In vitro effects of heparin-binding epidermal growth factor on adhesion stage of implantation.

A member of the epidermal growth factor (EGF) family, the heparin-binding EGF (HB-EGF) is expressed in the uteri of both humans and mice during the implantation process. To study the effects of HB-EGF on adhesion stage, we developed an in vitro implantation model employing Ishikawa cell line and JAR cell line, which may attach to Ishikawa cells. For 1, 6, 12, and 24 hours, co-cultures of JAR spheroids grown on Ishikawa monolayers were treated with 1, 10, and 100 ng∕mL doses of HB-EGF. Using immunocytochemistry and Western blot analysis, the effects of HB-EGF on the protein expressions of E-cadherin, Erb-B2 receptor tyrosine kinase 4 (ErbB4), and integrin ανß3 in Ishikawa and JAR cells were examined semi-quantitatively and quantitatively. Ultrastructural changes of in vitro implantation model were investigated by transmission electron microscopy. We revealed that HB-EGF influenced trophoblast cell adhesion to endometrial cells by upregulating the expression of the proteins ErbB4 and trophoblastic integrin ανß3. Decrease in trophoblastic E-cadherin expression and increase in endometrial E-cadherin expression were demonstrated accompanying morphological variations in cells required for the invasion. We discovered ultrastructurally that Ishikawa cells acquired uterodome-like appearance, including the organelles, when 10 and 100 ng∕mL dosages of HB-EGF were administered for 12 and 24 hours. However, following additional hours of adhesion and invasion, their intercellular spaces enlarged. The trafficking of vesicular transport was enhanced by JAR spheroids. We therefore discovered that in this implantation paradigm, HB-EGF may enhance the receptivity of Ishikawa cells and the adherence of JAR cells.

Antitumor activity of irinotecan with ellagic acid in C6 glioma cells.

OBJECTIVE: Irinotecan-based combination chemotherapies in malignant gliomas need to be examined. The aim of this study was to investigate the synergetic effect of ellagic acid, a natural polyphenolic antioxidant compound, with irinotecan, an inhibitor of topoisomerase I enzyme, on the growth, cadherin switch, and angiogenic processes of a glioma cell line. METHODS: A combination of 100 µM ellagic acid and 100 µM irinotecan was applied to rat C6 glioma cells for 24th, 48th, and 72nd h. The cell proliferation was evaluated by 5-bromo-2'-deoxyuridine immunocytochemistry. The expression levels of vascular endothelial growth factor, E-cadherin, and N-cadherin were measured using real-time polymerase chain reaction and their immunoreactivities using immunocytochemistry. RESULTS: The treatment of irinotecan with combining ellagic acid enhanced antitumor activity and the synergistic effect of these reduced the cell proliferation of C6 glioma by inhibiting the cadherin switch and promoting the antiangiogenic processes. CONCLUSIONS: Further research is required to prove a negative relationship between C6 glial cell proliferation and irinotecan with ellagic acid application. Our preliminary data suggest that even with the extremely short-term application, irinotecan with ellagic acid may affect glioma cells at the level of gene and protein expression.

Antitumor activity of irinotecan with ellagic acid in C6 glioma cells

SUMMARY OBJECTIVE: Irinotecan-based combination chemotherapies in malignant gliomas need to be examined. The aim of this study was to investigate the synergetic effect of ellagic acid, a natural polyphenolic antioxidant compound, with irinotecan, an inhibitor of topoisomerase I enzyme, on the growth, cadherin switch, and angiogenic processes of a glioma cell line. METHODS: A combination of 100 μM ellagic acid and 100 μM irinotecan was applied to rat C6 glioma cells for 24th, 48th, and 72nd h. The cell proliferation was evaluated by 5-bromo-2′-deoxyuridine immunocytochemistry. The expression levels of vascular endothelial growth factor, E-cadherin, and N-cadherin were measured using real-time polymerase chain reaction and their immunoreactivities using immunocytochemistry. RESULTS: The treatment of irinotecan with combining ellagic acid enhanced antitumor activity and the synergistic effect of these reduced the cell proliferation of C6 glioma by inhibiting the cadherin switch and promoting the antiangiogenic processes. CONCLUSIONS: Further research is required to prove a negative relationship between C6 glial cell proliferation and irinotecan with ellagic acid application. Our preliminary data suggest that even with the extremely short-term application, irinotecan with ellagic acid may affect glioma cells at the level of gene and protein expression.

Cordycepin prevents the esophageal stricture formation in the alkali-burn rat model by exerting anti-inflammatory and antifibrotic effects.

PURPOSE: To investigate the efficacy of cordycepin, an adenosine analogue, on prevention of esophageal damage and stricture formation due to esophageal caustic burns in rat model comparing with prednisolone. METHODS: Caustic esophageal burn was introduced by 37.5% of NaOH to distal esophagus. Thirty-two Wistar albino rats were divided in four groups: sham rats undergone laparotomy, treated with 0.9% NaCl; control rats injured with NaOH without cordycepin treatment; cordycepin group injured with NaOH, treated with 20 mg/kg cordycepin; prednisolone group injured with NaOH, treated with 1 mg/kg prednisolone for 28 days. Efficacy was assessed by histopathological and immunohistochemical analysis of esophageal tissues. RESULTS: Cordycepin treatment significantly decreased inflammation, granulation tissue and fibrous tissue formation and prevented formation of esophageal strictures shown by histopathological damage score and stenosis indexes compared to control group (p < 0.01). These effects are relatively more substantial than prednisolone, probably based on attenuation of elevation of proinflammatory cytokines hypoxia-inducible factor 1-alpha (HIF-1?), tumor necrosis factor alpha (TNF-?), proliferative and fibrotic factor fibroblast growth factor 2 (FGF2) and angiogenic factor vascular endothelial growth factor A (VEGFA) (p < 0.05). CONCLUSIONS: The findings suggest that cordycepin has a complex multifactorial healing process in alkali-burned tissue, more successful than prednisolone in preventing the formation of esophageal strictures and may be used as a therapeutic agent in the acute phase of esophageal alkali-burn.

Cordycepin prevents the esophageal stricture formation in the alkali-burn rat model by exerting anti-inflammatory and antifibrotic effects

ABSTRACT Purpose To investigate the efficacy of cordycepin, an adenosine analogue, on prevention of esophageal damage and stricture formation due to esophageal caustic burns in rat model comparing with prednisolone. Methods Caustic esophageal burn was introduced by 37.5% of NaOH to distal esophagus. Thirty-two Wistar albino rats were divided in four groups: sham rats undergone laparotomy, treated with 0.9% NaCl; control rats injured with NaOH without cordycepin treatment; cordycepin group injured with NaOH, treated with 20 mg/kg cordycepin; prednisolone group injured with NaOH, treated with 1 mg/kg prednisolone for 28 days. Efficacy was assessed by histopathological and immunohistochemical analysis of esophageal tissues. Results Cordycepin treatment significantly decreased inflammation, granulation tissue and fibrous tissue formation and prevented formation of esophageal strictures shown by histopathological damage score and stenosis indexes compared to control group (p < 0.01). These effects are relatively more substantial than prednisolone, probably based on attenuation of elevation of proinflammatory cytokines hypoxia-inducible factor 1-alpha (HIF-1?), tumor necrosis factor alpha (TNF-?), proliferative and fibrotic factor fibroblast growth factor 2 (FGF2) and angiogenic factor vascular endothelial growth factor A (VEGFA) (p < 0.05). Conclusions The findings suggest that cordycepin has a complex multifactorial healing process in alkali-burned tissue, more successful than prednisolone in preventing the formation of esophageal strictures and may be used as a therapeutic agent in the acute phase of esophageal alkali-burn.

Ellagic Acid Enhances Antitumor Efficacy of Temozolomide in an in vitro Glioblastoma Model.

AIM: To observe the effect of combining ellagic acid (EA), a natural phenol present in fruits and vegetables, and temozolomide (TMZ) on the proliferation and expression profile of C6 glioma cell line. MATERIAL AND METHODS: Rat C6 glioma cells were treated with 100-?M EA combined with 100 ?M TMZ for 24, 48, and 72 hours (h). Cell proliferation and p53 and caspase-3 protein levels were evaluated using immunocytochemistry. Multi drug resistance 1 (MDR1), O6-methylguanine-DNA methyltransferase (MGMT), and apoptotic protein (caspase-3 and p53) expressions were assessed using reverse transcription polymerase chain reaction (RT-PCR). RESULTS: EA combined with TMZ conspicuously reduced the cell viability at all incubation times (p < 0.001). EA significantly downregulated MGMT expression regardless of the presence of TMZ even at early hours (p < 0.001). The combination therapy reduced MDR1 expression only on 48 h in comparison with TMZ alone. EA alone upregulated caspase-3 at 48 h but upregulated p53 at 48 and 72 h. The combined therapy enhanced the immunoreactivities of p53 and caspase-3 proteins independent of the treatment durations but not of the genes. CONCLUSION: EA combined with TMZ may have a potential antiproliferative efficacy by inhibiting MGMT expression and activating apoptotic protein, p53 and caspase-3, expression.

Ellagic Acid Enhances the Antitumor Efficacy of Bevacizumab in an In Vitro Glioblastoma Model.

BACKGROUND: The anticarcinogenic effect of ellagic acid (EA), a natural phenol of fruits and vegetables, has been investigated in several types of tumors. The combined effect of EA with bevacizumab (BEV), a common drug used in treatment of recurrent glioma, on glioblastoma has not been reported. This study observed the combined effect of EA with BEV on the expression profile of the C6 glioma cell line. METHODS: Rat C6 glioma cells were treated with EA at 100 µmol/L concentration in combination with BEV at 100 ng/mL concentration for 24, 48, and 72 hours. Cell proliferation was detected by 5-bromo-2'-deoxyuridine immunohistochemistry, and p53 and caspase-3 protein levels were determined by immunohistochemistry and assessed by the H-Score. Expression profiles for P-glycoprotein (MDR1), O6-methylguanine DNA methyltransferase (MGMT), caspase-3, and p53 related proteins were detected by reverse transcriptase polymerase chain reaction after EA treatment with or without BEV. RESULTS: EA combined with BEV conspicuously reduced the cell viability of C6 glioma cells for all incubation times. EA significantly downregulated expression of MGMT regardless of combination with BEV even in the early hours after treatment. Combined EA and BEV reduced MDR1 expression only at 72 hours. EA affected the apoptotic proteins of p53 and caspase-3 at protein level in a time-dependent manner, but not at gene level. CONCLUSIONS: This study suggests successful antiproliferative efficacy of EA combined with BEV, probably through inhibition of MGMT expression and time-dependent inhibition of MDR1. EA combined with BEV may be an alternative treatment for drug-resistant gliomas.

Combining Ellagic Acid with Temozolomide Mediates the Cadherin Switch and Angiogenesis in a Glioblastoma Model.

OBJECTIVE: We aimed to evaluate the combined effect of ellagic acid (EA) and temozolomide (TEM) on the cadherin switch and angiogenesis in the C6 glioma cell line. METHODS: A total of 100 µM EA and 100 µM TEM were applied to rat C6 glioma cells for 24, 48, and 72 hours. Cell proliferation was detected by 5-bromo-2'-deoxyuridine immunohistochemistry. The messenger RNA and protein levels of E-cadherin, N-cadherin, and vascular endothelial growth factor (VEGF) were determined by real-time polymerase chain reaction and their immunohistochemistry, respectively, subsequent to EA treatment combined with TEM. RESULTS: EA in combination with TEM conspicuously reduced the viability of C6 glioma cells at all incubation times (P < 0.001). EA upregulated the expression of E-cadherin at the gene and protein levels in a time-independent manner (P < 0.05 and P < 0.001, respectively). By the presence of TEM, the increase was exaggerated at 24-hour incubation (P < 0.01). Conversely, EA reduced N-cadherin expression and immunoreactivity in a time-independent manner (P < 0.05 and P < 0.001, respectively), and combination with TEM enhanced this effect at the 24th hour (P < 0.001). Combination also downregulated the gene expression (P < 0.001) and immunoreactivity of VEGF only at 72 hours (P < 0.001). CONCLUSIONS: A successful therapeutic efficacy of EA combined with TEM is suggested probably by inhibiting the cadherin switch and angiogenesis.

Comparison of incisional delay patterns on a rat random flap model.

One of the simplest form of surgical delay can be performed by placing an incision around the flap without undermining, prior to flap elevation. In this study, we have compared the efficiency of different patterns of skin incision to improve flap survival. Twenty-eight animals were used in four groups. Incisional delay was performed prior to flap elevation in the three experiment groups. Complete incision of the three flap edges was performed in the all experiment groups with the exception of an intact skin section on the middle 1/3rd of the bilateral edges in group 1 (bilateral skin edge preserved delay: BSEPD), of a unilateral edge in group 2 (unilateral skin edge preserved delay: USEPD) and of the superior edge in group 3 (superior skin edge preserved delay: SSEPD) without any undermining. Two weeks following the delay procedure, dorsal skin flaps were raised and reinserted back to their place. The results were evaluated with the measurement of necrotic flap area, microangiographic imaging and histological evaluation. The mean percentage of necrotic flap area to whole flap area was 16.94%, 7.54%, 23.34% and 50.6% in the BSEPD, USEPD, SSEPD and control groups, respectively. In selected microangiographic images, vessels were more prominent in the delay groups. The results of the study indicate that three sided incision with an intact skin on the superior edge is not effective in providing a sufficient delay and flap survival improvement when compared to incisions with intact skin on the unilateral and bilateral edges.

The beneficial effects of subcutaneous Allium cepa injection on random flap survival in rats.

Random flaps are frequently used in the practice of reconstructive surgery. The aim of this experimental study was to investigate the effects of Allium cepa on random flap survival in rats. Fourteen Wistar rats were used for this experimental study. The subjects were divided into experiment and control groups. Rats in the experiment group received daily injections of A. cepa extract for 7 d before the elevation of the flaps. Following the treatment period, elevation and reinsertion of the dorsal flaps were performed. Necrotic and total flaps areas were marked and calculated 7 d after the flap elevation. Histological examinations and microangiography were performed to evaluate the results. The mean value of the proportion of necrotic flap areas to the total flap area was 25.06 and 50.6% in the A. cepa and control group, respectively (p = .0079). In the histological examination, number of vessels identified in the dermis were 23.75 ± 0.7 and 33.75 ± 9 in the A. cepa and control group, respectively (p = .7457). In angiographic images, vessels formations were more noticeable in the A. cepa group. We conclude that preoperative subcutaneous A. cepa injection increases dorsal flap survival in rats.

Subdermal nitrous oxide delivery increases skin microcirculation and random flap survival in rats.

Random skin flaps are essential tools in reconstructive surgery. In this study, we investigated the effect of subdermal nitrous oxide (N2O) application on random flap survival. In this experimental study, we used 21 female rats in three groups. In the N2O and air groups, gases were administrated under the proposed dorsal flap areas daily for seven days. Following the treatment period, flaps were raised and inserted back into their place from the dorsal skin. In the control group, the flaps were elevated and inserted back to their place without any pretreatment. Calculation of necrotic flap areas, histological examination and microangiography was performed to evaluate the results 7 days after the flap surgery. The average of necrotic flap area in the N2O, air and control group was 13.45%, 37.67% and 46.43%, respectively. (N2O vs air p = .044; N2O vs control p = .003). The average number of capillary formations identified in the histological analysis was 7.0 ± 1.58, 3.75 ± 2.36 and 4.4 ± 0.54 in the N2O, air and control group, respectively. (N2O vs air p = .017; N2O vs control p = .037). The average number of capillary structures identified in the angiography images were 6.3 ± 1.52, 1.6 ± 1.15 and 1.3 ± 0.57 in the N2O, air and control group, respectively. (N2O vs air p = .04; N2O vs control p = .02). We conclude that subdermal N2O application increases random flap survival through an increase in the skin microcirculation and could be promising for future clinical applications.

Comparison of subdermal and perforator delay techniques on a rat flap model.

BACKGROUND: In this study, we investigated the subdermal and perforator delay phenomena as a method to improve flap survival. MATERIALS AND METHODS: In this experimental study, we used 24 rats in three groups. In the control group, the dorsal flaps were elevated and reinserted back to their place. In the experimental groups, we practiced the delay phenomena with two different techniques. In the first experimental group, cranial and lateral side incisions were performed; however, the flaps were not cut-off from the underlying fascia. In the second experimental group, we placed a silicon sheet under the planned flap to cut-off the circulation from the perforator vessels. Four weeks after the delay procedure, the flaps were raised completely and reinserted back to their place. RESULTS: The average of necrotic area in the control group was 21.9% (±7.70). There was no necrosis in both experimental groups (P < 0.0001). Histological examination revealed that collagen density in both of the experimental groups was increased in comparison to the control group, it has only been found a significant first experimental group (P = 0.0315). We have not found any significant difference in lymphocyte density between the groups. Angiographic imaging has showed an increase in the vascular density in the flaps of the first experimental group. CONCLUSION: We believe that both of these delay techniques can be adapted to clinical applications and used safely to increase flap survival.

Preoperative subcutaneous sildenafil injection increases random flap survival in rats.

PURPOSE: To investigate the effect of subcutaneous sildenafil on random flap survival. METHODS: Fourteen Wistar rats, which were divided in to two groups, were used for this experimental study. Rats in the sildenafil group received subcutaneous sildenafil injections daily for seven days before flap elevation. At the end of the treatment period, 9x3 cm dorsal skin flaps were elevated and reinserted back into their place in all of the animals. Necrotic and whole flaps areas were recorded on graph papers. Seven days after the flap elevation samples for histological examination were taken and angiographies were performed to visualize the flap vascularization. RESULTS: The calculated average percentage of necrotic flap areas were 18.29% and 42.26% in the sildenafil and control group respectively.(p=0.0233). In selected angiography images, vessels were found to be more prominent in the sildenafil group. The average number of capillary formations under light microscopy was higher in the sildenafil group (p= 0.0286). CONCLUSION: The subdermal high dose sildenafil has a positive effect on flap survival.

Preoperative subcutaneous sildenafil injection increases random flap survival in rats

Abstract Purpose: To investigate the effect of subcutaneous sildenafil on random flap survival. Methods: Fourteen Wistar rats, which were divided in to two groups, were used for this experimental study. Rats in the sildenafil group received subcutaneous sildenafil injections daily for seven days before flap elevation. At the end of the treatment period, 9x3 cm dorsal skin flaps were elevated and reinserted back into their place in all of the animals. Necrotic and whole flaps areas were recorded on graph papers. Seven days after the flap elevation samples for histological examination were taken and angiographies were performed to visualize the flap vascularization. Results: The calculated average percentage of necrotic flap areas were 18.29% and 42.26% in the sildenafil and control group respectively.(p=0.0233). In selected angiography images, vessels were found to be more prominent in the sildenafil group. The average number of capillary formations under light microscopy was higher in the sildenafil group (p= 0.0286). Conclusion: The subdermal high dose sildenafil has a positive effect on flap survival.

Differences between Solution and Membrane Forms of Chitosan on the In Vitro Activity of Fibroblasts.

BACKGROUND: Chitosan, a linear polysaccharide, has been recently used in biomedical applications. In vitro studies have demonstrated its effect on cellular growth and its stimulatory action on cellular layer formation. AIMS: The present study aims to compare the proliferative effects of chitosan in two forms, membranous and solution forms, on Swiss 3T3 mouse embryonic fibroblasts. STUDY DESIGN: In vitro study. METHODS: Three experimental groups were formed: cells were cultured in a normal medium without chitosan (Control Group); cells were cultured either in a medium containing 2.0% chitosan in membranous form (Membrane Group) or chitosan solution at a concentration of 2.0% (Solution Group). Two different methods were used in the experiments: cells cultured on the medium containing chitosan in solution or membranous forms (method 1); and chitosan solution or membranous forms were added into the medium containing previously cultured cells (method 2). RESULTS: Scanning electron microscopic investigations of the experimental groups revealed cells with well-defined cellular projections, intact cellular membranes and tight intercellular junctions. They were especially prominent in the membrane group of method 1 and in the membrane and solution groups of method 2. Mouse monoclonal anti-collagen 1 primary antibody was used to indicate collagen synthesis. Prominent collagen synthesis was detected in the membrane groups on the 10(th) day of culture for both methods. Bromodeoxyuridine (BrdU) and MTT assays were performed in order to assess cellular proliferation and viability, respectively. BrdU labelling tests indicated a higher proliferation index in the membrane group of method 1 on the 5(th) and 10(th) days. For the second method, the membranous form on the 10(th) day and solution form on the 5(th) day were the most effective groups in terms of cellular proliferation. MTT results reflected a high cellular viability in method 1 on the 5(th) day of treatment with the membranous form, whereas cellular viability was highest in the solution form of method 2 on the 5(th) day. CONCLUSION: The membranous form of chitosan induced a significant proliferative effect and increased the ratio of cell-to-cell junctions of Swiss 3T3 mouse embryonic fibroblasts. Conveniently, the solution form also resulted in enhanced cell proliferation and viability compared to the control group. As the solution form is easy to prepare and apply to cells compared to the membrane form, the application of Chitosan directly to media appears to be a convenient alternative for tissue engineering approaches.

The effects of protein kinase C activator phorbol dibutyrate on traumatic brain edema and aquaporin-4 expression.

BACKGROUND: Aquaporin-4 (AQP4) is the major water channel in the central nervous system. Brain edema emerges from increased AQP4 expression in traumatic brain injury (TBI). Cell line studies have shown that the protein kinase activator phorbol ester exerts a suppressive effect on AQP4 and water permeability. The aim of this study was to investigate the effects of a phorbol ester, phorbol dibutyrate (PDBu), on increased TBI AQP4 expression and accompanying brain edema. METHODS: Fifty-six male Wistar rats were first divided into two groups: the edema group, in which the percentage of water in brain tissue would be evaluated, and the immunohistochemical group, allowing AQP4 expression to be determined. Both groups were further sub-divided into four groups consisting of 7 subjects. These four groups were as follows: sham-operated control group, severe diffuse TBI group, 0.9% saline-treated diffuse TBI group, and the PDBu-treated diffuse TBI group (2300 µg/kg, iv). The results were evaluated statistically. RESULTS: PDBu treatment significantly reduced brain water concentration (p<0.001). Furthermore, PDBu was found to reduce trauma-induced AQP4 upregulation (p<0.05). CONCLUSION: This study showed that traumatic brain edema was prevented by intravenous PDBu administration via AQP4 downregulation, supporting the idea emphasizing the importance of AQP4 expression control in TBI.