Methyl Thiazolyl Tetrazolium Colorimetric Assay Research Articles

Acetyl-11-keto-β-boswellic acid inhibits proliferation and induces apoptosis of gastric cancer cells through the phosphatase and tensin homolog /Akt/ cyclooxygenase-2 signaling pathway.

BACKGROUNDGastric cancer is one of the most common malignant tumors of the digestive system worldwide, posing a serious danger to human health. Cyclooxygenase (COX)-2 plays an important role in the carcinogenesis and progression of gastric cancer. Acetyl-11-keto-β-boswellic acid (AKBA) is a promising drug for cancer therapy, but its effects and mechanism of action on human gastric cancer remain unclear.AIMTo evaluate whether the phosphatase and tensin homolog (PTEN)/Akt/COX-2 signaling pathway is involved in the anti-tumor effect of AKBA in gastric cancer.METHODSHuman poorly differentiated BGC823 and moderately differentiated SGC7901 gastric cancer cells were routinely cultured in Roswell Park Memorial Institute 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. Gastric cancer cell proliferation was determined by methyl thiazolyl tetrazolium colorimetric assay. Apoptosis was measured by flow cytometry. Cell migration was assessed using the wound-healing assay. Expression of Bcl-2, Bax, proliferating cell nuclear antigen, PTEN, p-Akt, and COX-2 were detected by Western blot analysis. A xenograft nude mouse model of human gastric cancer was established to evaluate the anti-cancer effect of AKBA in vivo.RESULTSAKBA significantly inhibited the proliferation of gastric cancer cells in a dose- and time-dependent manner, inhibited migration in a time-dependent manner, and induced apoptosis in a dose-dependent manner in vitro; it also inhibited tumor growth in vivo. AKBA up-regulated the expression of PTEN and Bax, and down-regulated the expression of proliferating cell nuclear antigen, Bcl-2, p-Akt, and COX-2 in a dose-dependent manner. The PTEN inhibitor bpv (Hopic) reversed the high expression of PTEN and low expression of p-Akt and COX-2 that were induced by AKBA. The Akt inhibitor MK2206 combined with AKBA down- regulated the expression of p-Akt and COX-2, and the combined effect was better than that of AKBA alone.CONCLUSIONAKBA inhibits the proliferation and migration and promotes the apoptosis of gastric cancer cells through the PTEN/Akt/COX-2 signaling pathway.

The effect of different cross-linking conditions of EDC/NHS on type II collagen scaffolds: an in vitro evaluation.

The purpose of this paper is to analyze the properties of porcine cartilage type II collagen scaffolds crosslinked with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxy-succinamide (EDC/NHS) under different conditions. The porous EDC/NHS-crosslinked scaffolds were obtained through a two-step freeze-drying process. To determine the optimal crosslinking condition, we used different solvents and various crosslinking temperatures to prepare the scaffolds. Three crosslinking solutions were prepared with different solvents, photographs were taken with a flash in the darkroom, and light transmission was observed. Type II collagen was crosslinked on a horizontal shaker at a speed of 60r/min according to the above grouping conditions, and then the structural change of the scaffold in each group was observed. To investigate the swelling ratio and the in vitro degradation of the collagen scaffold, tests were also carried out by immersion of the scaffolds in a PBS solution and digestion in type II collagenase, respectively. The influence of the scaffolds on the proliferation of chondrocytes was assessed by the methyl thiazolyl tetrazolium colorimetric assay. The morphology of the crosslinked scaffolds cocultured with chondrocytes was characterized by a scanning electron microscope. The results proved that 75% alcohol and a crosslinking temperature of 37°C are recommended. Collagen fibrils are more densely packed after crosslinking with EDC/NHS and have a more uniform structure than that of noncrosslinked ones. The EDC-crosslinked scaffolds possessed excellent mechanical property and biocompatibility.

Assessment of the hepatocyte protective effects of gypenoside and its phosphorylated derivative against DHAV-1 infection on duck embryonic hepatocytes

BackgroundDuck viral hepatitis (DVH) is an acute disease of young ducklings with no effective veterinary drugs for treatment. Gynostemma pentaphyllum is a well-known traditional Chinese medicine that plays an important role in the treatment of various diseases. Gypenoside (GP), one of the main ingredients of Gynostemma pentaphyllum, was reported with good hepatoprotective effects. However, its low solubility limits its application in the clinics. To improve its solubility and bioactivity, a phosphorylated derivative of gypenoside (pGP) was prepared by the sodium trimetaphosphate-sodium tripolyphosphate (STMP-STPP) method. An infrared spectroscopy method was applied to analyse the structures of GP and pGP. Then, a methyl thiazolyl tetrazolium (MTT) colorimetric assay was applied to study the hepatocyte protective efficacy of these two drugs against duck hepatitis A virus type 1 (DHAV-1) infection, and qPCR, TUNEL labelling and flow cytometry methods were used to study the relevant hepatocyte protective in vitro.ResultsThe infrared spectroscopy detection results showed that the phosphorylation modification of GP was successful. The MTT colorimetric assay results showed that both GP and pGP possessed good hepatocyte protective efficacy in vitro, and pGP performed better than GP when the drug was added before or after virus inoculation. Furthermore, the qPCR results revealed that both drugs could effectively inhibit the adsorption (when adding GP and pGP pre-virus inoculation), replication and release of DHAV-1, and the viral inhibition rate of pGP was greater than that of GP. The subsequent TUNEL labelling and flow cytometry assays showed that both GP and pGP could significantly inhibit duck embryo hepatocyte apoptosis induced by DHAV-1, and the inhibition effect of pGP was much stronger than that of GP.ConclusionsGP exerts good hepatocyte protective efficacy not only by inhibiting the proliferation of DHAV-1 but also by inhibiting duck embryonic hepatocyte apoptosis induced by DHAV-1, and phosphorylation modification significantly improves the antiviral and the anti-apoptotic effects of GP. Therefore, pGP has the potential to be developed into a novel drug against DHAV-1 infection.

Effects of Clostridium�difficile toxin A on K562/A02 cell proliferation, apoptosis and multi-drug resistance

The aim of the present study was to investigate the cytotoxic effect and multi-drug resistance (MDR) of Clostridium difficile toxin A (TcdA) on K562/A02 cells, and understand its underlying molecular pathways. K562/A02 cells were treated with TcdA at different concentrations for 24, 48 and 72 h, and the inhibition effect and drug resistance of TcdA on K562/A02 cell proliferation was assessed by methyl thiazolyl tetrazolium colorimetric assay. Furthermore, cell cycle-apoptosis was analyzed by flow cytometry, P-glycoprotein (P-gp) expression was determined by western blot analysis and caspase-3 activity was measured using a caspase-3 activity kit. TcdA inhibited K562/A02 cell proliferation in a time- and dose-dependent manner. The inhibition rate of K562/A02 cells reached 8.76±0.76, 28.55±0.43, 47.89±0.27, 58.08±0.06 and 57.70±0.79% following treatment with 50, 100, 200, 400 and 800 ng/ml TcdA, respectively, for 24 h. K562/A02 cells in the G0/G1 phase increased and cells in the S phase decreased following treatment with TcdA (P<0.05), and the apoptotic rates in the 200 and 400 ng/ml concentration groups were 14.05 and 22.89%, respectively. In addition, TcdA (50 ng/ml) significantly inhibited the proliferation of K562/A02 cells and reduced the half maximal inhibitory concentration of these drugs in combination with chemotherapy drugs. The reversal folds were 3.09, 2.89 and 2.79, respectively. Furthermore, TcdA treatment was associated with the upregulation of P-gp in K562/A02 cells, and caspase-3 activity was observed to increase in K562/A02 cells following TcdA treatment, when compared with untreated controls (P<0.05). These findings suggested that TcdA may be able to inhibit K562/A02 cell growth, induce cell apoptosis by decreasing P-gp levels and caspase-3 activation, and partially reverse MDR. Further studies are required to evaluate the potential of TcdA as a candidate for the chemotherapy of hematologic malignancies.

Study on the expression of TRIP13 mRNA in chronic lymphocytic leukemia B lymphocyte and the molecular mechanism of TRIP13 mediated JVM-2 cell proliferation and apoptosis

目的探讨甲状腺激素受体相互作用分子13（TRIP13）基因表达和慢性淋巴细胞白血病（CLL）的相关性，验证TRIP13基因在CLL发生发展中发挥的生物学功能，探讨TRIP13基因调控CLL发生发展的下游分子机制。方法①应用实时荧光定量PCR检测30例CLL患者和12名造血干细胞供者（正常对照组）外周血CD19+ B淋巴细胞TRIP13 mRNA的表达水平。②使用慢病毒介导的shRNA干扰JVM-2细胞TRIP13 mRNA和蛋白表达水平，同时使用随机序列shRNA处理JVM-2细胞作为对照组，分别应用MTT法和流式细胞术检测TRIP13干扰组、对照组JVM-2细胞的增殖和凋亡情况。③Western blot法检测TRIP13干扰对JVM-2细胞凋亡相关蛋白表达的影响。结果①30例CLL患者外周血B淋巴细胞TRIP13 mRNA表达水平（2−ΔCt=0.014 89）高于正常对照组（2−ΔCt=0.000 19）（P<0.001）。②在JVM2细胞中筛选出有效干扰shRNA靶点，下调TRIP13的表达能够有效抑制JVM-2细胞增殖并促进细胞凋亡。③干扰TRIP13后JVM-2细胞Myc和Bcl-2蛋白表达下调（P<0.001），Bax、caspase3、Bad蛋白表达上调（P<0.001）。结论CLL患者外周血CD19+ B淋巴细胞TRIP13 mRNA表达上调；TRIP13基因通过影响细胞增殖和凋亡相关蛋白表达而调控JVM2细胞的增殖与凋亡。

Combination of basic fibroblast growth factor and epidermal growth factor enhances proliferation and neuronal/glial differential of postnatal human enteric neurosphere cells in vitro.

Human enteric neural stem cells (hENSCs) proliferate and differentiate into neurons and glial cells in response to a complex network of neurotrophic factors to form the enteric nervous system. The primary aim of this study was to determine the effect of basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) on in-vitro expansion and differentiation of postnatal hENSCs-containing enteric neurosphere cells. Enteric neurosphere cells were isolated from rectal polyp specimens of 75 children (age, 1-13 years) and conditioned with bFGF, EGF, bFGF+EGF, or plain culture media. Proliferation of enteric neurosphere cells was examined using the methyl thiazolyl tetrazolium colorimetric assay over 7 days of culture. Fetal bovine serum (10%) was added to induce the differentiation of parental enteric neurosphere cells, and differentiated offspring cells were immunophenotyped against p75 neutrophin receptor (neural stem cells), peripherin (neuronal cells), and glial fibrillary acidic protein (glial cells). Combining bFGF and EGF significantly improved the proliferation of enteric neurosphere cells compared with bFGF or EGF alone (both P<0.01) throughout 7 days of culture. The addition of bFGF drove a significantly greater proportion of enteric neurosphere cells to differentiate into neuronal cells than that of EGF (P<0.01), whereas addition of EGF resulted in significantly more glial differentiation compared with addition of bFGF (P<0.01). Combining bFGF and EGF drove enteric neurosphere cells to differentiate into neuronal cells in a proportion similar to glial cells. Our results showed that the combination of bFGF and EGF significantly enhanced the proliferation and differentiation of postnatal hENSCs-containing enteric neurosphere cells in vitro.

Astragalus Polysaccharide Inhibits Autophagy and Apoptosis from Peroxide-Induced Injury in C2C12 Myoblasts.

The aim is to study the effects and underlying mechanisms of astragalus polysaccharide (APS) on the peroxide-induced injury in C2C12 myoblasts in vitro. Cell viability in the presence or absence of APS was detected by the methyl thiazolyl tetrazolium colorimetric assay. The autophagosomes were observed by electron microscopy to examine the influence of APS on autophagy caused by H2O2 in C2C12 cells, and the percentage of apoptosis cells was measured by flow cytometry. To further confirm the effect of H2O2 on C2C12 cells, the protein expression of LC3 and RARP, which are the markers of autophagy and apoptosis, respectively, was analyzed by Western blot, as well as the expression levels of p-p70S6K, p70S6K, Bcl-2, Bax, cyto-C, and Caspase-3, to reveal the underlying mechanisms. We observed multiple effects of APS on C2C12 functionality. APS treatment of C2C12 cells at 1mg/mL reduced cell viability to less than 70%, and analysis by electron microscopy revealed that APS also reduced the number of H2O2-induced autophagosome formation. Similarly, APS abated the H2O2-mediated increase in cell apoptosis, which was accompanied by the inhibition of LC3 II and RARP that are normally upregulated by H2O2. The expression of p-p70S6K and p70S6K, however, remained unchanged in C2C12 cells in the Control, H2O2 and H2O2+APS groups. In addition, APS promoted the expression of protein Bcl-2 in H2O2-treated C2C12 cells, but did not change Bax, thus reducing the Bax/Bcl-2 ratio that in turn prevented the release of cytochrome c and the activation of caspase-3. APS inhibits the autophagy and apoptosis induced by peroxide injury in C2C12 myoblasts through two independent signaling pathways: the mTOR-independent pathway for the inhibition of autophagy, and the caspase-3-dependent pathway for the suppression of apoptosis.

Effects of herbal compound 861 on collagen synthesis and degradation in rat mesangial cells exposed to high glucose

To investigate the effects of Herbal Compound 861 (Cpd 861) on collagen synthesis and degradation in rat mesangial cells exposed to high glucose. The third to fifth passage of rat mesangial cells were exposed to high glucose and Cpd 861 at a concentration of 0.25-4.00 g/L for 24, 48 and 72 h, respectively. Benazepril (10(-7)-10(-3) mmol/L) was selected as positive control. The methyl thiazolyl tetrazolium colorimetric assay was used to evaluate the effect of Cpd 861 on cell proliferation. After incubation with Cpd 861 at a concentration of 2.00 g/L for 48 h, the protein secretions of collagen type IV, matrix metallopeptidase 9 (MMP-9), tissue inhibitor of metalloproteinase-1 (TIMP-1), transforming growth factor beta 1 (TGF-β1), and hepatocyte growth factor (HGF) were detected by enzyme-linked immunosorbent assay method. And rat mesangial cells were harvested to determine MMP-9, TIMP-1, TGF-β1 and HGF mRNA expression by reverse transcription polymerase chain reaction. Cpd 861 inhibited cell proliferation induced by high glucose in a dose- and time-dependent manner. Compared with high glucose, collagen type IV production was decreased significantly by Cpd 861 (P<0.01). Cpd 861 increased the protein secretions and mRNA expressions of MMP-9 and HGF, whereas the protein secretions and mRNA expressions of TIMP-1 and TGF-β1 were reduced markedly (P<0.05). The ratio of MMP-9 to TIMP-1 was enhanced by Cpd 861 significantly. There was no significant difference in all above-mentioned effects between Cpd 861 (2.00 g/L) and benazepril (10(-5) mmol/L). The anti-glomerulosclerosis mechanisms of Cpd 861 were partly attributed to its effects of inhibiting mesangial cell proliferation, decreasing collagen synthesis and enhancing collagen degradation.

Effects of Nickel-Smelting Fumes on the Regulation of NIH/3T3 Cell Viability, Necrosis, and Expression of hMLH1 and RASSF1A

Nickel is widely used and distributed in various industries. This study investigated the effect of nickel-smelting fumes on the regulation of NIH/3T3 cell viability, apoptosis, and necrosis and the expression of the tumor suppressor genes hMLH1 and RASSF1A. Cell viability was determined using a methylthiazolyl tetrazolium colorimetric assay. NIH/3T3 cell viability was reduced after exposure to different concentrations of nickel-smelting fumes, but cell apoptosis and necrosis were induced. Moreover, cell morphology changed significantly after exposure to different concentrations of nickel-smelting fumes, as determined using an inverted microscope or transmission electron microscope. Real-time RT-PCR and Western blot analyses showed that exposure of cells to concentrations of ≥100 µg/mL of nickel-smelting fumes upregulated the expression of hMLH1 and RASSF1A compared to the negative controls. These data suggest that nickel-smelting fumes could be toxic to cells, upregulating the expression of hMLH1 and RASSF1A and in turn inducing cell apoptosis and necrosis.

Impact of the biological function on epithelial ovarian cancer with ITIH4 gene expression down-regulating in vitro

To investigate the efficiency of biology function of ITIH4 gene silenced by small interfering RNA (siRNA) on ovarian cancer. The four pairs ITIH4 gene siRNA interference fragments (ITIH4-546, ITIH4-795, ITIH4-917 and ITIH4-1568) were designed respectively, and transfected into HO8910pm cells with ITIH4 mRNA high expression by liposomal method transiently. Quantitative PCR method was used to detect the ITIH4 mRNA expression in HO8910pm cells transfected with interference fragment. The ITIH4 917 was selected as the best silencing effect of siRNA interference fragment and then the recombinant plasmid expression vector pGPU6/GFP/Neo-shRNA-ITIH4-917 was constructed and transfected into HO8910pm cells. The stably transfected cells- pGPU6/GFP/Neo-shRNA-ITIH4-917-HO8910pm cells was obtained by screening of aminoglycoside antibiotics (G418). The experiment was divided into three groups, namely ITIH4-917 transfection group, the HO8910pm cell group transfected with pGPU6/GFP/Neo-shRNA plasmid (empty vector group), and the HO8910pm cell group transfected with pGPU6/GFP/Neo-shRNA-ITIH4-NC the plasmid (negative control group). Fluorescence quantitative reverse transcription (RT) PCR and western blot were used to detect the ITIH4 mRNA and protein expression. The cell proliferation, the cell cycle, colony formation of cells, cells migration and invasion in vitro were determined by using methyl thiazolyl tetrazolium (MTT), flow cytometry, colony formation assay and transmembrane (transwell) small chamber method [value represented by absorbance (A)], respectively. The fluorescent quantitative PCR results showed that the ITIH4 mRNA expression levels in ITIH4-917 HO8910pm cells was significantly lower than that in the control cells, the relative copy number was only 0.26 ± 0.15. Also the relative copy number of ITIH4 mRNA in ITIH4-917 transfection group cells was 0.34 ± 0.10, it significantly lower than that in empty vector group (1.87 ± 0.12, P = 0.008) and negative control group (1.58 ± 0.21, P = 0.032); Western blot results showed that the ITIH4 relative expression levels of the protein in ITIH4-917 HO8910pm group cells, empty vector group and negative control group were 0.51, 1.64 and 1.74, respectively, there were statistically significant differences (0.51 vs. 1.64, P = 0.012; 0.51 vs. 1.74, P = 0.014). MTT colorimetric assay showed that the proliferation of ITIH4-917 HO8910pm group cells was significantly faster than that in the empty vector group and negative control group, and there were statistically significant differences among them (P = 0.001). The S + G2/M phase cell ratio in ITIH4-917 HO8910pm group cells was 54.2%, which was significantly higher than that in the empty vector group or negative control group (26.3% and 31.3%, respectively, all P < 0.05). The colony formation rate (55.7 ± 0.7)% in ITIH4-917 HO8910pm group cells was also significantly higher than that in empty vector group (29.7 ± 0.9)% (P = 0.037) and negative control group (31.4 ± 0.3)% (P = 0.043). Migration and invasion experiments showed that cell migration in ITIH4-917 HO8910pm group cells was 0.40 ± 0.18, whicht was significantly higher than that in the negative control group or empty vector (0.30 ± 0.03, P = 0.031;0.25 ± 0.03, P = 0.028, respectively). Although the invasive ability of ITIH4-917 HO8910pm group cells (1.31 ± 0.34) was higher than that in the control cells (1.05 ± 0.68) and empty vector group (1.14 ± 0.08), while there were not significant difference (P > 0.05). It would be to promote the cell doubling time and increase the migration capability in HO8910pm cells that ITIH4 expression was down-regulating by ITIH4 mRNA interference.

Effect of curcumin on the proliferation and apoptosis of hepatic stellate cells

This study was designed to investigate the effect of curcumin (diferuloylmethane) on the proliferation and apoptosis of hepatic stellate cells (HSC). The cell line HSC-T6 (1.25 x 10(5) cells/mL) was incubated with curcumin and HSC proliferation was detected by a methyl thiazolyl tetrazolium colorimetric assay. HSC apoptosis was detected by flow cytometry, transmission electron microscope and agarose gel electrophoresis. HSC proliferation was significantly inhibited in a concentration-dependent manner (10.6 to 63.5%) after incubation with 20-100 microM curcumin, compared with a control group. At 20, 40, and 60 microM, after 24 h of incubation, curcumin was associated with a significant increase in the number of HSC in the G2/M phase, and a significant decrease in cell numbers in the S phase (P < 0.05). At these concentrations, curcumin was also associated with an increase in the apoptosis index of 15.3 +/- 1.9, 26.7 +/- 2.8, and 37.6 +/- 4.4%, respectively, compared to control (1.9 +/- 0.6%, P < 0.01). At 40 microM, the curcumin-induced apoptosis index at 12, 24, 36, and 48 h of incubation was 12.0 +/- 2.4, 26.7 +/- 3.5, 33.8 +/- 1.8, and 49.3 +/- 1.6%, respectively (P < 0.01). In conclusion, curcumin inhibits the in vitro proliferation of HSCs in the G2/M phase of the cell cycle and also induces apoptosis in a concentration- and time-dependent manner. The in vivo effect of curcumin on HSCs requires further investigation.

Effects of extracellular signal‐regulated kinase on rat cultured hepatic stellate cells stimulated by acetaldehyde

To investigate the effects of PD98059 on the cell cycle, cell proliferation, the secretion of type I collagen and expression of transforming growth factor-beta-1 mRNA in rat hepatic stellate cells stimulated by acetaldehyde. Rat hepatic stellate cells stimulated by acetaldehyde were incubated with different concentrations of PD98059. The cell cycle was analyzed by flow cytometry. Cell proliferation was assessed by methyl thiazolyl tetrazolium colorimetric assay. The mRNA expression of transforming growth factor-beta-1 was examined by reverse transcriptase polymerase chain reaction. Type I collagen of the culture medium was detected by enzyme-linked immunoadsorbent assay. Twenty, 50 and 100 micromol/L PD98059 could significantly inhibit the proliferation and provoke a G0/G1-phase arrest of hepatic stellate cells stimulated by acetaldehyde in a dose-dependent manner. The secretion of type I collagen and transforming growth factor-beta-1 mRNA expression of acetaldehyde-induced hepatic stellate cells were markedly inhibited by 50 and 100 micromol/L PD98059, respectively. Extracellular signal-regulated kinase signal transduction pathway could regulate cell proliferation, the secretion of type I collagen and transforming growth factor-beta-1 mRNA expression of rat hepatic stellate cells stimulated by acetaldehyde. This is most likely related to its regulative effect on the cell cycle.