Muse Cell Analyzer Research Articles

Effect of silibinin on GAS6/sAXL and JAK/STAT pathways in human cholangiocarcinoma cell line

Abstract Objectives Cholangiocarcinoma (CCA) is a highly heterogeneous biliary malignant tumor. Studies have demonstrated that JAK/STAT signaling is activated in many types of cancer. In addition, JAK/STAT is activated downstream of AXL, and the AXL receptor is activated by its ligand, GAS6. In this study, we investigated the anticarcinogenic effect of silibinin and its relationship with the GAS6/AXL ve JAK/STAT pathway in the human EGI-1 cell line. Methods Cell viability, apoptosis, and cell cycle were measured by Muse Cell Analyzer. All the protein levels were determined by the ELISA method. Results We observed that silibinin significantly reduced cell proliferation and colony formation (p<0.05, p<0.001, respectively). Silibinin also significantly induced total apoptosis and the G0/G1 phase of the cell cycle (p<0.01). We observed that silibinin significantly decreased JAK2 levels while increased STAT1 levels compared to the controls respectively (p<0.001; p<0.001). Besides, silibinin statistically decreased the levels of sAXL; however, numerically, but not statistically, it increased the level of GAS6 (p>0.05). Conclusions Silibinin reduces colony formation, inducing apoptosis and arresting cancer cells in the G0/G1 phase, which is an indicator of its anticancer activity. In addition, silibinin decreased the levels of JAK2 and sAXL, which contributes to the development of cancer, and increased the levels of STAT1, suggesting silibinin’s antiproliferative effects.

Rhus coriaria induces autophagic and apoptotic cell death in pancreatic cancer cells.

Background:Pancreatic cancer is a leading cause of cancer-related mortality worldwide with increasing global incidence. We previously reported the anticancer effect of Rhus coriaria ethanolic extract (RCE) in triple negative breast and colon cancer cells. Herein, we investigated the anticancer effect of RCE on human pancreatic cancer cells. Methods: Cell viability was measured using Cell Titer-Glo and staining of viable and dead cells based on differential permeability to two DNA binding dyes. Cell cycle distribution and annexin V staining was carried out in Muse cell analyzer. Protein level was determined by Western blot. Tumor growth was assessed by in ovo chick embryo chorioallantoic membrane assay. Results: We found that RCE significantly inhibited the viability and colony growth of pancreatic cancer cells (Panc-1, Mia-PaCa-2, S2-013, AsPC-1). The antiproliferative effects of RCE in pancreatic cancer cells (Panc-1 and Mia-PaCa-2) were mediated through induction of G1 cell cycle arrest, Beclin-1-independent autophagy, and apoptosis. RCE activated both the extrinsic and intrinsic pathways of apoptosis and regulated the Bax/Bcl-2 apoptotic switch. Mechanistically, we found that RCE inhibited the AKT/mTOR pathway, downstream of which, inactivation of the cell cycle regulator p70S6K and downregulation of the antiapoptotic protein survivin was observed. Additionally, we found that RCE-induced autophagy preceded apoptosis. Further, we confirmed the anticancer effect of RCE in a chick embryo xenograft model and found that RCE inhibited the growth of pancreatic cancer xenografts without affecting embryo survival. Conclusion: Collectively, our findings demonstrate that Rhus coriaria exerts potent anti-pancreatic cancer activity though cell cycle impairment, autophagy, and apoptosis, and is hence a promising source of anticancer phytochemicals.

Protective effect of lithium pyruvate against oxidative damage to peripheral blood mononuclear cells

IntroductionIn recent years, there has been renewed interest in lithium therapy due to emerging evidence of the protective effects of lithium against neuronal death caused by a wide range of neurotoxic effects. Oxidative stress is a common pathway that is involved in various pathologies. In this regard, the development and study of new lithium compounds with combined antioxidant effects becomes relevant. Pyruvate has many potential benefits due to its positive effects on cellular metabolism.ObjectivesThe purpose of this study was to study lithium pyruvate on blood cells of healthy donors under conditions of induced oxidative stress.Methods The study used blood from 20 healthy control group volunteers, aged 25 to 54 years. Venous blood was taken at baseline and then used for PBMCs extraction. After that cells were incubated during 24 hours in RPMI 1640 medium at 37°С and 5% carbon dioxide concentration. For oxidative stress induction hydroperoxide of trisubstituted butyl (HTB) was used in concentration of 50 μM. Cells were also incubated with lithium pyruvate in final concentration of lithium ions of 1.2 mM with or without HTB. Level of oxidative stress in culture was assessed by flow cytometer «Muse Cell Analyzer» (Merck Millipore, Germany) using «Oxidative stress» reagents kit (Merck Millipore, Germany). Statistical analysis was performed using the SPSS software, release 20.0 for Windows.Results Percentage of cells with reactive oxygen species (ROS) cultivated with HTB (65,33 (41,95-79,30) %) was statistically significant higher compared to intact cells (11,03 (7,93-15,53) %) (p=0.001). After addition of lithium pyruvate in culture statistically significant antioxidant effects were observed. In PBMCs incubated with HTB and lithium pyruvate statistically significant decreased percentage of cells with ROS (42,70 (16,73-58,70) %) (p=0.001)Conclusions A pronounced antioxidant effect of lithium pyruvate under induced oxidative stress on human peripheral blood mononuclear cells has been established. Lithium pyruvate can be considered as a promising psychotropic antioxidant for further experiments.Disclosure of InterestNone Declared

Anti-inflammatory effect of luteoloside against methylglyoxal induced human dental pulp cells.

The aim of this study was to investigate whether luteoloside, a flavonoid, could protect human dental pulp cells (HDPCs) against inflammation and oxidative stress induced by methylglyoxal (MGO), one of the advanced glycated end products (AGE) substances. HDPCs were stimulated with MGO and treated with luteoloside. MTT assay was used to determine cell viability. Protein expression was measured via western blotting. Reactive oxygen species (ROS) were measured with a Muse Cell Analyzer. Alkaline phosphatase activity (ALP) and Alizarin red staining were used for mineralization assay. Luteoloside down-regulated the expression of inflammatory molecules such as ICAM-1, VCAM-1, TNF-α, IL-1β, MMP-2, MMP-9, and COX-2 in MGO-induced HDPCs without showing any cytotoxicity. It attenuated ROS formation and enhanced osteogenic differentiation such as ALP activity and Alizarin red staining in MGO-induced HDPCs. Overall, luteoloside showed protective actions against inflammation and oxidative stress in HDPCs induced by MGO through its anti-inflammatory, anti-oxidative, and osteogenic activities by down-regulating p-JNK in the MAPK pathway. These results suggest that luteoloside might be a potential adjunctive therapeutic agent for treating pulpal pathological conditions in patients with diabetes mellitus.

Investigation of proapoptotic and cytotoxic effects of 2-aminobenzothiazole on human laryngeal carcinoma cells.

In the present study, we investigated the effects of 2-aminobenzothiazole application on human laryngeal carcinoma cells. Human larynx epidermoid carcinoma (HEp-2) (ATCC® CCL-23™) cells were purchased from American Type Culture Collection (ATCC, USA). Human larynx epidermoid carcinoma HEp-2 cells were cultured in complete Dulbecco's Modified Eagle's Medium (DMEM) supplemented with fetal bovine serum (FBS) (10%) and penicillin/streptomycin (1%) in a CO2 (5%) incubator under standard cell culture conditions. 2-aminobenzothiazole was prepared, and further dilutions ranging from 3.13 to 100 μM were prepared in fresh culture DMEM. HEp-2 cells on 96 well plates were incubated with the prepared dilutions of 2-aminobenzothiazole for 24, 48, and 72 hours. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test performed cytotoxicity evaluation and viability percentages. The annexin-V staining technique detected 2-aminobenzothiazole-triggered apoptosis of HEp-2 cells. The activated caspases 3/7 on HEp-2 cells after 2-aminobenzothiazole exposure were evaluated with flow cytometric analysis. The membrane potential changing of HEp-2 cells was measured following the Muse™ MitoPotential kit manufacturer instructions. MTT cytotoxicity test results showed that the viability of human laryngeal carcinoma cells decreased with an increase in the application of 2-aminobenzothiazole for 24 hours. The highest growth inhibition by 2-aminobenzothiazole for short-term application of 24 hours was detected at the highest concentration of 2-aminobenzothiazole (100 µM). The results underline that the cytotoxic effect of 2-aminobenzothiazole is dose-dependent. Cytotoxicity test results for an application time of 48 hours showed that the cytotoxicity of 2-aminobenzothiazole is dose-dependent on HEp-2 cells. The required dose of 2-aminobenzothiazole to decrease the cell viability to 50 percent has been 9-fold augmented. Annexin-V findings showed that after exposure to IC50 concentration of 2-aminobenzothiazole for 24 hours, HEp-2 cells underwent the early apoptotic stage (25.99%) and late apoptotic (16.69%), whereas 56.93% of the treated cells were alive. Only 0.39% of 2-aminobenzothiazole treated cells were necrotic. All study results showed that 2-aminobenzothiazole triggered apoptosis on HEp-2 cells with a percentage of total apoptotic cells 42.62 compared to untreated HEp-2 cells. Caspase 3/7 activation results showed that only 0.65% of control HEp-2 cells were with activated caspase 3/7, and 99.35% live cells. The analysis data from the Muse cell analyzer revealed that the percentage of cells with intact mitochondrial membranes was 21.30 after 2-aminobenzothiazole application, and 79.9% were cells with depolarized mitochondrial membranes. It has been understood that the depolarization of the inner mitochondrial membrane has been considered a dysfunction in mitochondria as a sign of apoptosis and drug toxicity. Based on all study findings, 2-aminobenzothiazole has cytotoxicity on human laryngeal carcinoma cells in a dose and time-dependent manner. That means that it decreased viability via inducing caspase-dependent apoptosis. Consequently, it was concluded that 2-aminobenzothiazole has good potential to lead to cytotoxicity and apoptosis on human laryngeal carcinoma cells and, after deeper in vitro and in vivo investigations, can be a good candidate for designing anticancer drugs with high efficiency.

MiR‑186‑5p regulates the inflammatory response of chronic obstructive pulmonary disorder by targeting HIF‑1α.

Chronic obstructive pulmonary disorder (COPD) is a chronic respiratory disease that is a major cause of morbidity and mortality worldwide. Previous studies have shown that miR‑186‑5p expression is significantly increased in COPD and is involved in multiple physiological and pathological processes. However, the role of miRNA‑186‑5p in the inflammatory response of COPD remains unclear. In this study, an invitro model of COPD was established using lipopolysaccharide (LPS)‑induced human bronchial epithelial cells (BEAS‑2B). CCK‑8 assays, flow cytometry, and a Muse cell analyzer were used to determine cell viability, cell cycle distribution, and apoptosis, respectively. The production of TNF‑α and IL‑6 were measured by ELISA. Reverse‑transcription‑quantitative PCR and western blotting were used to analyze mRNA and protein expression levels. The targeting relation between miR‑186‑5p and HIF‑1α was discovered using dual‑luciferase reporter assays. The results showed that transfection of miR‑186‑5p inhibitor inhibited cell proliferation and promoted cell apoptosis in the LPS‑induced BEAS‑2B cells. Inhibition of miR‑186‑5p markedly increased the levels of TNF‑α and IL‑6. miR‑186‑5p directly targeted and negatively regulated HIF‑1α expression. In addition, inhibition of miR‑186‑5p increased the expression of the NF‑κB pathway protein p‑p65. In conclusion, it was found that inhibiting miR‑186‑5p may improve inflammation of COPD through HIF‑1α in LPS‑induced BEAS‑2B cells, possibly by regulating NF‑κB signaling. These findings provide a novel potential avenue for the clinical management of COPD. Future research is required to determine the mechanism of the interaction between miR‑186‑5p and HIF‑1α in COPD.

The detection of pomegranate (Punica granatum L.) peel apoptotic effects using AgNOR staining in MDA-MB-231.

In present study, it was purposed to determine the in vitro effect of the extract obtained from the pomegranate (Punica granatum L.) peel on the breast cancer cell line. MDA-MB-231 cells were exposed to pomegranate peel extract (PoPx) at 37°C and 5% CO2 for varying durations (24 and 48h) and doses (25 and 50μg/mL). At the end of the incubation periods, argyrophilic nucleolus organizer regions (AgNOR) protein status, cell viability/apoptosis and cell cycle of MDA-MB-231 cells were examined in the Muse Cell Analyzer device. Cell viability was observed to be decreased when the groups treated with PoPx were compared with the control group. The group in which apoptosis was observed with the highest value was 50μg/mL PoPx group (p<0.05). In the cell cycle test, the number of cells in the G0/G1 stage was found to be significantly higher in the 25μg/mL PoPx group compared to the control and 50μg/mL PoPx groups at the end of the 24-h incubation period (p<0.05) The results also supported cell cycle and apoptosis, and at the end of 24h, Total AgNOR area(TAA)/Total nuclear area (NA) ratio and AgNOR numbered decreased on the 50μg/mL PoPx group and were found to be statistically significant compared to the control group (p<0.05). Consequently, it was determined that PoPx increased apoptosis on breast cancer cells by various mechanisms and inhibited cell viability/cell growth. This study showed that the widespread consumption of PoPx may be effective in preventing cancer formation and slowing its progression.

Quinacrine enhances the efficacy of cisplatin by increasing apoptosis and modulating cancer survival proteins in a colorectal cancer cell line.

Cisplatin and platinum-based compounds have been used successfully to treat various cancers. However, their use is often restricted due to the acquired resistance by cancer cells. Over-expression of p53 and inhibition of NF-kB sensitize several cancer cells towards cisplatin-induced apoptosis. Quinacrine, a cytotoxic drug with predictable safety revealed to concurrently suppress NF-kB and activate p53, which may be an attractive adjuvant in cisplatin chemotherapy. Therefore, the objective of the present study was to establish the role of quinacrine as an adjuvant in lowering the dose of cisplatin during cancer therapy to circumvent its toxic effects. The colon cancer (HCT-8) cells were cultured and cell survival assays were performed using standard procedures. Cell cycle arrest and the extent of apoptosis were determined using a muse cell analyzer. Cancer survival proteins were analyzed using western blotting techniques. We demonstrated that concomitant use of quinacrine with cisplatin increased cell apoptosis, suppressed cell proliferation and inhibited colony formation in a colorectal cancer cell line. Moreover, cell cycle arrest in the G0/G1 and G2/M phases and upregulation of p53 expression were observed. There was also downregulation of NF-kB and Bcl-xL protein expressions, both of which are associated with enhanced cell apoptosis and an increase in the sensitivity of cancer cells to cisplatin, overcoming its chemoresistance. Overall, the results of the present study and available literature clearly indicate that the use of quinacrine as an adjuvant with cisplatin may enhance its anti-cancer activity and reduce chemoresistance.

Does broccoli (brassica oleracea var. italica) extract have a potential therapeutic targets on human breast cancer (MDA-MB-231) using AgNOR detection method?

In this study, it was aimed to determine the in vitro effect of the extract obtained from mature broccoli on the breast cancer cell line. MDA-MB231 breast cancer cell line was used in the study. MDA-MB-231 cells were exposed to broccoli extract at 37°C and 5% CO2 for varying durations (24 and 48 hours) and doses (125 and 250 µl/ml).At the end of the incubation period, viability, apoptosis, cell cycle and AgNOR protein status of MDA-MB-231 cells were examined in the Muse Cell Analyzer. In the groups containing broccoli extract, a decrease in the percentage of viable cells and a significant increase in the percentage of early and total apoptosis were observed for both doses compared to the control. In the cell cycle test, the number of cells in the S phase increased in all groups.It was observed that the groups containing broccoli extract slowed down the cell cycle in the transition to the S checkpoint. AgNOR staining results also supported cell cycle and apopitosis, and AgNOR number and TAA/NA ratio decreased in the 125 µl/ml broccoli extract group after 24-48 hours and were found to be statistically significant compared to the control group. It was determined that broccoli increased apoptosis on breast cancer cells by various mechanisms and inhibited cell viability/cell growth. The results were similar to the results of AgNOR protein synthesis. The study showed that the regular and correct consumption of broccoli could be effective in preventing cancer formation and slowing its progression.

Doxorubicin-induced senescence promotes resistance to cell death by modulating genes associated with apoptotic and necrotic pathways in prostate cancer DU145 CD133+/CD44+ cells

Cancer stem cells (CSCs) are the most important cause of cancer treatment failure. Traditional cancer treatments, such as chemotherapy and radiotherapy, damage healthy cells alongside malignant cells, leading to severe adverse effects. Therefore, inducing cellular senescence without triggering apoptosis, which further damages healthy cells, may be an alternative strategy. However, there is insufficient knowledge regarding senescence induction in CSCs that show resistance to treatment and stemness properties. The present study aims to elucidate the effects of senescence induction on proliferation, cell cycle, and apoptosis in prostate CSCs and non-CSCs. Prostate CSCs were isolated from DU145 cancer cells using the FACS method. Subsequently, senescence induction was performed in RWPE-1, DU145, prostate CSCs, and non-CSCs by using different concentrations of Doxorubicin (DOX). Cellular senescence was detected using the senescence markers SA-β-gal, Ki67, and senescence-associated heterochromatin foci (SAHF). The effects of senescence on cell cycle and apoptosis were evaluated using the Muse Cell Analyzer, and genes in signaling pathways associated with the apoptotic/necrotic pathway were analyzed by real-time PCR. Prostate CSCs were isolated with 95.6 ± 1.4% purity according to CD133+/CD44+ characteristics, and spheroid formation belonging to stem cells was observed. After DOX-induced senescence, we observed morphological changes, SA-β-gal positivity, SAHF, and the lack of Ki67 in senescent cells. Furthermore; we detected G2/M cell cycle arrest and downregulation of various apoptosis-related genes in senescent prostate CSCs. Our results showed that DOX is a potent inducer of senescence for prostate CSCs, inhibits proliferation by arresting the cell cycle, and senescent prostate CSCs develop resistance to apoptosis.

MDA-MB-231 Human Breast Cancer Cell Line Treated with Ginseng (Panax Quinquefolius): Evaluation by Annexin V and AgNOR Staining

Aim: In this study, it was aimed to examine the time and dose dependent effects of Ginseng (Panax quinquefolius) on MDA-MB-231 cell lines. Material and Methods: MDA-MB-231 breast cancer cell line was used in the study. MDA-MB-231 cells were exposed to ginseng at 37°C and 5% CO2 for varying durations (24 and 48 hours) and doses (1 and 2 μg/ml ginseng). At the end of the incubation period, viability, apoptosis, cell cycle and Argyrophilic nucleolar organizing region (AgNOR) protein status of MDA-MB-231 cells were examined in the Muse Cell Analyzer. Results: It was observed that the dose inducing apoptosis was 1 μg/ml ginseng for 24 and 48 hours, and 2 μg/ml ginseng for 48 hours in the group that stopped the cell cycle in the G0/G1 phase. When comparing the two groups; while no difference was determined between the control and 1 µg/ml ginseng groups, the significant differences were detected between the control and 2 µg/ml ginseng groups for mean AgNOR number in 48 hours incubation. However, there was no significant difference for the TAA/NA ratio, in the groups for 48 hours. Conclusion: The current study showed that ginseng had a crucial function against cancer development. Also, both AgNOR values might be used as biomarkers for detection of the most reliable therapeutic dose selection for cancer and it has been shown that correct consumption of Ginseng can be effective in preventing cancer formation and slowing its progression.

Abstract 3822: Novel evidence for the role of the p53 signaling pathway in mediating the anticancer effects of aronia berry extract in colorectal cancer cells

Abstract Background: The development of safe and effective novel agents remains a critical challenge for improving the prognosis of patients with unresectable colorectal cancer (CRC). Natural products containing polyphenols can be a good source of anti-cancer agents for their potential effect on multiple signaling pathways with anti-cancer effect. Due to its rich polyphenols content, Aronia berry extract (ABE) is gaining increased attention as an anti-cancer agent with its protective effect against cancer cells, including CRC, exerting potent antioxidant and antiproliferative effects. Thus, we hypothesized that ABE could be a novel complementary therapeutic option in patients with CRC by virtue of its ability to modulate multiple key cancer-related pathways. Methods: First, an MTT assay was performed to prove the antiproliferative effect of ABE in two CRC cells, SW480 and HCT116, and IC25 and IC50 of ABE in both CRC cells were identified. Next, the anti-cancer effects of ABE were examined by wound healing, colony formation, and invasion assays. Apoptosis studies were performed using the Muse Annexin V &amp; Dead Cell Assay Kit on the muse cell analyzer. RNA-sequencing was performed to clarify the critical regulatory genes and pathways modulated by ABE by comparing genome-wide profiling between CRC cells with and without ABE treatment. Results: Results of the MTT assay revealed that ABE decreased the viability of both CRC cells, and the IC25 and IC50 of ABE were 80.0 µg/mL and 130.0 µg/mL for SW480 and 80.0 µg/mL and 140.0 µg/mL for HCT116. ABE inhibited proliferation, migration, cellular survival, and invasion of CRC cells and induced apoptosis in those cells (SW480 control vs. IC25: p&amp;lt;0.05, control vs. IC50: p&amp;lt;0.01, HCT116 control vs. IC25: p&amp;lt;0.01, control vs. IC50: p&amp;lt;0.01). After ABE treatment, the genome-wide transcriptomic analysis identified 439 differentially expressed genes (p&amp;lt;0.05 and |log2FC|&amp;gt;0.5) in both CRC cells. These differentially expressed genes were used for pathway analysis and some critical cancer-related pathways, including the p53 signaling pathway (p=0.005, fold enrichment 3.8), NF-kappa B signaling pathway (p=0.030, fold enrichment 2.7), Hippo signaling pathway (p=0.037, fold enrichment 2.2), and MAPK signaling pathway (p=0.011, fold enrichment 2.0), were detected as significantly activated or suppressed pathways. Conclusions: We have investigated the anti-cancerous effect of ABE in CRC cells and identified multiple critical cancer-related pathways modulated by ABE, with the p53 signaling pathway as the key mediator for growth suppressive anti-cancer efficacy of this naturally occurring phytochemical. Our findings provide evidence for the use of ABE as a safe and effective complementary and integrative medicine approach against CRC. Citation Format: Yoh Asahi, Keisuke Okuno, Caiming Xu, Aknobu Taketomi, Ajay Goel. Novel evidence for the role of the p53 signaling pathway in mediating the anticancer effects of aronia berry extract in colorectal cancer cells. [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 3822.

The Effects of Orcinol on Proliferation and Apoptosis of SW480 Human Colorectal Cancer Cells

Objective: Colorectal cancer is a rapidly increasing disease worldwide, and almost half of the diagnosed patients die from this disease each year. The methods used in the treatment of colorectal cancer, including traditional treatment methods such as surgery, radiotherapy and current chemotherapy options, have low effectiveness and have many side effects. Because of all these problems, the importance of developing new agents for the treatment of colorectal cancer is increasing. Orsinol is a secondary metabolite isolated from lichens, and there are findings regarding the antioxidant, antimicrobial and antidepressant activity of the compound. In recent years, research on the anticancer activity of the compound has also started to take place in the literature. In this study, it was aimed to investigate the efficacy of orcinol on cell proliferation and apoptosis in human SW480 colorectal cancer cells.&#x0D; Material and Method: Within the scope of the study, SW480 human colorectal cancer cells were used and cultured in DMEM medium. Orcinol was dissolved with dimethylsulfoxide to prepare a stock solution and applied to the cells in a concentration range of 1-25 mM. The effect of orcinol on cell viability was determined by MTT test. The apoptotic activity of the compound was evaluated with Annexin V binding assay using the Muse Cell Analyzer.&#x0D; Result and Discussion: The results of MTT analysis showed that orcinol significantly decreased cell viability at 5 mM and above (p

Capparis zeylanica L. root extract promotes apoptosis and cell cycle arrest, inhibits epithelial-to-mesenchymal transition and triggers E-cadherin expression in breast cancer cell lines.

Capparis zeylanica L. is a climbing shrub distributed in Indian subcontinent and Mediterranean region. Almost all parts of the plant are used in folk medicine and traditional practices to treat several human ailments. The present study was aimed to investigate the role of C. zeylanica L. root extract in preventing cancerous cells growth and proliferation, as well as promoting apoptosis and cell cycle arrest in MDA-MB-231 and MCF-7 breast cancer cells. Methanolic extract of C. zeylanica L. (MECz) was prepared and characterized by LC-ESI-MS/MS analysis. In vitro cytotoxicity and anti-proliferative activity of MECz was evaluated by MTT assay, while cell viability, apoptosis and cell cycle progression by Muse Cell analyzer. Furthermore, the mRNA and protein expressions of EMT markers were assessed using qRT-PCR and western blotting techniques, respectively. The MECz was found to be rich in phenolic compounds including chlorogenic acid, 6-gingerol, and certain triterpenes like ursolic acid etc. The apparent anti-metastasis activity of MECz was evident from IC50 value of 19.12 and 24.22μg/mL, respectively, on MDA-MB-231 and MCF-7 cells in MTT assay. An absolute decrease in cell viability (78.1-53.4% and 89.9-49.0%), augmented apoptosis (90.98-48.25% and 88.25-47.70%) and S phase, G2/M phase cell cycle arrest was found by MECz treatment on MDA-MB-231 and MCF-7 cells. The gene expression studies revealed that MECz could significantly (p < 0.001) regulate the expression of EMT markers such as snail, slug, zeb-1, twist-1, fibronectin, vimentin and E-cadherin at molecular level. These findings demonstrate that C. zeylanica L. root extract inhibits breast cancer cells growth and proliferation through regulating the expression of key EMT marker genes and proteins. Thus, MECz may be suggested as a potential anti-metastasis agent in the treatment of breast cancer.

PSIII-3 Late-Breaking: Diisobutyl Phthalate (DIBP) at Low Doses Alters Gene Expression of Epigenetic Regulators in Bovine Granulosa Cells

Abstract Phthalates are ubiquitous environmental contaminants and impact reproduction and epigenetic mechanisms. The use of diisobutyl phthalate (DIBP) has increased in the last decade. However, studies about DIBP's effects on female reproductive physiology are limited. Methylation of DNA and RNA are epigenetic processes that regulate gene expression, cell biology, and reproductive functions. How DIBP exposure regulates these epigenetic mechanisms in ovarian follicular cells remains to be unveiled. We hypothesize that DIBP at low doses alters the expression of RNA and DNA methylation regulators in bovine granulosa cells (GC). Bovine GC was isolated from small antral follicles (1-5 mm on surface diameter) from ovaries obtained at a slaughterhouse. Cells were cultured in vitro until reaching an average 70% confluence and treated with DIBP at levels detected in human blood (0 ng/mL, 1 ng/mL, 10 ng/mL, 100 ng/mL) combined with hormones that regulate ovarian folliculogenesis: FSH or FSH+IGF1. Cells were cultured for 24h for total RNA isolation (n = 5 pools) or for 48h for cell counting (n=5 pools). Relative mRNA abundance of RNA N6 methyladenine (m6A) methylation machinery (m6A writers: METTL3, METTL14; m6A erasers: ALKBH5, FTO), RNA 5-methylcytosine (m5C) methylation regulators (NSUN2, TRDMT1), and DNA methylation players (DNA methyltransferases: DNMT1, DNMT3A, DNMT3B; DNA demethylases: TET1, TET2, TET3) was determined via qPCR using 18S as housekeeping gene and expressed as 2-ΔΔCt. Cell proliferation was analyzed by cell counting using a MUSE cell analyzer. Data were analyzed via one-way ANOVA with GLM procedures of SAS for Windows. Normality was tested using Shapiro-Wilk test procedures and data considered not normal were transformed to log2 (Y + 0.6). Data sets that showed non-gaussian distribution after log-transformation was analyzed via the Kruskal-Wallis test using GraphPad Prism 9.2.0. Cell proliferation was not affected by DIBP treatments in combination with FSH or with FSH+IGF1. In GC treated with FSH in combination with DIBP, there was no significant effect on relative mRNA abundance of RNA or DNA methylation players. In combination with FSH+IGF1, GC treated with DIBP had lower (P &amp;lt; 0.05) METTL14 relative mRNA abundance at 1 ng/mL and 10 ng/mL than at 0 ng/mL and 100 ng/mL. TRDMT1 relative mRNA abundance was lower (P &amp;lt; 0.05) in the DIBP group at 10 ng/mL in comparison to groups 0 ng/mL and 1 ng/mL, but no 100 ng/mL. In addition, there was no significant effect on the relative mRNA abundance of DNA methylation regulators. In summary, GC treated with FSH+IGF1 and DIBP at low doses alters the gene expression of RNA methylation players in m6A and m5C RNA modifications in a non-monotonic dose-response, but does not alter mRNA abundance of DNA methylation regulators.

Linifanib induces apoptosis in human ovarian cancer cells via activation of FOXO3 and reactive oxygen species

Linifanib is known as an inhibitor of receptor tyrosine kinase. Even though it has been widely recognized as efficient inhibitor of receptor tyrosine kinases, anti-carcinogenic effect has not been investigated enough in ovarian cancer. In this study, we investigated the anti-cancer effect of linifanib on human ovary cancer SKOV3 cells. WST-1, cell counting assay, and observation of morphological changes were performed to evaluate the cytotoxic effect of linifanib in SKOV3 cells. We analyzed SKOV3 cells treated with linifanib using Muse cell analyzer. We focused on investigating the effect of linifanib on DNA damage in nucleus. Additionally, intracellular reactive oxygen species (ROS) level was measured through Muse cell analyzer. Western blotting was performed to evaluate the protein expression level related to apoptosis. We found that linifanib inhibited proliferation of SKOV3 cells. Our results showed that linifanib induced apoptosis in SKOV3 cells. Additionally, linifanib induced DNA damage in SKOV3 cells. We found that intracellular ROS level increased after treatment of linifanib in SKOV3 cells. Interestingly, FOXO3 was transferred from cytosol into nucleus after linifanib treatment. Taken together, our results supported that linifanib inhibited the proliferation of human ovary cancer SKOV3 cells, which suggested that linifanib might have the potential to be developed as drugs for ovarian cancer treatment.

INVESTIGATION OF URSODEOXYCHOLIC ACID EFFECTS ON SIROLIMUS TREATED ADIPOSE TISSUE-DERIVED MESENCHYMAL STEM CELLS

Objective&#x0D; The usage of mesenchymal stem cells (MSC) with&#x0D; immunosuppressive drugs after organ transplantation&#x0D; is becoming remarkable in clinical applications.&#x0D; However, the drugs negatively affect MSCs.&#x0D; Ursodeoxycholic acid (UDCA), which is an antioxidant&#x0D; molecule, may reverse these effects. The study aims&#x0D; that to determine the effects of sirolimus and UDCA&#x0D; on human adipose tissue-derived MSCs (ADMSCs)&#x0D; individually and in combination.&#x0D; Material and Method&#x0D; The cytotoxicity of the agents was evaluated by&#x0D; WST-1 test in time and dose-dependent manner.&#x0D; The combinational effects were determined using&#x0D; isobologram analysis. Muse cell analyzer was used for&#x0D; the evaluation of apoptosis and cell cycle. Oxidative&#x0D; stress markers were measured by biochemical methods.&#x0D; Results&#x0D; IC50 dose of sirolimus was determined as 18.58μM&#x0D; in the 48th hour. Because no cytotoxic effect was&#x0D; observed at the studied doses of UDCA, the apoptosis,&#x0D; cell cycle, and oxidative stress indicator analyses&#x0D; were continued with a safe dose of 100 μM. Sirolimus&#x0D; promoted apoptosis and inhibited cell proliferation.&#x0D; It was determined that UDCA reduced the apoptotic&#x0D; and anti-proliferative effects of sirolimus on ADMSCs&#x0D; with its anti-oxidant property.&#x0D; Conclusion&#x0D; The UDCA treatment in combination with&#x0D; immunosuppressive therapy after organ and tissue&#x0D; transplantation may have positive effects on ADMSCs.

Exenatide increases CTRP3 gene expression in adipose cells by inhibiting adipogenesis and induces apoptosis

Considering the rapidly increasing prevalence of obesity worldwide, the number of weight control drugs is very few. Incretin-based therapies are currently being developed to achieve weight control, and Glucagon-Like Peptide-1 Receptor Agonists (GLP-1RA) are used in incretin-based therapies. This study aimed to investigate the cytotoxicity of exenatide, a GLP-1A, on 3T3-L1 adipocytes and the effect of exenatide on the expression of adipogenesis-related genes, insulin and glucose levels, and apoptosis. Cytotoxic activity of exenatide on 3T3-L1 adipocytes was determined by MTT method. Gene expression levels were determined by qPCR. Apoptosis studies were performed on the Muse Cell Analyzer. C1q/TNF-related protein-3 (CTRP3) expression levels were found to be higher in exenatide treated adipocyte cells than in control cells (p < 0.001). Adipocyte cells treated with exenatide were found to have lower PPAR-γ gene expression levels when compared to control adipocyte cells (p < 0.001). Intracellular insulin (p < 0.001) and glucose levels were higher in 3T3-L1 adipocytes treated with exenatide compared to control adipocyte cells. Total apoptosis increased approximately 1.5 times as a result of exenatide administration. The increase in CTRP3 gene expression, which is thought to be a new biomarker for obesity, and the decrease in PPAR-γ gene expression indicate that exenatide is a promising new pharmacotherapeutic agent in the treatment of obesity by regulating the expression of genes related to adipogenesis and lipogenesis and inducing apoptosis.

Cellular analysis on the radiation induced bystander effects due to bismuth oxide nanoparticles with 6 MV photon beam radiotherapy

BackgroundThe therapeutic strategies in killing the cancerous tissue while keeping the normal healthy tissue uninterrupted can be further improved by introducing nanoparticles as radiosensitizer in radiotherapy. Nevertheless, healthy cells might still be affected by radiation-induced bystander effect (RIBE) response due the presence of nanoparticles. Thus, the potential negative effects in non-irradiated adjacent cells must be investigated. AimThe current study aims to explore the cellular effects of bismuth oxide nanoparticles (Bi2O3 NPs) on human breast cancer (MCF-7) and human foetal osteoblast (hFOB 1.19) cells due to the RIBE after irradiation with 6 MV clinical photon beam. Materials and methodsThe irradiated-cell conditioned medium (ICCM) treated with and without Bi2O3 NPs from the irradiated cells were collected and then transferred into the non-targeted cells. The ability of the bystander cells to proliferate and their ROS generation were analyzed. DNA status of the cells and the possibility of apoptosis were evaluated using FTIR spectroscopy and MUSE Cell Analyzer, respectively. ResultsThe current study revealed that MCF-7 and hFOB 1.19 bystander cells may continue to proliferate and survive following short and long-term incubation with ICCM treatment with Bi2O3 NPs. The survival rate of the treated bystander cells increased approximately 3%–8% compared to the untreated group. Although the bystander cells exhibited an increase in ROS and apoptosis, the addition of Bi2O3 NPs had no significant detrimental influence on the cells. Variation in the DNA vibrational amplitude detected by FTIR in bystander cells was less than 5% compared to the control group. ConclusionThis study discovered that the application of Bi2O3 NPs as a radiosensitizer did not give a negative impact on the bystander cells. This outcome may give an advantage to the application of Bi2O3 NPs as radiosensitizer in radiotherapy to enhance cancer cells death without introducing further detrimental effects to the non-targeted cells.

Abstract 817: microRNA-26a-5p is a prognostic factor that regulates cancer stemness

Abstract Background: Cancer stem cells (CSCs) identified in lung cancer exhibit resistance to chemotherapy, radiotherapy, and targeted therapy. Therefore, a technology to control of CSCs is needed to overcome such resistance to cancer therapy. Various evidences about the association between epithelial-mesenchymal transition related transcriptomic alteration and acquisition of CSC phenotype have been proposed recently. In our previous research, down-regulated miR-26a-5p is closely related to mesenchymal-like lung cancer cell lines. These findings suggest that miR-26a-5p might be involved in lung cancer stemness. Methods: RNA polymerase III subunit G (POLR3G) was selected as a candidate target of miR-26a-5p related to cancer stemness. its quantitative relationship was investigated by polymerase chain reaction, western blot after transfection of miR-26a-5p. luciferase assay were done for investigating the direct regulation of miR-26a-5p on POLR3G expression. After transfection of miR-26a-5p, colony formation assay and sphere formation assay were performed to evaluate the effect on cancer stemness. By treating cancer cell by miR-26a-5p and paclitaxel, cell viability was checked by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay and Muse cell analyzer. Expression level of each gene and its impact on survival were revealed by the cancer genome atlas pancancer database. Results: miR-26a-5p regulated the expression of POLR3G directly. Overexpression of miR-26a-5p induced down regulation of POLR3G and a marked reduction of colony formation and sphere formation. Co-treatment of miR-26a-5p with paclitaxel decreased cell growth, suggesting that miR-26a-5p might play a role as a chemotherapy sensitizer. In the cancer genome atlas data, down-regulated miR-26a-5p and up-regulated POLR3G were shown compared to adjacent normal tissue. High miR-26a-5p and low POLR3G expression were also related to higher survival rate of patients with lung adenocarcinoma. Conclusions: Overexpression of miR-26a-5p can suppress lung cancer stemness and make cancer cell become sensitive to chemotherapy. This finding provides a novel insight into a potential lung cancer treatment by regulating stemness. Citation Format: Daeun Kang, Chang Ryul Park, Minhyeok Lee, Su Yel Lee, Se Jin Park, Wan Jin Hwang, Gwan Woo Ku, Seong Lan Yu, In Beom Jeong, Sun Jung Kwon, Jaeku Kang, Eung Bae Lee, Ji Woong Son. microRNA-26a-5p is a prognostic factor that regulates cancer stemness [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 817.