Proliferating Cell Nuclear Antigen Staining Research Articles

Antineoplastic Activity Evaluation of Brazilian Brown Propolis and Artepillin C in Colorectal Area of Wistar Rats.

The study's aim was to evaluate Brazilian Brown Propolis (BBP) and Artepillin C (ARC) chemopreventive action in Wistar rats' colons. Fifty male Wistar rats were divided into ten experimental groups, including control groups, groups with and without 1,2-dimethylhydrazine (DMH) induction, and BBP, ARC, and ARC enriched fraction (EFR) treatments, for sixteen weeks. Aberrant crypt foci (ACF) were classified as hyperplastic or dysplastic, and proliferating cell nuclear antigen (PCNA) expression was quantified. ACF amounts in experimental groups (induced or not) decreased in both colon portions, while the isolated Aberrant Crypt (AC) number increased. Experimental groups of animals showed higher hyperplasia and dysplasia amounts compared with control groups. The ACF dysplastic amount present in groups induced and treated, in both colon portions, had similar values to IDMH (DMH induction group without treatment). In addition, DMH was effective in ACF inducing and there was positive staining for PCNA in basal and upper dysplastic foci portions in all experimental groups, in the mitotic index (MI) evaluation. To conclude, considering all the experimental groups, the one treated with EFR (fraction enriched with ARC) had the lowest rates of cell proliferation. BBP and its derivatives prevented crypt cell clonal expansion.

P183 Therapeutic Effects of Curcumin and Ginsenoside Combination in a Animal Model of Radiation Proctitis

Abstract Background Although various preventive and therapeutic modalities have been proposed to manage radiation-induced proctitis, there are no universally accepted preventive measures for effective treatment. This study aimed to investigate using an animal model whether the combination of curcumin and ginsenoside could be an effective treatment or preventive agent for progressive rectal inflammation caused by radiation. Methods C57BL/6 mice were exposed to gamma irradiation at 27 Gy (3 Gy/min) in the lower abdominal (rectal) region, establishing an animal model with acute radiation proctitis. A combination hydrogel containing 5 mM curcumin and 1 mM ginsenoside Rh1 was prepared and administered into the inflammed rectum of the murine model. This administration was repeated every 2 to 3 days after radiation exposure for a total of 6 times. Two weeks after radiation exposure, the mice were sacrificed to obtain the irradiated tissue samples. The histological and molecular evaluations were performed using H&E staining, Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analysis, Proliferating Cell Nuclear Antigen (PCNA) staining, immunohistochemistry, and qPCR. Results The murine group treated with the combination hydrogel of curcumin and ginsenoside showed significant restoration of the damaged rectal mucosa induced by radiation, including recovery of the mucosal layer and villi, mitigation of epithelial loss, and repair of crypt damage, compared to control group. Additionally, at the cellular level, a comparative analysis of apoptosis and proliferation within the tissues through TUNEL and PCNA staining demonstrated the combination hydrogel not only significantly inhibited cell death but also significantly increased cell proliferation. The gene expression of inflammatory cytokines in rectal tissue, including IL-6 and IFN-γ was significantly decreased in the treatment group. The infiltration of immune cells including T cells (CD4) and macrophages (CD68), which are important components in the pathogenesis of radiation proctitis, as shown by immunohistochemistry of tissue samples, was significantly reduced in the treatment group compared to the control group. Conclusion These promising results suggest that the combination of curcumin and ginsenosides has therapeutic potential for the management of radiation proctitis and provide valuable insights into new therapeutic strategies in clinical applications.

The influence of zinc on apoptosis and cell proliferation in palatal shelves during the fusion phase in mice and identification of a special protein family based on gene expression in cleft palate

Objective: To investigate the effect of the trace element zinc (Zn) on apoptosis and cell proliferation in palate shelvesduring the fusion phase, and to screen candidate genes of the Zn-finger special protein (Sp) family that were differentially expressed between the cleft palate and the normal palate to explore the mechanism of Zn in the development of cleft palate. Methods: Zn-rich, normal-Zn, low-Zn, and Zn-deficient diets were fed to female mice and, for the resultant fetuses, paraffin slices of their heads were made at embryonicdays 14.5 and 16.5. Using terminaldeoxynucleotidyltransferase-mediated dUTP nick-end labeling, the number of apoptotic cells in the palatal shelves was counted, and cell proliferation activity was detected using proliferating cell nuclear antigen staining. Total RNA from the palatal shelves of fetal mice was extracted from the Zn-rich diet, normal Zn-diet, and Zn-deficient-diet groups. We used microarray analysis to examine the expression of genes to identify intergroup differential gene expression and polymerase chain reaction tests to validate the results. Results: At ED14.5, the incidence of cleft palate in the regular zinc group, zinc rich group, low zinc group, and zinc deficient group was 8% (3/36), 2% (1/39), 29% (12/41), and 39% (15/38), respectively. The HE staining results at ED14.5 showed that both the left and right palatal processes in the zinc group had been lifted up and were in contact and connected with each other. In the zinc deficiency group, the left and right palatine processes remained vertically downwards on both sides of the tongue, ultimately forming cleft palate; In the low zinc group, the left and right palatine processes were raised but not in contact, ultimately resulting in cleft palate. There is no significant difference between the zinc rich group and the regular zinc group. At ED14.5, the positive rates of proliferative cells in the palatal process of fetal mice in the regular zinc group (80.29% ± 7.39%) and the zinc rich group (87.69% ± 6.62%) were significantly higher than those in the zinc deficient group (56.05% ± 16.13%) and the low zinc group (56.22% ± 9.61%) (t=4.32, P<0.05). The apoptosis index of fetal rat palatal process cells in the zinc deficient group (38.80% ± 3.10%) and the low zinc group (28.80% ± 6.19%) were significantly higher than those in the regular zinc group (16.80% ± 1.82%) (t=19.35, P<0.001; t=5.81, P<0.001). There were 663 differentially expressed genes in the zinc rich group and the zinc deficient group, with 513 up-regulated genes and 150 down-regulated genes, among which Sp5 was found to be located. The real time PCR results showed that compared with the regular zinc group (2.22 ± 0.36), the expression level of Sp5mRNA in the palatal process tissue of the zinc deficient group (1.23 ± 0.38) significantly increased (P<0.05), while the zinc rich group (3.68 ± 0.90) significantly decreased (P<0.05). Conclusions: Trace element Zn content was found to be closely related to the occurrence of cleft palate in mice offspring, with a lack of Zn leading to cleft palate.

Comparison of angiogenic potential in vitrified vs. slow frozen human ovarian tissue

Vitrification of ovarian tissue is a promising alternative approach to the traditional slow freezing method. Few empirical investigations have been conducted to determine the angiogenic profiles of these two freezing methods. In this study we aimed to answer the question whether one of the cryopreservation methods should be preferred based on the secretion of angiogenic factors. Tissue culture with reduced oxygen (5%) was conducted for 48 h with samples of fresh, slow frozen/thawed and vitrified/rapid warmed ovarian cortex tissue from 20 patients. From each patient, tissue was used in all three treatment groups. Tissue culture supernatants were determined regarding cytokine expression profiles of angiogenin, angiopoietin-2, epidermal growth factor, basic fibroblast growth factor, heparin binding epidermal growth factor, hepatocyte growth factor, Leptin, Platelet-derived growth factor B, placental growth factor and vascular endothelial growth factor A via fluoroimmunoassay. Apoptotic changes were assessed by TUNEL staining of cryosections and supplemented by hematoxylin and eosin and proliferating cell nuclear antigen staining. Comparing the angiogenic expression profiles of vitrified/rapid warmed tissue with slow frozen/thawed tissue samples, no significant differences were observed. Detection of apoptotic DNA fragmentation via TUNEL indicated minor apoptotic profiles that were not significantly different comparing both cryopreservation methods. Vitrification of ovarian cortical tissue does not appear to impact negatively on the expression profile of angiogenic factors and may be regarded as an effective alternative approach to the traditional slow freezing method.

Ellipticine induces apoptosis and mitochondrial dysfunction in human endometriosis cell lines by activating MAPK signaling pathway

Purpose: To assess the effect of ellipticine (EPT), an alkaloid isolated from the Oleaceae family, on endometriosis, and to identify its possible mechanisms of action.&#x0D; Methods: Human endometriosis-like cell lines exposed to EPT were subjected to bromodeoxyuridine/5-bromo-2´-deoxyuridine and proliferating cell nuclear antigen staining. Flow cytometry and immunoblot analyses were used to assess the effect of EPT on cell apoptosis. Mitochondrial damage was determined by JC-1 staining and immunoblotting. Immunoblot assays were performed to determine the effects of EPT on the MAPK pathway.&#x0D; Results: Ellipticine inhibited the viability of human endometriosis cell lines and stimulated cell apoptosis (p &lt; 0.01). It further induced mitochondrial damage in human endometriosis cell lines (p &lt; 0.01). Mechanistically, EPT acted on MAPK pathway, and induced apoptosis and mitochondrial dysfunction (p &lt; 0.01) in human endometriosis cells.&#x0D; Conclusion: Ellipticine is a potential treatment strategy for the management of endometriosis. However, further exploration of this potential should be explored via in vivo studies.

Cyclic High Negative-pressure External Volume Expansion Reduces Daily Device Application Time with Similar Effects on Recipient Site Preparation in a Murine Model

PURPOSE: Recipient site preparation using external volume expansion (EVE) increases graft survival in large-volume fat grafting. To improve patient compliance with using the device, we tested a new cyclic high negative-pressure (CHNP) mode that involves 1 h/day at -55 mmHg, cycled between 1-s negative-pressure activation, followed by a 2-s deactivation period in an animal model. METHOD: A miniaturized EVE device was applied to thirty 8-week-old male Sprague-Dawley rats. The rats were assigned to three groups (no pressure for the control group, conventional -25 mmHg for 8 h/day for conventional EVE, and cyclic high negative-pressure for CHNP group). After 28 days, micro-computed tomography (CT) was performed, and skin biopsy specimens were obtained. RESULTS: CHNP group showed a 6.6-fold increase in volume as compared to the control group, which showed a 4.4-fold increase compared to conventional EVE group. Hematoxylin and eosin staining showed a similar increase in subcutaneous tissue thickness in both EVE groups, compared to the control group. Masson’s trichome and proliferating cell nuclear antigen staining showed significantly higher collagen deposition and subdermal adipocytes in EVE groups. Immunohistochemistry against platelet endothelial cell adhesion molecule 1 showed 2.5- and 2.7-times higher vessel density in the conventional and CHNP EVE groups, respectively. There was no statistically significant difference in subcutaneous tissue thickness, collagen deposition, subdermal adipocyte proliferation, and vessel density between the two EVE groups. CONCLUSION: Cyclic high negative-pressure produced comparable results in recipient site preparation (subcutaneous tissue thickening and angiogenesis) compared to the conventional protocol, while markedly reduced the daily wear-time from 8 h to 1 h. Although further clinical data must be acquired, our new pressure setting seems promising and provides a more patient-friendly pre-expansion environment.

Effects of neferine on retinal tissue in experimental diabetic rat model.

To investigate vascular endothelial growth factor (VEGF) and proliferating cell nuclear antigen (PCNA) immunoreactivities, as well as apoptosis and oxidative stress levels in Streptozotocin (STZ)-induced diabetic rats, and determine how neferine affected these parameters. Thirty-five male Sprague Dawley rats were divided into five groups of seven. Fasting blood glucose was measured 72h after diabetes mellitus (DM) induction in 21 rats using 60mg/kg STZ dissolved in 0.4ml (0.1M) sodium-citrate buffer (pH:4.5), with values > 250mg/dl considered diabetic. Group 1 received no treatment. Group 3 (healthy rats) received daily intraperitoneal (IP) 4mg/kg neferine. Following DM induction: Group 2 (sham) received daily IP 0.25ml/kg 0.9% normal saline; Group 4 received single IP 0.01mL (2.5mg/kg) bevacizumab, followed by daily IP 0.25mL/kg 0.9% normal saline; and Group 5 received daily IP 4mg/kg neferine. Total antioxidant capacity (TAC) and total oxidative stress (TOS) levels in serum and ocular tissue homogenates were evaluated using ELISA. TUNEL method was used for determining apoptosis and immuno-histochemical staining for PCNA and VEGF immunoreactivities. Group 5 had significantly higher TAC and lower TOS in serum and ocular tissue homogenates than Group 4 (p < 0.05). Despite significantly lower VEGF levels and apoptosis (p < 0.05), there was no significant change in PCNA immunoreactivity in Group 5 (p > 0.05). DM was associated with lower TAC, higher TOS and apoptotic cells, as well as VEGF and PCNA immunoreactivities in the retina. Neferine altered parameters other than PCNA in the opposite direction, demonstrating reductive effects on DM.

Blocking the PCNA/NKp44 Checkpoint to Stimulate NK Cell Responses to Multiple Myeloma.

Simple SummaryMembrane-associated PCNA is expressed on the surface of human MM cell lines and primary MM cells. Mab 14-25-9 interacts with membrane-associated PCNA and blocks its binding to NK-expressed NKp44, thus activating NK function. We showed that mAb 14-25-9 can serve as an immune checkpoint blocker, enhancing the function of NK cells on target human MM cell lines and primary cells.Multiple Myeloma (MM) is a devastating malignancy that evades immune destruction using multiple mechanisms. The NKp44 receptor interacts with PCNA (Proliferating Cell Nuclear Antigen) and may inhibit NK cells’ functions. Here we studied in vitro the expression and function of PCNA on MM cells. First, we show that PCNA is present on the cell membrane of five out of six MM cell lines, using novel anti-PCNA mAb developed to recognize membrane-associated PCNA. Next, we stained primary bone marrow (BM) mononuclear cells from MM patients and showed significant staining of membrane-associated PCNA in the fraction of CD38+CD138+ BM cells that contain the MM cells. Importantly, blocking of the membrane PCNA on MM cells enhanced the activity of NK cells, including IFN-γ-secretion and degranulation. Our results highlight the possible blocking of the NKp44-PCNA immune checkpoint by the mAb 14-25-9 antibody to enhance NK cell responses against MM, providing a novel treatment option.

Proliferating Cell Nuclear Antigen (PCNA) Localization in the Ovaries of Buffalo, Dog and Pig by Immunohistochemistry

Background: It was expected from the previous studies that the immune histochemical staining for PCNA within follicular cells altered depending on the stage of follicular growth. Therefore, we aimed at determining the expression of proliferating cell nuclear antigen protein (PCNA) in the buffalo, dog and pig ovaries. Methods: The localization of PCNA was demonstrated in formalin fixed, paraffin embedded tissue sections of buffalo, dog and pig ovaries using primary mouse monoclonal anti-PCNA antibody. Result: Staining for PCNA was not observed in pregranulosa cells but observed in oocytes of primordial follicles. In primary to early secondary follicles, in oocytes and in few granulosa cells the positive staining was detected. The late secondary preantral follicles and actively growing small to large antral follicles showed strong PCNA labelling in the layers of granulosa and theca cells. PCNA staining was expressed in nuclei of oocytes in preantral and small antral follicles. The follicles demonstrating advanced atresia showed mild to no PCNA labelled granulosa and theca cells. The results of our study demonstrated that follicular growth and development in ovaries may be effectively monitored by determining the rate of granulosa cell proliferation of PCNA.

Cyclic High Negative-Pressure External Volume Expansion Reduces Daily Device Application Time With Similar Effects on Recipient Site Preparation in a Murine Model.

Introduction: Recipient site preparation using external volume expansion (EVE) increases graft survival in large-volume fat grafting. To improve patient compliance with using the device, we tested a new cyclic high negative-pressure (CHNP) mode that involves 1 h/day at -55 mm Hg, cycled between 1-second negative-pressure activation, followed by a 2-second deactivation period in an animal model. Material and Method: A miniaturized EVE device was applied to 30 8-week-old male Sprague-Dawley rats. The rats were assigned to 3 groups (no pressure for the control group, conventional -25 mm Hg for 8 h/day for conventional EVE, and CHNP mode for the CHNP group). After 28 days, micro-computed tomography was performed and skin biopsy specimens were obtained. Results: The CHNP group showed a 6.6-fold increase and the conventional EVE group showed a 4.4-fold increase in volume compared to the control group. Hematoxylin and eosin staining showed a similar increase in subcutaneous tissue thickness in both EVE groups, compared to the control group. Masson's trichome and proliferating cell nuclear antigen staining showed significantly higher collagen deposition and subdermal adipocytes in EVE groups. Immunohistochemistry against platelet endothelial cell adhesion molecule 1 showed 2.5- and 2.7-times higher vessel density in the conventional and CHNP EVE groups, respectively. There was no statistically significant difference in subcutaneous tissue thickness, collagen deposition, subdermal adipocyte proliferation, and vessel density between the 2 EVE groups. Conclusion: CHNP produced comparable results in recipient site preparation (subcutaneous tissue thickening and angiogenesis) compared to the conventional protocol, while markedly reducing the daily wear-time from 8 hours to 1 hour. Although further clinical data must be acquired, our new pressure setting seems promising and provides a more patient-friendly pre-expansion environment.

Spatiotemporal Expression of SHH/GLI Signaling in Human Fetal Bladder Development.

Objectives: Sonic hedgehog (SHH) signaling is important in bladder development. Mice with defective hedgehog signaling develop bladder anomalies. Clinically, urinary tract malformations are reported in human fetuses and infants with mutations of SHH and related signaling pathway genes. Information on the expression of SHH and associated signaling genes in normal human bladder development is fragmentary. This study determined the temporal and spatial expression patterns of SHH signaling pathway components in human fetal bladders by immunohistochemistry (IHC).Material and Methods: Twenty-four bladder specimens from 16 male and 8 female human fetuses aged 12- to 36-week (wk) were obtained from the First Affiliated Hospital of Xi'an Jiaotong University. The tissue slides were processed for IHC staining with SHH, Patched1 (PTC-1), Patched2 (PTC-2), Smoothened (SMO), GLI1 and proliferating cell nuclear antigen (PCNA). The expression levels of each gene were analyzed by semi-quantitative histological scoring system.Results: High intensity of SHH and SMO expression was detected in developing bladder urothelial cells, with no staining in lamina propria (LP), but with minimal expression of SMO in differentiating smooth muscle (SM) layers. The spatial distribution pattern of PTC1 and GLI1 was more complex with minimal expression in the LP layer, moderate expression in the SM layer, and high expression in the urothelium. PTC2 expression was mainly localized in the urothelium and LP, but no expression in the SM layer. All of the SHH signaling components were detected in fetal bladder tissues throughout the development, with expression peaks at 12- and 23-wk, coinciding with high cell proliferation as indicated by PCNA staining in the cell nuclei of urothelium and SM.Conclusions: The autocrine SHH signaling in the developing urothelium, and paracrine SHH signaling in the developing smooth muscle layer, mediated by SMO, PTC-1 and GLI1 were demonstrated during human bladder development. Expression of SHH signaling components peaked at 12-and 23-wk. The first expression peak at 12-wk may relate to urothelium growth, SM induction, and dilation of the bladder cavity. The second expression peaked at 23-wk may relate to urothelium and SM layer differentiation.

Harnessing the Power of Kiwifruit for Radiosensitization of Melanoma.

Melanoma is the deadliest variant of skin cancer and its incidence continues to increase. There are limited treatment options for advanced and metastatic cases of melanoma, despite advances in immunotherapy and chemotherapy. Melanoma is notorious as a radioresistant tumor. Previous studies found that phytochemicals, such as resveratrol and those found in green tea and blueberry, can sensitize various cancer cells, including melanoma, to radiotherapy. Our previous study also revealed that kiwifruit extract (KE) has antitumor activity to melanoma cells. This study was designed to expand upon our previous investigation and determine KE's potential as a radiosensitizer on CRL-11147 melanoma cancer cells and elucidate the possible mechanisms behind its potential. Proliferation and apoptosis of CRL-11147 melanoma cells under radiation therapy (RT) plus KE versus RT alone were investigated using Proliferative cell nuclear antigen (PCNA) staining, quick cell proliferation assay, clonogenic assay, and caspase-3 activity assay. Reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC) were then used to investigate the mechanisms behind the observed results. The percentage of CRL-11147 colonies, PCNA staining intensity, and the optic density value of CRL-11147 cells decreased with RT/KE vs. RT alone. Relative caspase-3 activity was increased with RT/KE vs. RT alone. Increased expression of the anti-proliferative molecule p27 and pro-apoptotic molecule TRAILR1 correlated with the anti-tumor effect seen in the RT/KE group versus the RT alone group. KE augments radiosensitivity of CRL-11147 by up-regulating both p27 and TRAILR1 to inhibit proliferation and increase apoptosis, respectively.

Proliferating cell nuclear antigen presentation, as a marker of folliculogenesis, in the transplanted ovarian tissue.

One of the most important ways to understand the ovarian biology is studding the initiation of primordial follicle development and subsequent folliculogenesis control. In this study, proliferating cell nuclear antigen (PCNA) presentation was used as a marker of follicular development in the thawed ovarian tissue (OT) following transplantation onto chick embryo chorioallantoic membrane (CAM) using two methods of freezing of slow freezing and vitrification. Samples of OT from 10 patients were subjected to slow freezing and vitrification. After warming, CAM transplantation was done and PCNA proliferation index (PI; percent of PCNA-positive granulosa cells) was calculated for each follicle stage. Image J software was used to determine the mean staining intensity. PCNA was positive for granulosa cells and oocytes nuclei, but negative for ooplasm. There were no remarkable PCNA staining in the granulosa cells of primordial follicles, but increased significantly as follicle progression (p< 0.05). Proliferation rate was also insignificantly higher in the vitrified than slow freezing group, before and after transplantation (p< 0.05). Lower PCNA presentation index was observed after CAM transplantation (p< 0.05). The earliest stage of follicular recruitment took place in the transitional follicles, before squamous cells transform to cuboidal cells. PCNA showed that follicles had proliferation power after cryopreservation. Higher presentation after vitrification may indicate accelerated folliculogenesis in the thawed OT.

Antitumor Activity of Nitazoxanide against Colon Cancers: Molecular Docking and Experimental Studies Based on Wnt/β-Catenin Signaling Inhibition.

In colon cancer, wingless (Wnt)/β-catenin signaling is frequently upregulated; however, the creation of a molecular therapeutic agent targeting this pathway is still under investigation. This research aimed to study how nitazoxanide can affect Wnt/β-catenin signaling in colon cancer cells (HCT-116) and a mouse colon cancer model. Our study included 2 experiments; the first was to test the cytotoxic activity of nitazoxanide in an in vitro study on a colon cancer cell line (HCT-116) versus normal colon cells (FHC) and to highlight the proapoptotic effect by MTT assay, flow cytometry and real-time polymerase chain reaction (RT-PCR). The second experiment tested the in vivo cytotoxic effect of nitazoxanide against 1,2-dimethylhydrazine (DMH) prompted cancer in mice. Mice were grouped as saline, DMH control and DMH + nitazoxanide [100 or 200 mg per kg]. Colon levels of Wnt and β-catenin proteins were assessed by Western blotting while proliferation was measured via immunostaining for proliferating cell nuclear antigen (PCNA). Treating HCT-116 cells with nitazoxanide (inhibitory concentration 50 (IC50) = 11.07 µM) revealed that it has a more cytotoxic effect when compared to 5-flurouracil (IC50 = 11.36 µM). Moreover, it showed relatively high IC50 value (non-cytotoxic) against the normal colon cells. Nitazoxanide induced apoptosis by 15.86-fold compared to control and arrested the cell cycle. Furthermore, nitazoxanide upregulated proapoptotic proteins (P53 and BAX) and caspases but downregulated BCL-2. Nitazoxanide downregulated Wnt/β-catenin/glycogen synthase kinase-3β (GSK-3β) signaling and PCNA staining in the current mouse model. Hence, our findings highlighted the cytotoxic effect of nitazoxanide and pointed out the effect on Wnt/β-catenin/GSK-3β signaling.

Network pharmacology-based evaluation of natural compounds with paclitaxel for the treatment of metastatic breast cancer

Metastatic breast cancer is a prevalent life-threatening disease. Paclitaxel (PTX) is widely used in metastatic breast cancer therapy, but the side effects limit its chemotherapeutic application. Multidrug strategies have recently been used to maximize potency and decrease the toxicity of a particular drug by reducing its dosage. Therefore, we have evaluated the combined anti-cancerous effect of PTX with tested natural compounds (andrographolide (AND), silibinin (SIL), mimosine (MIM) and trans-anethole (TA)) using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, trypan blue dye exclusion assay, proliferating cell nuclear antigen (PCNA) staining, network pharmacology, molecular docking, molecular dynamics (MD) and in vivo chick chorioallantoic membrane (CAM) angiogenesis assay. We observed a reduction in the IC50 value of PTX with tested natural compounds. Further, the network pharmacology-based analysis of compound-disease-target (C-D-T) network showed that PTX, AND, SIL, MIM and TA targeted 55, 61, 56, 31 and 18 proteins of metastatic breast cancer, respectively. Molecular docking results indicated that AND and SIL inhibited the C-D-T network's core target kinase insert domain receptor (KDR) protein more effectively than others. While MD showed that the binding of AND with KDR was stronger and more stable than others. In trypan blue dye exclusion assay and PCNA staining, AND and SIL along with PTX were found to be more effective than PTX alone. CAM assay results suggested that AND, SIL and TA increase the anti-angiogenic potential of PTX. Thus, natural compounds can be used to improve the anti-cancer potential of PTX.

Promotive action of 2-acetylaminofluorene on hepatic precancerous lesions initiated by diethylnitrosamine in rats: Molecular study.

BACKGROUNDDiethylnitrosamine (DEN) induces hepatic neoplastic lesions over a prolonged period.AIMTo investigate the promotive action of 2-acetylaminofluorene (2-AAF) when combined with DEN in order to develop a rat model for induction of precancerous lesion and investigate the molecular mechanism underlying the activity of 2-AAF. METHODSThe pre-precancerous lesions were initiated by intraperitoneal injection of DEN for three weeks consecutively, followed by one intraperitoneal injection of 2-AAF at three different doses (100, 200 and 300 mg/kg). Rats were separated into naïve, DEN, DEN + 100 mg 2-AAF, DEN + 200 mg 2-AAF, and DEN + 300 mg 2-AAF groups. Rats were sacrificed after 10 wk and 16 wk. Liver functions, level of alpha-fetoprotein, glutathione S-transferase-P and proliferating cell nuclear antigen staining of liver tissues were performed. The mRNA level of RAB11A, BAX, p53, and Cyclin E and epigenetic regulation by long-noncoding RNA (lncRNA) RP11-513I15.6, miR-1262 (microRNA), and miR-1298 were assessed in the sera and liver tissues of the rats.RESULTS2-AAF administration significantly increased the percent area of the precancerous foci and cell proliferation along with a significant decrease in RAB11A, BAX, and p53 mRNA, and the increase in Cyclin E mRNA was associated with a marked decrease in lncRNA RP11-513I15.6 expression with a significant increase in both miR-1262 and miR-1298. CONCLUSION2-AFF promoted hepatic precancerous lesions initiated through DEN by decreasing autophagy, apoptosis, and tumor suppression genes, along with increased cell proliferation, in a time- and dose-dependent manner. These actions were mediated under the epigenetic regulation of lncRNA RP11-513I15.6/miR-1262/miR-1298.

Expression of p63 and Proliferating Cell Nuclear Antigen in Oral Submucous Fibrosis.

ABSTRACTAim:Oral squamous cell carcinoma (OSCC) may be preceded by potentially malignant disorders such as oral submucous fibrosis (OSMF). p63 can detect genetic changes in OSMF and it facilitates early detection of malignant transformation, whereas proliferating cell nuclear antigen (PCNA) is a marker of proliferation and may prove to be a useful objective indicator of the biological behavior of various tumors. The aim of this study was to investigate the expression and pattern of the intensity of p63 protein and PCNA in normal oral mucosa and OSMF using immunohistochemistry (IHC) and to correlate the expression of these biomarkers.Materials and Methods:A total of 15 archival paraffin-embedded blocks obtained from our department, which were histopathologically diagnosed early OSMF (n = 4), intermediate OSMF (n = 4), and advanced OSMF (n = 2) and normal mucosa (n = 5), were taken as the standard for comparison. p63 and PCNA positivity was analyzed using Kruskall-Wallis test followed by pairwise comparison using Mann-Whitney U test. The pattern of staining and intensity was compared using Chi-Square test for which Statistical Package for the Social Sciences (SPSS, v 22.0, IBM Corporation, Armonk, New York) was used.Results:All samples showed positive staining for p63 and PCNA. A statistically significant difference (P < 0.05) was seen between the frequency of occurrence of p63 and the PCNA pattern of expression among all the groups. The intensity of staining was mild to intense in the basal layer, as there was a progression toward the severity of the disease. Almost 75.4% correlation existed between p63 and PCNA, with high correlation and marked relationship.Conclusions:The OSMF is considered a potentially malignant disorder that has the potential to get transformed into OSCC. The malignant transformation is often associated with changes at the genetic level, and these are reflected by the altered expression of proteins. Our results showed that biomarkers such as p63 and PCNA are significant in predicting the malignant transformation in OSMF, so in future they may serve as a prognostic tool in the early detection of malignancies.

Increased myofiber size and reduced satellite cell numbers in medial rectus muscle of patients with intermittent exotropia

ABSTRACT Purpose To elucidate the differences in muscle bundle and satellite cells in medial rectus muscle through histological and Immunofluorescence studies of intermittent exotropia patients and normal controls. Materials and Methods: From January 2015 to December 2017, 15 patients who underwent medial rectus resection surgery at Kosin University Gospel Hospital were enrolled. Four medial recti muscles collected from two brain-dead men without strabismus were used as controls and compared with the intermittent exotropia group. Hematoxylin and eosin (HE) staining were performed, and all muscle bundle diameters were measured with the Image J program and compared to the mean value. Immunological staining for MyoHC (Myosin Heavy Chain), PAX7 (Transcription Factor), and PCNA (Proliferating Cell Nuclear Antigen) were performed to analyze the distribution of myocytes and PAX7-positive and PCNA-positive cells. Results: The mean ages of the strabismus and control groups were 17.33 ± 13.05 and 22.0 ± 5.85 years, respectively, and the male to female ratio was 7:2 and 2:0. The average angle of deviation in the exotropia patients was 36.0 ± 16.83 prism diopters. The mean muscle bundle diameter under light microscopy was 60.21 ± 1.48 in the exotropia group and 52.27 ± 0.74 in the control group. The exotropia group showed significantly greater mean muscle bundle diameter (p < .001) and diameter regularity than the control group. The PAX7(+)/muscle bundle number ratio was 0.016 ± 0.014 and 0.056 ± 0.015 in the exotropia group and control group, respectively (p < .001), and the PCNA(+)/muscular bundle number ratio was 0.015 ± 0.017 and 0.182 ± 0.102 (p < .001). Both were significantly higher in the control group compared to that in the exotropia patients. Conclusion: In the exotropia group, mean diameter of medial rectus muscle bundle was significantly larger than that of control group. The ratios of PAX7 and PCNA to muscle bundle number were significantly higher in the control group than intermittent extropia group. We found the negative relationship between activation of satellite cells and muscle bundle diameter, and it might take one step forward to elucidate the pathogenesis of intermittent extropia.

Inhibition of ERK or Akt ameliorates intimal hyperplasia via up-regulation of Cx37 and down-regulation of Cx43 in balloon injury rat model.

Connexins (Cxs) are reported to participate in atherosclerosis associated intimal hyperplasia (IH), while their function involved in the balloon injury (BI) induced IH and restenosis is less reported. Forty-eight male Sprague-Dawley rats were randomly assigned to not injured (NI) group and BI group, which were further administrated with ERK-inhibitor U0216 and Akt-inhibitor MIK2206. Western blot and RT-PCR were utilized to detect the expression of Cx30, Cx37, Cx40, and Cx43 at 6 hours, 24 hours, 7 days, and 14 days post-surgery. H&E staining and related intima area, media area, and luminal area measurement were applied to indicate neointima formation and IH. ERK and Akt phosphorylation levels and proliferating cell nuclear antigen (PCNA) immunostaining were also detected. Among the four Cxs detected, Cx37 showed down-regulated, and Cx43 showed up-regulated temporal expression pattern in BI rats with confirmed neointima formation. Up-regulated p-ERK (P<0.01) and p-Akt (P<0.01) can be detected in BI rats compared with NI rats. Meanwhile, U0216 and MIK2206 can significantly reduce Cx43 expression and increase CX37 expression accompanied with reduced neointima formation and PCNA staining (P<0.05 or P<0.01) in BI rats. ERK or Akt inhibition can alleviate BI-induced IH via up-regulation of Cx37 and down-regulation of Cx43.

Adipose-Derived Stem Cells Promote Intussusceptive Lymphangiogenesis by Restricting Dermal Fibrosis in Irradiated Tissue of Mice.

Currently, there is no definitive treatment for lymphatic disorders. Adipose-derived stem cells (ADSCs) have been reported to promote lymphatic regeneration in lymphedema models, but the mechanisms underlying the therapeutic effects remain unclear. Here, we tested the therapeutic effects of ADSC transplantation on lymphedema using a secondary lymphedema mouse model. The model was established in C57BL/6J mice by x-irradiation and surgical removal of the lymphatic system in situ. The number of lymphatic vessels with anti-lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) immunoreactivity increased significantly in mice subjected to transplantation of 7.5 × 105 ADSCs. X-irradiation suppressed lymphatic vessel dilation, which ADSC transplantation could mitigate. Proliferative cell nuclear antigen staining showed increased lymphatic endothelial cell (LEC) and extracellular matrix proliferation. Picrosirius red staining revealed normal collagen fiber orientation in the dermal tissue after ADSC transplantation. These therapeutic effects were not related to vascular endothelial growth factor (VEGF)-C expression. Scanning electron microscopy revealed structures similar to the intraluminal pillar during intussusceptive angiogenesis on the inside of dilated lymphatic vessels. We predicted that intussusceptive lymphangiogenesis occurred in lymphedema. Our findings indicate that ADSC transplantation contributes to lymphedema reduction by promoting LEC proliferation, improving fibrosis and dilation capacity of lymphatic vessels, and increasing the number of lymphatic vessels via intussusceptive lymphangiogenesis.