Caspase-independent Cell Death Research Articles

Buparvaquone Induces Ultrastructural and Physiological Alterations Leading to Mitochondrial Dysfunction and Caspase-Independent Apoptotic Cell Death in Leishmania donovani.

Leishmaniasis is a neglected tropical disease (endemic in 99 countries) caused by parasitic protozoa of the genus Leishmania. As treatment options are limited, there is an unmet need for new drugs. The hydroxynaphthoquinone class of compounds demonstrates broad-spectrum activity against protozoan parasites. Buparvaquone (BPQ), a member of this class, is the only drug licensed for the treatment of theileriosis. BPQ has shown promising antileishmanial activity but its mode of action is largely unknown. The aim of this study was to evaluate the ultrastructural and physiological effects of BPQ for elucidating the mechanisms underlying the in vitro antiproliferative activity in Leishmania donovani. Transmission and scanning electron microscopy analyses of BPQ-treated parasites revealed ultrastructural effects characteristic of apoptosis-like cell death, which include alterations in the nucleus, mitochondrion, kinetoplast, flagella, and the flagellar pocket. Using flow cytometry, laser scanning confocal microscopy, and fluorometry, we found that BPQ induced caspase-independent apoptosis-like cell death by losing plasma membrane phospholipid asymmetry and cell cycle arrest at sub-G0/G1 phase. Depolarization of the mitochondrial membrane leads to the generation of oxidative stress and impaired ATP synthesis followed by disruption of intracellular calcium homeostasis. Collectively, these findings provide valuable mechanistic insights and demonstrate BPQ's potential for development as an antileishmanial agent.

Identification of a novel form of caspase-independent cell death triggered by BH3-mimetics in diffuse large B-cell lymphoma cell lines

BH3-mimetics represent promising anti-cancer agents in tumors that rely on the anti-apoptotic function of B-Cell Lymphoma 2 (BCL2) proteins, particularly in leukemia and lymphoma cells primed for apoptosis. Mechanistically, BH3-mimetics may displace pro-apoptotic binding partners thus inducing BAX/BAK-mediated mitochondrial permeabilization followed by cytochrome c release, activation of the caspase cascade and apoptosis. Here, we describe a novel mode of caspase-independent cell death (CICD) induced by BH3-mimetics in a subset of diffuse large B-cell lymphoma (DLBCL) cells. Of note, rather than occurring via necroptosis, CICD induced immediately after mitochondrial permeabilization was associated with transcriptional reprogramming mediated by activation of c-Jun N-terminal Kinase (JNK) signaling and Activator Protein 1 (AP1). Thereby, CICD resulted in the JNK/AP1-mediated upregulation of inflammatory chemokines and increased migration of cytotoxic Natural Killer (NK) cells. Taken together, our study describes a novel mode of CICD triggered by BH3-mimetics that may alter the immune response towards dying cells.

Nano-flow cytometry unveils mitochondrial permeability transition process and multi-pathway cell death induction for cancer therapy

Mitochondrial permeability transition (mPT)-mediated mitochondrial dysfunction plays a pivotal role in various human diseases. However, the intricate details of its mechanisms and the sequence of events remain elusive, primarily due to the interference caused by Bax/Bak-induced mitochondrial outer membrane permeabilization (MOMP). To address these, we have developed a methodology that utilizes nano-flow cytometry (nFCM) to quantitatively analyze the opening of mitochondrial permeability transition pore (mPTP), dissipation of mitochondrial membrane potential (Δ\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\Delta$$\\end{document}Ψm), release of cytochrome c (Cyt c), and other molecular alternations of isolated mitochondria in response to mPT induction at the single-mitochondrion level. It was identified that betulinic acid (BetA) and antimycin A can directly induce mitochondrial dysfunction through mPT-mediated mechanisms, while cisplatin and staurosporine cannot. In addition, the nFCM analysis also revealed that BetA primarily induces mPTP opening through a reduction in Bcl-2 and Bcl-xL protein levels, along with an elevation in ROS content. Employing dose and time-dependent strategies of BetA, for the first time, we experimentally verified the sequential occurrence of mPTP opening and Δ\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\Delta$$\\end{document}Ψm depolarization prior to the release of Cyt c during mPT-mediated mitochondrial dysfunction. Notably, our study uncovers a simultaneous release of cell-death-associated factors, including Cyt c, AIF, PNPT1, and mtDNA during mPT, implying the initiation of multiple cell death pathways. Intriguingly, BetA induces caspase-independent cell death, even in the absence of Bax/Bak, thereby overcoming drug resistance. The presented findings offer new insights into mPT-mediated mitochondrial dysfunction using nFCM, emphasizing the potential for targeting such dysfunction in innovative cancer therapies and interventions.

4′-O-methylbavachalcone alleviates ischemic stroke injury by inhibiting parthanatos and promoting SIRT3

Cerebral ischemia-reperfusion injury (CIRI) can induce massive death of ischemic penumbra neurons via oxygen burst, exacerbating brain damage. Parthanatos is a form of caspase-independent cell death involving excessive activation of PARP-1, closely associated with intense oxidative stress following CIRI. 4′-O-methylbavachalcone (MeBavaC), an isoprenylated chalcone component in Fructus Psoraleae, has potential neuroprotective effects. This study primarily investigates whether MeBavaC can act on SIRT3 to alleviate parthanatos of ischemic penumbra neurons induced by CIRI. MeBavaC was oral gavaged to the middle cerebral artery occlusion-reperfusion (MCAO/R) rats after occlusion. The effects of MeBavaC on cerebral injury were detected by the neurological deficit score and cerebral infarct volume. In vitro, PC-12 cells were subjected to oxygen and glucose deprivation/reoxygenation (OGD/R), and assessed cell viability and cell injury. Also, the levels of ROS, mitochondrial membrane potential (MMP), and intracellular Ca2+ levels were detected to reflect mitochondrial function. We conducted western blotting analyses of proteins involved in parthanatos and related signaling pathways. Finally, the exact mechanism between the neuroprotection of MeBavaC and parthanatos was explored. Our results indicate that MeBavaC reduces the cerebral infarct volume and neurological deficit scores in MCAO/R rats, and inhibits the decreased viability of PC-12 cells induced by OGD/R. MeBavaC also downregulates the expression of parthanatos-related death proteins PARP-1, PAR, and AIF. However, this inhibitory effect is weakened after the use of a SIRT3 inhibitor. In conclusion, the protective effect of MeBavaC against CIRI may be achieved by inhibiting parthanatos of ischemic penumbra neurons through the SIRT3-PARP-1 axis.

Abstract 440: BAP1 deubiquitinates ACLY to promote lipogenesis yet predisposing cholangiocarcinoma to iron overload and ferroptosis

Abstract Background: Cholangiocarcinoma (CCA) is a highly lethal and aggressive epithelial cell malignancy of the liver and biliary tract. CCA survival remains poor and response to chemotherapy or immunotherapy is limited. We AIM to identify metabolic vulnerabilities that render CCA susceptible to ferroptosis, an iron-dependent and caspase-independent cell death driven by lipid peroxidation. Materials and Methods: We treated human and mouse CCA cell lines with ferroptosis inducers (RSL3, Erastin), inhibitors (Fer-1, Lip-1), necroptosis inhibitor and a pan-caspase inhibitor. Cell viability, intracellular and mitochondrial iron, lipid peroxidation, mitochondrial structure, and function by cell-based and biochemical assays. Gain- and loss-of-function strategies, TURBO proximity-dependent biotin identification, and metabolic techniques were used for mechanistic studies. In vivo, orthotopically implanted C57B/6 mice were treated with RSL3 or vehicle intraperitoneally, followed by assessment of tumor burden and immunophenotyping analysis. Epcam-aptamer coated nanoparticles (tMNPs) packaged with siRNA against GPX4 were used for selective ferroptosis induction. Results: We identified ferroptosis-sensitive and -resistant human and mouse CCA cells. Ferroptosis-sensitive cells, rescued by ferroptosis inhibitors but not by necroptosis or pan-caspase inhibitors, exhibited elevated intracellular free iron and mitochondrial iron overload. This led to significant mitochondrial dysfunction and structural changes associated with ferroptosis. Mitochondrial free iron triggered reactive oxygen species generation and lipid peroxidation. We identified BRCA-associated protein 1 (BAP1) as a potential driver of ferroptosis sensitivity. BAP1, mutated in 22-25% of human CCAs, is a tumor suppressor and deubiquitinase (DUB). BAP1 knockdown by siRNA protected cells from ferroptosis, while re-expression of BAP1 sensitized cells to ferroptosis. Subcellular fractionation identified BAP1 in extranuclear compartments, including mitochondria and cytosol. We found that BAP1 interacts and deubiquitinates ATP citrate lyase (ACLY), which converts iron chelator citrate to oxaloacetate and acetyl-CoA. Inhibition of mitochondrial citrate export, protected cells from iron overload and ferroptosis. In vivo, RSL3-induced ferroptosis significantly reduced tumor burden, as compared to vehicle-treated mice. RSL3-induced ferroptosis promoted an immunogenic tumor immune microenvironment. Lastly, tMNPs with siGPX4 induced marked ferroptosis of mouse CCA cells and showed maximal enrichment in liver orthotopic tumors in vivo. Conclusions: Our findings indicate that BAP1 deubiquitination of ACLY predisposes CCA cells to iron overload by exhausting citrate to support lipogenesis. CCA vulnerability to ferroptosis opens up novel therapeutic opportunities for CCA. Citation Format: Peyton Classon, Sophia Jaramillo, Danielle Carlson, Irene Yan, Tushar Patel, Sumera I. Ilyas, Rory L. Smoot, Gregory J. Gores, Davide Povero. BAP1 deubiquitinates ACLY to promote lipogenesis yet predisposing cholangiocarcinoma to iron overload and ferroptosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 440.

CHCHD4 binding affects the active site of apoptosis inducing factor (AIF): Structural determinants for allosteric regulation

Apoptosis-inducing factor (AIF), which is confined to mitochondria of normal healthy cells, is the first identified caspase-independent cell death effector. Moreover, AIF is required for the optimal functioning of the respiratory chain machinery. Recent findings have revealed that AIF fulfills its pro-survival function by interacting with CHCHD4, a soluble mitochondrial protein which promotes the entrance and the oxidative folding of different proteins in the inner membrane space. Here, we report the crystal structure of the ternary complex involving the N-terminal 27-mer peptide of CHCHD4, NAD+, and AIF harboring its FAD (flavin adenine dinucleotide) prosthetic group in oxidized form. Combining this information with biophysical and biochemical data on the CHCHD4/AIF complex, we provide a detailed structural description of the interaction between the two proteins, validated by both chemical cross-linking mass spectrometry analysis and site-directed mutagenesis.

Intracellular calcium links milk stasis to lysosome-dependent cell death during early mammary gland involution

Involution of the mammary gland after lactation is a dramatic example of coordinated cell death. Weaning causes distension of the alveolar structures due to the accumulation of milk, which, in turn, activates STAT3 and initiates a caspase-independent but lysosome-dependent cell death (LDCD) pathway. Although the importance of STAT3 and LDCD in early mammary involution is well established, it has not been entirely clear how milk stasis activates STAT3. In this report, we demonstrate that protein levels of the PMCA2 calcium pump are significantly downregulated within 2–4 h of experimental milk stasis. Reductions in PMCA2 expression correlate with an increase in cytoplasmic calcium in vivo as measured by multiphoton intravital imaging of GCaMP6f fluorescence. These events occur concomitant with the appearance of nuclear pSTAT3 expression but prior to significant activation of LDCD or its previously implicated mediators such as LIF, IL6, and TGFβ3, all of which appear to be upregulated by increased intracellular calcium. We further demonstrate that increased intracellular calcium activates STAT3 by inducing degradation of its negative regulator, SOCS3. We also observed that milk stasis, loss of PMCA2 expression and increased intracellular calcium levels activate TFEB, an important regulator of lysosome biogenesis through a process involving inhibition of CDK4/6 and cell cycle progression. In summary, these data suggest that intracellular calcium serves as an important proximal biochemical signal linking milk stasis to STAT3 activation, increased lysosomal biogenesis, and lysosome-mediated cell death.

TNFRSF19 promotes endoplasmic reticulum stress-induced paraptosis via the activation of the MAPK pathway in triple-negative breast cancer cells.

TNFRSF19 is a member of the tumor necrosis factor receptor superfamily, and its function exhibits variability among different types of cancers. The influence of TNFRSF19 on triple-negative breast cancer (TNBC) has yet to be definitively established. In this study, bioinformatics analyses revealed that lower TNFRSF19 was associated with the poorer prognosis, higher lymph node metastasis and lower immune infiltration. Subsequently, data obtained from the TCGA database and collection of tissue samples revealed that the mRNA and protein expression levels of TNFRSF19 were observed to be significantly reduced in TNBC tissue compared to normal tissue. Additionally, the results of in vitro experiments have demonstrated that TNFRSF19 possessed the ability to inhibit the proliferation, migration and invasive capabilities of TNBC cells. In vivo trials elucidated that TNFRSF19 could suppress tumor xenografts growth. Mechanistically, TNFRSF19 initiated caspase-independent cell death and induced paraptosis. Moreover, rescue assays demonstrated that TNFRSF19 induced-paraptosis was facilitated by MAPK pathway-mediated endoplasmic reticulum (ER) stress. In conclusion, our findings demonstrated that the upregulation of TNFRSF19 functioned as a tumor suppressor in TNBC by stimulating paraptosis through the activation of the MAPK pathway-mediated ER stress, highlighting its potential to be a new therapeutic target for TNBC.

Vibrio parahaemolyticus thermostable direct haemolysin induces non-classical programmed cell death despite caspase activation.

Thermostable direct haemolysin (TDH) is the key virulence factor secreted by the human gastroenteric bacterial pathogen Vibrio parahaemolyticus. TDH is a membrane-damaging pore-forming toxin. It evokes potent cytotoxicity, the mechanism of which still remains under-explored. Here, we have elucidated the mechanistic details of cell death response elicited by TDH. Employing Caco-2 intestinal epithelial cells and THP-1 monocytic cells, we show that TDH induces some of the hallmark features of apoptosis-like programmed cell death. TDH triggers caspase-3 and 7 activations in the THP-1 cells, while caspase-7 activation is observed in the Caco-2 cells. Interestingly, TDH appears to induce caspase-independent cell death. Higher XIAP level and lower Smac/Diablo level upon TDH intoxication provide plausible explanation for the functional inability of caspases in the THP-1 cells, in particular. Further exploration reveals that mitochondria play a central role in the TDH-induced cell death. TDH triggers mitochondrial damage, resulting in the release of AIF and endonuclease G, responsible for the execution of caspase-independent cell death. Among the other critical mediators of cell death, ROS is found to play an important role in the THP-1 cells, while PARP-1 appears to play a critical role in the Caco-2 cells. Altogether, our work provides critical new insights into the mechanism of cell death induction by TDH, showing a common central theme of non-classical programmed cell death. Our study also unravels the interplay of crucial molecules in the underlying signalling processes. Our findings add valuable insights into the role of TDH in the context of the host-pathogen interaction processes.

THU548 The Role Of Sirtuin 3 In Endocrine Therapy Resistant Breast Cancer Cells Response To Fulvestrant

Abstract Disclosure: K.A. de Oliveira: None. F.D. Andrade: None. M. Ozgul-Onal: None. A. Yadav: None. T. Onal: None. L. Jin: None. S. Sengupta: None. R. Clarke: None. Endocrine therapy can lead to cellular nutrient deprivation by reducing glucose and glutamine uptake and total cellular ATP production in breast cancer cells. The ability to bypass this metabolic stress is fundamental to how breast cancer cells regulate growth and acquire endocrine resistance. SIRT3, which can be activated by calorie restriction, modulates mitochondrial adaptation to low energy input. It has a key role in mitochondrial integrity and function, regulating cell survival, death, and metabolism, regulating the shift to amino-acid and fatty-acid catabolism. SIRT3 can maintain ROS levels at the appropriate levels for sustaining a proliferative phenotype, preventing apoptosis and promoting carcinogenesis. A role of SIRT3 in epigenetic regulation has also been reported. The ability of SIRTs to sense and respond to changes in energy, coupled with their intrinsic deacetylase/deacylase functions, could provide a mechanism for the cell to rewire signaling and maintain a newly acquired drug-resistant phenotype. Using antiestrogen sensitive (LCC1) and cross-resistant (LCC9) breast cancer cells, we confirmed higher expression of SIRT3 in LCC9, compared with LCC1 cells. Upregulation of SIRT3 expression within 24 hrs of Fulvestrant (ICI 1 µM) treatment (p=0.0176) in LCC1 was shown, whereas no effect was observed in LCC9 cells. Inhibition of SIRT3 activity, using the inhibitor LC0296 (15 µM), reduced growth rate in both LCC1 and SIRT3-silenced LCC9 cells under treatment with ICI (p<0.0001, p<0.001 respectively). Treatment with SIRTUIN 3 inhibitor and ICI induced caspase-independent cell death in LCC1 and SIRT3-silenced LCC9 cells compared to the untreated control and ICI only treated cells (p<0.001). The combination of SIRT3 inhibition (LC0296, 15 µM) and ICI treatment (0.5 µM) increased ROS production in LCC9-SIRT3 silenced cells in 24 hrs compared with the untreated control and ICI only treated cells (p<0.001). The combination of LC0296 and ICI induced a decrease in mitochondrial membrane potential and ATP production, and a decrease in glycolytic capacity and glycolytic reserve in both LCC1 and SIRT3-silenced LCC9 cells, compared with the untreated control (p<0.001). Moreover, ICI significantly reduced ATP production in the mitochondria of the SIRT3-silenced LCC9 cells (p<0.001) but not in the non-silenced (control) LCC9 cells. Taken together, these results show that SIRT3 may have a key role in determining the response to fulvestrant response in ER+ breast cancer cells; a more detailed understanding of this role for SIRT3 is under investigation in our laboratory. Presentation: Thursday, June 15, 2023

Disruption of caspase-independent cell proliferation pathway on spheroids (HeLa cells) treated with curcumin

ABSTRACT Curcumin is an antiproliferative phytochemical extracted from Curcuma longa L and which has been studied in preclinical drug screening using cell monolayers and animal models. However, several limitations of these culture systems may be overcome by performing screening with three-dimensional (3-D) cell culture. The aim of this study was to investigate the effects of curcumin on cytotoxicity and genotoxicity as well as spheroid growth using cervical adenocarcinoma HeLa cell spheroids by performing RT-PCR mRNA expression of genes involved in cell death (CASP3, CASP8, CASP9, PARP1, BBC3, BIRC5, BCL2, TNF), autophagy (BECN1, SQSTM1), cell cycle regulation (TP53, C-MYC, NF-kB, CDKN1A, m-TOR, TRAF-2), DNA damage repair (H2AFX, GADD45A, GADD45G), oxidative stress (GPX1), reticulum stress (EIF2AK3, ERN1), and invasion (MMP1, MMP9) was investigated. Curcumin was cytotoxic in a concentration-dependent manner. Curcumin-treated spheroids exhibited lower proliferative recovery and cell proliferation attenuation, as observed in the clonogenic assay. Further, no marked genotoxicity was detected. Curcumin-treated spheroids displayed reduced expression of BECN1 (2.9×), CASP9 (2.1×), and PARP1 (2.1×) mRNA. PARP1 inhibition suggested disruption of essential pathways of proliferation maintenance. Downregulated expression of CASP9 mRNA and unchanged expression of CASP3/8 mRNA suggested caspase-independent cell death, whereas downregulated expression of BECN1 mRNA indicated autophagic disruption. Therefore, curcumin exhibits the potential for drug development with antiproliferative activity to be considered for use in cancers.

Transcriptional pausing factor M1BP regulates cellular homeostasis by suppressing autophagy and apoptosis in Drosophila eye.

During organogenesis cellular homeostasis plays a crucial role in patterning and growth. The role of promoter proximal pausing of RNA polymerase II, which regulates transcription of several developmental genes by GAGA factor or Motif 1 Binding Protein (M1BP), has not been fully understood in cellular homeostasis. Earlier, we reported that M1BP, a functional homolog of ZKSCAN3, regulates wingless and caspase-dependent cell death (apoptosis) in the Drosophila eye. Further, blocking apoptosis does not fully rescue the M1BPRNAi phenotype of reduced eye. Therefore, we looked for other possible mechanism(s). In a forward genetic screen, members of the Jun-amino-terminal-(NH2)-Kinase (JNK) pathway were identified. Downregulation of M1BP ectopically induces JNK, a pro-death pathway known to activate both apoptosis and caspase-independent (autophagy) cell death. Activation of JNK pathway components can enhance M1BPRNAi phenotype and vice-versa. Downregulation of M1BP ectopically induced JNK signaling, which leads to apoptosis and autophagy. Apoptosis and autophagy are regulated independently by their genetic circuitry. Here, we found that blocking either apoptosis or autophagy alone rescues the reduced eye phenotype of M1BP downregulation; whereas, blocking both apoptosis and autophagy together significantly rescues the M1BP reduced eye phenotype to near wild-type in nearly 85% progeny. This data suggests that the cellular homeostasis response demonstrated by two independent cell death mechanisms, apoptosis and autophagy, can be regulated by a common transcriptional pausing mechanism orchestrated by M1BP. Since these fundamental processes are conserved in higher organisms, this novel functional link between M1BP and regulation of both apoptosis and autophagy can be extrapolated to humans.

A Missense Variant in AIFM1 Caused Mitochondrial Dysfunction and Intolerance to Riboflavin Deficiency.

AIFM1 is a mitochondrial flavoprotein involved in caspase-independent cell death and regulation of respiratory chain complex biogenesis. Mutations in the AIFM1 gene have been associated with multiple clinical phenotypes, but the effectiveness of riboflavin treatment remains controversial. Furthermore, few studies explored the reasons underlying this controversy. We reported a 7-year-old boy with ataxia, sensorimotor neuropathy and muscle weakness. Genetic and histopathological analyses were conducted, along with assessments of mitochondrial function and apoptosis level induced by staurosporine. Riboflavin deficiency and supplementation experiments were performed using fibroblasts. A missense c.1019T > C (p. Met340Thr) variant of AIFM1 was detected in the proband, which caused reduced expression of AIFM1 protein and mitochondrial dysfunction as evidenced by downregulation of mitochondrial complex subunits, respiratory deficiency and collapse of ΔΨm. The proportion of apoptotic cells in mutant fibroblasts was lower than controls after induction of apoptosis. Riboflavin deficiency resulted in decreased AIFM1 protein levels, while supplementation with high concentrations of riboflavin partially increased AIFM1 protein levels in variant fibroblasts. In addition, mitochondrial respiratory function of mutant fibroblasts was partly improved after riboflavin supplementation. Our study elucidated the pathogenicity of the AIFM1 c.1019T > C variant and revealed mutant fibroblasts was intolerant to riboflavin deficiency. Riboflavin supplementation is helpful in maintaining the level of AIFM1 protein and mitochondrial respiratory function. Early riboflavin treatment may serve as a valuable attempt for patients with AIFM1 variant.

Limocitrin increases cytotoxicity of KHYG-1 cells against K562 cells by modulating MAPK pathway.

Natural killer (NK) cells are gaining popularity in the field of cancer immunotherapy. The present study was designed to investigate the effect of a natural flavonol compound limocitrin in increasing cytotoxicity of a permanent NK leukemia cell line KHYG-1 against an aggressive leukemia cell line K562. The findings revealed that limocitrin increased the expressions of cytolytic molecules perforin, granzymes A and B, and granulysin in KHYG-1 cells by inducing phosphorylation of transcription factor CREB, leading to increased lysis of K562 cells. Mechanistically, limocitrin was found to increase the expressions of t-Bid, cleaved caspase 3, and cleaved PARP to induce K562 cell apoptosis. Moreover, limocitrin reduced the expressions of SET and Ape1 to inhibit DNA repair mechanism, leading to caspase-independent K562 cell death. At the molecular level, limocitrin was found to increase the phosphorylation of ERK, p38, and JNK to increase granzyme B expression in KHYG-1 cells. Taken together, the study indicates that limocitrin increases cytotoxicity of NK cells against a range of cancer cells.

Targeting sphingolipid metabolism with the sphingosine kinase inhibitor SKI-II overcomes hypoxia-induced chemotherapy resistance in glioblastoma cells: effects on cell death, self-renewal, and invasion

BackgroundGlioblastoma patients commonly develop resistance to temozolomide chemotherapy. Hypoxia, which supports chemotherapy resistance, favors the expansion of glioblastoma stem cells (GSC), contributing to tumor relapse. Because of a deregulated sphingolipid metabolism, glioblastoma tissues contain high levels of the pro-survival sphingosine-1-phosphate and low levels of the pro-apoptotic ceramide. The latter can be metabolized to sphingosine-1-phosphate by sphingosine kinase (SK) 1 that is overexpressed in glioblastoma. The small molecule SKI-II inhibits SK and dihydroceramide desaturase 1, which converts dihydroceramide to ceramide. We previously reported that SKI-II combined with temozolomide induces caspase-dependent cell death, preceded by dihydrosphingolipids accumulation and autophagy in normoxia. In the present study, we investigated the effects of a low-dose combination of temozolomide and SKI-II under normoxia and hypoxia in glioblastoma cells and patient-derived GCSs.MethodsDrug synergism was analyzed with the Chou-Talalay Combination Index method. Dose–effect curves of each drug were determined with the Sulforhodamine B colorimetric assay. Cell death mechanisms and autophagy were analyzed by immunofluorescence, flow cytometry and western blot; sphingolipid metabolism alterations by mass spectrometry and gene expression analysis. GSCs self-renewal capacity was determined using extreme limiting dilution assays and invasion of glioblastoma cells using a 3D spheroid model.ResultsTemozolomide resistance of glioblastoma cells was increased under hypoxia. However, combination of temozolomide (48 µM) with SKI-II (2.66 µM) synergistically inhibited glioblastoma cell growth and potentiated glioblastoma cell death relative to single treatments under hypoxia. This low-dose combination did not induce dihydrosphingolipids accumulation, but a decrease in ceramide and its metabolites. It induced oxidative and endoplasmic reticulum stress and triggered caspase-independent cell death. It impaired the self-renewal capacity of temozolomide-resistant GSCs, especially under hypoxia. Furthermore, it decreased invasion of glioblastoma cell spheroids.ConclusionsThis in vitro study provides novel insights on the links between sphingolipid metabolism and invasion, a hallmark of cancer, and cancer stem cells, key drivers of cancer. It demonstrates the therapeutic potential of approaches that combine modulation of sphingolipid metabolism with first-line agent temozolomide in overcoming tumor growth and relapse by reducing hypoxia-induced resistance to chemotherapy and by targeting both differentiated and stem glioblastoma cells.

Lysophosphatidylserine induces necrosis in pressure overloaded male mouse hearts via G protein coupled receptor 34

Heart failure is a leading cause of mortality in developed countries. Cell death is a key player in the development of heart failure. Calcium-independent phospholipase A2β (iPLA2β) produces lipid mediators by catalyzing lipids and induces nuclear shrinkage in caspase-independent cell death. Here, we show that lysophosphatidylserine generated by iPLA2β induces necrotic cardiomyocyte death, as well as contractile dysfunction mediated through its receptor, G protein-coupled receptor 34 (GPR34). Cardiomyocyte-specific iPLA2β-deficient male mice were subjected to pressure overload. While control mice showed left ventricular systolic dysfunction with necrotic cardiomyocyte death, iPLA2β-deficient mice preserved cardiac function. Lipidomic analysis revealed a reduction of 18:0 lysophosphatidylserine in iPLA2β-deficient hearts. Knockdown of Gpr34 attenuated 18:0 lysophosphatidylserine-induced necrosis in neonatal male rat cardiomyocytes, while the ablation of Gpr34 in male mice reduced the development of pressure overload-induced cardiac remodeling. Thus, the iPLA2β—lysophosphatidylserine—GPR34—necrosis signaling axis plays a detrimental role in the heart in response to pressure overload.

Galectin-9 has non-apoptotic cytotoxic activity toward acute myeloid leukemia independent of cytarabine resistance

Acute myeloid leukemia (AML) is a malignancy still associated with poor survival rates, among others, due to frequent occurrence of therapy-resistant relapse after standard-of-care treatment with cytarabine (AraC). AraC triggers apoptotic cell death, a type of cell death to which AML cells often become resistant. Therefore, therapeutic options that trigger an alternate type of cell death are of particular interest. We previously identified that the glycan-binding protein Galectin-9 (Gal-9) has tumor-selective and non-apoptotic cytotoxicity towards various types of cancer, which depended on autophagy inhibition. Thus, Gal-9 could be of therapeutic interest for (AraC-resistant) AML. In the current study, treatment with Gal-9 was cytotoxic for AML cells, including for CD34+ patient-derived AML stem cells, but not for healthy cord blood-derived CD34+ stem cells. This Gal-9-mediated cytotoxicity did not rely on apoptosis but was negatively associated with autophagic flux. Importantly, both AraC-sensitive and -resistant AML cell lines, as well as AML patient samples, were sensitive to single-agent treatment with Gal-9. Additionally, Gal-9 potentiated the cytotoxic effect of DNA demethylase inhibitor Azacytidine (Aza), a drug that is clinically used for patients that are not eligible for intensive AraC treatment. Thus, Gal-9 is a potential therapeutic agent for the treatment of AML, including AraC-resistant AML, by inducing caspase-independent cell death.

Cremastranone-Derived Homoisoflavanes Suppress the Growth of Breast Cancer Cells via Cell Cycle Arrest and Caspase-Independent Cell Death.

Breast cancer is the most common cancer and a frequent cause of cancer-related deaths among women wordlwide. As therapeutic strategies for breast cancer have limitations, novel chemotherapeutic reagents and treatment strategies are needed. In this study, we investigated the anti-cancer effect of synthetic homoisoflavane derivatives of cremastranone on breast cancer cells. Homoisoflavane derivatives, SH-17059 and SH-19021, reduced cell proliferation through G2/M cell cycle arrest and induced caspase-independent cell death. These compounds increased heme oxygenase-1 (HO-1) and 5-aminolevulinic acid synthase 1 (ALAS1), suggesting downregulation of heme. They also induced reactive oxygen species (ROS) generation and lipid peroxidation. Furthermore, they reduced expression of glutathione peroxidase 4 (GPX4). Therefore, we suggest that the SH-17059 and SH-19021 induced the caspase-independent cell death through the accumulation of iron from heme degradation, and the ferroptosis might be one of the potential candidates for caspase-independent cell death.

A risk signature of necroptosis-related lncRNA to predict prognosis and probe molecular characteristics for male with bladder cancer.

Bladder cancer (BC) is a frequently diagnosed cancer with high mortality. Male patients have a higher risk of developing BC than female patients. As a type of caspase-independent cell death, necroptosis plays a significant role in the occurrence and progression of BC. The aberrant function of long non-coding RNAs (lncRNAs) plays an indispensable role in GI. However, the relationship between lncRNA and necroptosis in male patients with BC remains unclear. The clinical information and RNA-sequencing profiles of all BC patients were retrieved from The Cancer Genome Atlas Program. A total of 300 male participants were selected for the study. We conducted to identify the necroptosis-related lncRNAs (NRLs) by Pearson correlation analysis. Subsequently, least absolute shrinkage and selection operator Cox regression were conducted to establish a risk signature with overall survival-related NRLs in the training set and to validate it in the testing set. Finally, we verified the effectiveness of the 15-NRLs signature in prognostic prediction and therapy via survival analysis, receiver operating characteristic curve analysis, and Cox regression. Furthermore, we analyzed the correlation between the signature risk score and pathway enrichment analysis, immune cell infiltration, anticancer drug sensitivity, and somatic gene mutations. We developed 15-NRLs (AC009974.1, AC140118.2, LINC00323, LINC02872, PCAT19, AC017104.1, AC134312.5, AC147067.2, AL139351.1, AL355922.1, LINC00844, AC069503.1, AP003721.1, DUBR, LINC02863) signature, and divided patients into a high-risk group and low-risk group through the median risk score. Kaplan-Meier and receiver operating characteristic curves showed that the prognosis prediction had satisfactory accuracy. Cox regression analysis indicated that the 15-NRLs signature was a risk factor independent of various clinical parameters. Additionally, immune cell infiltration, half-maximal inhibitory concentration, and somatic gene mutations differed significantly among different risk subsets, implying that the signature could assess the clinical efficacy of chemotherapy and immunotherapy. This 15-NRLs risk signature may be helpful in assessing the prognosis and molecular features of male patients with BC and improve treatment modalities, thus can be further applied clinically.

Artemisia sieberi Besser essential oil inhibits the growth and migration of breast cancer cells via induction of S-phase arrest, caspase-independent cell death and downregulation of ERK

Ethnopharmacological relevanceArtemisia sieberi Besser is a medicinal herb that has been traditionally used across the Middle East for the treatment of cancer. Further pharmacological studies on its extracts revealed that they possess cytotoxic activity against certain cancer cells, however, there were no studies conducted on the anticancer potential of Artemisia sieberi essential oil (ASEO). Aim of the studyTo evaluate the anticancer potential of ASEO, elucidate the oil's mode of action for the first time and investigate its chemical composition. Materials and methodsArtemisia sieberi was collected from Hail, Saudi Arabia, and its essential oil was obtained via hydrodistillation. The oil's activity against HCT116, HepG2, A549 and MCF-7 cells was assessed using SRB assay, while its anti-metastatic potential was assessed via a migration assay. Cell-cycle analysis and apoptosis assay were conducted via flow cytometry, while protein expression levels were investigated using Western blotting. The oil's chemical constituents were identified using GCMS. ResultsASEO exerted its highest cytotoxic activity against MCF-7 with an IC50 value of 38.7 μg/ml. Further studies showed that the oil inhibited MCF-7 cells' migration, induced S-phase arrest and apoptosis. Western blot analysis showed no change in the expression level of caspase-3 after treatment, indicating the induction of caspase-independent apoptosis-like cell death in MCF-7. Treatment of MCF-7 with the oil resulted in downregulation of the protein expression levels of total ERK and its downstream target, LC3, indicating that any potential activation of the ERK signalling pathway during the cancer cells' growth would be inhibited. Finally, GCMS analysis identified the oil's major components as cis-crysanthenyl acetate (48.56%), davanone (10.28%), 1,8-cineole (6.81%) and caryophyllene diepoxide (5.34%), whereby it is suggested that these compounds might be responsible for the oil's bioactivity. ConclusionASEO possessed in vitro anticancer activity and modulated the ERK signalling pathway. This is the first study to explore the anticancer potential of ASEO in detail and reflects the significance of investigating essential oils from medicinal plants that have been traditionally used against cancer. This work might pave the way for further in vivo studies that could result in developing the oil into a natural effective anticancer treatment.