Augmented Apoptosis Research Articles

Nuclear factor-Y mediates pancreatic β-cell compensation by repressing reactive oxygen species-induced apoptosis under metabolic stress.

Pancreatic β-cells elevate insulin production and secretion through a compensatory mechanism to override insulin resistance under metabolic stress conditions. Deficits in β-cell compensatory capacity result in hyperglycemia and type 2 diabetes (T2D). However, the mechanism in the regulation of β-cell compensative capacity remains elusive. Nuclear factor-Y (NF-Y) is critical for pancreatic islets' homeostasis under physiological conditions, but its role in β-cell compensatory response to insulin resistance in obesity is unclear. In this study, using obese ( ob/ob ) mice with an absence of NF-Y subunit A (NF-YA) in β-cells ( ob , Nf-ya βKO) as well as rat insulinoma cell line (INS1)-based models, we determined whether NF-Y-mediated apoptosis makes an essential contribution to β-cell compensation upon metabolic stress. Obese animals had markedly augmented NF-Y expression in pancreatic islets. Deletion of β-cell Nf-ya in obese mice worsened glucose intolerance and resulted in β-cell dysfunction, which was attributable to augmented β-cell apoptosis and reactive oxygen species (ROS). Furthermore, primary pancreatic islets from Nf-ya βKO mice were sensitive to palmitate-induced β-cell apoptosis due to mitochondrial impairment and the attenuated antioxidant response, which resulted in the aggravation of phosphorylated c-Jun N-terminal kinase (JNK) and cleaved caspase-3. These detrimental effects were completely relieved by ROS scavenger. Ultimately, forced overexpression of NF-Y in INS1 β-cell line could rescue palmitate-induced β-cell apoptosis, dysfunction, and mitochondrial impairment. Pancreatic NF-Y might be an essential regulator of β-cell compensation under metabolic stress.

SARS-CoV-2 SUD2 and Nsp5 Conspire to Boost Apoptosis of Respiratory Epithelial Cells via an Augmented Interaction with the G-Quadruplex of BclII.

The molecular mechanisms underlying how SUD2 recruits other proteins of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to exert its G-quadruplex (G4)-dependent pathogenic function is unknown. Herein, Nsp5 was singled out as a binding partner of the SUD2-N+M domains (SUD2core) with high affinity, through the surface located crossing these two domains. Biochemical and fluorescent assays demonstrated that this complex also formed in the nucleus of living host cells. Moreover, the SUD2core-Nsp5 complex displayed significantly enhanced selective binding affinity for the G4 structure in the BclII promoter than did SUD2core alone. This increased stability exhibited by the tertiary complex was rationalized by AlphaFold2 and molecular dynamics analysis. In line with these molecular interactions, downregulation of BclII and subsequent augmented apoptosis of respiratory cells were both observed. These results provide novel information and a new avenue to explore therapeutic strategies targeting SARS-CoV-2. IMPORTANCE SUD2, a unique protein domain closely related to the pathogenesis of SARS-CoV-2, has been reported to bind with the G-quadruplex (G4), a special noncanonical DNA structure endowed with important functions in regulating gene expression. However, the interacting partner of SUD2, among other SARS-CoV-2 Nsps, and the resulting functional consequences remain unknown. Here, a stable complex formed between SUD2 and Nsp5 was fully characterized both in vitro and in host cells. Moreover, this complex had a significantly enhanced binding affinity specifically targeting the Bcl2G4 in the promoter region of the antiapoptotic gene BclII, compared with SUD2 alone. In respiratory epithelial cells, the SUD2-Nsp5 complex promoted BclII-mediated apoptosis in a G4-dependent manner. These results reveal fresh information about matched multicomponent interactions, which can be parlayed to develop new therapeutics for future relevant viral disease.

Mechanical injury accentuates lipid deposition in ApoE-/- mice and advance aortic valve stenosis: A novel modified aortic valve stenosis model.

Current mouse models still have limitations in studying aortic valve stenosis (AVS). A suitable animal model bearing a close resemblance to the pathophysiological processes of humans needs to be developed. Here, we combined two risk factors to create a mouse model that mimics the pathological features of human AVS. We combined WI and hyperlipidemia in ApoE-/- mice to explore the synergistic effect on the stenosis of the aortic valve. Transthoracic echocardiography revealed progressively increased peak velocity with age in ApoE-/- mice to velocities above C57 mice when fed a high-fat diet after wire injury. Moreover, ApoE-/- mice demonstrated lower cusp separation and lower aortic valve area after 8 weeks vs. C57 mice. Gross morphology and MRI showed advanced thickening, sclerosis aortic valve, narrowing of the orifice area, and micro-CT showed obvious calcification in the aortic valves in the hyperlipidemia group after wire injury. Histopathology studies showed thickening and fibrosis of aortic valve leaflets in the hyperlipidemia group after wire injury. Notably, lipid deposition was observed in ApoE-/- mice 8 weeks after wire injury, accompanied by overexpressed apoB and apoA proteins. After wire injury, the hyperlipidemia group exhibited augmented inflammation, ROS production, and apoptosis in the leaflets. Moreover, the combination group exhibited advanced fibro-calcific aortic valves after wire injury. Overall, we present the synergistic effect of wire injury and hyperlipidemia on lipoproteins deposition in the development of AVS in ApoE-/- mice, this model bear close resemblance to human AVS pathology.

EIF4A1 Is a Prognostic Marker and Actionable Target in Human Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a primary liver tumor with high lethality and increasing incidence worldwide. While tumor resection or liver transplantation is effective in the early stages of the disease, the therapeutic options for advanced HCC remain limited and the benefits are temporary. Thus, novel therapeutic targets and more efficacious treatments against this deadly cancer are urgently needed. Here, we investigated the pathogenetic and therapeutic role of eukaryotic initiation factor 4A1 (eIF4A1) in this tumor type. We observed consistent eIF4A1 upregulation in HCC lesions compared with non-tumorous surrounding liver tissues. In addition, eIF4A1 levels were negatively correlated with the prognosis of HCC patients. In HCC lines, the exposure to various eIF4A inhibitors triggered a remarkable decline in proliferation and augmented apoptosis, paralleled by the inhibition of several oncogenic pathways. Significantly, anti-growth effects were achieved at nanomolar concentrations of the eIF4A1 inhibitors and were further increased by the simultaneous administration of the pan mTOR inhibitor, Rapalink-1. In conclusion, our results highlight the pathogenetic relevance of eIF4A1 in HCC and recommend further evaluation of the potential usefulness of pharmacological combinations based on eIF4A and mTOR inhibitors in treating this aggressive tumor.

MicroRNA-141-3p mediates epithelial cell proliferation, apoptosis, and epithelial-mesenchymal transition and alleviates pulmonary fibrosis in mice via Spred2.

This study probed the mechanism of microRNA (miR)-141-3p in the progression of pulmonary fibrosis (PF). Mice were intratracheally administered with bleomycin (BLM) to establish a PF mouse model. To investigate the effects of miR-141-3p/Spred2 on PF in mice, PF mice received tail vein injections with agomir-141-3p and/or adenovirus vectors overexpressing Spred2 one week after BLM treatment. Then, the pathological changes of lung tissues were analyzed with H&E and Masson's trichrome staining and hydroxyproline contents in lung tissues were measured. For cell experiments, after loss- and gain-of-function assays, the role of miR-141-3p/Spred2 in the apoptosis and viability of TGF-β1-stimulated MLE-12 cells was examined by flow cytometry and CCK-8 assay. miR-141-3p, Spred2, COl 1, and α-SMA expression was determined in cells and mice. Then, the binding of miR-141-3p to Spred2 was tested with a dual-luciferase reporter assay. There were abnormally upregulated Spred2 and downregulated miR-141-3p in lung tissues of PF mice. TGF-β1 decelerated viability and augmented apoptosis and COl 1 and α-SMA expression in MLE-12 cells. Spred2 knockdown diminished apoptosis and α-SMA and COl 1 expression while enhancing proliferation in TGF-β1-treated MLE-12 cells. Mechanistically, Spred2 was a target gene of miR-141-3p. miR-141-3p upregulation accelerated proliferation and repressed apoptosis and α-SMA and COl 1 expression in TGF-β1-treated MLE-12 cells, which was nullified by further overexpressing Spred2. miR-141-3p alleviated PF in mice by targeting Spred2. miR-141-3p negatively modulates Spred2 to promote proliferation and repress epithelial-mesenchymal transition and apoptosis of epithelial cells, as well as ameliorating PF in mice.

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.

Activation of Sphingomyelin Phosphodiesterase 3 in Liver Regeneration Impedes the Progression of Colorectal Cancer Liver Metastasis Via Exosome-Bound Intercellular Transfer of Ceramides

The machinery that prevents colorectal cancer liver metastasis (CRLM) in the context of liver regeneration (LR) remains elusive. Ceramide (CER) is a potent anti-cancer lipid involved in intercellular interaction. Here, we investigated the role of CER metabolism in mediating the interaction between hepatocytes and metastatic colorectal cancer (CRC) cells to regulate CRLM in the context of LR. Mice were intrasplenically injected with CRC cells. LR was induced by 2/3 partial hepatectomy (PH) to mimic the CRLM in the context of LR. The alteration of corresponding CER-metabolizing genes was examined. The biological roles of CER metabolism invitro and invivo were examined by performing a series of functional experiments. Induction of LR augmented apoptosis but promoted matrix metalloproteinase 2 (MMP2) expression and epithelial-mesenchymal transition (EMT) to increase the invasiveness of metastatic CRC cells, resulting in aggressive CRLM. Up-regulation of sphingomyelin phosphodiesterase 3 (SMPD3) was determined in the regenerating hepatocytes after LR induction and persisted in the CRLM-adjacent hepatocytes after CRLM formation. Hepatic Smpd3 knockdown was found to further promote CRLM in the context of LR by abolishing mitochondrial apoptosis and augmenting the invasiveness in metastatic CRC cells by up-regulating MMP2 and EMT through promoting the nuclear translocation of β-catenin. Mechanistically, we found that hepatic SMPD3 controlled the generation of exosomal CER in the regenerating hepatocytes and the CRLM-adjacent hepatocytes. The SMPD3-produced exosomal CER critically conducted the intercellular transfer of CER from the hepatocytes to metastatic CRC cells and impeded CRLM by inducing mitochondrial apoptosis and restricting the invasiveness in metastatic CRC cells. The administration of nanoliposomal CER was found to suppress CRLM in the context of LR substantially. SMPD3-produced exosomal CER constitutes a critical anti-CRLM mechanism in LR to impede CRLM, offering the promise of using CER as a therapeutic agent to prevent the recurrence of CRLM after PH.

Molecular mechanisms behind ROS regulation in cancer: A balancing act between augmented tumorigenesis and cell apoptosis.

ROS include hydroxyl radicals (HO.), superoxide (O2..), and hydrogen peroxide (H2O2). ROS are typically produced under physiological conditions and play crucial roles in living organisms. It is known that ROS, which are created spontaneously by cells through aerobic metabolism in mitochondria, can have either a beneficial or detrimental influence on biological systems. Moderate levels of ROS can cause oxidative damage to proteins, DNA and lipids, which can aid in the pathogenesis of many disorders, including cancer. However, excessive concentrations of ROS can initiate programmed cell death in cancer. Presently, a variety of chemotherapeutic drugs and herbal agents are being investigated to induce ROS-mediated cell death in cancer. Therefore, preserving ROS homeostasis is essential for ensuring normal cell development and survival. On account of a significant association of ROS levels at various concentrations with carcinogenesis in a number of malignancies, further studies are needed to determine the underlying molecular mechanisms and develop the possibilities for intervening in these processes.

DUT enhances drug resistance to proteasome inhibitors via promoting mitochondrial function in multiple myeloma.

Acquired chemoresistance to proteasome inhibitors (PIs), such as bortezomib (BTZ), becomes an intractable obstacle in the management of multiple myeloma (MM) in the clinic, but the underlying mechanisms are still not well elucidated. In the current study, we established bortezomib-resistant (BR) myeloma cells and performed stable isotope labeling by amino acids in cell culture (SILAC) assay to screen profiled protein expression. The level of deoxyuridine triphosphatase (DUT), an important enzyme of nucleotide metabolism, increased in the BR MM cells. Retrospective analysis indicated patients with higher DUT expression had poorer responses to PI-based treatment and clinical outcomes. DUT knockdown by RNAi effectively minimized BTZ resistance in MM cells. Moreover, DUT knockdown was accompanied with the downregulation of proliferating cell nuclear antigen (PCNA), contributing to decelerating cell growth, as well as augmented apoptosis due to bortezomib treatment. In contrast, DUT overexpression in parental MM.1S and LP-1 cells enhanced BTZ resistance. Furthermore, acquired resistance to BTZ could trigger the modulation of mitochondrial metabolism and function, as evidenced by elevated expression of genes associated with mitochondrial metabolism, as well as altered oxygen consumption rate and adenosine triphosphate (ATP) production in BR MM cells. DUT inhibition partially attenuated mitochondrial modulation, and instead favored an early impairment of mitochondrial integrity upon BTZ exposure so as to restrict MM progression and overcome drug resistance to BTZ treatment both in vitro and in vivo. In conclusion, we unveiled previously unrecognized effects of DUT on acquired drug resistance of MM, thus manipulating DUT may be efficacious for sensitizing MM cells to PIs.

Plumbagin downregulates UHRF1, p-Akt, MMP-2 and suppresses survival, growth and migration of cervical cancer CaSki cells

Plumbagin is a natural compound known to impede growth of cancerous cells. However, anti-cervical cancer effects of plumbagin and its underlying molecular mechanism still remains elusive. In this study, plumbagin reduced the viability of CaSki cells in a concentration dependent manner and suppressed their colony formation potential. It led to G2/M phase arrest with downregulation of E2F1 and upregulation of p21. Plumbagin reduced mitochondrial membrane potential and concomitantly increased the percentage of apoptotic cells as revealed by annexin V-propidium iodide staining. Real Time PCR and western blotting confirmed that plumbagin induced apoptosis by reducing the expression of pAkt, procaspase 9 and full-length PARP. Furthermore, scratch assay showed that plumbagin suppressed migratory potential of CaSki cells which could be due to the reduced expression and activity of MMP-2 and upregulation of TIMP2. Interestingly, plumbagin also downregulated UHRF1 expression. Transient silencing of UHRF1 like plumbagin, induced G2/M phase arrest, enhanced apoptosis and suppressed metastasis of CaSki cells suggesting the role of UHRF1 in mediating anti-cancer activities of plumbagin. Plumbagin at IC20 (1 μM) interacted synergistically with cisplatin and reduced its IC50 value by 13.23 fold with improved effectivity as revealed by augmented apoptosis in CaSki cells.

Systemic GDF11 replenishment ignites myocardial injury through diminishing anti-apoptotic activity of cardiac progenitor cells

Abstract Background Tissue damage due to acute myocardial infarction is caused by both the ischemic insult and subsequent reperfusion injury (I/R). Restoration of coronary blood flow accelerates cardiomyocyte death, a phenomenon referred to as reperfusion injury, the extent of which is partly modulated by cardiac progenitor cells (CPC). Development of novel therapies to reduce infarct size, the main determinant of outcome, represent a huge unmet medical need (1). Systemic levels of growth differentiation factor 11 (GDF11), a TGF-β superfamily member that shares 90% homology with myostatin, decline with age, and GDF11 replenishment by heterochronic parabiosis or systemic recombinant GDF11 (rGDF11) delivery was postulated to have rejuvenating effects (2). Purpose We aimed to probe the effects of systemic GDF11 replenishment on I/R injury and deepen insights into the molecular mechanisms involved. Methods We designed a vehicle-controlled study in which young (3–4 months) and old (22–24 months) C57Bl/6 mice were randomly assigned to either daily systemic rGDF11 or control treatment over 30 days before myocardial I/R injury was induced. Dissected hearts were subjected to in-depth profiling followed by IPA-guided -omics to identify key regulatory mechanisms. Finally, in vitro experiments on human CPCs and HL-1 cardiomyocytes were performed. Results Myocardial Gdf11 expression declined with age, whereas myostatin (Mstn) showed an opposing expression pattern (Fig. 1A), a trend similarly observed upon I/R (Fig. 1B). Surprisingly, after the 30-day study period (Fig. 1C), young and aged rGDF11-treated mice showed higher I/R-induced infarct size and serum cardiac troponin I levels than controls, despite comparable areas at risk (Fig. 1D). Importantly, while proxies of necroptosis/pyroptosis remained unchanged, rGDF11-treated animals showed reduced cardiomyocyte viability irrespective of their age (Fig. 2A). Targeted transcriptomics applied on cardiac tissues of both groups identified the CPC-marker Nkx2–5 to be differentially regulated (Fig. 2B-C), an expression pattern validated in an independent cohort at both mRNA and protein levels (Fig. 2D). In the adult myocardium, the expression of both Nkx2–5 and its cofactor Gata4 is mainly confined to CPCs; indeed, similar reductions in Nkx2–5 and Gata4 expression were observed in CPCs exposed to rGDF11 (Fig. 2E) which coincided with accelerated cardiomyocyte death if cultured in conditioned media obtained from CPCs treated with rGDF11 (Fig. 2F), pointing toward a paracrine signalling pathway. Conclusions Myocardial expression of GDF11 declines with age, and is blunted upon I/R injury, thereby opposing the expression pattern of myostatin. Surprisingly, however, systemic GDF11 replenishment by rGDF11 supplementation enhances rather than reduces myocardial infarct size through augmented apoptosis, a phenomenon mediated by diminished cardioprotective function of CPCs. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): Foundation for Cardiovascular Research – Zurich Heart House

Long noncoding RNA CDKN2B-AS1 silencing protects against esophageal cancer cell invasion and migration by inactivating the TFAP2A/FSCN1 axis.

Esophageal cancer (EC) is the most aggressive malignancy in the gastrointestinal tract. Long noncoding RNA cyclin-dependent kinase inhibitor 2 B antisense RNA 1 (CDKN2B-AS1) is implicated in EC development. However, the specific mechanisms involved remain poorly defined. Therefore, this research aimed to explore the mechanism of action of CDKN2B-AS1 in EC. Quantitative real-time polymerase chain reaction was conducted to measure CDKN2B-AS1 expression in EC cells and western blotting was utilized to evaluate transcription factor AP-2 alpha (TFAP2A) and fascin actin-bundling protein 1 (FSCN1) expression. After gain-of-function and loss-of-function assays, cell proliferation, migration, invasion, apoptosis, and apoptosis-related protein expression were assessed using cell counting kit-8, scratch tests, Transwell assays, flow cytometry, and western blotting, respectively. The binding relationship between CDKN2B-AS1 and TFAP2A was assessed by RNA immunoprecipitation and RNA pull-down assays. The binding relationship between TFAP2A and FSCN1 was evaluated using dual-luciferase reporter and chromatin immunoprecipitation assays. Tumor xenografts from nude mice were used for in vivo verification. CDKN2B-AS1, TFAP2A, and FSCN1 were upregulated in EC cells. Mechanistically, CDKN2B-AS1 transcriptionally activated FSCN1 by recruiting TFAP2A to the FSCN1 promoter. Silencing CDKN2B-AS1 or TFAP2A suppressed EC cell proliferative, migrating, and invasive properties and augmented apoptosis. TFAP2A was bound to CDKN2B-AS1 and the FSCN1 promoter. Overexpression of TFAP2A or FSCN1 abolished the effects of CDKN2B-AS1-silencing on EC cell function. CDKN2B-AS1 silencing curtailed tumorigenesis in nude mice, which was nullified by the upregulation of TFAP2A or FSCN1. Our findings demonstrated the antioncogenic effects of silencing CDKN2B-AS1 in EC through inactivation of the TFAP2A/FSCN1 axis.

Roles of autophagy in angiotensin II-induced cardiomyocyte apoptosis.

Autophagy is a self-degradation process of cytoplasmic components and occurs in the failing heart. Angiotensin II plays a critical role in the progression of heart failure and induces autophagy. We investigated the mechanism underlying angiotensin II-enhanced autophagy and examined the role of autophagy in angiotensin II-induced cardiomyocyte injury. Neonatal rat cardiomyocytes were treated with angiotensin II (1-100 nmol/L). Angiotensin II dose-dependently increased autophagy indicators of microtubule-associated protein 1 light chain (LC) 3-II and monodansylcadaverine-labelled vesicles. It also enhanced the intracellular production of reactive oxygen species (ROS), assessed by H2DCFDA, an intracellular ROS indicator. NADPH oxidase- and mitochondria-derived ROS production was increased by angiotensin II, while angiotensin II-induced LC3-II expression was suppressed by inhibitors of these sources of ROS. Confocal microscopy revealed that superoxide-producing mitochondria colocalized with lysosomes after the angiotensin II stimulation. Myocyte apoptosis was assessed by nuclear staining with DAPI and caspase-3 activity. A 6-h stimulation with angiotensin II did not affect myocyte apoptosis, while a co-treatment with 3-methyl-adenine (3MA), an autophagy inhibitor, augmented apoptosis. These results indicate that autophagy suppressed apoptosis because it removed damaged mitochondria in the early stages of the angiotensin II stimulation. A longer angiotensin II stimulation for 24 h induced apoptosis and propidium iodide-positive lethal myocytes, while the co-treatment with 3MA did not lead to further increases. In conclusion, angiotensin II-induced autophagy removes ROS-producing mitochondria. Autophagy is a beneficial phenomenon against myocyte apoptosis in the early phase, but its benefit was limited in the late phase of angiotensin II stimulation.

Atomically dispersed 3d metal bimetallic dual-atom catalysts and classification of the structural descriptors

Solid atomic catalysts with well-defined and complex structures are believed to effectively bridge homogeneous and heterogeneous catalysis. Nonetheless, the current limited capacity of “precise engineering” in solid atomic catalysts has led to structural heterogeneity and thus unsatisfactory catalytic selectivity. Here, we show that late 3 d metal cations, such as Co 2+ , Ni 2+ , Cu 2+ , and Zn 2+ , can be assembled to afford combinations of “dual atoms” within zeolitic micropores, and this clearly avoids issues like uncontrolled metal aggregation during synthesis. In this work, by the quantitative evaluation of the structural descriptors over a probe superoxide dismutation reaction, we demonstrate the unique synergistic advantage between (i) neighboring bimetallic active motifs, (ii) tertiary structure around the zeolitic support, and (iii) the local coordination environment. The identification and tunability of the structural descriptors shown in this work unravel a reliable approach to the precise engineering of next-generation solid dual-atom catalysts. • Atom-precise synthesis of supported bimetallic dual-atom catalysts • Reliable approach for precise microenvironment engineering in solid catalysts • Discovery of the interplay between the structural descriptors and catalytic properties • Demonstration of synergistic effect with sorafenib for augmented tumor cell apoptosis We aim to present a creative approach to afford dual-atom bimetallic catalysts in a controlled and modular manner. This directly allows the combinations of different late 3 d metals (forming M 1 -M 2 -supported dual atoms) and shows extensive possibility not limited to catalytic applications. Through the unique synergistic advantage between the two metal species and the tertiary structure around the zeolitic support, we demonstrated the feasibility of developing next-generation dual-atom bimetallic catalysts in a controlled manner as well as the identification of the structural descriptors. The manipulation and control of materials and phenomena at atomic and molecular dimensions are achieved by utilizing the fundamental concepts of coordination and solid-state chemistries. The findings presented in this work reveal the precise engineering of supported dual-atom catalysts (such as combinations of 3 d /4 d metals) over support materials not only for biochemical applications but also for more extensive applications. This shall genuinely bring heterogeneous catalysis toward the molecular frontier that addresses the critical challenges in the field.

Clusterin Binding Modulates the Aggregation and Neurotoxicity of Amyloid-β(1-42).

Alzheimer's disease (AD) is the most common neurodegenerative disorder characterized by the accumulation of amyloid-β (Aβ) aggregates in the brain. Clusterin (CLU), also known as apolipoprotein J, is a potent risk factor associated with AD pathogenesis, in which Aβ aggregation is essentially involved. We observed close colocalization of CLU and Aβ(1-42) (Aβ42) in parenchymal amyloid plaques or vascular amyloid deposits in the brains of human amyloid precursor protein (hAPP)-transgenic Tg2576 mice. Therefore, to elucidate the binding interaction between CLU and Aβ42 and its impact on amyloid aggregation and toxicity, the two synthetic proteins were incubated together under physiological conditions, and their structural and morphological variations were investigated using biochemical, biophysical, and microscopic analyses. Synthetic CLU spontaneously bound to different possible variants of Aβ42 aggregates with very high affinity (Kd = 2.647nM) in vitro to form solid CLU-Aβ42 complexes. This CLU binding prevented further aggregation of Aβ42 into larger oligomers or fibrils, enriching the population of smaller Aβ42 oligomers and protofibrils and monomers. CLU either alleviated or augmented Aβ42-induced cytotoxicity and apoptosis in the neuroblastoma-derived SH-SY5Y and N2a cells, depending on the incubation period and the molar ratio of CLU:Aβ42 involved in the reaction before addition to the cells. Thus, the effects of CLU on Aβ42-induced cytotoxicity were likely determined by the extent to which it bound and sequestered toxic Aβ42 oligomers or protofibrils. These findings suggest that CLU could influence amyloid neurotoxicity and pathogenesis by modulating Aβ aggregation.

Chitosan IR806 dye-based polyelectrolyte complex nanoparticles with mitoxantrone combination for effective chemo-photothermal therapy of metastatic triple-negative breast cancer

Chemo-photothermal therapy is one of the emerging therapies for treating triple-negative breast cancer. In this study, we have used ionotropic gelation method to fabricate chitosan and IR806 dye-based polyelectrolyte complex (CIR-PEx) nanoparticles. These nano-complexes were in size range of 125 ± 20 nm. The complexation of IR 806 dye with chitosan improved photostability, photothermal transduction, and showed excellent biocompatibility. Cancer cells treated with CIR-PEx NPs enhanced intracellular uptake within 5 h of incubation and also displayed mitochondrial localization. With the combination of CIR-PEx NPs and a chemotherapeutic agent (i.e., mitoxantrone, MTX), a significant decline in cancer cell viability was observed in both 2D and 3D cell culture models. The chemo-photothermal effect of CIR-PEx NPs + MTX augmented apoptosis in cancer cells when irradiated with NIR light. Furthermore, when tested in the 4 T1-tumor model, the chemo-photothermal therapy showed a drastic decline in tumor volume and inhibited metastatic lung nodules. The localized hyperthermia caused by photothermal therapy reduced the primary tumor burden, and the chemotherapeutic activity of mitoxantrone further complemented by inhibiting the spread of cancer cells. The proposed chemo-photothermal therapy combination could be a promising strategy for treating triple-negative metastatic breast cancer.

Abstract 5667: EZH2 inhibition targets B7-H3 in Myc amplified medulloblastoma

Abstract Medulloblastoma (MB) is the most common malignant pediatric brain tumor. The most aggressive MB subtype Group 3, is characterized by Myc amplifications. However, targeting Myc has proven unsuccessful, necessitating alternative therapies for MB. The histone methyltransferase, enhancer of zeste homolog 2 (EZH2), is well-known for its role in catalyzing the tri-methylation of H3K27 (H3K27me3). Recent studies have demonstrated that EZH2 is overexpressed in Group 3 MB and that Myc and EZH2 regulate the expression of the B7 homolog 3 (B7-H3). As we previously showed the oncogenic functions of B7-H3 in Group 3 MB, this study aims to investigate the efficacy of EZH2 inhibition to reduce B7-H3 levels in an effort to improve treatment options and patient survival. Datamining studies conducted using MB patient datasets showed a positive correlation between EZH2 and B7-H3 transcripts. Additionally, we showed that inhibition of EZH2 using EPZ005687 in Myc-amplified MB cell lines downregulated B7-H3 and Myc expression. Furthermore, targeting EZH2 through shRNA and pharmacological inhibition reduced the viability and augmented apoptosis, as indicated by LDH, MTT, clonogenic, and FACS assays. We additionally tested the consequences of EZH2 inhibition on the C17.2, murine neural stem cells, and C17.2 NSC transformed with EZH2 oncogene (C17.2_EZH2OX). Furthermore, the C17.2_EZH2OXcells were susceptible to EPZ005687 treatment compared to C17.2 control cells, as demonstrated by MTT and FACS. Collectively, our study highlights the therapeutic benefits of targeting EZH2 in Myc-amplified MB cell survival. Citation Format: Katherine Shishido, Sujatha Venkataraman, Rajeev Vibhakar, Kiran Velpula, Swapna Asuthkar. EZH2 inhibition targets B7-H3 in Myc amplified medulloblastoma [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 5667.

Abstract 3501: Induction of tumor cell apoptosis &amp; immunogenic cell death by CD40L-armed oncolytic adenoviruses

Abstract Low tumor immunogenicity and a cold tumor microenvironment are two main characteristics occurring in different cancer types responsible for immunotherapy resistance. Immunostimulatory gene therapy using oncolytic viruses (OVs) has been developed as a new promising cancer therapy for its capacity to induce an anti-tumor immune response and sensitize the non-responding patients to checkpoint blockade therapy. Lokon adenoviruses (LOAds) are serotype 5/35 oncolytic adenoviruses that in addition to their oncolytic capacity encode for immunostimulatory molecules. Here we have evaluated the anti-tumor effects of trimerized membrane-bound (TMZ)-CD40L-armed LOAd700 and TMZ-CD40L/4-1BBL-armed LOAd703 in a variety of solid tumors to understand the underlying tumor modulating mechanisms. A panel of eight different human solid cancer cell lines (T24, PEA2, Mel526, H727, Skov3, MiaPaca2, H29 and A549) was infected with LOAd(-), a virus without transgenes, LOAd700 or LOAd703 and assessed for their response in different assays. MTS assay was used to measure the cell viability, flow cytometry for evaluating the cell surface markers and luminescent assay for measuring apoptosis (caspase 3/7 activity). LOAd viruses efficiently killed all tested cell lines. However, the oncolytic capacity of CD40L-armed LOAd700 and LOAd703 was significantly higher than LOAd(-) in two cell lines, T24 and PEA2, which were found to express the CD40 receptor. Death receptor TRAIL-R1 was significantly upregulated in T24 infected with LOAd703 and PEA2 infected with either LOAd700 or LOAd703. TRAIL-R2 was upregulated post LOAd infection in 7/8 cell lines where the upregulation was significant in T24, PEA2 and H29. Interestingly, the immunogenic cell death marker calreticulin, a damage-associated molecular pattern (DAMP) known as an “eat-me” signal to dendritic cells, was upregulated post LOAd infection in all cell lines. The increase in calreticulin level was statistically significant in 4/8 cell lines (T24, PEA2, Mel526 and H29) post LOAd700 and/or LOAd703 infection. Furthermore, LOAd700 and LOAd703 increased the effector caspase 3/7 activity in 5/8 cell lines (T24, PEA2, Mel526, Skov3 and MiaPaCa2) indicating an augmented apoptosis level. In conclusion, CD40L-armed LOAd viruses were, in addition to their high oncolytic capacity, demonstrated to induce apoptosis. Upregulation of cell surface calreticulin as well as the death receptors TRAIL-R1 and -R2 indicated generation of a more immunogenic phenotype of cancer cells. Thus, our findings suggest that CD40L-armed LOAd viruses could be inducers of apoptosis and immunogenic cell death in a variety of cancer types with an augmented effect in cancer cells expressing the CD40 receptor. Citation Format: Sedigheh Naseri, Mariela Mejia Cordova, Jessica Wenthe, Ann-Charlotte Hellström, Tanja Lövgren, Angelica Loskog, Emma Eriksson, Gustav Ullenhag. Induction of tumor cell apoptosis &amp; immunogenic cell death by CD40L-armed oncolytic adenoviruses [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 3501.

Mechanism Study on Radiosensitization Effect of Curcumin in Bladder Cancer Cells Regulated by Filamin A.

ObjectiveTo study the radiosensitization effect of curcumin, a natural product with anti-inflammatory and anti-cancer properties, in bladder cancer cells and identify the specific role of FLNA gene in that process.MethodsCCK-8 method was initially adopted to identify the proper interventional concentration of curcumin. T24 bladder cancer cells were subjected to CCK-8, flow cytometry, and colony formation assay to study the cell biological behaviors under different interventions. γ-H2AX test was performed to test the level of damage in T24 cells. RT-qPCR and Western blot were conducted to measure FLNA mRNA and protein levels.ResultsLow-dose curcumin (10, 20 μM) following X-ray exposure resulted in increased DNA damage, augmented apoptosis, and reduced proliferation of T24 cells. Certain radiosensitization was demonstrated when curcumin was applied at 10 μM. Additionally, elevation of FLNA gene and protein levels was also indicated upon combination treatment.ConclusionLow-dose curcumin has certain radiosensitization effect in bladder cancer, where FLNA plays a certain regulatory role.

Kaempferol Regresses Carcinogenesis through a Molecular Cross Talk Involved in Proliferation, Apoptosis and Inflammation on Human Cervical Cancer Cells, HeLa

Kaempferol, a flavonoid, contains a plethora of therapeutic properties and has demonstrated its efficacy against cancer. This study aims to unravel the molecular targets that are being modulated by kaempferol on HeLa cells. Various assays were performed, namely: MTT assay, flow cytometry to analyze DNA content and quantitate apoptosis. Quantitative PCR and protein profiling were performed to evaluate the modulated manifestation of different genes involved in apoptosis, cell growth and inflammation. Kaempferol exhibited reduction in cell viability of HeLa cells (IC50 = 50 µM 48 h), whereas it did not show any significant effect on viability of the AC-16 cell line. Kaempferol-impacted apoptosis was definitive, as it induced DNA fragmentation, caused disruption of membrane potential, accumulation of cells in the G2-M phase and augmented early apoptosis. Consistently, kaempferol induced apoptosis in HeLa cells by modulating the expression of various genes at both transcript and protein levels. It upregulated the expression of pro-apoptotic genes, including APAF1, BAX, BAD, Caspases 3, and 9, etc., at the transcript level and Bad, Bax, p27, p53, p21, Caspases 3 and 8 etc. at the protein level, while it downregulated the expression of pro-survival gene BCL-2, BIRC8, MCL-1, XIAP, and NAIP at the transcript level and Bcl-2, XIAP, Livin, clap-2 at the protein level. Kaempferol attenuated oxidative stress by upregulating GSH activity and anti-inflammatory response by suppressing NF-kB pathways. Moreover, kaempferol averted rampant cell division and induced apoptosis by modulating AKT/MTOR and MAP kinase pathways. Hence, kaempferol can be considered as a natural therapeutic agent with a differential profile.