A-fen had a binding affinity (Ka) of 6.68 × 106 M-1 for the COX-2 enzyme, which was 2.1-fold higher than that of fenbufen, the original NSAID. In vivo evaluation of anti-inflammatory activity was performed in two animal models. In the turpentine oil-induced model, the mRNA expression levels of inflammatory parameters such as COX-2, TNF-α, IL-1ß, and IL-6 were reduced, and in the carrageenan-induced edema model, swelling was suppressed by 72% and there was a 2.88-fold inhibition compared with the saline group. Correlation analysis between in vitro, in silico, and in vivo studies revealed that Gd-DO3A-fen acts as an anti-inflammatory theragnostic agent by directly binding to COX-2.
Subject(s)
Organometallic Compounds , Animals , Mice , Organometallic Compounds/chemistry , Gadolinium/chemistry , Cyclooxygenase 2/genetics , Turpentine , Contrast Media/chemistry , Magnetic Resonance Imaging/methods , Anti-Inflammatory Agents/pharmacology , Anti-Inflammatory Agents/therapeutic use , Anti-Inflammatory Agents, Non-Steroidal/pharmacologyABSTRACT
In this study, we designed, synthesized, and evaluated gadolinium compounds conjugated with flavonoids as potential theranostic agents for the treatment of inflammation. These novel theranostic agents combine a molecular imaging agent and one of three flavonoids (galangin, chrysin, and 7-hydroxyflavone) as anti-inflammatory drugs as a single integrated platform. Using these agents, MR imaging showed contrast enhancement (>10 in CNR) at inflamed sites in an animal inflammation model, and subsequent MR imaging used to monitor the therapeutic efficacy of these integrated agents revealed changes in inflamed regions. The anti-inflammatory effects of these agents were demonstrated both in vitro and in vivo. Furthermore, the antioxidant efficacy of the agents was evaluated by measuring their reactive oxygen species scavenging properties. For example, Gd-galangin at 30 µM showed a three-fold higher ROS scavenging of DPPH. Taken together, our findings provide convincing evidence to indicate that flavonoid-conjugated gadolinium compounds can be used as potentially efficient theranostic agents for the treatment of inflammation.
ABSTRACT
We determined the effects of two extracts from Acer palmatum Thumb. leaves (hot water extract KIOM-2015EW and 25% ethanol extract KIOM-2015EE) in a benzalkonium chloride (BAC)-induced dry eye mouse model. Dry eye was induced by 0.2% BAC for 2 weeks, followed by treatment three times (eye drop) or once (oral administration) daily with KIOM-2015E for 2 weeks. Treatment with both KIOM-2015EE and KIOM-2015EW resulted in a marked increase in tear volume production for the 4 days of treatment. The Lissamine Green staining score, TUNEL-positive cells, and inflammatory index were significantly decreased after 2 weeks. Topical KIOM-2015EE administration exhibited a greater improvement in decreasing the ocular surface staining scores, inflammation, dead cells, and increasing tear production in a dose-dependent manner compared with the other groups. Furthermore, KIOM-2015E significantly reduced the phosphorylation of NF-κB, which was activated in the BAC-treated cornea. Topical administration was much more effective than oral administration for KIOM-2015E and KIOM-2015EE was more effective than KIOM-2015EW. Application of KIOM-2015E resulted in clinical improvement, inhibited the inflammatory response, and alleviated signs of dry eye. These results indicate that KIOM-2015E has potential as a therapeutic agent for the clinical treatment of dry eye.
Subject(s)
Acer , Dry Eye Syndromes , Mice , Animals , Benzalkonium Compounds , Mice, Inbred BALB C , Dry Eye Syndromes/chemically induced , Dry Eye Syndromes/drug therapy , Disease Models, Animal , Plant Extracts/pharmacology , Plant Extracts/therapeutic use , TearsABSTRACT
There has been an immense effort by global pharmaceutical companies to develop anti-COVID-19 drugs, including small molecule-based RNA replication inhibitors via drug repositioning and antibody-based spike protein blockers related to cell entry by SARS-CoV-2. However, several limitations to their clinical use have emerged in addition to a lack of progress in the development of small molecule-based cell entry inhibitors from natural products. In this study, we tested the effectiveness of kuwanon C (KC), which has mainly been researched using in silico docking simulation and can serve as an effective building block for developing anti-COVID-19 drugs, in blocking the spike S1 RBD:ACE2 receptor interaction. KC is a natural product derived from Morus alba L., commonly known as mulberry, which has known antiviral efficacy. Molecular interaction studies using competitive ELISA and the BLItz system revealed that KC targets both the spike S1 RBD and the ACE2 receptor, successfully disrupting their interaction, as supported by the in silico docking simulation. Furthermore, we established a mechanism of action by observing how KC prevents the infection of SARS-CoV-2 spike pseudotyped virus in ACE2/TPRSS2-overexpressing HEK293T cells. Finally, we demonstrated that KC inhibits clinical isolates of SARS-CoV-2 in Vero cells. Future combinations of small molecule-based cell entry inhibitors, such as KC, with the currently prescribed RNA replication inhibitors are anticipated to significantly enhance the efficacy of COVID-19 therapies.
Subject(s)
Biological Products , COVID-19 Drug Treatment , Morus , Chlorocebus aethiops , Animals , Humans , SARS-CoV-2 , Angiotensin-Converting Enzyme 2 , Spike Glycoprotein, Coronavirus/metabolism , Morus/metabolism , Vero Cells , HEK293 Cells , Peptidyl-Dipeptidase A/metabolism , Protein Binding , Antiviral Agents/pharmacology , Pharmaceutical Preparations , RNA/metabolismABSTRACT
Many preclinically tested nanoparticles in existing animal models fail to be directly translated into clinical applications because of their poor resemblance to human cancer. Herein, the enhanced permeation and retention (EPR) effect of glycol chitosan nanoparticles (CNPs) in different tumor microenvironments (TMEs) was compared using different pancreatic tumor models, including pancreatic cancer cell line (BxPC3), patient-derived cancer cell (PDC), and patient-derived xenograft (PDX) models. CNPs were intravenously injected into different tumor models, and their accumulation efficiency was evaluated using non-invasive near-infrared fluorescence (NIRF) imaging. In particular, differences in angiogenic vessel density, collagen matrix, and hyaluronic acid content in tumor tissues of the BxPC3, PDC, and PDX models greatly affected the tumor-targeting efficiency of CNPs. In addition, different PDX models were established using different tumor tissues of patients to predict the clinical EPR effect of CNPs in inter-patient TMEs, wherein the gene expression levels of PECAM1, COL4A1, and HAS1 in human tumor tissues were observed to be closely related to the EPR effect of CNPs in PDX models. The results suggested that the PDX models could mimic inter-patient TMEs with different blood vessel structures and extracellular matrix (ECM) content that critically affect the tumor-targeting ability of CNPs in different pancreatic PDX models. This study provides a better understanding of the heterogeneity and complexity of inter-patient TMEs that can predict the response of various nanoparticles in individual tumors for personalized cancer therapy.
Subject(s)
Nanoparticles , Neoplasms , Animals , Humans , Heterografts , Nanoparticles/chemistry , Neoplasms/diagnostic imaging , Neoplasms/drug therapy , Neoplasms/metabolism , Tumor Microenvironment , Extracellular Matrix/metabolism , Disease Models, Animal , Xenograft Model Antitumor AssaysABSTRACT
Here, we describe the synthesis, characterization, and in vitro biological evaluation of a series of transition metal complexes containing benzothiazole aniline (BTA). We employed BTA, which is known for its selective anticancer activity, and a salen-type Schiff-based ligand to coordinate several transition metals to achieve selective and synergistic cytotoxicity. The compounds obtained were characterized by NMR spectroscopy, mass spectrometry, Fourier transform infrared spectroscopy, and elemental analysis. The compounds L, MnL, FeL, CoL, and ZnL showed promising in vitro cytotoxicity against cancer cells, and they had a lower IC50 than that of the clinically used cisplatin. In particular, MnL had synergistic cytotoxicity against liver, breast, and colon cancer cells. Moreover, MnL, CoL, and CuL promoted the production of reactive oxygen species in HepG2 tumor cell lines. The lead compound of this series, MnL, remained stable in physiological settings, and docking results showed that it interacted rationally with the minor groove of DNA. Therefore, MnL may serve as a viable alternative to platinum-based chemotherapy.
ABSTRACT
The use of cancer-derived exosomes has been studied in several cancer types, but the cancer-targeting efficacy of glioma-derived exosomes has not been investigated in depth for malignant glioblastoma (GBM) cells. In this study, exosomes were derived from U87MG human glioblastoma cells, and selumetinib, a new anticancer drug, was loaded into the exosomes. We observed the tropism of GBM-derived exosomes in vitro and in vivo. We found that the tropism of GBM-derived exosomes is in contrast to the behavior of non-exosome-enveloped drugs and non-GBM-specific exosomes in vitro and in vivo in an animal GBM model. We found that the tropism exhibited by GBM-derived exosomes can be utilized to shuttle selumetinib, with no specific targeting moiety, to GBM tumor sites. Therefore, our findings indicated that GBM-derived exosomes loaded with selumetinib had a specific antitumor effect on U87MG cells and were non-toxic to normal brain cells. These exosomes offer improved therapeutic prospects for glioblastoma therapy.
ABSTRACT
Hepatocellular carcinomas (HCCs) are aggressive tumors with a poor prognosis. Approved first-line treatments include sorafenib, lenvatinib, and a combination of atezolizumab and bevacizumab; however, they do not cure HCC. We investigated MBP-11901 as a drug candidate for HCC. Cell proliferation and cytotoxicity were evaluated using normal and cancer human liver cell lines, while Western blotting and flow cytometry evaluated apoptosis. The anticancer effect of MBP-11901 was verified in vitro through migration, invasion, colony formation, and JC-1 MMP assays. In mouse models, the tumor volume, tumor weight, and bodyweight were measured, and cancer cell proliferation and apoptosis were analyzed. The toxicity of MBP-11901 was investigated through GOT/GPT and histological analyses in the liver and kidney. The signaling mechanism of MBP-11901 was investigated through kinase assays, phosphorylation analysis, and in silico docking simulations. Results. MBP-11901 was effective against various human HCC cell lines, leading to the disappearance of most tumors when administered orally in animal models. This effect was dose-dependent, with no differences in efficacy according to administration intervals. MBP-11901 induced anticancer effects by targeting the signaling mechanisms of FLT3, VEGFR2, c-KIT, and PDGFRß. MBP-11901 is suggested as a novel therapeutic agent for the treatment of advanced or unresectable liver cancer.
ABSTRACT
We describe the synthesis, characterization, molecular modeling, and in vitro anticancer activity of three benzothiazole aniline (BTA) ligands and their corresponding platinum (II) complexes. We designed the compounds based on the selective antitumor properties of BTA, along with three types of metallic centers, aiming to take advantage of the distinctive and synergistic activity of the complexes to develop anticancer agents. The compounds were characterized using nuclear magnetic resonance spectrometry, Fourier transform infrared spectroscopy, mass spectrometry, elemental analysis, and tested for antiproliferative activity against multiple normal and cancerous cell lines. L1, L2, and L1Pt had better cytotoxicity in the liver, breast, lung, prostate, kidney, and brain cells than clinically used cisplatin. Especially, L1 and L1Pt demonstrated selective inhibitory activities against liver cancer cells. Therefore, these compounds can be a promising alternative to the present chemotherapy drugs.
ABSTRACT
Purpose: The present study aimed to determine whether the administration of Acer palmatum thumb. leaf extract (KIOM-2015E) protects against the degeneration of rat retinal ganglion cells after ischemia/reperfusion (I/R) induced by midbrain cerebral artery occlusion (MCAO). Methods: Sprague-Dawley rats were subjected to 90 min of MCAO, which produces transient ischemia in both the retina and brain due to the use of an intraluminal filament that blocks the ophthalmic and middle cerebral arteries. This was followed by reperfusion under anesthesia with isoflurane. The day after surgery, the eyes were treated three times (eye drop) or one time (oral administration) daily with KIOM-2015E for five days. Retinal histology was assessed in flat mounts and vertical sections to determine the effect of KIOM-2015E on I/R injury. Results: A significant loss of brain-specific homeobox/POU domain protein 3A (Brn3a) and neuron-specific class III beta-tubulin (Tuj-1) fluorescence and a marked increase in glial fibrillary acidic protein (GFAP) and glutamine synthetase (GS) expression were observed after five days in the PBS-treated MCAO group compared to the sham-operated control group. However, KIOM-2015E treatment reduced (1) MCAO-induced upregulation of GFAP and GS, (2) retinal ganglion cell loss, (3) nerve fiber degeneration, and (4) the number of TUNEL-positive cells. KIOM-2015E application also increased staining for parvalbumin (a marker of horizontal cell associated calcium-binding protein and amacrine cells) and recoverin (a marker of photoreceptor expression) in rats subjected to MCAO-induced retinal damage. Conclusions: Our findings indicated that KIOM-2015E treatment exerted protective effects against retinal damage following MCAO injury and that this extract may aid in the development of novel therapeutic strategies for retinal diseases, such as glaucoma and age-related macular disease.
Subject(s)
Acer/metabolism , Apoptosis/drug effects , Neuroprotective Agents/pharmacology , Plant Extracts/pharmacology , Reperfusion Injury/metabolism , Retinal Degeneration/prevention & control , Retinal Ganglion Cells/drug effects , Acer/chemistry , Animals , Chromatography, High Pressure Liquid , Down-Regulation , Glial Fibrillary Acidic Protein/metabolism , Glutamate-Ammonia Ligase/metabolism , Male , Nerve Fibers/pathology , Plant Leaves/chemistry , Plant Leaves/metabolism , Rats , Rats, Sprague-Dawley , Reperfusion Injury/complications , Reperfusion Injury/mortality , Retinal Degeneration/complications , Retinal Degeneration/metabolism , Retinal Ganglion Cells/cytology , Retinal Ganglion Cells/pathology , Transcription Factor Brn-3B/metabolism , Tubulin/metabolism , Up-RegulationABSTRACT
Rosmarinic acid (RosA), an important polyphenol, is known for its antioxidant and anti-inflammatory activities. However, its application in theranostics has been rarely reported. Therefore, a new single-molecule anti-inflammatory theranostic compound containing RosA would be of great interest. A gadolinium (Gd) complex of 1,4,7,10-tetraazacyclododecane-1,4,7-trisacetic acid (DO3A) and RosA (Gd(DO3A-RosA)(H2O)) was synthesized and examined for use as a single-molecule theranostic agent. Its kinetic stability is comparable to that of clinically used macrocyclic magnetic resonance imaging contrast agents. In addition, its relaxivity is higher than that of structurally analogous Gd-BT-DO3A. This agent was evaluated for inflammatory targeting magnetic resonance contrast and showed strong and prolonged enhancement of imaging in inflamed tissues of mice. The theranostic agent also possesses antioxidant and anti-inflammatory activities, as evidenced by reactive oxygen species scavenging, superoxide dismutase activity, and inflammatory factors. The novel RosA-conjugated Gd complex is a promising theranostic agent for the imaging of inflamed tissues, as well as for the treatment of inflammation and oxidative stress.
ABSTRACT
Advancements in recanalization therapies have rendered reperfusion injury an important challenge for stroke management. It is essential to work toward effective therapeutics that protect the ischemic brain from reperfusion injury. Here, we report a new concept of neuroprognostic agents, which combine molecular diagnostic imaging and targeted neuroprotection for treatment of reperfusion injury after stroke. These neuroprognostic agents are inflammation-targeted gadolinium compounds conjugated with nonsteroidal anti-inflammatory drugs (NSAIDs). Our results demonstrated that gadolinium-based MRI contrast agents conjugated with NSAIDs suppressed the increase in cyclooxygenase-2 (COX-2) levels, ameliorated glial activation, and neuron damage that are phenotypic for stroke by mitigating neuroinflammation, which prevented reperfusion injury. In addition, this study showed that the neuroprognostic agents are promising T1 molecular MRI contrast agents for detecting precise reperfusion injury locations at the molecular level. Our results build on this new concept of neuroprognostics as a novel management strategy for ischemia-reperfusion injury, combining neuroprotection and molecular diagnostics.
Subject(s)
Cyclooxygenase 2/metabolism , Gadolinium/chemistry , Magnetic Resonance Imaging , Neuroprotective Agents/pharmacology , Reperfusion Injury/complications , Reperfusion Injury/prevention & control , Stroke/complications , Animals , Anti-Inflammatory Agents, Non-Steroidal/chemistry , Contrast Media/chemistry , Cyclooxygenase 2/chemistry , Male , Molecular Docking Simulation , Neuroprotective Agents/chemistry , Protein Conformation , Rats , Rats, Sprague-DawleyABSTRACT
ETHNOPHARMACOLOGICAL RELEVANCE: Astragali Radix (AR), the root of Astragalus mongholicus Bunge, is widely applied in traditional medicine to promote skin health and tissue regeneration. AIM OF THE STUDY: This study investigated the effects of AR and its active compound, formononetin (FMT), on skin barrier defects in keratinocytes exposed to diesel particulate matter (PM). MATERIALS AND METHODS: HaCaT cells and three-dimensional (3D) human skin reconstructed model were pre-treated with AR (50, 100⯵g/ml) and FMT (30, 50⯵M), then treated with PM (200⯵g/ml). RESULTS: AR and FMT significantly enhanced the expression of Keratin (KRT) 16 in PM stimulated HaCaT cells. PM increased p53 and Bax expression as well as the subsequent cleavage of caspase 3 and PARP in HaCaT cells, while this was inhibited by AR and FMT treatment. In vitro studies using the PM stimulated 3D human skin reconstructed model revealed that AR and FMT increased the expression of KRT 16 and KRT 17. Histological examination of the 3D human skin reconstructed model showed that AR and FMT up-regulated the expression of Ki67, but down-regulated the expression of cleaved caspase 3. Both AR and FMT significantly inhibited phosphorylation of ERK, but not JNK and p38 MAPK in PM stimulated HaCaT cells. CONCLUSIONS: These results suggest that AR and FMT act as anti-pollution agents and alleviate PM induced skin barrier defects through regulation of apoptosis and proliferation in keratinocytes.
Subject(s)
Drugs, Chinese Herbal/pharmacology , Isoflavones/pharmacology , Keratinocytes/drug effects , Particulate Matter/toxicity , Protective Agents/pharmacology , Skin/drug effects , Vehicle Emissions/toxicity , Apoptosis/drug effects , Astragalus propinquus , Cell Line , Cell Proliferation/drug effects , Extracellular Signal-Regulated MAP Kinases/metabolism , Humans , In Vitro Techniques , Keratinocytes/metabolism , Phosphorylation/drug effects , Skin/metabolismABSTRACT
In this study, we designed and synthesized a highly stable manganese (Mn2+)-based hepatobiliary complex by tethering an ethoxybenzyl (EOB) moiety with an ethylenediaminetetraacetic acid (EDTA) coordination cage as an alternative to the well-established hepatobiliary gadolinium (Gd3+) chelates and evaluated its usage as a T1 hepatobiliary magnetic resonance imaging (MRI) contrast agent (CA). This new complex exhibits higher r1 relaxivity (2.3 mM-1 s-1) than clinically approved Mn2+-based hepatobiliary complex Mn-DPDP (1.6 mM-1 s-1) at 1.5 T. Mn-EDTA-EOB shows much higher kinetic inertness than that of clinically approved Gd3+-based hepatobiliary MRI CAs, such as Gd-DTPA-EOB and Gd-BOPTA. In addition, in vivo biodistribution and MRI enhancement patterns of this new Mn2+ chelate are comparable to those of Gd3+-based hepatobiliary MRI CAs. The diagnostic efficacy of the new complex was demonstrated by its enhanced tumor detection sensitivity in a liver cancer model using in vivo MRI.
Subject(s)
Biliary Tract/diagnostic imaging , Contrast Media/chemical synthesis , Edetic Acid/chemistry , Liver/diagnostic imaging , Magnetic Resonance Imaging/methods , Manganese/chemistry , Animals , Cell Line , Chelating Agents/chemistry , Chelating Agents/pharmacokinetics , Contrast Media/chemistry , Edetic Acid/pharmacokinetics , Female , Gadolinium DTPA/chemistry , Heterografts , Humans , Hydrogen-Ion Concentration , Kinetics , Liver Neoplasms/diagnostic imaging , Liver Neoplasms/pathology , Male , Mice , Molecular Docking Simulation , Proton Magnetic Resonance Spectroscopy , Spectrometry, Mass, Electrospray Ionization , Spectrometry, Mass, Fast Atom BombardmentABSTRACT
The aim of this study was to assess the anti-inflammatory and anti-apoptotic effects of KIOM-2015EW, the hot-water extract of maple leaves in hyperosmolar stress (HOS)-induced human corneal epithelial cells (HCECs). HCECs were exposed to hyperosmolar medium and exposed to KIOM-2015EW with or without the hyperosmolar media. Tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-6 production and apoptosis were observed, and the activation of mitogen-activated protein kinases (MAPKs) including extracellular signal regulated kinase (ERK), p38 and c-JUN N-terminal kinase (JNK) signaling and nuclear factor (NF)-κB was confirmed. Compared to isomolar medium, the induction of cell cytotoxicity significantly increased in HCECs exposed to hyperosmolar medium in a time-dependent manner. KIOM-2015EW-treatment significantly reduced the mRNA and protein expression of pro-inflammatory mediators and apoptosis. KIOM-2015EW-treatment inhibited HOS-induced MAPK signaling activation. Additionally, the HOS-induced increase in NF-κB phosphorylation was attenuated by KIOM-2015EW. The results demonstrated that KIOM-2015EW protects the ocular surface by suppressing inflammation in dry eye disease, and suggest that KIOM-2015EW may be used to treat several ocular surface diseases where inflammation plays a key role.
Subject(s)
Acer , Anti-Inflammatory Agents/pharmacology , Apoptosis/drug effects , Epithelium, Corneal/drug effects , Osmotic Pressure , Plant Extracts/pharmacology , Xerophthalmia/prevention & control , Acer/chemistry , Anti-Inflammatory Agents/isolation & purification , Cells, Cultured , Dose-Response Relationship, Drug , Epithelium, Corneal/metabolism , Epithelium, Corneal/pathology , Humans , Inflammation Mediators/metabolism , Interleukin-1beta/metabolism , Interleukin-6/metabolism , Mitogen-Activated Protein Kinases/metabolism , Phosphorylation , Phytotherapy , Plant Extracts/isolation & purification , Plant Leaves , Plants, Medicinal , Signal Transduction/drug effects , Time Factors , Transcription Factor RelA/metabolism , Tumor Necrosis Factor-alpha/metabolism , Xerophthalmia/etiology , Xerophthalmia/metabolism , Xerophthalmia/pathologyABSTRACT
We examined the connection between matrix metalloproteinase (MMP) expression/activity and pterygium fibroblast migration, and how these were affected by bevacizumab and/or cyclosporine A (CsA). Fibroblasts were obtained from 20 pterygia and 6 normal conjunctival specimens. Expression levels of MMP-3 and MMP-13 were examined after bevacizumab administration. Immunofluorescence staining was used to examine expression of both MMPs in fibroblasts migrating out from explanted pterygium tissues. Rates of cell migration from explant-cultured pterygia tissues and scratch-wounded confluent pterygium fibroblasts were examined in the presence of MMP-3 or MMP-13 inhibitors, as well as bevacizumab and/or CsA. A scratch wound healing migration assay was performed to determine the effects of bevacizumab and/or CsA. Protein expression of both MMPs in pterygium tissues and in cells migrating from organ-cultured pterygium tissues was greater than that observed in normal cells. Inhibition of the activities of both MMPs decreased their expression levels; these were also significantly reduced in bevacizumab-injected pterygium tissues. Bevacizumab significantly reduced the expression of both MMPs and cell migration. Pretreatment with CsA prior to bevacizumab exposure markedly inhibited cell migration and the expression of both MMPs. CsA enhanced the inhibitory effects of bevacizumab on pterygium fibroblast migration in vitro, possibly by inhibiting expression of both MMPs. These findings suggest that combined CsA and bevacizumab treatment may provide a potential therapeutic strategy for reducing the rate of pterygium recurrence.
Subject(s)
Bevacizumab/pharmacology , Cyclosporine/pharmacology , Fibroblasts/drug effects , Fibroblasts/metabolism , Matrix Metalloproteinase 13/metabolism , Matrix Metalloproteinase 3/metabolism , Pterygium/metabolism , Cell Movement/drug effects , Cell Movement/genetics , Down-Regulation , Female , Gene Expression Regulation/drug effects , Humans , Immunohistochemistry , Male , Matrix Metalloproteinase 13/genetics , Matrix Metalloproteinase 3/geneticsABSTRACT
Cyclosporine A (CsA), an immunomodulatory drug, and is increasingly used to treat moderate dry eye syndrome and ocular surface inflammation. However, any inhibitory effect on differentiation of fibroblasts to myofibroblasts remains unclear. Here, we show that the inhibitory effect of CsA on transforming growth factor-beta2 (TGF-ß2)-induced myofibroblasts in primary cultured human pterygium fibroblasts. CsA significantly decreased mRNA and protein expression of myofibroblast-related markers including α-SMA, laminin, and fibronectin. These findings were supported by the results from immunofluorescence staining. Taken together, these results indicate the therapeutic potential of CsA against pterygium progression. Further studies are necessary to elucidate the precise intracellular signal mechanism responsible for CsA-induced downregulation of myofibroblast markers in pterygium fibroblasts.
Subject(s)
Cyclosporine/pharmacology , Fibroblasts/metabolism , Pterygium/drug therapy , Pterygium/metabolism , Actins/metabolism , Cell Differentiation , Cells, Cultured , Female , Fibronectins/metabolism , Humans , Immunosuppressive Agents/pharmacology , Inflammation , Laminin/metabolism , Male , Microscopy, Fluorescence , Muscle, Smooth/metabolism , Myofibroblasts/metabolism , Oligonucleotides/chemistry , Pterygium/surgery , Signal Transduction , Software , Transforming Growth Factor beta2/pharmacologyABSTRACT
This study aimed to investigate the neuroprotective properity of staurosporine (STS) and identify the neuroprotective mechanism of staurosporine in mouse retina ganglion cell after optic nerve injured. Mice (C57BL/6) were anaesthetised with a mixture of 5 mg/kg xylazine hydrochloride and 40 mg/kg tiletamine/zolazepam (Zoletil®). Optic nerves of the mice were crushed (Templeton JP et al., 2012). With micro-forceps, the bulbar conjunctiva was grasped and retracted, rotating the globe nasally. The exposed optic nerve was grasped approximately 1-3 mm from the globe with Dumont #N7 cross-action forceps for 10 s. One day after crushing, intravitreal injections of STS (500 nM) were administered using a Narishige IM-300 air pressure regulator. For analysing the change in ganglion cell number, the mice were allowed to live for 30 days, after which they were killed and the ganglion cell survival was assessed. A significant and marked loss of fluorescent spots was found after 30 days, with fewer 4',6-diamidino-2-phenylindole (DAPI)-expressing retinal ganglion cells (RGCs) remaining in the injured and phosphate buffered saline (PBS)-injected group than those in non-injured PBS-injected controls. However, RGC cell numbers dramatically increased in the STS intravitreal injection group. Moreover, degradation of nerve fibre (NF) was markedly reduced in the STS injection group compared with the injured and PBS-injected group by inducing astrocyte expression of Bcl-2. Our data suggested that injection of STS into the vitreous may have a potential therapeutic effect in retinal diseases such as glaucoma.
ABSTRACT
PURPOSE: The purpose of this study was to compare the cytotoxicity and antiinflammatory effect of preserved and unpreserved 0.1% fluorometholone (FML). METHODS: Drug-induced morphological changes and cytotoxicity were examined in human corneal epithelial cells. Dry eye was induced in mice by treatment with 0.2% benzalkonium chloride (BAC) for the first 2 weeks, and then, the eyes (4 groups; Normal saline, BAC, preserved FML, and unpreserved FML) were treated thrice daily with each formulation for the next 2 weeks. Corneal tissues were embedded in paraffin and stained with hematoxylin and eosin for histopathological examination. Immunofluorescence staining was performed for tumor necrosis factor-α, interleukin-6, and human leukocyte antigen-DR. Terminal deoxynucleotidyl transferase dUTP nick end labeling assay was performed to evaluate drug-induced cytotoxicity. RESULTS: BAC and preserved FML caused cell shrinkage and detachment from the plate in a dose-dependent manner, and cell viability decreased significantly. However, cytotoxicity was reduced on treatment with unpreserved FML. Hematoxylin-eosin staining revealed surface desquamation, irregular surface, loss of cell borders, and stromal shrinkage in the group treated with BAC. On BAC exposure, tumor necrosis factor-α, interleukin-6, and human leukocyte antigen-DR were strongly detected, and cytotoxicity was markedly increased, as evidenced by a positive result in the terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Ocular surface damage and inflammation were slightly reduced on treatment with preserved FML. In comparison, unpreserved FML did not induce morphological changes; moreover, decreased cell cytotoxicity and ocular surface inflammation were observed. CONCLUSIONS: The cytotoxicity of antiinflammatory eye drops evaluated in this study was induced by the preservative BAC. Accordingly, unpreserved FML is more effective than preserved eye drops in decreasing ocular inflammation.