Development of a Pancreatic Cancer Specific Binding Peptide Using Phage Display / 대한소화기학회지

BACKGROUND/AIMS: Pancreatic cancer has a very poor prognosis, and early diagnosis is a way to increase the survival rate of patients. The purpose of this study was to develop pancreatic cancer-specific peptides for imaging studies.METHODS: Three pancreatic cancer cell lines, MIA PaCa-2, UACC-462, and BxPC-3, and a control cell line, CCD841, were used. Biopannings were performed on MIA PaCa-2 using a phage display library. After this, the peptides were synthesized and labeled with fluorescein isothiocyanate (FITC). Immunocytochemistry (ICC), enzyme-linked immunosorbent assay (ELISA), and fluorescence-activated cell sorter (FACS) were performed to examine the specific binding. To examine its therapeutic applications, a photosensitizer, chlorin e6 (Ce6), was conjugated on the peptide and photodynamic therapy was performed. Cell survival was investigated using a [3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide] assay.RESULTS: After three biopannings, the phages were amplified from 1.4×104 to 3.2×105 plaque-forming units. The most strongly binding phage was selected from the ELISA and ICC results. FITC-labeled peptide, M5, in the three pancreatic cancer cell lines showed significantly higher immunofluorescence in the ICC experiments than that of CCD841. The higher binding ability to MIA PaCa-2 cells was confirmed from FACS analysis, which showed a right shift compared to CCD841. M5 bound to Ce6 showed a significantly lower cell survival rate than that of Ce6 alone in photodynamic therapy, which was observed consistently as a change in the tumor size and fluorescence intensity in MIA PaCa-2 cell-implanted animal models.CONCLUSIONS: This study showed that the noble peptide, M5, binds specifically to the pancreatic cancer cell line, MIA PaCa-2. The M5 peptide has potential use in future optical diagnostic and therapeutic purposes.

Development of a Pancreatic Cancer Specific Binding Peptide Using Phage Display / 대한소화기병학회잡지

BACKGROUND/AIMS: Pancreatic cancer has a very poor prognosis, and early diagnosis is a way to increase the survival rate of patients. The purpose of this study was to develop pancreatic cancer-specific peptides for imaging studies. METHODS: Three pancreatic cancer cell lines, MIA PaCa-2, UACC-462, and BxPC-3, and a control cell line, CCD841, were used. Biopannings were performed on MIA PaCa-2 using a phage display library. After this, the peptides were synthesized and labeled with fluorescein isothiocyanate (FITC). Immunocytochemistry (ICC), enzyme-linked immunosorbent assay (ELISA), and fluorescence-activated cell sorter (FACS) were performed to examine the specific binding. To examine its therapeutic applications, a photosensitizer, chlorin e6 (Ce6), was conjugated on the peptide and photodynamic therapy was performed. Cell survival was investigated using a [3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide] assay. RESULTS: After three biopannings, the phages were amplified from 1.4×104 to 3.2×105 plaque-forming units. The most strongly binding phage was selected from the ELISA and ICC results. FITC-labeled peptide, M5, in the three pancreatic cancer cell lines showed significantly higher immunofluorescence in the ICC experiments than that of CCD841. The higher binding ability to MIA PaCa-2 cells was confirmed from FACS analysis, which showed a right shift compared to CCD841. M5 bound to Ce6 showed a significantly lower cell survival rate than that of Ce6 alone in photodynamic therapy, which was observed consistently as a change in the tumor size and fluorescence intensity in MIA PaCa-2 cell-implanted animal models. CONCLUSIONS: This study showed that the noble peptide, M5, binds specifically to the pancreatic cancer cell line, MIA PaCa-2. The M5 peptide has potential use in future optical diagnostic and therapeutic purposes.

Characterization of allergic response induced by repeated dermal exposure of IL-4/Luc/CNS-1 transgenic mice to low dose formaldehyde / 한국실험동물학회지

Although formaldehyde (FA) is known to be a major allergen responsible for allergic contact dermatitis, there are conflicting reports regarding correlation between FA exposure and interleukin (IL-4) expression. To investigate whether allergic responses including IL-4 expression were induced by repeated dermal exposure to low dose FA, alterations in the luciferase signal and allergic phenotypes were measured in IL-4/Luc/CNS-1 transgenic (Tg) mice containing luciferase cDNA under control of the IL-4 promoter after exposure to 4% FA for 2 weeks. High levels of luciferase were detected in the abdominal region of the whole body and submandibular lymph node (SLN) of FA treated mice. Additionally, the ear thickness and IgE concentration were significantly upregulated in the FA treated group when compared with the acetone olive oil (AOO) treated group. FA treated mice showed enhanced auricular lymph node (ALN) weight, epidermis and dermis thickness, and infiltration of inflammatory cells. Furthermore, the expression of IL-6 among T helper 2 cytokines was higher in the FA treated group than the AOO treated group, while vascular endothelial growth factor (VEGF) levels remained constant. Overall, the results presented herein provide additional evidence that various allergic responses may be successfully induced in IL-4/Luc/CNS-1 Tg mice after exposure to low dose FA for 2 weeks. The luciferase signal under the IL-4 promoter may reflect general indicators of the allergic response induced by exposure to low dose FA.

Metabolomics approach to serum biomarker for loperamide-induced constipation in SD rats / 한국실험동물학회지

Loperamide has long been known as an opioid-receptor agonist useful as a drug for treatment of diarrhea resulting from gastroenteritis or inflammatory bowel disease as well as to induce constipation. To determine and characterize putative biomarkers that can predict constipation induced by loperamide treatment, alteration of endogenous metabolites was measured in the serum of Sprague Dawley (SD) rats treated with loperamide for 3 days using 1H nuclear magnetic resonance (1H NMR) spectral data. The amounts and weights of stool and urine excretion were significantly lower in the loperamide-treated group than the No-treated group, while the thickness of the villus, crypt layer, and muscle layer was decreased in the transverse colon of the same group. The concentrations of aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and creatinine (Cr) were also slightly changed in the loperamide-treated group, although most of the serum components were maintained at a constant level. Furthermore, pattern recognition of endogenous metabolites showed completely separate clustering of the serum analysis parameters between the No-treated group and loperamide-treated group. Among 35 endogenous metabolites, four amino acids (alanine, glutamate, glutamine and glycine) and six endogenous metabolites (acetate, glucose, glycerol, lactate, succinate and taurine) were dramatically decreased in loperamide-treated SD rats. These results provide the first data pertaining to metabolic changes in SD rats with loperamide-induced constipation. Additionally, these findings correlate the changes in 10 metabolites with constipation.

Toxicity of fermented soybean product (cheonggukjang) manufactured by mixed culture of Bacillus subtilis MC31 and Lactobacillus sakei 383 on liver and kidney of ICR mice / 한국실험동물학회지

To investigate the toxic effects of cheonggukjang (CKJ) manufactured using mixed cultures of Bacillus subtilis MC31 and Lactobacillus sakei 383 on the liver and kidney of ICR mice, an alteration on the related markers including body weight, organ weight, urine composition, liver pathology and kidney pathology were analyzed after oral administration at dosage of 25, 50 and 100 mg/kg body weight/day of CKJ for 14 days. Any significant toxicity was not observed on the body and organ weight, clinical phenotypes, urine parameters and mortality in the CKJ-treated group compared with the vehicle-treated group. Also, liver toxicity analysis revealed no significant increase in alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST) or lactate dehydrogenase (LDH) in response to CKJ. Additionally, the specific pathological features induced by most toxic compounds were not observed upon liver histological analysis. Furthermore, kidney toxicological analysis revealed that blood urea nitrogen (BUN) and the serum creatinine (Cr) levels and pathological features on histological sections did not differ significantly between the vehicle- and CKJ-treated groups. Overall, these results suggest that CKJ does not induce any specific toxicity in liver and kidney organs of ICR at dose of 100 mg/kg body weight/day as no observed adverse effect level (NOAEL).

Precautionary effects of Red Liriope platyphylla on NGF secretion and Abeta42 deposition under the preclinical stage of Alzheimer's disease in Tg2576 mice / 한국실험동물학회지

Red Liriope platyphylla (RLP) has been manufactured from Liriope platyphylla (L. platyphylla, LP) roots using steaming process and investigated as a curative agent for treatment of diabetes, obesity and neurodegenerative disorders. To examine the precautionary effects of aqueous extract RLP (AEtRLP) on the preclinical stages of Alzheimer's Disease (AD), alterations of the key factors influencing AD were investigated in Tg2576 mice after AEtRLP7 treatment for 4 months. Abeta-42 peptides level was significantly decreased in the brain of AEtRLP7-treated Tg2576 mice compared to vehicle-treated Tg2576 mice, although significant differences on improving behavioral defects were not observed in the same group. The concentration of nerve growth factor (NGF) in serum was also higher in AEtRLP7-treated Tg2576 mice than vehicle-treated Tg2576 mice. However, the phosphorylation of TrkA and Erk among the downstream effectors of the high affinity NGF receptor was significantly lower in AEtRLP7-treated Tg2576 mice. A similar pattern was observed in the expression level of downstream effectors within low affinity NGF receptor. Overall, these results suggest that AEtRLP7 can contribute to preventing the production and deposition of Abeta-42 peptides during the early progression stage of AD in the brain of Tg2576 mice through increased NGF secretion.

Red Liriope platyphylla stimulated the insulin secretion through the regulation of calcium concentration in rat insulinoma cells and animal models / 한국실험동물학회지

The aim of this study was to investigate the effects of Red L. platyphylla (RLP) on calcium and glucose levels during insulin secretion. To achieve this, alteration of insulin and calcium concentrations was measured in rat insulinoma-1 (INS-1) cells and animal models in response to RLP treatment. In INS-1 cells, maximum secretion of insulin was detected upon treatment with 200 microg/mL of RLP for 20 min. Nifedipine, an L-type calcium channel blocker, effectively inhibited insulin secretion from INS-1 cells. Regarding calcium levels, the maximum concentration of intracellular calcium in INS-1 cells was obtained by treatment with 100 microg/mL of RLP, whereas this level was reduced under conditions of 200 microg/mL of RLP. Further, RLP-treated INS-1 cells showed a higher level of intracellular calcium than that of L. platyphylla (LP), Korea White Ginseng (KWG), or Korea Red Ginseng (KRG)-treated cells. This RLP-induced increase in intracellular calcium was abrogated but not completely abolished upon treatment with 40 microM nifedipine in a dose-dependent manner. Furthermore, the insulin level was dramatically elevated upon co-treatment with high concentrations of glucose and RLP, whereas it was maintained at a low level in response to glucose and RLP co-treatment at low concentrations. In an animal experiment, the serum concentration of calcium increased or decreased upon RLP treatment according to glucose level compared to vehicle treatment. Therefore, these results suggest that insulin secretion induced by RLP treatment may be tightly correlated with calcium regulation, which suggests RLP is an excellent candidate for diabetes treatment.

Therapeutic effects of fermented soycrud on phenotypes of atopic dermatitis induced by phthalic anhydride / 한국실험동물학회지

Atopic dermatitis (AD), which is known as the most common pruritic skin disease, is caused by epidermal barrier dysfunction, allergies, microwave radiation, histamine intolerance, and genetic defects. To investigate the therapeutic effects of fermented soycrud (FSC) on AD pathology, alteration of AD phenotypes induced by phthalic anhydride (PA) treatment was assessed by ear thickness analysis, measurement of immune-related organ weights, ELISA, and histological and pathological analyses of ICR mice after FSC treatment for 2 weeks. Except for water content, the concentrations of most major components were lower in FSC compared to common tofu (CMT). Thymus and lymph node weights were significantly reduced in ICR mice treated with PA+CMT or PA+FSC, whereas spleen and body weights were maintained. Elevation of ear thickness induced by PA treatment was rapidly diminished in the CMT- and FSC-treated groups, although there was no significant difference between the two groups. Furthermore, significant reduction of epidermal thickness was detected in both the PA+CMT- and PA+FSC-treated groups. However, IgE concentration and dermal thickness were reduced only by PA+FSC treatment, whereas PA+CMT treatment maintained levels comparable to PA+vehicle treatment. The number of infiltrated mast cells was higher in the PA+vehicle-treated group compared to the untreated control. Following CMT or FSC treatment, mast cell infiltration was slightly reduced, although the CMT-treated group showed greater cell numbers. These results indicate that FSC may significantly relieve the phenotypes of AD induced by PA treatment and should be considered as a potential candidate for AD therapy.

In vitro and in vivo study of effects of fermented soybean product (chungkookjang) on NGF secretion ability and NGF receptor signaling pathway / 한국실험동물학회지

In order to investigate the effects of a fermented soybean product (Chungkookjang, CKJ) on nerve growth factor (NGF) metabolism, NGF secretion ability and its related signaling pathway were analyzed in B35 neuronal cells and the Tg2576 mouse model of Alzheimer's disease (AD). In B35 cells, the concentration of NGF significantly increased upon treatment with Taegwang (TG)-CKJ and Shinhwa (SH)-CKJ extracts compared with vehicle. Further, a significant increase in PC12 cell length as well as the phsophorylation levels of TrkA and Akt, which are members of a high affinity NGF receptor signaling pathway, were observed after treatment with TG-CKJ and SH-CKJ conditional medium (CM). On the other hand, there was no difference in activation of the NGF receptor p75NTR signaling pathway between vehicle and all CKJ treated groups. In Tg2576 mice showing early stage of AD, the concentrations of NGF in the serum and brain were reduced compared with those in Non-Tg mice. Treatment of Tg2576 mice with SH-CKJ, which contains high concentrations of total flavonoids and phenolic compounds, for 8 weeks dramatically recovered the NGF level to that of Non-Tg mice. Furthermore, the low phosphorylation levels of TrkA and Erk in the NGF receptor TrkA signaling pathway were rapidly recovered to those of Non-Tg mice after SH-CKJ treatment in vehicle treated Tg2576 mice, whereas the phosphorylation level of Akt was maintained at a constant level. These results suggest that CKJ may stimulate NGF secretion ability as well as the NGF receptor TrkA signaling pathway in PC12 cells and Tg2576 mice.

Toxicity of red Liriope platyphylla manufactured by steaming process on liver and kidney organs of ICR mice / 한국실험동물학회지

Red Liriope platyphylla (RLP) produced by steaming process has been reported to enhance the secretion of insulin and nerve growth factor (NGF). However, there has been no report on the toxicity of RLP in the specific organs of mice. To investigate the toxic effect of RLP, we tried to observe a significant alteration on body weight, food/water intake, organ weight, liver pathology and kidney pathology in female ICR mice received 12.5, 25.0 and 50.0 mg/kg body weight/day of RLP via gavage for 10 days. Out of seven organs including brain, heart, lung, liver, kidney, spleen and ovary, two organs (heart and lung) showed significantly decreased weights in the medium dosage RLP-treated group, whereas weights of other organs were maintained at constant levels in all dosage groups. In the liver toxicity analysis, no significant increase of alkaline phosphatase (ALP), alanine aminotransferase (ALT), and aspartate amino-transferase (AST) were detected in any RLP-treated group compared to vehicle-treated group. The specific pathological changes induced by most of toxic compounds were not observed in the liver in microscopic examination. Furthermore, in the kidney toxicological analysis, a significant enhancement of the blood urea nitrogen (BUN) concentration was detected in the high dosage RLP-treated group compared to the vehicle-treated group. However, the serum creatinine (CA) concentration on the serum biochemistry as well as the pathological changes in microscopic examination were not significantly different between the vehicle- and RLP-treated groups. Therefore, these results suggest that RLP does not induce any specific toxicity in liver or kidney tissues of mice, although the BUN level slightly increased in 50.0 mg/kg of RLP-treated group.