Development and characterization of cancer stem cell-based tumoroids as an osteosarcoma model.

Three-dimensional (3D) cancer tumor models are becoming vital approaches for high-throughput drug screening, drug targeting, development of novel theranostic systems, and personalized medicine. Yet, it is becoming more evident that the tumor progression and metastasis is fueled by a subpopulation of stem-like cells within the tumor that are also called cancer stem cells (CSCs). This study aimed to develop a tumoroid model using CSCs. For this purpose CD133+ cells were isolated from SaOS-2 osteosarcoma cell line with magnetic-activated cell sorting. To evaluate tumoroid formation ability, the cells were incubated in different cell numbers in agar gels produced by 3D Petri Dish® method. Subsequently, CD133+ cells and CD133- cells were co-cultured to investigate CD133+ cell localization in tumoroids. The characterization of tumoroids was performed using Live&Dead staining, immunohistochemistry, and quantitative polymerase chain reaction analysis. The results showed that, CD133+ , CD133- and SaOS-2 cells were all able to form 3D tumoroids regardless of the initial cell number, but, while 72 hr were needed for CD133+ cells to self-assemble, 24 hr were enough for CD133- and SaOS-2 cells. CD133+ cells were located within tumoroids randomly with high cell viability. Finally, when compared to two-dimensional (2D) cultures, there were 5.88, 4.14, 6.95, and 1.68-fold higher messenger RNA expressions for Sox2, OCT3/4, Nanog, and Nestin, respectively, in CD133+ cells that were cultured within 3D tumoroids, showing longer maintenance of stem cell phenotype in 3D, that can allow more relevant screening and targeting efficiency in pharmaceutical testing. It was concluded that CSC-based tumoroids are propitious as 3D tumor models to fill the gap between conventional 2D in vitro culture and in vivo animal experiments for cancer research.

Novel Simple Strategy for Cartilage Tissue Engineering Using Stem Cells and Synthetic Polymer Scaffold.

Cartilage created by tissue engineering is a promising new development in facial reconstructive surgery. The purpose of this study was to evaluate the histological results of implantation of synthetic polymer scaffold with chondrocytes differentiated from adipose-derived mesenchymal stem cells. Adipose tissue obtained from Wistar albino rats was dissociated, incubated and placed in culture medium. After a sufficient level of stem cell proliferation, the differentiation phase was started. Cells were collected on the 7th and 21st day of culture for chondrogenic characterization. After the 21st day of the differentiation phase of chondrocytes, they were transferred onto poly(dl-lactide-epsilon-caprolactone) synthetic polymer and culture continued for 24 hours. The scaffold with chondrocytes was then implanted into a subcutaneous area of skin on the back of the neck of the rat. Six weeks after implantation, all rats were sacrificed and the implantation areas were analyzed. Chondrocytes derived from adipogenic mesenchymal stem cells were stained positively with collagen II, aggrecan and Sox-9 after the differentiation stages. Histological examination of the excised material showed that chondrocytes were present, and the scaffold had been completely absorbed. The results of this study indicate that the differentiation method from mesenchymal stem cells to chondrogenic lineage was straightforward and scaffold with cells was easily accessible. This technique may be a good option for cartilage tissue engineering.

13-93B3 Bioactive Glass: a New Scaffold for Transplantation of Stem Cell-Derived Chondrocytes.

Research using animal models gives human trials hope for recovery in many fields of regenerative medicine, although they are sometimes poor predictors for human experiences. Our goal was to investigate whether rat chondrocytes, differentiated from adipose-derived stem cells, could be transplanted using a new, easily shaped, bioactive glass scaffold, and to show the immunohistochemical results. Intraperitoneal and retroperitoneal adipose tissue was extracted from 6 male Wistar albino type rats. The fatty tissue samples were fragmented and incubated. Chondrogenic differentiation was carried out and collagen type II, bFGF, and Sox-9 immunohistochemical characterization analysis was performed. Differentiated chondrocytes were implanted on 13-93B3 bioactive glass scaffolds and transplanted into the right ears of the rats. As control, only the biomaterial was transplanted into the left ears of the rats. After 1 month, the rats were sacrificed and transplantation areas were examined immunohistochemically. Histological examination of control samples from the left ears revealed that the biomaterial was covered with connective tissue, its general structure was preserved, and resorption of the scaffold had started. In specimens from the right ears, the biomaterial was covered with connective tissue, its structure was preserved, cartilage cells were present around the biomaterial, and the presence of cartilage tissue was demonstrated immunohistochemically. In conclusion, 13-93B3 bioactive glass scaffold contributed to the formation of new collagen and the survival of chondrocytes, and is a promising new biomaterial that will prove very useful in regenerative medicine.

Role of Proopiomelanocortin in Preventing Miscarriage.

Objective: To compare the distribution of proopiomelanocortin (POMC) in decidua and placenta samples from missed abortion and voluntary termination cases in order to research the effects in the etiology of missed abortion. Study Design: Decidual materials were collected from patients who were diagnosed with missed abortion (n=19) and legal voluntary termination cases (n=15) under 10 gestational weeks. Materials were divided into 2 groups for examination. For all samples, POMC primary antibody was performed by immunohistochemical staining. The number of stained cells was calculated by using the H-score technique. Results: In the missed abortion group the mean age was 28.7 (18­41), and in the control group the mean age was 27.5 (21­37). POMC immunoreactivity was determined to be lower in the parenchyma and placenta of the missed abortion group than those of the control group. POMC immunoreactivities were found to be higher in both the syncytiotrophoblast and cytotrophoblast cells of the missed abortion group than those of the control group (p<0.005). Conclusion: POMC has become a paradigmatic polypeptide precursor and has a role in the parturition process. Local production of POMC in placenta and decidua may influence pregnancy and may have a role in missed abortion pathogenesis.

Fatty acid composition and anticancer activity in colon carcinoma cell lines of Prunus dulcis seed oil.

CONTEXT: Almond oil is used in traditional and complementary therapies for its numerous health benefits due to high unsaturated fatty acids content. OBJECTIVES: This study investigated the composition and in vitro anticancer activity of almond oil from Northern Cyprus and compared with almond oil from Turkey. MATERIALS AND METHODS: Almond oil from Northern Cyprus was obtained by supercritical CO2 extraction and analyzed by GC-MS. Almond oil of Turkey was provided from Turkish pharmacies. Different concentrations of almond oils were incubated for 24 and 48 h with Colo-320 and Colo-741 cells. Cell growth and cytotoxicity were measured by MTT assays. Anticancer and antiprolifetarive activities of almond oils were investigated by immunocytochemistry using antibodies directed against to BMP-2, ß-catenin, Ki-67, LGR-5 and Jagged 1. RESULTS: Oleic acid (77.8%; 75.3%), linoleic acid (13.5%; 15.8%), palmitic acid (7.4%; 6.3%), were determined as the major compounds of almond oil from Northern Cyprus and Turkey, respectively. In the MTT assay, both almond oils were found to be active against Colo-320 and Colo-741 cells with 1:1 dilution for both 24 h and 48 h. As a result of immunohistochemical staining, while both almond oils exhibited significant antiproliferative and anticancer activity, these activities were more similar in Colo-320 cells which were treated with Northern Cyprus almond oil. DISCUSSION AND CONCLUSION: Almond oil from Northern Cyprus and Turkey may have anticancer and antiproliferative effects on colon cancer cells through molecular signalling pathways and, thus, they could be potential novel therapeutic agents.

Analysis of the association of chronic spontaneous urticaria with interlekin-4, -10, transforming growth factor-ß1, interferon-γ, interleukin-17A and -23 by autologous serum skin test.

AIM: To contribute to the understanding of the pathogenesis of chronic spontaneous urticaria (CSU) by identifying its relationship with autoimmunity and cytokines using the autologous serum skin test (ASST) and peripheral blood mononuclear cell culture (PBMC) method. MATERIAL AND METHODS: Interleukins (IL)-4, IL-10, transforming growth factor (TGF-ß1), interferon (IFN)-γ, IL-17A, and IL-23 levels in cell supernatants obtained by the PBMC method were measured using ELISA. Disease activity was assessed by determining the urticaria activity score (UAS). RESULTS: A total of 40 patients diagnosed with CSU participated in this study. Twenty patients had positive ASST results, and 20 had negative results. The control group included 20 healthy volunteers. We found that the IL-23 (p = 0.01), IL-10 (p = 0.04) and IL-4 (p = 0.04) levels of the patient groups were significantly lower compared with those of the control group. The IL-23 (p = 0.009), IL-10 (p = 0.009), IL-4 (p = 0.001), and IL-17 (p = 0.05) levels of the ASST(-) patient group were significantly lower compared with those of the control group. In addition, the IL-4 (p = 0.03) and IFN-γ (p = 0.05) levels of the ASST(+) patient group were significantly lower compared with those of the control group, and the ASST(+) patients had a significantly higher UAS than the ASST(-) patients (p = 0.021). CONCLUSIONS: These results, when considered together with current reports in the literature, indicate that immune dysregulation occurs in the pathogenesis of CSU, causing cytokine imbalance.

Montelukast is effective in preventing of ovarian hyperstimulation syndrome; an experimental study.

OBJECTIVES: To determine the efficacy of montelukast in comparison with cabergoline in the prevention of ovarian hyperstimulation syndrome (OHSS) in rats. MATERIAL AND METHODS: An experimental OHSS model was formed in 35 female Wistar rats. Rats (22 days old) were randomized into 5 groups, each containing 7 animals. The control group received no therapy; the mild OHSS group was administered pregnant mare serum gonadotropin (PMSG) 10 IU for 4 days, hCG 10 IU on the 5th day; the severe OHSS group received PMSG 10 IU for 4 days, hCG 30 IU on the 5th day The montelukast group: received montelukast 10 mg/kg/day and the cabergoline group was administered cabergollne 100 microg/kg/day via oral gavage for 6 days (days 22-27), in addition to those of severe OHSS. All groups were sacrificed on 28th day Body weight, ovarian diameter and weight, vascular permeability vascular endothelial growth factor (VEGF), semiquantitative VEGF receptor-1, and VEGF receptor-2 (VEGFR-2) immunohistochemistry were evaluated. RESULTS: Ovarian diameter and VEGF expression were significantly lower in the montelukast and cabergoline groups than in the severe OHSS group. While montelukast was more effective in limiting vascular permeability in the severe OHSS, cabergoline was superior to montelukast with respect to the limiting effect on increased body weight and VEGFR-2 expression. CONCLUSIONS: The VEGF/VEGFR-2 interaction plays an important role in OHSS pathogenesis. Montelukast limits VEGF expression, and cabergoline reduces both VEGF and VEGFR-2 expressions; they are both effective therapies for the prevention of severe OHSS.

Regulatory-T, T-helper 1, and T-helper 2 cell differentiation in nasal mucosa of allergic rhinitis with olive pollen sensitivity.

BACKGROUND: Allergic rhinitis (AR) is a disease in which T-helper (Th)2 response is predominant and its pathogenic mechanism is still poorly understood. AIM: To evaluate the possible role of Th1, Th2 and regulatory-T (Treg) cells in the pathogenesis of AR. METHODS: This case-control study enrolled 41 patients with seasonal AR (10-62 years old), sensitive to olive pollens, and 15 healthy controls (18-60 years old). Nasal biopsy was performed and specimens of nasal lavage fluid were obtained from all participants. The levels of interleukin (IL)-4, IL-10, interferon (IFN)-γ and transforming growth factor-ß (TGF-ß) were measured in nasal lavage fluid specimens. The expression of FOXP3, GATA-3 and T-bet was measured by immunohistochemical methods in the nasal biopsy specimens. RESULTS: The levels of IFN-γ in the group with AR were significantly lower than those in the control group (p = 0.008). The levels of IL-4, IL-10 and TGF-ß did not differ between the two groups. The expression of FOXP3 and T-bet in patients with AR was significantly lower than that in the control group (both p = 0.001). Expression of GATA-3 in the nasal mucosa was similar between the groups (p = 0.2). The ratios of T-bet/GATA-3 and FOXP3/GATA-3 in the AR group were significantly lower than those in the control group (p = 0.001). CONCLUSION: Insufficient Treg and Th1 cells may be associated with the allergic inflammation that may be attributed to the Th2 immune response in patients suffering from AR who are sensitive to olive pollen.

The Role of TREK-1 and AQP5 in Gonadocorticoid-Related Voice Disorders.

OBJECTIVES: TWIK-related potassium channel-1 (TREK-1) and Aquaporin 5 (AQP5) are involved in epithelial integrity and fluid transport, respectively. In this study, we aimed to compare physiological and gestational patterns of TREK-1 and AQP5 location and expression in rat larynx. Our secondary objective was to reveal the effect of estradiol (E2) and progesterone (PG) on these two biomolecules. METHODS: This study was conducted on 20 Wister albino female rats which were assigned as control (group A) and pregnant group (group B). The rats were sacrificed at 20th day of pregnancy. Blood was obtained directly from the ventricle for detection of serum E2 and PG levels. Larynx was resected for immunohistochemical analyses and real-time polymerase chain reaction testing for detection of TREK-1 and AQP5 staining and expression, respectively. RESULTS: Relative TREK-1 (P = 0.035) and AQP5 (P = 0.019) expression was found to be significantly high in group B when compared with group A. We found positive correlation between serum E2 levels and both biomolecules (TREK-1; P = 0.018, AQP5; P = 0.016). We also found positive correlation between serum PG levels and both biomolecules (TREK-1; P = 0.001, AQP5; P = 0.019). TREK-1 immunostaining was found to be higher in surface epithelium and lamina propria of vocal cord mucosa. AQP5 was particularly found to be located in basement membrane and adjacent superficial lamina propria. We revealed the physiological and gestational pattern of laryngeal TREK-1 and AQP5 expression for the first time. Gestational expression of both TREK-1 and AQP5 was found to be increased. Stimulatory effect of E2 and PG on laryngeal TREK-1 and AQP5 expression was also revealed. CONCLUSIONS: We revealed upregulatory effect of E2 and PG on laryngeal TREK-1 and AQP5 expression. Based on this finding, it can be suggested that TREK-1 and AQP5 play role in biomolecular processes leading gonadocorticoid-related voice changes.

Role of VPAC1 and VPAC2 receptors in the etiology of pregnancy rhinitis: an experimental study in rats.

INTRODUCTION: Pregnancy rhinitis is a common sex hormone-related otorhinolaryngological disorder. There are some epidemiological and physiological studies on pregnancy rhinitis, but histopathological and biomolecular changes have not been studied thoroughly. OBJECTIVES: The receptors VPAC1 and VPAC2 are known for their roles in allergic rhinitis. On the other hand, activation of subclinical allergy has been suggested in the pathophysiology of pregnancy rhinitis. Therefore, we aimed to compare the physiological and gestational pattern of VPAC1 and VPAC2 expression in rat nasal mucosa. METHODS: Twenty adult Wister albino female rats were enrolled into the study. Two groups constituted as 10 control (group A) and 10 pregnant (group B) rats. They were fed ad libitum and sheltered at room temperature (22°±2°C). The rats were sacrificed at the 20th day of gestation by intraperitoneal injection of 400mg/kg Na-pentobarbitone. Then, 10-15mL of blood was taken, and samples were reserved for the detection of serum estradiol and progesterone levels by ELISA test. The nasal septum was resected and divided in half for immunohistochemical analyses and real time polymerase chain reaction testing of VPAC1 and VPAC2. RESULTS: VPAC1 and VPAC2 were found to be in all layers of septal specimens, but the immunostaining of surface epithelium was more distinct in specimens of both groups. We demonstrated higher overall staining intensity in the pregnant group. PCR revealed significant increase in expression of VPAC1 (p=0.023) and VPAC2 (p=0.021) in pregnant group when compared with control group. In addition, we demonstrated upregulatory effect of estradiol and progesterone on the vasoactive intestinal peptide receptor expression. CONCLUSIONS: Gestational up-regulation of nasal VPAC1 and VPAC2 was shown both by PCR and immunohistochemical analysis. These findings support the hypothesis that PR is caused by the activation of subclinical allergy that is present before pregnancy.

Impact of Pregnancy and Glucocorticoid Treatment on NF-κB and MUC5AC in Mucosa of Rat Larynx.

OBJECTIVE: The aim of this study is to reveal physiological expression and distribution of nuclear factor-kappa B (NF-κB) and MUC 5 subtype AC (MUC5AC) in rat laryngeal mucosa and to find out the effect of pregnancy and glucocorticoid treatment on these biomolecules. METHODS: This animal experiment was done in Experimental Animals Research and Application Center of Manisa Celal Bayar University in accordance with the accepted policy on the use of animals. A total of 30 young, adult Wister albino female rats were randomized into a control group (group A), a pregnant group (group B), and a steroid administered group (group C). Sacrification was done by injection of sodium-pentobarbitone (400 mg/kg) solution via intraperitoneal route in all groups. Serum estradiole (E2) and progesterone (PG) were determined by enzyme-linked immunosorbent assay. The relative expression and distribution of NF-κB and MUC5AC in laryngeal mucosa was studied both by immunohistochemistry (IHC) and polymerase chain reaction testing. Expression and immunohistochemical localization of NF-κB and MUC5AC was evaluated by light microscopy (Olympus BX41). In statistical analyses; relative expression of NF-κB and MUC5AC were compared on group basis. The effect of E2 and PG levels on these biomolecules was also evaluated. RESULTS: NF-κB was found to be significantly low both in group B (P < 0.05) and C (P < 0.001) when compared with group A, while MUC5AC was found to be significantly high both in group B (P < 0.05) and group C (P < 0.05) when compared with group A. Concerning IHC; NF-κB was found to be expressed in epithelium and lamina propria. MUC5AC was found to be expressed particularly in the epithelial layer in all groups. Statistically significant negative correlation between PG and NF-κB expression (P = 0.048), but no correlation between PG and MUC5AC expression (P = 0.487) were revealed. On the other hand, no correlation was found between E2 and the expression of relevant biomolecules (NF-κB [P = 0.270], MUC5AC [P = 0.829]). We also did found a significant negative correlation between the expression of NF-κB and MUC5AC (P = 0.031). CONCLUSIONS: In this study, the physiological expression of NF-κB and MUC5AC in rat laryngeal mucosa was shown for the first time both by polymerase chain reaction and IHC. The impact of pregnancy and glucocorticoid treatment on the expression and distribution of these biomolecules was also revealed. The expression of NF-κB was found to be decreased while the expression of MUC5AC was found to be increased both by pregnancy and glucocorticoid treatment. The inhibitory effect of serum PG on NF-κB expression in rat laryngeal mucosa was also shown for the first time. The expression of MUC5AC was found to be increased both in pregnant and glucocorticoid administered group. Negative correlation between NF-κB and MUC5AC expression was also revealed in rat larynx for the first time. These findings may partially unclose the histochemical background of voice changes caused by pregnancy and as well as by glucocorticoid treatment.

Antibiotic treatment is superior to ursodeoxycholic acid on total parenteral nutrition associated hepatic dysfunction.

PURPOSE: This study aimed to investigate the apoptotic mechanisms, oxidative stress, and mechanisms of effect of antibiotics and ursodeoxycholic acid (UDCA) in total parenteral nutrition (TPN)-associated liver injury. METHODS: Four groups of young rabbits were used in the study as follows: Group 1 (n: 7): TPN + Metronidazole (30 mg/kg IV) + Gentamicin (6 mg/kg IV); Group 2 (n: 7): TPN + UDCA (15 mg/kg per oral); Group 3 (n: 6): TPN only; and Group 4 (n: 7): Control group. After 10 days, the animals were killed and livers were removed. Hepatic apoptosis, apoptotic proteins, malondialdehyde (MDA) and myeloperoxidase (MPO) levels were studied in liver, and direct bilirubin values were assessed in the blood samples. RESULTS: Direct bilirubin increased with TPN, and antibiotic combination, as the most effective group, significantly lowered its levels (p < 0.01). MDA values also showed significant differences in comparisons between G1 and G3 (p < 0.05) and G1-G4 (p < 0.01). An increased number of apoptotic cells was detected particularly in G2 and G3, whereas the lowest levels, other than in the control group, were found in G1. All TUNEL-positive cell number data were statistically significant except between G2 and G3(p < 0.05). Caspase-3 and Bax immunoreactivities were greatest in G2. Significant differences were shown in caspase-3 immunoreactivity between the groups (p < 0.01), except between G1 and G3 (p > 0.05). All comparisons between the groups were significant for Bax (p < 0.01). In contrast, Bcl-2 immunoreactivity was moderate and highest in G1: comparisons between G1 and the other groups demonstrated statistically significant differences (p < 0.01). Fas-L immunoreactivity was greatest in G2, and all comparisons between the groups were statistically significant (p < 0.01). CONCLUSIONS: Metronidazole and gentamicin combination is effective on TPN-induced liver injury by the Bcl-2 anti-apoptotic pathway, total anti-apoptotic effect and by decreasing bilirubin levels. Oxidative injury in the liver increased with therapy. UDCA seems less effective on TPN-associated liver injury.

Levosimendan up-regulates transforming growth factor-beta and smad signaling in the aorta in the early stage of sepsis.

BACKGROUND: This prospective, controlled experimental study was planned to investigate the effects of levosimendan on transforming growth factor (TGF)-beta3 and Smad1, Smad2 and Smad3 expression in the early stages of sepsis. METHODS: Twenty-four rats were randomized into four groups: (1) sham-operated controls, (2) dobutamine group--subjected to abdominal hypertension and peritonitis-induced sepsis using cecal ligation and puncture (CLP), then treated with 10 microg x kg(-1) min(-1) intravenous (IV) dobutamine infusion, (3) levosimendan group--as in 2, then treated with levosimendan IV bolus 200 microg x kg(-1) followed by 200 microg x kq(-1) min(-1) IV infusion, and (4) a control group as in 2, with no treatment. All rats were killed 8 hours after CLP. Aorta tissue samples were analyzed by immunohistochemical staining. RESULTS: CLP caused mild interleukin (IL)-1 immunostaining in both control and dobutamine groups. Immunoreactivity of tumor necrosis factor (TNF)-alpha was mild in both sham and control groups. TGF-beta3 immunostaining was mildly increased in groups sham, control and dobutamine, whereas it was found moderate in group levosimendan. Smad1, Smad2 and Smad3 were found moderately increased only in group levosimendan. CONCLUSION: Beneficial effects of levosimendan on hemodynamics and global oxygen transport were reported in experimental and clinical trials. Besides its potency on C++ ion sensitivity, it should influence inflammatory cytokine production by diminishing TGF-beta3 and Smad1, Smad2 and Smad3 expression.

Obtaining Stem Cell Spheroids from Foreskin Tissue and the Effect of Corchorus olitorius L. on Spheroid Proliferation.

OBJECTIVES: Mesenchymal stem cells are self-renewing stem cells. The human foreskin has potential to be used as a source of stem cells. The aim of the study was to obtain spheroid formation of human foreskin cells (hnFSSCs) isolated from newborn human foreskin tissue. In addition, the apoptotic and proliferative effects of a traditional plant, Corchorus olitorius L. (C. olitorius), on hnFSSC spheroids were investigated. MATERIALS AND METHODS: After a routine circumcision procedure the cells were isolated and cultured in suitable medium. The plant leaves was extracted with ethanol and their composition was analyzed by liquid chromatography coupled with mass spectrometry (LC-MS/MS). The foreskin stem cells were characterized immunocytochemically by CD45, CD34, and CD90 antibodies. hnFSSC spheroids were formed using the hanging drop technique. Immunofluorescence staining was used on the obtained spheroids to determine the distribution of caspase-3 and Ki-67 after being treated with C. olitorius extract for 48 h. RESULTS: Immunostaining analysis showed that hnFSSCs were positive for CD45 and CD34 and negative for CD90. According to LC-MS/MS C. olitorius was rich in flavanols and hydrocinnamic acid derivatives. Although the spheroids obtained were loose and floating, the cells interacted with each other. Caspase-3 activity was higher in the control group than in the extract-treated group and Ki-67 was higher in the extract-treated group than in the control group, suggesting that the plant might have the capacity to increase stem cell proliferation due to its rich polyphenolic content. CONCLUSION: The results suggest that hnFSSCs and spheroids might be used in stem cell generation, tissue repair and renewal as human foreskin tissue has potential to be used as a stem cell source. C. olitorius also increased proliferation of hnFSSCs, showing that polyphenols might increase proliferation of stem cells.

The effect of dietary and topical celecoxib on 4-nitroquinoline-1-oxide-induced lingual epithelium alternations in rat.

OBJECTIVE: To compare the preventive effect of dietary and topical Celecoxib (CCB), a potent inhibitor of COX-2 on 4-NQO-induced tongue SCC in rat. METHODS: Fifty male Sprague Dawley adult 3- 3.5 months old rats were used as animal model in this study. The tongue SCC was induced by a daily administration of 30 ppm 4-NQO, in drinking water, for 8 months. The rats in case groups received dietary or topical CCB. Tongue Specimens were prepared for histopathological and immunohistochemical (Ki-67) staining and or TUNEL assay were examined. Values are expressed as mean +/- SEM and analyzed with Npar Kruscal Wallis and one-way ANOVA tests. p < 0.05 was considered significant. RESULTS: The incidence of tongue precancer lesions, judged by morphological and morhometrical criteria and apoptosis/proliferation ratio, was significantly (p < 0.01) reduced by CCB. The effect of topical CCB use, at high doses, was comparable to the effect of dietary CCB. CONCLUSION: Both topical and dietary CCB have inhibitory effect on 4-NQO induced SCC on tongue. The effect of CCB is probably mediated by suppression of cell proliferation and induction of apoptosis.

The effect of Colchicum pusillum in human colon cancer cells via Wnt/ß-catenin pathway.

OBJECTIVE: Colchicum pusillum belongs to the family Colchicaceae that particularly rich in tropolonic alkaloids. The aim of this study was to investigate the cytotoxicity and in vitro anticancer activity of Colchicum pusillum ethanolic extract on Colo-320 primer and Colo-741 metastatic colon adenocarcinoma cell lines. MATERIALS AND METHODS: Colchicum pusillum was collected and extracted with ethanol. Different concentrations of Colchicum pusillum extract were incubated for 24 h and 48 h with Colo-320 and Colo-741 cells. Cell growth and cytotoxicity were measured by 3­(4,5­dimethylthiazol­2­yl)­2,5­diphenyltetrazolium bromide (MTT) assays. Anticancer and antiproliferative activities of Colchicum pusillum were investigated by immunocytochemistry using antibodies directed against to ß-catenin, Ki-67, LGR-5 Ki-67, DKK1, Frizzled-4, Wnt4, Wnt7a and caspase3 in Colo-741 cells. RESULTS: All concentrations of Colchicum pusillum extract had toxic effect in Colo-320 cells. Because of this, we used Colchicum pusillum extract at 20 µg/ml for evaluate anticancer activities only in Colo-741 cells. As a result of immunohistochemical staining, ß-catenin, LGR-5 and caspase-3 immunoreactivities were significantly increased while Wnt7a immunostaining intensity was decreased in Colo-741 cells. Conclusion We conclude that Colchicum pusillum extract increased ß-catenin and LGR-5 via Wnt/ß-catenin pathway in colon cancer cells. Interestingly, it decreased other signaling molecule, Wnt7a which is assumed to play protective role during carcinogenesis. Also, it increased significantly caspase-3 immunoreactivity showing that apoptotic pathways were triggered.

Effects of adipose and bone marrow-derived mesenchymal stem cells on vaginal atrophy in a rat menopause model.

BACKGROUND & OBJECTIVES: Vaginal atrophy is characterized by thinning of vaginal epithelial layers and decreased local blood flow. We aimed to evaluate the regenerative effects of Adipose derived mesenchymal stem cells (ADMSC) and Bone marrow derived mesenchymal stem cells (BMDSC) on vaginal atrophy in rat menopause model. MATERIALS AND METHODS: Rats were randomly divided into 4 (four) groups: sham, control, ADMSC, BMDSC. Vaginal epithelial thickness, structure of the lamina propria, blood vessels in the lamina propria, collagen deposition, and muscle structure were evaluated. Anti ER α, VEGF, VEGFR 1, Bax and bcl-2 antibodies were analyzed. Beta actin gene was used as endogenous control. Genetical differences among the groups were compared by using Kruskal Wallis and Mann Whitney U test. p < 0.05 was regarded as statistically significant. RESULTS: Epithelial thickness of ADMSC group was higher than control group, but less than sham group Epithelial thickness of BMDSC group was less than sham group. Lamina propria and muscle tissue of ADMSC and BMDSC groups were found to be similar to sham group. VEGFR-1, VEGF, Bax and ER-α staining levels were higher in ADMSC and BMDSC groups than control group. ADMSC group stained stronger with VEGFR-1 and VEGF than BMDSC group. Bcl-2 staining level was increased in ADMSC applied group. No statistically significant difference was detected in Bax and Bcl-2 genes and Bax-/Bcl-2 ratio. CONCLUSIONS: Although genetic expression might have ended and could not be significantly demonstrated, histological and immunohistochemical results favor ADMSC application in vaginal atrophy rather than BMDSC.

Effects of silymarin and pentoxifylline on matrix metalloproteinase-1 and -2 expression and apoptosis in experimental hepatic fibrosis.

BACKGROUND: Many therapeutic strategies have been proposed to treat liver fibrosis, but no drugs have been proved effective. Matrix metalloproteinases (MMPs) have been reported to play a role in some cellular cascades of hepatic inflammation and fibrosis. OBJECTIVE: The purpose of this study was to investigate whether silymarin and pentoxifylline (PTX) have hepatoprotective and antifibrotic effects in experimental hepatic fibrosis. METHODS: Sprague-Dawley rats were divided into 4 groups: silymarin group (silymarin 4 mg/kg · d(-1) orally, common bile duct ligation [CBDL]); PTX group (PTX 2 mg/kg · d(-1) intraperitoneally, CBDL); sham group (common bile duct [CBD] exploration only); and control group (saline 1 mL/d orally, CBDL). The CBD was explored and dissected sufficiently to allow passage of a 3/0 silk suture via midline laparotomy. On day 10, all animals were euthanized via cervical dislocation. Then, 5-cm(3) liver samples from the right lobe were removed for histomorphologic evaluation and 3-mL blood samples were taken via cardiac puncture for biochemical analyses. Apoptosis was determined using the terminal deoxynucleotidyltransferase-biotin nick end-label (TUNEL) staining method. Plasma levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and γ-glutamyltransferase; total and indirect bilirubin concentration; hepatic MMP-1 and -2 and tissue inhibitor of MMP (TIMP)-l and -2 activity; and transforming-growth factor (TGF)-ß1 concentration were measured. Collagen content was determined by measuring hydroxyproline in liver samples. Malondialdehyde (MDA) was used to estimate lipid peroxidation. RESULTS: Thirty-two adult male Sprague-Dawley rats were divided into 4 groups: silymarin group (n = 7), PTX group (n = 7), sham group (n = 9), and control group (n = 9). Compared with the control group (14.6 [2.44]), mean (SD) hepatocyte apoptosis (as measured by the ratio of TUNEL-positive cells) was significantly suppressed in the silymarin group (1.2 [0.13]; P = 0.001) and the PTX group (3.8 [0.34]; P = 0.001). Mean (SD) MMP-2 activity in the silymarin group (57.35 [9.89] µg/mL; P = 0.04) and the PTX group (46.88 [9.56] µg/mL; P = 0.04) was significantly lower than that observed in the control group (232.32 [79.76] µg/mL). Compared with the control group (1.37 [0.38] µg/mL), TIMP-2 activity was significantly lower in the silymarin group (0.55 [0.13] µg/mL; P = 0.04) and the PTX group (0.42 [0.09] µg/mL; P = 0.01). Compared with the control group (909.17 [117.35] µg/mL), TGF-ß1 was significantly lower in the silymarin group (518.24 [30.34] µg/mL; P = 0.01) and the PTX group (519.57 [47.27] µg/mL; P = 0.01). Histomorphologic changes were significantly greater in the sham group than in the silymarin and PTX groups: hemorrhage (2.44 [0.29] vs 1.29 [0.18] and 1.57 [0.20], respectively; both, P = 0.04); sinusoidal dilatation (2.22 [0.22] vs 1.57 [0.20] and 1.71 [0.18]; both, P = 0.04); presinusoidal polymorphonuclear cell infiltration (3-44 [0.24] vs 2.57 [0.20] and 2.14 [0.26]; P = 0.03 and P = 0.008, respectively); and inflammation (3.44 [0.24] vs 2.57 [0.20] and 2.14 [0.26]; P = 0.03 and P = 0.008, respectively). In the control group, all biochemical markers were elevated, supporting the presence of liver injury. Compared with the control group (630.00 [46.80] U/L), plasma AST activity was significantly lower in the silymarin group (443.11 [78.73]; P = 0.04) and the PTX group (349.42 [34.00]; P = 0.03). Compared with the control group (191.12 [32.93] U/L), plasma ALT activity was significantly lower in the silymarin group (86.14 [4.97]; P = 0.04) and the PTX group (84.14 [11.21]; P = 0.04). MDA concentration was significantly lower in the silymarin group compared with the control group (0.08 [0.01] vs 0.22 [0.03] nmol/mL; P = 0.004); MDA was also significantly lower in the silymarin group than in the PTX group (0.11 [0.02]; P = 0.03). CONCLUSIONS: Silymarin and PTX were associated with lower histopathologic liver damage, hepatocyte apoptosis, and regulation of extracellular matrix proteins. Lipid peroxidation in hepatocytes was significantly lower in the silymarin group compared with the PTX group. Silymarin and PTX appeared to have hepatoprotective effects in this experimental liver fibrosis model, but further clinical and experimental studies are needed.

The role of PRP and adipose tissue-derived keratinocytes on burn wound healing in diabetic rats.

Introduction: Diabetic burn wounds and ulcers are significant complications of diabetic patients. The aim of this study is to investigate the use of platelet rich-plasma (PRP) and/or keratinocyte-like cells (KLCs) in diabetic thermal wound rat model and to evaluate EGF, FGF-2, TGF-ß1, COL1α2, MCP-1 and VEGF-α as wound healing markers at gene expression level. Method: In this study, we used adipose tissue as the source of mesenchymal stem cells (MSCs) and differentiated MSCs into KLCs. KLCs were characterized and transferred to the burn areas on the dorsum of streptozotocine (STZ)-induced diabetic rats. We prepared PRP from rat blood and evaluated its effect alone or in combination with KLCs. On 3rd, 7th, 10th and 14th days after treatment, wound areas were measured and biopsy samples were excised from the wound areas of the KLCs and/or PRP-treated and untreated diabetic rats to analyze gene expression levels of wound healing markers by qPCR. Results: We observed that, wound contraction started earlier in the PRP and/or KLCs-treated groups in comparison to the control group. However, PRP and KLCs when applied in combination showed additive affect in wound healing. In all groups treated with KLCs and/or PRP, the gene expression levels of evaluated growth factors and COL1α2 increased, while MCP-1 levels decreased when compared to the untreated diabetic rats. In addition, the most prominent difference in qPCR results belongs to combined PRP and KLCs-treated group. Conclusion: We demonstrated that applying PRP and KLCs in combination has a greater potential for treatment of diabetic burn wounds.

The effect of Misoprostol, a prostaglandin E1 analog, on apoptosis in ischemia-reperfusion-induced intestinal injury.

The aim of this study was to investigate whether Misoprostol, a synthetic prostaglandin (PG) E1 analog, has any effect on the prevention of apoptosis in ischemia-reperfusion (I/R)-induced intestinal injury. Thirty adult male Wistar albino rats were divided into three groups: group I=sham operated+saline; group II=I/R+saline; and group III=I/R+Misoprostol. Misoprostol (50microg/kg/d) was administered as an intragastric meal for 3 days. The terminal ileum was collected for histological and biochemical investigations. Apoptotic cells were detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelled (TUNEL) reaction. Immunohistochemical analysis was performed to determine the distribution of inducible nitric oxide synthase (iNOS) and endothelial NOS (eNOS). Samples were also analyzed for malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px). The number of TUNEL-positive cells was higher in group II when compared to the other two groups (p<0.05). In group III this value was higher when compared to group I, but lower than group II (p<0.05). iNOS immunoreactivity was not detected in ileum sections of group I animals, but moderate immunoreactivity was seen in group II and mild immunoreactivity in group III. The immunoreactivity of eNOS was moderate in ileum sections of all three groups. In ileum tissue, MDA was found to be higher in group II compared to group I (p<0.05), but there was no difference in group III. SOD was not different between groups I and III, but was significantly higher in group II (p<0.05). In our experimental model of I/R-induced intestinal injury, apoptosis is induced in enterocytes, whereas Misoprostol decreases enterocyte apoptosis in this experimental model. Our results indicate that Misoprostol may play a key role in the pathophysiologic events leading to failure of the intrinsic gut barrier defense mechanisms of intestinal epithelium.