Hoechst 33342 Research Articles

Anti-tumor effect of Huaier extract supernatant on human gastric cancer HGC-27 and MGC-803 cells

The purpose of this study was to investigate the effect of Huaier extract supernatant(HES) on the proliferation, apoptosis, autophagy, and migration of human gastric cancer HGC-27 and MGC-803 cells and its molecular mechanisms. The main components in HES were preliminarily analyzed by high-performance liquid chromatography-mass spectrometry(HPLC-MS). Methyl thiazolyl tetrazolium(MTT) assay, colony formation assay, and 5-ethynyl-2'-deoxyuridine(EdU) staining assay were used to explore the effect of HES on the proliferation of human gastric cancer HGC-27 and MGC-803 cells. Hoechst staining and flow cytometry assay were used to determine the effect of HES on apoptosis of human gastric cancer HGC-27 and MGC-803 cells. Acridine orange staining and cell scratch assay were used to determine the effect of HES on autophagy and migration of human gastric cancer HGC-27 and MGC-803 cells, respectively. Western blot was used to investigate the regulatory effect of HES on the expression levels of proteins related to apoptosis, epithelial-mesenchymal transition(EMT), and signaling pathways in human gastric cancer HGC-27 and MGC-803 cells. The results showed that HES mainly contained some components with high polarities. HES significantly reduced the cell viability of human gastric cancer cells in a dose-and time-dependent manner. The IC_(50 )values after 48 h of HES treatment in human gastric cancer HGC-27 and MGC-803 cells were 7.56 and 10.77 g·L~(-1), respectively. Meanwhile, HES inhibited the colony-forming ability and short-term proliferation of human gastric cancer cells. The apoptosis rates of HGC-27 and MGC-803 cells treated with 8 g·L~(-1) HES for 72 h were 62.13%±8.92% and 54.50%±3.26%, respectively. HES also promoted autophagy in human gastric cancer cells and impaired their migration ability in vitro. Moreover, HES up-regulated the cleavage of the apoptosis marker poly ADP-ribose polymerase(PARP) and the protein expression level of the epithelial cell marker E-cadherin, and down-regulated the protein levels of phosphorylated-mammalian target of rapamycin(p-mTOR), phosphorylated-S6(p-S6), and phosphorylated-extracellular signal-regulated kinase(p-ERK) in human gastric cancer cells. Therefore, HES is one of the effective anti-tumor components of Huaier, which inhibits the proliferation and migration of human gastric cancer cells, and induces apoptosis and autophagy. Moreover, the mTOR signal and ERK signal may be involved in the anti-gastric cancer effect of HES. This study provides novel references for the in-depth research and clinical application of Huaier. It is also of great significance to promote the scientific development and utilization of Huaier.

Lymphocyte Classification from Hoechst Stained Slides with Deep Learning.

Multiplex immunofluorescence and immunohistochemistry benefit patients by allowing cancer pathologists to identify proteins expressed on the surface of cells. This enables cell classification, better understanding of the tumour microenvironment, and more accurate diagnoses, prognoses, and tailored immunotherapy based on the immune status of individual patients. However, these techniques are expensive. They are time consuming processes which require complex staining and imaging techniques by expert technicians. Hoechst staining is far cheaper and easier to perform, but is not typically used as it binds to DNA rather than to the proteins targeted by immunofluorescence techniques. In this work we show that through the use of deep learning it is possible to identify an immune cell subtype without immunofluorescence. We train a deep convolutional neural network to identify cells expressing the T lymphocyte marker CD3 from Hoechst 33342 stained tissue only. CD3 expressing cells are often used in key prognostic metrics such as assessment of immune cell infiltration, and by identifying them without the need for costly immunofluorescence, we present a promising new approach to cheaper prediction and improvement of patient outcomes. We also show that by using deep learning interpretability techniques, we can gain insight into the previously unknown morphological features which make this possible.

Anticancer activity of pure and silver doped copper oxide nanoparticles against A549 Cell line

In day-to-day life, green synthesis of metal oxide nanoparticles has still now achieved excellent heed in the field of anticancer activity. In this present study, Moringa oleifera leaf extract functions as reducing, capping, and stabilizing agents for the preparation of pure and silver doped copper oxide nanoparticles of various (5%, 10%, 15%, and 20%) concentrations. The synthesized materials were subjected to various characterization techniques. XRD, reveals that nanoparticles are in the monoclinic phase and the mean crystallite size for pure and Ag-doped CuO nanoparticles of various concentrations is found to be in the range of 15.22–3.67 nm. SEM images show that nanoparticles are relieved from particle size enlargement and EDAX analysis confirmed the existence of elements like Cu, O, and Ag. The biomolecules present in the leaf extract that helps for the bio-reduction of CuO nanoparticles were identified by FT-IR spectrometer. From UV–vis absorption spectra, peaks from 210 to 240 nm were observed. Furthermore, it also exhibits that if the concentration of silver doping increases there will be an increase in the energy bandgap. By MTT assay, the powerful cytotoxic activity for the synthesized pure and Ag-doped CuO nanoparticles of different concentrations has been probed against human lung cancer A549 cells. Among that, 20% of Ag-doped CuO nanoparticles showed a decrease in cell viability with an IC50 value of 15 µg/mL. After treatment with green synthesized nanoparticles, the cell death was assessed through three types of assessments which include AO/EB dual staining, Hoechst staining, and DCFH-DA staining assays in the cell line. The results betrayed that through apoptosis induction, the tested samples treated against human lung cancer A549 cells showed inhibition in cell proliferation and enhances morphological, and cytological alterations via an increased level of ROS accumulation in the NSCLC cell line.

Bisphenol A-induced neurobehavioral transformation is associated with augmented monoamine oxidase activity and neurodegeneration in zebrafish brain

As bisphenol A (BPA) effortlessly crosses the blood-brain barrier, its serious impacts on the neuronal microenvironment towards precocious induction of oxidative stress and neuromorphological alteration can’t be ignored. Incidentally, a symmetric study establishing the possible link of transformed neurobehavior with heightened monoamine oxidase (MAO) activity and neuromorphological alteration in zebrafish brain subsequent to BPA-exposure is limiting in the literature. The study was conducted to delineate the role of BPA towards the genesis of aggressive behaviour in zebrafish and its correlation with brain MAO activity. Mirror biting test and open field test were conducted to evaluate the aggressive and explorative behaviour respectively. Biochemical studies were performed to delineate the modulation of the antioxidant defence system. Cresyl violet staining and Hoechst staining in the periventricular grey zone of the zebrafish brain were conducted to evaluate neuronal pyknosis and chromatin condensation. Our study showed that BPA exposure is associated with the genesis of aggressive neurobehavioral response. Moreover, the brain MAO activity, oxidative stress and chromatin condensation were increased with increase in exposure duration. The results of the present study gave conclusive evidence that BPA act as a potent neurotoxicant in transforming the native neurobehavioral response of zebrafish through heightened oxidative stress, MAO activity and altered neuromorphology.

Withania somnifera deploys immunomodulation and exerts anticancer effects on lung adenocarcinoma cells

Immunomodulation using phytochemicals is the alternate therapeutic modality to treat cancer. Withania somnifera (L.) Dunal is known to possess several pharmacological activities such as anti-inflammatory, anti-arthritic, neuroprotective and antitumor activity . In this study, we have evaluated the immunomodulatory and anticancer effects of W. somnifera (L.) Dunal ethanolic extract on lung adenocarcinoma cells. Anti- inflammatory effect was assessed using albumin denaturation method. Cell viability of murine macrophage cells was evaluated using MTT assay and nitric oxide scavenging capacity was assessed using Griess method. Compatibility of the extract on RAW 267.4 and THP 1 cells was assessed using morphological assessment, AO-EB and Hoechst staining methods. Further, RAW 267.4 (macrophage) and THP 1 (monocyte) cells were treated with the extract to assess the arginase, phagocytic and pinocytic activity. W. somnifera (L.) Dunal extract was administered to lung adenocarcinoma cells and evaluated for the induction of ROS to indicate the apoptotic effects. Further, immunofluorescence and immunoblotting was performed using the cell lysates to study the effect of extract on the expression of CTLA 4, HIF-1 α , TNF- α , and PECAM-1. In this study we found that, W. somnifera (L.) Dunal extract showed a dose dependent inhibition of protein denaturation and the extract did not show any cytotoxicity on RAW 267.4 cells. Griess assay performed with the culture supernatants revealed that the extract exhibited a dose dependent inhibition of NO production. RAW 267.4 and THP 1 cells showed a significant inhibition of arginase activity with ⁎⁎⁎ p <0.0005. At higher concentration of treatment, both the immune cells displayed a significant increase in the phagocytic and pinocytic effect. W. somnifera (L.) Dunal treatment increased the ROS production in lung adenocarcinoma cells and downregulated the expressions of CTLA-4, HIF-1 α , TNF- α , and PECAM-1. This study confirmed that W. somnifera (L.) Dunal ethanol extract possesses immunomodulatory effect, to enhance the cell death in lung cancer cells via ROS generation. • Ashwagandha has the ability to enhance nitric oxide scavenging activity, along with decreased activity of arginase, which is responsible for the cancer cell invasion and metastasis. • Ashwagandha regulates the phagocytic and pinocytic property in macrophages and monocytes, indicating the cellular defense against abnormal cells. • ROS generation is responsible for the apoptosis of the cells, upon treatment of ashwagandha A549 lung adenocarcinoma cells showed apoptosis by increasing the expression of ROS. • Decreased expression of HIF-1 α & TNF- α promotes cancer cell death.

High-Efficiency Multi-Drug Functional Profiling in a Microfluidic Device Towards Personalized Predictions in Pediatric Leukemia

Treatment of pediatric acute lymphoblastic leukemia (ALL) has seen dramatic improvements over the last several decades, leading to overall survival rates of over 85%. However, for patients with relapsed/refractory disease, achieving complete remission remains extremely challenging. Improving treatment outcomes for these patients will require increasingly personalized treatment strategies that make use of functional screens in addition to molecular profiling technologies for treatment selection. Given the past success of combination chemotherapy in cancer treatment and recent promise for newly developed targeted agents, functional assays that are able to evaluate patient response to combinations of therapies will likely be most effective in identifying clinically actionable treatment protocols. Moreover, assays that can achieve this with minimal patient sample and reduced experimental workload will have the greatest potential for clinical translation and integration into clinical workflows. To that end, this work aims to develop a microfluidic assay to directly test candidate combinations of therapeutics in patient derived ALL cells. We aim for this to serve as a more effective way to screen for drug combination response and higher order interactions relative to standard wellplate formats. Once validated, this assay can be used to identify biomarkers of response to standard and experimental combination regimens in pediatric ALL, and ultimately, could be used to prospectively guide therapy in newly diagnosed patients. The developed device achieves this by enabling simultaneous exposure to 3 superimposed small molecule drug gradients across a 3D culture of leukemia cells such that each location in the device comprises a unique combination of all 3 drugs within a simplex (Fig 1A). Single cell drug concentration exposure is determined from experimentally validated steady state computational fluid dynamics (CFD) model predictions, and single cell viability is analyzed via Calcein (live), PI (dead) and Hoechst (nuclear) staining following 48 hours of treatment. Using this system, we then develop a novel experimental and computational framework for evaluating drug combination efficacy and synergy across the full range of combination ratios captured in our device. Specifically, we evaluate response across multiple devices at optimally spaced dose levels, increasing coverage of the 3-drug combination space and allowing for custom implementation of established synergy models. This enables synergy assessment in a continuous gradient of drug combination ratios, as opposed to the discrete combination ratios commonly evaluated in standard assays, and may further improve detection of drug synergy and identification of the most potent and efficacious combination ratios. To demonstrate this, we analyze response to a combination of daunorubicin, vincristine, and nelarabine in Jurkats, a T-ALL cell line, across 7 different devices at 7 different dose levels (Fig 1B). We quantify viability as a function of concentration across each device and each dose level, and generate dose response curves at distinct zones within the device. Specifically, we generate dose response curves in zones dominated by daunorubicin, vincristine, and nelarabine alone and find half maximal effective concentration values (EC50 values) similar to those reported in literature. We further evaluate response at varying ratios of each 2-drug combination, and generate dose response curves as shown in Fig 1B. Analyzing the 2-drug combination dose responses relative to single-drug dose responses allows for quantification of 2-drug combination synergy based on Loewe and DiaMOND synergy models, and future work will extend our developed method to analyze emergent 3-drug combination synergy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Abstract 12398: CD151 Expression Reflects Different Features in Cardiac Subtype Specification From Human Pluripotent Stem Cells

Introduction: In conventional cardiac differentiation methods, a heterogeneous cardiac population is generated from human induced pluripotent stem cells (hiPSCs). Purifying ventricular- or atrial cardiomyocytes (VCMs or ACMs, respectably) is valuable for drug discovery for region-specific heart diseases. Previously, we have identified a novel marker, CD151, which can separate advanced specified subtypes; CD151 high VCMs in ventricular inducing condition (VIC) and CD151 low ACMs in atrial inducing condition (AIC). Although CD151 is known as a cell adhesion molecule, the function in cardiac subtype specification is unclear. In this study, we addressed the mechanisms of how CD151 distinguishes cardiomyocyte subtypes in cardiac differentiation from hiPSCs. Methods: We performed RNA sequencing in VIC- and AIC- CD151 high/low CMs. Pathway analysis using differential expression genes between CD151 high/low CMs was conducted to identify prominent pathways in subtype specification. Results: In VIC, mitosis-related genes were enriched in CD151 high VCMs. To clarify whether those genes affect cell proliferation or multi-nucleation, we performed the flow cytometric analysis of CD151 high/low VCMs with KI-67 proliferation marker and Hoechst staining. Although the percentage of KI67-positive cells was almost the same, binucleated cells were significantly more in CD151 high VCMs than in CD151 low VCMs. In AIC, transcriptome analysis suggested that Notch signaling was activated in CD151 high ACMs. To reveal that Notch signaling inhibits atrial specification, we analyzed ACMs treated with LY411575, a Notch signaling inhibitor. LY411575 induced atrial marker gene expression and increased the number of cells showing action potentials of ACMs. CD151 low ACMs after LY411575 treatment contained ACMs with the highest efficiency. Conclusion: Our results demonstrate that the CD151 expression indicates mitotic activity in VCMs. CD151 high VCMs are more advanced specified VCMs with binucleation without cell proliferation. On the other hand, in ACMs, CD151 works as an indicator of Notch signaling, which suppresses atrial specification. Our study provides a novel approach to generating and purifying hiPSCs-ACMs by Notch inhibition and CD151 separation.

3D-Printed Impedance Micropump for Continuous Perfusion of the Sample and Nutrient Medium Integrated with a Liver-On-Chip Prototype.

In recent decades, organ-on-chip devices have gained substantial interest as an alternative for studying the pathophysiological processes relevant to drug screening. Micropumps are being utilized to simulate the in vivo physiological fluid flow more realistically in these organ-on-chip devices. Micropumps play a crucial role in pumping, perfusion, and circulation of fluids in various microdevices such as on-chip PCR, DNA microarrays, miniature bioreactor cell separation, and lab-on-chip biosensing platforms. With the rapid growth in technology, efficient pumping for proper circulation of media and nutrients has become imperative. In this study, we have described the design and development of an open-source impedance micropump for continuous perfusion of nutrient medium in a liver-on-chip prototype. This micropump is controlled via an integrated microcontroller board, with an observed flow rate ranging from 0.2 to 2 mL/min. Google Sketchup 2020 and DLP 3D printing were used to fabricate small precise parts of the impedance micropump. The flow rate was measured to characterize the actuating performance of the micropump. The poly-dimethyl siloxane-based liver-on-chip prototype has been fabricated using a soft photolithography procedure. Further, a study of continuous perfusion of culture medium through the liver-on-chip containing the Hepg2 cell line was successfully performed by integrating it with the impedance micropump. Hoechst staining and Alamar Blue observed cell viability to confirm the healthy cell growth inside the liver-on-chip microfluidic chip. The compactness of the overall setup allows it to fit in a Petri plate, eliminating chances of contamination while cell handling.

Comparison of Wound Healing Effect of Skin Micrograft Impregnated into Two Kinds of Artificial Dermis in a Murine Wound Model.

Skin MG was prepared from the skin of C57BL/6JJcl mice using Rigeneracons and administered onto Pelnac Gplus and Integra sheets. The amount of MG suspension impregnated in Pelnac Gplus and Integra was evaluated. Pelnac Gplus and Integra sheets combined with MG were applied to murine defects, and wound area, neoepithelium length, granulation tissue formation, and newly formed capillaries were compared with the control groups on days 7 and 14. The weight percentage of the MG absorbed by Pelnac Gplus and Integra was 88.8% ± 3.5% and 28.2% ± 7.0%, respectively (P < 0.05). In the in vivo experiment, the area of newly formed granulation tissue and both the number and area of newly formed capillaries in the PelnacG + MG group were significantly larger than those in the control group at 14 days after implantation (P < 0.05). Skin MG was successfully impregnated into Pelnac Gplus by simple administration but not into Integra. Administration of skin MG into the Pelnac Gplus promoted granulation formation and angiogenesis. Pelnac Gplus was more suitable than Integra in the combination therapy.

WITHDRAWN: Marsdenia tenacissima extract activates FoxO3a nuclear translocation to promote apoptosis via inhibiting autophagy in prostate cancer

• MTE inhibits the proliferation of PCa cells by inducing apoptosis • MTE reduces Atg5/Atg12 protein expression and promotes nuclear translocation of FoxO3a • MTE-induced apoptosis relies on the inhibition of autophagy and FoxO3a activation in PCa cells Prostate cancer (PCa) is one of the most commonly diagnosed malignant tumors in older men, causing high morbidity and mortality along with significant economic and medical costs. Marsdenia tenacissima extract (MTE) has been approved for the treatments of esophageal, gastric, lung, and liver cancers in China. Recently, it has been reported that MTE exhibits a promising therapeutic effect on PCa, but the underlying mechanism is unclear. To investigate the action mechanisms of MTE against PCa. HPLC-CAD-QTOF-MS/MS technology was performed to analyze the component characterization of MTE. Effects of MTE on PCa cell proliferation, apoptosis, and autophagy were assessed using CCK-8 assays, Hoechst staining, TUNEL staining, transmission electron microscopy, and acridine orange staining. The key molecules and important signaling pathways regulated by MTE in PCa cells were explored using proteomics and qRT-PCR technology. FoxO3a expression and localization were monitored by western blot, immunofluorescence, and immunohistochemistry assays. Rapamycin, an inducer of autophagy, was used to investigate the relationship between MTE-induced autophagy and apoptosis. The PC3 xenograft mouse model was employed to verify the anti-PCa effect and underlying mechanism of MTE in vivo . In total, HPLC-CAD-QTOF-MS/MS analysis identified 21 components of MTE. MTE significantly inhibited the cell proliferation of several PCa cell lines. In PC3 and C4-2 cells, MTE treatment significantly induced apoptosis, increased cleaved PARP and Bax expressions, and decreased Bcl-2 expression. Proteomic and qRT-PCR analyses revealed that the anti-PCa activity of MTE was closely related to the autophagic degradation of FoxO3a. Moreover, MTE significantly inhibited autophagy via downregulating the expression of Atg5/Atg12. More importantly, MTE significantly increased the expression and nuclear translocation of FoxO3a. Interestingly, Rapamycin reversed MTE-induced apoptosis and FoxO3a protein accumulation. Finally, in PC3 xenograft mice, MTE treatments significantly suppressed tumor growth, induced tumor cell apoptosis, inhibited tumor cell autophagy, and activated FoxO3a nuclear translocation. The present study demonstrated that MTE inhibits the autophagic degradation of FoxO3a, which promotes FoxO3a nuclear translocation and ultimately induces PCa apoptosis. Our data provide new insights into the role of MTE as a potential treatment for PCa.

Abiraterone Acetate, in Combination with Apigenin, Attenuates the Survival of Human Castration-Sensitive Prostate Cancer Cells.

Abiraterone acetate (AA) is a selective inhibitor of CYP17 α-hydroxylase, which is crucial for androgen biosynthesis. Apigenin (Api) is a natural plant-derived flavonoid with potent antiproliferative and antimigration effects. We aimed to investigate the possible role of Api in combination with the androgen receptor inhibitor AA in the treatment of androgen-sensitive human prostate cancer LNCaP cells. The cells were either exposed to 10 μM AA, 25 μM Api, or in combination for 48 hours, then the viability rate was determined by the MTT test, whilst apoptosis and cell cycle phases were assessed by image-based cytometry. The expression of selected mRNA and proteins were evaluated by RT-qPCR and Western blot, respectively. The combination of AA and Api significantly inhibited LNCaP as well as androgen-insensitive PC3 cell survival in a manner more marked than observed with either single treatment. Co-administration of Api with AA triggered apoptosis. This effect was demonstrated by Hoechst staining, and up-regulation of Bax, cytochrome c, caspase -3, and - 8 and down-regulation of Bcl-2 expression confirmed the effect. AA and Api each individually arrested the cell cycle in the G1 phase, with dual applications, leading to no further increase in the effect produced. The expression of NF-κB p105/p50 and the phosphorylation of AKT markedly decreased after apigenin treatment, with combination treatment leading to a favourable effect in terms of further augmenting the reduction. The co-administration of Api with AA strongly enhanced the efficacy of AA therapy in the treatment of prostate cancer cells. These data suggested that the combination of AA and Api would be a potential chemotherapeutic strategy against prostate cancer.

Effects of Salidroside Combined with Paclitaxel on Proliferation, Migration, and Epithelial Mesenchyme of Colorectal Cancer Cells.

Colorectal cancer (CRC) is a multifactorial disease and one of the most common malignancies worldwide. Salidroside (Sal) is a plant with a wide range of pharmacological effects and plays an important role in the treatment of many diseases, and is considered a new hope for the treatment of tumors. The purpose of this study was to investigate the effect of the combination of Sal and paclitaxel (Pac) on colorectal cancer cells and its mechanism of action. The effects of different mass concentrations of Sal, Pac, and the combination intervened in the cells for 48 h were examined using the CCK8 method. The inhibition rate was obtained, and the optimal concentration of the respective drug group was screened. The proliferative capacity of the respective group was obtained. Subsequently, the results of apoptosis, cloning, migration, invasion, and angiogenesis were observed through cell morphological analysis (shape observation and Hoechst staining), colony formation assay, cell scratching assay, Transwell, angiogenesis assay, and protein immunoblotting (Western blotting) to detect the expression of epithelial-mesenchymal transition (EMT)-associated proteins and PI3K pathway-associated proteins. Different concentrations of Sal, Pac, and the combined application had significant effects in inhibiting cells in a concentration-dependent manner. Compared with the control group, the Sal group, the Pac group, and the combination group significantly inhibited the clonal number, migration, invasion, and tube-forming ability of colorectal cancer cells. Besides, the combined application had a better effect than the Sal and Pac groups. The apoptosis level was up-regulated in all drug groups, and the up-regulation was more significant in the combination group. The expression of E-cad protein was up-regulated, the expression of N-cad and Vim protein was down-regulated, and the expression of PI3K and AKT phosphorylation was down-regulated in the respective group, and the difference was more significant in the combination group compared with the group of individual drugs. The combined application of Sal and Pac significantly can decrease the survival rate of colorectal cancer cells, and the mechanism may be correlated with the blocking of the PI3K/AKT pathway, thus inhibiting EMT.

Direct labeling of milk cells without centrifugation for counting total and differential somatic cells using flow cytometry.

Somatic cell count (SCC) in milk is an essential indicator for defining and managing udder health. However, analyzing differential SCC (dSCC) can be helpful in determining the type or evolution stage of mastitis. A high abundance of polymorphonuclear cells (PMN) is associated with acute mastitis; however, the status of a chronic disease is less well characterized. A method capable of analyzing SCC and dSCC can prove to be a helpful tool for monitoring the status of evolution of mastitis disease in a better way. Therefore, a new direct-flow cytometry method was developed to count and differentiate somatic cells in milk without the steps of centrifugation or washing, avoiding variabilities that occur due to enrichment or loss of specific cell types. In this new method, SCC is analyzed using the method of DNA staining with Hoechst stain, whereas dSCC are analyzed using specific antibodies targeting 2 main cell types associated with mastitis: PMN cells and antigen-presenting cells, which are associated with innate and adaptive immunity. Equivalent SCC values were obtained between the new method and the routine ISO 13366-2 method in a comparison of 240 raw milk samples. Furthermore, dSCC results were confirmed by microscopy after May-Gründwald-Giemsa staining in 165 quarter milk samples from healthy and diseased cows. The method was verified with fluorescence microscopy on the 2 targeted cell types and in raw milk samples. The newly developed method is independent of any instrument and can be further designed to differentiate other cell types and animal species by selecting appropriate antibodies.

Copper Nanoparticles Induce Apoptosis and Oxidative Stress in SW480 Human Colon Cancer Cell Line.

Cu nanoparticles (CuNPs) have various applications in biomedicine, owing to their unique properties. As the effect of CuNPs on the induction of oxidative stress and apoptosis in the human colorectal cancer cell line SW480 has not yet been studied, we investigated the toxicity and mechanism of action of these NPs in SW480 cells. MTT assay was performed to assess the effect of the particles on the viability of SW480 cells. The levels of oxidative stress were assessed after 24h of treatment with CuNPs by evaluating the Reactive Oxygen Specious (ROS) production. The antioxidant enzyme activity was assessed using a colorimetric method. To investigate the effect of NPs on cellular apoptosis, Hoechst33258 staining was performed, and the expression of Bax, Bcl-2, and p53 was evaluated by qRT-PCR. The MTT assay results showed that CuNPs inhibited the viability of SW480 cells. Moreover, the increase in ROS production at all three concentrations (31, 68, and 100μg/ml) was significant. It has been observed that CuNPs lead to increased expression of Bax and p53, and decreased expression of Bcl-2. Hoechst staining was performed to confirm apoptosis. In conclusion, the induction of apoptosis demonstrated the anticancer potential of the CuNPs.

The Impact of Chemoradiotherapy on Biophysical Properties of Glioblastoma Utilizing a Novel Murine Model

<h3>Purpose/Objective(s)</h3> Glioblastoma, historically viewed as a single disease entity, is now known to be composed of various subtypes. Here, we present our work evaluating the effects of chemoradiotherapy on cellular biophysical processes using a novel murine model of glioblastoma developed by our group. <h3>Materials/Methods</h3> Primary tumor cell lines were established from a murine glioma model generated from a synthetic DNA transposon system delivered oncogenic drivers SV40-LgTA and either NRASV12 or PDGFB. We have previously shown these cell lines, termed NRAS and PDGF, respectively, recapitulate the mesenchymal and proneural tumor subtypes. Treatment was administered with temozolomide (TMZ) to a maximum of 100µM, single fraction radiation therapy (RT) to a maximum of 10 Gy, or both. Cells were stained with Hoechst dye, imaged with an epifluorescence microscope with 10X objective lens, and counted with an image segmentation algorithm daily for 4 days to estimate cell survival and proliferation. On post-treatment day 4, surviving cells were plated on Matrigel coated 9.3kPa polyacrylamide gel and imaged with an inverted, transmitted light microscope with 10X objective lens every 5 minutes for 4 hours. Single cell mean squared displacement (MSD) was measured from time lapse images via an image segmentation algorithm. The random motility coefficient (µ) was calculated by the relationship MSD(t) = 4µt, where t is time. Data was compared via the Kruskal-Wallis test with Bonferroni correction for multiple comparisons when appropriate. <h3>Results</h3> There was no significant difference in cellular death or proliferation between treatment and control arms for the PDGF cell lines. In the NRAS cell lines, TMZ alone did not induce cell death while RT (P < 0.01) and combined modality therapy (P < 0.01) yielded cellular killing in a dose dependent manner. Furthermore, RT-treated NRAS cells proliferated more slowly than non-treated cells (0.05 vs. 0.20 hr-1, P=0.04). Control NRAS cell lines migrated faster than their PDGF counterparts at baseline (3.82 vs. 0.73 µm2/hr, P<1E-5). Within each cell line, the use of combined modality therapy significantly reduced cellular motility (PDGF: 0.73 vs. 0.21 µm2/hr, P=0.008; NRAS: 0.65 vs. 3.82 µm2/hr, P<1E-5). RT monotherapy additionally reduced motility in NRAS cell lines (1.07 vs. 3.82 µm2/hr, P<1E-5) while no difference was observed in PDGF cells. TMZ monotherapy did not affect motility within either cell line. <h3>Conclusion</h3> We evaluated biophysical properties of a novel murine glioma model utilizing oncogenic drivers overexpressing NRASV12 or PDGFB which we have previously shown recapitulates the mesenchymal and proneural subtypes of human glioblastoma, respectively. Here, we observed phenotypic differences in cellular migration following RT and TMZ regimens which could have subtype-specific ramifications for radiotherapy dose and target volume delineation.

Delphinidin induces autophagic flux blockage and apoptosis by inhibiting both multidrug resistance gene 1 and DEAD-box helicase 17 expressions in liver cancer cells.

To investigate the function and regulatory mechanisms of delphinidin in the treatment of hepatocellular carcinoma. HepG2 and HuH-7 cells were treated with different concentrations of delphinidin. Cell viability was analysed by 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The cell autophagy and autophagic flux were analysed by LC3b-green fluorescent protein (GFP)-Adv and LC3b-GFP-monomeric red fluorescent protein-Adv transfected HepG2 and HuH-7 cells, respectively. Cell apoptosis was analysed by Hoechst33342 staining, terminal deoxynucleotidyl transferase dUTP nick end labeling staining and DNA laddering. Cell autophagy, apoptosis and survival related protein expressions were detected by Western blotting. After treatment with different concentrations of delphinidin, the cell survival rate was significantly decreased. Delphinidin could block the autophagic flux, resulting in a significant increase in autophagosomes, and led to an increase in cell apoptosis. The combined application of delphinidin and cisplatin could promote the antitumour effect and reduce the dose of cisplatin in tumour cells. Further mechanism studies reveal that delphinidin could inhibit the multidrug resistance gene 1 (MDR1) and the tumour-promoting transcription cofactor DEAD-box helicase 17 (DDX17) expression in tumour cells. Overexpression of DDX17 could reverse delphinidin's antitumor function in tumour cells. Delphinidin has a strong anti-tumour effect by inducing tumour cell autophagic flux blockage and apoptosis by inhibiting of both MDR1 and DDX17 expression.

Biointerfacial Analysis of HEK293 Cells in Contact with Hydrogels based on Poly‐N‐Isopropylacrylamide and Copolymers

AbstractSeveral researches have demonstrated that synthetic hydrogels can mimic the mechanical and physicochemical properties of native extracellular matrices and act as cell‐scaffold. The biointerface can influence over tissue activities such as adhesion, signaling, cell–cell communications, and proliferation. In this work, the behavior of human embryonic kidney cells (HEK293) in contact with hydrogel surfaces based on poly‐N‐isopropylacrylamide (PNIPAM) and copolymers is studied. Ionic and neutral hydrogel surfaces are synthesized by free radical polymerization and characterized by Fourier‐transform infrared spectroscopy, swelling capacity, and wettability at culture conditions. Viability, proliferation, and bioadhesive capacity are analyzed by 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide and Neutral Red assays, [3H]‐thymidine technique, Acridine Orange and Hoechst dye. Noncytotoxic and proliferative effects are observed in all cases. HEK293 cells are mainly adapted and adhered on neutral and low ionic charge surfaces showing typical cell morphology, and normal mitotic rates. While, lower adhesion, abnormal nuclear and cytoplasmic morphologies with mitotic/fragmentation processes are observed after contact with high ionic surfaces. The cellular cycle after cultive on PNIPAM, assessed by flow cytometry, is not affected regarding control surfaces (polystyrene). Therefore, hydrogel surfaces with neutral and low ionic charges can be apt to in vitro tissue development and possible applications of biomedical treatments.

Regulation of survivin and caspase/Bcl-2/Cyto-C signaling by TDB-6 induces apoptosis of colorectal carcinoma LoVo cells.

Colorectal carcinoma (CRC) treatment remains severe. Survivin is aberrantly overexpressed in CRC tissues and might be a potential target for CRC treatment. TDB-6 is a new taspine derivative. The purpose of this study is to investigate the inhibitory effect of TDB-6 on CRC and its underlying mechanism. The MTT assay and xenograft model were utilized to investigate the inhibitory effect of TDB-6 on LoVo cells in vitro and in vivo. Hoechst staining and Annexin-V FITC/PI analysis were conducted to study the effect of TDB-6 on LoVo cell apoptosis. Mitochondrial membrane potential (Δψm) assay was conducted to demonstrated whether TDB-6 could induce mitochondrial-mediated apoptosis of LoVo cells. Western blotting was conducted to investigate the effect of TDB-6 on survivin protein and caspase/Bcl-2/Cyto-C signaling. The results indicated that TDB-6 induced mitochondria-mediated apoptosis and inhibited the proliferation and growth of LoVo cells in vitro and in vivo. Mechanistic investigation utilizing western blotting indicated that TDB-6 inhibited survivin protein expression, and the inhibitory effect was augmented by TDB-6 and YM-155 co-administration, which revealed that TDB-6 might induce apoptosis of LoVo cells by targeted regulation of survivin. TDB-6 also regulated survivin downstream signaling. It significantly increased the protein level of cleaved caspase-3, cleaved caspase-7, cleaved caspase-9, cleaved-PARP, and Cyto-C, and decreased the protein level of Bcl-2. TDB-6 might be a promising survivin inhibitor with great potential for CRC treatment.

Protective effect of Tongqiao Huoxue Decoction containing cerebrospinal fluid on OGD/R-damaged HT22 cells via regulation of ASK1/MKK4/JNK signaling pathway

To investigate the protective effect of Tongqiao Huoxue Decoction containing cerebrospinal fluid(TQHXD-CSF) on HT22 cells damaged by oxygen-glucose deprivation/reoxygenation(OGD/R) and whether the mechanism is related to the regulation of ASK1/MKK4/JNK signaling pathway. HT22 cells were subjected to OGD/R to simulate cerebral ischemia-reperfusion injury(CIRI). Then the cells were randomly divided into five groups: blank cerebrospinal fluid(control group), OGD/R group, TQHXD-CSF group, Z-VAD-FMK group(20 μmol·L~(-1)) and TQHXD-CSF+Z-VAD-FMK group. Except the control group, cells in the other groups were reoxygenated for 12 h after 6 h of oxygen and glucose deprivation for modeling OGD/R, and group administration was performed. Cell viability and cytotoxicity were detected by CCK8 and LDH assay kit, respectively and the morphology of HT22 cells was observed by inverted microscope. Western blot and qRT-PCR were employed to detect the protein and mRNA expression levels of Bax, Bcl-2 and caspase-3, respectively. Then HT22 cells were assigned into the control group, OGD/R group, si-NC group, si-ASK1 group, TQHXD-CSF group and TQHXD-CSF+si-ASK1 group. Cell viability, proliferation and apoptosis were determined by CCK8, electric cell-substrate impedance sensing(ECIS), and Hoechst staining and flow cytometry, respectively. The protein expression of MKK4, p-MKK4, JNK, p-JNK, c-Jun, p-c-Jun, Cyt C, Bax, Bcl-2 and caspase-3 was tested by Western blot. The results showed that compared with OGD/R group, TQHXD-CSF significantly enhanced cell viability, improved cell morphology and reduced the protein and mRNA expression levels of Bax, Bcl-2 and caspase-3. In addition, when ASK1 was silenced, compared with OGD/R group, TQHXD-CSF remarkably improved cell viability, and decreased apoptosis rate and the protein expression levels of p-MKK4, p-JNK, p-c-Jun, Cyt C, Bax/Bcl-2 and caspase-3, but the effect was not as good as that of TQHXD-CSF+si-ASK1 group. In conclusion, TQHXD-CSF can inhibit apoptosis mediated by ASK1/MKK4/JNK signaling pathway in OGD/R-damaged HT22 cells, and has protective effect on ischemia-reperfusion injury.

High-content phenotyping of Parkinson's disease patient stem cell-derived midbrain dopaminergic neurons using machine learning classification.

SummaryCombining multiple Parkinson's disease (PD) relevant cellular phenotypes might increase the accuracy of midbrain dopaminergic neuron (mDAN) in vitro models. We differentiated patient-derived induced pluripotent stem cells (iPSCs) with a LRRK2 G2019S mutation, isogenic control, and genetically unrelated iPSCs into mDANs. Using automated fluorescence microscopy in 384-well-plate format, we identified elevated levels of α-synuclein (αSyn) and serine 129 phosphorylation, reduced dendritic complexity, and mitochondrial dysfunction. Next, we measured additional image-based phenotypes and used machine learning (ML) to accurately classify mDANs according to their genotype. Additionally, we show that chemical compound treatments, targeting LRRK2 kinase activity or αSyn levels, are detectable when using ML classification based on multiple image-based phenotypes. We validated our approach using a second isogenic patient-derived SNCA gene triplication mDAN model which overexpresses αSyn. This phenotyping and classification strategy improves the practical exploitability of mDANs for disease modeling and the identification of novel LRRK2-associated drug targets.