Trypan Blue Dye Exclusion Assay Research Articles

Therapeutic potential of xanthones from Swertia chirata in breast cancer cells.

Background & objectives:Medicinal plants like Swertia chirata are rich sources of different xanthones. This study was aimed to assess the cytotoxic potential of four most abundant xanthones present in S. chirata both in vivo and in vitro in Ehrlich ascites carcinoma (EAC), a mouse transplantable breast carcinoma cell line and two human breast carcinoma cell lines (MCF-7 and MDA-MB-231).Methods:Four xanthones derived from S. chirata namely 1-hydroxy-3,7,8-trimethoxyxanthone (XA), 1,8-dihydroxy-3,5-dimethoxyxanthone (XB), 1-hydroxy-3,5,8-trimethoxyxanthone (XC) and 1,5,8-trihydroxy-3-methoxyxanthone (XD) were used for determination of sub-lethal dose on the cell lines EAC, MCF-7, MDA-MB-231 and verified toxicity of sub-lethal dose on normal murine fibroblast cells. Cytotoxicity was measured in vivo and survivability of mice was plotted accordingly. Therapeutic efficacy of XD was evaluated both in vivo and in vitro by determination of lipid peroxidation (LPO), reactive oxygen species (ROS) generation and by quantitating the enzyme status (GSH, catalase, superoxide dismutase) in treated and untreated samples. DNA damage was evaluated using comet and DNA fragmentation assays. Furthermore, apoptotic effect was analyzed by flow cytometry and validated by TUNEL assay and Western blotting.Results:Among all the xanthones tested XD showed IC50 at the lowest dose, and normal cells were unaffected at this dose. Survivability of mice increased significantly when treated with XD compared to other xanthones and cisplatin. Significantly increased ROS and LPO were found in cancer cells as a result of XD treatment which was unaltered in normal cell line. XD induced DNA damage and apoptosis in cancer cell lines.Interpretation & conclusions:Our experimental data indicate that XD may potentially act as a chemotherapeutic agent by enhancing ROS in breast cancer cells thereby leading to apoptosis.

Overactivation of Akt Contributes to MEK Inhibitor Primary and Acquired Resistance in Colorectal Cancer Cells.

RAS and BRAF-mutated colorectal cancers are associated with resistance to chemotherapy and poor prognosis, highlighting the need for new therapeutic strategies. Although these cancers sometimes respond to mitogen activated protein kinase kinase (MEK) inhibitor treatment, they often acquire resistance via mechanisms, which are poorly understood. Here, we investigated the mechanism of MEK inhibitor resistance in primary- and acquired-resistant cells. Cell viability was examined using the trypan blue dye exclusion assay. Protein expression was analyzed by western blotting. Somatic mutations in colorectal cancer cells were investigated using the polymerase chain reaction array. PD0325901 and trametinib induced cell death in LoVo and Colo-205 cells but not in DLD-1 and HT-29 cells, which have a PIK3CA mutation constitutively activating Akt and NF-κB. Treatment with PD0325901 and trametinib suppressed ERK1/2 activation in all four cell lines but only induced Akt and NF-κB activation in DLD-1 and HT-29 cells. Inhibition of Akt but not NF-κB, overcame MEK inhibitor resistance in DLD-1 and HT-29 cells. Acquired-resistant LoVo/PR, Colo-205/PR and LoVo/TR cells have constitutively active Akt due to a M1043V mutation in the kinase activation loop of PIK3CA and Akt inhibitor resensitized these cells to MEK inhibitor. These results demonstrate that the overactivation of Akt plays a critical role in MEK inhibitor primary and acquired resistance and implicate combined Akt/MEK inhibition as a potentially useful treatment for RAS/BRAF-mutated colorectal cancer.

The effects of IGF-I on prostate cancer progression and the influence of hyperglycaemia

Epithelial to mesenchymal transition (EMT) is a necessary process in the conversion of benign tumor to aggressive and highly invasive cancer. Dysregulation of the IGF system and impaired metabolic regulation have been implicated in the progression of prostate cancer. However, the mechanisms underlying these effects require further investigation. We used normal prostate epithelial cells PNT2 and DU145 prostate cancer cells. Western immunoblotting was used to determine changes in protein abundance. Trypan blue dye exclusion assay was employed to assess cell proliferation and transwell migration assays to assess cells migration. Under normal glucose conditions, IGF-I inhibited EMT in PNT2 cells demonstrated by an upregulation in the epithelial marker E-cadherin together with loss of mesenchymal markers; vimentin and fibronectin. In contrast to PNT2 cells, IGF-I induced EMT in DU145 cells, as shown by the reduction of E-cadherin level and upregulation of vimentin and fibronectin. We observed that exposure to hyperglycaemia (25mM glucose concentration) alone induced EMT in both PNT2 and DU145 cells. The changes in EMT markers induced by hyperglycaemia (loss of epithelial marker and increase of mesenchymal markers) associated with increased cell proliferation and migration. In high glucose conditions, IGF-I was still able to inhibit EMT in PNT2 cells, whereas in DU145 cancer cells, the addition of IGF-I could not enhance EMT any further. In conclusion, IGF-I and hyperglycaemia play important roles in promoting prostate cancer cell progression through the regulation of EMT programme.

Investigation of the Antibacterial Activity and in vivo Cytotoxicity of Biogenic Silver Nanoparticles as Potent Therapeutics.

Biogenic nanoparticles are the smartest weapons to deal with the multidrug-resistant “superbugs” because of their broad-spectrum antibacterial propensity as well as excellent biocompatibility. The aqueous biogenic silver nanoparticles (Aq-bAgNPs) and ethanolic biogenic silver nanoparticles (Et-bAgNPs) were synthesized using aqueous and ethanolic extracts of Andrographis paniculata stem, respectively, as reducing agents. Electron microscopic images confirmed the synthesis of almost spherical shaped biogenic silver nanoparticles (bAgNPs). The zeta potentials of the nanoparticles were negative and were −22 and −26 mV for Aq-bAgNPs and Et-bAgNPs, respectively. The antibacterial activity of bAgNPs was investigated against seven pathogenic (i.e., enteropathogenic Escherichia coli, Salmonella typhi, Staphylococcus aureus, Vibrio cholerae, Enterococcus faecalis, Hafnia alvei, Acinetobacter baumannii) and three nonpathogenic (i.e., E. coli DH5α, E. coli K12, and Bacillus subtilis) bacteria at different time points (i.e., 12, 16, 20, and 24 h) in a dose-dependent manner (i.e., 20, 40, and 60 μg) through broth dilution assay, disk diffusion assay, CellToxTM Green uptake assay, and trypan blue dye exclusion assay. The lowest minimum inhibitory concentration value for both the bAgNPs was 0.125 μg. Et-bAgNPs showed the highest antibacterial activity against S. aureus at 60 μg after 16 h and the diameter of inhibited zone was 28 mm. Lipid peroxidation assay using all the bacterial strains revealed the formation of malondialdehyde–thiobarbituric acid adduct due to the oxidation of cell membrane fatty acids by bAgNPs. The bAgNPs showed excellent hemocompatibility against human as well as rat red blood cells. Furthermore, there was no significant toxicity observed when the levels of rat serum ALT, AST, γ-GT (i.e., liver function biomarkers), and creatinine (i.e., kidney function biomarker) were determined.

Screening In vitro Anticancer Activity of Alseodaphne semecarpifolia Nees Stem Bark Extracts against some Cancer Cell lines

Introduction: Cancer is considered as the prime lethal disease that affects different organs of the body. Even with the rapid developments in the medical sciences, there are no proper medicines to cure specific kind of cancer without side effects. The inhibition of tumour cell growth without side effects either by the use herbal or synthetic drugs is considered as an important target in cancer therapy. In traditional medicinal system A. semecarpifolia stem bark is the prime source of herbal drug to treat lymphatic and skin cancers. Objective: The purpose of this study is to evaluate the anticancer potential of A. semecarpifolia stem bark extracts against some cancer cell lines. Methods: The in vitro anticancer activity was evaluated against DLA, EAC, HeLa, HepG2 and L929 cell lines by trypan blue dye exclusion assay and SRB assay. Results: The results of the anticancer activity revealed that, when compared to standard drug Cyclophosphamide, SBPEE and SBCE of A. semecarpifolia showed significant anticancer activity against DLA and EAC cell lines, without causing any toxicity to the normal mouse fibroblast cells L929. Whereas, none of the three extracts showed cytotoxicity against HeLa, HepG2 and L929 cell lines. Conclusion: The present study suggested that, SBPEE and SBCE possesses significant cytotoxic activity against DLA and EAC cell lines, which confirms the traditional medicinal claim of A. semecarpifolia as a potent anticancer plant against lymphatic and skin cancer.

Chitosan And N, N, N-Trimethyl Chitosan Nanoparticle Encapsulation Of Ocimum Gratissimum Essential Oil: Optimised Synthesis, In Vitro Release And Bioactivity.

BackgroundThe encapsulation of plant essential oils (EOs) with polymeric materials (e.g. chitosan (CS) and N, N, N-trimethyl chitosan (TMC)) and the further reduction of the polymers into their nano sizes are gaining research interest in nanotechnology due to potential applications in medical drug delivery systems as well as the food and pharmaceutical industry. The present study reports a novel approach for the synthesis of Ocimum gratissimum essential oil (OGEO)-loaded CS and TMC nanoparticles with distinct bioactive and physiochemical properties.MethodsThe OGEO-loaded CS and TMC nanoparticles were characterised using various microscopic and spectroscopic techniques. The bioactive compounds in Ocimum gratissimum methanolic extract (OG-MeOH) and EOs was evinced with gas chromatography-mass spectrometry (GC-MS). Total phenolic content (TPC) of OGEO and OG-MeOH was determined using the Folin-Ciocalteu method. The in vitro drug release kinetic pattern was ascertained by membrane dialysis, while antioxidant activity was determined by the 2,2-diphenyl-1picrylhydrozyl (DPPH) free radical scavenging method. The disc diffusion method was used for antibacterial activity evaluation, while MTT and a trypan blue dye exclusion assay were used to assess cytotoxic activity on MDA-MB-231 breast cancer cells.ResultsGC-MS analysis revealed components that have not been previously reported for Ocimum gratissimum. The maximum OGEO cumulative drug release percentage in vitro was observed at pH 3 for both OGEO-loaded chitosan nanoparticles (OGEO-CSNPs) and OGEO-loaded N, N, N-trimethyl chitosan nanoparticles (OGEO-TMCNPs). The antioxidant activity of OGEO-CSNPs and OGEO-TMCNPs never reached a steady state after 75 h. OGEO-TMCNPs exhibited antibacterial activity at a lower concentration for both Gram-negative and Gram-positive food pathogens. In vitro cytotoxicity revealed the increased toxicity of OGEO-TMCNPs on MDA-MB-231 breast cancer cell lines.ConclusionOGEO-loaded CS and TMC nanoparticles were synthesised using a novel material optimisation approach. The synthesised nanoparticles have shown a promising application in the pharmaceutical and food industries.

Controlled Release and Photothermal Behavior of Multipurpose Nanocomposite Particles Containing Encapsulated Gold-Decorated Magnetite and 5-FU in Poly(lactide-co-glycolide).

Nowadays, many research studies have been conducted to prepare multidisciplinary probes in drug delivery systems and cancer therapy with high performance and minimum side effects. Here, poly(lactide-co-glycolide) (PLGA) nanocomposite particles containing 5-fluorouracil (5-FU) and gold-decorated magnetite nanoparticles with a raspberry-like morphology were designed and prepared as a novel and anticancer probe. For this reason, Fe3O4 nanoparticles were synthesized by a coprecipitation method and modified with (3-mercaptopropyl) trimethoxysilane for the deposition of gold nanoparticles. Then, they were embedded in the PLGA matrix alone and accompanied by 5-FU with 92 and 88% loading efficiencies, respectively, through a multiple emulsion solvent evaporation method. Chemical structure and composition of the prepared samples in each step were completely characterized by several techniques. The morphology of the nanocomposite particles was assessed by field emission scanning electron microscopy, high-resolution transmission electron microscopy, and selected area electron diffraction patterns, and their particle size and colloidal stability after 1 week were evaluated by dynamic light scattering. Because of the coexistence of gold and Fe3O4 nanoparticles, the final probe provided enhanced dual magneto and photothermal responses by increasing the temperature up to 42.7 °C under 5 min external alternating magnetic field and to 42.1 °C within just 1 min near-infrared irradiation at 808 nm. Trypan blue dye exclusion assays showed that they are biocompatible with reasonable toxicity (IC50 of 0.62 mg/mL) with respect to DU145 prostate cancer cells. Drug release profile of the 5-FU-loaded nanocomposite particles demonstrated their controlled release at 37 °C in phosphate-buffered saline solution. These indicate multidisciplinary characteristics of such particles in cancer therapy by photothermal, magnetic hyperthermia, and chemotherapy according to the presence of various active components.

Protective effects of noradrenaline on benzo[a]pyrene-induced oxidative stress responses in brain tumor cell lines.

Benzo[a]pyrene (B[a]P) is an ubiquitous environmental pollutant that is generated during combustion of fossil fuels. We examine the effect of noradrenaline (NA) on B[a]P-induced neurotoxicity in brain tumor cell lines like neuroblastoma (Neuro2a) and glioma (C6). We pre-treated tumor cells with NA for 6h, followed by addition of B[a]P for additional 24h. Cell viability was measured using trypan blue dye-exclusion assay and comet assay was performed to measure DNA damage. Cell cycle status was analyzed using flow cytometry and oxidative DNA damage (8-oxodG) production was examined by immunostaining. The intracellular Ca2+ concentration was analyzed using Fura-2AM. Our results showed viability of Neuro2a and C6 cells declined (24% and 20%) in B[a]P-treated groups. However, pre-treating with NA increased viability of cells by reducing percentage of cell death in both. Furthermore, B[a]P-induced deregulation of cell cycle (G2/M and S phase cell arrest) was significantly restored by pre-treatment with NA in Neuro2a cells as compared to C6 cells. We further observed increased 8-oxodG production in B[a]P-treated cells; however, NA pre-treatment significantly (p < 0.05) reduced the 8-oxodG production in Neuro2a, while C6 cells were less affected possibly due to better protective machinery. B[a]P-induced intracellular Ca2+ influx was significantly reduced in both the cell lines due to co-treatment of NA possibly by reducing Ca2+ influx. NA protects brain tumor cells against B[a]P-induced neurotoxicity may be by decreasing percentage of G2 cell arrest, oxidative DNA damage, and reducing intracellular Ca2+ influx. These findings suggested that NA may be considered as a natural potential protective agent against B[a]P-induced neurotoxicity. Graphical abstract Graphical abstract showing differential protective mechanism of NA against B[a]P-induced toxicity through antioxidant mechanism maintaining homeostasis for oxidative stress in Neuro2a and C6 cell lines. The schematic graph showed the biological significance of the NA that regulates the induction of metabolic processes of cell cycle after exposure to the environmental pollutants. B[a]P increases the intracellular levels of Ca2+, but also induces damage to cellular molecules including DNA causing cell cycle arrest. The B[a]P-induced DNA damage due to base lesions generated in the genome, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) is one of the most abundant because of guanine's lowest redox potential among DNA bases through intracellular calcium homoeostasis.

Induction of apoptosis and modulation of homologous recombination DNA repair pathway in prostate cancer cells by the combination of AZD2461 and valproic acid.

Cancer therapies using defects in homologous recombination (HR) DNA repair pathway of tumor cells are not yet approved to be applicable in patients with malignancies other than BRCA1/2-mutated tumors. This study was designed to determine the efficacy of combination therapy of a histone deacetylase inhibitor, valproic acid (VPA) and a novel PARP inhibitor AZD2461 in both PC-3 (PTEN-mutated) and DU145 (PTEN-unmutated) prostate cancer cell lines. The Trypan blue dye exclusion assay and the tetrazolium-based colorimetric (MTT) assay were performed to measure the cytotoxicity while combination effects were assessed based on Chou-Talalay's principles. Flow-cytometric assay determined the type of cell death. The real-time PCR analysis was used to evaluate the alterations in mRNA levels of HR-related genes while their protein levels were measured using the ELISA method. γ-H2AX levels were determined as a marker of DNA damage. We observed a synergistic relationship between VPA and AZD2461 in all affected fractions of PC-3 cells (CI<0.9), but not in DU145 cells (CI>1.1). Annexin-V staining analysis revealed a significant induction of apoptosis when PC-3 cells were treated with VPA+AZD2461 (p<0.05). Both mRNA and protein levels of Rad51 and Mre11 were significantly decreased in PC-3 cells co-treated with VPA+AZD2461 while enhanced H2AX phosphorylation was found in PC-3 cells after 12 and 24 hours of co-treatment (p<0.05). Our findings established a preclinical rationale for selective targeting of HR repair pathways by a combination of VPA and AZD2461 as a mechanism for reducing the HR pathway sufficiency in PTEN-mutated prostate cancer cells.

Lupeol from Nyctanthes arbor-tristis Inhibits Matrix Metalloproteinase Activity, Angiogenesis and Proliferation of Glioma Cells

Angiogenesis plays a critical role in cancer progression and, hence, inhibiting angiogenesis is considered as a key strategy in cancer therapy. In this study, we screened the antiangiogenic and cytotoxic properties of Nyctanthes arbor-tristis Linn. plant of family Oleaceae, which is used in traditional medicine for the treatment of cancer, arthritis and inflammation. The antiangiogenic activity of ethanolic extract of Nyctanthes arbor-trsitis (ENA) was assessed using Chick Chorioallantoic Membrane (CAM) assay. The mechanism of antiangiogenic action was evaluated via gelatin digestion assay for matrix metalloproteinase (MMP) inhibitory activity. Cytotoxic activity of the extract on glioma cells was assessed using MTT and trypan blue dye exclusion assays. ENA impaired capillary formations in chick CAM model and inhibited MMPactivity on gelatin gels in a concentration dependent manner. The extract was found to be cytotoxic on glioma cells at higher concentrations. Bioactivity guided purification of ENA using column chromatography and thin layer chromatography afforded lupeol as the active principle. Lupeol exhibits significant MMPinhibitory activity at a concentration of 2 μg/mL and cytotoxic activity on glioma with an IC50 value of 10.75 μg/mL. The results of this study hold promise for developing this plant as a source of lupeol, a highly bioactive compound against angiogenesis.

High levels of EGFR prevent sulforaphane-induced reactive oxygen species-mediated apoptosis in non-small-cell lung cancer cells

BackgroundSulforaphane (SFN) has been shown to induce the production of reactive oxygen species (ROS) and inhibit epidermal growth factor receptor (EGFR)–mediated signaling in non-small-cell lung cancer (NSCLC). NSCLC cells harboring constitutively active EGFR mutations are more sensitive to SFN treatment than cells with wild-type EGFR, but whether NSCLC cells with high levels of EGFR expression are more resistant or sensitive to SFN treatment is not known. Study designWe employed a pair of cell lines, CL1-0 and CL1-5, which have the same genetic background but different levels of EGFR expression, to examine the effects of high EGFR level in the sensitivity to SFN. MethodsThe effect of SFN on cell viability and tumorigenicity was examined by trypan blue dye-exclusion assay, clonogenic assays, flow cytometry, and immunoblotting in vitro as well as tumorigenicity study in vivo. ROS levels in cells were assessed by flow cytometry using the ROS-reactive fluorescent indicator CM-H2DCFDA. Knockdown of EGFR in CL1-5 cells was infected with an EGFR-targeting small hairpin (interfering) RNA (shRNA)–containing lentivirus. ResultsWe present evidence that cells with high-level EGFR expression (CL1-5) are more resistant to SFN treatment than those with low-level expression (CL1-0). SFN treatment produced a similar increase in ROS and caused arrest of a cell population at S-phase accompanied by the induction of γH2AX, a DNA damage-response marker, in both cell sublines. However, SFN induced apoptosis only in the high-EGFR-expressing CL1-0 subline. Pretreatment with the antioxidant N-acetyl-L-cysteine prevented SFN-induced apoptosis in CL1-0 cells and production of γH2AX in both CL1-0 and CL1-5 cells. shRNA-mediated knockdown of EGFR in CL1-5 cells rendered the cells susceptible to SFN-induced apoptosis. ConclusionThe cellular effects produced by SFN in NSCLC cells are largely mediated by SFN-induced production of ROS. Cells with higher levels of EGFR were more resistant to SFN treatment and showed resistance to SFN-induced apoptosis, suggesting that high EGFR levels protect cells from SFN-induced apoptosis. Despite this, we found that SFN retained the ability to inhibit the growth of NSCLC tumors with high-level EGFR expression in vivo.

Mild antagonistic effect of Valproic acid in combination with AZD2461 in MCF-7 breast cancer cells.

Background: Breast cancer (BC) is a complex disease, but current treatments are not efficient enough considering increased relapse and decreased survival rate among patients. Poly (ADP-ribose) polymerase inhibitors are recently developed anticancer agents which target cells with defects in homologous recombination (HR) pathway. This study wishes to assess whether the combination of AZD2461 as a newly developed PARP1 inhibitor and valproic acid (VPA), a histone deacetylase inhibitor could effectively reduce the growth of MCF-7 cells with no fundamental DNA repair defect. Methods: Both trypan blue dye exclusion assay and MTT viability test were used to evaluate cell death. γ-H2AX levels, as a marker of DNA repair, were measured using in cell ELISA method. The Student's t-test and non-parametric analysis of variance (ANOVA) were applied for our data analyses where p-value <0.05 was considered statistically significant. Results: As calculated by CompuSyn software, IC50 values for VPA and AZD2461 were 4.89 mM and 42.83 µM respectively following 48 hours treatment. Also, the trypan blue exclusion assay results showed a concentration- and time-dependent decrease when MCF-7 cells were treated with both agents (p<0.05). Combination analysis showed a mild antagonism (CI>1.1) while γ-H2AX levels found not to be significantly increased in MCF-7 cells co-treated with VPA+AZD2461 compared to each agent alone (p=0.29). Conclusion: Our findings revealed that the combination of VPA and AZD2461 could decrease cell viability of MCF-7 cells, but it was not able to significantly increase unrepaired DNA damage sites. The mechanism responsible for drugs combination was not of synergism or addition. Determining the type of involved cell death mechanisms might be followed in further studies.

Anti‐biofilm and cytoprotective activities of quercetin againstPseudomonas aeruginosaisolates

Increase in infection with multidrug resistant Pseudomonas aeruginosa is a serious global challenge in healthcare. Pseudomonas aeruginosa is capable of causing human infection in various sites and complicates the infection due to its virulence factors. This study was aimed to investigate the effect of quercetin, a dietary flavonoid against the virulence factors of P. aeruginosa and its cell protective effects on epithelial cells. Bactericidal activity, anti-biofilm activity and effect on different virulence factors were carried out using standard methods by using five P. aeruginosa isolates. Cytotoxicity and cell protective effect of quercetin was evaluated by trypan blue dye exclusion assay. All the tested isolates were completely inhibited (100%) by quercetin at a concentration of 500μgml-1 . It showed significant (P<0·05) inhibitory effect on virulence factors including biofilm formation and showed significant protective effect on HEK 293T cells infected with P. aeruginosa strains. This study supports the role of quercetin against P. aeruginosa, by inhibiting virulence factors as well as its cytoprotective activity during bacterial infection either by attenuating the virulence or providing direct protective effect to the host cells. SIGNIFICANCE AND IMPACT OF THE STUDY: The increase in infections caused by opportunistic pathogen Pseudomonas aeruginosa is a serious concern in the health care system. This study describes the beneficial effects of a dietary flavonoid, quercetin against pathogenic P. aeruginosa strains and its protective effect against the P. aeruginosa infection in HEK 293T cells invitro.

Bufalin induces apoptosis via mitochondrial ROS-mediated caspase-3 activation in HCT-116 and SW620 human colon cancer cells

Objective: Bufalin has been reported to kill various types of cancer including human colorectal cancer. Our previous study demonstrated that bufalin induced cell death via autophagy in HT-29 and Caco-2 colon cancer cells, but the action of bufalin remains unclear. This study was conducted to investigate the role of bufalin in other colon cancer HCT-116 and SW620 cells as well as its potential mechanism.Methods: The effect of bufalin in HCT-116 and SW620 colon cancer cells was detected by assessing cell viability and cell death. Apoptotic cells were analyzed by Western blot and trypan blue dye exclusion assay. Mitochondrial ROS production was analyzed by flow cytometry after DCFDA and DHR-123 staining. The potential mechanism was investigated via pharmacological inhibitors.Results: Bufalin had high potency against HCT-116 and SW620 cells with IC50 values of 12.823 ± 1.792 nM and 26.303 ± 2.498 nM in HCT-116 and SW620 cells, respectively. Bufalin decreased cell viability, increased cell death as well as caspase-3 downstream target (cleaved PARP) accumulation, and these actions were significantly blocked by pan-caspase inhibitor zVAD-FMK. Mechanistically, ROS production, but neither the NAD(P)H oxidase, AMPK, ERK nor p38, is responsible for bufalin-induced apoptotic cell death. Moreover, bufalin-induced ROS generation is derived from mitochondria.Conclusion: Bufalin significantly induces apoptosis in HCT-116 and SW620 colon cancer cells via mitochondrial ROS-mediated caspase-3 activation. We believe that our novel findings will greatly alter our current understanding on the anti-cancer mechanism of bufalin in colon cancer cells and will pave the way for further exploiting the clinical application.

Cytotoxicity and genotoxicity of size-fractionated particulate matter collected in underground workplaces

Road tunnel construction involves the use of explosives and diesel-powered machines in a very limited indoor space. Tunnel construction workers are thus exposed to a variety of toxic substances, including dust, asbestos, silica, concrete, diesel fumes, and oil mist. In this study, the low noise miniaturized Sioutas Cascade Impactor (SKC) has been used to collect PM samples as a function of particle size (i.e., aerodynamic diameter). Airborne particulates were sampled into a road tunnel (final length 2391 m) under construction near the village of Pale, Municipality of Foligno, Umbrian Apennines (State Highway 77 “Val di Chienti”). Three sampling sessions were performed: (i) in the West yard, during dislodging of rocks; (ii) in the East yard during drilling of rocks, and (iii) during scaling and grouting with quick-drying shotcrete. The aim of this study was to characterize size-fractionated PM samples for their cytotoxic/genotoxic potentials. Toxicological evaluation was carried out in vitro on A549 lung carcinoma cells: cytotoxicity was assessed by Trypan blue dye exclusion assay, whereas genotoxicity was assessed by comet assay (primary DNA damage) and cytokinesis-block micronucleus (CBMN) Cytome assay (cytogenetic effects). Primary DNA damage (i.e., strand breakage) was mainly caused by coarse fractions A (O > 2.5 μm) sampled in the West yard (sample i) and in the East yard (sample iii). Cytogenetic effects were mainly caused by the fine fractions AF (aerodynamic O < 0.25 μm) sampled in the East yard (sample ii). Results reported in this article show that particulate matter (PM) samples collected underground (i.e., during road tunnel construction) may trigger various cytotoxic/genotoxic effects. The recent classification of air pollution and PM itself as carcinogenic to humans (group 1) by the IARC makes indoor/outdoor air pollution a growing matter of concern. Our results highlight the need in this occupational setting for a reduction, if applicable, of PM emission (prevention), or an implementation of work practices by stressing the importance of proper use of protective equipment (protection).

Antiproliferative and apoptogenic effects of myosmine on erythroleukemia and hepatocellular carcinoma cells

Myosmine, 3-(1-pyrroline-2-yl) pyridine is a minor tobacco alkaloid that has also been found in various widely used foods. Recently, this phytochemical has been gaining an increasing interest as a potential risk factor for the development of oesophageal adenocarcinoma. This study aimed to examine the effects of myosmine on the cell viability and proliferative activity of erythroleukemia and hepatocellular carcinoma cells and to obtain additional information about the mechanisms underlying its cytotoxic activity. The in vitro cytotoxic effect of myosmine on the HepG2 and MEL tumour cell lines was assessed by MTT dye reduction and trypan blue dye exclusion assays. The alterations in the tumour cell morphology induced by myosmine were analysed by fluorescent microscopy after staining with acridine orange (AO)/ethidium bromide (EtBr) and 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI). Annexin V-FITC/propidium iodide (PI) staining was used to assess the apoptosis-inducing ability of myosmine. The modulating action of antioxidant treatment on myosmine-induced cytotoxicity against the HepG2 tumour cell line was also examined. The cell viability tests indicated that myosmine induced a significant dose-dependent reduction of the viability and proliferative activity of both tumour cell lines. Fluorescent microscopy studies revealed marked alterations in the morphology of myosmine-treated tumour cells with signs of cell cycle arrest and apoptosis. The results of the simultaneous treatment with myosmine and vitamin C showed modulating activity of vitamin C on the cytotoxic effect of myosmine with concentration- and time-dependent variations. The presented results could contribute to the assessment of the potential health risks associated with the dietary myosmine exposure. Abbreviations AO: acridine orange; DAPI: 4‘,6-diamidine-2‘-phenylindole dihydrochloride; DMEM: Dulbecco's modified Eagle medium; DMSO: dimethyl sulfoxide; EtBr: ethidium bromide; FITC: fluorescein isothiocyanate; GERD: gastroesophageal reflux disease; MEL: murine erythroleukemia; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NNN: N'-nitrosonornicotine; PBS: phosphate buffered saline; PI: propidium iodide

Studies on Genotoxic Effects of Mobile Phone Radiation on A375 Cells

Introduction: Radiation from cell phones has been associated with an increased risk of cancer. The literature has reported evidence of certain biological effects resulting from exposure to various wavelengths, doses, and intensities of radiofrequency radiation. The present study aimed to evaluate the possible adverse effects of radiation from a GSM mobile phone operating at 900 MHz on human melanoma A375 cells. Material and Methods: Cellular morphology was observed under an inverted phase contrast microscope. Cell viability was determined through trypan blue dye exclusion and clonogenic assay. Moreover, flow cytometry was applied to detect DNA damage, cell cycle arrest, and reactive oxygen species (ROS) production. Cellular reduced glutathione (GSH) content was estimated by measuring the total soluble thiol. In addition, the physico-chemical changes were assessed using spectrophotometer and viscometer. Results: This study revealed that there was no change in cellular morphology and necrotic cell killing; although a small effect was observed on delayed cell death. Depletion in GSH content was noted, but ROS generation was not significantly different from that of the control group. No DNA damage was found during such exposure and there was no alteration in cell cycle distribution. In vitro evaluation of radiation effect on calf thymus DNA showed a slight perturbation in absorption spectra that was completely reversible with time. On the other hand, viscometric analysis showed no changes. Conclusion: From the findings, it can be concluded that this range of mobile phone radiation for 60 min of continuous exposure has no genotoxic impact on A375 cells.

Contraction Dynamics of Rod Microtissues of Gingiva-Derived and Periodontal Ligament-Derived Cells.

Tissue engineering strategies using microtissues as “building blocks” have high potential in regenerative medicine. Cognition of contraction dynamics involved in the in vitro self-assembly of these microtissues can be conceived as the bedrock of an effective periodontal tissue regenerative therapy. Our study was directed at evaluating the shrinkage in the rod-shaped structure of a directed self-assembly of human gingiva-derived cells (GC) and periodontal ligament-derived cells (PDLC) and developing insights into the potential mechanisms responsible for the shrinkage. GC and PDLC were seeded in non-adherent agarose molds to form rod microtissues. Cells used for the experiments were characterized using fluorescence-activated cell sorting (FACS). To assess the viability, resazurin-based cytotoxicity assays, trypan blue dye exclusion assay, MTT and live/dead staining, and histological evaluation of rods based on hematoxylin and eosin staining were performed. Rod contraction was evaluated and measured at 0, 2, 6, and 24 h and compared to L-929 cells. The role of transforming growth factor (TGF)-β signaling, phosphoinositide 3-kinase (PI3K)/AKT, and mitogen activated protein kinase (MAPK) signaling was analyzed. Our results show that the rod microtissues were vital after 24 h. A reduction in the length of rods was seen in the 24 h period. While the recombinant TGF-β slightly reduced contraction, inhibition of TGF-β signaling did not interfere with the contraction of the rods. Interestingly, inhibition of phosphoinositide 3-kinase by LY294002 significantly delayed contraction in GC and PDLC rods. Overall, GC and PDLC have the ability to form rod microtissues which contract over time. Thus, approaches for application of these structures as “building blocks” for periodontal tissue regeneration should consider that rods have the capacity to contract substantially. Further investigation will be needed to unravel the mechanisms behind the dynamics of contraction.

APRICOXIB

Background: Breast cancer is most frequently diagnosed cancer globally but there is not any ideal economical and safer agent that not only decreases the progression but also resolve complexities associated with breast cancer such as inflammatory conditions. There was strong link between inflammation and cancer specially breast cancer. Thus by inhibiting the COX enzyme may inhibit the progression of cancer beside of its role in inflammatory conditions of breast. Study Design: Interventional In Vitro trial. Setting: Department of Pharmacology in alliance with PCMD. Period: The duration of study from April 2016 to February 2017. Methodology: For this purpose we used five cancerous lines MCF-7, MDA-MB-231, MCF-10, HT-29 and Hela cell lines. For demonstrating the cytotoxic effects of Apricoxib we used MTT assay (for all cell Lines) and Trypan blue dye exclusion assay (Primarily for MCF-7 cell lines). For calculation of minimum dose required for exert cytotoxic effects of Apricoxib and its selectivitytowards cancerous cells of breast tissue we calculated its IC50 value and Selectivity Index (SI) by MTT assay. Results: Apricoxib significantly reduce the viability of MCF-7, MDA-MB-231, Hela, HT-29 as assessed by MTT assay in dose dependent manner (χ2 (2) = 26.483, p&lt;0.001), (χ2 (2) = 26.49, p&lt;0.001), (χ2 (2) = 26.062, p&lt;0.001) and (χ2 (2) = 26.062, p&lt;0.001) respectively. However Apricoxib had non-significant effects on % viability of MCF-10 cell line (χ2 (2) = 4.167, p=0.654) as assessed by MTT assay. Furthermore Apricoxib had lowest IC50 value against MCF-7 cell line. Conclusion: This study demonstrated that beside of primarily anti-inflammatory effects Apricoxib have additional benefits in term of exerting the cytotoxic effects (in vitro) on cancerous cell lines as indicated by reducing the % viability and reducing the Absorbance value of test sample as compare to control. This opens the newer path for researcher to evaluate different aspects of Apricoxib in field of chemotherapy.

The Impact of Hyperthermia in Radiosensitivity of Human Cervical Carcinoma Cell Line HeLa

Introduction: The objective of this work was to investigate the therapeutic effect of a combination of hyperthermia at 43oC exposured for 20, 40, 60 min with 6MeV electron beams at different radiation doses (2, 4, 6 Gy) compared to radiation alone. Materials and Methods: In this in vitro study, HeLa cells were exposed to either: 6MeV electron beams at different doses (2, 4, 6 Gy) and hyperthermia at 43oC for 20, 40, 60 min alone or electron beam followed by hyperthermia with 20-minute interval. The effect of hyperthermia and radiation alone as well as a combination of radiation with hyperthermia on cell viability and cell survival were estimated using the trypan blue dye exclusion assay and MTT assay immediately and 48 h after treatments, respectively. Results: The data obtained by trypan blue exclusion test showed that no significant reduction on cell viability when cells were treated RT (6 MeV, 2, 4, 6 Gy) and HT (43oC, 20, 40, 60 min) alone as well as RT followed by HT in comparison with control group (cells without treatment with the RT and HT). In addition, the data obtained by MTT assay showed significant cytotoxicity in the radiated groups compared to the control group. Also, our results showed HT alone did not effect on cytotoxicity. Moreover, our results showed that RT in combination with HT at 43oC for 20 min did not create significance cytotoxicity on HeLa cells in comparison with control group (group treated with radiation) (p>0.05). In contrast, RT in combination with HT at 43oC for 40,60 min had significant cytotoxicity on HeLa cells in comparison with control group (group treated with radiation) (p<0.05). Conclusion: Our data suggest that combined treatment of 6MeV electron beams (RT) and hyperthermia (HT) can result in radiosensitization of cervical cancer cells.