Methyl Thiazol Tetrazolium Assay Research Articles

The effect of silencing alpha - fetoprotein expression in HepG2 cell on the cellular function

Objective To construct lentiviral vectors with small interfering RNA (siRNA) targeted alpha-fetoprotein (AFP) and investigate the effect of silencing AFP expression on proliferation, invasion and metastasis of HepG2 cells. Methods Synthetic siRNA-AFP and its negative control were transfected into HepG2 cell by lentiviral vector, then getting a stable cell line with AFP expression silenced by puromycin. Real-time fluorescent quantitative polymerase chain reaction (FQ-PCR) and Western blotting was used for detecting the effects of interference at mRNA and protein levels. In addition, methyl thiazol tetrazolium (MTT) assay was used to measure the proliferation of HepG2 cell. The invasion ability changes were analyzed by Transwell and the migration of HepG2 cell was detected by wound healing assay. Results The results of FQ-PCR and Western blotting demonstrated that the AFP expression was significantly decreased about 90% and (62.53±6.17)%, respectively both at mRNA and protein levels after transfection (P<0.05). The proliferation abilities of HepG2 cell were apparently decreased (31.17±1.16)% by silencing AFP expression after culturing 6 days (P<0.05). The inhibition of silencing AFP could induce suppression of invasion and migration (P<0.05). Conclusion Silencing of AFP expression can effectively inhibit proliferation, invasion and metastasis process in HepG2 cell. Key words: Alpha-fetoprotein; RNA interference; HepG2 cell; Invasion; Metastasis

Long non-coding RNA maternally expressed gene 3 expression in non-small cell lung cancer and its role in proliferation and migration

Objective To evaluate the maternally expressed gene 3 (MEG3) expression in non-small cellu luang cancer and its role proliferation and migration. Methods Fourty eight paties diagnosed of non-small cell lung cancer were included in this study. The MEG3 expression of the cancer tissue and normal lung tissue for the 48 cases were tested by real-time quantitative polymerase chain reaction (Real-time PCR) assay. The lung cancer cell line of A549 and H460 were transfected with long non-coding RNA MEG3 over expression plasmid (Pc-DNA3.1-MEG3). After transfection of Pc-DNA3.1-MEG3, the ablity of prolifieration and migration were tested by methyl thiazol tetrazolium (MTT) and wound assay. The associated protein fore prolifieration and migration were tested by Western blotting assay. Results Compared with normal lung tissue, 25 (52.08%) cancer tissue were showed low expression of MEG3 with statistical difference (P<0.05); The expression of MEG3 were 0.48±0.04, 0.62±0.05 and 10.36±1.78, 11.66±2.01 in A549 AND H460 lung cancer cell line for transfection of pcDNA3.1 and pcDNA3.1-MEG3 with statistical difference (P<0.05); The proliferation and migration ability was significant decreased after transfection of pcDNA3.1-MEG3 for A549 and H460 lung cancer cells (P<0.05). And the cyclin D1 protein expression was significant inhibit after transfection of pcDNA3.1-MEG3 (P<0.05). Conclusion MEG3 was relative low expressed in non-small cell lung cancer (NSCLC) and it inhibits proliferation and migration through inbibiting Cyclin D1 expression. Key words: Non-samll cell lung cancer; Long non-coding RNA; Maternally expressed gene 3; Proliferation; Migration

Pluronic-based micelle encapsulation potentiates myricetin-induced cytotoxicity in human glioblastoma cells.

As one of the natural herbal flavonoids, myricetin has attracted much research interest, mainly owing to its remarkable anticancer properties and negligible side effects. It holds great potential to be developed as an ideal anticancer drug through improving its bioavailability. This study was performed to investigate the effects of Pluronic-based micelle encapsulation on myricetin-induced cytotoxicity and the mechanisms underlying its anticancer properties in human glioblastoma cells. Cell viability was assessed using a methylthiazol tetrazolium assay and a real-time cell analyzer. Immunoblotting and quantitative reverse transcriptase polymerase chain reaction techniques were used for determining the expression levels of related molecules in protein and mRNA. The results indicated that myricetin-induced cytotoxicity was highly potentiated by the encapsulation of myricetin. Mitochondrial apoptotic pathway was demonstrated to be involved in myricetin-induced glioblastoma cell death. The epidermal growth factor receptor (EGFR)/PI3K/Akt pathway located in the plasma membrane and cytosol and the RAS-ERK pathway located in mitochondria served as upstream and downstream targets, respectively, in myricetin-induced apoptosis. MiR-21 inhibitors interrupted the expression of EGFR, p-Akt, and K-Ras in the same fashion as myricetin-loaded mixed micelles (MYR-MCs) and miR-21 expression were dose-dependently inhibited by MYR-MCs, indicating the interaction of miR-21 with MYR-MCs. This study provided evidence supportive of further development of MYR-MC formulation for preferentially targeting mitochondria of glioblastoma cells.

Determination of the optimum conditions for lung cancer cells treatment using cold atmospheric plasma

Cold atmospheric plasmas (CAPs) can affect live cells and organisms due to the production of different reactive species. In this paper, the effects of various parameters of the CAP such as the treatment time, gas mixture, gas flow rate, applied voltage, and distance from the nozzle on the LL/2 lung cancer cell line have been studied. The probable effect of UV radiation has also been investigated using an MgF2 filter. Besides the cancerous cells, the 3T3 fibroblast cell line as a normal cell has been treated with the CAP. The Methylthiazol Tetrazolium assay showed that all parameters except the gas flow rate could impress effectively on the cancer cell viability. The cell proliferation seemed to be stopped after plasma treatment. The flow cytometry assay revealed that apoptosis and necrosis were appreciable. It was also found that treating time up to 2 min will not exert any effect on the normal cells.

Surface characterization and biological properties of regular dentin, demineralized dentin, and deproteinized dentin.

Bone autografts are often used for reconstruction of bone defects; however, due to the limitations of autografts, researchers have been in search of bone substitutes. Dentin is of particular interest for this purpose due to high similarity to bone. This in vitro study sought to assess the surface characteristics and biological properties of dentin samples prepared with different treatments. This study was conducted on regular (RD), demineralized (DemD), and deproteinized (DepD) dentin samples. X-ray diffraction and Fourier transform infrared spectroscopy were used for surface characterization. Samples were immersed in simulated body fluid, and their bioactivity was evaluated under a scanning electron microscope. The methyl thiazol tetrazolium assay, scanning electron microscope analysis and quantitative real-time polymerase chain reaction were performed, respectively to assess viability/proliferation, adhesion/morphology and osteoblast differentiation of cultured human dental pulp stem cells on dentin powders. Of the three dentin samples, DepD showed the highest and RD showed the lowest rate of formation and deposition of hydroxyapatite crystals. Although, the difference in superficial apatite was not significant among samples, functional groups on the surface, however, were more distinct on DepD. At four weeks, hydroxyapatite deposits were noted as needle-shaped accumulations on DemD sample and numerous hexagonal HA deposit masses were seen, covering the surface of DepD. The methyl thiazol tetrazolium, scanning electron microscope, and quantitative real-time polymerase chain reaction analyses during the 10-day cell culture on dentin powders showed the highest cell adhesion and viability and rapid differentiation in DepD. Based on the parameters evaluated in this in vitro study, DepD showed high rate of formation/deposition of hydroxyapatite crystals and adhesion/viability/osteogenic differentiation of human dental pulp stem cells, which may support its osteoinductive/osteoconductive potential for bone regeneration.

Sodium alginate conjugated graphene oxide as a new carrier for drug delivery system

The biomedical applications of graphene-based materials, including drug delivery, have grown rapidly in the past few years. The aim of this present study is to enhance the efficiency and specificity of anticancer drug delivery and realize intelligently controlled release and targeted delivery. Graphene oxide (GO) was first prepared from purified natural graphite according to a modified Hummers’ method. Then GO was functionalized with adipic acid dihydrazide to introduce amine groups, and sodium alginate (SA) was covalently conjugated to GO by the formation of amide bonds. The resulting GO–SA conjugate was characterized and used as a carrier to encapsulate the anticancer drug doxorubicin hydrochloride (DOX·HCl) to study in vitro release behavior. The maximum loading capacity of DOX on GO–SA was 1.843mg/mg and the drug release rate under tumor cell microenvironment of pH 5.0 was significantly higher than that under physiological conditions of pH 6.5 and 7.4. Methylthiazol tetrazolium (MTT) assay was applied to evaluate the Hela cells and NIH-3T3 cells cytotoxicity of GO–SA. Results showed that GO–SA had no obvious toxicity and GO–SA/DOX exhibits notable cytotoxicity to Hela cells. Cell uptake studies indicated that GO–SA could specifically transport the DOX into Hela cells over-expressing CD44 receptors and showed enhanced toxicity.

Inhibition of PTTG1 expression by microRNA suppresses proliferation and induces apoptosis of malignant glioma cells.

The present study aimed to investigate the role of pituitary tumor-transforming gene 1 (PTTG1) in the proliferation, invasion and apoptosis of human malignant glioma U251 cells. Firstly, 2 microRNAs (miRNAs) targeting PTTG1 messenger (m)RNA were ligated into a pcDNA6.2-GW/EmGFP-miR expression vector. The recombinant plasmids, miRNA-1 and miRNA-2 (miR-2), were transfected into U251 cells using the liposome method. PTTG1 mRNA and protein levels were evaluated using quantitative polymerase chain reaction and western blot analysis. The proliferation and invasion abilities of U251 cells were determined using methylthiazol tetrazolium and Matrigel assays. Flow cytometry analysis with Annexin V/propidium iodide double staining was used to determine the percentage of apoptotic cells. PTTG1 expression was effectively suppressed by miR-2. U251 cell growth was inhibited between 10.7 and 34.7% in the miR-2 group compared with the blank group. The Matrigel assay demonstrated that the percentage of infiltrating U251 cells was significantly lower in the miR-2 group (12.3±1.0%) compared to the blank group (24.7±1.4%; P<0.001) and the negative control group (24.0±2.0%; P<0.05). A higher percentage of apoptotic U251 cells were observed in the miR-2 group compared with the blank group (53.6 vs. 32.4%) using flow cytometry due to cycle arrests at the G2/M phase. The miR-2-transfected U251 cells were subcutaneously injected into nude mice, and these mice possessed a decreased tumor tissue growth rate and higher percentage of apoptotic cells compared with the blank and negative control groups. In conclusion, PTTG1 gene expression in human malignant glioma U251 cells was effectively suppressed by exogenous miR-2. The downregulation of PTTG1 induced glioma cell apoptosis and cell cycle arrest at the G2/M phase, which inhibited cell proliferation, reverse invasion and infiltration of glioma cells.

Minocycline, a putative neuroprotectant, co-administered with doxorubicin-cyclophosphamide chemotherapy in a xenograft model of triple-negative breast cancer

Minocycline is purported to have neuroprotective properties in experimental models of some human neurologic diseases, and has therefore been identified as a putative neuroprotectant for chemotherapy-induced cognitive impairment (CICI) in breast cancer patients. However, because its mechanism of action is believed to be mediated through anti-inflammatory, anti-apoptotic, and anti-oxidant pathways, co-administration of minocycline with chemotherapeutic agents has the potential to reduce the efficacy of anticancer drugs. The objective of this study is to evaluate the effect of minocycline on the activity of the AC chemotherapeutic regimen (Adriamycin [doxorubicin], Cytoxan [cyclophosphamide]) in in vitro and in vivo models of triple-negative breast cancer (TNBC). Clonogenic and methylthiazol tetrazolium (MTT) assays were used to assess survival and viability in two TNBC cell lines treated with increasing concentrations of AC in the presence or absence of minocycline. Biomarkers of apoptosis, cell stress, and DNA damage were evaluated by western blot. The in vivo effects of AC and minocycline, each alone and in combination, were assessed in a xenograft model of TNBC in female athymic nude mice by weekly tumor volume measurement, body and organ weight measurement, and histopathology. Apoptosis and proliferation were characterized by immunohistochemistry in the xenografts tumors. Brains from tumor-bearing mice were evaluated for microglial activation, glial scars, and the proportion of neural progenitor cells. Data from these in vitro and in vivo studies demonstrate that minocycline does not diminish the cytotoxic and tumor-suppressive effects of this chemotherapeutic drug combination in TNBC cells. Moreover, minocycline appeared to prevent the reduction in doublecortin-positive neural progenitor cells observed in AC-treated mice. We posit that minocycline may be useful clinically for its reported neuroprotective activity in breast cancer patients receiving AC without loss of chemotherapeutic efficacy.

Natural compound oblongifolin C inhibits autophagic flux, and induces apoptosis and mitochondrial dysfunction in human cholangiocarcinoma QBC939 cells.

The compounds, which are obtained from natural plants or microbes may offer potential as one of the strategies for the management of cholangiocarcinoma. Oblongifolin C (OC), a natural small molecule compound extracted and purified from Garcinia yunnanensis Hu, can activate the mitochondrial apoptotic pathway in human cervical cancer cells. However, the direct effects of OC on cholangiocarcinoma cells are not well defined. The effect of OC on cell apoptosis and its underlying mechanisms were investigated in cultured QBC939 cells by the methyl thiazol tetrazolium assay, mitochondrial membrane potential, ATP content and western blot analysis. The present study reported that the in vitro treatment of human cholangiocarcinoma QBC939 cells with different concentrations (5, 10, 20 and 40 μM) of OC decreased cell viability and induced apoptosis in a dose-dependent manner. The results of the present study also showed that OC-induced QBC939 cell apoptosis was mediated through the inhibition of autophagy and mitochondrial dysfunction (MtD). Additionally, inhibiting autophagy increased OC-induced apoptosis and MtD, whereas exposure to the autophagy inducer, rapmycin, attenuated these changes. Together, the results of the present study are the first, to the best of our knowledge, to identify OC as a chemotherapeutic agent against human cholangiocarcinoma QBC939 cells in vitro via the regulation of autophagy and MtD.

The impact of RNA interference-induced ADAR1 down-regulation on cell proliferation of liver cancer

Objective To observe the impact of RNA interference-induced ADAR1 down-regulation on cell proliferation of liver cancer. Methods Small interfering RNA (siRNA) was transfected into liver cancer cell line SMMC-7721. ADAR1 expression was detected by RT-PCR and Western blotting. Cell proliferation was determined by methyl thiazol tetrazolium (MTT) assay. Results After transfection for 24, 48, and 72 h, ADAR1 mRNA expression was 0.612±0.086, 0.264±0.018, 0.156±0.063 in experimental group and 1.032±0.107, 0.898±0.092, 0.968±0.074 in control group, respectively. Experimental group had significantly lower ADAR1 mRNA than the other groups (P 0.05). ADAR1 protein relative expression was 0.684±0.079, 0.324±0.042, 0.145±0.058 in experimental group and 1.002±0.092, 0.917±0.068, 0.972±0.073 in control group, respectively, which was statistically significant (P<0.05). After transfection with siRNA, the proliferation ability of SMMC-7721 cells was enormously inhibited (P<0.05). Conclusion ADAR1 mRNA and protein level could be significantly decreased by specific RNA interfe-rence, and cell proliferation in SMMC-7721 cells were also greatly inhibited. Key words: RNA interference; Gene, ADAR1; Liver cancer

Abstract 3246: Phytotherapy boosts innate immunity to maximize cancer survival

Abstract We studied surgery, radiation, and chemotherapy in animals and found all these modalities suppressed cancer growth but they also impaired the immune functions of the animal host. We also studied phytotherapy (use of plant chemicals) and found it to have no side effects and with more lasting benefits. Using in vitro cell culture system with both malignant and non-malignant cells, and immune cells (macrophages and natural killer cells), we compared crude extract of Pacific yew and the chemotherapeutic drug Paclitaxel derived from Pacific yew. The crude extract or the drug was incubated separately with: human malignant ovarian cells, nonmalignant ovarian cells, macrophages, and natural killer cells. Cell viability was determined by MTT (methylthiazol tetrazolium) assay. Results show the drug damaged all these four types of cells while the crude plant extract only damaged the cancer cells showing its selective toxicity for cancer cells. Study with other phytochemicals also shows selective toxicity for cancer cells while enhancing the activities of immune cells. Phytotherapy is thus a non-toxic chemotherapy and immunotherapy. In our clinical observation, 160 men and women diagnosed with five types of cancer (72 with breast cancer, 28 with prostate cancer, 18 with colon cancer, 14 with lung cancer, 28 with ovarian cancer) chose to use phytotherapy (a plant-based diet) plus lifestyle modification. The five year survival of these patients were 90%, 89%, 83%, 86%, and 82%, respectively. Compared with five year survival data from National Cancer Institute (www.seer.cancer.gov) for these five types of cancer: 82%, 50%, 62%, 15%, and 45%, respectively, the phytotherapy group exhibits greater cancer survival. Since numerous studies have now revealed that cancer is attributable to our lifestyle habits – the way we eat, and the way we live, our data suggest that regardless of treatment choices, a change in diet and lifestyle is likely to improve cancer survival. Citation Format: Benjamin H. Lau. Phytotherapy boosts innate immunity to maximize cancer survival. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3246.

Abstract 1703: Expression of Hedgehog signals and growth inhibition by itraconazole in endometrial cancer

Abstract Background: There are limited therapeutic opportunities for the treatment of endometrial cancer (EC). Itraconazole, a common antifungal agent and a potent inhibitor of the Hedgehog pathway, has been shown to be clinically effective for various types of cancer, but its clinical efficacy for EC is unknown. Herein, we evaluated the efficacy of itraconazole in treating EC. Patients and Methods: We reviewed cases of patients with serous endometrial intraepithelial carcinoma (SEIC) and patients who were diagnosed with EC with endometrioid adenocarcinoma grade 1 or 2 at FIGO stage IA with myometrial invasion and negative cytology of ascites and who recurred at our institution from January 2005 to December 2013. We also reviewed cases with endometrioid adenocarcinoma grade 1 or 2 at FIGO stage IA with myometrial invasion and negative cytology of ascites without recurrence over 5 years after surgery during 2006 and 2007. Immunohistochemistry was performed on EC tumour samples including SEIC for sonic Hedgehog (sHh), glioma-associated oncogene homolog 1 (GLI1), Progesterone receptor (PR), and chemokine ligand 18 (CCL18). Furthermore, we examined the effects of itraconazole on human EC cell lines (Ishikawa and HEC-1A cells) using a modified methylthiazol tetrazolium (MTT) assay and a migration assay. Results: SHh and GLI1 were expressed in SEIC and endometrioid adenocarcinoma. Immunohistochemical evaluation of sHh and GLI1 did not distinguish between recurrent and non-recurrent low-grade FIGO stage IA EC. The MTT assay revealed that itraconazole significantly inhibited the growth of Ishikawa and HEC-1A cells in a dose-dependent and time-dependent manner. However, migration was not significantly effected for either Ishikawa or HEC-1A cells. HEC-1A cells exhibited dose-dependent migration retardation. Conclusion: Hedgehog signalling plays a role in carcinogenesis and malignant progression in EC. Itraconazole at a physiological dose may suppress progression of EC. Citation Format: Kayo Inoue, Hiroshi Tsubamoto, Hiroaki Shibahara, Kazuo Tamura. Expression of Hedgehog signals and growth inhibition by itraconazole in endometrial cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1703.

Calcium Hydroxide–induced Proliferation, Migration, Osteogenic Differentiation, and Mineralization via the Mitogen-activated Protein Kinase Pathway in Human Dental Pulp Stem Cells

IntroductionCalcium hydroxide has been extensively used as the gold standard for direct pulp capping in clinical dentistry. It induces proliferation, migration, and mineralization in dental pulp stem cells (DPSCs), but the underlying mechanisms are still unclear. The aim of this study was to investigate the role of the mitogen-activated protein (MAP) kinase pathway in calcium hydroxide–induced proliferation, migration, osteogenic differentiation, and mineralization in human DPSCs. MethodsHuman DPSCs between passages 3 and 6 were used. DPSCs were preincubated with inhibitors of MAP kinases and cultured with calcium hydroxide. The phosphorylated MAP kinases were detected by Western blot analysis. Cell viability was analyzed via the methylthiazol tetrazolium assay. Cell migration was estimated using the wound healing assay. Alkaline phosphatase (ALP) expression was analyzed using the ALP staining assay. Mineralization was studied by alizarin red staining analysis. ResultsCalcium hydroxide significantly promoted the phosphorylation of the c-Jun N-terminal kinase (JNK), p38, and extracellular signal–regulated kinase. The inhibition of JNK and p38 signaling abolished calcium hydroxide–induced proliferation of DPSCs. The inhibition of JNK, p38, and extracellular signal–regulated kinase signaling suppressed the migration, ALP expression, and mineralization of DPSCs. ConclusionsOur study showed that the MAP kinase pathway was involved in calcium hydroxide–induced proliferation, migration, osteogenic differentiation, and mineralization in human DPSCs.

Role of long non-coding RNA maternally expressed gene 3 on cell proliferation and apoptosis of clear cell renal cell carcinoma

Objective To examine the expression of long non-coding RNA maternally expressed gene 3 (MEG3) in human clear cell renal cell carcinoma tissues (ccRCC) and cell lines. And to analyze the effects of MEG3 overexpression on the proliferation and apoptosis of ccRCC cells. Methods Real-time quantitative polymerase chain reaction (Real-time PCR) was carried out to measure the expression level of MEG3 in ccRCC specimens and cell lines compared with respective controls. After MEG3 was stabilized overexpressed in 786-O cells, methyl thiazol tetrazolium (MTT) and flow-cytometric assays were performed to evaluate the effects of MEG3 on cell proliferation and apoptosis. Protein levels of growth-or apoptosis-related molecules were then determined by Western blotting. Results MEG3 expression was decreased in ccRCC specimens and cell lines compared with respective controls. Overexpression of MEG3 in ccRCC cell line 786-O [0.51±0.02 vs. 11.26±1.89 for pcDNA3.1 and pcDNA3.1-MEG3 cell lie respectively with statistical difference (P<0.05)] resulted in delayed cell proliferation rate and induction of cell apoptosis[(7.02±1.21)% vs. (13.95±5.48)%] in vitro. Cyclin D1 and B cell lymphoma/leukemia-2 associated X protein (bax) protein levels were affected by MEG3 overexpression in these cell lines. Conclusion MEG3 was down-regulated in ccRCC, in which it functions as a potent tumor suppressor by regulating cell proliferation and apoptosis processes. Key words: Long non-coding RNA; Maternally expressed gene 3; Clear cell renal cell carcinoma; Cell proliferation; Apoptosis

Synergistic anti-pancreatic cancer immunological effects by treatment with reduced expression in immortalized cells/dickkopf-3 protein and peripheral blood mononuclear cells.

Reduced expression in immortalized cells/dickkopf-3 (REIC/DKK3) is a reported tumor suppressor gene and has potential to become an innovative therapy for various cancers. We examined the antitumor immunological effects of human REIC/DKK3 protein against pancreatic cancer. Activation of extracellular signal-regulated kinases 1 and 2, mammalian target of rapamycin, and signal transducer and activator of transcription 3 by REIC/DKK3 protein was assessed in human peripheral blood mononuclear cells using immunoblotting. Pancreatic cancer cell lines (AsPC-1 and MIA Paca-2) were cocultured with peripheral blood mononuclear cells, and the anticancer effects of REIC/DKK3 protein were assessed using the methyl thiazole tetrazolium, cytotoxicity, and enzyme-linked immunospot assays. The antitumor immunological effects of the combined treatment with REIC/DKK3 protein and peripheral blood mononuclear cells were also assessed in a pancreatic cancer model using non-obese diabetic/severe combined immunodeficiency mice. The REIC/DKK3 protein activated extracellular signal-regulated kinases 1 and 2, mammalian target of rapamycin, and signal transducer and activator of transcription 3 in peripheral blood mononuclear cells. REIC/DKK3 protein inhibited in vitro cancer cell viability and enhanced cytotoxicity when incubated with peripheral blood mononuclear cells. REIC/DKK3 protein induced significant production of interferon gamma from lymphocytes incubated with pancreatic cancer cells, indicating that CD8+ T cells were activated in the peripheral blood mononuclear cells when cocultured with AsPC-1 and MIA Paca-2 in the presence of REIC/DKK3 protein. Combined treatment with REIC/DKK3 protein and peripheral blood mononuclear cells produced in vivo anticancer immunostimulatory effects on pancreatic cancer cells. The REIC/DKK3 protein and peripheral blood mononuclear cells synergistically enhanced anticancer immunological effects against pancreatic cancer cells. The observed immunomodulatory effect of combined treatment likely occurs in adenovirus-mediated REIC/DKK3 gene therapy and provides important clues to the therapeutic mechanisms involving immune cells.

Effect of filamin A on proliferation and invasion of human gastric cancer MKN28 cells

Objective To explore the effect of filamin A (FLNa) gene on the proliferation and invasion of human gastric cancer MKN28 cells and to elucidate the possible mechanism. Methods Recombinant vector plasmid of pcDNA3.1/V5-His-TOPO/FLNa was transfected into MKN28 cells by lipofectAMINE. The expression of FLNa protein in MKN28/FLNa cells was detected by Western blotting. The proliferation rate of MKN28/FLNa cells was determined by methyl thiazol tetrazolium (MTT) assay. The invasion ability of MKN28/FLNa cells was determined by Transwell invasion assay. The expression of Cyclin D1 and matrix metalloproteinase-9 (MMP-9) protein in MKN28/FLNa cells was detected by Western blotting. Results The expression of FLNa protein was enhanced in MKN28 cells transfected with FLNa (P<0.05). Compared to control group, the proliferation of MKN28/FLNa cells on the 3rd day and 4th day was significantly inhibited (0.45±0.04 vs. 0.62±0.02, and 0.65±0.03 vs. 0.81±0.03, P<0.05). Compared to control group, the invasion ability of MKN28/FLNa cells was significantly decreased [(266±12) cells vs. (405±16) cells vs. (426±13) cells, P<0.05]. The expression levels of Cyclin D1 and MMP-9 protein in MKN28/FLNa cells were downregulated (P<0.05). Conclusion The FLNa gene could inhibit the proliferation and invasion of human gastric cancer MKN28 cells, which may be associated with down-regulation of Cyclin D1 and MMP-9 expression. Key words: Gastric cancer; Filamin A; Cyclin D1; Matrix metalloproteinases-9

MicroRNA-27a functions as a tumor suppressor in renal cell carcinoma by targeting epidermal growth factor receptor

Numerous studies have suggested that microRNAs (miRNAs) are vital in the development of various types of human cancers, including renal cell carcinoma (RCC), and the regulation of tumor progression and invasion. However, the effect of miRNA-27a (miR-27a) on the tumorigenesis of RCC is unclear. The aim of the present study was to investigate the function of miR-27a and identify its possible target genes in RCC cells. In the present study, cell proliferation, migration and invasion and the percentage of apoptotic cells were detected by methylthiazol tetrazolium assays, Annexin V analysis, wound-healing assays and Transwell invasion assays. Western blot analysis was performed to validate the protein expression level and assess whether the epidermal growth factor receptor (EGFR) was a target gene of miR-27a. A tumor xenograft animal model was used to detect the role of miR-27a on RCC cell growth in vivo. The present study demonstrated that miR-27a significantly suppressed human RCC 786-O cell proliferation and induced cell apoptosis. Restoration of miR-27 also resulted in 786-O cell migration and invasion inhibition. Furthermore, upregulated miR-27a attenuated RCC tumor growth in the tumor xenograft animal model. The present results suggested that miR-27a functions as a tumor suppressor in RCC. The western blot analysis assay revealed that EGFR was a novel target of miR-27a. The growth suppression of RCC cells was attributed partly to the downregulation of the cell cycle by ERFR inhibition. The present findings may aid in the understanding of the molecular mechanism of miR-27a in the tumorigenesis of RCC, and may provide novel diagnostic and therapeutic options for RCC.

Improving the Efficacy of Cryopreservation of Spermatogonia Stem Cells by Antioxidant Supplements.

Cryopreservation of spermatogonial stem cells (SSCs) is an applicable method for young males seeking fertility preservation before starting a treatment. It increases reactive oxygen species (ROS) formation and oxidative stress, which damages cellular structures. In this study, we added two antioxidants, catalase and α-tocopherol (α-TCP), to the basic freezing medium to evaluate their effects on the efficiency of SSCs. SSCs were isolated from testes of 3- to 6-day-old male mice using enzymatic digestion. The enrichment of isolated cells was evaluated by flow cytometry and Stra8 antibody. Catalase (40 μg/mL), or α-TCP (200 μg/mL) was added to the basic freezing medium. The cell viability was evaluated by the methylthiazoltetrazolium (MTT) assay. After thawing, cells were cultured for 1 month, and the expression pattern of specific genes of SSCs and the ability of the cells to restore spermatogenesis were used to determine the efficiency of the cryopreservation method. The survival rate of the frozen cells in the presence of catalase or α-TCP was significantly higher than the control group (p < 0.05). The number of colonies and their diameter measured after 1 month were significantly higher in the antioxidant groups than in the control group (p < 0.05). Gene expression and resumption of spermatogenesis also followed the same pattern. Thus, adding antioxidants to the basic freezing medium can be helpful in increasing the quality and viability of SSCs after cryopreservation. This new approach to stem cells cryopreservation can also be a promising strategy for fertility preservation in patients who suffer from malignancy.

Morphology, proliferation, and gene expression of gingival fibroblasts on Laser-Lok, titanium, and zirconia surfaces.

Soft tissue seal plays a critical role in long-term success of dental implants, and the effects of implant surface treatments such as laser ablation have been a topic of particular interest in this respect. Considering the existing controversy regarding soft tissue behavior in contact with implant surfaces, this study sought to assess the morphology, proliferation, and gene expression of human gingival fibroblasts (HGFs) on different abutment surfaces. In this in vitro, experimental study, HGFs were cultured on 45 discs (Laser-Lok, titanium, and zirconia). Cell morphology, proliferation rate, and interleukin 10 (IL-10), tumor necrosis factor alpha (TNFα), fibronectin, and integrin gene expressions were assessed by electron microscopy, methyl thiazol tetrazolium (MTT) assay, and real-time polymerase chain reaction (PCR), respectively. Data were analyzed using ANOVA and the Kruskal-Wallis H test. Fibroblast attachment was noted in all the three groups. Spindle-shaped cells with pseudopod-like processes were more frequently seen in the Laser-Lok group. Cell proliferation was significantly higher in the Laser-Lok group compared to those in the other groups (P = 0.0002). Significant differences were found in the expression of IL-10, TNFα, fibronectin, and integrin genes among the groups (P < 0.01). Within the limitations of this study, HGFs on Laser-Lok surfaces had a more mature morphology and greater proliferation and differentiation as compared to those on zirconia and titanium surfaces. This indicates better attachment of these cells to laser-modified surfaces, creating a more efficient soft tissue seal around dental implants.

Preparation and characterization of aminoethyl hydroxypropyl methyl cellulose modified with nisin

Nisin grafted aminoethyl hydroxypropyl methyl cellulose (AEHPMC) was prepared by an enzyme-catalyzed reaction in the presence of microbial transglutaminase (MTGase). AEHPMC was synthesized with 2-chloroethylamine hydrochloride (CEH) which was as an intermediate reactant. The parameters, which influenced the NH2% and the degree of substitution (DS), including reaction time, reaction temperature and the mass ratio of the reactants were investigated. Antioxidant activities of AEHPMC-nisin were evaluated by the scavenging activity of hydroxyl and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical. With the DS and concentration increasing of AEHPMC-nisin, the radical scavenging activity increased. The Methylthiazol tetrazolium (MTT) assay indicated that AEHPMC-nisin had low-toxicity to L929 cells. Therefore, the derivative of HPMC may show a promising potential application in biomedical, food and pharmaceutical fields.