Methyl Thiazol Tetrazolium Assay Research Articles

The Influence of Streptococcus mutans Biofilm Formation in a Polymicrobial Environment (Streptococcus gordonii & Porphyromonas gingivalis).

Biofilms play a vital role in the occurrence or worsening of an infectious disease. Streptococcus mutans is a bacterium with the ability to form biofilms that plays a key role in the development of infectious diseases such as dental caries. The formation of biofilms in S. mutans is mediated by quorum sensing. Inhibiting quorum sensing can be considered as one of the approaches to prevent caries. This study aims to investigate the ability of Streptococcus gordonii and Porphyromonas gingivalis bacteria to inhibit the formation of S. mutans biofilm. This research was conducted to analyze bacterial biofilm formation and metabolism. The bacteria used are S. mutans (serotype C), S. gordonii (ATCC 5165), and P. gingivalis (ATCC 33277). Biofilm formation was analyzed using the crystal violet assay. Bacterial metabolism was analyzed using the methylthiazol tetrazolium (MTT) assay. The results of the crystal violet assay indicate a decrease in biofilm formation in S. mutans when in the presence of S. gordonii and S. mutans in the presence of P. gingivalis. The results of the MTT assay show no significant change in the bacterial metabolism of S. mutans in the presence of S. gordonii and S. mutans in the presence of P. gingivalis. However, S. mutans with the presence of S. gordonii and P. gingivalis show an increase in biofilm formation and bacterial metabolism. S. gordonii and P. gingivalis are each capable of inhibiting the formation of S. mutans biofilm in a polymicrobial environment.

Characteristics and biological responses of selective laser melted Ti6Al4V modified by micro-arc oxidation

Background/purposeAdditive manufacturing (AM) technology, such as selective laser melting (SLM), has been used to fabricate medical devices of Ti-6wt.% Al-4wt.%V (Ti6Al4V) alloys in dentistry. Strontium (Sr) has been shown to have the potential to treat osteoporosis. The aim of this study was to investigate the physicochemical and biological properties of strontium-containing coatings on selective laser melted Ti6Al4V (SLM-Ti6Al4V) substrate. Materials and methodsThe disk of Ti6Al4V was prepared by SLM method. The strontium-containing coatings were prepared by micro-arc oxidation (MAO) in aqueous electrolytes. The surface topography, chemical composition, and phase of strontium-containing MAO (SrMAO) coatings were performed by scanning electron microscope (SEM), energy dispersive X-ray spectrometer (EDS), and thin film X-ray diffraction (TF-XRD), respectively. The apatite-forming ability of the MAO coatings was conducted in simulating body fluid (SBF), and the cell proliferation was determined by methylthiazoletetrazolium (MTT) assay. ResultsThe microstructure of SLM-Ti6Al4V displays acicular α-phase organization. The TF-XRD results indicated that the phase of SrMAO coating was anatase, rutile, and titanium. The calcium, phosphorus, and strontium were detected in the coatings by EDS. Using the SEM, the surface morphology of SrMAO coatings exhibited a uniform 3D porous structure. The SrMAO coatings could induce a bone-like apatite layer after immersion in SBF, and presented significantly higher cell proliferation than untreated specimens in in-vitro experiments. ConclusionAll findings in this study indicate that SrMAO coatings formed on SLM-Ti6Al4V surfaces exhibit a benefit on biological responses and thereby are suitable for biomedical applications.

Examining the Effects of Asiaticoside on Dental Pulp Stem Cell Viability and Proliferation: A Promising Approach to Root Canal Treatment.

This study aims to evaluate the impact of asiaticoside (AC) on the viability and proliferation of dental pulp stem cells (DPSCs), considering the known negative effects of routinely used intracanal medicaments. This evaluation will be compared with the outcomes from using traditional intracanal medicaments, specifically triple antibiotic paste (TAP) and calcium hydroxide [Ca(OH)2]. The DPSCs were obtained from the third molars of an adult donor. The application of flow cytometry was employed to do a phenotypic analysis on DPSCs using CD90, CD73, CD105, CD34, CD14, and CD45 antibodies. The methylthiazol tetrazolium (MTT) assay was employed to assess cellular viability. The cells were treated with different concentrations of TAP and Ca(OH)2 (5, 2.5, 1, 0.5, and 0.25 mg/mL), along with AC (100, 50, 25, 12.5, and 6.25 µM). A cell proliferation rate was performed at 3, 5, and 7 days. The characterization of DPSCs was conducted by flow cytometry analysis, which verified the presence of mesenchymal cell surface antigen molecules (CD105, CD73, and CD90) and demonstrated the absence of hematopoietic markers (CD34, CD45, and CD14). Cells treated with concentrations over 0.5 mg/mL of TAP and Ca(OH)2 showed a notable reduction in cell viability in comparison to the untreated cells (p < 0.05). Additionally, the cells treated with different concentrations of AC 12.5, 6.25, 25, and 50 µM did not differ significantly from the untreated cells (p > 0.05). Nevertheless, cells treated with concentrations of 100 µM showed a significant reduction in viability compared to the untreated cells (p < 0.05). After a period of 7 days, it was noted that cells exposed to three different concentrations of AC (50, 25, and 12.5 µM) had a notable rise in cell density in comparison to TAP and Ca(OH)2 (p < 0.05). Furthermore, cells that were exposed to a concentration of 12.5 µM exhibited the highest cell density. The cellular viability of the AC-treated cells was superior to that of the TAP and Ca(OH)2-treated cells. Moreover, the AC with a concentration of 12.5 µM had the highest degree of proliferation. This study underscores the importance of evaluating alternative root canal medicaments and their effects on DPSCs' growth and vitality. The findings on AC, particularly its influence on the survival and proliferation of DPSCs, offer valuable insights for its probable use as an intracanal medication. This research contributes to the ongoing efforts to identify safer and more effective intracanal treatments, which are crucial for enhancing patient outcomes in endodontic procedures. How to cite this article: Alazemi MJ, Badawi MF, Elbeltagy MG, et al. Examining the Effects of Asiaticoside on Dental Pulp Stem Cell Viability and Proliferation: A Promising Approach to Root Canal Treatment. J Contemp Dent Pract 2024;25(2):118-127.

Effect of Boswellic Acid on Viability of Dental Pulp Stem Cells Compared to the Commonly Used Intracanal Medications: An In Vitro Study.

This study was aimed at evaluating the effect of acetyl-11-keto-β-boswellic acid (AKBA) on dental pulp stem cells (DPSCs) viability and proliferation to be used as a potential root canal medicament. Dental pulp stem cells were isolated from human third molars. The phenotypic characterization of DPSCs was verified by flow cytometry analysis. The viability assay was performed using the methyl-thiazoltetrazolium (MTT) assay. Cells were treated with different concentration of triple antibiotic paste (TAP) and calcium hydroxide Ca(OH2) (5, 2.5, 1, 0.5, and 0.25 mg/mL), AKBA (10, 5, 1, 0.1, and 0.01 µM). All experiments were done in separate triplicate experiments. Results: Dental pulp stem cells were characterized by flow cytometry. Cells treated with Ca(OH)2 (1, 2.5, and 5 mg/mL) showed significantly reduced viability compared with the control cells (p < 0.05). Dental pulp stem cells treated with 1, 2.5, and 5 mg/mL TAP resulted in a significant decrease in viability (p < 0.05). Cells treated with AKBA in concentrations (1, 0.1, and 0.01 µM) demonstrated higher viability than the control group (p < 0.05), while AKBA in concentrations (5 and 10 µM) showed equal or decreased viability than the control group. (p > 0.05). Regarding cell density assay, AKBA showed significant increase in cell density after 5 and 7 days compared with cells medicated with TAP and Ca(OH)2 while TAP revealed marked reduction in cell density in all the tested intervals. Acetyl-11-keto-β-boswellic acid in lower concentrations (0.01, 0.1, and 1 µM) demonstrated superior cell viability than TAP and Ca(OH)2, and it may possess the potential to be an intracanal medicament in regenerative endodontics. Studying the effect of different potential root canal medicaments and their capability to induce DPSCs proliferation might be of value. The influence of AKBA on the viability and proliferation of DPSCs tested in this study sheds light on its use as a potential intracanal medication especially in regenerative endodontics. How to cite this article: Amer NA, Badawi MF, Elbeltagi MG, et al. Effect of Boswellic Acid on Viability of Dental Pulp Stem Cells Compared to the Commonly Used Intracanal Medications: An In Vitro Study. J Contemp Dent Pract 2023;24(12):957-966.

Efficient Delivery of Recombinant Human Bone Morphogenetic Protein (Rhbmp-2) With Cockle Shell Derived Calcium Carbonate Nanoparticles (CaCO3NPs)

Bone morphogenetic protein-2 (BMP-2) has a significant function in the formation of cartilage and bones. Notably, dosing of only BMP-2 protein intravenously is ineffective. Persistent transportation of the stabilized BMP-2 through a carrier has been seen to be essential for enhancing the osteogenesis im pact of BMP-2. The current research built a new system of drug delivery by utilising cockle shell derived calcium carbonate nanoparticles (CaCO3NPs) and studied the efficacy of the delivery system on the recombinant human bone morphogenetic protein (rhBMP-2). rhBMP-2-CaCO3NPs nanoparticles were synthesised by means of a modest precipitation procedure along with mechanical grinding. Fourier-tran sform infrared spectroscopy, UV–Vis spectrophotometer, scanning electron microscope, X-ray powder diffraction, transmission electron microscope, and zeta potential were u tilised for characterising the conjugated rhBMP-2-CaCO3NPs . Cytotoxicity of rhBMP-2, CaCO3NPs and rhBMP-2-CaCO 3NPs was studied by utilising methylthiazol tetrazolium assay against fibroblast (Rat-1) cells in comparison to rhBMP-2 and CaCO3NPs. The outcomes signified bio-stability of CaCO3NPs and lower toxicity for Rat-1 cells. In summary, CaCO3NPs were prepared by a simp le precipitation process. The ensuing nanoparticles could competently entrap rhBMP-2 and generated stable rhBMP-2-CaCO3NPs. A sustained discharge of rhBMP-2 from t he CaCO3NPs was seen. CaCO3NPs loaded with r hBMP-2 demonstrated reasonable bio-compatibility. The outcomes indicated that CaCO3NPs may have significant ability as carrier of therapeutic proteins within bone tissue en gineering.

Effective Combination of Targeted Therapies with Sonodynamic Treatment for Use in Exploring Differences in Therapeutic Efficacy across Organelle Targets.

Acoustic kinetic therapy systems that target specific organelles can improve the precision of a sonosensitizer, which is a perfect combination of targeted therapy and sonodynamic therapy (SDT) and plays an important role in current acoustic kinetic therapy. In this study, we loaded PpIX, a sonosensitizer, on targeted-functional carbon dots (CDs) via an amide reaction and then generated the mitochondria-targeted system (Mit-CDs-PpIX) and nucleus-targeted system (Nuc-CDs-PpIX), respectively, to deliver the sonosensitizer. Both systems exhibited minimal cytotoxicity in the absence of ultrasound stimulation. The efficacy of the targeted SDT systems was investigated using methylthiazol tetrazolium (MTT) assays, live/dead staining, flow cytometry, etc. Compared with the free PpIX and mitochondria-targeted system, the nucleus-targeted system is more potent in killing effect under ultrasound stimulation and induces apoptosis with higher intensity. To achieve the equal killing effect, the effective concentration of Nuc-CDs-PpIX is just one third of that of Mit-CDs-PpIX.

Nanohybrid Based on (Mn, Zn) Ferrite Nanoparticles Functionalized With Chitosan and Sodium Alginate for Loading of Curcumin Against Human Breast Cancer Cells

This study reports on the synthesis of Mn1 − xZnxFe2O4 (Mn, Zn ferrite) magnetic nanoparticles (MNPs) as drug delivery carriers for effective therapeutic outcomes. The MNPs were prepared using the coprecipitation method, and their magnetic properties were investigated based on their composition. Among the compositions tested, Mn0.8Zn0.2Fe2O4 MNPs exhibited superparamagnetic properties with a saturation magnetization moment of 34.6 emu/g at room temperature (25°C). To enhance the water solubility of curcumin (Cur), known for its hydrophobic nature, it was successfully loaded onto alginate (Alg)/chitosan (Chit)@Mn0.8Zn0.2Fe2O4 nanoparticles (NPs). The nanocomposite was characterized by field emission scanning electron microscopy (FE-SEM) which revealed a particle size of approximately 20 nm. The crystalline structure of the NPs was analyzed using X-ray diffraction, while Fourier-transform infrared (FTIR), energy-dispersive X-ray, and map analysis techniques were employed for further characterization. In terms of drug release, there was an initial burst release of Cur (around 18%) within the first hour, followed by a slower release (approximately 61%) over the next 36 h. The anti-tumor properties of the Cur-loaded NPs were evaluated using the Methyl Thiazol Tetrazolium (MTT) assay and quantitative real-time polymerase chain reaction. The MTT assay confirmed a higher cytotoxic effect of Cur-loaded Alg/Chit@Mn0.8Zn0.2Fe2O4 NPs on the MCF-7 breast cancer cell line compared to free Cur, highlighting the significance of incorporating Cur into nano-sized carrier systems.Graphical

Phytogenic Synthesis and Characterization of Silver Metallic/Bimetallic Nanoparticles Using Beta vulgaris L. Extract and Assessments of Their Potential Biological Activities

The synthesis of novel nanomedicines through eco-friendly protocols has been applied on a large scale with the prediction of discovering alternate therapies. The current work attained phytogenic synthesis of Ag-mNPs, AgSeO2-bmNPs, and Ag-TiO2-bmNPs through bio-reduction using an aqueous extract of Beta vulgaris (red beetroot). The phytochemical profile of the eco-friendly synthesized metallic/bimetallic nanoparticles was studied. The optical properties of nano-solutions were studied via UV-visible spectroscopy. The Fourier-transform infrared spectroscopy (FT-IR) spectral analyses revealed that stretching vibrations at wavenumbers 3303.81–3327.81 cm−1 attributed to phenolic hydroxyl groups documented shifts in the values in this range owing to proton dissociation through the bio-reduction of the metal ions. The surface morphology and the charge of the nanoparticles were investigated using a Transmission Electron Microscope (TEM) and zeta potential analyses. The prepared nano-solutions showed lower antioxidant activity (1,1-Diphenyl-2-picrylhydrazyl (DPPH•) and phosphomolybdate assays) than the plant extract. These results together with phytochemical analyses support the participation of the reactive species (phenolic contents) in the bio-reduction of the metal ions in the solutions through the formation of metallic/bimetallic nanoparticles. Ag-mNPs, AgSeO2-bmNPs, and Ag-TiO2-bmNPs showed antibacterial potentiality. AgSeO2-bmNPs were superior with inhibitory zone diameters of 34.7, 37.7, 11.7, and 32.7 mm against Enterococcus faecalis, Staphylococcus aureus, Escherichia coli, and Salmonella enterica, respectively. Applying the Methylthiazole Tetrazolium (MTT) assay, the Ag-TiO2 bmNPs revealed potent cytotoxicity against the HePG2 tumor cell line (IC50 = 18.18 ± 1.5 µg/mL), while Ag-SeO2 bmNPs revealed the most potent cytotoxicity against the MCF-7 cell line (IC50 = 17.92 ± 1.4 µg/mL).

Evaluation of Chemical Compositions and the Antioxidant and Cytotoxic Properties of the Aqueous Extract of Tri-Yannarose Recipe (Areca catechu, Azadirachta indica, and Tinospora crispa).

Tri-Yannarose is a Thai traditional herbal medicine formula composed of Areca catechu, Azadirachta indica, and Tinospora crispa. It possesses antipyretic, diuretic, expectorant, and appetite-stimulating effects. This study aimed to evaluate the antioxidant activities, cytotoxicity, and chemical constituents of an aqueous extract following a Tri-Yannarose recipe and its plant ingredients. The phytochemical analysis was performed using LC-QTOF-MS. Antioxidant activities were determined using DPPH, ABTS, TPC, TFC, FRAP, NBT, MCA, and ORAC assays. Cytotoxicity was investigated using a methyl thiazol tetrazolium (MTT) assay. In addition, the relationship between the chemical composition of Tri-Yannarose and antioxidant activities was investigated by examining the structure-activity relationship (SAR). The results of the LC-QTOF-MS analysis revealed trigonelline, succinic acid, citric acid, and other chemical constituents. The aqueous extract of the recipe showed significant scavenging effects against ABTS and DPPH radicals, with IC50 values of 1054.843 ± 151.330 and 747.210 ± 44.173 µg/mL, respectively. The TPC of the recipe was 92.685 mg of gallic acid equivalent/g of extract and the TFC was 14.160 mg of catechin equivalent/g of extract. All extracts demonstrated lower toxicity in the Vero cell line according to the MTT assay. In addition, the SAR analysis indicated that prenyl arabinosyl-(1-6)-glucoside and quinic acid were the primary antioxidant compounds in the Tri-Yannarose extract. In conclusion, this study demonstrates that Tri-Yannarose and its plant ingredients have potent antioxidant activities with low toxicity. These results support the application of the Tri-Yannarose recipe for the management of a range of disorders related to oxidative stress.

Ferulago angulata Methanolic Extract Protects PC12 Cells Against Beta-amyloid-induced Toxicity.

Alzheimer's disease (AD) is an age-dependent neurodegenerative disease. Beta-amyloid (Aβ)-induced neurotoxicity has a pivotal role in AD pathogenesis; therefore, the modulation of Aβ toxicity is the promising therapeutic approach to control the disease progression. Medicinal plants because of their multiple active ingredients are effective in complex diseases, such as AD. Therefore, several studies have studied medicinal plants to find an effective treatment for AD. Ferulago angulata is a medicinal plant with antioxidant and neuroprotective activity. The present study was done to assess the protective effect of the methanolic extract of Ferulago angulate on Aβ-induced toxicity and oxidative stress in PC12 cells. The methanolic extract of aerial parts of the plant was prepared by the maceration method. PC12 cells were cultured according to a standard protocol. PC12 cells were incubated for 24 hours with Aβ alone, and Aβ in combination with various concentrations of the F. angulata extract. Cell viability was determined by the methyl thiazole tetrazolium (MTT) assay. Also, reactive oxygen species (ROS) production and the activity of acetylcholine esterase (AChE), glutathione peroxidase (GPx), and caspase-3 enzymes were measured. The extract dose-dependently protected PC12 cells against Aβ-induced cell death. Also, Aβ increased ROS production, AChE, and caspase-3 activity, and decreased the GPx activity, which all were ameliorated by F. angulata extract. F. angulata extract protects against Aβ-induced oxidative stress and apoptosis. These effects may be due to the antioxidant and anticholinesterase activity of the extract. It is recommended to assess F. angulata extract as an anti-AD agent. Ferulago angulata extract dose-dependently ameliorates Aβ-induced cytotoxicity in PC12 cells.Aβ induced oxidative stress in PC12 cells, which was attenuated by the F. angulata extract.Aβ increased acetylcholinesterase activity in PC12 cells, which was prevented by the F. angulata extract. Alzheimer's disease (AD) is a common form of dementia in the elderly with a complex pathophysiology. Beta-amyloid (Aβ)- induced neurotoxicity plays a pivotal role in AD progression. So far, there is no cure for AD. Medicinal plants contain various pharmacologically active compounds that make them suitable for the treatment of complex diseases. In this study, the anti-AD effect of F. angulata extract was investigated by assessing its protective effect against Aβ-induced toxicity in PC12 cells F. angulata extract improved Aβ-induced toxicity by diminishing oxidative stress and apoptosis. Therefore, F. angulata extract merits further studies for use in the treatment of AD.

In vitro wound healing effects of combinations of Aloe vera gel with different extracts of Bulbine frutescens

Many skin conditions, including wounds, have been treated traditionally with plant-based medicines such as Aloe vera and Bulbine frutescens. The gel from the leaves of A. vera has shown potential in burn wound treatment and other skin conditions, while B. frutescens exhibited accelerated wound healing properties in previous in vitro and in vivo studies. This study investigated whether the skin wound healing properties of A. vera gel could be enhanced by combining it with each of two different B. frutescens extracts (aqueous and gel). Firstly, the in vitro cytotoxicity of the selected plant materials was measured on HaCaT and 84BR cells by means of methylthiazol tetrazolium (MTT) assays. The in vitro re-epithelialization potential or wound healing properties were determined by means of the scratch assay on HaCaT cell layers. The wound closure percentage and migration rate values were measured in an untreated control group as well as after 24 h treatment with the individual plant materials as well as combinations thereof. The scratch assay results showed that the wound closure percentage and migration rate values for each of the selected plant materials individually were higher than that of the untreated control group. When A. vera gel (0.25 mg/ml) was combined with B. frutescens aqueous extract in three different concentrations (0.25, 0.50 and 0.75 mg/ml), all the combination ratios exhibited higher wound closure percentage and migration rate values than that of the control group and it was also higher than the effects of each individual plant material alone. On the other hand, combination of A. vera gel with B. frutescens gel extract did not show increased wound healing effects as compared to that of the control group nor the individual plant materials. It can be concluded that the wound healing properties of A. vera gel can be increased by combining it with B. frutescens aqueous extract.

Knocking down the expression of the molecular motors, myosin A, C and F genes in Toxoplasma gondii to decrease the parasite virulence

Toxoplasmosis is a serious parasitic infection and novel therapeutic options are highly demanded to effectively eliminate it. In current study, Toxoplasma gondii myosin A, C and F genes were knocked down using small interference RNA (siRNA) method and the parasite survival and virulence was evaluated in vitro and in vivo. The parasites were transfected with specific siRNA, virtually designed for myosin mRNAs, and co-cultured with human foreskin fibroblasts. The transfection rate and the viability of the transfected parasites were measured using flow cytometry and methyl thiazole tetrazolium (MTT) assays, respectively. Finally, the survival of BALB/c mice infected with siRNAs-transfected T. gondii was assessed. It was demonstrated that a transfection rate of 75.4% existed for siRNAs, resulting in 70% (P = 0.032), 80.6% (P = 0.017) and 85.5% (P = 0.013) gene suppression for myosin A, C and F in affected parasites, respectively, which was subsequently confirmed by Western blot analysis. Moreover, lower parasite viability was observed in those with knocked down myosin C with 80% (P = 0.0001), followed by 86.15% (P = 0.004) for myosin F and 92.3% (P = 0.083) for myosin A. Considerably higher mouse survival (about 40 h) was, also, demonstrated in mice challenged with myosin siRNA-transfected T. gondii, in comparison with control group challenged with wild-type parasites. In conclusion, myosin proteins knock down proposes a promising therapeutic strategy to combat toxoplasmosis.

Mitochondrial reactive oxygen species promote mitochondrial damage in high glucose-induced dysfunction and apoptosis of human dental pulp cells

Background/purposeHigh glucose (HG)-induced aberrant proliferation, apoptosis and odontoblastic differentiation of dental pulp cells (DPCs) have been implicated in the pathogenesis of impaired diabetic pulp healing; however, the underlying mechanism remains unclear. This study aimed to investigate the role of mitochondrial reactive oxygen species (mtROS) and mitochondria in HG-induced dysfunction and apoptosis of DPCs. Materials and methodsHuman DPCs (hDPCs) were cultured in a low-glucose, high-glucose, mannitol, and MitoTEMPO medium in vitro. Methylthiazol tetrazolium assay, Annexin V-FITC/PI staining and scratch-wound assay were used to analyze cell proliferation, apoptosis and migration, respectively. Alkaline phosphatase staining and alizarin red S staining were used to evaluate cell differentiation. DCF-DA staining, MitoSOX staining, MitoTracker Red staining, JC-1 staining, and adenosine triphosphate (ATP) kit assay were performed to investigate total ROS and mtROS generation, mitochondrial density, mitochondrial membrane potential (MMP), and ATP synthesis, respectively. Quantitative PCR assay was performed to detect the mRNA expression of mitochondrial biogenesis- and dynamics-related markers. Transmission electron microscopy was used to observe the mitochondrial ultrastructure. ResultsHG augmented the production of total ROS and mtROS, and triggered mitochondrial damage in hDPCs, as reflected by decreased mitochondrial density, depolarized MMP, reduced ATP synthesis, altered mRNA expression of mitochondrial biogenesis- and dynamics-related markers, and abnormal mitochondrial ultrastructure. Supplementation of MitoTEMPO alleviated the mitochondrial damage and reversed the aberrant proliferation, apoptosis, migration and odontoblastic differentiation of HG-stimulated hDPCs. ConclusionHG triggers mitochondrial damage via augmenting mtROS generation, resulting in the inhibited proliferation, migration, and odontoblastic differentiation of hDPCs and enhanced their apoptosis.

PDCD6 Promotes Hepatocellular Carcinoma Cell Proliferation and Metastasis through the AKT/GSK3β/β-catenin Pathway

ObjectiveProgrammed cell death 6 (PDCD6), a Ca2+-binding protein, has been reported to be aberrantly expressed in all kinds of tumors. The aim of this study was to explore the role and mechanism of PDCD6 in hepatocellular carcinomas (HCCs). MethodsThe expression levels of PDCD6 in liver cancer patients and HCC cell lines were analyzed using bioinformatics and Western blotting. Cell viability and metastasis were determined by methylthiazol tetrazolium (MTT) and transwell assays, respectively. And Western blotting was used to test related biomarkers and molecular pathway factors in HCC cell lines. LY294002, a PI3K inhibitor inhibiting AKT, was used to suppress the AKT/GSK3β/β-catenin pathway to help evaluate the role of this pathway in the HCC carcinogenesis associated with PDCD6. ResultsThe analysis of The Cancer Genome Atlas Database suggested that high PDCD6 expression levels were relevant to liver cancer progression. This was consistent with our finding of higher levels of PDCD6 expression in HCC cell lines than in normal hepatocyte cell lines. The results of MTT, transwell migration, and Western blotting assays revealed that overexpression of PDCD6 positively regulated HCC cell proliferation, migration, and invasion. Conversely, the upregulation of PDCD6 expression in the presence of an AKT inhibitor inhibited HCC cell proliferation, migration, and invasion. In addition, PDCD6 promoted HCC cell migration and invasion by epithelial-mesenchymal transition. The mechanistic investigation proved that PDCD6 acted as a tumor promoter in HCC through the AKT/GSK3β/β-catenin pathway, increasing the expression of transcription factors and cellular proliferation and metastasis. ConclusionPDCD6 has a tumor stimulative role in HCC mediated by AKT/GSK3β/β-catenin signaling and might be a potential target for HCC progression.

Inhibition of MDM2 Using Idasanutlin (RG-7388) enhances the Chemo-sensitivity of B-ALL Cells to Daunorubicin

Background: Although significant advances have been made in the treatment of cancer, a significant number of patients with acute lymphoblastic leukemia (ALL) show resistance to treatment. Thus, it is necessary to seek novel therapeutic agents to overcome this problem. Studies have indicated that the expression level of mouse double minute 2 (MDM2), a negative regulator of p53, is markedly elevated in patients with refractory or recurrent ALL. Thus, targeting MDM2 using a specific inhibitor, Idasanutlin, can increase the activity of p53. This study evaluated the possible synergistic effect of Idasanutlin and Daunorubicin on the induction of apoptosis in NALM-6 cells.&#x0D; Materials and methods: In this fundamental study, the anti-proliferative effects of Idasanutlin on NALM-6 cells, either alone or in combination with Daunorubicin, were confirmed by MTT(methyl-thiazol-tetrazolium) assay, Annexin/PI apoptosis assay, and cell cycle analysis. Quantitative reverse transcription-PCR (qRT-PCR) and Western blot analyses were applied to elucidate the molecular mechanisms underlying the anti-leukemic activity of Idasanutlin.&#x0D; Results: Idasanutlin synergistically enhanced Daunorubicin-induced apoptosis and activated caspase-3, thereby activating programmed cell death in a dose-dependent manner (P&lt;0.001). The treatment of NALM-6 cells with Idasanutlin caused cell cycle arrest in the G1 phase by an increase in the expression of p21 (P&lt;0.001). Moreover, a significant increase was detected in the expression of pro-apoptotic genes (P&lt;0.001), as well as a remarkable decrease in the expression of anti-apoptotic (P&lt;0.01) and multidrug resistance1 (MDR1) genes (P&lt;0.01).&#x0D; Conclusions: It seems that Idasanutlin can cooperatively promote daunorubicin-induced apoptosis in NALM-6 cells. These findings open up a new horizon in the application of Idasanutlin in combination with Daunorubicin to overcome drug resistance in patients with ALL.

Effects of Electronic Cigarette E-Liquids on the Adhesion and Growth of Osteoblast Cells

Electronic cigarettes (E-cigarettes) were designed to replace traditional cigarettes to decrease the harmful effects of smoking. The liquid in e-cigarettes contains different chemicals, such as Propylene Glycol (PG) and Vegetable Glycerin (VG), and may contain nicotine. To be more attractive, e-liquids are supplemented with flavorings. Because vaped e-liquids are shown to be damaging to oral tissues, similar effects could occur in the oral cavity with non-vaped e-liquids. We evaluated the effects of non-vaped e-liquid constituents on osteoblast behavior. For this purpose, human osteoblast cells (MG-63) were exposed to e-liquid containing 70% PG -30% VG with and without tobacco flavor and with or without nicotine at 12, 18 mg/mL, or no nicotine content. The e-liquids were used at various concentrations (0, 1, and 5%). To evaluate the effect of the e-liquids on osteoblast morphology and adhesion, optical microscope observations and viable cell counting using trypan blue exclusion were used. Cell growth was also analyzed using the Methyl Thiazol Tetrazolium (MTT) assay, while cytotoxicity was tested by measuring Lactate Dehydrogenase (LDH) levels after cell exposure for 24 h to the e-liquids. Our results show that e-liquids induced significant morphological changes evidenced by round cell forms, with no contact between cells. Adhesion was significantly reduced, particularly in the presence of nicotine. E-liquids at a concentration of 5% significantly reduced osteoblast growth. This effect was observed with both flavored and non-flavored e-liquid with or without nicotine. The decreased osteoblast adhesion and growth after exposure to e-liquid was confirmed by increased levels of LDH. Overall results indicate the potentially harmful effects of e-liquid non-vaped chemicals on bone cells, which could lead to the impairment of bone regeneration and tissue remodeling processes.

ALK5 Inhibition of Subconjunctival Scarring From Glaucoma Surgery: Effects of SB-431542 Compared to Mitomycin C in Human Tenon's Capsule Fibroblasts.

The gold standard for managing postoperative ocular fibrosis in glaucoma surgery is the chemotherapeutic mitomycin C (MMC) despite its association with significant adverse effects. This study compares in vitro the antifibrotic efficacy and cytotoxicity of the small-molecule TGFβ1 inhibitor SB-431542 (SB) to MMC. To measure collagen contraction, human Tenon's capsule fibroblasts (HTCFs) embedded in a three-dimensional collagen lattice were exposed to 0.2 mg/mL MMC or 20 µM SB followed by incubation with 2 ng/mL TGFβ1. Total protein extracted from experimentally treated HTCFs underwent immunoblotting for α-smooth muscle actin (α-SMA), matrix metallopeptidase 9 (MMP-9), and EDA splice-variant fibronectin (EDA-FN) expression. Cytotoxicity and cell metabolism were assessed using LIVE/DEAD staining, lactate dehydrogenase (LDH) assay, and methylthiazole tetrazolium (MTT) assay. Collagen lattice contraction in TGFβ1-induced HTCFs was significantly lowered by SB and MMC. Pretreatment with SB and MMC significantly lowered protein expression of α-SMA, MMP-9, and EDA-FN in HTCFs relative to TGFβ1 alone. HTCF viability in collagen lattices was significantly reduced with MMC pretreatment but not SB pretreatment. MMC-pretreated HTCFs had a significant increase in LDH release after 3 hours and a decrease in MTT activity after 20 minutes, while SB-pretreated HTCFs showed no significant changes via MTT or LDH assay during the same treatment period. SB shows comparable efficacy to MMC in reducing expression of fibrosis-promoting proteins in HTCFs and in vitro scarring activity. SB distinguishes itself from MMC by exhibiting less cytotoxicity in both two-dimensional and three-dimensional in vitro assays. This study demonstrates in vitro the potential of SB as a safer alternative ocular antifibrotic agent.

Chalcone-3 Inhibits the Proliferation of Human Breast Cancer MDA-MB-231 Cell Line.

Chalcone-3 has been shown to be cytotoxic and selective against luminal subtype breast cancer cell lines, which are suspected to occur through the mechanism of epidermal growth factor receptors (EGFR) inhibition. However, the cytotoxic effect has never been tested on cell strains from patients with triple negative breast cancer (TNBC), where EGFR expression is known to increase. This study aimed to identify the role of chalcone-3 in one of the downstream targets of EGFR as an antiproliferative agent. Chalcone-3 was examined for its effect on proliferation in human breast cancer MDA-MB-231 cell lines. The percentage of proliferation inhibition was analyzed using methyl-thiazol tetrazolium assay. Flow cytometry was used to analyze the population of cell cycle distribution and the expression of cyclin-D1 and pEGFR. Chalcone-3 inhibited the proliferation of MDA-MB-231 cells in a dose and time-dependent manner with an IC50 value of 17.98±6.36 µg/mL by inducing cell cycle arrest at the G2/M phase. Flow cytometry assays showed that chalcone-3 significantly reduced the expression of pEGFR and cyclin-D1, contributing to cell cycle arrest. Chalcone-3 might have potential as an anti-proliferative drug to treat TNBC.

In-vitro cytotoxicity and in-vivo antitumor activity of two platinum complexes with 1,3-dimethyl pentyl glycine ligand against breast cancer

Platinum (Pt) derivatives are good candidates for discovering new anti-tumor agents. The present research aims to explore the in-vivo and in-vitro anticancer activity of two platinum complexes with 1,3-dimethyl pentyl glycine ligand (DMPG), [Pt(bpy)(13DMPG)]NO3 and [Pt(dach)(13DMPG)]NO3, against breast cancer cells. The present study was conducted to investigate the cytotoxic potential of these compounds (2–400 μM) compared to standard drugs (cisplatin, oxaliplatin, and carboplatin) on SKBR3 cells using the methyl thiazol-tetrazolium (MTT) assay. Furthermore, the gene expression changes of Bak, Bim, Bcl-2, Caspase-3, and Caspase-9 were carried out by real-time polymerase chain reaction (PCR), and flow cytometric analysis was performed to confirm the cell apoptosis in the presence of the compounds. For more validation, in-vivo anticancer activities of both compounds were investigated against breast transplanted tumors in the BALB/c mice model. The cytotoxic studies by MTT assay revealed the anti-proliferative potential of both derivatives. [Pt(dach)(13DMPG)]NO3 with an IC50 value of 15 μM, exhibited higher cytotoxicity against SKBR3 cells as compared to [Pt(bpy)(13DMPG)]NO3, oxaliplatin, and carboplatin. Based on the flow cytometry analysis, both derivatives demonstrated apoptotic effects. Also, real-time PCR analysis revealed an up-regulation of Bak, Bim, Bax, Caspases-3, and Caspase-9 genes and a significant reduction in Bcl-2 gene expression in treated cells with both compounds compared to the control group. In-vivo results validated in-vitro analysis and showed the anticancer activity of compounds against breast transplanted tumors in the BALB/c mice model. According to the results, [Pt(dach)(13DMPG)]NO3 displayed a significant anticancer activity.

Autophagic-Related Anticancer Effect of Grapes Extract and Tomatoes Extract: Ex-Vivo Study

Cells undergo autophagy to save themselves from injury, but progressive autophagy can cause cell death. This study characterized and compared the effect of grape (resveratrol) and tomato (lycopene) extracts and their combination on modulating autophagy-related miRNA and its target gene in squamous cell carcinoma cell line. Docking analysis for extracts and selected genes was performed. Methyl Thiazol Tetrazolium assays were used to assess the cytotoxicity of extracts and their combination toward HEp-2 cells. qRT-PCR was used to quantify changes in gene expression. Data were statistically analyzed. miRNA-20a was identified as a potential effector in laryngeal cancer, and sequestosome-1 (SQSTM1) was its target gene. Docking analysis showed that resveratrol interacted with miRNA-20a and showed less affinity toward SQSTM1. Hydrogen bonds and hydrophobic interactions were predicted. In contrast, lycopene showed less affinity toward miRNA-20a than resveratrol. Increasing doses of resveratrol, lycopene, and their combination induced a statistically significant reduction in mean percent viability and mean fold changes of miRNA-20a and SQSTM1 expression in treated HEp-2 cells. Pearson’s correlation showed a statistically significant positive correlation between miRNA-20a and SQSTM1 (R=0.812, p≤0.001). Grape and tomato extracts and their combination display promising cytotoxicity against HEp-2 cells in a dose- and time-dependent fashion. Both extracts reduce the expression of miRNA-20a and SQSTM1 with subsequent inhibition autophagy and promotion of apoptosis in HEp-2 cells.&#x0D;