Methyl-thiazoldiphenyl Tetrazolium Research Articles

Graphene-Functionalized Titanium Carbide Synthesis and Characterization and Its Cytotoxic Effect on Cancer Cell Lines.

Background Graphene is a versatile material with promising applications in various fields such as electronics, energy, biomedicine, and the environment due to its exceptional mechanical strength, thermal and electrical conductivity, transparency, and chemical stability. Graphene has been extensively used in biological and medical settings. MXene is a two-dimensional (2D) material that exhibits a strong affinity for water and electrical conductivity because of its surface terminations (oxygen {-O}, fluorine {-F}, and hydroxyl {-OH}) andtransition metal carbide or nitride. MXene has attracted significant attention recently for its wide range of applications and unique properties. This study focuses on the synthesis and characterization of graphene-functionalized MXene. Furthermore, we investigated its cytotoxic effects on cancer cell lines. The characterization of graphene-functionalized MXene is carried out using scanning electron microscopy (SEM), X-ray diffraction(XRD), and Fourier transform infraredspectroscopy (FTIR) assays. Materials and methods Graphene powder was finely ground in isopropyl alcohol and then sonicated for two hours to produce solution A. MXene was synthesized by reacting titanium aluminum carbide (Ti3AlC3) with hydrofluoric acid (HF). A mixture of Ti3AlC3 and HF was heated to 40°C with continuous stirring for 24 hours to form solution B. Subsequently, solutions A and B were combined and stirred for 30 minutes. The resulting mixture was transferred to a hydrothermal reactor and maintained at 180°C for 12 hours. After the completion of the reaction, the resulting material was cooled to room temperature and purified through washing with distilled water, ethanol, and acetone. The sample was then dried at 80°C for 12 hours. Results The X-ray diffraction (XRD) study confirms the formation of graphene-functionalized titanium carbide (Ti3C2). The sharp peaks indicate a highly crystalline nature. Graphene is a sheet-like structure with numerous gaps. Particles exhibit a multitude of voids and pores on their surfaces. Upon incorporation, graphene displays a small sheet-like structure. Graphene-functionalized titanium carbide confirms the presence of distinct layered or sheet-like structures stacked together. Following the addition of the material, some cancer cells are eradicated, and they exhibit increased biocompatibility, demonstrating anticancer activity. Conclusion Graphene-functionalized titanium carbide has been successfully synthesized and characterized, as evidenced by various analytical methods such as X-ray diffraction (XRD), scanning electron microscopy (SEM), and methyl-thiazoldiphenyl-tetrazolium (MTT) assays. The cytotoxic impact of the synthesized graphene-functionalized titanium carbide on cancer cell lines was examined. The findings reveal a notable cytotoxic effect, indicating its potential as an anticancer agent. Further research in collaboration with experts from diverse fields will be crucial to advance and translate this technology into practical applications for cancer patients. Future scope Graphene and titanium carbide are promising materials for cancer research, biomedical applications, and imaging. Nevertheless, additional research is required to comprehend their mechanisms, enhance their properties, assess their safety and efficacy, and conduct clinical trials.

The Cytotoxic Assessment of Antibacterial-Enhanced Mineral Trioxide Aggregate Compared to Commercially Available Bioceramic Cements by Using Methyl-Thiazoldiphenyl-Tetrazolium (MTT) Assay on Human Dental Pulp Stem Cells: An In Vitro Study.

Background and objective Preserving the vitality of the tooth is of prime significance during therapies such as direct pulp capping and pulpotomy that promote tertiary dentine formation and healing of pulp stumps. Procedures like apexogenesis and apexification also stimulate dentin and bone formation for root growth and closure. Conventional mineral trioxide aggregate (MTA) has good biocompatible and physical properties like longer setting time, presence of a cytotoxic component, i.e., tricalcium aluminate (TCA), moderate compressive strength, and moderate antimicrobial activity. Eliminating TCA and the addition of antibacterial components would improve the properties of the cement. In this study, we aimed to assess the cytotoxicity of MTA Angelus, Biodentine, and two antibacterial-enhanced MTAs by using methyl-thiazoldiphenyl-tetrazolium (MTT) assay. Materials and methods Human dental pulp was extirpated from extracted third molars, and human dental pulp stem cells(HDPSCs)were isolated and characterized by flow cytometry. HDPSCs were treated with MTA, Biodentine, or two antibacterial-enhanced MTAs depending on the study group. The control group constituted the untreated HDPSCs. The cell viability of HDPSCs was assessed using an MTT assay on days one, three, and seven. Results Varied levels of cytotoxicity were noticed atdifferent time periods assessed using the tested materials, which was statistically significant (p=0.01). At alltime periods assessed, the highest cell viability was noticed with Biodentine (88.7% on the first day, 80.4% on the third day, and 91.8% on the seventh day). Antibacterial-enhanced MTAs, either added with metronidazole or doxycycline, had more mean viable cells compared to conventional MTA on the third and seventh day (p=0.043 and 0.018 respectively). Conclusion Antibacterial-enhanced MTAsshowed reduced cytotoxic properties when compared to conventional MTA. Biodentine was associated with the highestcell viability at all time periods.

Anticancer effect of polyphenolic acid enriched fractions from Grewia bracteata Roth on tumor cells and their p53 gene independent ROS mediated apoptosis in colon cancer cells

It is the first report on leaves of Grewia bracteata Roth for its anticancer effect. In this study, three polarity-guided solvent extracts of Grewia bracteata leaves from n-hexane (GLH), ethyl acetate (GLE), and methanol (GLM) were screened for anticancer effects on HeLa, HCT-116, MCF-7, HCT-116 p53−/− and PC-3 cells via methyl thiazoldiphenyltetrazolium bromide (MTT) assay. Based on the results, GLM was fractionated, and the obtained fractions were tested on HCT-116 cells. Further, FT-IR, HPLC analysis, clonogenic assay, wound healing assay, DCFDA, and cell cycle experiments were conducted on HCT-116 cells. The extracts from methanol (GLM) and ethyl-acetate (GLE) demonstrated a more selective and promising inhibition on HCT-116 cells than others. Notably, GLM recorded superior inhibition on HCT-116 p53−/− than GLE. Amongst, the methanol column fraction (GMCF) showed prominent inhibition on HCT-116 (IC50:63.55 ± 0.61 μg/ml) and HCT-116 p53−/− (IC50: 84.51 ± 0.58 μg/ml) cells. Further, the test on normal cells (NKE) revealed minimal toxicity of GMCF. The phytochemical test, FT-IR, HPLC, and LC-HRMS analyses confirmed the high abundance of polyphenolic acid/polyphenols in GMCF. Further, the clonogenic and wound healing assays on HCT-116 cells were also performed. Later, the probable cell death mechanism was identified using DCFDA and cell cycle experiments. These experiments disclosed that GMCF induced HCT-116 cell death was probably due to reactive oxygen species (ROS) upregulation and cells cycle arrest at SubG0 phase. It inferred that the activity is most probably p53 independent, a tumor suppressor gene responsible for drug resistance in colon cancer.

Tinospora sinensis (Lour.) Merr alkaloid rich extract induces colon cancer cell death via ROS mediated, mTOR dependent apoptosis pathway: “an in-vitro study”

BackgroundColorectal cancer (CRC) is the second most mortality rate causing disease after lung cancer. Though there is a significant improvement in the treatment schedule offered to CRC. However, there is no notable decrease in terms of cases as well as death rate. Hence, there is an urgent need to discover novel cancer therapeutics to treat CRC. Since ancient times, the use of phytochemicals has drawn huge attention as chemo-preventive and chemotherapeutic agents. Earlier studies on Tinospora sinensis (TS) revealed the cytotoxic effect on human colorectal carcinoma (HCT-116) cells, yet the mechanism is to be uncovered. Therefore, the present study was designed to study the cell death mechanism of TS in HCT-116 cells.MethodDifferent extracts such as n-hexane, ethyl acetate, and ethanol extracts from the root part of TS were prepared using a cold maceration process. The extracts were screened against cancer cell lines by methyl thiazoldiphenyltetrazolium bromide (MTT) assay. From the result, the most active extract was subjected to gas chromatography-mass spectrometry (GC-MS) and Fourier-Transform infrared spectroscopy (FTIR) analyses to identify the major constituents. Finally, the mechanism of cytotoxicity to cancer cells for the most active extract was evaluated using various experiments such as cell cycle analysis, Annexin-V assay, and Western blot.ResultsThe results from the MTT assay indicated that the n-hexane extract of TS inhibits the growth of HCT-116 cells more effectively than other cancer cells like Henrietta Lacks cervical cancer cells (Hela), and Michigan cancer foundation-breast cancer (MCF-7). The GC-MS and FT-IR analyses revealed the presence of alkaloids in the n-hexane extract and were responsible for the apoptosis activity in HCT-cells via reactive oxygen species (ROS) generation, and phosphoinositide 3-kinase (PI3K)/ protein Kinase B (Akt)/ mammalian target of rapamycin (mTOR) down-regulation.ConclusionThis study concludes that this finding is unique of its kind, and for the first time. The anticancer effect of TS root is specific to colon cancer cells (HCT-116). This distinctive finding helps the researchers to investigate further, and to identify a novel source for anti-colon cancer drug candidates in near future.

Analysis of Pulp Tissue Viability and Cytotoxicity of Pulp Capping Agents.

The present research study assessed the cell viability and cytotoxic effect of mineral tri-oxide aggregate (MTA), Tetric N-Bond Universal bonding agent, Theracal PT (pulpotomy treatment), and platelet-rich fibrin (PRF) as pulp capping agents on human dental pulp stem cells (hDPSCs). The cells were isolated from the pulp tissue of an extracted healthy permanent third molar. After four passages in Dulbecco’s Modified Eagle’s Medium, the primary cells were employed for the investigation. The test materials and untreated cells (negative control) were subjected to an Methylthiazol-diphenyl-tetrazolium (MTT) cytotoxicity assay and assessed at 24-, 48-, and 72-h intervals. The Wilcoxon matched-paired t-test and Kruskal−Wallis analysis of variance (ANOVA) test were applied (p < 0.05). PRF imparted the highest cell viability at 48 h (p < 0.001), followed by MTA, Theracal PT, and Tetric N-Bond. Similarly, PRF had the highest potential to enhance cell proliferation and differentiation (p < 0.001), followed by Theracal PT, MTA, and the bonding agent at the end of 24 h and 72 h, respectively. Finally, PRF sustained the viability of human primary dental pulp stem cells more effectively than Theracal PT and MTA; however, the application of a Tetric N-Bond as a pulp capping agent was ineffective.

Pendulone induces apoptosis via the ROS-mediated ER-stress pathway in human non-small cell lung cancer cells

PurposePendulone, an isoflavone compound, is known to act against human cancer cells. However, its role in human non-small cell lung cancer (NSCLC) and the exact molecular mechanisms of action have never been reported. MethodsWe investigated the effects of pendulone on cell proliferation and apoptosis in human NSCLC H1299 cells. Cell viability was examined using the methyl-thiazol-diphenyl-tetrazolium (MTT) assay. Flow cytometry was employed to evaluate apoptotic indices such as the cell cycle, mitochondrial membrane potential, cytochrome c release, caspase activity, and death receptor expression. The expression of proteins related to the cell cycle and apoptosis were analyzed by Western blot analysis. ResultsPendulone significantly decreased H1299 cell viability by inducing endoplasmic reticulum (ER) stress through the accumulation of reactive oxygen species (ROS). Pendulone induced the expression of ER stress-associated proteins, such as ATF4 and CHOP, which promoted the expression of death receptors. Activation of caspase 8 induced extrinsic pathway apoptosis. Pendulone also caused the loss of mitochondrial membrane potential, inhibited the anti-apoptotic proteins BCL-2 and activated the pro-apoptotic protein BAX, which promoted the release of cytochrome c to activate caspase 9. Antioxidant N-acetylcysteine (NAC), with its ROS-suppressive property, reversed pendulone-induced ER stress and cell apoptosis. ConclusionOur findings provide evidence that pendulone induces apoptosis by inducing ER stress through ROS accumulation and mitochondrial dysfunction in NSCLC cell lines.

Effect of two commercial acellular dermal scaffolds on biological behavior of human gingival fibroblasts.

Background Periodontal regeneration is an essential goal of periodontal therapy. Acellular dermal matrix (ADM) has been recommended as an alternative to autogenous grafts. However, since it is devoid of cells and vasculature, there are concerns regarding the biological behavior of cells on ADM. This study aimed to assess the effects of two commonly used ADMs on biological behavior, i.e., attachment and proliferation, of human gingival fibroblasts (HGFs). Methods This in vitro, experimental study was conducted on explanted and cultured HGFs. ADM types 1 and 2 (n=26; measuring 10×15 mm) were rinsed with saline solution, adapted to the bottom of 52 wells, exposed to HGFs with a cell density of 16,000 cells/mL, and incubated at 37°C for 12, 24, and 84 hours and seven days. Cell attachment was assessed 12 hours after incubation using 4›,6-diamidino-2-phenylindole (DAPI) and methyl-thiazol-diphenyl-tetrazolium (MTT) assay under a fluorescence microscope. Cell viability was assessed at 24 and 84 hours and one week using the MTT assay. Cells were then platinum-coated, and their morphology was evaluated under a scanning electron microscope (SEM). Data were analyzed using ANOVA. Results HGFs were evaluated in 60 samples in three groups (n=20). Cell attachment was the same in the three groups, as shown by the MTT assay and DAPI test (P=0.6). Cell viability at one week was 3.73±0.02, 2.88±0.29, and 2.13±0.24 in the control, ADM 1, and ADM 2 groups, respectively. The difference was statistically significant (P=0.01). Conclusion Both scaffolds were the same in terms of attachment of HGFs. However, ADM 1 was superior to ADM2 in terms of cell viability and morphology at one week. It was concluded that the quality of acellular dermal scaffolds could significantly influence cellular behaviors and tissue maturation.

In vitro anticancer activity of Eclipta alba whole plant extract on colon cancer cell HCT-116

BackgroundsColon cancer is the third most deadly and one of the most diagnosed diseases in the world. Although routine screening and early detection during last decades has improved the survival, colon cancer still claims hundreds of thousands lives each year worldwide. Surgery and chemotherapy is mainstay of current treatment, nevertheless toxicity associated with this treatment underscores the urgency of demand of a better therapeutics. Close to 50% of current chemotherapeutic drugs are direct or indirect descendants compounds isolated from medicinal plants, which indicate plants are great potential sources of novel therapeutics. In our literature review we found Eclipta alba to posses many pharmacological activities, including those with anticancer potentials. However, no study on anticancer activity of this kind has been reported.MethodsPhytochemicals were extracted by maceration method from shade dried whole plant of Eclipta alba using methanol as a solvent. The anticancer effect of extract was investigated on various cancer cell lines like human colorectal carcinoma (HCT-116), human prostate cancer (PC-3), Michigan cancer foundation-breast cancer (MCF-7) and renal cell carcinoma (RCC-45). We have also studied the effects on normal human embryonic lung fibroblast cell (WI-38) using MTT (methyl thiazoldiphenyltetrazolium bromide) assay, clonogenic (colony formation) and migration assay. Finally obtained results were analyzed using ANNOVA and Dunnett’s test.ResultsResults obtained from MTT assay revealed that the methanolic extract of Eclipta alba carried significant (p < 0.005) specificity against HCT-116 cells as compared to the other cancer cells. This extract also showed minimal or nontoxicity to WI-38 cells. Migration as well as clonogenic assays also confirmed the anticancer potential of the extract against HCT-116 cells.ConclusionThis is a unique finding of its kind because the specific anticancer effect with minimal toxicity on normal cells has not been reported on Eclipta alba extract. Finally this finding opens up a great possibility to develop a novel antitumor drug candidate against deadly colon cancer in the future.

Cytotoxicity and Mineralization Potential of Four Calcium Silicate-Based Cements on Human Gingiva-Derived Stem Cells

The aim of this study was to evaluate the cytotoxicity and mineralization potential of four calcium silicate-based cements on human gingiva-derived stem cells (GDSCs). The materials evaluated in the present study were ProRoot MTA (Dentsply Tulsa Dental Specialties), Biodentine (Septodont), Endocem Zr (Maruchi), and RetroMTA (BioMTA). Experimental disks of 6 mm in diameter and 3 mm in height were produced and placed in a 100% humidified atmosphere for 48 h to set. We evaluated the cytotoxic effects of the cements using methyl-thiazoldiphenyl-tetrazolium (MTT) and live/dead staining assays. We used a scratch wound healing assay to evaluate cell migratory ability. Mineralization potential was determined with an Alizarin red S (ARS) staining assay. In the MTT assay, no significant differences were found among the ProRoot MTA, Biodentine, and control groups during the test period (p &gt; 0.05). The Endocem Zr and RetroMTA groups showed relatively lower cell viability than the control group at day 7 (p &lt; 0.05). In the wound healing assay, no significant differences were found among the ProRoot MTA, Biodentine, Endocem Zr, and control groups during the test period (p &gt; 0.05). The RetroMTA group had slower cell migration compared to the control group at days 3 and 4 (p &lt; 0.05). In the ARS assay, the ProRoot MTA, Biodentine, and RetroMTA groups exhibited a significant increase in the formation of mineralized nodules compared to the Endocem Zr and control groups on day 21 (p &lt; 0.05). In conclusion, the four calcium silicate-based cements evaluated in the present study exhibited good biological properties on GDSCs. ProRoot MTA, Biodentine, and RetroMTA showed higher mineralization potential than the Endocem Zr and control groups.

Biocompatibility and Mineralization Activity of Three Calcium Silicate-Based Root Canal Sealers Compared to Conventional Resin-Based Sealer in Human Dental Pulp Stem Cells.

The purpose of this study was to compare the cytotoxic effects and mineralization activity of three calcium silicate-based root canal sealers to those of a conventional resin-based sealer. Experiments were performed using human dental pulp stem cells grown in a monolayer culture. The root canal sealers tested in this study were EndoSequence BC Sealer (Brasseler), BioRoot RCS (Septodont), Endoseal MTA (Maruchi), and AH Plus (Dentsply DeTrey). Experimental disks 6 mm in diameter and 3 mm in height were made and stored in a 100% humidity chamber at 37 °C for 72 h to achieve setting. The cytotoxicity of various root canal sealers was evaluated using a methyl-thiazoldiphenyl-tetrazolium (MTT) assay. To evaluate cell migration ability, a scratch wound healing method was used, and images of the scratch area were taken using a phase-contrast microscope. Cell morphology was evaluated by a scanning electron microscope after direct exposure for 72 h to each sealer disk. In the cell viability assay, there were no significant differences between the EndoSequence BC, BioRoot RCS, Endoseal MTA, and control groups in any experimental period (p > 0.05). In the cell migration assay, there were no significant differences between the EndoSequence BC, Endoseal MTA, and control groups in any experimental period (p > 0.05). BioRoot RCS exhibited slower cell migration relative to EndoSequence BC and Endoseal MTA for up to 72 h (p < 0.05). Conversely, it showed a similar wound healing percentage at 96 h (p > 0.05). In an evaluation of cell morphology, cells in direct contact with EndoSequence BC, BioRoot RCS, and Endoseal MTA disks showed superior spreading compared to those in contact with the AH Plus disk. In an Alizarin red staining assay, EndoSequence BC, BioRoot RCS, and Endoseal MTA showed a significant increase in mineralized nodule formation compared to the AH Plus group (p < 0.05). In conclusion, all calcium silicate-based root canal sealers tested in this study showed good biological properties and mineralization activity compared to conventional resin-based sealer.

Impact of Nanostructured Lipid Carriers as an Artificial Tear Film in a Rabbit Evaporative Dry Eye Model.

To characterize formulations of nanostructured lipid carriers (NLCs) as an artificial tear film and evaluate their efficacy in protecting the ocular surface epithelial cells from desiccating stress in vivo. The physicochemical properties of NLCs, produced with components similar to the tear film such as phosphatidylcholine and squalene, were determined. In vitro cytotoxicity of NLCs was evaluated by a short-time exposure test in porcine corneal epithelial cells using a methyl thiazol diphenyl-tetrazolium bromide assay. The residence time of NLCs in rabbit eyes and the efficacy of NLCs eye drops in protecting the rabbit corneal epithelium from desiccating stress were assessed. Nanosized NLCs with a mean size of ∼39 ± 5 nm and a zeta potential of -30 mV could be produced and formulated into eye drop with a pH of 6.90 ± 0.01, osmolarity of 273 ± 1 mOsm/L, and surface tension of 39 ± 1 mN/m (for air interface). Eye drop formulations of NLCs were nontoxic to porcine corneal epithelial cells. NLCs drops showed higher ocular surface retention and formed a stable corneal film compared with a saline solution. Moreover, NLCs eye drops showed greater efficacy in protecting the corneal surface against desiccating stress compared with a polymer-based commercial artificial tear. NLCs eye drops are biocompatible in rabbit eyes and show potential as a tear replacement vehicle for the treatment of dry eye disease.

In vitro evaluation of cytotoxicity of Emblica officinalis (amla) on cultured human primary dental pulp fibroblasts

The dental pulp tissue is capable of healing after surgical amputation of infected/inflamed tissue during vital pulp therapy, when in contact with a suitable medicament. Emblica officinalis (amla), a traditional medicine, is one such medicament which has never been evaluated for its healing potential in pulp therapy. The aim of the study was to evaluate the cytotoxicity of E. officinalis (amla) against human primary dental pulp fibroblasts. This was in vitro study. Human dental pulp fibroblasts were obtained from dental pulp tissue of extracted over-retained primary incisors. The primary cells were cultured using the Dulbecco's Modified Eagle's Medium and used for the study after the fourth passage. The test medicament was E. officinalis with mineral trioxide aggregate (MTA) (100%) and untreated cells as positive and negative controls, respectively. Methyl-thiazol-diphenyl-tetrazolium (MTT) cytotoxicity assay was performed, and the cell survival was observed and analyzed at intervals of 24, 48, and 72 h. Cell survival within groups was compared with Wilcoxon matched-paired t-test and in between groups at each point interval was analyzed with the Kruskal-Wallis ANOVA test. The level of significance was set at 0.05. Within the groups, across the time periods of evaluation, there was a decline in cell survival in both the groups but was statistically significant in the MTA group. On interval-wise comparison, the decline in cell survival was statistically significant between the three groups at 72 h (P = 0.001). E. officinalis preserved the vitality of the human primary dental pulp fibroblasts and has the potential to be developed into vital pulp therapy medicament.

A Soft Coral-Derived Compound, 11-Dehydrosinulariolide, Induces G2/M Cell Cycle Arrest and Apoptosis in Small Cell Lung Cancer.

11-Dehydrosinulariolide, an active compound that is isolated from the cultured soft coral Sinularia flexibilis, has been suggested to show anti-tumor biological characteristics according to previous studies. However, its potential effect on small cell lung cancer (SCLC) remains unknown. The present study investigates the underlying mechanism for the treatment of SCLC in vitro and in vivo. Cell viability was examined using the methyl-thiazol-diphenyl-tetrazolium (MTT) assay. Flow cytometry was applied to evaluate cell cycle distribution and apoptosis. The expression of proteins related to the cell cycle and apoptosis was analyzed by Western blot analysis. Additionally, an in vivo study was performed to determine the anti-SCLC effect on an H1688 subcutaneous tumor in a BALB/c nude mouse model. 11-Dehydrosinulariolide inhibited cell growth, triggered G2/M arrest and induced H1688 cell apoptosis in a dose- and time-dependent manner. Additionally, 11-dehydrosinulariolide caused the accumulation of p53 and Bax, accompanied by the activation of DNA damage-inducing kinases, including ataxia-telangiectasia mutated (ATM) and checkpoint kinase 2 (CHK2). Moreover, 11-dehydrosinulariolide increased the activity of caspase-3 and -7, suggesting that caspases are involved in 11-dehydrosinulariolide-induced apoptosis. 11-Dehydrosinulariolide also increased the level of tumor suppressor phosphatase and tensin homolog (PTEN) and inhibited the expression of phosphorylated Akt. In the in vivo study, the intraperitoneal injection of 11-dehydrosinulariolide at a dosage of 10 mg/kg significantly inhibited tumor growth compared with the control treatment. Together, the data indicate that 11-dehydrosinulariolide induces G (2)/M cell cycle arrest and apoptosis through various cellular processes, including the upregulation of p53 and Bax, activation of ATM and Chk2, activation of caspase-3 and -7, and accumulation of PTEN, leading to inhibition of the Akt pathway. These findings suggest that 11-dehydrosinulariolide might serve as a promising chemotherapy drug in the treatment of SCLC.

Levistilide A inhibits angiogenesis in liver fibrosis via vascular endothelial growth factor signaling pathway.

Levistilide A (C24H28O4, molecular weight = 380.48) derived from Angelica sinensis (Danggui) has been reported to inhibit hepatic stellate cell proliferation. This study investigated the effects of levistilide A on liver fibrosis relating to angiogenesis, particularly on the characteristic change in liver sinusoidal endothelial cells. LX-2 cells were activated by TGF-β1, and the human hepatic sinusoidal endothelial cells (HHSECs) were induced by endothelial cell growth supplement. Cell viability was detected using a methylthiazoldiphenyl-tetrazolium bromide assay; F-actin was visualized through the fluorescence probe method; cell proliferation was examined using the EdU kit; antiangiogenesis activity was assessed using the tube formation assay and transgenic zebrafish model. To verify the results invivo, rats were subcutaneously injected with CCl4 twice a week for six weeks to duplicate the liver fibrosis model and then treated with 10 mL/kg of normal saline, 4 mg/kg of sorafenib, and 3 and 6 mg/kg of levistilide A for three weeks from the fourth week. Collagen deposition was detected through Sirius Red staining; liver microvasculature was examined through vWF labeling and X-ray 2D imaging; sinusoidal fenestrations were observed through scanning electron microscopy; collagen I, α-SMA, CD31, vascular endothelial growth factor (VEGF), and VEGF-R2 were detected through Western blotting. Our results indicated that levistilide A attenuated LX-2 cell activation and HHSEC proliferation. The ability of HHSECs to form tubelike structures in Matrigel was inhibited, and the number of functional vessels in transgenic zebrafish decreased. In invivo experiments, levistilide A reduced collagen deposition and the number of new microvessels; ameliorated sinusoid capillarization; and downregulated the expression of CD31, VEGF, and VEGF-R2. These findings suggest that levistilide A can inhibit liver fibrosis through antiangiogenesis by alleviating sinusoid capillarization via the VEGF signaling pathway. Impact statement Levistilide A has been reported to inhibit hepatic stellate cell (HSC) proliferation. In this study, we further investigated the mechanisms of levistilide A on liver fibrosis relating to angiogenesis, particularly on the characteristic change in liver sinusoidal endothelial cells. The cell models of HSC and liver sinusoidal endothelial cell and CCl4 induced liver fibrosis model were used. These results suggest that levistilide A can inhibit liver fibrosis through antiangiogenesis by alleviating sinusoid capillarization via the vascular endothelial growth factor signaling pathway. The effect of levistilide A on liver fibrosis was confirmed, and its detailed mechanism was also discussed. These findings suggest that levistilide A may be a great potential drug for treating liver fibrosis through antiangiogenesis, and this effect will be verified in other fibrotic animal model studies or by clinical trials.

White mineral trioxide aggregate mixed with calcium chloride dihydrate: chemical analysis and biological properties.

ObjectivesThis study aimed to evaluate the chemical and biological properties of fast-set white mineral trioxide aggregate (FS WMTA), which was WMTA combined with calcium chloride dihydrate (CaCl2·2H2O), compared to that of WMTA.Materials and MethodsSurface morphology, elemental, and phase analysis were examined using scanning electron microscope (SEM), energy dispersive X-ray microanalysis (EDX), and X-ray diffraction (XRD), respectively. The cytotoxicity and cell attachment properties were evaluated on human periodontal ligament fibroblasts (HPLFs) using methyl-thiazol-diphenyltetrazolium (MTT) assay and under SEM after 24 and 72 hours, respectively.ResultsResults showed that the addition of CaCl2·2H2O to WMTA affected the surface morphology and chemical composition. Although FS WMTA exhibited a non-cytotoxic profile, the cell viability values of this combination were lesser than WMTA, and the difference was significant in 7 out of 10 concentrations at the 2 time intervals (p < 0.05). HPLFs adhered over the surface of WMTA and at the interface, after 24 hours of incubation. After 72 hours, there were increased numbers of HPLFs with prominent cytoplasmic processes. Similar findings were observed with FS WMTA, but the cells were not as confluent as with WMTA.ConclusionsThe addition of CaCl2·2H2O to WMTA affected its chemical properties. The favorable biological profile of FS WMTA towards HPLFs may have a potential impact on its clinical application for repair of perforation defects.

Cytocompatibility of calcium silicate-based sealers in a three-dimensional cell culture model.

The aim of the present study was to evaluate cytotoxic effects and cytokine production of calcium silicate-based sealers (EndoSeal, EndoSequence BC Sealer, and MTA Fillapex) using an in vitro root canal filling model and three-dimensional (3D) cell culture. AH Plus as a reference was compared to contemporary calcium silicate cements regarding cell viability and cytokine production. Root canals of 30 human maxillary incisors were prepared using a single-file reciprocating technique. The samples were randomly distributed and canals filled with either AH Plus, EndoSeal, EndoSequence BC Sealer, and MTA Fillapex (n=6). In the negative control group, the root canal remained unfilled. Sealers were placed into the canals along with a gutta-percha cone placed to working length. Balb/c 3T3 fibroblasts, cultured in a type I collagen 3D scaffold, were exposed to filling material and the respective root apex for 24h. Cytocompatibility of the materials was evaluated using the methyl-thiazoldiphenyl-tetrazolium (MTT) assay. The production of IL-1β, IL-6, and IL-8 was analyzed using enzyme-linked immunosorbent assay (ELISA). One-way analysis of variance was performed, and when the F-ratios were significant, data were compared by Duncan's multiple-range test. The alpha-type error was set at 0.05. EndoSeal, Endosequence BC Sealer and AH Plus showed cell viability that was similar to the negative control group (P>0.05), while MTA Fillapex sealer was cytotoxic (P<0.05). Varying production of IL-1β, IL-6, and IL-8 was detected in all samples. In an in vitro root canal filling model with 3D cell culture, AH Plus, EndoSeal, and EndoSequence BC Sealer were cytocompatible. These results may suggest that AH Plus, EndoSeal and EndoSequence BC Sealer may achieve better biological response when compared to MTA Fillapex.

Cytocompatibility of Biodentine using a three-dimensional cell culture model.

To evaluate the cytotoxic effects of Biodentine, using a three-dimensional (3D) cell culture associated with an insitu root-end filling experimental model. White mineral trioxide aggregate (MTA) and zinc oxide cement were used as reference for comparison. IL-1α and TNF-α cytokine production were also evaluated. The root canals of 24 human maxillary incisor teeth were prepared using a single-file reciprocating technique. After root filling, a 3-mm root-end resection was performed and 3mm of gutta-percha was removed from the canal. The teeth were randomly distributed to receive one of the following root-end filling materials: Biodentine, white MTA or zinc oxide cement (positive control group). In the negative control group, the root canal was not retro-filled. The cytocompatibility of the materials was evaluated using the methyl-thiazol-diphenyl-tetrazolium (MTT) assay in an insitu root-end filling experimental model. Balb/c 3T3 fibroblasts, cultured in rat tail collagen type I 3D scaffold, were exposed to the root apex for 24h, and cell viability was measured by means of reduction MTT salt. IL-1α and TNF-α production were analysed using enzyme-linked immunosorbent assay. One-way analysis of variance was performed and, when the F-ratios were significant, data were compared by Duncan's multiple-range test. The alpha-type error was set at 0.05. Biodentine and MTA groups had similar cell activity to the negative control group (P>0.05), indicating low cytotoxicity for both materials. The stronger cytotoxicity effect was identified on the zinc oxide cement (P<0.05). Zinc oxide cement caused a significant up-regulation in IL-1α and TNF-α (P<0.05). No significant differences amongst MTA, Biodentine and the negative control group were observed for TNF-α (P>0.05); however, both MTA and Biodentine were associated with overproduction of IL-1α when compared to the control group (P<0.05). Biodentine and MTA had similar cytocompatibility in a 3D cell culture model associated with an insitu root-end filling model. The methodology could be used as an alternative to assess the cytocompatibility of endodontic cements because it is more closely related to the invivo situation.

Antisense oligodeoxynucleotide targeting HER2 mRNA sensitized docetaxel in breast cancer treatment

Context: Human epidermal growth factor receptor 2 (HER2) is one of the oncogenes closely associated with the development and prognosis of breast carcinoma. Down-regulation of HER2 mRNA by antisense oligodeoxynucleotide (ASO) HER2 has been suggested to be a feasible treatment for patients with breast carcinoma.Objective: The antitumor effects of ASO HA6722 were investigated in vitro and in vivo.Materials and methods: In this study, SK-BR-3, a HER2-overexpressing breast carcinoma cell line, was used as the model for in vitro experiments. Inhibitory effects of the ASO HA6722 were detected by methyl-thiazoldiphenyl tetrazolium (MTT) assay. Meanwhile, HER2 mRNA levels were monitored by reverse transcription polymerase chain reaction (RT-PCR). The in vivo antitumor effects were evaluated in nude mice xenograft model.Results: Our results showed that HA6722 alone could inhibit the growth of SK-BR-3 cells in a dose-dependent manner with the IC50 value of 41.8 ± 8.1 nM. In addition, the antitumor effect of docetaxel (TXT) could be sensitized by low dose of HA6722 both in vitro and in vivo, suggesting that ASO HA6722 could inhibit the growth of breast cancer cells and enhance the cytotoxic effects of TXT.Discussion and conclusion: The combination treatment of TXT and HA6722 could be a more effective approach for breast cancer treatment. The future study should focus on the antitumor effect in other models.

Meglumine Eicosapentaenoic acid (MeEPA) a new soluble omega-3 fatty acid formulation: In vitro bladder cancer cytotoxicity tests in combination with epirubicin and mitomycin

To determine if Meglumine-Eicosapentaenoic Acid (MeEPA) acts synergistically with epirubicin and mitomycin to enhance cytotoxicity towards bladder cancer cell lines in vitro. Bladder cancer cells were exposed to MeEPA in combination with epirubicin or mitomycin. Residual viable cell biomass was estimated with the methyl-thiazoldiphenyl tetrazolium (MTT) assay following drug exposure. Drug interaction was analysed using median effect analysis to determine levels of synergism. Most combinations of MeEPA with both epirubicin and mitomycin showed a high-level of synergism. At high doses, drug precipitation adversely affected MTT assay analysis suggesting antagonism of action. However, the predominant pattern was of synergism for most dose combinations tested. Bladder cancer treated by endoscopic resection alone is subject to high recurrence rates. Post-operative intravesical instillation of epirubicin and mitomycin can halve recurrence rates, but there is no evidence that disease progression to invasive bladder cancer is altered. Thus, optimisation of current treatment strategies is required. The anti-tumour activity of fatty acids is well established and MeEPA is a new, soluble formulation with the potential to enhance intravesical drug efficacy.

Preclinical evaluation of antisense bcl-2 as a chemosensitizer for patients with gastric carcinoma.

Because bcl-2 is a critical factor for anticancer drug-induced apoptosis, the authors conducted a preclinical evaluation of antisense (AS) bcl-2 as an enhancer of the chemotherapeutic effect in the treatment of patietns with gastric carcinoma. AS bcl-2 was used with 18-mer phosphorothiated oligonucleotides in the MKN-45 gastric carcinoma cell line. Drug sensitivity in vitro was evaluated using the methyl-thiazoldiphenyl tetrazolium assay, and antitumor effects in vivo were evaluated using the nude mouse xenograft. Apoptosis was determined with the terminal deoxyuridine triphosphate nick-end labeling assay. AS bcl-2 in vitro was treated with lipofectin, whereas it was administered intraperitoneally for 6 consecutive days twice every 2 weeks in vivo. Anticancer drugs were administered intraperitoneally four times per week. bcl-2 was down-regulated to 60% of its initial value after treatment with 1.0 muM AS bcl-2 compared with the controls of random and mismatched oligonucleotides. Drug sensitivity to doxorubicin, cisplatin, and paclitaxel (TXL) was increased 3-4-fold when used in combination with AS bcl-2, which was determined with 50% inhibitory concentration values, compared with the control group. Increased drug sensitivity was associated with apoptosis, which increased in Bax and poly-adenosine diphosphate (ADP-ribose) polymerase and decreased in phosphorylated Akt (pAkt). The antitumor effect of cisplatin and TXL in vivo was enhanced significantly in combination with AS bcl-2. Down-regulation of bcl-2 was observed on Day 4 after the treatment with AS bcl-2. Combination treatment with AS bcl-2 and anticancer drugs, including cisplatin and TXL, may be a new strategy for enhancing chemotherapeutic effects in the treatment of gastric carcinoma.