Decitabine combined with cold atmospheric plasma induces pyroptosis via the ROS/Caspase-3/GSDME signaling pathway in Ovcar5 cells.

BACKGROUND: High methylation of the DFNA5 gene results in the absence of GSDME, a key protein that mediates pyroptosis, while decitabine demethylates the DFNA5 gene, resulting in high expression of the GSDME protein. Cold atmospheric plasma (CAP) is a novel anti-cancer method that induces tumor cell death. METHODS: The pyroptosis induced by decitabine in combination with CAP in Ovcar5 cells was evaluated. In particular, mitochondrial membrane potential was estimated by JC-1 staining, dehydrogenase (LDH) release was assessed by ELISA, Annexin V/PI staining was detected by flow cytometry, the cell cycle changes were evaluated using PI staining followed by detection by flow cytometry, and Caspase-9 cleavage, Caspase-3 cleavage and GSDME expression were evaluated by western blot. RESULTS: Decitabine resulted in high expression of the GSDME in Ovcar5 in a concentration-dependent manner and increased tumor cell sensitivity to CAP. CAP induced mitochondrial damage and activated the Caspase-9/Caspase-3 pathway. Therefore, decitabine combined with CAP induced Ovcar5 cell pyroptosis through Caspase-3 mediated GSDME cleavage. Reactive oxygen species (ROS) generated by CAP treatment played an important role in the CAP/decitabine combination-induced production of ROS, activation of Caspase-9/Caspase-3, GSDME cleavage and pyroptosis that ROS scavenger NAC inhibited all these processes. CONCLUSIONS: CAP combined with decitabine induced Caspase-3 activation, which cleaved decitabine-upregulated GSDME and ediated pyroptosis.

AMPK inhibition sensitizes acute leukemia cells to BH3 mimetic-induced cell death.

BH3 mimetics, including the BCL2/BCLXL/BCLw inhibitor navitoclax and MCL1 inhibitors S64315 and tapotoclax, have undergone clinical testing for a variety of neoplasms. Because of toxicities, including thrombocytopenia after BCLXL inhibition as well as hematopoietic, hepatic and possible cardiac toxicities after MCL1 inhibition, there is substantial interest in finding agents that can safely sensitize neoplastic cells to these BH3 mimetics. Building on the observation that BH3 mimetic monotherapy induces AMP kinase (AMPK) activation in multiple acute leukemia cell lines, we report that the AMPK inhibitors (AMPKis) dorsomorphin and BAY-3827 sensitize these cells to navitoclax or MCL1 inhibitors. Cell fractionation and phosphoproteomic analyses suggest that sensitization by dorsomorphin involves dephosphorylation of the proapoptotic BCL2 family member BAD at Ser75 and Ser99, leading BAD to translocate to mitochondria and inhibit BCLXL. Consistent with these results, BAD knockout or mutation to BAD S75E/S99E abolishes the sensitizing effects of dorsomorphin. Conversely, dorsomorphin synergizes with navitoclax or the MCL1 inhibitor S63845 to induce cell death in primary acute leukemia samples ex vivo and increases the antitumor effects of navitoclax or S63845 in several xenograft models in vivo with little or no increase in toxicity in normal tissues. These results suggest that AMPK inhibition can sensitize acute leukemia to multiple BH3 mimetics, potentially allowing administration of lower doses while inducing similar antineoplastic effects.

Apoptotic stress causes mtDNA release during senescence and drives the SASP.

Senescent cells drive age-related tissue dysfunction partially through the induction of a chronic senescence-associated secretory phenotype (SASP)1. Mitochondria are major regulators of the SASP; however, the underlying mechanisms have not been elucidated2. Mitochondria are often essential for apoptosis, a cell fate distinct from cellular senescence. During apoptosis, widespread mitochondrial outer membrane permeabilization (MOMP) commits a cell to die3. Here we find that MOMP occurring in a subset of mitochondria is a feature of cellular senescence. This process, called minority MOMP (miMOMP), requires BAX and BAK macropores enabling the release of mitochondrial DNA (mtDNA) into the cytosol. Cytosolic mtDNA in turn activates the cGAS-STING pathway, a major regulator of the SASP. We find that inhibition of MOMP in vivo decreases inflammatory markers and improves healthspan in aged mice. Our results reveal that apoptosis and senescence are regulated by similar mitochondria-dependent mechanisms and that sublethal mitochondrial apoptotic stress is a major driver of the SASP. We provide proof-of-concept that inhibition of miMOMP-induced inflammation may be a therapeutic route to improve healthspan.

Eltrombopag directly activates BAK and induces apoptosis.

Small molecule direct BAK activators can potentially be used for the development of anti-cancer drugs or as tools to study BAK activation. The thrombopoietin receptor agonist eltrombopag (Eltro) inhibits BAX activation and BAX-mediated apoptosis. Here we report that, in contrast to its function as a BAX inhibitor, Eltro directly binds BAK but induces its activation in vitro. Moreover, Eltro induces or sensitizes BAK-dependent cell death in mouse embryonic fibroblasts (MEFs) and Jurkat cells. Chemical shift perturbation analysis by NMR indicates that Eltro binds to the BAK α4/α6/α7 groove to initiate BAK activation. Further molecular docking by HADDOCK suggests that several BAK residues, including R156, F157, and H164, play an important role in the interaction with Eltro. The introduction of an R156E mutation in the BAK α4/α6/α7 groove not only decreases Eltro binding and Eltro-induced BAK activation in vitro but also diminishes Eltro-induced apoptosis. Thus, our data suggest that Eltro directly induces BAK activation and BAK-dependent apoptosis, providing a starting point for the future development of more potent and selective direct BAK activators.

AKT inhibition sensitizes acute leukemia cells to S63845-induced apoptosis.

The MCL1 inhibitors are undergoing clinical testing for multiple leukemia. However, because that MCL1 inhibition has on-target hematopoietic, hepatic and cardiac toxicities, there is substantial interest in finding agents can sensitize leukemia cells to the MCL1 inhibitors. Here we describe that the AKT inhibitors MK-2206 and Gsk690693 sensitize multiple leukemia cells to the MCL1 inhibitor S63845. Further experiments demonstrate that MK-2206 and Gsk690693 sensitize S63845 through the mitochondrial apoptosis pathway. Moreover, MK-2206 downregulates the anti-apoptotic protein BCLXL and induces the BH3-only pro-apoptotic protein BAD dephosphorylation and mitochondrial translocation. Knockdown of BAD significantly inhibits MK-2206-induced sensitization to S63845. Thus, our results suggest that MK-2206 sensitizes multiple leukemia cells to S63845-induced apoptosis, with the mechanisms involving BAD dephosphorylation and BCLXL downregulation.

The mtDNA-STING pathway plays an important role in both navitoclax- and S63845-induced autophagy and enhances cell death.

Targeting BCL2 family proteins to induce cancer cell death has been successful in the treatment of cancer. BH3 mimetics such as ABT-737 not only induce cell death, but also activate autophagy. The molecular mechanism by which the BH3 mimetics induce autophagy is still controversial. In this study, we show that the BCL2/BCLXL/BCLw inhibitor navitoclax and the MCL1 inhibitor S63845 induce both apoptosis and autophagy in mouse embryonic fibroblasts (MEFs) and leukemia cell lines, while autophagy induced by navticlax and S63845 in leukemia cell lines requires the inhibition of caspase activities. Further experiments demonstrate that the autophagy induced by navitoclax or S63845 does not depend on Beclin 1, but downstream of Bax/Bak. Moreover, both navitoclax and S63845 treatment induce mtDNA release in MEFs, which activates STING and thereby induces autophagy, while STING KO inhibits both navitoclax- and S63845-induced autophagy. Furthermore, STING KO diminishes navitoclax- or S63845-induced apoptosis, suggesting that STING activation enhances rather than inhibits apoptosis. Thus, our findings provide new insights into the regulations of navitoclax- or S63845-induced autophagy and cell death.

The Tumorigenic Effect of the High Expression of Ladinin-1 in Lung Adenocarcinoma and Its Potential as a Therapeutic Target.

The oncogenic role of Ladinin-1 (LAD1), an anchoring filament protein, is largely unknown. In this study, we conducted a series of studies on the oncogenic role of LAD1 in lung adenocarcinoma (LUAD). Firstly, we analyzed the aberrant expression of LAD1 in LUAD and its correlation with patient survival, tumor immune infiltration, and the activation of cancer signaling pathways. Furthermore, the relationship between LAD1 expression and K-Ras and EGF signaling activation, tumor cell proliferation, migration, and colony formation was studied by gene knockout/knockout methods. We found that LAD1 was frequently overexpressed in LUAD, and high LAD1 expression predicts a poor prognosis. LAD1 exhibits promoter hypomethylation in LUAD, which may contribute to its mRNA upregulation. Single-sample gene set enrichment analysis (ssGSEA) showed that acquired immunity was negatively correlated with LAD1 expression, which was verified by the downregulated GO terms of "Immunoglobulin receptor binding" and "Immunoglobulin complex circulating" in the LAD1 high-expression group through Gene Set Variation Analysis (GSVA). Notably, the Ras-dependent signature was the most activated signaling in the LAD1 high-expression group, and the phosphorylation of downstream effectors, such as ERK and c-jun, was strongly inhibited by LAD1 deficiency. Moreover, we demonstrated that LAD1 depletion significantly inhibited the proliferation, migration, and cell-cycle progression of LUAD cells and promoted sensitivity to Gefitinib, K-Ras inhibitor, and paclitaxel treatments. We also confirmed that LAD1 deficiency remarkably retarded tumor growth in the xenograft model. Conclusively, LAD1 is a critical prognostic biomarker for LUAD and has potential as an intervention target.

A prediction model for prognosis of gastric adenocarcinoma based on six metabolism-related genes.

Background: The study of tumor metabolism is of great value to elucidate the mechanism of tumorigenesis and predict the prognosis of patients. However, the prognostic role of metabolism-related genes (MRGs) in gastric adenocarcinoma (GAD) remains poorly understood. Methods: We downloaded the gene chip dataset GSE79973 (n = 20) of GAD from the Gene Expression Omnibus (GEO) database to compare differentially expressed genes (DEGs) between normal and tumor tissues. We then extracted MRGs from these DEGs and systematically investigated the prognostic value of these differential MRGs for predicting patients' overall survival by univariable and multivariable Cox regression analysis. Six metabolic genes (ACOX3, APOE, DIO2, HSD17B4, NUAK1, and WHSC1L1) were identified as prognosis-associated hub genes, which were used to build a prognostic model in the training dataset GSE15459 (n = 200), and then validated in the dataset GSE62254 (n = 300). Results: Patients were divided into high-risk and low-risk subgroups based on the model's risk score, and it was found that patients in the high-risk subgroup had shorter overall survival than those in the low-risk subgroup, both in the training and testing datasets. In addition, for the training and testing cohorts, the area under the ROC curve of the prognostic model for one-year survival prediction was 0.723 and 0.667, respectively, indicating that the model has good predictive performance. Furthermore, we established a nomogram based on tumor stage and risk score to effectively predict the overall survival (OS) of GAD patients. The expression of 6 MRGs at the protein level was confirmed by immunohistochemistry (IHC). Kaplan-Meier survival analysis further confirmed that their expression influenced OS in GAD patients. Conclusion: Collectively, the 6 MRGs signature might be a reliable tool for assessing OS in GAD patients, with potential application value in clinical decision-making and individualized therapy.

SLFN5-mediated chromatin dynamics sculpt higher-order DNA repair topology.

Repair of DNA double-strand breaks (DSBs) elicits three-dimensional (3D) chromatin topological changes. A recent finding reveals that 53BP1 assembles into a 3D chromatin topology pattern around DSBs. How this formation of a higher-order structure is configured and regulated remains enigmatic. Here, we report that SLFN5 is a critical factor for 53BP1 topological arrangement at DSBs. Using super-resolution imaging, we find that SLFN5 binds to 53BP1 chromatin domains to assemble a higher-order microdomain architecture by driving damaged chromatin dynamics at both DSBs and deprotected telomeres. Mechanistically, we propose that 53BP1 topology is shaped by two processes: (1) chromatin mobility driven by the SLFN5-LINC-microtubule axis and (2) the assembly of 53BP1 oligomers mediated by SLFN5. In mammals, SLFN5 deficiency disrupts the DSB repair topology and impairs non-homologous end joining, telomere fusions, class switch recombination, and sensitivity to poly (ADP-ribose) polymerase inhibitor. We establish a molecular mechanism that shapes higher-order chromatin topologies to safeguard genomic stability.

A BAK subdomain that binds mitochondrial lipids selectively and releases cytochrome C.

How BAK and BAX induce mitochondrial outer membrane (MOM) permeabilization (MOMP) during apoptosis is incompletely understood. Here we have used molecular dynamics simulations, surface plasmon resonance, and assays for membrane permeabilization in vitro and in vivo to assess the structure and function of selected BAK subdomains and their derivatives. Results of these studies demonstrate that BAK helical regions α5 and α6 bind the MOM lipid cardiolipin. While individual peptides corresponding to these helical regions lack the full biological activity of BAK, tandem peptides corresponding to α4-α5, α5-α6, or α6-α7/8 can localize exogenous proteins to mitochondria, permeabilize liposomes composed of MOM lipids, and cause MOMP in the absence of the remainder of the BAK protein. Importantly, the ability of these tandem helices to induce MOMP under cell-free conditions is diminished by mutations that disrupt the U-shaped helix-turn-helix structure of the tandem peptides or decrease their lipid binding. Likewise, BAK-induced apoptosis in intact cells is diminished by CLS1 gene interruption, which decreases mitochondrial cardiolipin content, or by BAK mutations that disrupt the U-shaped tandem peptide structure or diminish lipid binding. Collectively, these results suggest that BAK structural rearrangements during apoptosis might mobilize helices involved in specific protein-lipid interactions that are critical for MOMP.

Targeted Inhibition of LPL/FABP4/CPT1 fatty acid metabolic axis can effectively prevent the progression of nonalcoholic steatohepatitis to liver cancer.

Rationale: Nonalcoholic steatohepatitis (NASH), as one of the key stages in the development of nonalcoholic fatty liver disease (NAFLD), can directly progress to HCC, but the underlying mechanism is not fully understood. Methods: Differentially expressed genes (DEGs) in each stage of disease development were studied through a GEO dataset deriving from a Stelic Animal Model (STAM), which can simulate the evolution of NAFLD/NASH to HCC in humans. GSVA analysis was performed to analyze the differentially expressed oncogenic signatures in each stage. A human NAFLD-related dataset from GEO database was utilized for gene expression verification and further validated in the protein level in STAM mice. Small molecule inhibitors were applied to STAM mice for investigating whether inhibition of the LPL/FABP4/CPT1 axis could prevent the occurrence of NASH-related HCC in vivo. Microsphere formation and clonal formation assays in vitro were applied to study if inhibition of the LPL/FABP4/CPT1 axis can reduce the viability of liver cancer stem cells (LCSCs). Results: We found that upregulation of the LPL/FABP4/CPT1 molecular axis, as a fatty acid metabolic reprogramming process, occurred specifically during the NASH phase. GSVA analysis showed widespread activation of a large number of oncogenic signals, which may contribute to malignant transformation during NASH. Furthermore, inhibition of the LPL/FABP4/CPT1 axis could effectively delay the tumor growth in STAM mice. Cell assays revealed inhibitors targeting this axis can significantly reduce the sphere-forming, proliferation, and clonality of LCSCs. Conclusion: These results suggest that activation of the LPL/FABP4/CPT1 axis is essential for LCSCs maintenance, which acts synergistically with a variety of up-regulated oncogenic signals that drive the hepatocyte-LCSCs transdifferentiation during NASH to HCC progression. Thus, targeting the LPL/FABP4/CPT1 axis may provide a potential direction for NASH-related HCC prevention.

An Immune-Associated Genomic Signature Effectively Predicts Pathologic Complete Response to Neoadjuvant Paclitaxel and Anthracycline-Based Chemotherapy in Breast Cancer.

Breast cancer is now the leading cause of cancer morbidity and mortality among women worldwide. Paclitaxel and anthracycline-based neoadjuvant chemotherapy is widely used for the treatment of breast cancer, but its sensitivity remains difficult to predict for clinical use. In our study, a LASSO logistic regression method was applied to develop a genomic classifier for predicting pathologic complete response (pCR) to neoadjuvant chemotherapy in breast cancer. The predictive accuracy of the signature classifier was further evaluated using four other independent test sets. Also, functional enrichment analysis of genes in the signature was performed, and the correlations between the prediction score of the signature classifier and immune characteristics were explored. We found a 25-gene signature classifier through the modeling, which showed a strong ability to predict pCR to neoadjuvant chemotherapy in breast cancer. For T/FAC-based training and test sets, and a T/AC-based test set, the AUC of the signature classifier is 1.0, 0.9071, 0.9683, 0.9151, and 0.7350, respectively, indicating that it has good predictive ability for both T/FAC and T/AC schemes. The multivariate model showed that 25-gene signature was far superior to other clinical parameters as independent predictor. Functional enrichment analysis indicated that genes in the signature are mainly enriched in immune-related biological processes. The prediction score of the classifier was significantly positively correlated with the immune score. There were also significant differences in immune cell types between pCR and residual disease (RD) samples. Conclusively, we developed a 25-gene signature classifier that can effectively predict pCR to paclitaxel and anthracycline-based neoadjuvant chemotherapy in breast cancer. Our study also suggests that the immune ecosystem is actively involved in modulating clinical response to neoadjuvant chemotherapy and is beneficial to patient outcomes.

Constitutive BAK/MCL1 complexes predict paclitaxel and S63845 sensitivity of ovarian cancer.

We previously found that preformed complexes of BAK with antiapoptotic BCL2 proteins predict BH3 mimetic sensitivities in lymphohematopoietic cells. These complexes have not previously been examined in solid tumors or in the context of conventional anticancer drugs. Here we show the relative amount of BAK found in preformed complexes with MCL1 or BCLXL varies across ovarian cancer cell lines and patient-derived xenografts (PDXs). Cells bearing BAK/MCL1 complexes were more sensitive to paclitaxel and the MCL1 antagonist S63845. Likewise, PDX models with BAK/MCL1 complexes were more likely to respond to paclitaxel. Mechanistically, BIM induced by low paclitaxel concentrations interacted preferentially with MCL1 and displaced MCL1-bound BAK. Further studies indicated that cells with preformed BAK/MCL1 complexes were sensitive to the paclitaxel/S63845 combination, while cells without BAK/MCL1 complexes were not. Our study suggested that the assessment of BAK/MCL1 complexes might be useful for predicting response to paclitaxel alone or in combination with BH3 mimetics.

Down-regulation of BCL2L13 renders poor prognosis in clear cell and papillary renal cell carcinoma.

BACKGROUND: BCL2L13 belongs to the BCL2 super family, with its protein product exhibits capacity of apoptosis-mediating in diversified cell lines. Previous studies have shown that BCL2L13 has functional consequence in several tumor types, including ALL and GBM, however, its function in kidney cancer remains as yet unclearly. METHODS: Multiple web-based portals were employed to analyze the effect of BCL2L13 in kidney cancer using the data from TCGA database. Functional enrichment analysis and hubs of BCL2L13 co-expressed genes in clear cell renal cell carcinoma (ccRCC) and papillary renal cell carcinoma (pRCC) were carried out on Cytoscape. Evaluation of BCL2L13 protein level was accomplished through immunohistochemistry on paraffin embedded renal cancer tissue sections. Western blotting and flow cytometry were implemented to further analyze the pro-apoptotic function of BCL2L13 in ccRCC cell line 786-0. RESULTS: BCL2L13 expression is significantly decreased in ccRCC and pRCC patients, however, mutations and copy number alterations are rarely observed. The poor prognosis of ccRCC that derived from down-regulated BCL2L13 is independent of patients' gender or tumor grade. Furthermore, BCL2L13 only weakly correlates with the genes that mutated in kidney cancer or the genes that associated with inherited kidney cancer predisposing syndrome, while actively correlates with SLC25A4. As a downstream effector of BCL2L13 in its pro-apoptotic pathway, SLC25A4 is found as one of the hub genes that involved in the physiological function of BCL2L13 in kidney cancer tissues. CONCLUSIONS: Down-regulation of BCL2L13 renders poor prognosis in ccRCC and pRCC. This disadvantageous factor is independent of any well-known kidney cancer related genes, so BCL2L13 can be used as an effective indicator for prognostic evaluation of renal cell carcinoma.

Enrichment and detection of circulating tumor cells by immunomagnetic beads and flow cytometry.

OBJECTIVE: The purpose of the article is to establish a quick enrichment and detection method using immunomagnetic beads and flow cytometry to analyze circulating tumor cells (CTCs) in the peripheral blood. RESULTS: After incubation with CD326-PE and CD45-APC antibodies, more than 60% MCF7 cells in M-Buffer could be detected while less than 10% of the same cells could be detected by flow cytometry (FCM) if spiked into blood. However, in combination with CD326 and CD45 immunomagnetic beads, detection rate of MCF7 cells in blood reached 57%. For circulating tumor cells, enrichment by CD326 and CD45 immunomagnetic beads improve the detection rate from nearly undetectable to more than 24.14%. CONCLUSIONS: Live CTCs in peripheral blood can be effectively and sensitively detected by using a combination of immunomagnetic beads (CD45 and CD326) and flow cytometry.

BCL2L13: physiological and pathological meanings.

BCL2L13 is a BCL2-like protein. It has been discovered for two decades, now on the way to be a hotspot of research with its physiological and pathological meanings found in recent years. Start with the pro-apoptotic activity, there have been reported consecutively that BCL2L13 could also induce mitochondrial fragmentation, inhibit cell death and promote mitophagy. Similar to BNIP3, BCL2L13 cannot be indiscriminately categorized into pro- or anti-apoptotic proteins. It anchors in the mitochondrial outer membrane, and expresses in various cells and tissues. This article reviews for the first time that BCL2L13 functions in physiological processes, such as growth and development and energy metabolism, and its dysregulation participating in pathological processes, including cancer, bacterial infection, cardiovascular diseases and degenerative diseases, suggesting its important roles in these events.