Trypan Blue Method Research Articles

Antioxidant and Anti-Proliferative Effects of Ornithogalum balansae Extracts Collected in Black Sea Region Against Lung Cancer Cells

Ornithogalum is a genus of wild herb species widely used as food and in traditional medicine. This study investigated some of the biologically active properties of Ornithogalum balansae grown in the eastern Black Sea region of Türkiye. The investigations were carried out using the methanolic extracts of the plant’s aerial and bulbs (B1 and B2) parts. The phenolic composition was examined as total phenolic content (TPC), total flavonoid content (TFC), and HPLC-PDA. The antioxidant capacity of the extracts was tested based on ferric reducing/antioxidant power (FRAP) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging methods. The anti-proliferative activity was tested against metastatic cell lines H1299 and H209, non-small-cell lung A549, and fibroblast MRC-5 cell lines using MTT and trypan blue methods. Wound-healing and invasion chamber assays were used to determine the inhibitory effects of the extracts on migration and invasion, respectively. The extract of the aerial part contained a large number of phenolic substances and high antioxidant capacity. The extract exhibited a significant anti-proliferative effect on the human lung cancer cells (A549 and H209), with IC50 values of 0.97 ± 0.04 and 1.06 ± 0.07 µg/mL, respectively. Moreover, the aerial part exhibited inhibition of migratory and invasive capacities in A549 cells at a concentration of 1.50 µg/mL. The findings associated with O. balansae suggest a promising therapeutic potential against lung cancer cells.

The Influence of Bioceramic Cements on the Activity of Macrophages of Different Lineages: A Laboratory Study

IntroductionThe repair process of periradicular tissues depends, among other factors, on the properties of endodontic cements. Macrophages are among the main cells involved in this process. Materials and MethodsMurine peritoneal macrophages obtained from C57BL/6 (MBL6) and BALB/c (MBalb) mice, respectively, were cultured with capillaries containing or not Endosequence BC Sealer (BC), Sealer Plus BC (MK), Bio-C Sealer (Ang), and mineral trioxide aggregate (MTA). Cell viability was measured by Trypan blue and MTT methods at 24, 48, and 72 hours. Cell adhesion, phagocytosis of Saccharomyces boulardii, production of reactive oxygen species (ROS), nitric oxide (NO), and the cytokines tumor necrosis factor-α and transforming growth factor (TGF)-β were also evaluated. The data were analyzed using the analysis of variance test (P < .05). ResultsCell viability was similar between bioceramic sealers and MTA (P > .05). There was no statistical difference between both macrophages when adherence and phagocytose were assayed. The presence of inflammation stimulus significantly altered the production of ROS by MBL6 macrophages in contact with the cements. The production of TGF-β was similar for both lineages of macrophages. ConclusionsThis study shows that the evaluated bioceramic cements do not interfere with MBL6 and MBalb macrophage adhesion, phagocytic capacity, or TGF-β production. The cements stimulated the production of ROS by MBL6 macrophages in response to induced inflammation, potentially favoring the elimination of residual pathogens.

Essential oil of oregano (Origanum vulgare L.) reduces infection and proliferation of Toxoplasma gondii in BeWo cells with induction of autophagy and death of tachyzoites through a mechanism similar to necrosis.

Toxoplasmosis poses a global health threat, ranging from asymptomatic cases to severe, potentially fatal manifestations, especially in immunocompromised individuals and congenital transmission. Prior research suggests that oregano essential oil (OEO) exhibits diverse biological effects, including antiparasitic activity against Toxoplasma gondii. Given concerns about current treatments, exploring new compounds is important. This study was to assess the toxicity of OEO on BeWo cells and T. gondii tachyzoites, as well as to evaluate its effectiveness in in vitro infection models and determine its direct action on free tachyzoites. OEO toxicity on BeWo cells and T. gondii tachyzoites was assessed by MTT and trypan blue methods, determining cytotoxic concentration (CC50), inhibitory concentration (IC50), and selectivity index (SI). Infection and proliferation indices were analyzed. Direct assessments of the parasite included reactive oxygen species (ROS) levels, mitochondrial membrane potential, necrosis, and apoptosis, as well as electron microscopy. Oregano oil exhibited low cytotoxicity on BeWo cells (CC50: 114.8µg/mL ± 0.01) and reduced parasite viability (IC50 12.5 ± 0.06µg/mL), demonstrating 9.18 times greater selectivity for parasites than BeWo cells. OEO treatment significantly decreased intracellular proliferation in infected cells by 84% after 24h with 50μg/mL. Mechanistic investigations revealed increased ROS levels, mitochondrial depolarization, and lipid droplet formation, linked to autophagy induction and plasma membrane permeabilization. These alterations, observed through electron microscopy, suggested a necrotic process confirmed by propidium iodide labeling. OEO treatment demonstrated anti-T. gondii action through cellular and metabolic change while maintaining low toxicity to trophoblastic cells.

Single cell analysis of Chinese hamster ovary cells during a bioprocess using a novel dynamic imaging system.

Reliable monitoring of mammalian cells in bioreactors is essential to biopharmaceutical production. Trypan blue exclusion is a method of determining cell density and viability that has been used for over one hundred years to monitor cells in culture and is the current standard method in biomanufacturing. This method has many disadvantages however and there is a growing demand for more detailed and in-line measurements of cell growth in bioreactors. This article assesses a novel dynamic imaging system for single cell analysis. This data shows that comparable total cell density, viable cell density and percentage viability data shown here, generated by the imaging system, aligned well with conventional trypan blue counting methods for an industrially relevant Chinese Hamster Ovary (CHO) cell line. Furthermore, detailed statistical analysis shows that the classification system used by the PharmaFlow system can reveal trends of interest in monitoring the health of mammalian cells over a 6-day bioreactor culture. The system is also capable of sampling at-line, removing the necessity for taking samples off-line and enabling real time monitoring of cells in a bioreactor culture.

Development and characterization of stable proanthocyanidin‐loaded PLAROsomes as a potential drug carrier system for augmenting anticancer activity

AbstractThe aim of the present study was to develop potential drug carrier system formulations, namely PLAROsomes for the delivery of Proanthocyanidin (PACY), isolated from the extract of dried grape seeds. The PLAROsomes loaded with PACY were prepared using the thin film hydration method. The influence of various process parameters and material attributes was investigated using the design of experiments (DoE). The PACY‐loaded PLAROsomes were characterized using hyphenated tools. The in‐vitro anticancer activities of PACY‐loaded PLAROsomes were confirmed using the Trypan blue method, MTT method, and flow cytometric analysis on MCF‐7 cells. The polymer‐to‐lipid ratio, among various process parameters and material attributes, significantly influenced the average particle size. Additionally, it played a crucial role in determining the percentage of PACY released from PACY‐loaded PLAROsomes. The size of the PACY‐loaded PLAROsomes ranged from 40 to 300 nm, and the optimized batch demonstrated a drug entrapment efficiency of 86.38±0.22%. PACY‐loaded PLAROsomes exhibited improved in vitro anticancer activities against MCF‐7 breast cancer cell lines compared to PACY. PACY‐loaded PLAROsomes showed greater activity at lower concentrations in cytotoxicity studies, as supported by apoptosis analysis. Therefore, PLAROsomes could present a promising drug carrier system for delivering PACY in breast cancer treatment, offering a sustained release effect.

Effect of Shoutai Pills on expression of key glycolytic proteins and apoptosis related factors at maternal fetal interface in mouse model of threatened abortion with syndrome of kidney deficiency

This study aims to explore the mechanism of Shoutai Pills in treating threatened abortion. According to the random number table method, ICR female mice were randomized into a normal group, a model group, a dydrogesterone group, and a Shoutai Pills group, with 15 mice in each group. Mice were administrated with normal saline(normal and model groups) or the suspension of Shoutai Pills or dydrogesterone by gavage at 9:00 am every day. At 16:00 every day, mice in the normal group were administrated with an equal volume of distilled water, while those in the model, Shoutai Pills, and dydrogesterone groups were administrated with hydrocortisone solution by gavage for 4 consecutive days. ICR female and male mice were caged in a ratio of 2∶1 during the pre-estrous or estrous period. From the first day of pregnancy, drug administration was continued for 5 consecutive days. On day 6, mice were administrated with mifepristone by gavage to establish the model of kidney deficiency-induced abortion. On day 6 of pregnancy, 10 female ICR mice were randomly selected from each group, and the uterus was collected for observation of the pathological changes of trophoblasts at the maternal-fetal interface by hematoxylin-eosin(HE) staining. The protein levels of key enzymes of glycolysis, hexokinase 2(HK2), enolase 1(ENO1), pyruvate kinase M2(PKM2), and lactate dehydrogenase A(LDHA), were determined by Western blot and immunofluorescence. The expression of apoptosis-related proteins including B cell lymphoma-2(Bcl-2), Bcl-2-associated protein X(Bax), and cysteinyl aspartate-specific proteinase-3(caspase-3) was determined by Western blot and real-time PCR. Terminal-deoxynucleoitidyl transferase-mediated nick-end labeling was employed to examine apoptosis. The embryo loss rate of the remaining five female mice was calculated by trypan blue staining method on day 14 of pregnancy. On day 14 of pregnancy, the embryo loss rate of the normal group was 5.00%, which was lower than that(27.78%) in the model group(P&lt;0.05). Dydrogesterone and Shoutai Pills groups showed reduced embryo loss rates(10.26% and 7.50%, respectively) compared with the model group. On day 6 of pregnancy, compared with the normal group, the model group showed down-regulated expression of HK2, ENO1, PKM2, LDHA, and Bcl-2 and up-regulated expression of Bax and caspase-3(P&lt;0.05). Compared with the model group, dydrogesterone and Shoutai Pills up-regulated the expression of HK2, ENO1, PKM2, LDHA, and Bcl-2 and down-regulated the expression of Bax and caspase-3(P&lt;0.05). Compared with that in the normal group, the apoptosis rate in the model group increased(P&lt;0.05). Compared with the model group, dydrogesterone and Shoutai Pills reduced the apoptosis rate(P&lt;0.05). In conclusion, Shoutai Pills can reduce the embryo loss rate and protect embryos by promoting aerobic glycolysis at the maternal-fetal interface and inhibiting the apoptosis of trophoblasts in mice.

Alpha-Pinene and Tannic Acid Inhibit Trichomonas vaginalis Protozoan Cells by Inducing Apoptosis.

Trichomoniasis is one of the most common sexually transmitted infections worldwide. The growing concern of drug resistance of this infection has cautioned the need for new drug development. We evaluated the potential antiproliferative and apoptotic effect of α-pinene and tannic acid (TA) on Trichomonas vaginalis cells. In addition, the cytotoxicity of agents on Vero cells was investigated. Trichomonas cells were axenically cultured in TYI-S-33 medium. In vitro antiproliferative activity of α-pinene, TA, and metronidazole was investigated against Trichomonas cells. The assays were carried out in triplicate using microtiter plate and trypan blue staining method. Annexin V/PI staining with flow cytometry was used to evaluate apoptosis induction. In addition, the cytotoxic effect was measured by MTT assay. α-Pinene and TA exhibited significant inhibition of the Trichomonas cells and the lowest IC50 values were 22.9 μg/ml and 140 μg/ml at 48 hours' incubation, respectively. The CC50 was found at 116 μg/ml for α-pinene and 473 μg/ml for TA, after 48 hours of treatment. The flow cytometry study demonstrated that the natural compounds induced apoptosis in Trichomonas cells. After 24 hours of treatment, the induction of apoptosis was 5.2% - 36.6% at concentrations of 3.9 - 62.5 μg/ml for α-pinene and TA induced-apoptosis was 6.1% - 53.8% at concentrations of 125-2000 μg/ml. Although the results show the antiproliferative and apoptotic effect of α-pinene and TA on Trichomonas cells, in vivo studies are needed to further clarify the effects of these compounds.

Combinatorial Optimization of Chemotherapeutic Agents on T-Cell Acute Lymphoblastic Leukemia

Introduction: Up to now, treatment options for T-cell acute lymphoblastic leukemia (T-ALL) remain quite limited. This study aims to assess the efficacy of monotherapies and combination of chemotherapeutic agents for T-ALL. Jurkat, CCRF-CEM are late T-cell progenitor (LTP) cell lines, while Loucy is early T-cell progenitor (ETP) cell line characterized by an immature phenotype. The drugs under evaluation include venetoclax, a selective BCL-2 inhibitor; cytarabine, a cytosine analog with established antineoplastic activity; bendamustine, an alkylating agent; and azacitidine, a DNA methyltransferase inhibitor. Given the notable tumor heterogeneity observed in T-ALL, the comprehensive assessment of these therapeutic agents on Jurkat, CCRF-CEM, and Loucy may render valuable insights for treatment strategies in T-ALL. Methods: Cell lines were cultured in RPMI-1640 medium supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO2. Venetoclax, bendamustine, and azacitidine were prepared as stock solutions in DMSO, while cytarabine was prepared in distilled water. Monotherapies (venetoclax, cytarabine, bendamustine and azacitidine) and combination therapies (venetoclax plus cytarabine, venetoclax plus bendamustine and venetoclax plus azacitidine) were administered. A control group received DMSO or distilled water only. Cell viability was assessed after 48 hours by using Trypan blue and colorimetric method (MTS assay) on cells treated with various drug concentrations. Statistical analysis was conducted using the SynergyFinder Plus and drc R package, with significance set at p &amp;lt; 0.05. Results: Our study demonstrated that T-ALL cell lines displayed a different pattern of drug sensitivities according to combination of chemotherapeutic agents. Loucy showed greater sensitivity to venetoclax while Jurkat and CCRF-CEM exhibited higher sensitivity to cytarabine, bendamustine, and azacitidine. When conducting drug combinations on Jurkat and CCRF-CEM, the addition of venetoclax to cytarabine, bendamustine, or azacitidine resulted in an additive effect, with Loewe synergic scores ranging from -10 to 10 for each combination. On the other hand, Loucy demonstrated a distinct pattern of response; the combination of venetoclax with cytarabine showed additivity (Loewe synergic score: 8.45 and 5.82 with MTS and trypan blue assays, respectively), while venetoclax combined with bendamustine or azacitidine showed a synergistic effect (Loewe synergic score &amp;gt; 10 with MTS assay). Notably, among these combination treatments on Loucy, the pairing of venetoclax with bendamustine showed the most significant synergistic effects, with a Loewe score of 16.6. Conclusion: Venetoclax-based chemotherapeutic combinations exhibited various responses in T-ALL cell lines. Of note, the combination of venetoclax plus bendamustine showed potent synergistic effects in all T-ALL cell lines. These findings warrant further study to develop fundamentally curative therapeutic strategies in T-ALL.

New aryl-/heteroarylpiperazine derivatives of 1,7-dimethyl-8,9-diphenyl-4-azatricyclo[5.2.1.02,6]dec-8-ene-3,5,10-trione: Synthesis and preliminary studies of biological activities

Compounds containing dicarboximide skeleton such as succinimides, maleimides, glutarimides, and phthalimides possess broad biological properties including anti-fungal, antibacterial, antidepressant, or analgesic activities. The piperazine ring is found in a wide range of molecules that have demonstrated a variety of biological functions such as anticancer action and 5-HT receptors agonist/antagonist activity. In the present study, we combined both structures to develop new antitumor agents, a series of piperazine derivatives of 1,7-dimethyl-8,9-diphenyl-4-azatricyclo[5.2.1.02,6]dec-8-ene-3,5,10-trione and evaluated their biological activity. The structures of all tested compounds were confirmed by 1H and 13C NMR and by ESI MS spectral analysis. Their cytotoxicity was assessed in vitro against eight human cancer cell lines, namely prostate (PC3), colon (HCT116, SW480, SW620), leukemia (K562), liver (HepG2), lung (A549) and breast (MDA-Mb-231) in contrast to normal HMEC-1 cell line, by using MTT and Trypan blue method. The tested compounds showed significant activity toward cancer cells. The most pronounced cytotoxic effect was observed in K562 and HCT116 with IC50 values below 10 μM for all studied compounds. Importantly, the most promising derivatives for each cancer cell line (IC50 < 10 μM) exerted a weaker cytotoxic effect toward normal HMEC-1 cells than cancer cells. The evaluation of proapoptotic and inhibitory effects on IL-6 release showed that K562 and HCT116 cells were more sensitive to studied compounds than other cancer cell lines. Furthermore, for all piperazine derivatives, the functional activities at the 5‐HT1A, D2 receptors as well as their binding affinities at the 5‐HT2A, H1 and M receptors, were determined. The current investigation was able to successfully design compounds with both serotoninergic and anticancer properties. It serves as a good starting point for a multimodal approach for the management of cancer and cancer-related symptoms.

Distinct effects of Fgf7 and Fgf10 on the terminal differentiation of murine bladder urothelium revealed using an organoid culture system

BackgroundDysregulation of the terminal differentiation of bladder urothelium is associated with the pathogenesis of urinary tract disorders. Fibroblast growth factor (Fgf)7 and Fgf10 stimulate urothelial proliferation; however, their roles in cellular differentiation remain unclear. In this study, we used an organoid system to investigate the roles of these Fgfs in regulating bladder urothelium differentiation and identify their distribution patterns in the mouse bladder.MethodsAdult bladder epithelia (AdBE) isolated from adult mouse bladder tissues (AdBTs) were used to culture adult bladder organoids (AdBOs) in the presence of Fgf7 and Fgf10. The differentiation status of the cells in AdBTs, AdBEs, AdBOs, and neonatal bladder tissues (NeoBTs) was analyzed via quantitative real-time-PCR for the presence of undifferentiated cell markers (Krt5, Trp63, and Krt14) and differentiated cell markers (Krt20, Upk1a, Upk2, and Upk3a). Organoid cell proliferation was assessed by counting cell numbers using the trypan blue method. The effects of Fgf7 and Fgf10 on organoid differentiation were assessed using different doses of Fgfs, and the involvement of peroxisome proliferator-activated receptor γ (PPARγ) signaling in these processes was tested by introducing a PPARγ agonist (Rosiglitazone) and antagonist (T0070907) to the culture. The expression patterns of Fgf7 and Fgf10 were examined via in situ hybridization of AdBTs.ResultsAdBOs showed higher expression of undifferentiated cell markers and lower expression of differentiated cell markers than AdBTs, NeoBTs, and AdBEs, indicating the relatively immature state of AdBOs. Differentiation of AdBOs was enhanced by Rosiglitazone and Fgf7, suggesting an interplay of intracellular signals between Fgf7 and PPARγ. Co-addition of T0070907 suppressed Fgf7-mediated differentiation, demonstrating that PPARγ is activated downstream of Fgf7 to promote cellular differentiation into umbrella cells. Furthermore, we found that Fgf7 is predominantly expressed in the umbrella cells of the urothelium, whereas Fgf10 is predominantly expressed in the urothelium and stroma of AdBTs.ConclusionsWe demonstrated that unlike Fgf10, Fgf7 induces cellular differentiation via PPARγ activity and has a unique tissue distribution pattern in the adult bladder. Further studies on the Fgf7-PPARγ signaling axis would provide insights into the differentiation mechanisms toward functional umbrella cells and the pathogenesis of several urinary tract diseases.

Biocompatibility of graphene oxide nanosheets functionalized with various amino acids towards mesenchymal stem cells

Graphene and its derivatives have gained popularity due to their numerous applications in various fields, such as biomedicine. Recent reports have revealed the severe toxic effects of these nanomaterials on cells and organs. In general, the chemical composition and surface chemistry of nanomaterials affect their biocompatibility. Therefore, the purpose of the present study was to evaluate the cytotoxicity and genotoxicity of graphene oxide (GO) synthesized by Hummer's method and functionalized by different amino acids such as lysine, methionine, aspartate, and tyrosine. The obtained nanosheets were identified by FT-IR, EDX, RAMAN, FE-SEM, and DLS techniques. In addition, trypan blue and Alamar blue methods were used to assess the cytotoxicity of mesenchymal stem cells extracted from human embryonic umbilical cord Wharton jelly (WJ-MSCs). The annexin V staining procedure was used to determine apoptotic and necrotic death. In addition, COMET and karyotyping techniques were used to assess the extent of DNA and chromosome damage. The results of the cytotoxicity assay showed that amino acid modifications significantly reduced the concentration-dependent cytotoxicity of GO to varying degrees. The GO modified with aspartic acid had the lowest cytotoxicity. There was no evidence of chromosomal damage in the karyotyping method, but in the comet assay, the samples modified with tyrosine and lysine showed the greatest DNA damage and rate of apoptosis. Overall, the aspartic acid-modified GO caused the least cellular and genetic damage to WJ-MSCs, implying its superior biomedical applications such as cell therapy and tissue engineering over GO.

Graphene Quantum Dot‐PEI‐Cyclodextrin Nanocarrier for Simultaneous miR21a Delivery and Cell Imaging in Cancer Therapy

AbstractOver‐expression of miR21 plays an important role in several cancers by promoting cancer cell proliferation, migration, invasion, and metastasis. Here, we attempted to prepare a beta cyclodextrin‐polyethyleneimine‐graphene quantum dot (βCD‐PEI‐GQD) nanocarrier for cellular delivery of miR21a. For this purpose, tosylated‐βCD and GQD were conjugated to branched PEI. The product was characterized by FTIR, 1H‐NMR, and fluorescence spectroscopy. The morphology, particle size distribution, and ζ‐potential of miR21a were examined by TEM and DLS following overnight incubation with βCD‐PEI‐GQD in aqueous media. The miR21 silencing was measured by stem‐loop RT‐PCR in HepG2 human hepatoma cell line. Cellular uptake and cell toxicity assays were determined by fluorescence microscopy and Trypan blue staining method, respectively. The formation of miR21a/CD‐PEI‐GQD Nanoplex with a decreased average size of 114 nm and a ζ‐potential (+36.1 mV) lower than CD‐PEI‐GQD nanocarrier by adding miR21a was confirmed at optimum C/P ratio =8.7. RT‐PCR revealed that miR21a/βCD‐PEI‐GQD Nanoplex significantly downregulated miR21 expression levels effectively. Overall, miR21a delivery using CD‐PEI‐GQD is presented as a novel trackable nanocarrier for cancer therapy.

INVESTIGATING THE FORMATION OF 3D BONE CELL SPHEROIDS FOR SCAFFOLD-FREE BONE TISSUE ENGINEERING

3D spheroid culture is a bridge between standard 2D cell culture and in vivo research which mimics the physiological microenvironment in scaffold-free conditions. Here, this 3D technique is being investigated as a potential method for engineering bone tissue in vitro. However, spheroid culture can exhibit limitations, such as necrotic core formation due to the restricted access of oxygen and nutrients. It is therefore important to determine if spheroids without a sizeable necrotic core can be produced. This study aims to understand necrotic core formation and cell viability in 3D bone cell spheroids using different seeding densities and media formulations.Differentiated rat osteoblasts (dRObs) were seeded in three different seeding densities (1×104, 5×104, 1×10 cells) in 96 well U-bottom cell-repellent plates and in three different media i.e., Growth medium (GM), Mineralisation medium 1 (MM1) and MM2. Spheroids were analysed from day 1 to 28 (N=3, n=2). Cell count and viability was assessed by trypan blue method. One way ANOVA and post-hoc Tukey test was performed to compare cell viability among different media and seeding densities. Histological spheroid sections were stained with hematoxylin and eosin (H&amp;E) to identify any visible necrotic core.Cell number increased from day 1 to 28 in all three seeding densities with a notable decrease in cell viability. 1×104 cells proliferated faster than 5×104 and 1×105 cells and had proportionately similar cell death. The necrotic core area was relatively equivalent between all cell seeding densities. The larger the spheroid size, the larger is the size of the necrotic core.This study has demonstrated that 3D spheroids can be formed from dRobs at a variety of seeding densities with no marked difference in necrotic core formation. Future studies will focus on utilising the bone cell spheroids for engineering scalable scaffold-free bone tissue constructs.

Abstract 2855: Epithelial-mesenchymal transition profile after cisplatin-resistance induction in OSCC cell line

Abstract Oral cancer is among the most common types of cancer in humans. Chemotherapy, mainly based on cisplatin, is utilized in the treatment of advanced cases. However, a loss of chemotherapy effectiveness is frequent and can be related to both inherent or acquired resistance to the chemotherapy agent, resulting in high relapse rates. Epithelial-mesenchymal transition (EMT) plays an important role in different stages of cancer, and it is correlated to resistance to therapy, but the process is still unclear. The purpose of this study is to explore EMT markers in a cisplatin resistant-based model. After determination of IC50, SCC9 cells were intermittently exposed to stepwise increasing concentrations of cisplatin, and Resistance Index (RI) was calculated. Population Doubling (PD) and Population Doubling Time (PDT) were calculated counting the number of viable cells using Trypan Blue method. Scratch assay was performed to investigate the impact on cellular migration. EMT-correlated markers were accessed by RT-qPCR and western blot. After 4 intermittent exposures to cisplatin, we obtained different clones with cisplatin resistance level, SCC9/CPR5 (intermediate cisplatin resistance) and SCC9/CPR11 (final cisplatin resistance) the SCC9/CPR5 clone was obtained showing a mixed morphology between polygonal and fusiform. The complete fusiform morphology resembling mesenchymal cells was seen in SCC9/CPR11, which demonstrated 2-fold of cisplatin resistance in comparison to parental cell line. SCC9/CPR11 showed a difference of less than 1.16 PD and more than 15.5 hours PDT in relation to SCC9. Interestingly, the migratory profile was increased in SCC9/CPR5. Snail1 and Vimentin mRNA levels and protein expression were increased at SCC9/CPR11, but surprisingly E-cadherin was overexpressed but not in protein level. Others markers such as Zeb1, N-cadherin, Vimentin, Snail2 and Twist showed an increase in mRNA levels only in SCC-9-CPR11, however only Twist exhibited high protein expression. Our study showed the establishment of cisplatin resistant SCC9 cells in different moments in relation to a resistance level based on the main two protein markers of EMT, Snail1 and Vimentin. Our data need to be further validated in larger well characterized techniques and we further envision investigating the epigenetic in DNA damage pathways correlating with cisplatin resistance and EMT status. Citation Format: Dieila Giomo De Lima, Gabriell Bonifácio Borgato, Gustavo Narvaes Guimarães, Débora Campanella Bastos, Ricardo Della Coletta, Ana Paula De Souza. Epithelial-mesenchymal transition profile after cisplatin-resistance induction in OSCC cell line [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2855.

Effect of Mir-4270 Inhibitor and Mimic on Viability and Stemness in Gastric Cancer Stem-Like Cells Derived from MKN-45 Cell Line.

MicroRNAs (miRNAs) are significant regulatory factors in stem cell proliferation, and change in miRNA expression influences the cancer stem cell viability and gene expression. Herein, we evaluated the effect of the hsa-miR-4270 inhibitor and its mimic on the expression of stem cell markers in gastric cancer (GC) stem-like cells. GC stem-like cells were isolated from the MKN-45 cell line by a non-adherent surface system. The cells were confirmed by differentiation assays using dexamethasone and insulin as adipogenesis-inducing agents and also Staurosporine as a neural-inducing agent. Isolated GC stem-like cells were treated with different concentrations (0, 15, 20, 25, 30, 40, 50, and 60 nM) of hsa-miR-4270 inhibitor and its mimic. The quantity of cell viability was determined by trypan blue method. Transcription of the stem cell marker genes, including CD44, OCT3/4, SOX2, Nanog, and KLF4, was evaluated by real-time RT-PCR. The results showed that GC stem-like cells were differentiated into both adipose cells using dexamethasone and insulin and neural cells by Staurosporine. Treatment of GC stem-like cells with hsa-miR-4270 inhibitor decreased cell viability and downregulated OCT3/4, CD44, and Nanog to 86%, 79%, and 91% respectively. Also, SOX2 and KLF4 were overexpressed to 8.1- and 1.94-folds, respectively. However, hsa-miR-4270 mimic had opposite effects on the cell viability and gene expression of the stem cell markers. The effect of hsa-miR-4270 inhibitor and its mimic on the expression of the stem cell markers in GCSCs indicated that hsa-miR-4270 stimulates the stemness property of GCSCs, likely through stimulating the development of gastric stem cells.

Activation of ERK1/2 by MOS and TPL2 leads to dasatinib resistance in chronic myeloid leukaemia cells.

The development of BCR::ABL1 tyrosine kinase inhibitors (TKIs), such as dasatinib, has dramatically improved survival in cases of chronic myeloid leukaemia (CML). However, the development of resistance to BCR::ABL1 TKIs is a clinical problem. BCR::ABL1 TKI resistance is known to have BCR::ABL1-dependent or BCR::ABL1-independent mechanisms, but the mechanism of BCR::ABL1 independence is not well understood. In the present study, we investigated the mechanism of BCR::ABL1-independent dasatinib resistance. The expression and activation level of genes or proteins were evaluated using array CGH, real time PCR, or western blot analysis. Gene expression was modulated using siRNA-mediated knockdown. Cell survival was assessed by using trypan blue dye method. We found that dasatinib-resistant K562/DR and KU812/DR cells did not harbour a BCR::ABL1 mutation but had elevated expression and/or activation of MOS, TPL2 and ERK1/2. In addition, MOS siRNA, TPL2 siRNA and trametinib resensitized dasatinib-resistant cells to dasatinib. Moreover, expression levels of MOS in dasatinib non-responder patients with CML were higher than those in dasatinib responders, and the expression of TPL2 tended to increase in dasatinib non-responder patients compared with that in responder patients. Our results indicate that activation of ERK1/2 by elevated MOS and TPL2 expression is involved in dasatinib resistance, and inhibition of these proteins overcomes dasatinib resistance. Therefore, MOS, TPL2 and ERK1/2 inhibitors may be therapeutically useful for treating BCR::ABL1-independent dasatinib-resistant CML.

Evaluating the Magnolol Anticancer Potential in MKN-45 Gastric Cancer Cells.

Background and Objectives: Combination therapy improves the effect of chemotherapy on tumor cells. Magnolol, used in treating gastrointestinal disorders, has been shown to have anti-cancer properties. We investigated the synergistic effect of cisplatin and magnolol on the viability and maintenance of MKN-45 gastric cancer cells. Materials and Methods: The toxicity of magnolol and/or cisplatin was determined using the MTT technique. The trypan blue method was used to test magnolol and/or cisplatin's effect on MKN-45 cell growth. Crystal violet staining was used to assess the treated cells' tendency for colony formation. The expression of genes linked to apoptosis, cell cycle arrest, and cell migration was examined using the qPCR method. Results: According to MTT data, using magnolol and/or cisplatin significantly reduced cell viability. The ability of the treated cells to proliferate and form colonies was also reduced considerably. Magnolol and/or cisplatin treatment resulted in a considerable elevation in Bax expression. However, the level of Bcl2 expression was dramatically reduced. p21 and p53 expression levels were significantly increased in the treated cells, while MMP-9 expression was significantly reduced. Conclusions: These findings show that magnolol has a remarkable anti-tumor effect on MKN-45 cells. In combination with cisplatin, magnolol may be utilized to overcome cisplatin resistance in gastric cancer cells.

Combination synergy between β-carotene and lupeol against breast adenocarcinoma in vitro and in vivo using combination index

BackgroundBreast adenocarcinoma is one of the leading causes of death among women regardless of development in the field of diagnosis, treatment and prevention. Synergistic approaches are emerging as a strategy to overcome monotherapy resistance in cancer treatment. PurposeEvaluation of the combinatory activity of β-carotene (BC) and lupeol (LUP) for anti-proliferative activity by median effect equation and combination index. Validation was performed by fixed dose combination against murine mammary adenocarcinoma namely Ehrlich ascites carcinoma (EAC). MethodsCytotoxicity assay was performed using the MTT and Trypan blue methods. Isobologram and combination index analysis were used to analyze the type of interaction between β-carotene (BC) and lupeol (LUP). Based on the synergistic dose combination determination of in-vivo antitumor activity was performed using EAC cells inoculated in mice treated with BC, LUP and their combination (BC+LUP). Tumor volume, cell count, weight along with hematological, biochemical and antioxidant parameters were evaluated. ResultsThe combination (BC+LUP) exhibited the highest inhibition (IC50=54.8 ± 1.84) compare to the standard inhibitor, wher eas BC and LUP showed moderate inhibition with an IC50 of 101.5 ± 2.66, and 84.08±3.56 respectively. The hematological, biochemical, and tissue antioxidant parameters were reverted to normal levels in the treated animal groups. BC and Lupeol reduced the volume, weight, and viable cell count of the tumor; increased the life span of EAC tumor-bearing mice. Conclusionβ-Carotene and lupeol were found to be more effective in combination for the anti-proliferative activity. Thus combination techniques must be employed to find out the synergistic activity of the molecules.

Evaluation of fluorescence-based viability stains in cells dissociated from scleractinian coral Pocillopora damicornis

The application of established cell viability assays such as the commonly used trypan blue staining method to coral cells is not straightforward due to different culture parameters and different cellular features specific to mammalian cells compared to marine invertebrates. Using Pocillopora damicornis as a model, we characterized the autofluorescence and tested different fluorescent dye pair combinations to identify alternative viability indicators. The cytotoxicity of different representative molecules, namely small organic molecules, proteins and nanoparticles (NP), was measured after 24 h of exposure using the fluorescent dye pair Hoechst 33342 and SYTOX orange. Our results show that this dye pair can be distinctly measured in the presence of fluorescent proteins plus chlorophyll. P. damicornis cells exposed for 24 h to Triton-X100, insulin or titanium dioxide (TiO2) NPs, respectively, at concentrations ranging from 0.5 to 100 µg/mL, revealed a LC50 of 0.46 µg/mL for Triton-X100, 6.21 µg/mL for TiO2 NPs and 33.9 µg/mL for insulin. This work presents the approach used to customize dye pairs for membrane integrity-based cell viability assays considering the species- and genotype-specific autofluorescence of scleractinian corals, namely: endogenous fluorescence characterization followed by the selection of dyes that do not overlap with endogenous signals.

MiR-19b-3p regulated by BC002059/ABHD10 axis promotes cell apoptosis in myocardial infarction

BackgroundRecently, microRNAs (miRNAs), have been extensively investigated in diseases. The upregulated expression of miR-19b-3p has been validated in patients with hypertrophic cardiomyopathy. Nonetheless, it regulatory mechanism in myocardial infarction (MI) is still unclear.PurposeThis research aimed to investigate the role and molecular regulation mechanism of miR-19b-3p in MI.MethodsQRT-PCR and western blot assays measured RNA and protein expression. Cell apoptosis were tested by flow cytometry and TUNEL assays. Cell viability was measured by trypan blue staining method. RIP and luciferase report assays examined gene interaction. The assays were performed under hypoxia condition.ResultsMiR-19b-3p was highly expressed in myocardial cell line H9C2, primary cardiomyocytes, and tissues from MI mouse model. MiR-19b-3p inhibition suppressed the apoptosis of cardiomyocytes. BC002059 could up-regulate ABHD10 through sequestering miR-19b-3p. BC002059 upregulation was observed to repress cell apoptosis. Rescue experiments demonstrated that miR-19b-3p overexpression abrogated the suppressive impact of BC002059 on the apoptosis of MI cells, and infarct size, area at risk as well as CK-MB and LDH release of MI mouse model tissues, which was further abolished via ABHD10 increment.ConclusionMiR-19b-3p regulated by BC002059/ABHD10 axis promotes cell apoptosis in MI, which might provide a novel perspective for MI alleviation research.