PCNA Protein Expression Research Articles

CCL2 and IL18 expressions may associate with the anti-proliferative effect of noncontact electro capacitive cancer therapy in vivo.

Background: Noncontact Electro Capacitive Cancer Therapy (ECCT) is a novel treatment modality in cancer. Chemokine (C-C motif) ligand 2 (CCL2) has a major role in the outgrowth of metastatic breast cancer. Interleukin 18 (IL18) plays a role in macrophage alteration, which leads to excessive angiogenesis. This study aims to elaborate on the association of CCL2, IL18, IL23α, and TNF-α (tumor necrosis factor-alpha) expression with the anti-proliferative effect of ECCT in rat breast tumor tissue. Methods: Low intensity (18 Vpp) and intermediate frequency (150 kHz) alternating current-electric field (AC-EF) between two capacitive electrodes were exposed as external EF to a rat cage. Twenty-four rats were divided into four groups of six replicates. Breast tumor tissues were collected from 7, 12-dimethylbenz[a]anthracene (DMBA)-induced rats. Two groups were none DMBA-induced rats without ECCT exposure (NINT) and with (NIT). The other two groups were DMBA-induced rats without ECCT exposure (INT) and with (IT). Mammary glands and breast tumor tissues were collected from each group and preserved. Hematoxylin-eosin and immunohistochemistry staining were performed on paraffin sections of tissues using anti-PCNA, anti-ErbB2, anti-Caspase3, and anti-CD68. CCL2, IL18, IL23α, and TNF-α mRNA relative expressions were analyzed using qRT-PCR. Results: ECCT exposure may cause the reduction of PCNA protein expression as well as ErbB2 on breast tumor tissues, but it causes the increase of Caspase3 and macrophage CD68 protein. In rat breast tumor tissues of IT groups, the mRNA expression of CCL2 and IL18 are significantly down-regulated, in contrast with the up-regulated expression of these cytokines in tumor tissues of the INT group. IL23α and TNF- α expression remained similar in both groups. Conclusion: CCL2 and IL18 expressions have an association with the inhibition of breast tumor cell proliferation affected by ECCT exposure.

Downregulated lncRNA UCA1 acts as ceRNA to adsorb microRNA-498 to repress proliferation, invasion and epithelial mesenchymal transition of esophageal cancer cells by decreasing ZEB2 expression

ABSTRACT Objective Esophageal cancer (EC) is one of the most general malignant tumors in humans. There were few studies researching the connections between lncRNA UCA1 and EC. This study is to research the effect of lncRNA UCA1 adsorbing microRNA-498 (miR-498) as a ceRNA to regulate ZEB2 expression on epithelial mesenchymal transition (EMT), invasion and migration of EC cells. Methods UCA1, miR-498 and ZEB2 expression in EC tissues and cells was detected by RT-qPCR or western blot analysis. EC cells were transfected with siRNA-UCA1, miR-498 mimics or their controls to determine cell colony, proliferation, cycle distribution, apoptosis, migration and invasion by colony formation assay, CCK-8 assay, flow cytometry, and Transwell assay, respectively. The protein expression of PCNA, c-Myc, E-cadherin, N-cadherin, MMP-2 and MMP-9 was detected by Western blot analysis. The growth rate and weight of transplanted tumor in nude mice were observed. Results There were overly expressed UCA1 and ZEB2 and lowly expressed miR-498 in EC tissues and cells. LncRNA UCA1 acted as ceRNA to inhibit miR-498 expression and thereby increasing ZEB2 expression. With down-regulated UCA1 and up-regulated miR-498, ZEB2 expression, cell proliferation, colony formation, invasion, migration ability, EMT, tumor growth rate and weight in nude mice were apparently reduced. Conclusion This study demonstrates that inhibited UCA1 up-regulated miR-498 and down-regulated ZEB2, thereby repressing proliferation activity, invasion, migration, and EMT of EC cells.

Mfn2 inhibits proliferation and cell-cycle in Hela cells via Ras-NF-\u03baB signal pathway

BackgroundMitofusin 2 (Mfn2) is outer membrane protein, as the inhibitor of Ras protein. This study aimed to investigate the effect of Mfn2 on cell proliferation, and cell-cycle in Hela cervical carcinoma cell lines.MethodsAfter treated with Adv-mfn2 or Adv-control for 48 h and 60 h, the RNA and protein of Mfn2 in Hela cells were detected by qRT-PCR and western blot. The immunofluorescence assay was performed to observe the expression and sub-location of Mfn2 in Hela cells. The flow cytometry was performed to detect the cell cycle of Hela cells, while western blots were performed to observe the Ras-NF-κB signal pathway. Then, the xenografted cervix carcinoma mouse model was used to confirm the effect of Mfn2 in Hela cells in vivo and the expression of Ras-NF-κB signaling pathway in vivo.ResultsIn immunofluorescence detection, Mfn2 was located in cytoplasmic, not in the nucleus. In addition, Mfn2 inhibited cell proliferation of Hela cells through reducing PCNA protein expression. Mfn2 induced arrest in G0/G1 phase of the cell cycle in Hela cells. Meanwhile, Mfn2 reduced Cyclin D1 protein expression. Moreover, Mfn2 decreased the Ras signal pathway proteins such as Myc, NF-κB p65, STAT3 in a dose-dependent manner. Then, the in vivo experiment also confirmed that Mfn2 could inhibit the tumor growth, and depress the Cyclin D1, Ras, Myc, NF-κB p65, Erk1/2 and mTOR protein expression.ConclusionsMfn2 could significantly inhibit cell proliferation in Hela cells. It might be acted as an potential anti-cancer target through inducing cell cycle arrest in human cervical carcinoma cells.

Dendrobium officinale Polysaccharides Inhibit 1-Methyl-2-Nitro-1-Nitrosoguanidine Induced Precancerous Lesions of Gastric Cancer in Rats through Regulating Wnt/β-Catenin Pathway and Altering Serum Endogenous Metabolites.

Dendrobium officinale is a herb in traditional Chinese medicine where D. officinale polysaccharides (DOP) are the main active ingredient. This study aimed at evaluating DOP efficiency at inhibiting 1-Methyl-2-nitro-1-nitrosoguanidine (MNNG) induced precancerous lesions of gastric cancer (PLGC) in rats through the Wnt/b-catenin pathway and analyzing the variations of serum endogenous metabolites. PLGC was established in male Sprague-Dawley (SD) rats by administering 150 μg/mL MNNG in drinking water for 7 months and giving 0.1 mL of 10% NaCl once weekly during the initial 20 weeks. Treatment with DOP inhibited the progress of PLGC through decreasing the expression of β-catenin by immunohistochemical analysis. The futher study indicated DOP downregulated gene expression of Wnt2β, Gsk3β, PCNA, CyclinD1, and β-catenin, as well as protein expression of Wnt2β, PCNA, and β-catenin. On the other hand, there were nine endogenous metabolites identified after the DOP treatment. Among these, the most significant one is betaine because of its strong antioxidant activity, leading to an anti-tumor effect. DOP can inhibit MNNG-induced PLGC models via regulating Wnt/β-catenin pathway and by changing endogenous metabolites.

MON-218 Protective role of TV-induced autophagy in an in vivo model of unilateral ureteral obstruction-induced renal cell injury

Toxicodendron vernicifluum is used as a traditional herbal medicine in Asia. The physiological properties and antioxidative effects of Toxicodendron vernicifluum extract (TV) have been demonstrated in several experimental studies. Unilateral ureteral obstruction (UUO) is a well-established experimental model of renal injury leading to obstructive nephropathy. Toxicodendron vernicifluum is used as a traditional herbal medicine in Asia. The physiological properties and antioxidative effects of Toxicodendron vernicifluum extract (TV) have been demonstrated in several experimental studies. Unilateral ureteral obstruction (UUO) is a well-established experimental model of renal injury leading to obstructive nephropathy. Oxidative stress resulting from UUO may be aggravated by increased ROS production. Furthermore, oxidative stress may induce renal apoptosis and contribute to the pathogenesis of kidney disease caused by UUO. This study evaluated the possible renoprotective effects of TV on UUO-induced tubular damage, as well as the mechanism through which it exerts antioxidative and anti-apoptotic effects against UUO-induced cell death. Male Sprague–Dawley rats weighing 180–200 g each were assigned to one of two groups. The first group (UUO+TV) of rats drank TV for 2 weeks before surgery. The second group (UUO) of rats drank water for 2 weeks. Three days later, a morphologic evaluation of renal injury was conducted using hematoxylin and eosin and TUNEL staining. The renal protein expression of PCNA, caspase-3, Nrf2, catalase, and phosphorylated p38 as markers of autophagy was determined by immunoblotting. Obstruction injury caused marked apoptosis and oxidative stress in the UUO group. It also increased the level of phosphorylated p38 and decreased the level of PCNA, suggesting delayed recovery from damage. The number of TUNEL-positive cells, which were detected based on DNA fragmentation, was increased in the UUO group. Notably, there was a significant relationship with increased expression of cleaved caspase-3, which was counteracted by TV treatment. Moreover, a comparison with the UUO group revealed that TV significantly enhanced the regulation of autophagy and autophagic flux. Taken together, our findings suggest that the induction of autophagy protects against UUO-induced apoptotic damage via ROS and a p38-regulated pathway in this in vivo model. TV-induced autophagy may be a useful target for therapeutic intervention in ischemia-reperfusion renal injury.

1,25 Dihydroxyvitamin D3 Enhances the Antifibroid Effects of Ulipristal Acetate in Human Uterine Fibroids.

Both European and American trials showed superior effect of ulipristal acetate (UPA) to placebo. However, the latter, which included black patients with known higher vitaminD3 deficiency risk, showed lower rate of amenorrhea responders and insignificant uterine fibroid (UF) size reduction. Our objective is to investigate whether adding vitamin D3 to UPA can enhance UPA potency on UF phenotype in vitro. We screened the antiproliferative effect of different (UPA/vitaminD3) combinations on UF cell proliferation using dimethylthiazolyl diphenyltetrazolium bromide assay. Cells then were treated with UPA 100 nM in the presence or absence of vitamin D3 100 nM, and expression level of several markers related to proliferation, apoptosis, fibrosis, inflammation, and angiogenesis was measured on RNA or at protein level using quantitative real-time polymerase chain reaction, Western blot, immunofluorescence, or multiplex enzyme-linked immunosorbent assay techniques. Significant dose- and time-dependent growth inhibitory effects of UPA/vitamin D3 combinations were observed compared to untreated cells at 2 and 4 days (P < .05). Importantly, vitamin D3/UPA combination significantly reduced cell proliferation as compared to UPA at 2, 4, 6, and 8 days (P < .05). Combination treatment significantly decreased protein expression of proliferation markers Ki-67, PCNA, and CyclinD1 by more than 50% compared to UPA (P < .05) along with a significant increase in apoptosis induction. Combination treatment resulted in a 2-fold decrease in protein levels of extracellular matrix markers collagen-1 and fibronectin besides pro-fibrogenic cytokine transforming growth factor β3 (P < .05). Moreover, it significantly decreased the production of pro-inflammatory cytokines interleukins 6, 8, 1α, and 1β compared to UPA (P < .05). Combination of vitamin D3 with UPA exhibits additional and orchestrated anti-UF effects, therefore might offer a more favorable clinical option.

Physicochemical properties and laxative effects of polysaccharides from Anemarrhena asphodeloides Bge. in loperamide-induced rats

Ethnopharmacological relevanceAs a traditional Chinese herbal medicine, Anemarrhena asphodeloides Bge. possesses the effects of nourishing yin, moistening dryness, clearing lungs and relieving fire. Simultaneously, it has been used to treat constipation for more than one thousand years in China. However, modern medical studies are limited and lacking on its therapeutic mechanism.Aim of the study: This current study was aimed to investigate the laxative activities and explore the potential mechanism of Anemarrhena asphodeloides Bge. polysaccharides (AABP) in loperamide-induced constipation rats. Materials and methodsThe structure of AABP was determined by using infrared spectrum, high performance gel permeation chromatography (HPGPC), and high performance liquid chromatography (HPLC). Real-time quantitative polymerase chain reaction (PCR), multitudinous methods were adopted to explore the underlining therapeutic mechanism of AABP in treating constipation, including enzyme-linked immunosorbent assay (ELISA), histopathological, immunohistochemistry and western blotting. ResultsIn the present study, the average molecular weight of AABP was determined as 1.11 × 103 kDa. The primary monosaccharide compositions were analyzed including D-mannose, L-rhamnose, D-galacturonic acid, D-glucose, D-galactose and L-arabinose (1, 0.04, 0.53, 0.11, 0.33, 0.25, respectively) by high-performance liquid chromatography (HPLC). AABP significantly increased the levels of gastrin (Gas), motilin (MTL), substance P (SP), 5-hydroxytryptamine (5-HT) and vasoactive intestinal peptide (VIP), and decreased the NO content of loperamide-induced rats to ameliorate constipation in the rats. Whilst, AABP repaired the damaged colons by regulating PCNA and ICAM-1 protein expressions. Additionally, AABP up-regulated the levels of SCF, c-Kit, AQP3 and VIP as well as down-regulated the expressions of AQP8, AQP4 and PGE2. ConclusionThe present findings suggested that AABP were the laxative active ingredients isolated from Anemarrhena asphodeloides Bge., which could treat constipation through regulating the gastrointestinal hormones and neurotransmitters to improve the intestinal motility and water metabolism.

The expression of long non-coding RNA-LINC01410 in pancreatic cancer and its effect on proliferation and migration of pancreatic cancer cells

Objective: To explore the expression of long non-coding RNA-LINC01410 in the pancreatic cancer tissues, corresponding paracancerous tissues, human pancreatic cancer cell lines and normal pancreatic ductal epithelial cell line, and analyze the effect of LINC01410 on pancreatic cancer cell proliferation and migration. Methods: Real-time fluorescent quantitative PCR (RT-qPCR) was used to detect the expression level of LINC01410 in 16 cases of pancreatic cancer tissue and its adjacent tissues. RT-qPCR was performed to analyze LINC01410 expression in the pancreatic cancer cell lines AsPC-1, CAPAN-1, SW1990, BxPC-3 and CFPAC-1, and human normal pancreatic ductal epithelial cell line HPDE6-C7. Transfection of interference plasmid (shRNA) in the pancreatic cancer cell line with the highest expression level of LINC01410 were used to knock-down the expression of LINC01410. CCK-8 assay, colony formation assay, and transwell chamber assay were performed to detect the proliferation and migration of pancreatic cancer cells. The complementary paired miRNAs and downstream genes of LINC01410 were predicted by bioinformatics. The expression of miRNA and downstream genes was detected by RT-qPCR, and the protein expression of downstream genes was determined by Western blot. Results: The expression of LINC01410 in the pancreatic cancer tissues was significantly higher than that in the adjacent tissues [(3.46±0.32) vs (0.65±0.08), P<0.01]. The expression levels of LINC01410 in the pancreatic cancer cell lines were significantly higher than that in the normal human pancreatic ductal epithelial cells (P<0.05). The expression of LINC01410 was highest in BxPC-3 cells (P<0.01). After knock-down of the LINC01410 expression in the pancreatic cancer cell line BxPC-3, the cell proliferation was significantly inhibited (P<0.05), and the cell migration ability was decreased (P<0.05). LINC01410 complementarily paired with miR-497-5p, and miR-497-5p complementarily paired with IFITM3. After inhibiting the expression of LINC01410, the expression of miR-497-5p was increased [(1.04±0.17) vs (5.79±0.43), P<0.01], the mRNA expression of IFITM3 was decreased [(0.39±0.05) vs (1.00±0.03), P<0.01], and the protein expression of IFITM3, CDK6, Cyclin D2, PCNA, Vimentin, and N-cadherin was decreased. Conclusions: The expression of LINC01410 was increased in pancreatic cancer tissues and cell lines. Down-regulation of LINC01410 expression inhibits the proliferation and migration of pancreatic cancer BxPC-3 cells, and its mechanism may be closely related to regulating the miR-497-5p and IFITM3 gene expression.

The effects of neuromedin S on the hypothalamic-pituitary-testicular axis in male pigs in vitro

Evidence has shown that neuromedin S (NMS) and its receptor (NMU2R) are expressed in the hypothalamus, pituitary, and testis of pigs. To determine the potential mechanisms of NMS, we systematically investigated the direct effects of NMS on the hypothalamic-pituitary-testicular (HPT) axis of male pigs in vitro. We initially confirmed that NMU2R distributed in isolated hypothalamic cells, anterior pituitary cells and Leydig cells using immunocytochemistry. Subsequently we investigated the direct effects of NMS on hormone secretion from cells (anterior pituitary cells and Leydig cells) treated with different doses of NMS. The results showed that NMS increase the release of LH and FSH from anterior pituitary cells and testosterone from Leydig cells. NMS up-regulated the expression of NMU2R and GnRH mRNAs in hypothalamic cells, NMU2R, LH and FSH mRNAs in anterior pituitary cells, and NMU2R, STAR, P450 and 3β-HSD mRNAs and the expression of PCNA and Cyclin B1 protein in Leydig cells; moreover, it down-regulated the expression of GnIH mRNA in hypothalamic cells. Using immunofluorescence staining and confocal microscopy, we also demonstrated the colocalization of NMU2R and AR or GnIH in Leydig cells. These data in vitro indicated that NMS may regulate the release and/or synthesis of LH, FSH and testosterone at different levels of the reproductive axis through NMU2R, which provided novel evidence of the potential roles of NMS in regulation of pig reproduction.

CD40L inhibits cell growth of THP-1 cells by suppressing the PI3K/Akt pathway.

IntroductionAcute myeloid leukemia (AML), the hematological malignant tumor with high mortality, is still difficult to treat. CD40L is a type II transmembrane protein, which has been reported to have the potential to inhibit growth of some cancer cells.Materials and methodsIn order to determine the role of CD40L on AML-M5 cell line THP-1, we overexpressed CD40L in the cells using a lentiviral vector system (pHBLV-CMVIE-Zs Green-T2A-puro vector); overexpression was confirmed by the detection of green fluorescent protein and CD40L protein expression.ResultsCellular apoptosis, proliferation, and cycle assays showed that CD40L could promote the apoptosis of, suppress the proliferation of, and stimulate the arrest of the G1/S phase of THP-1 cells. Finally, the protein expression of P53, Bax/Bcl-2, cyclinD1, PCNA, PTEN, and p-Akt illustrated that CD40L may partly influence cell growth of THP-1 cells through those genes, which was confirmed by immunohistochemistry and a PI3K/Akt activator.ConclusionTaken together, CD40L could inhibit cell growth of THP-1 cells through the PI3K/Akt pathway, indicating that the overexpression of CD40L may be a potential target to treat the AML-M5 disease.

Hesperidin inhibits cell proliferation and induces mitochondrial-mediated apoptosis in human lung cancer cells through down regulation of β-catenin/c-myc

Abstract The aim of this study is to evaluate the mechanism of actions involved in the anticancer effect of hesperidin against cell proliferation and inducing apoptosis in human lung cancer A549 cells. MTT assay were performed, by treating the cells with different concentrations of hesperidin ranges from 0, 6.25, 12.5, 25, 50 and 100 μM for 0, 6, 12, 24, and 48 h. Hesperidin treatment significantly inhibited the cell viability based on concentration and time dependent manner, which shows optimal LC 50 effective, dose as 12.5 and 25 μM for 24 h and this LC50 doses and optimized time point used for further studies. Hesperidin treated cells significantly inhibited the cell proliferation through down regulation of c-myc, β-catenin, PCNA protein expressions. An additional studies also shows that hesperidin significantly increased the p53 and p21 tumor suppressor proteins which subsequently inhibited the cyclin dependent kinase 4 (cdk4) and cyclin D protein expressions. Further studies supported that restoring of tumor suppressor proteins by hesperidin treatment directly affects the Bcl-2/Bax ratio, which limits the Bcl-2 levels and increased the pro-apoptotic Bax protein levels. Mitochondrial transmembrane potential studies, increased levels of cytochrome c, APAF-1, caspase-3 and DNA fragmentation was observed in hesperidin treated cells which clearly indicates apoptosis induction. Taken together, these findings suggest the anticancer effect of hesperidin in human lung cancer A549 cells through up regulation of p53, which triggers the proliferation arrest and mitochondrial dependent apoptosis activation.

The role of Parvimonas micra in intestinal tumorigenesis in germ-free and conventional APCmin/+ mice.

531 Background: Our in-house meta-analysis of fecal shotgun metagenomic sequences from colorectal cancer (CRC) and control subjects from four cohorts of various ethnicities identified a higher abundance of Parvimonas micra in CRC. We aimed to investigate the effect of P. micra in colon tumor formation and development. Methods: We collected 309 fecal samples and 259 colon biopsies from patients with CRC, advanced adenoma and healthy subjects. P. micra strain was isolated from the feces of a CRC patient. APC min/+ mice and germ-free (GF) mice were orally gavaged with P. micra, or Esherichia coli. Colon epithelial cell line NCM460 and cancer cell lines HT-29 and Caco-2 were exposed to P. micra or E. coli conditional medium. Results: P. micra was significantly enriched both in the feces (n = 207, p &lt; 0.01) and tissue biopsies (n = 99, p &lt; 0.01) of CRC patients compared with controls (n = 102 for fecal samples, n = 160 for tissues biopsies). APCmin/+ mice gavaged with P. micra exibited significantly higher tumor burden ( p &lt; 0.01) and tumor load ( p &lt; 0.01), compared to mice gavaged with either E. coli or non-bacterial control. Consistently, cell proliferation was significantly higher in the colon tissues of P. micra gavaged GF mice relative to control mice evidenced by increased Ki-67 positive cells ( p &lt; 0.05) and PCNA protein expression ( p &lt; 0.01) at weeks 20 and 32. In line with this, colon cell lines NCM460, HT-29 and Caco-2 exposed to P. micra conditional medium significantly increased proliferation than control group (all p &lt; 0.05). Flow cytometry analyses showed that Th2 and Th17 cells were markedly increased, while Th1 were reduced in the lamina propria of the colon tissues of P. micra gavaged mice compared to control mice (all p &lt; 0.01) . Consistently, P. micra colonization in GF mice was associated with increased expression of pro-inflammatory cytokines, including TNF-α, IL-6 and IL-12 (all p &lt; 0.01). Conclusions: The abundance of P. micra was significantly increased in the feces and tissue biopsies of CRC patients. P. micra promotes intestinal carcinogenesis in APC min/+ mice and increase cell proliferation in GF mice. The tumor promoting effect of P. micra is associated with altered immune responses and enhanced inflammation in the gut.

GALNT6 Promotes Tumorigenicity and Metastasis of Breast Cancer Cell via β-catenin/MUC1-C Signaling Pathway.

Polypeptide N-acetylgalactosaminyl transferase-6 (GALNT6), a member of the N-acetyl-D-galactosamine transferase family, was shown to be over-expression in mammary cancer and could be used as a biomarker. However, its roles and underlying mechanisms in the pathogenesis of breast cancer are still unclear. In this study, we reported that GALNT6 was up-expression in breast cancer, and it was not associated with tumor stage. The expression level of GALNT6 and menopause status was associated with patient survival. Biological function results illustrated that knockdown of GALNT6 inhibited proliferation, migration and invasion of MDA-MB-231 cells, and increased cell apoptosis. Knockdown of GALNT6 in breast cancer cell attenuated the protein expression of PCNA, cyclin D1, C-myc and β-catenin, and increased the expression of E-cadherin, caspase 3 and cleaved PARP1. Cell fractionation assay showed that knockdown of GALNT6 reduced the levels of β-catenin and MUC1-C in nucleus. Simultaneously knockdown of GALNT6 and β-catenin significantly reduced the level of C-myc. Co-IP experiments indicated that GALNT6 interacted with MUC1-N, β-catenin interacting with MUC1-C in breast cancer cells. Together, our study reveals that GALNT6 promotes tumorigenicity and metastasis through β-catenin/MUC1-C signaling pathway.

Effects of boron on the proliferation, apoptosis and immune function of splenic lymphocytes through ERα and ERβ

ABSTRACT This experiment aimed to investigate the role of ERα and ERβ in boron's effect on splenic lymphocyte of rat. After inhibition of ERα and ERβ using MPP and PHTPP, the effects of 0.4 mmol/L boron on the splenic CD4+/CD8+ T-cell ratio, lymphocyte proliferation rate, IFN-γ concentration and PCNA protein expression disappeared. Furthermore, after inhibiting ERβ with PHTPP alone, the decreasing effects of 40 mmol/L boron on the splenic CD8+ T-cell proportion, lymphocyte proliferation rate, the concentrations of IgG and cytokines, as well as the expression of Bcl-2 also disappeared. However, when ERα was inhibited by MPP alone, the effect of 40 mmol/L boron on splenic lymphocyte was unchanged. The results suggest that ERα mainly mediate the effects of low-dose boron (0.4 mmol/L) on splenic T-cell subsets, lymphocyte proliferation and apoptosis, and immune function, while ERβ may mediate the effects of both low-dose and high-dose boron (40 mmol/L) on splenic lymphocytes.

Effect of RAB31 silencing on osteosarcoma cell proliferation and migration through the Hedgehog signaling pathway.

Osteosarcoma (OS) is a prevalent cancer that plagues people worldwide. Identifying prognostic markers would be useful in treating human OS. In this study, we aimed to explore the functions of Ras-related protein Rab-31 (RAB31) in OS-cell proliferation, migration, and invasion as well as its roles in the Hedgehog signaling pathway for better understanding of the mechanism. To assess the detailed regulatory mechanism of RAB31 silencing on OS, both RT-qPCR and Western blot analysis were employed to evaluate the expressions of RAB31 as well as the Hedgehog signaling pathway-related genes. Besides, we also investigated the effects of silenced RAB31 both in vitro and in vivo. First, we found that in OS tissues, both mRNA and protein expressions of RAB31 and PCNA had a significant increase. Second, the Hedgehog signaling pathway was detected to play an integral role in OS progression. Finally, after transfection of RAB31-siRNA to reduce the expression of RAB31, the Hedgehog signaling pathway was suppressed, along with cell proliferation, invasion, and migration. Therefore, we conclude that RAB31 plays an important role in OS development and its silencing delays the OS progression via suppression of the Hedgehog signaling pathway.

Toxicological effects of 3-methyl-4-nitrophenol on mouse ovarian and testicular cell proliferation, apoptosis and oocyte maturation

The aim of the present study was to reveal the effects of 3-methyl-4-nitrophenol (PNMC) on mouse gonadal cell proliferation and apoptosis. Immature female and male mice were intramuscularly injected with 100 mg/kg PNMC or vehicle every five days. One-month later, ovarian rather than testicular weights were significantly decreased. The positive terminal deoxyribonucleotidyl transferase-mediated dUTP–digoxigenin nick end-labelling staining was enhanced in both testicular seminiferous epithelium and ovarian corpus luteum. Further study in ovaries showed that PNMC treatment increased the protein expressions of PCNA, p21 and p27, and decreased the expression of Cyclin D2. Whereas PNMC had no significantly influence on those protein expressions in testes. In addition, we demonstrated that 100 nM PNMC significantly suppressed mouse oocyte meiotic resumption and cumulus cell expansion in vitro. These findings suggest that the reproductive toxicities of PNMC involve affecting the processes of gonadal cell apoptosis and proliferation.

A18072 Effect of transplanting adipose tissue-derived mesenchymal stem cells on intimal hyperplasia after carotid balloon injury

Objectives: To investigate the effects of transplanting adipose tissue-derived mesenchymal stem cells on intimal hyperplasia after carotid balloon injury and the mechanism Methods: 45 male Sprague Dawley rats were randomly divided into three groups. The balloon catheter was inserted into the left carotid to produce intimal injury. The injured carotid artery and normal carotid artery were used for HE-stained. The mRNA expressions of eNOS were determined by RT-PCR. and the protein expressions of p21, p27 and PCNA were determined by Western Blotting. Serum concentration of NO were detected by ELISA Results: Enhanced green fluorescent protein positive cells were localized to the traumatized arterial segment after transplantation. W/L ratios of ADMSC group were lower than of experimental group. The protein expression of p27, p21 and PCNA and significantly increased after balloon injury, In comparison with injury group, ADMSC treatment significantly increased the protein expression of p27, p21 and decreased PCNA protein expression. The mRNA expressions of eNOS were significantly increased than the experimental group after four weeks treatment, and the serum level of NO were significantly increased after four weeks treatment, compared with experimental group Conclusion: ADMSC could localize to the traumatized arterial segment and inhibit the process of intimal hyperplasia by upregulating the expression of protein p21, p27 and eNOS mRNA, as well as the level of NO in plasma

Induction of HRR genes and inhibition of DNMT1 is associated with anthracycline anti-tumor antibiotic-tolerant breast carcinoma cells.

The aim of the study was to understand the role of homologous recombination repair (HRR) pathway genes in development of chemotolerance in breast cancer (BC). For this purpose, chemotolerant BC cells were developed in MCF-7 and MDA MB 231 cell lines after treatment with two anthracycline anti-tumor antibiotics doxorubicin and nogalamycin at different concentrations for 48h with differential cell viability. The drugs were more effective in MCF-7 (IC50: 0.214-0.242µM) than in MDA MB 231 (IC50: 0.346-0.37µM) as shown by cell viability assay. The drugs could reduce the protein expression of PCNA in the cell lines. Increased mRNA/protein expression of the HRR (BRCA1, BRCA2, FANCC, FANCD2, and BRIT1) genes was seen in the cell lines in the presence of the drugs at different concentrations (lower IC50, IC50, and higher IC50) irrespective of the cell viability (68-41%). Quantitative methylation assay showed an increased percentage of hypomethylation of the promoters of these genes after drug treatment in the cell lines. Similarly, chemotolerant neoadjuvant chemotherapy (NACT) treated primary BC samples showed significantly higher frequency of hypomethylation of the genes than the pretherapeutic BC samples. The drugs in different concentrations could reduce m-RNA and protein expression of DNMT1 (DNA methyltransferase 1) in the cell lines. Similar phenomenon was also evident in the NACT samples than in the pretherapeutic BC samples. Thus, our data indicate that reduced DNMT1 expression along with promoter hypomethylation and increased expression of the HRR genes might have importance in chemotolerance in BC.

The enhanced effect of tetrahydrocurcumin on radiosensitivity of glioma cells.

To evaluate the effects of tetrahydrocurcumin (THC) on the radiosensitivity of glioma cells and the possible molecular mechanism. MTT assay, colony forming and wound healing assays were performed to detect the proliferation, radiosensitivity and migration of cells with various treatments. Cell apoptosis, cell cycle and GHS level were determined for exploring potent sensitization mechanism of THC. Meanwhile, protein expressions of cyclin D1 and PCNA were also measured. Furthermore, both orthotopic C6 mouse models and C6 subcutaneously grafted mouse models were established to test the tumour inhibitory effects of combined treatment in vivo. Cells treated with combined THC and radiation demonstrated lower cell viability and higher apoptosis rate as compared to radiation group. Moreover, the intracellular GSH was also decreased in the THC co-treated C6 cells. More importantly, combinatorial treatment group significantly induced G0/G1 cell cycle arrest and a decrease in the S phase cell through the down-regulation of cyclin D1 and PCNA. The in-vivo therapeutic efficacy assay indicated that the growth of tumour was greatly inhibited in combinatorial group. Tetrahydrocurcumin can synergistically enhance the radiosensitivity of glioma cells by inhibiting the expressions of cyclin D1 and PCNA.

Effect of PAK1 gene silencing on proliferation and apoptosis in hepatocellular carcinoma cell lines MHCC97-H and HepG2 and cells in xenograft tumor.

This study intends to explore the effect of the PAK1 gene silencing on apoptosis and proliferation of hepatocellular carcinoma (HCC) MHCC97-H and HepG2 cells and cells in xenograft tumor. MHCC97-H and HepG2 cells and mice with xenograft tumor in vivo were randomly divided into control, empty vector and PAK1 shRNA groups. Morphology and the expression of green fluorescent protein of MHCC97-H and HepG2 cells and cells in xenograft tumor were observed. MTT assay and flow cytometry were used to detect proliferation, cell cycle and apoptosis of MHCC97-H and HepG2 cells and cells in xenograft tumor. The expressions of PAK1, PCNA, Ki67, Cyclin E, CDK2, p21, p53, Bax and Bcl-2 were measured using the quantitative reverse transcription polymerase chain reaction and western blotting. Compared with the control and empty vector groups, number of adherent cells of MHCC97-H and HepG2 cells and cells in xenograft tumor was reduced, and green fluorescent cells became round and reduced in the PAK1 shRNA group. Cell proliferation, the cells at S phase, the mRNA and protein expressions of PAK1, PCNA, Ki67, Cyclin E, CDK2 and Bcl-2 of MHCC97-H and HepG2 cells and cells in xenograft tumor were decreased, while the cells at G1 phase, apoptosis rate, the mRNA and protein expressions of p21, p53 and Bax of MHCC97-H and HepG2 cells and cells in xenograft tumor were increased in the PAK1 shRNA group. PAK1 gene silencing decreases proliferation of MHCC97-H cells, HepG2 cells and cells in xenograft tumor through the p53/p21 pathway.