Semi-quantitative Western Blot Research Articles

WW domain-containing E3 ubiquitin protein ligase 1 depletion evokes antitumor effect in cutaneous squamous cell carcinoma by inhibiting signal transducer and activator of transcription 3 signaling pathway.

ObjectivesWW domain-containing E3 ubiquitin protein ligase 1 (WWP1) has been implicated in tumor progression. We aimed to investigate the role of WWP1 in cutaneous squamous cell carcinoma (CSCC).MethodsWWP1 gene and protein levels were detected using semi-quantitative reverse transcription-polymerase chain reaction, immunohistochemistry and western blotting. The effects of WWP1 on cell cycle, apoptosis, cell migration and invasion were examined by flow cytometry, wound healing and Transwell assays, respectively. The antitumor efficacy of WWP1 small interfering RNA was determined in CSCC tumor xenografts in mice.ResultsWWP1 expression was significantly higher in CSCC tissues and cells than in normal skin and cells, respectively. WWP1 expression was significantly associated with histological grade, invasion depth and lymph node metastasis in patients with CSCC. High expression predicted metastatic potential and an unfavorable prognosis. WWP1 downregulation suppressed tumor growth in vitro and in vivo, reduced cell migration and invasion, arrested the cell cycle in G0/G1 and induced apoptosis in A431 cells. WWP1 depletion also decreased phosphorylated signal transducer and activator of transcription 3 (STAT3), matrix metalloproteinase-2, cyclin D1 and Bcl-2, but did not affect total STAT3.ConclusionsWWP1 is a potential target for the diagnosis, prognosis and therapy of patients with CSCC.

Inhibitory effect of icariin on osteosarcoma cell proliferation via the Wnt/β-catenin signaling pathway.

Osteosarcoma, is a kind of malignant tumor derived from malignant interstitial cells. The pathogenesis of osteosarcoma remains unclear and despite use of chemotherapy drugs, resistance to these drugs affects the success of treatment. The present study was conducted to investigate the effects of icariin (ICA) on osteosarcoma cell proliferation and to investigate the role of the Wnt/β-catenin signaling pathway in the inhibition process of ICA on osteosarcoma cell proliferation. Different concentrations of ICA were selected to treat the osteosarcoma cell line 143B for 24 h, and then the onset concentration of ICA when it inhibited the growth of osteosarcoma cancer cell line 143B was detected via an MTT assay. The effect of ICA on the apoptosis of colon cancer cell line 143B under this concentration was detected using a flow cytometer. RNA in osteosarcoma cell line 143B was extracted, followed by reverse transcription. The expression levels of related and apoptotic proteins in the Wnt/β-catenin signaling pathway using ICA were detected by semi-quantitative PCR and western blot analysis, respectively. The expression quantities of vascular endothelial growth factor (VEGF) and MMP-9 were detected by ELISA. MTT assay showed that ICA inhibited the growth of 143B when its concentration was 5 µM (p<0.01). Flow cytometry showed that the number of apoptotic cells after ICA treatment was significantly higher than that in control group (p<0.01). RNA in osteosarcoma cell line 143B was extracted, followed by reverse transcription. Semi-quantitative PCR and western blot analysis revealed that the expression levels of p-GSK3β, β-catenin, c-Myc and cyclin D1 in cells after ICA treatment were significantly downregulated (p<0.01), while the expression level of caspase-3 was significantly increased (p<0.01). ELISA showed that the expression quantities of VEGF and MMP-9 were significantly decreased (p<0.01). Thus, ICA can significantly inhibit osteosarcoma cell proliferation and promote osteosarcoma cell apoptosis, which may be realized by affecting the expression of the Wnt/β-catenin signaling pathway and blocking the expression of related proteins.

Validation of Neurotensin Receptor 1 as a Therapeutic Target for Gastric Cancer.

Gastric cancer is the fifth most common type of malignancy worldwide, and the survival rate of patients with advanced-stage gastric cancer is low, even after receiving chemotherapy. Here, we validated neurotensin receptor 1 (NTSR1) as a potential therapeutic target in gastric cancer. We compared NTSR1 expression levels in sixty different gastric cancer-tissue samples and cells, as well as in other cancer cells (lung, breast, pancreatic, and colon), by assessing NTSR1 expression via semi-quantitative real-time reverse transcription polymerase chain reaction, immunocytochemistry and western blot. Following neurotensin (NT) treatment, we analyzed the expression and activity of matrix metalloproteinase-9 (MMP-9) and further determined the effects on cell migration and invasion via wound-healing and transwell assays. Our results revealed that NTSR1 mRNA levels were higher in gastric cancer tissues than non-cancerous tissues. Both of NTSR1 mRNA levels and expression were higher in gastric cancer cell lines relative to levels observed in other cancer-cell lines. Moreover, NT treatment induced MMP-9 expression and activity in all cancer cell lines, which was significantly decreased following treatment with the NTSR1 antagonist SR48692 or small-interfering RNA targeting NTSR1. Furthermore, NT-mediated metastases was confirmed by observing epithelial-mesenchymal transition markers SNAIL and E-cadherin in gastric cancer cells. NT-mediated invasion and migration of gastric cancer cells were reduced by NTSR1 depletion through the Erk signaling. These findings strongly suggested that NTR1 constitutes a potential therapeutic target for the inhibition of gastric cancer invasion and metastasis.

Genistein and daidzein induce apoptosis of colon cancer cells by inhibiting the accumulation of lipid droplets.

AimThe purpose of this study was to investigate the possible mechanisms of genistein (GEN) and daidzein (DAI) in inducing apoptosis of colon cancer cells by inhibition of lipid droplets (LDs) accumulation.MethodsHT-29 cells were used and treated by GEN or DAI in this paper. LDs accumulation was induced and inhibited by oleic acid (OA) and C75, respectively. The expression changes of LDs-related markers were confirmed by semiquantitative real time-PCR (RT–PCR), Western blotting, and immunofluorescence staining.ResultsGEN and DAI effectively reduced the LDs accumulation and downregulated the expression of Perilipin-1, ADRP and Tip-47 family proteins and vimentin levels. GEN and DAI significantly induced the mRNA expression of PPAR-γ, Fas, FABP, glycerol-3-phosphate acyltransferase (GPAT3), and microsomal TG transfer protein (MTTP), and reduced the mRNA expression of UCP2. Furthermore, the results showed a decrease of PI3K expression by GEN and DAI when compared with OA treatment, and both GEN and DAI can increase the expression of FOXO3a and caspase-8 significantly when these proteins were decreased by OA treatment. GEN is more effective than DAI in inducing cell apoptosis.ConclusionOur results demonstrated that GEN and DAI inhibit the accumulation of LDs by regulating LDs-related factors and lead to a final apoptosis of colon cancer cells. These results may provide important new insights into the possible molecular mechanisms of isoflavones in anti-obesity and anti-tumor functions.

Expression of MMP-2 and TIMP-3 with incidence and prognosis of giant-cell tumor of the bone.

The expression of matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of matrix metalloproteinase-3 (TIMP-3) in giant-cell tumor of bone (GCT), and the correlation of their expression with the clinicopathologic features and prognosis were investigated. A total of 70 GCT patients treated in our hospital from September, 2013 to September, 2015, were selected, and the tumor and para-carcinoma tissues were obtained by surgery. The expression levels of MMP-2 and TIMP-3 in GCT and para-carcinoma tissues were detected via semi-quantitative polymerase chain reaction (PCR) and western blot analysis, as well as immunohistochemical staining. Moreover, the clinicopathological data of the GCT patients were collected to study the correlation of MMP-2 and TIMP-3 with clinicopathological features and prognosis of GCT. The results of semi-quantitative PCR and western blot analysis revealed that the expression level of MMP-2 in tissues of the 70 GCT patients was significantly higher than that in para-carcinoma tissues, and the difference was statistically significant (P<0.01), while the expression level of TIMP-3 was obviously lower than that in para-carcinoma tissues (P<0.01). The results of immunohistochemical staining revealed that the positive expression rate of MMP-2 was 57.6% in GCT tissues and 18.9% in para-carcinoma tissues, while that of TIMP-3 was 63.2% in GCT tissues and 13.8% in para-carcinoma tissues, and the differences were statistically significant (P<0.01). The expression levels of MMP-2 and TIMP-3 were correlated with the diameter of tumor, clinical staging, lymph node metastasis and relapse of GCT (P<0.01), but were not correlated with the age and sex of GCT patients (P>0.05). There was a negative correlation between MMP-2 and TIMP-3 expression levels (r=−0.258, P<0.05). The expression levels of MMP-2 and TIMP-3 are closely related to the clinicopathological features and prognosis of patients, which can be used as one of the clinical examination indexes of GCT and also provide new insights for the clinical treatment of GCT.

High C-X-C motif chemokine5 expression is associated with malignant phenotypes of prostate cancer cells via autocrine and paracrine pathways.

The present study aimed to examine the effects and mechanisms of exogenous C-X-C motif chemokine5(CXCL5) and lentiviral CXCL5 overexpression on the regulation of malignant behaviors of prostate cancer cells invitro and in a nude mouse xenograft model. The expression levels of CXCL5 and a number of tumor-related genes were assessed by using semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR), western blotting, ELISA, or immunohistochemistry in normal and cancerous prostate cells and tissues. Cell proliferation, colony formation, and Transwell assays were performed to determine the effects of exogenous, autocrine, and paracrine CXCL5 on prostate cancer cell proliferative and migratory capacity. The results indicated that CXCL5 expression was upregulated in PC‑3 and DU145 prostate cancer cells, in WPMY‑1 normal prostate stromal cells, and in RWPE‑1 prostate epithelial cells, as well as in prostate cancer tissue specimens. Exogenous CXCL5 exposure resulted in increase in prostate cancer cell proliferation, colony formation, and migration. In cells transfected with a CXCL5 overexpression vector, in cells cultured in conditioned medium from CXCL5-overexpressing WPMY cells, and in cells co-cultured with CXCL5‑OE WPMY cells prostate cancer cell malignant phenotypes were induced in an autocrine/paracrine fashion invitro; similar results were observed in nude mouse xenografts. CXCL5 overexpression also regulated expression of tumor-related genes, including BAX, N-Myc downstream-regulated gene3, extracellular signal-regulated kinase1/2, C-X-C chemokine receptor type 2, interleukin18, Bcl‑2, and caspase‑3. These data demonstrated that CXCL5 expression was upregulated in prostate cancer tissues and that exogenous CXCL5 protein exposure or CXCL5 overexpression promoted malignant phenotypes of prostate cancer cells invitro and invivo.

Sleep-wake rhythm after extracorporeal circulation in New Zealand rabbits

To evaluate the change of sleep-wake rhythm after extracorporeal circulation (ECC) in New Zealand rabbits, and to explore the role of clock genes in sleep-wake rhythm disorder by ECC. Methods: A total of 54 New Zealand rabbits were randomly divided into 3 groups: a normal group (Group N), a sham group (Group S) and a model group (Group ECC). Electrocorticogram (ECOG), electroophthalmogram (EOG) and electromyogram (EMG) were respectively recorded by multipurpose EEG recorder, and the sleep-wake rhythm was also recorded. The mRNA and protein expressions of period1 (Per1) and cryptochrome1 (Cry1) were detected by semi-quantitative reverse transcriptase PCR (RT-PCR) and Western blot in pineal gland of rabbits. The differences between the 3 groups were compared. Results: 1) Compared with the Group N and Group S at 24, 48 h respectively, the total amount of sleep (TAS), light time, slow wave sleep (SWS) in the Group ECC at 24, 48 h were significantly reduced (all P<0.05), and the proportion of light sleep increased (all P<0.05), the proportion of SWS decreased (all P<0.05); 2) Compared with the Group N and Group S, the expression of Per1 mRNA in the Group ECC at 24, 48 h and Cry1 mRNA at 24 h significantly increased (all P<0.05); 3) Compared with the Group N and Group S, the expression of Per1 protein in the Group ECC at 48 h and Cry1 protein at 24 h significantly increased (all P<0.05); 4) In the Group ECC, the sleep-wake rhythm disorder and clock genes expression were ameliorated at 72 h after surgery. Conclusion: ECC can cause sleep-wake rhythm disorder in New Zealand rabbits, which may be related to the abnormal expression of Per1 and Cry1, and their transcription proteins.

Identification of novel indole based heterocycles as selective estrogen receptor modulator

In the present study, we have designed and synthesized indole derivatives by coalescing the indole nucleus with chromene carbonitrile and dihydropyridine nucleus. Two compounds 5c and 6d were selected from series I and II after sequential combinatorial library generation, docking, absorption, distribution, metabolism and excretion (ADME) filtering, anti-proliferative activity, cytotoxicity, and ER-α competitor assay kit by utilizing estrogen receptor-α (ER-α) dominant T47D BC cells line and PBMCs (Peripheral Blood Mononuclear Cells). Cell imaging experiment suggested that both the compounds successfully cross cellular biomembrane and accumulate in nuclear, cytoplasmic and plasma membrane region. Semiquantitative RT-PCR and Western blotting experiments further supported that both compounds reduced the expression of mRNA and receptor protein of ER-α, thereby preventing downstream transactivation and signaling pathway in T47D cells line. Current findings imply that 5c and 6d represent novel ER-α antagonists and may be used in the development of chemotherapy for the management of BC.

Association of High Mobility Group Box Chromosomal Protein 1 and Receptor for Advanced Glycation End Products Serum Concentrations With Extraglandular Involvement and Disease Activity in Sjögren's Syndrome.

To assess serum levels of high mobility group box chromosomal protein 1 (HMGB-1) and the soluble receptor for advanced glycation end products (sRAGE) in patients with Sjögren's syndrome (SS) and explore correlations with disease activity. Thirty-nine patients with SS and 21 healthy controls were included in this cross-sectional study. Clinical and laboratory values were obtained from all patients. Disease activity was assessed using the European League Against Rheumatism SS Disease Activity Index (ESSDAI). Serum samples were collected and HMGB-1 and sRAGE levels were measured using enzyme-linked immunosorbent assay (ELISA), and HMGB-1 concentrations were semiquantified by Western blotting. In ELISA, HMGB-1 serum levels did not differ between healthy controls and patients with SS (P = 0.783). When measured by semiquantitative Western blotting, HMGB-1 levels were increased in patients with SS compared to healthy controls (P = 0.012). HMGB-1 serum levels detected by Western blotting were higher in patients with extraglandular manifestations (P = 0.003) and were correlated with ESSDAI disease activity (r = 0.544, P < 0.0001). Furthermore, sRAGE was elevated in the sera of patients with SS (P = 0.003) compared to healthy controls and was also correlated with the ESSDAI (r = 0.545, P = 0.002). Serum levels of total HMGB-1 and sRAGE were elevated in patients with SS compared to healthy controls and correlated with disease activity as measured by the ESSDAI. Patients with extraglandular involvement had high serum levels of HMGB-1.

Effect of MeCP2 on TGF- β1-induced Extracellular Matrix Production in Nasal Polyp-derived Fibroblasts.

Purpose Methyl-CpG-binding protein 2 (MeCP2), known as a transcriptional regulator, has been suggested to play an important role in myofibroblast differentiation in the lung. The purpose of this study was to investigate the role of MeCP2 in transforming growth factor (TGF)- β1-induced myofibroblast differentiation and extracellular matrix (ECM) production in nasal polyp-derived fibroblasts (NPDFs). Methods To identify the expression of MeCP2 in nasal polyp tissues, immunohistochemistry staining and Western blot were performed. TGF- β1-induced NPDFs were treated with 5-azacytidine, a DNA methylation inhibitor, and the expression levels of α-SMA and fibronectin were determined by semiquantitative reverse transcription polymerase chain reaction, immunofluorescent staining, and Western blotting. The total soluble collagen was analyzed by the Sircol collagen assay. MeCP2 silenced by MeCP2-specific small interference ( si) RNA was verified by Western blot. Results The expression levels of MeCP2 increased in nasal polyp tissues compared to normal inferior turbinate tissues. 5-Azacytidine significantly inhibited the expression of α-SMA and fibronectin mRNA in a dose-dependent manner. In addition, 5-azacytidine suppressed collagen production and the expression of MeCP2 in the same manner. The expression levels of a-SMA and collagen production were significantly blocked by MeCP2 silencing in TGF- β1-induced NPDFs. Conclusions Our data suggest that MeCP2 plays an essential role in TGF- β1-induced myofibroblast differentiation and ECM production in NPDFs.

Molecular architecture of mouse and human pancreatic zymogen granules: protein components and their copy numbers

A molecular model of pancreatic zymogen granule (ZG) is critical for understanding its functions. We have extensively characterized the composition and membrane topology of rat ZG proteins. In this study, we report the development of targeted proteomics approaches to quantify representative mouse and human ZG proteins using LC-SRM and heavy isotope-labeled synthetic peptides. The absolute quantities of mouse Rab3D and VAMP8 were determined as 1242 ± 218 and 2039 ± 151 (mean ± SEM) copies per ZG. The size distribution and the averaged diameter of ZGs 750 ± 23 nm (mean ± SEM) were determined by atomic force microscopy. The absolute quantification of Rab3D was then validated using semi-quantitative Western blotting with purified GST-Rab3D proteins as an internal standard. To extend our proteomics analysis to human pancreas, ZGs were purified using human acini obtained from pancreatic islet transplantation center. One hundred and eighty human ZG proteins were identified for the first time including both the membrane and the content proteins. Furthermore, the copy number per ZG of human Rab3D and VAMP8 were determined to be 1182 ± 45 and 485 ± 15 (mean ± SEM). The comprehensive proteomic analyses of mouse and human pancreatic ZGs have the potential to identify species-specific ZG proteins. The determination of protein copy numbers on pancreatic ZGs represents a significant advance towards building a quantitative molecular model of a prototypical secretory vesicle using targeted proteomics approaches. The identification of human ZG proteins lays a foundation for subsequent studies of altered ZG compositions and secretion in pancreatic diseases.

Ibuprofen cases Proteasome dysfunction in mice liver

Introduction and objectivesPrevious clinical studies have suggested that nonsteroidal anti‐inflammatory drugs (NSAIDs) are associated with increased risk of stroke and cardiovascular disease. Acetaminophen, the active ingredient in Tylenol, is not an NSAID and is associated with increased risk of liver toxicity. The common use of NSAIDs for pain and fever relief is a concern but it is unclear if NSAIDs is also causing adverse effects on other tissues such as the liver, kidney and brain. Previous research in our laboratory has suggested that NSAIDs cause proteasome dysfunction in cardiac tissue. To investigate the possibility that NSAIDs also influence liver function, ibuprofen treated mice were used to determine if this NSAID affects the ubiquitin proteasome system (UPS). The UPS is critical for cell function as it is responsible for degradation of 60–80% of intracellular proteins.Method/ResultsEight‐week‐old male and female C57BL/6J mice were randomly assigned to one of 4 groups: (A) control (B) Group treated with ibuprofen (C) Group treated with ibuprofen + vitamin C (D) Group treated with vitamin C alone. Mice were treated with water or ibuprofen (100 mg/kg) and vitamin C (150 mg/kg) dissolved in drinking water for 6 weeks. Liver from 6 week treated animals were homogenized and cytosol fractions obtained. The three independent proteolytic proteasome activities, β1 caspase‐like activity, β2 trypsin‐like activity, and β5 chymotrypsin‐like activity were measured in the different cytosolic liver fractions. The proteasome inhibitor bortezomib was utilized to ensure the activity measured was due to the proteasome.Semi‐quantitative western blotting suggests that the 20S proteasome amount was similar in all 4 groups but the immunoproteasome subunit β5i was increased in both male and female ibuprofen treated and ibuprofen with vitamin C groups. The β1 and β2 proteasome activities were significantly decreased in both male and female mice treated with ibuprofen. Although inclusion of vitamin C with the ibuprofen did not fully restore proteasome activity, it resulted in an improvement in the β1 proteasome activity compared to ibuprofen treated mice. The β5 proteasome activity was also significantly decreased in both male and female mice treated with ibuprofen. However, this decrease was significantly more pronounced in female mice than in male mice suggesting gender‐specific effects of ibuprofen on proteasome function.ConclusionThese results suggest that ibuprofen significantly disrupts the UPS and causes proteasome dysfunction in liver tissue. The experimental data also suggests that proteasome dysfunction in livers from ibuprofen treated mice differs in female and male mice.Support or Funding InformationAmerican Heart Association Award #16GRNT31350040This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Investigating Intracellular RecA Concentrations in the Emerging Pathogen Acinetobacter baumannii

Acinetobacter baumannii is an opportunistic pathogen responsible for many hospital‐acquired infections. A. baumannii is known for its ability to rapidly gain antibiotic resistances and survive desiccation. This ability to survive adverse conditions has been linked to A. baumannii's unique DNA‐damage response (DDR), which is modulated by the evolutionarily conserved recombinase, RecA. We have previously shown that in A. baumannii, RecA is needed to upregulate the expression of error‐prone DNA polymerase genes, which in turn results in higher frequency of mutagenesis.A. baumannii lacks a functional homologue for the highly conserved global Escherichia coli DDR transcriptional repressor, indicating that their DDRs are regulated differently. In contrast to E. coli, the DDR in A. baumannii is bimodal. That is, when DNA damage is induced and expression of DDR genes is tracked, two distinct subpopulations are evident, one with low expression of DDR genes and one with high. The two populations survive differently, suggesting that the bacterium can fall back on the population that does survive. The details of the mechanism underlying this regulation remain largely unknown. Our lab has recently shown that the recA gene is regulated by a 5′untranslated region that is important for both its stability and expression. Moreover, preliminary evidence suggests that differential recA expression is needed to maintain the bimodal nature of the DDR. Because of RecA's role in the DDR, we sought to compare the levels of RecA in A. baumannii and E. coli. We hypothesized that RecA intracellular concentration in A. baumannii would be lower than E. coli's, consistent with the differences in regulatory roles. To test this hypothesis, we performed a semiquantitative Western blot using anti‐E. coli RecA polyclonal antibody. We first purified His‐tagged RecA from each species. The purified proteins were then used to prepare a standard curve for the Western blot. As expected, we observed a difference in affinity of the anti‐recA antibody based on differences in the primary protein sequence of RecA from the two species. (A. baumannii and E. coli RecA are approximately 74% identical). Remarkably, and in agreement with our hypothesis, we find that the levels of basal intracellular RecA were at least three times lower in A. baumannii than in E. coli. This difference in RecA protein concentration may have profound effects on A. baumannii physiology and is consistent with the different regulatory role for RecA in A. baumannii. These results show that the intracellular RecA concentration in bacteria may provide insights into the regulation of the DDR and the mechanism by which A. baumannii is able to survive adverse conditions and readily acquire antibiotic resistances.Support or Funding InformationFunding was provided by the National Institute of General Medical Sciences (RO1GM088230 to Veronica Godoy).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Bi-directional solid fermentation products of Trametes robiniophila Murr with Radix Isatidis inhibit proliferation and metastasis of breast cancer cells

BackgroundThe bi-directional solid fermentation product extract of Trametes robiniophila Murr (Huaier) with Radix Isatidis (TIF) has been shown to have good anti-tumor activity. However, the mechanisms of this activity are still unknown. In the present study, we aimed to investigate its inhibitory effect on both SK-BR-3 and MDA-MB-231 cells, and explore the possible mechanisms of its anti-cancer effect in vitro. MethodsThe experiment comprised a control group, Radix Isatidis group, Huaier group, and TIF group. The cell viability was measured by MTT and the distribution of cell cycle and apoptosis levels were analyzed by flow cytometry. Cell scratch, Transwell, and adhesion assays were used to measure the effects of the test compounds on the migration, invasion, and adhesion capability of SK-BR-3 and MDA-MB-231 cells. The effects of TIF on the mRNA and protein expression related to apoptosis and migration were measured by using semi-quantitative RT-PCR and western blotting. ResultsTIF strongly inhibited the cell proliferation of the SK-BR-3 and MDA-MB-231 cells in a time-dependent manner and induced G2/M arrest and apoptosis. Furthermore, TIF significantly inhibited the proliferation, migration, invasion, and adhesion capabilities of SK-BR-3 and MDA-MB-231 cells. Compared with other treatments, the anticancer effect of TIF were stronger in MDA-MB-231 cells. Semi-quantitative RT-PCR suggested that TIF may upregulate the expression of p53 and caspase-3 to inhibit cell proliferation, and downregulate the expression of MMP-9/Snail and MMP-9/MMP-2 to inhibit the migration, invasion, and adhesion capabilities of SK-BR-3 and MDA-MB-231 cells. Western blotting results showed that TIF increased the expression of p53 protein and decreased the expression of MMP-9 protein in SK-BR-3 and MDA-MB-231 cells. ConclusionThe results indicated that the bi-directional solid fermentation may enhance the efficacy of Huaier in MDA-MB-231 cells and that TIF may be an effective complementary medicine for cancer treatment.

Correlation between the expression of Drp1 in vascular endothelial cells and inflammatory factors in hypertension rats.

The objective of this study was to investigate the expression level of dynamin-related protein 1 (Drp1) in vascular endothelium of hypertension rats and its correlation with expression of inflammatory factors. Twenty spontaneous hypertension rats (SHR) were randomly divided into SHR group (n=10) and inhibition group (MD group, n=10), and the Sprague Dawley rats were enrolled as the control group (C group, n=10). For rats in the MD group, Mdivi-1, a mitochondrial division inhibitor, was given in dosage of 25 mg/kg. After 4 weeks of administration, blood pressure was measured via tail-artery blood pressure measurement. The blood samples collected from the abdominal aorta of rats were used to assay the C-reaction protein (CRP) concentration in serum through radioimmunoassay. Hematoxylin and eosin (H&E) staining was performed for sections of thoracic aorta for morphological observation and measurement of medial thickness. Enzyme-linked immunosorbant assay (ELISA), semi-quantitative real-time polymerase chain reaction (RT-PCR) and western blotting was carried out for detecting the expression levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). Drp1 and monocyte chemotactic protein 1 (MCP-1). After 4 weeks of drug administration, the blood pressure in the MD group was significantly higher (P<0.01). The medial thickness of the thoracic aorta in the MD group was significantly decreased in comparison with the SHR group (P<0.01). The results of ELISA showed that compared with the SHR group, the expression levels of IL-6 and TNF-α in the MD group were remarkably decreased (P<0.01). Semi-quantitative RT-PCR results indicated that the mRNA expression levels of Drp1 and MCP-1 in the MD group were significantly lower than those in the SHR group (P<0.05). In the SHR rats, after administration of Mdivi-1, the expression of Drp1 is decreased, which contributes to the alleviation in inflammatory reactions and protects the vessels in SHR rats.

Up-regulation of p21WAF1/CIP1 by small activating RNA inhibits the in vitro and in vivo growth of pancreatic cancer cells.

To study the inhibitory effect of p21WAF1/CIP1 activation by saRNA on the growth of human pancreatic cancer cells PANC-1 in vitro and in vivo. A dsRNA (dsP21) targeting the p21WAF1/CIP1 gene promoter at position-322 relative to the transcription start site was transfected into PANC-1 cells. Expression of mRNA and protein was evaluated by semiquantitative RT-PCR and Western blotting. Proliferation of PANC-1 cells was measured by the MTT method, and the apoptosis rate was detected by flow cytometry. PANC-1 cells were transplanted subcutaneously in nude mice, and the inhibitory effect of dsP21 on tumor growth was observed. The introduction of dsP21 was shown to efficiently up-regulate expression of the p21WAF1/CIP1 gene in PANC-1 cells according to the results of RT-PCR and Western blotting (P <0.01, compared with controls). The inhibitory effect on cell proliferation was confirmed by the MTT test (P <0.05, compared with controls). The apoptosis rate of PANC-1 cells treated with dsP21 was significantly higher than that of the control cells (P <0.01). Our experimental data showed that dsP21-mediated up-regulation of p21 expression exerted an apparent growth inhibitory effect on PANC-1 cells in vivo. dsP21 targeting the p21WAF1/CIP1 gene promoter can specifically up-regulate expression of the p21WAF1/CIP1 gene in PANC-1 cells. It therefore has a substantially inhibitory effect on cell proliferation in vitro and in vivo and can be used as a new method and material for the gene therapy of pancreatic cancer.

Mechanism of gastrodin in cell apoptosis in rat hippocampus tissue induced by desflurane.

This study investigated the protective effect ofgastrodin on cell apoptosis in rats hippocampus tissues induced by desflurane to explore its mechanism. A total of 36 rats were randomly divided into three groups: Blank control group (C group, n=12), desflurane anesthesia group (DF group, n=12) and gastrodin treatment group (GT group, n=12). Rats in DF group were treated with anesthesia using desflurane. Rats in GT group were treated with gavage using gastrodin and the same treatment as DF group. After the experiment, novel object recognition test and water maze test were performed. The hippocampus tissues were taken from the rat after the behavioral experiment; then the number of apoptotic cells was detected using the terminal deoxynucleotidyltransferase-mediated dUTP nick end labelling (TUNEL) kit, and the mRNA and protein expression levels of p38 and interleukin-1 (IL-1) were detected via semi-quantitative polymerase chain reaction (PCR) and western blot analysis. After the desflurane anesthesia, novel object recognition showed that compared with that in DF group, the exploration capacity of novel objects in GT group was increased (P<0.01). The water maze test showed that the escape latencies in DF group, T1 in GT group was significantly shortened, but T2 was significantly prolonged (P<0.01). TUNEL assay showed that the number of apoptotic cells in hippocampus tissues in GT group was significantly fewer than that in group DF (P<0.01). Semi-quantitative PCR and western blot analysis showed that the expression levels of p38 and IL-1β in GT group were lower than those in DF group (P<0.01). The results show that gastrodin has a protective effect on the apoptosis of hippocampus cells of rats induced by desflurane. Its protection mechanism may be realized through decreasing the increased p38 and IL-1β expression levels induced by desflurane, thus blocking the p38 mitogen-activated protein kinase (p38 MAPK) pathway.

Effects of carbamazepine combined with vitamin B12 on levels of plasma homocysteine, hs-CRP and TNF-α in patients with epilepsy.

The efficacy of carbamazepine combined with vitamin B12 in epilepsy treatment by comparing levels of plasma homocysteine (Hcy), serum TNF-α and hs-CRP in patients with epilepsy before and after treatment was investigated. Fifty-eight patients with epilepsy who were admitted and received treatment in The First People's Hospital of Xuzhou were recruited as subjects, and fifty-eight healthy volunteers were recruited as the control group. Patients were treated with carbamazepine combined with vitamin B12 for a period of three months. The mRNA and protein levels of TNF-α and hs-CRP in serum were measured before and after treatment using semi-quantitative RT-PCR and western blotting, respectively. The plasma Hcy levels were measured as well. Within one year after the 3-month treatment, the frequency and duration of seizure were tracked. After treatment with carbamazepine combined with vitamin B12 for patients with epilepsy, the Hcy level was significantly higher than that before treatment and that in the control group (P<0.01). The mRNA and protein levels of TNF-α and hs-CRP in serum were significantly higher in patients than that in healthy people (P<0.01). After treatment these levels were reduced (P<0.01), but still higher than those in healthy people (P<0.05, P<0.01). After treatment, the frequency and duration of seizures were all reduced (P<0.05, P<0.01). The results suggested that carbamazepine combined with vitamin B12 was effective in treatment of epilepsy by reducing levels of TNF-α and hs-CRP in the serum, but had a risk of increasing the Hcy level.

In vitro study on reversal of ovarian cancer cell resistance to cisplatin by naringin via the nuclear factor-κB signaling pathway.

The aim of the present study was to investigate the mechanism of action by which naringin reverses the resistance of ovarian cancer cells to cisplatin. Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and western blotting assays were used to detect the effects of different concentrations of naringin on the expressions of nuclear factor (NF)-κB and P-glycoprotein (P-gp) in the SKOV3/CDDP cell line. Small interfering RNA (siRNA) targeting NF-κB was designed and synthesized to silence NF-κB, and recombinant plasmid vectors overexpressing NF-κB were constructed to transfect cells. RT-qPCR and western blotting assays were subsequently performed to detect the effects of NF-κB on the expression of P-gp at the mRNA and protein levels. Naringin was added to the NF-κB-overexpressing SKOV3/CDDP cells and cultured for 48 h, followed by the detection of the expression of P-gp. RT-PCR and western blotting results demonstrated that the gene and protein expressions of NF-κB and P-gp were significantly decreased in a dose-dependent manner by naringin treatment (P<0.05). In cells overexpressing NF-κB, P-gp expression was significantly elevated (P<0.05), and the expression of P-gp was significantly decreased when NF-κB was silenced (P<0.05). Treatment with naringin was able to significantly ameliorate the NF-κB-induced overexpression of P-gp (P<0.05). These results indicate that naringin is able to inhibit the expression of NF-κB and P-gp in SKOV3/CDDP cells. Such an inhibitory effect may increase gradually with concentration, and is associated with blockade of the NF-κB signaling pathway. This pathway may represent one of the mechanisms of action by which Naringin reverses resistance to platinum-based agents in ovarian cancer cells.

Bone Regeneration using Silk Hydroxyapatite Hybrid Composite in a Rat Alveolar Defect Model.

Background: To overcome the limited source of autogenous bone in bone grafting, many efforts have been made to find bone substitutes. The use of hybrid composites of silk and hydroxyapatite to simulate natural bone tissue can overcome the softness and brittleness of the individual components.Methods: Critical-sized, 7 x 4 x 1.5 mm alveolar defects were created surgically in 36 Sprague-Dawley rats. Three treatment groups were tested: an empty defect group (group I), a silk fibrin scaffold group (group II), and a hydroxyapatite-conjugated silk fibrin scaffold group (group III). New bone formation was assessed using computed tomography and histology at 4, 8, and 12 weeks, and semi-quantitative western blot analysis was done to confirm bone protein formation at 12weeks. Statistical analysis of new bone formation was done using the Kruskal-Wallis test.Results: Radiomorphometric volume analysis revealed that new bone formation was 64.5% in group I, 77.4% in group II, and 84.8% in group III (p=0.027) at 12 weeks. Histologically, the osteoid tissues were surrounded by osteoblasts not only at the border of the bone defect but in the center of the scaffold implanted area in group III from week 8 on. Semi-quantitative western blotting revealed that osteocalcin expression in group III was 1.8 times higher than group II and 2.6 times higher than group I.Conclusions: New bone formation was higher in hybrid scaffolds. Both osteoconduction at the defect margin and osteoinduction at the center of the defect were confirmed. There were no detected complications related to foreign body implantation.