OBJECTIVETo determine the effect of aspirin on cell proliferation, alkaline phosphatase (ALP) activity, cell cycle and apoptosis in murine bone marrow stromal cells, so as to explore an appropriate dose range to improve bone regeneration in periodontal treatment.

METHODSST2 cells were stimulated with aspirin (concentrations of 1, 10, 100 and 1 000 μmol/L) for 1, 2, 3, 5 and 7 d. Cell proliferation was measured by methyl thiazolyl tetrazolium (MTT) assay. After ST2 cells were treated for 1, 3 and 7 d, ALP activity was measured by ALP kit, cell cycle and apoptosis were measured by flow cytometry (FCM) after treated for 48 h.

RESULTSMTT assays showed that various doses of aspirin have different effects on the cell growth. Briefly, lower concentrations (1, 10 μmol/L) of aspirin promoted the cell growth, the A value of 0, 1 and 10 μmol/L aspirin 7-day-treated cells were 0.313±0.012, 0.413±0.010 and 0.387±0.017 respectively (P <0.01 vs control), and so did the ALP level ([4.3±0.9], [6.0±0.3] and [7.7±0.4] μmol·min(-1)·g(-1), P <0.05 vs control), while higher concentrations, especially 1000 μmol/L of aspirin might inhibit the cell growth with time going, A value and ALP level were 0.267±0.016, (4.3±1.3) μmol·min(-1)·g(-1) respectively (P <0.05 vs control). Cell cycle analysis revealed no changes in comparison to control cells after treatment with 1 or 10 μmol/L aspirin, but it was observed that cell mitosis from S phase to G2/M phase proceeded at higher concentrations of 100 μmol/L aspirin, and the cell cycle in phase G0/G1 arrested at 1000 μmol/L. Parallel apoptosis/necrosis studies showed that the percentage of cells in apoptosis decreased dramatically at all doses of aspirin, the apoptosis rates of ST2 cells responded to 0, 1, 10, 100 and 1000 μmol/L aspirin were (11.50±0.90)%, (5.30±0.10)%, (5.50±0.10)%, (4.90±0.90)% and (7.95±0.25)% respectively (P<0.05 vs control).

CONCLUSIONSThis study demonstrated that lower dosage of aspirin can promote ST2 cells growth, osteogenic activity and inhibit its apoptosis. Aspirin maybe used for the bone reconstruction with a proper concentration.

Alkaline Phosphatase ; metabolism ; Animals ; Apoptosis ; drug effects ; Aspirin ; administration & dosage ; pharmacology ; Bone Regeneration ; Cell Cycle ; drug effects ; Cell Division ; Cell Line, Tumor ; Cell Proliferation ; Flow Cytometry ; Formazans ; Mesenchymal Stromal Cells ; cytology ; drug effects ; enzymology ; Mice ; Periodontics ; Tetrazolium Salts ; Time Factors

OBJECTIVETo investigate the effect of CAL-101, a selective inhibitor of PI3Kδ, in combination with bortezomib on the proliferation and apoptosis in human mantle cell lymphoma cell lines Z138, HBL-2 and Jeko-1 in vitro, to explore its mechanisms and provide the foundation for effective treatment strategies against mantle cell lymphoma.

METHODSMTT assay was applied to detect the inhibitory effects of CAL-101 and bortezomib either alone or combined on Z138, HBL-2 and Jeko-1 cells. Calcusyn software was used to analyze the synergistic cytotoxicity. Western blot was used to detect the expression of PI3K-p110σ and p-Akt, Akt, p-ERK and ERK proteins after the cells were exposed to different concentrations of CAL-101. Flow cytometry was employed to assess the apoptosis rate. NF-κB kit was used to determine the changes of location of NF-κB P65, and Western blot was applied to detect the level of caswpase-3 and the phosphorylation of Akt in different groups.

RESULTSCAL-101 and BTZ inhibited the proliferation of Z138, HBL-2 and Jeko-1 cells in a dose- and time-dependent manner. CAL-101/BTZ combination induced significantly synergistic cytotoxicity in the MCL cells. The results of Western blot assay showed that CAL-101 significantly blocked the phosphorylation of Akt and ERK in the MCL cell lines. In addition, CAL-101 combined with BTZ induced pronounced apoptosis (P < 0.01). For example, after the Z138 cells exposed to the drugs for 48 h, the apoptosis rates of the control, CAL-101, BTZ and CAL-101 + BTZ groups were: (2.6 ± 1.8)%, (40.0 ± 3.0)%, (34.0 ± 1.0)%, and (67.4 ± 1.0)%, respectively; and when drug treatment was given to HBL-2 cells over 96 h, the apoptosis rates of these four cell groups were (7.4 ± 0.6)%, (30.7 ± 5.7)%, (12.0 ± 1.0)%, and (85.0 ± 4.0)%, respectively. The combination therapy contributed to the enhanced inactivity of nuclear factor-κB (NF-κB) and Akt inactivation in the MCL cell lines (P < 0.05), however, the casepase-3 activity was up-regulated.

CONCLUSIONSThe combination of CAL-101 and bortezomib is muchmore effective in inhibiting proliferation and promoting apoptosis of mantle cell lymphoma cell lines (Z138, HBL-2 and Jeko-1), which may be mediated through inhibiting PI3K/Akt signaling pathway and the transcription of NF-κB.

Antineoplastic Agents ; pharmacology ; Antineoplastic Combined Chemotherapy Protocols ; pharmacology ; Apoptosis ; drug effects ; Blotting, Western ; Boronic Acids ; Bortezomib ; pharmacology ; Caspase 3 ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Class Ia Phosphatidylinositol 3-Kinase ; antagonists & inhibitors ; Dose-Response Relationship, Drug ; Drug Synergism ; Formazans ; Humans ; Lymphoma, Mantle-Cell ; drug therapy ; pathology ; MAP Kinase Signaling System ; drug effects ; NF-kappa B ; metabolism ; Neoplasm Proteins ; metabolism ; Phosphatidylinositol 3-Kinases ; metabolism ; Phosphorylation ; drug effects ; Proto-Oncogene Proteins c-akt ; metabolism ; Purines ; administration & dosage ; pharmacology ; Pyrazines ; Quinazolinones ; administration & dosage ; pharmacology ; Signal Transduction ; Software ; Tetrazolium Salts

OBJECTIVETo study the molecular mechanism of cisplatin to enhance the ability of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in reversing multidrug resistance in vincristine-resistant human gastric cancer SGC7901/VCR cells.

METHODSMTT assay was used to measure the 50% inhibiting concentration (IC₅₀) and cell survival in SGC7901 and SGC7901/VCR cells after different treatments. SGC7901/VCR cells were treated with different concentrations of DDP, different concentrations of TRAIL alone or in combination, and then the mRNA and protein levels of several genes were determined by RT-PCR, RT-qPCR and Western-blot analysis. After targeted silencing with specific siRNA and transfection of recombinant plasmid c-myc into the SGC7901/VCR cells, the mRNA and protein levels of DR4, DR5 and c-myc were determined by RT-PCR and Western-blot analysis.

RESULTSAfter combined treatment with TRAIL and DDP of the SGC7901/VCR cells, the IC₅₀ of VCR, DDP, ADM, and 5-Fu treatment was significantly decreased compared with the control group or TRAIL-treated group (P < 0.05). After treatment with 0, 10, 50 ng/ml TRAIL in combination with 0.4 µg/ml DDP, the SGC7901/VCR cells showed significantly higher activation of caspase 3, down-regulation of DNA-PKcs/Akt/GSK-3β signaling pathway, and higher inhibition of MDR1(P-gp) and MRP1 than those treated with TRAIL alone (P < 0.01 for all). The mRNA and protein levels of DR4, DR5, c-myc were significantly decreased after silencing c-myc with specific siRNA in the SGC7901/VCR cells (P < 0.01 for all), and were significantly increased after transfection of recombinant plasmid c-myc into the SGC7901/VCR cells (P < 0.01 foe all). After the treatment with 10 ng/ml TRAIL, 0.25 µg/ml DDP + 10 ng/ml TRAIL and 0.5 µg/ml DDP + 10 ng/ml TRAIL, the relative expression level of c-myc protein in the SGC7901/VCR cells was 0.314 ± 0.012, 0.735 ± 0.026, and 0.876 ± 0.028, respectively, and the relative expression of cytochrome C was 0.339 ± 0.036, 0.593 ± 0.020 and 0.735 ± 0.031, respectively, and the relative expression levels of DR4, DR5, active-caspase 3 and active-caspase 9 in the SGC7901/VCR cells were also increased along with increasing DDP concentrations.

CONCLUSIONSThe activation of DNA-PKcs/Akt/GSK-3β signaling pathway and high expression of MDR1 and MRP1 play an important role in the multi-drug resistance properties of SGC7901/VCR cells. After combining with TRAIL, DDP can enhance the expression of DR4 and DR5 through up-regulating c-myc and enhancing the activation of caspase 3 and caspase 9 by facilitating mitochondrial release of cytochrome C. It may be an important molecular mechanism of DDP-induced sensitization of TRAIL to reverse the multidrug resistancein SGC7901/VCR cells.

ATP-Binding Cassette, Sub-Family B, Member 1 ; metabolism ; Antineoplastic Agents ; administration & dosage ; pharmacology ; Antineoplastic Combined Chemotherapy Protocols ; administration & dosage ; pharmacology ; Caspase 3 ; metabolism ; Caspase 9 ; metabolism ; Cell Line, Tumor ; Cisplatin ; administration & dosage ; pharmacology ; Down-Regulation ; Drug Resistance, Multiple ; drug effects ; Drug Resistance, Neoplasm ; drug effects ; Fluorouracil ; administration & dosage ; pharmacology ; Formazans ; Genes, myc ; Glycogen Synthase Kinase 3 ; metabolism ; Glycogen Synthase Kinase 3 beta ; Humans ; Inhibitory Concentration 50 ; Multidrug Resistance-Associated Proteins ; metabolism ; Neoplasm Proteins ; metabolism ; Plasmids ; Proto-Oncogene Proteins c-myc ; metabolism ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; pharmacology ; Receptors, TNF-Related Apoptosis-Inducing Ligand ; metabolism ; Stomach Neoplasms ; drug therapy ; pathology ; TNF-Related Apoptosis-Inducing Ligand ; administration & dosage ; pharmacology ; Tetrazolium Salts ; Transfection ; methods

OBJECTIVETo investigate the role of mitochondrial calcium uniporter in cardioprotection elicited by ischemic postconditioning (Postcond).

METHODSMale Sprague-Dawley rats were used for Langendorff isolated heart perfusion. The hearts subjected to global ischemia for 30 min followed by 120 min of reperfusion. Left ventricular developed pressure (LVDP), maximal rise/fall rate of left ventricular pressure (± dP/dtmax) were measured. The level of lactate dehydrogenase (LDH) in the coronary effluent was measured spectrophotometrically, the content of formazan of myocardium was also measured at the end of reperfusion.

RESULTCompared to I/R group, Postcond had an significant increase in the mechanical function of the left ventricle, with LDH release reduced and the content of formazan increased. Spermine, the opener of mitochondrial calcium uniporter, deteriorated the mechanical function of left ventricle and decreased the formazan content, and increased LDH release. Ruthenium red, the inhibitor of mitochondrial calcium uniporter, increased the mechanical function of the left ventricle, decreased the LDH release, but the content of formazan was not increased.

CONCLUSIONThe inhibition of mitochondrial calcium uniporter is involved in the mechanisms of ischemic postconditioning.

Animals ; Calcium Channels ; metabolism ; physiology ; Disease Models, Animal ; Formazans ; analysis ; Heart ; physiopathology ; Ischemic Postconditioning ; L-Lactate Dehydrogenase ; analysis ; Male ; Myocardial Reperfusion Injury ; metabolism ; physiopathology ; prevention & control ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo determine the effects of organic amine diphenhydramine on the 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide dye (MTT) reduction assay.

METHODSThe primarily cultured cortical astrocytes were incubated with various concentrations of diphenhydramine for 24 h. To analyze the effects of diphenhydramine and other organic amines on the MTT assay, the data obtained from the MTT assay were compared with the results obtained from morphological observation and hoechst 33342 and propidium iodide (PI) nucleus double staining.

RESULTThe MTT assay showed that diphenhydramine (10(-4)mol/L), pyrilamine (10 (-4)mol/L) and zolantidine (10 (-5)mol/L) caused a significant increase in MTT reduction in astrocytes. However there was no proliferation, apoptosis or necrosis detected by hoechst and PI nucleus double staining. Light microscopy revealed that exocytosis of formazan granules was inhibited by diphenhydramine.

CONCLUSIONDiphenhydramine and other organic amines may enhance MTT reduction by suppression of MTT formazan exocytosis in astrocytes, which may affect the results of cell viability studies.

Animals ; Astrocytes ; drug effects ; metabolism ; physiology ; Cell Survival ; Cells, Cultured ; Diphenhydramine ; pharmacology ; Drug Interactions ; Formazans ; pharmacokinetics ; Rats ; Rats, Sprague-Dawley ; Tetrazolium Salts ; pharmacokinetics

PURPOSE: Avulsed tooth can be completely recovered, if sound periodontal ligament (PDL) of tooth is maintained. Although a lot of storage solutions have been explored for the better storage of avulsed tooth, there is a shortcoming that the preservation time is much short. On the other hand, there has been studies that (-)-epigallocatechin-3-gallate (EGCG), the most abundant polyphenol in green tea, which is related to the anti inflammatory, antioxygenic, and antibacterial effects, allows the successful preservations of tissues and cells. This study evaluated the effect of EGCG on avulsed-teeth preservation of Beagle dogs for a period of time. METHODS: The atraumatically extracted teeth of Beagle dogs were washed and preserved with 0/10/100 microM of EGCG at the time of immediate, period 1 (4 days in EGCG-contained media and additional 1 day in EGCG-free media), period 2 (8 days in EGCG-contained media and additional 2 days in EGCG-free media) and period 3 (12 days in EGCG-contained media and additional 2 days in EGCG-free media). Then, the cell viabilities of preserved teeth was calculated by dividing optical density (OD) of 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay with OD of eosin assay to eliminate the measurement errors caused by the different tissue volumes. RESULTS: From the results, the immediately analyzed group presented the highest cell viability, and the rate of living cells on teeth surface decreased dependent on the preservation period. However, the 100 microM of EGCG-treated group showed statistically significant positive cell activity than EGCG-free groups throughout preservation periods. CONCLUSIONS: Our findings showed that 100 microM EGCG could maintain PDL cell viability of extracted tooth. These results suggest that although EGCG could not be a perfect additive for tooth preservation, it is able to postpone the period of tooth storage. However, further in-depth studies are required for more plausible use of EGCG.
Animals ; Catechin ; Cell Survival ; Dogs ; Eosine Yellowish-(YS) ; Formazans ; Hand ; Organ Preservation ; Periodontal Ligament ; Tea ; Tetrazolium Salts ; Thiazoles ; Tooth ; Tooth Avulsion ; Tooth Replantation

PURPOSE: Avulsed tooth can be completely recovered, if sound periodontal ligament (PDL) of tooth is maintained. Although a lot of storage solutions have been explored for the better storage of avulsed tooth, there is a shortcoming that the preservation time is much short. On the other hand, there has been studies that (-)-epigallocatechin-3-gallate (EGCG), the most abundant polyphenol in green tea, which is related to the anti inflammatory, antioxygenic, and antibacterial effects, allows the successful preservations of tissues and cells. This study evaluated the effect of EGCG on avulsed-teeth preservation of Beagle dogs for a period of time. METHODS: The atraumatically extracted teeth of Beagle dogs were washed and preserved with 0/10/100 microM of EGCG at the time of immediate, period 1 (4 days in EGCG-contained media and additional 1 day in EGCG-free media), period 2 (8 days in EGCG-contained media and additional 2 days in EGCG-free media) and period 3 (12 days in EGCG-contained media and additional 2 days in EGCG-free media). Then, the cell viabilities of preserved teeth was calculated by dividing optical density (OD) of 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay with OD of eosin assay to eliminate the measurement errors caused by the different tissue volumes. RESULTS: From the results, the immediately analyzed group presented the highest cell viability, and the rate of living cells on teeth surface decreased dependent on the preservation period. However, the 100 microM of EGCG-treated group showed statistically significant positive cell activity than EGCG-free groups throughout preservation periods. CONCLUSIONS: Our findings showed that 100 microM EGCG could maintain PDL cell viability of extracted tooth. These results suggest that although EGCG could not be a perfect additive for tooth preservation, it is able to postpone the period of tooth storage. However, further in-depth studies are required for more plausible use of EGCG.
Animals ; Catechin ; Cell Survival ; Dogs ; Eosine Yellowish-(YS) ; Formazans ; Hand ; Organ Preservation ; Periodontal Ligament ; Tea ; Tetrazolium Salts ; Thiazoles ; Tooth ; Tooth Avulsion ; Tooth Replantation

The methanol extract from the leaves of Petasites japonicus Maxim (PJ) was studied for its (anti-)mutagenic effect with the SOS chromotest and reverse mutation assay. The (anti-)carcinogenic effects were evaluated by the cytotoxicity on human cancer line cells and by the function and the expression of gap junctions in rat liver epithelial cell. PJ extracts significantly decreased spontaneous beta-galactosidase activity and beta-galactosidase activity induced by a mutagen, ICR, in Salmonella (S.) typhimurium TA 1535/pSK 1002. All doses of the extract (0.08-100 mg/plate) decreased the reversion frequency induced by benzo (alpha)pyrene (BaP) in S. typhimurium TA 98. It decreased not only the spontaneous reversion frequency but also that induced by BaP in S. typhimurium TA 100. PJ extract showed greater cytotoxic effects on human stomach, colon and uterus cancer cells than on other cancer cell types and normal rat liver epithelial cells. Dye transfers though gap junctions were significantly increased by PJ extracts at concentrations greater than 200 microg/mL and the inhibition of dye transfer by 12-O-tetradecanoylphorobol-13-acetate (TPA) was obstructed in all concentrations of PJ. PJ significantly increased the numbers of gap junction protein connexin 43, and increased the protein expression decreased by TPA in a dose-dependent manner. Based on these findings, PJ is suggested to contain antimutagenic and anticarcionogenic compounds.
Animals ; Cell Line, Tumor ; Cell Survival/*drug effects ; Formazans/chemistry ; Gap Junctions/*metabolism ; Humans ; Mutagenicity Tests ; Petasites/*metabolism ; Plant Extracts/*pharmacology ; Plant Leaves/metabolism ; Rats ; Tetrazolium Salts/chemistry

OBJECTIVETo introduce a rapid colorimetric method for assessing the viability of osteosarcoma cells after chemotherapy.

METHODSColorimetric assay and automatic microplate scanning spectrophotometer were used for assaying the viability of osteosarcoma cells.

RESULTSClose correlation was found between the absorbance at 570 nm of the formazan products and the number of viable osteosarcoma cells.

CONCLUSIONAn effective, sensitive and convenient colorimetric assay has been established to assess the survival of osteosarcoma cells following chemotherapy.

Antineoplastic Agents ; therapeutic use ; Bone Neoplasms ; drug therapy ; pathology ; Cell Survival ; drug effects ; Colorimetry ; methods ; Formazans ; Humans ; Osteosarcoma ; drug therapy ; pathology ; Sensitivity and Specificity ; Tetrazolium Salts

A normal prion protein (PrPc) is converted to a proteaseresistant isoform by an apparent self-propagating activity in transmissible spongiform encephalopathy, a neurodegenerative disease. The cDNA encoding open reading frame (ORF) of the bovine prion protein gene (Prnp) was cloned from Korean cattle by PCR, and was transfected into Chinese hamster ovary (CHO-K1) cells using lipofectamine. The gene expression of the cloned cDNA was confirmed by RT-PCR and Western blotting with the monoclonal antibody, 6H4. Cellular changes in the transfected CHO-K1 cells were investigated using parameters such as MTT, lactate dehydrogenase (LDH), and superoxide dismutase (SOD) activities, as well as nitric oxide (NO) production, and an apoptosis assay. In the MTT and LDH assays, the bovine PrnP-transfectant showed a lower proliferation rate than the wild-type (p < 0.05). Production of NO, after LPS or ConA stimulation, was not detected in either transfectants or CHO-K1 cells. In SOD assay under ConA stimulation, the SOD activity of transfectants was 10 times higher than that of CHO-K1 cells at 6 h after treatment (p < 0.05). The genomic DNA of both the transfectants and control cells began to be fragmented at 6 h after treatment with cyclohexamide. Caspase-3 activity was reduced by transfection with the bovine Prnp (p < 0.05). Conclusively, the viability of transfectants expressing exogenous bovine Prnp was decreased while the capacities for cellular protection against antioxidative stress and apoptosis were increased.
Animals ; Apoptosis/physiology ; CHO Cells/cytology/enzymology/*physiology ; Caspase 3/metabolism ; Cattle ; Cell Growth Processes/physiology ; Cloning, Molecular ; Cricetinae ; Cricetulus ; Encephalopathy, Bovine Spongiform/genetics/*pathology ; Formazans ; Hydro-Lyases/metabolism ; Nitric Oxide/metabolism ; Prions/biosynthesis/genetics/*physiology ; Superoxide Dismutase/metabolism ; Tetrazolium Salts ; Transfection