Low Levels Of Apoptosis Research Articles

Helicobacter pylori infection delays neutrophil apoptosis and exacerbates inflammatory response.

Aim: Understanding molecular mechanisms of Helicobacter pylori (H. pylori)-induced inflammation is important for developing new therapeutic strategies for gastrointestinal diseases.Materials & methods: We designed an H. pylori-neutrophil infection model and explored the effects of H. pylori infection on neutrophils.Results: H. pylori infected neutrophils showed a low level of apoptosis. H. pylori stimulation activated the NACHT/LRR/PYD domain-containing protein 3 (NLRP3)-gasdermin-D (GSDMD) pathway for interleukin (IL)-1β secretion. However, IL-1β secretion was not completely dependent on GSDMD, as inhibition of autophagy significantly reduced IL-1β release, and autophagy-related molecules were significantly upregulated in H. pylori-infected neutrophils.Conclusion: Therefore, H. pylori infection inhibits neutrophils apoptosis and induces IL-1β secretion through autophagy. These findings may be utilized to formulate therapeutic strategies against H. pylori mediated chronic gastritis.

RIPK1 is dispensable for cell death regulation in β-cells during hyperglycemia

ObjectiveReceptor-interacting protein kinase 1 (RIPK1) orchestrates the decision between cell survival and cell death in response to tumor necrosis factor (TNF) and other cytokines. Whereas the scaffolding function of RIPK1 is crucial to prevent TNF-induced apoptosis and necroptosis, its kinase activity is required for necroptosis and partially for apoptosis. Although TNF is a proinflammatory cytokine associated with β-cell loss in diabetes, the mechanism by which TNF induces β-cell demise remains unclear. MethodsHere, we dissected the contribution of RIPK1 scaffold versus kinase functions to β-cell death regulation using mice lacking RIPK1 specifically in β-cells (Ripk1β-KO mice) or expressing a kinase-dead version of RIPK1 (Ripk1D138N mice), respectively. These mice were challenged with streptozotocin, a model of autoimmune diabetes. Moreover, Ripk1β-KO mice were further challenged with a high-fat diet to induce hyperglycemia. For mechanistic studies, pancreatic islets were subjected to various killing and sensitising agents. ResultsInhibition of RIPK1 kinase activity (Ripk1D138N mice) did not affect the onset and progression of hyperglycemia in a type 1 diabetes model. Moreover, the absence of RIPK1 expression in β-cells did not affect normoglycemia under basal conditions or hyperglycemia under diabetic challenges. Ex vivo, primary pancreatic islets are not sensitised to TNF-induced apoptosis and necroptosis in the absence of RIPK1. Intriguingly, we found that pancreatic islets display high levels of the antiapoptotic cellular FLICE-inhibitory protein (cFLIP) and low levels of apoptosis (Caspase-8) and necroptosis (RIPK3) components. Cycloheximide treatment, which led to a reduction in cFLIP levels, rendered primary islets sensitive to TNF-induced cell death which was fully blocked by caspase inhibition. ConclusionsUnlike in many other cell types (e.g., epithelial, and immune), RIPK1 is not required for cell death regulation in β-cells under physiological conditions or diabetic challenges. Moreover, in vivo and in vitro evidence suggest that pancreatic β-cells do not undergo necroptosis but mainly caspase-dependent death in response to TNF. Last, our results show that β-cells have a distinct mode of regulation of TNF-cytotoxicity that is independent of RIPK1 and that may be highly dependent on cFLIP.

Exercise Inhibits Doxorubicin-Induced Cardiotoxicity via Regulating B Cells.

Doxorubicin is an effective chemotherapeutic agent, but its use is limited by acute and chronic cardiotoxicity. Exercise training has been shown to protect against doxorubicin-induced cardiotoxicity, but the involvement of immune cells remains unclear. This study aimed to investigate the role of exercise-derived B cells in protecting against doxorubicin-induced cardiotoxicity and to further determine whether B cell activation and antibody secretion play a role in this protection. Mice that were administered with doxorubicin (5 mg/kg per week, 20 mg/kg cumulative dose) received treadmill running exercise. The adoptive transfer of exercise-derived splenic B cells to μMT-/- (B cell-deficient) mice was performed to elucidate the mechanism of B cell regulation that mediated the effect of exercise. Doxorubicin-administered mice that had undergone exercise training showed improved cardiac function, and low levels of cardiac apoptosis, atrophy, and fibrosis, and had reduced cardiac antibody deposition and proinflammatory responses. Similarly, B cell pharmacological and genetic depletion alleviated doxorubicin-induced cardiotoxicity, which phenocopied the protection of exercise. In vitro performed coculture experiments confirmed that exercise-derived B cells reduced cardiomyocyte apoptosis and fibroblast activation compared with control B cells. Importantly, the protective effect of exercise on B cells was confirmed by the adoptive transfer of splenic B cells from exercised donor mice to μMT-/- recipient mice. However, blockage of Fc gamma receptor IIB function using B cell transplants from exercised Fc gamma receptor IIB-/- mice abolished the protection of exercise-derived B cells against doxorubicin-induced cardiotoxicity. Mechanistically, we found that Fc gamma receptor IIB, an important B cell inhibitory receptor, responded to exercise and increased B cell activation threshold, which participated in exercise-induced protection against doxorubicin-induced cardiotoxicity. Our results demonstrate that exercise training protects against doxorubicin-induced cardiotoxicity by upregulating Fc gamma receptor IIB expression in B cells, which plays an important anti-inflammatory role and participates in the protective effect of exercise against doxorubicin-induced cardiotoxicity.

CCRG-07. GOLD NANOPARTICLES DEMONSTRATE FAVORABLE EFFECTS ON THE BEHAVIOR OF MESENCHYMAL STEM CELLS, THEREBY AUGMENTING THEIR OVERALL BIOLOGICAL PERFORMANCE

Abstract Gold nanoparticles (AuNPs) have been extensively studied for their interaction with cells, particularly mesenchymal stem cells (MSCs), and their potential to influence various cellular behaviors such as proliferation and differentiation. In this research, AuNP solutions at concentrations ranging from 1.25 to 10 ppm were prepared to investigate their biocompatibility. The presence of AuNPs was confirmed using ultraviolet-visible spectroscopy, while Dynamic Light Scattering assay characterized their size and scanning electron microscopy revealed their shape. MSCs were treated with different concentrations of AuNPs to assess biocompatibility. The results indicated that 1.25 and 2.5 ppm concentrations of AuNPs significantly enhanced MSC proliferation, reduced reactive oxygen species generation, and attenuated platelet/monocyte activation. Furthermore, the presence of filopodia and lamellipodia suggested improved adhesion ability of MSCs when incubated with 1.25 and 2.5 ppm AuNPs. The expression of matrix metalloproteinase (MMP-2/9) was also upregulated under these concentrations, indicating enhanced cell migration ability. Lower levels of apoptosis were observed in MSCs treated with 1.25 and 2.5 ppm AuNPs compared to higher concentrations, as assessed by Annexin-V/PI double staining and Fluorescence Activated Cell Sorting (FACS). Western blotting analysis showed increased expression of anti-apoptotic proteins Cyclin-D1 and Bcl-2. Additionally, real-time PCR analysis demonstrated reduced expression of inflammatory cytokines (TNF-α, IL-1β, IFN-γ, IL-6, and IL-8) in MSCs treated with 1.25 and 2.5 ppm AuNPs. Animal model tests revealed that these concentrations of AuNPs exhibited better biocompatibility, including anti-inflammatory effects and endothelialization. Overall, 1.25 and 2.5 ppm AuNP solutions were shown to enhance the biological functions of MSCs, suggesting their potential as biocompatible nanomedicine for regenerative research.

Myelodysplastic Syndrome Red Blood Cell Physiology Abnormalities and Their Correlation with Disease Risk and Therapeutic Interventions

PV and KT as well as VP and MA contributed equally Introduction: Myelodysplastic syndromes (MDS) are clonal hematopoietic disorders characterized by ineffective hematopoiesis, different degree of cytopenias, and increased risk of transformation to AML. Anemia constitutes a main clinical manifestation contributing to increased morbidity and mortality. It is currently managed with red blood cell (RBC) transfusions, erythropoiesis stimulating agents (EPO), and recently,with luspatercept. The latter enhances late stage erythropoiesis and is indicated for Ring Sideroblasts (RS) MDS resistant to EPO. Several lines of evidence suggest that MDS pathophysiology is associated with structural and functional distortions of mature red cell (RBCs), such as hemolysis that in rare instances include acquired lesions like hemoglobinopathies and membranopathies.These abnormalities add variability to the clinical MDS phenotypes and could serve as biomarkers for early diagnosis or monitoring. However, they have been scarcely investigated. Patients and methods: We examined 40 MDS patients and 12 healthy sex- and age-matched controls. According to the IPSS-Rrisk stratification system, 8 patients were High Risk (HR) and 32 patients Low Risk (LR)MDS, of whom 8 under EPO (EPO-LR) and 4 under luspatercept (luspatercept-LR) treatment. None of them had received RBC transfusions in the past three months. Following full blood count analyses and RBC fractionations, we measured hemolysis (basal, osmotic, mechanical, and oxidative), eryptosis and oxidative stress, as well as the antioxidant capacity of the plasma through spectrophotometry,fluorometry and flow cytometry. Results: Compared to controls, the cohort of MDS patients was characterized by anemia (Hb 10.7 vs 13.6g/dL) and significantly higher HDWand RDW (17.5 vs. 13.4%) indexes. RBCs exhibited signs of eryptosis (RBC calcium concentration 7063±1443 vs. 6027±1541 RFU/mg; PS surface exposure 0.61±0.45 vs. 0.33±0.17%) and oxidative stress (higher metHb/oxyHb ratio; membrane-bound hemichromes 45.0±15.3 vs. 30.2±13.3). About 18% of the patients presented spontaneous hemolysis. Clinical severity correlated with the levels of cytopenias, and oxidative hemolysis. Anemia correlated positively with the age, and the levels of membrane-bound hemichromes, eryptosis and intracellular reactive oxygen species (ROS). According to ROC analysis, the combination of RDW, metHb/oxyHb ratio, and total Hb concentration is predictive of MDS, across all IPSS-R categories(area under the curve: 0.908; Figure 1). We then examined the subgroups of MDS patients. Apart from the RDW, the LR-MDS subgroup exhibited on average normal RBC features, in opposite to the more anemic and thrombocytopenicHR-MDS subgroup that showed pathologically increasedresistance to osmotic hemolysis, intracellular calcium (7946±1917 vs. 6026±1541RFU), and metHb/oxyHb ratio (18.5±7.3 vs. 10.4±3.9%), when compared to controls. Of note, similar abnormalities were observed in the EPO subgroup that further showed reticulocytosis, PS externalization, and membrane-bound hemichromes(p<0.05) Compared to the non-treated subgroup, the EPO-LR had lower WBC counts, but higher RDW, PS exposure (0.75±0.44 vs. 0.41±0.26%) and resistance to osmotic stress, aspreviously reported in other EPO-treated patients.Luspatercept-LR had higher WBC and PLT counts but exhibited EPO-like effects on RBCs, including pathological resistance to osmotic/mechanical lysis, ROS concentration (1347±453 vs. 960±160 RFU), Hb oxidation, and PS exposure (1.05±0.60 vs. 0.41±0.26%), compared to the non-treated LR subgroup. Conclusion: MDS RBCs are highly heterogeneous in terms of volume and Hb concentration and present signs of oxidative stress and eryptosis. Consequently, these easily accessible markers could be an accessory tool to the MDS diagnosis.In the context of ineffective hematopoiesis and increased apoptosis of precursor erythroid cells,the LR-MDS patients exhibit minimal RBC abnormalities. On the other hand, the expansion of BM blasts and the lower level of apoptosis in HR-MDS seem to affect RBCs in a hemoglobinopathy-like manner in terms of Hb oxidation, eryptosis signaling, and membrane ion channels. EPO and luspatercept treated LR patients exhibit improved cytopenias but worse RBC physiology most probably attributed to clinical factors like RS phenotype.

Optimization of ultrasound extraction technology of DTD from Ageratina adenophora, and the preparation and characterization of nanoemulsions: Insecticidal activity and safety evaluation

Due to the imperfect extraction process of 4,7-dimethyl-1-(propan-2-ylidene)− 1,4,4a,8a-tetrahydronaphthalene-2,6(1 H,7 H)-dione (DTD) and the nonconstruction of delivery systems, their practical application in the field of botanical pesticides is limited. To overcome these problems, ultrasound-assisted extraction technology was optimized using BoxBehnken design (BBD). The optimal ultrasound extraction conditions of DTD were as follows: time (39 min), solid to liquid ratio (1:31), and temperature (68 ℃). The extraction yield was 0.4528 ± 0.0182 %. Nanoemulsions (NEs) loaded with DTD were prepared and characterized, and the optimal formulation of DTD NEs was determined as follows: 1 wt% DTD, 1.5 wt% S-200, 1 wt% TW-80, and 96.5 wt% deionized water. The NEs prepared by the optimal formula had small contact angles and tension levels, indicating that they had good wetting properties. The larvicidal activity of DTD NEs is similar to that of azadirachtin EC. In the biosafety assessment, the DTD NE treatment group had low levels of cell apoptosis, the toxicity of the DTD NEs to earthworms was low, and the DTD NEs had no adverse effect on maize growth. Therefore, DTD NEs offer a high degree of biosafety. DTD NEs have the potential to serve as a safe, green alternative pesticide for IPM projects to control important agricultural pests.

IMMUNOCHEMICAL ANALYSIS OF BCL-2 AND BAX EXPRESSION IN BRONCHIAL EPITHELIUM IN RATS IN ACUTE BACLOFEN POISONING

Baclofen (also sold under trade names Lioresal, Baclosan) belongs to a group of drugs called antispasmodics. This drug is available in oral and intrathecal forms. Baclofen has a pronounced psychotropic effect and is often a subject to abuse, especially among young people. The aim of the study was to detect apoptosis in the bronchial epithelium in rats. The study was performed on 20 male Wistar rats (weight: 290-350 g., age: 20 weeks). The animals were divided into 4 groups. The group of controls included 5 intact rats, each of three experimental groups included 5 rats treated with baclofen at a dose of 85 mg/kg and euthanized by the displacement of the cervical vertebrae after 3, 4,5, and 24 hours, respectively. In the bronchial epithelium of the controls bax was weakly expressed, which shows a low level of apoptosis. 3 and 4,5 hours after baclofen administration bax was strongly expressed and 24 hours after baclofen administration bax was moderately expressed. In the bronchial epithelium of the controls bcl-2 was weakly expressed; 3 hours after baclofen administration bcl-2 was moderately expressed; 4,5 hours and 24 hours after baclofen administration bcl-2 was strongly expressed. The results from the present study suggest the involvement of apoptosis in the development in the lesion of bronchial epithelium. Furthermore, bax and bcl-2 expression in the regenerating epithelial cells may play a role in the recovery of this lesion. Keywords: baclofen, poisoning, immunohistichemistry (IHC), apoptosis, bcl-2, bax.

XLF/Cernunnos loss impairs mouse brain development by altering symmetric proliferative divisions of neural progenitors

XLF/Cernunnos is a component of the ligation complex used in classical non-homologous end-joining (cNHEJ), a major DNA double-strand break (DSB) repair pathway. We report neurodevelopmental delays and significant behavioral alterations associated with microcephaly in Xlf-/- mice. This phenotype, reminiscent of clinical and neuropathologic features in humans deficient in cNHEJ, is associated with a low level of apoptosis of neural cells and premature neurogenesis, which consists of an early shift of neural progenitors from proliferative to neurogenic divisions during brain development. We show that premature neurogenesis is related to an increase in chromatid breaks affecting mitotic spindle orientation, highlighting a direct link between asymmetric chromosome segregation and asymmetric neurogenic divisions. This study reveals thus that XLF is required for maintaining symmetric proliferative divisions of neural progenitors during brain development and shows that premature neurogenesis may play a major role in neurodevelopmental pathologies caused by NHEJ deficiency and/or genotoxic stress.

Sodium Butyrate Induces CRC Cell Ferroptosis via the CD44/SLC7A11 Pathway and Exhibits a Synergistic Therapeutic Effect with Erastin.

Human normal intestinal epithelial cell line FHT and colorectal tumor cell line HCT-116 were treated with NaB alone or in combination with different programmed cell death inhibitors. Cell activity was then assessed with MTT assays and PI staining; ferroptosis with Fe2+, glutathione (GSH), and lipid peroxidation assays; signaling pathway screening with PCR arrays; and CD44, SCL7A11, and GPX4 expression with Western blotting. A CD44-overexpressing HCT-116 cell line was constructed to determine the effect of the overexpression of CD44 on NaB-induced ferroptosis. The synergistic effect of co-treatment with NaB and Erastin was assessed by isobolographic analysis. NaB induced apoptosis and ferroptosis in HCT-116 cells but only induced low-level apoptosis in FHC cells. Moreover, NaB significantly increased intracellular Fe2+ and promoted GSH depletion and lipid peroxidation in HCT-116 cells. Ferroptosis-related qPCR array analysis identified CD44/SLC7A11 as a potential effector molecular of NaB-induced ferroptosis. NaB significantly inhibited the expression of CD44 and SLC7A11 in mouse CRC tissues. A CD44 overexpressed HCT-116 cell line was used to verify that CD44/SLC7A11 was a key signaling pathway that NaB-induced GSH depletion, lipid peroxidation accumulation, and ferroptosis in HCT-116 cells. Examination of whether NaB can increase the effect of ferroptosis agents showed that NaB, in combination with Erastin, a ferroptosis inducer, further promoted HCT-116 cell death and increased changes of ferroptosis markers. Our results suggest that NaB induces ferroptosis in CRC cells through the CD44/SLC7A11 signaling pathway and has synergistic effects with Erastin. These results may provide new insights into CRC prevention and the combined use of NaB and ferroptosis-inducing agents.

Polyploid/Multinucleated Giant and Slow-Cycling Cancer Cell Enrichment in Response to X-ray Irradiation of Human Glioblastoma Multiforme Cells Differing in Radioresistance and TP53/PTEN Status.

Radioresistance compromises the efficacy of radiotherapy for glioblastoma multiforme (GBM), the most devastating and common brain tumor. The present study investigated the relationship between radiation tolerance and formation of polyploid/multinucleated giant (PGCC/MGCC) and quiescent/senescent slow-cycling cancer cells in human U-87, LN-229, and U-251 cell lines differing in TP53/PTEN status and radioresistance. We found significant enrichment in MGCC populations of U-87 and LN-229 cell lines, and generation of numerous small mononuclear (called Raju cells, or RJ cells) U-87-derived cells that eventually form cell colonies, in a process termed neosis, in response to X-ray irradiation (IR) at single acute therapeutic doses of 2-6 Gy. For the first time, single-cell high-content imaging and analysis of Ki-67- and EdU-coupled fluorescence demonstrated that the IR exposure dose-dependently augments two distinct GBM cell populations. Bifurcation of Ki-67 staining suggests fast-cycling and slow-cycling populations with a normal-sized nuclear area, and with an enlarged nuclear area, including one resembling the size of PGCC/MGCCs, that likely underlie the highest radioresistance and propensity for repopulation of U-87 cells. Proliferative activity and anchorage-independent survival of GBM cell lines seem to be related to neosis, low level of apoptosis, fraction of prematurely stress-induced senescent MGCCs, and the expression of p63 and p73, members of p53 family transcription factors, but not to the mutant p53. Collectively, our data support the importance of the TP53wt/PTENmut genotype for the maintenance of cycling radioresistant U-87 cells to produce a significant amount of senescent MGCCs as an IR stress-induced adaptation response to therapeutic irradiation doses.

Imaging DNA damage response by γH2AX in vivo predicts treatment response to Lutetium-177 radioligand therapy and suggests senescence as a therapeutically desirable outcome.

Rationale: An effective absorbed dose response relationship is yet to be established for Lutetium-177 based radionuclide therapies such as 177Lu-DOTATATE and 177Lu-PSMA. The inherent biological heterogeneity of neuroendocrine and prostate cancers may make the prospect of establishing cohort-based dose-response relationships unobtainable. Instead, an individual-based approach, monitoring the dose-response within each tumor could provide the necessary metric to monitor treatment efficacy. Methods: We developed a dual isotope SPECT imaging strategy to monitor the change over time in the relationship between 177Lu-DOTATATE and 111In-anti-γH2AX-TAT, a modified radiolabelled antibody that allows imaging of DNA double strand breaks, in mice bearing rat pancreatic cancer xenografts. The dynamics of γH2AX foci, apoptosis and senescence following exposure to 177Lu-DOTATATE was further investigated in vitro and in ex vivo tumor sections. Results: The change in slope of the 111In-anti-γH2AX-TAT to 177Lu signal between days 5 and 7 was found to be highly predictive of survival (r = 0.955, P < 0.0001). This pivotal timeframe was investigated further in vitro: clonogenic survival correlated with the number of γH2AX foci at day 6 (r = -0.995, P < 0.0005). While there was evidence of continuously low levels of apoptosis, delayed induction of senescence in vitro appeared to better account for the γH2AX response to 177Lu. The induction of senescence was further investigated by ex vivo analysis and corresponded with sustained retention of 177Lu within tumor regions. Conclusions: Dual isotope SPECT imaging can provide individualized tumor dose-responses that can be used to predict lutetium-177 treatment efficacy. This bio-dosimeter metric appears to be dependent upon the extent of senescence induction and suggests an integral role that senescence plays in lutetium-177 treatment efficacy.

GENETIC CHARACTERISTICS OF PADDLEFISH (POLYODON SPATHULA (WALBAUM, 1792)) BROOD STOCKS

GENETIC CHARACTERISTICS OF PADDLEFISH (POLYODON SPATHULA (WALBAUM, 1792)) BROOD STOCKS

Bariatric Surgery Associates with Nonalcoholic Steatohepatitis/Hepatocellular Carcinoma Amelioration via SPP1 Suppression.

Nonalcoholic steatohepatitis (NASH) is one of the most common chronic liver diseases worldwide and no effective drugs or treatments have been approved for disease management. Recently, bariatric surgery (BS) is considered to be a novel disease-modifying therapy for NASH and liver metabolic diseases, according to clinical follow-up studies. Despite the revealment of physiopathological alterations, underlying mechanisms and key factors remain indeterminate. This study included multiple bulk RNA-sequencing datasets to investigate transcriptome variation in one-year follow-up BS and diet management (Diet) NASH patients' liver biopsies. Liver functions, fibrosis, and carcinogenesis were predicted in liver samples via hallmark-based function enrichment analysis. Key factors generated from multi-dataset comparison were further assessed with hepatocellular carcinoma (HCC) progression and prognosis. BS leads to active gene expression alterations in NASH liver in comparison to diet management (Diet). Both approaches reduce cell stress and immune response, whereas BS contributes to higher metabolic levels and lower apoptosis levels. The macrophage infiltration, adipose accumulation, and fibroblast activation were revealed to be lower in post-BS NASH livers, further demonstrating positive correlations mutually. Seven key genes (MNDA, ALOX5AP, PECAM1, SPP1, CD86, FGF21, CSTA) were screened out as potential macrophage-associated and carcinogenetic factors suppressed by BS. SPP1 was identified as a crucial factor participating in BS intervened NASH-HCC progression. This study determined that BS exerts potentially superior protective functions in NASH livers compared to diet management. SPP1 may serve as a novel factor to study the functionalities of BS on NASH patients.

Evaluation of targeted oxidative stress induced by oxygen-ozone in vitro after ischemic induction

ABSTRACT Encephalic vascular accident, or stroke, is the most common pathology of the central nervous system in humans, the second leading cause of death and physical and cognitive disabilities, in developing countries. It presents as an ischemic (more common) or hemorrhagic form. Ozone therapy has been shown to be effective in neuromodulation, neuroprotection, and nerve regeneration. The present study aimed to evaluate the effect of targeted mild ozone after inducing cerebral ischemia in vitro. Neuroblastoma lineage cells (SH-SY5Y) and canine amniotic membrane stem cells were subjected to 24 hours of hypoxia in an incubator culture chamber. The cells were evaluated by MTT assay, colorimetric assay spectrophotometry, fluorescence microscopy, and flow cytometry. Treatment with low concentrations of ozone (2–10 µg/mL), indicated a possible neuroregenerative effect at low concentrations, correlated with lower levels of apoptosis and oxidative stress compared to cells not subjected to hypoxia. High concentrations of ozone (18–30 µg/mL) promoted an increase in rate of apoptosis and cell death. We developed a novel protocol that mimics ozone therapy for ischemic stroke, using ozonized culture medium after hypoxia induction. Although more studies are needed, we conclude that ozone has a dose-dependent hormetic effect and can reverse the effect of ischemia in vitro at low concentrations.

Patulin and LL-Z1640-2 induce apoptosis of cancer cells by decreasing endogenous protein levels of Zic family member 5.

Zic family member 5 (ZIC5) is a transcription factor that promotes the survival of several cancer cell types. As ZIC5 is expressed at minimal levels in normal human adult tissues, it is a potential therapeutic target. In this study, we screened a chemical library containing 3398 compounds that includes pre-existing drugs and compounds with known effects to identify ZIC5 inhibitors. In the first screening, 18 hit compounds decreased GFP intensity in melanoma A375 cells overexpressing GFP-tagged ZIC5. In the second screening, five compounds that attenuated ZIC5 protein levels in A375 cells were identified. Among them, LL-Z1640-2 and patulin selectively induced apoptosis in melanoma cells expressing ZIC5, while only inducing very low levels of apoptosis in normal human melanocytes, which have no detectable ZIC5 expression. LL-Z1640-2 and patulin also induced apoptosis in BRAF inhibitor-resistant melanoma, pancreatic cancer, cholangiocarcinoma and colorectal cancer cells. LL-Z1640-2- and patulin-mediated suppression of melanoma proliferation were rescued by ZIC5 overexpression. These results suggest that LL-Z1640-2 and patulin are promising compounds that decrease ZIC5 expression to induce apoptosis in cancer cells.

Abstract 186: Advances in 3D cell culture analysis: Utilizing novel flow cytometry technology for rapid analysis of intact spheroids

Abstract Cancer cells grown in two dimensional (2D) monolayers are a convenient model to use for basic research and in generating lead candidates in the drug discovery process. However, cells grown in 2D cultures lack many of the structural, biological, and functional attributes of solid tumors, which contributes to the high clinical failure rates for biologic and small molecule drugs. Multicellular three-dimensional (3D) tumor spheroids are advanced models which more faithfully mimic the physiological tumor milieu and is more predictive of therapeutic efficacy than traditional monolayer cultures. However, the inability to generate and to analyze large numbers of intact spheroids with a simplified workflow remains a barrier in the adoption of 3D models for a wide range of applications. To overcome these barriers, we used the Velocyt® large particle flow cytometer (Bennubio) to evaluate spheroid health in a well-established small molecule spheroid killing assay. The Velocyt technology rapidly and gently analyzes particles from 3 to 300 microns and comes with a suite of novel informatics tools purpose built for large particle analysis. To exemplify the use of large particle flow cytometry for the analysis of intact spheroids we generated uniformed-sized spheroids (~180 microns) from the H1650 human lung and HCT116 human colon cancer cell lines using microwell technology. Two to three days after spheroid generation, the spheroids were treated with increasing concentrations of staurosporine and other compounds. Spheroids were harvested at one to three days post treatment (depending on drug treatment) and stained with fluorescent cell viability and apoptotic dyes. Intact spheroids were analyzed for fluorescence and size using the Velocyt flow cytometer and between one and two thousand spheroids were assessed for each treatment in approximately three minutes. In untreated control spheroids, fluorescence was detected in a subpopulation of spheroids consistent with expected low levels of background apoptosis. In treated spheroids, an increase in the mean fluorescence intensity (MFI) of each particle indicated an increase in the number of non-viable and apoptotic cells in each spheroid. MFI increased nearly 6-fold with high concentrations of staurosporine compared to control spheroids, and dead cells were detected in nearly 100% of spheroids that were administered the highest staurosporine concentration. Using the size-based measurement parameters of the Velocyt, we also showed a decrease in overall spheroid size concurrent with increased numbers of spheroid fragments and single cells with increasing drug concentration. These data highlight that the use of 3D cell models combined with the Velocyt large particle flow cytometer is a powerful tool to help advance basic and translational cancer research. Citation Format: John P. O'Rourke, Mathew Saunders, Valerie Sanchez. Advances in 3D cell culture analysis: Utilizing novel flow cytometry technology for rapid analysis of intact spheroids [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 186.

Preparation of CD52-targeted chimeric antigen receptor-modified T cells and their anti-leukemia effects

目的构建一种靶向CD52的嵌合抗原受体T细胞（CD52 CAR-T），探索其对CD52+白血病的治疗效果。方法应用分子克隆技术构建以CD52为抗原结合区的CD52 scFv、4-1BB为共刺激分子的二代CAR-T细胞。慢病毒载体包装后感染T细胞，制备CD52 CAR-T细胞，通过流式细胞术检测CD52 CAR-T细胞CD4、CD8细胞亚群及其分化状态。通过体外杀伤实验、脱颗粒实验及细胞因子的释放等功能实验，观察CD52 CAR-T细胞对CD52+白血病细胞的特异性细胞毒作用。结果①成功构建了pCDH-CD52 scFv-CD8α-4-1BB-CD3ζ-GFP表达载体，感染人T细胞，获得靶向CD52的CAR-T细胞。②感染CD52 CAR表达载体的第6天，T细胞中表达CD52的细胞可被清除［CD52 CAR-T组CD52+ T细胞为（4.48±4.99）％，Vector-T组为（56.58±19.8）％，P＝0.011］。③CD52 CAR-T的自我杀伤不会影响T细胞的增殖，培养后期CD52 CAR-T组的增殖速度高于Vector-T组。④T细胞表型检测发现CD52 CAR可以促进CAR-T细胞向中心记忆T细胞及效应记忆T细胞分化，减少CAR-T细胞向终末效应细胞分化。⑤CD52 CAR-T细胞与HuT78-19t及MOLT4-19t靶细胞以效靶比1∶1共培养24 h，HuT78-19t细胞可被清除，而对MOLT4-19t细胞无明显杀伤作用［（2.66±1.60）％对（56.66％±5.74）％，P<0.001］。⑥脱颗粒实验显示，HuT78-19t细胞对CD52 CAR-T细胞的激活作用显著高于MOLT4-19t细胞［（57.34±11.25）％对（13.06±4.23）％，P<0.001］。⑦CD52 CAR-T与HuT78-19t细胞共培养48 h上清中细胞因子分泌水平［IFN-γ（3706±226）pg/ml、TNF-α（1732±560）pg/ml］远高于MOLT4-19t细胞组［IFN-γ（1577±846）pg/ml、TNF-α（74±12）pg/ml］（P值均<0.01）。结论该研究成功制备了具有抗白血病作用的CD52 CAR-T细胞，为进一步的靶向CD52的免疫治疗奠定了基础。

ASPP2 Coordinates ERS-Mediated Autophagy and Apoptosis Through mTORC1 Pathway in Hepatocyte Injury Induced by TNF-α.

Background: Though ASPP2 plays an important role in regulating cell apoptosis and autophagy in case of liver injury, there remains a lack of clarity on the molecular mechanism of ASPP2 regulating autophagy and apoptosis. Methods: A hepatocyte injury model was constructed using HL7702 cell line and TNF-α. The cells were treated by ASPP2 overexpression adenovirus or short hairpin RNA lentivirus and endoplasmic reticulum stress (ERS) or the mammalian target of rapamycin (mTOR) inhibitor or agonist, respectively. The autophagy was detected by means of western blot and Green fluorescent protein-labeled- Microtubule-associated protein light chain 3 (GFP-LC3) plasmid transfection, while the apoptosis was detected through western blot, flow cytometry and TUNEL assay. Besides, the proteins related to ERS and mTOR were detected by western blot. Results: The low level of ASPP2 expression was accompanied by high-level autophagy and low-level apoptosis and vice versa in case of hepatocyte injury induce by TNF-α. By upregulating the proteins related to mTORC1 and ERS, ASPP2 induced apoptosis but inhibited autophagy. However, the effect of ASPP2 on autophagy and apoptosis can be reversed by the use of mTORC1 and ERS interfering agent, which indicates that ASPP2 regulated autophagy and apoptosis through mTORC1and ERS pathway. ERS treatment made no difference to the expression of ASPP2 and mTOR-related proteins, which suggests the possibility that the regulation of ERS on apoptosis and autophagy could occur in the downstream of ASPP2 and mTOR. Conclusion: ASPP2 could inhibit autophagy and induce apoptosis through mTORC1-ERS pathway in case of the hepatocyte injury induce by TNF-α. The role of ASPP2-mTORC1-ERS axis was verified in hepatocyte injury, which suggests the possibility that ASPP2 is an important regulatory molecule for the survival and death of hepatocyte.

The Influence of Human Astrocyte-Conditioned Media on Glioblastoma Multiforme Response to Temozolomide and Bay 11-7082

Glioblastoma Multiforme (GBM) cells interact with a complex, heterogeneous tumor microenvironment (TME). This TME consists of astrocytes, endothelial cells, microglia, and pericytes, which together play a role in GBM progression and resistance. However, there are not enough in vitro three-dimensional (3D) models to study the effect of the TME on GBM resistance to chemotherapeutics. In this study, we created a GBM TME by culturing GBM cells with media that had been conditioned by human astrocytes (HA) in 3D microwells. In order to investigate the effect of the TME on GBM resistance to chemotherapeutic agents, cells were treated with Temozolomide (TMZ) in combination with nuclear factor- <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex-math notation="LaTeX">$\kappa \text{B}$ </tex-math></inline-formula> (NF- <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex-math notation="LaTeX">$\kappa \text{B}$ </tex-math></inline-formula> ) inhibitor “Bay 11-7082”. We examined the influence of HA conditioned media (CM) on the expression of various genes and the response to TMZ and Bay 11–7082 in our 3D cultures. Our data suggested that proteins and metabolic factors produced by HA in CM can significantly alter GBM response to chemotherapeutics. Our results indicated lower levels of apoptosis- and drug resistance-related genes were detected in LN229 and U87 cultures in their respective cell culture media compared to HA CM. Our results confirmed HA affect GBM response to therapy.

Multiple Pathway-Dephosphorylated ASK-1 Confers Temozolomide-Resistance to Human Glioma Cells.

To elucidate the function of ASK-1 and the role of its modulators in the induction of Temozolamide (TMZ) resistance in glioma and the underlying mechanism. ASK-1 phosphorylation, the IC50 of TMZ, cell viability, and apoptosis were assessed in the U87 and U251 glioma cell lines and the derived TMZ-resistant cell lines U87-TR and U251-TR. We then blocked ASK-1 function, either with an inhibitor or by overexpression of multiple ASK-1 upstream modulators, to further explore the role of ASK-1 in TMZ-resistant glioma. TMZ-resistant glioma cells showed high IC50 values of TMZ, high survival, and low levels of apoptosis following the TMZ challenge. ASK-1 phosphorylation, but not protein expression, was higher in U87 and U251 cells than in TMZ-resistant glioma cells exposed to TMZ. The addition of the ASK-1 inhibitor selonsertib (SEL) resulted in the dephosphorylation of ASK-1 in U87 and U251 cells after the TMZ challenge. SEL treatment increased the TMZ resistance of U87 and U251 cells, as evidenced by the increased IC50 and cell survival rate and low apoptosis rate. Overexpression of some ASK-1 upstream suppressors [Thioredoxin (Trx), protein phosphatase 5 (PP5), 14-3-3, and cell division cycle 25C (Cdc25C)] led to various degrees of ASK-1 dephosphorylation and a TMZresistant phenotype in U87 and U251 cells. Dephosphorylation of ASK-1 induced TMZ resistance in human glioma cells, and several ASK-1 upstream suppressors, including Trx, PP5, 14-3-3, and Cdc25C, are involved in this phenotypic change induced by dephosphorylation of ASK-1.