Line Of Cells Research Articles

Ефективність вирощування молоді осетрових (Acipenseridae) видів риб за різної твердості води

Ефективність вирощування молоді осетрових (Acipenseridae) видів риб за різної твердості води

BLOC1S1 promotes proliferation of goat spermatogonial stem cells

With the rapid development of gene editing technology, the study of spermatogonial stem cells (SSCs) holds great significance in understanding spermatogenesis and its regulatory mechanism, developing transgenic animals, gene therapy, infertility treatment and protecting rare species. Biogenesis of lysosome-related organelles complex 1 subunit 1 (BLOC1S1) is believed to have anti-brucella potential. Exploring the impack of BLOC1S1 on goat SSCs not only helps investigate the ability of BLOC1S1 to promote SSCs proliferation, but also provides a cytological basis for disease-resistant breeding research. In this study, a BLOC1S1 overexpression vector was constructed by homologous recombination. The BLOC1S1 overexpression cell line of goat spermatogonial stem cells was successfully constructed by lentivirus packaging, transfection and puromycin screening. The overexpression efficiency of BLOC1S1 was found to be 18 times higher using real time quantitative PCR (RT-qPCR). Furthermore, the results from cell growth curve analysis, flow cytometry for cell cycle detection, and 5-ethynyl-2'-deoxyuridine (EdU) staining showed that BLOC1S1 significantly increased the proliferation activity of goat SSCs. The results of RT-qPCR, immunofluorescence staining and Western blotting analyses revealed up-regulation of proliferation-related genes (PCNA, CDK2, CCND1), and EIF2S3Y, a key gene regulating the proliferation of spermatogonial stem cells. These findings strongly suggest that the proliferative ability of goat SSCs can be enhanced through the EIF2S3Y/ERK pathway. In summary, this study successfully created a goat spermatogonial stem cell BLOC1S1 overexpression cell line, which exhibited improved proliferation ability. This research laid the groundwork for exploring the regulatory role of BLOC1S1 in goat spermatogonia and provided a cell platform for further study into the biological function of BLOC1S1. These findings also establish a foundation for breeding BLOC1S1 overexpressing goats.

Microarray evidence that 8-cell human embryos express some hormone family members including oxytocin.

This study is to discover hormone pathways active in early cleaving human embryos. A list of 152 hormones and receptors were compiled to query the microarray database of mRNAs in 8-cell human embryos, two lines of human embryonic stem cells plus human fibroblasts before and after induced pluripotency. Over half of the 152 hormones and receptors were silent on the arrays of all cell types, and more were detected at high or moderate levels on the 8-cell arrays than on the pluripotent cell or fibroblast arrays. Eight hormone family genes were uniquely detected at least 22-fold higher on the 8-cell arrays than the stem cell arrays: AVPI1, CCK, CORT, FSTL4, GIP, GPHA2, OXT, and PPY suggesting novel roles for these proteins in early development. Oxytocin was detected by pilot immunoassay in culture media collected from Day 3 embryos. Robust detection of CRHR1 and EPOR suggests the 8-cell embryo may be responsive to maternal CRH and EPO. The over-expression of POMC and GHITM suggests POMP peptide products may have undiscovered roles in early development and GHITM may contribute to mitochondrial remodeling. Under-detected on the 8-cell arrays at least tenfold were two key enzymes in steroid biosynthesis, DHCR24 and FDPS. The 8-cell human embryo may be secreting oxytocin, which could stimulate its own progress down the fallopian tube as well as play a role in early neural precursor development. The 8-cell embryo does not synthesize reproductive steroid hormones. As previously reported for growth factor families, the early embryo over-expresses more hormones than hormone receptors.

Effects of 2.4 GHz radiofrequency electromagnetic field (RF-EMF) on glioblastoma cells (U -118 MG).

Mobile phones and Wi-Fi are the most commonly used forms of telecommunications. Initiated with the first generation, the mobile telephony is currently in its fifth generation without being screened extensively for any biological effects that it may have on humans or on animals. Some studies indicate that high frequency electromagnetic radiation emitted by mobile phone and Wi-Fi connection can have a negative effect upon human health, and can cause cancer, including brain tumour. The aim of the study was to investigate the influence of 2.4 GHz radiofrequency electromagnetic field (RF-EMF) on the proliferation and morphology of normal (human embryonic kidney cell line Hek-293) and cancer cells (glioblastoma cell line U-118 MG). The cell cultures were incubated in RF-EMF at the frequency of 2.4 GHz, with or without dielectric screen, for 24, 48 and 72h. In order to analyse the influence of the electromagnetic field on cell lines, Cytotoxicity test Cell Counting Kit-8 was performed. To protect cells against emission of the electromagnetic field, a dielectric screen was used. It was found that 2.4 GHz RF electromagnetic field exposure caused a significant decrease in viability of U-118 MG and Hek-293 cells. The impact of the electromagnetic field was strongest in the case of cancer cells, and the decrease in their survival was much greater compared to the healthy (normal) cells of the Hek-293 line. Results of the study indicate that using a radio frequency electromagnetic field (2.4 GHz) has a clearly negative effect on the metabolic activity of glioblastoma cells. RF-EMF has much less impact on reducing the viability of normal cells (Hek -293) than cancer cells.

Anion efflux mediates transduction in the hair cells of the zebrafish lateral line.

Hair cells are the principal sensory receptors of the vertebrate auditory system, where they transduce sounds through mechanically gated ion channels that permit cations to flow from the surrounding endolymph into the cells. The lateral line of zebrafish has served as a key model system for understanding hair cell physiology and development, often with the belief that these hair cells employ a similar transduction mechanism. In this study, we demonstrate that these hair cells are exposed to an unregulated external environment with cation concentrations that are too low to support transduction. Our results indicate that hair cell excitation is instead mediated by a substantially different mechanism involving the outward flow of anions. Further investigation of hair cell transduction in a diversity of sensory systems and species will likely yield deep insights into the physiology of these unique cells.

Generation of novel in vitro flexible kidney organoid model to investigate the role of extracellular vesicles in induction of nephrogenesis

BackgroundDuring kidney organogenesis, metanephric mesenchyme (MM) and ureteric bud (UB) interact reciprocally to form nephrons. Signaling stimuli involved in these interactions include Wnts, growth factors and nano/micro particles. How UB and MM are interacting is not completely understood. Our study investigated the signaling and communication via extracellular vesicles (EVs) during nephrogenesis. Embryonic day (E) 11.5 mouse kidney UB and MM produce very low number of primary cells that have limited ability for proliferation in culture. Such limitations obstruct studying the role of EVs in induction of nephrogenesis. These issues necessitate to generate a nephrogenesis model allowing to study the comprehensive role of EVs during nephrogenesis.ResultsOur study generated a UB derived cell line-based in vitro flexible model of nephrogenesis allowing expandable cell culturing, in addition to performing characterization, tracking and blocking of EVs. UB cell line aggregation with E11.5 MM cells induced the formation of segmented nephrons. Most efficient nephrogenesis was obtained by the co-culturing of 30,000 cells of UB cell line with 50,000 MM cells. Results revealed that both the UB and the MM secrete EVs during nephrogenesis. UB cell line derived EVs were characterized by their size, morphology and expression of markers (CD63, TSG101, CD9 and CD81). Furthermore, proteomics data of UB cell line-derived EVs revealed large number of proteins involved in nephrogenesis-related signaling pathways. Palmitoylated GFP-tagged EVs from UB cell line were found in the nephron formation zone in the developing kidney organoid. UB cell line derived EVs did not induce nephrogenesis in MM cells but significantly contributed to the survival and nephrogenesis-competency of MM cells. The secretion of EVs was continuously inhibited during the ongoing nephrogenesis by the knockdown of RalA and RalB gene expression using short hairpin RNAs. This inhibition partially impaired the ability of UB cell line to induce nephrogenesis. Moreover, impaired nephrogenesis was partially rescued by the addition of EVs.ConclusionOur study established a novel in vitro flexible model of nephrogenesis that solved the limitations of primary embryonic kidney cells and mouse embryonic stem cell kidney organoids for the EV research. EVs were found to be an integral part of nephrogenesis process.Graphical 7F9YfcJgNbSGEsBfhqoDc7Video

Curcumin loaded as prepared electrospun titania nanofibers for post breast cancer surgeries

In the present study, curcumin-loaded titania nanofibers were synthesized by the conventional sol-gel method via the electrospinning technique using polyvinyl pyrrolidone and characterized via SEM, FTIR, and XRD. Moreover, their contact angle measurement, degradability, water uptake, and mechanical strength were investigated, besides their antibacterial activity, antioxidant activity, sustained drug release, and cytotoxicity against normal cells and breast cancer cell lines. It can be observed that as the concentration of curcumin increased, the mechanical characteristics of the samples were enhanced. The antibacterial results declared that the increase in curcumin concentration enhanced the inhibitory effect against the different pathogens. Moreover, the viability of cells of the normal cell line, seeded on the samples was in the range from 61.4 to 86.5% which indicated its safety on normal cells. However, the cell viability on the cancerous cell line was found to be 33.8, 19.7, 17.9 and 0% for 0, 1, 3, and 5% loaded TiO2/PVP respectively. This lethal effect of the prepared mats on breast cancer cells with nearly no cytotoxic effect on normal cells, favors the usage of the prepared 5% curcumin-loaded TiO2/PVP mat as anticancerous wound healing bandages for postoperative breast cancer surgeries.Graphical

Kremen1 regulates the regenerative capacity of support cells and mechanosensory hair cells in the zebrafish lateral line

Mechanosensory hair cells in the inner ear mediate the sensations of hearing and balance, and in the specialized lateral line sensory system of aquatic vertebrates, the sensation of water movement. In mammals, hair cells lack the ability to regenerate following damage, resulting in sensory deficits. In contrast, non-mammalian vertebrates, such as zebrafish, can renew hair cells throughout their lifespan. Wnt signaling is required for development of inner ear and lateral line hair cells and regulates regeneration. Kremen1 inhibits Wnt signaling and hair cell formation, though its role in regeneration is unknown. We used a zebrafish kremen1 mutant line to show overactive Wnt signaling results in supernumerary support cells and hair cell regeneration without increased proliferation, in contrast with the previously described role of Wnt signaling during hair cell regeneration. This work allows us to understand the biology of mechanosensory hair cells and how regeneration might be promoted following damage.

Abstract B070: Antitumor effect of AZD5305, a selective PARP1 inhibitor, in breast and gastric cancer cells

Abstract Background: Poly (ADP-ribose) polymerase (PARP) 1 has multiple cellular functions including its crucial role in DNA damage response. PARP inhibitor (PARPi) blocks the synthesis of PAR polymers and traps PARP1 on the damage sites. Currently approved PARPis target both PARP1 and PARP2, but the inhibition of PARP2 can cause undesirable hematological toxicity. AZD5305 is a novel selective PARP1 inhibitor that can reduce the hematological toxicity by off-targeting of PARP2. We evaluated the anti-tumor effects and mechanism of action of AZD5305 in breast and gastric cancer cells in vitro. Methods: To investigate the anti-tumor effects of AZD5305, we used 2 cell lines of breast cancer cells (HCC1395 and MCF7) and 3 cell lines of human gastric cancer cells (SNU-601, SNU-668 and KATO Ⅲ). The cytotoxicity of AZD5305 was evaluated by colony formation assay (CFA) for 14 days with increasing concentration of AZD5305 (doses range: 0-5nM). Cell cycle was analyzed by flow cytometry and apoptotic cell death was assessed by annexin-Ⅴ/PI staining. DNA damage was detected by comet assay and cell immunofluorescence assay. Results: BRCA1/2 mutated HCC1395 and RAD51C deficient SNU-601 were sensitive to AZD5305 (IC50: 0.25 nM), and SNU-668 was moderately sensitive (IC50: 2.05nM), while MCF7 and KATO III were less-sensitive to AZD5305 (IC50 > 5 nM). AZD5305 induced the G2/M cell cycle arrest in a dose-dependent manner in HCC1395 and SNU-601, but not in MCF7 and KATO Ⅲ. Moreover, AZD5305 increased sub-G1 population in HCC1395 and SNU-601 cells indicating apoptotic cell death, which was also confirmed by annexin-V assay and the cleaved PARP and caspase-7. Phosphorylation of RPA, γH2AX, and the expression of molecules related to DNA damage response were increased in the sensitive cells. Moreover, comet tails which indicate fragmented DNA were observed in the sensitive cells, but not in insensitive cells. Conclusions: AZD5305, a novel PARP1 selective inhibitor, shows cytotoxic effect in HR defective breast and gastric cancer cells in vitro. AZD5305 accumulates DNA damage which leads to apoptotic cell death in sensitive cell lines, but not in less-sensitive cell lines. Citation Format: Sujin Ham, So Hyeon Kim, Hae Min Hwang, Minyoung Lee, Youlim Noh, Changyun Lee, Yu-Jin Kim, Jinyong Kim, Dae-Won Lee, Kyung-Hun Lee, Seock-Ah Im. Antitumor effect of AZD5305, a selective PARP1 inhibitor, in breast and gastric cancer cells [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B070.

Abstract B064: Single-cell RNA sequencing reveals tumorigenic trajectories of mismatch repair deficient cells

Abstract Mismatch repair (MMR) deficiency is commonly found in tumors with high mutational burden. Although the mutational signatures of these deficiencies are determined, the initial trajectories these cells follow to reach a tumorigenic state with high mutational load have not been studied due to lack of isogenic model systems. For this reason, we generated three isogenic MMR deficient cell lines from a normally untransformed cell line with few genomic abnormalities and low mutational burden. After continued culturing of these MMR-deficient cell lines for several months, we profiled them by single cell RNA sequencing using the 10X Chromium platform. Next, we identified different clusters of cells in MMR deficient cell lines that acquire tumorigenic properties. In these clusters, we identified gene expression signatures that are responsible from the tumorigenic properties of these clusters of cells. After isolation of the single clones that bear these gene expression signatures, we characterized how MMR deficiency can give rise to tumors with particular gene expression trajectories. In addition, we performed CRISPR/Cas9-based genome-wide screens to understand the vulnerabilities of these clusters of cells. Altogether, our results shed light on the early steps of tumorigenesis in MMR deficiency. Citation Format: Alexandra Vitor, Salvatore Benfatto, Frank Zenke, Sebastian Waszak, Balca Mardin. Single-cell RNA sequencing reveals tumorigenic trajectories of mismatch repair deficient cells [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B064.

Human Umbilical Cord Perivascular Cells Prevent Tumor Growth in a Melanoma Tumor-Bearing Mouse Model and Modulate Breast Cancer and Melanoma Cells in a Cell Line-Dependent Manner In Vitro

First trimester (FTM) and term human umbilical cord perivascular cells are promising mesenchymal stromal cell candidates to mitigate side effects of oncotherapy, but their safety for cancer patients remains to be determined. This study was designed to determine if human umbilical cord perivascular cells modulate tumor growth when injected systemically in a tumor-bearing mouse model. Immunodeficient mice-bearing palpable subcutaneous SK-MEL-28 human melanoma tumors were randomized to receive a tail vein injection of three human umbilical cord perivascular cell lines resuspended in hank’s buffer saline solution (vehicle) or vehicle only, as a control. Fibroblast cells were included as a cell control in some experiments. Tumor size was monitored weekly and weighed at 3-weeks postinjection. Cell fate and tumor cell proliferation, apoptosis, vascularization as well as tumor-associated immune cells were assessed using immunostaining and flow cytometry. Serum tumor necrosis factor alpha and C-reactive protein levels were measured using enzyme-linked immunosorbent assays. Transwell coculture models were used to study the paracrine effects of multiple lines of human umbilical cord cells on human melanoma cell lines as well as breast cancer cell lines. Systemic administration of FTM and term human umbilical cord perivascular cells, but not fibroblast cells, prevented melanoma tumor growth in a tumor-bearing animal model by modulating tumor cell proliferation and systemic inflammatory mechanisms. Cancer cell- and donor-dependent paracrine effects on cancer cell growth were observed in vitro. Our preclinical studies thus suggest that, with regards to its effects on tumor growth, systemic administration of FTM and term human umbilical cord perivascular cells may be a safe cell therapy to address the side effects of cancer.

Zerumbone (ZER), a Potential Anticancer for Breast Mediates Cancer Cell Death Through Targeting β-catenin Signaling Pathway in MCF-7 Cell Line

Background: Zerumbone is a sesquiterpene which was found to delay progression of breast cancer through apoptosis induction via up-regulating Bax protein and down-regulating Bcl2 protein. Purpose: In this study, we aimed to investigate Zerumbone, a natural compound of medicinal herbal plant isolated from Zingiber zerumbet, for anti-cancer activity against MCF-7 cell line. Methods: Human cancer cell lines of breast MCF-7 cells were obtained from American Type Culture Collection (ATCC, Manassas, VA). ON target plus smart pool siRNA, the most efficient method is used for preparation of β-catenin siRNA. Cyclophilin B siRNA is considered as positive control. Zrumbone is an ethanol-soluble compound isolated from Zingiber zerumbet, extracted by the hydro-distillation (steam distillation) method. Purification of ZER was examined using HPLC. The MCF-7 cells transfected with 100 µM human β-catenin (CTNNB1) siRNA or cyclophilin B siRNA positive control. After transfection, the cells were harvested at 48hrs for mRNA analysis or 48 – 72 hrs for protein analysis. IC50 for ZER was determined using MTT assay. MCF-7 transfected with β-catenin siRNA were then treated with ZER IC50. The mRNA expression was analyzed using real-time to ensure the knock down of β-catenin. TUNNEL assay was used to confirm apoptosis and flow cytometry was applied for quantification of apoptotic cells. Western blot analysis was done to evaluate the effect of ZER and β-catenin (CTNNB1) siRNA on the expression of β-catenin and its functional impact on the proliferation and survival of MCF-7 cells. Results: ZER decreased β-catenin protein expression in breast cancer cell lines. ZER downregulated β-catenin mRNA level in breast cancer cell lines. Depletion of β-catenin by β-catenin siRNA and ZER induce cell apoptosis. Apoptosis caused by ZER was confirmed/examined using by annexin V (annexin V was used as a marker for drug action). Conclusion: Zerumbone possesses anti-breast cancer activity, which could be attributed to the reduction of β-catenin protein expression in MCF-7 breast cancer cell lines, downregulation of β-catenin mRNA level in breast cancer cell lines and through annexin V.

Nanoinjection of extracellular vesicles to single live cells by robotic fluidic force microscopy.

In the past decade, extracellular vesicles (EVs) have attracted substantial interest in biomedicine. With progress in the field, we have an increasing understanding of cellular responses to EVs. In this Technical Report, we describe the direct nanoinjection of EVs into the cytoplasm of single cells of different cell lines. By using robotic fluidic force microscopy (robotic FluidFM), nanoinjection of GFP positive EVs and EV-like particles into single live HeLa, H9c2, MDA-MB-231 and LCLC-103H cells proved to be feasible. This injection platform offered the advantage of high cell selectivity and efficiency. The nanoinjected EVs were initially localized in concentrated spot-like regions within the cytoplasm. Later, they were transported towards the periphery of the cells. Based on our proof-of-principle data, robotic FluidFM is suitable for targeting single living cells by EVs and may lead to information about intracellular EV cargo delivery at a single-cell level.

Application of State Transition Theory and Treatment Modeling for Predicting Response to Chemotherapy in a Mouse Model of Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a cancer of the myeloid line of blood cells, and the cancer originates in the bone marrow (BM) and circulates the body through the peripheral blood (PB), which can rapidly lead to BM failure and death. AML initiation and progression are composed of multiple biological properties, and we have previously shown that the state transition theory, state-space, and a model of the stochastic differential equation (SDE) could predict AML disease evolution using the mRNA of a conditional Cbfb-MYH11 (CM) knock-in mouse model. The model of the SDE represents AML evolution from health to disease as a state transition of the transcriptome state described as a particle undergoing Brownian motion in a double-well quasi-potential, where critical points define states of healthy state, c1, transition to AML, c2, and overt AML, c3. Along with the disease evolution of AML, prediction, and understanding of AML disease responses from chemotherapy aiming to provide optimized therapeutic strategies are required. Analyzing disease responses to chemotherapy from the state transition theory perspective will give us an understanding of post-chemotherapy disease dynamics and useful predictions which have the possibility to optimize treatment processes. To test the applicability of the AML state transition model, a time-series experiment using the conditional CM knock-in mouse model and state-transition modeling were performed. The mouse model was designed to recapitulate the human inv(16) AML, the leukemogenic fusion gene Cbfb-MYH11 (CM) in conditional CM knock-in mice (Cbfb56M/+/Mx1-Cre) cause the development of AML with a median survival of approximately 4 months after induction of CM. To induce CM expression, 6-8 weeks old CM knock-in mice were injected intraperitoneally with polyinosinic-polycytidylic acid [poly (I:C)] (InvivoGen, tlrl-picw-250) at 14 mg/kg/dose every other day for a total of 7 doses. CM leukemic mice were treated with a combination of cytarabine (50mg/kg/day; 5 days) and daunorubicin (1.5mg/kg/day; 3 days) after detection of overt leukemia, which is monitored by circulating leukemia blast (cKit+ > 20%) to model the 7+3 standard of care treatment for newly diagnosed AML. We used three independent experiments of mice as training and test cohorts (Group 1, Group 2, and Group 3). We collected 42 PBMC and 1 BM from CM-induced mice from Group 1 (4 AML and 1 control), 68 PBMC and 2 BM from Group 2 (4 AML), and 64 PBMC from Group 3 (3 AML and 2 control). The samples were collected weekly before, during, and following chemotherapy and subjected to RNA sequencing. The effects of chemotherapy were observed in the AML state-space with chronological time points as the transcriptome particle moving from leukemia state, c3, towards a state of healthy state, c1, crossing the unstable state, c2. The relapse was defined as the time of the first observation after the particle crosses back over unstable state, c2, towards the leukemic state, c3. The 10 AML mice out of 11 AML mice achieved a partial response with a mean time to relapse of 5 weeks. All 11 AML mice showed the anti-correlation between AML state in the state space was anti-correlated with the blast percentages (ckit%). We modified the state transition treatment model by introducing a parameter, γ, and the half-life of cytarabine (31.1 hours) and daunorubicin (21.9 hours). The gamma parameter is a force that works against the double-well quasi potential (γ >1) and represents the anti leukemogenic effects of cytarabine and daunorubicin. Gamma was determined by fitting the model to 6 AML mice data from the training cohort. The value was determined to be γ=2.7, providing a novel quantification of the treatment effect. We performed a mean arrival time analysis using simulations of the state transition treatment model, which accurately predicted the time to relapse with a prediction error of 6.6 days for the 3 AML mice from the test cohort. We could apply the state transition theory and propose the state transition treatment model to simulate the treatment process and the response from chemotherapy. Our model accurately predicted the time to respond to chemotherapy, from leukemia to health, and relapse, from health to leukemia, in the CM mouse model after chemotherapy. The state transition treatment model has implications for improving therapeutic strategies by targeting transcriptome state transition critical points in human AML.

Downregulation of MALAT1 in triple-negative breast cancer cells

BackgroundMALAT1 is one of the most abundant nuclear long non-coding RNAs, which has been found to be elevated in various types of cancers. However, conflicting reports on MALAT1 in breast cancer cell lines challenge understanding of MALAT1's involvement in breast cancer progression. AimMeasurement of normalized relative quantity (NRQ) of MALAT1 transcripts in cell lines representing triple-negative breast cancer (TNBC) and luminal breast cancer. Materials and methodsThe studies were performed using cell lines representing luminal breast cancer (T47D, MCF-7), TNBC (MDA-MB-468, CAL-51, MDA-MB-231), and MCF-10A cell line of normal breast epithelial cells. Total RNA was isolated from six independent cell cultures of each line, treated with DNase I, and used to synthesize complementary DNA, which was used in quantitative real-time PCR (qPCR) assays. Four MALAT1 fragments and reference genes CCSER2, ANKRD17, PUM1, GAPDH were amplified. ResultsGeometric means of the NRQ of MALAT1 in breast cancer cell lines had the shortest 95% confidence intervals when CCSER2 was used for normalization. MALAT1 major transcript levels thus estimated in TNBC cell lines were found to be statistically significantly reduced compared to levels in both MCF-10A cells and luminal breast cancer cell lines, while MALAT1 minority splice variants were found to be increased in almost all breast cancer cell lines. ConclusionCCSER2-normalized qPCR results indicate MALAT1 downregulation in cell lines representing the more aggressive breast cancer subtype compared to both the normal breast epithelial cell line and the estrogen receptor-positive breast cancer cell lines.

Inhibition of Lysine Acetyltransferases p300/CBP Overcomes BTK Inhibitor Resistance in Mantle Cell Lymphoma Cells

Background: Although Bruton's Tyrosine Kinase inhibitors (BTKis) are effective against relapsed refractory mantle cell lymphoma (MCL), the limited duration of therapeutic response and the emergence of resistance remain clinical issues. We conducted a CRISPR/Cas9 screen to identify the genes responsible for BTKi sensitivity and resistance. Materials and Methods: To identify the genes responsible for BTKi resistance, we used a CRISPR library (Kosuke Yusa, V3), targeting 18,740 human genes. We compared three BTKis, ibrutinib, acalabrutinib and tirabrutinib, using DMSO as a control. We transduced Granta519, an ibrutinib-resistant MCL cell line expressing Cas9 with the CRISPR library lentiviruses. Next, we counted sgRNA copy numbers using next generation sequencing after a two-week drug treatment. We identified 76 genes that sensitized cells to the BTKis upon knockout. After excluding common essential genes based on the Cancer Dependency Map portal (depmap.org/portal/), we ranked these genes according to the extent to which the combination of their knockout and BTKis inhibited cell proliferation. Results: Lysine acetyltransferases (KATs), p300 and CBP were ranked first and ninth, respectively. Knockout of p300 or CBP alone moderately decreased cell numbers, where as their combination with BTKi treatment significantly decreased cell numbers. To further validate this synergistic effect, we performed an MTS assay in combination with p300/CBP inhibitors (C-646 and A-485) and ibrutinib in MCL cell lines (Granta519 and Mino). The p300/CBP inhibitor increased ibrutinib sensitivity, leading to a up to four-fold greater suppression of cell viability. This effect was even greater than that of existing drugs, including venetoclax (a BCL-2 inhibitor), and idelalisib (a PI3K inhibitor), which are thought to have synergistic effects. The synergistic effect of a p300/CBP inhibitor and ibrutinib was also observed in long-term cultures for up to two weeks in both ibrutinib-resistant and ibrutinib-sensitive MCL cell lines. Furthermore, combination treatment resulted in an enhancement of G0/G1 cell cycle arrest and an increase in apoptotic cells in the MCL cell lines. To elucidate the mechanism underlying this synergistic effect, we measured the expression of IκBα mRNA as an indicator of NFκB activity by real-time PCR. In Granta519, ibrutinib alone caused an initial decrease in IκBα mRNA expression followed by a rebound increase. However, the addition of A-485 decreased the rebound and suppressed IκBα mRNA expression. These results indicate that ibrutinib and A-485 cooperatively suppress NFκB activity. The combination of ibrutinib and A-485 synergistically suppressed phosphorylation of PLCγ2 and of AKT as confirmed by Western blotting. This suggests that downstream PLCγ2 and AKT activation might be involved in ibrutinib resistance. Lentiviral overexpression of p300 in Granta519 cells enhanced phosphorylation of AKT. On the other hand, ibrutinib suppressed the expression of p300, and conversely, p300/CBP inhibitors suppressed the expression of BTK. These data suggest that the expression of p300 and BTK are dependent on each other. We hypothesize that direct or indirect interactions may occur through phosphorylation by BTK and acetylation by p300. Conclusion: Our CRISPR screen identified lysine acetyltransferases p300/CBP as BTK inhibitor resistance genes in human MCL cells. The combination of a p300/CBP inhibitor and a BTK inhibitor synergistically suppresses both BTK and AKT, leading to NFκB suppression. Taken together, we first identified that the combination of p300/CBP inhibition and BTK inhibition could be a promising strategy for treating patients with MCL.

Compatibility of Venetoclax and OPN51107 in Treatment of Mantle Cell Lymphoma

Introduction Mantle Cell Lymphoma (MCL) is the third most common form of B-Cell lymphoma and patients diagnosed tend to have poor long-term survival when compared to patients of other B-cell lymphoma subtypes. Deregulation of the apoptotic cellular machinery contributes to early acquirement of therapy resistance in MCL and contributes to the poor clinical outcomes observed in relapsed/refractory MCL patients. Developing therapeutic strategies using agents that favor a pro-apoptotic cellular environment are being investigated in pre-clinical and clinical studies. To this end, we evaluated the therapeutic effects of targeting the Bcl-2 related proteins using Venetoclax, a Bcl-2 inhibitor, and OPN51107, a BET inhibitor, in MCL pre-clinical models. OPN51107 targets BRD4 in B-cells, a protein involved in the preinitiation complex for transcription of miRNA inhibit the translation of BIM, a pro-apoptotic initiator in the Bcl-2 family. Methods Using a panel of MCL cell lines (HBL-2, Granta, Z-138, Mino, Jeko-1, and Rec-1) along with resistant cells of each cell line made to Cytarabine, we conducted pre-clinical targeting of Bcl-2 and BRD4 with Venetoclax and OPN51107 respectively. Single and double agent exposure experiments were performed at 72 hrs and coefficients of synergy were assessed using CalcuSyn. Synergy was determined from cellular viability using Cell Titer-Glo (Promega) and was read using a BioTek Synergy HTX multimode reader. During in vivo experiments, 6-week SCID mice were inoculated with 10 × 10 6 HBL-2 or Granta cells via tail vein injection. OPN51107 was administered at 5 and 20 mg/kg/dose PO Days (3-7, 10-14. And 17-21). Venetoclax was administered at 50 and 100 mg/kg/dose PO Days (3-7, 10-14, and 17-21). Bcl-2 family member protein expression was determined via western blot. Protein-protein interactions of members of the Bcl-2 family were also studied though Co-immunoprecipitation. Results In vitro exposure of MCL cells lines resulted in an up-regulation of BIM levels. Synergistic activity between Venetoclax and OPN51107 was observed in SCID mice bearing the Granta cells but not in animals inoculated with the HBL-2 cell line. The median survival for Granta bearing SCID mice treated with Venetoclax and OPN51107 was longer (26 days) when compared to animals treated with either Venetoclax (24 days) or OPN51107 (25 days) (P values: 0.000383 and 0.089 respectively). Ongoing studies are investigating protein to protein interactions between BIM and other Bcl-2 related proteins to further define the mechanisms responsible for the synergy observed between Venetoclax and OPN51107. Conclusion Previous in vitro activity presented a synergistic relationship on cell death from combination treatment of OPN51107 and Venetoclax. Our present research suggests that this relationship can also be replicated in vivo and further research has been done to help elucidate a possible mechanism of action responsible for the enhancement of activity seen. Continuing these directions to understand the synergy between OPN51107 and Venetoclax is necessary to advance therapies within MCL.

The Effects of Acute Bisphenol A Toxicity on the Hematological Parameters, Hematopoiesis, and Kidney Histology of Zebrafish (Danio rerio)

In this study, the results of evaluating the acute toxicity of Bisphenol A on Danio rerio are presented, encompassing peripheral blood parameters, the composition of hematopoietic cells of erythroid and myeloid lines in the head kidney, and data from histological studies. The LC50 values of Bisphenol A for adult zebrafish individuals for 12, 24, and 48–96 h were determined, which were 18.04, 7.55, and 6.22 mg/L, respectively. The study includes data on the morphology and quantitative frequency of specific cells in the hematopoietic tissue of the head kidney, along with the consideration of adaptive mechanisms in hematopoiesis under BPA exposure. The application of polynomial regression analysis to reveal the concentration–effect relationship for some hematological and histological parameters was demonstrated. Significant increases in the frequency of erythrocyte nuclear abnormalities were observed at BPA concentrations of 6 and 8 mg/L, which indicates a genotoxic effect. BPA’s impact on fish peripheral blood parameters manifested as an increase in the number of erythrocytes (RBC) and immature erythrocytes, as well as a decrease in the number of lymphocytes. The most notable pathological changes in the head kidney’s hematopoietic tissue included circulatory disturbances and the formation of inflammation/degradation foci, as confirmed by histopathologic indices. At BPA concentrations of 2 and 4 mg/L, the observed changes were compensated for by hematopoietic adaptation mechanisms; however, at concentrations of 6 and 8 mg/L, acute systemic toxicity was evident.

ID2 Promotes Leukemogenesis and Chemoresistance of Acute Myeloid Leukemia By Modulating Cellular Oxidative Metabolism

Acute myeloid leukemia (AML) relies on quiescent leukemia stem cells (LSCs) to evade chemotherapy and drive disease relapse. Recent research indicates that a specific subset of LSCs, characterized by elevated oxidative phosphorylation and/or increased senescence, is responsible for chemotherapy resistance and relapse. While numerous genes associated with LSC maintenance and resistance have been identified, the underlying molecular mechanisms remain elusive. Inhibitor of DNA binding (ID) proteins, a group of Helix-Loop-Helix (HLH) transcription regulators, play a dominant role in regulating other HLH proteins (E proteins) required for the normal development of hematopoietic, muscle, neuronal, and other cells. Our previous investigation revealed that ID2 protects quiescent hematopoietic stem cells (HSCs) activation and exhaustion. Analysis of TCGA data sets demonstrated that high levels of ID2 expression are linked to reduced overall survival in AML patients, suggesting ID2 may be required to maintain some LSCs. Consistent with murine cell findings, we found that knocking down (KD) ID2 expression in human cord blood cells resulted in a loss of CD34+CD38- progenitors. Similarly, ID2-KD in AML cell lines (UT-7, MB-02, and M-07e) significantly reduced their leukemogenic potential in NSG mice, indicating that ID2 is essential for AML cell growth in vivo. Conversely, overexpressing ID2 in the human AML cell line M-07e enhanced their leukemic potential and burden in NSG mice, supporting the notion that ID2 may preserve LSC quiescence. Experiments with primary AML-Patient Derived Xenograft (AML-PDX) cells, which are more clinically relevant, also showed reduced leukemic potential in transplanted NSG mice upon ID2 knockdown. Additionally, ID2-KD cells displayed significantly lower levels of reactive oxygen species (ROS) and mitochondrial activity compared to control cells in both AML cell lines and PDX cells. As ROS-low cells are more susceptible to cytarabine (AraC), we investigated whether ID2-KD renders AML cells more susceptible to AraC. Indeed, MB-02-ID2-KD and UT7-ID2-KD cells showed increased sensitivity to AraC, while M-07e cells overexpressing ID2 exhibited resistance to AraC. Furthermore, AraC effectively eliminated ROS-low cells, but ROS-high cells remained resistant, supporting previous findings. To further investigate the effect of ID2-KD in AraC resistance in vivo, UT7-ID-KD and MB-02-ID2-KD transplanted NSG mice were treated with AraC and monitored for their overall survival, and it was observed that ID2-KD transplanted mice have significantly higher overall survival compared to the control cell transplanted mice. In conclusion, it can be said that ID2 plays a critical role in maintaining normal murine and human HSCs, and it likely contributes to leukemia development and chemotherapy resistance. Therefore, targeting ID2 may offer a promising therapeutic approach to selectively tackle LSCs, followed by chemotherapy and bone marrow transplantation to achieve complete remission.

Unveiling the anticancer potentiality of single cell oils produced by marine oleaginous Paradendryphiella sp. under optimized economic growth conditions

Bioprospecting about new marine oleaginous fungi that produce advantageous bioproducts in a green sustainable process is the key of blue bioeconomy. Herein, the marine Paradendryphiella sp. was utilized for single cell oils (SCOs) production economically, via central composite design, the lipid content enhanced 2.2-fold by 5.5 g/L lipid yeild on seawater-based media supplemented with molasses concentration 50 g/L, yeast extract, 2.25 g/L at initial pH value (5.3) and 8 days of static incubation. Subsequently, the fatty acid methyl esters profiles of SCOs produced on optimized media under different abiotic conditions were determined; signifying qualitative and quantitative variations. Interestingly, the psychrophilic-prolonged incubation increased the unsaturation level of fatty acids to 59.34%, while ω-6 and ω-3 contents representing 23.53% and 0.67% respectively. Remarkably, it exhibited the highest EC100 dose by 677.03 µg/mL on normal human lung fibroblast Wi-38 cells. Meanwhile, it showed the highest inhibiting proliferation potential on cancer cell lines of A549, MDA-MB 231 and HepG-2 cells by 372.37, 417.48 and 365.00 µg/mL, respectively. Besides, it elevated the oxidative stress, the expression of key apoptotic genes and suppressed the expression of key oncogenes (NF-κB, BCL2 and cyclin D); implying its promising efficacy in cancer treatment as adjuvant drug. This study denoted the lipogenesis capacity of Paradendryphiella sp. under acidic/alkaline and psychrophilic/mesophilic conditions. Hereby attaining efficient and economic process under seasonal variation with different Egyptian marine sources to fill the gap of freshwater crisis and simultaneously preserve energy.