Activation Of Executioner Caspases Research Articles

Chitosan-encapsulated naringenin promotes ROS mediated through the activation of executioner caspase-3.

We previously reported that chitosan nanoparticle-encapsulated Naringenin (CS-NPs/NAR) could scavenge free radicals at lower doses and be cytotoxic to cancer cells. The current study continues to focus on the mechanism behind CS-NPs/NAR-induced breast cancer cell (MDA-MB-231) death. MDA-MB-231 cells were treated with higher concentrations (100, 200, and 200µg) of Chitosan nanoparticles (CS-NPs), naringenin (NAR), and chitosan-encapsulated naringenin (CS-NPs/NAR). The cell viability, proliferation, and oxidative stress parameters, such as nitric oxide [NO], xanthine oxidase (XOD), and xanthine dehydrogenase (XDH) levels, were analyzed. ROS levels were determined through DCFDA analysis. MTT-based cell cytotoxicity and BrdU cell proliferation analysis depicted the cytotoxicity effects (37% and 29% for 24 and 48h) and exhibited a reduction in the proliferation of MDA-MB-231 by CS-NPs/NAR. A significant increase in NO content, XOD, a decrease in XDH, and an increase in ROS levels were observed upon treatment with CS-NPs/NAR. Fluorescent images suggested the increase in the ROS level upon treatment with CS-NPs/NAR in cancer cells, and the results suggested that it could induce apoptosis. Further, to confirm this, the activity of caspase-3 was analyzed through western blotting, and the result suggested that the higher concentration of CS-NPs/NAR has increased the activation of procaspase3 when compared to free NAR. Hence, the current investigation concludes that high doses of CS-NPs/NAR induce and increase oxidative stress and so increased activation of procaspase3 may lead to cancer cell apoptosis and reduction in cell proliferation.

Embelin inhibits viability of cutaneous T cell lymphoma cell lines HuT78 and H9 by targeting inhibitors of apoptosis

Cutaneous T cell lymphoma (CTCL) is a varied group of neoplasms that affects the skin. Acquired resistance against chemotherapeutic drugs and associated toxic side effects are limitations that warrant search for novel drugs against CTCL. Embelin (EMB) is a naturally occurring benzoquinone derivative that has gained attention owing to its anticancer pharmacological actions and nontoxic nature. We assessed the anticancer activity of EMB against CTCL cell lines, HuT78, and H9. EMB inhibited viability of CTCL cells in a dose-dependent manner. EMB activated extrinsic and intrinsic pathways of apoptosis as shown by the activation of initiator and executioner caspases. EMB-induced apoptosis also involved suppression of inhibitors of apoptosis, XIAP, cIAP1, and cIAP2. PARP cleavage and upregulation of pH2AX indicated DNA damage induced by EMB. In conclusion, we characterized a novel apoptosis-inducing activity of EMB against CTCL cells, implicating EMB as a potential therapeutic agent against CTCL.

The BCL-2 family protein BCL-RAMBO interacts and cooperates with GRP75 to promote its apoptosis signaling pathway

The BCL-2 family protein BCL-RAMBO, also known as BCL2-like 13, anchors at the outer mitochondrial membrane and regulates apoptosis, mitochondrial fragmentation, and mitophagy. However, the mechanisms underlying the proapoptotic role of BCL-RAMBO remain unclear. In the present study, we demonstrated that BCL-RAMBO interacted with glucose-regulated protein 75 (GRP75), also known as heat shock protein family A member 9, and mortalin using co-immunoprecipitation and glutathione S-transferase-based pull-down assays. BCL-RAMBO interacted with GRP75 via its No BCL-2 homology domain. The interaction between BCL-RAMBO and GRP75 was confirmed by genetic interactions in Drosophila because a rough eye phenotype caused by the ectopic expression of BCL-RAMBO was partially suppressed by mutations in Hsc70-5, a mammalian GRP75 ortholog. In human embryonic kidney 293T cells, the co-expression of BCL-RAMBO and GRP75 facilitated an elevation in executioner caspase activity and poly (ADP-ribose) polymerase 1 (PARP-1) cleavage. In contrast, the knockdown of GRP75 suppressed elevated executioner caspase activity and PARP-1 cleavage in BCL-RAMBO-transfected cells. The mitochondrial release of cytochrome c induced by BCL-RAMBO was also attenuated by the knockdown of GRP75. These results indicate that GRP75 interacts with BCL-RAMBO and plays a crucial role in the BCL-RAMBO-dependent apoptosis signaling pathway.

Magnetospirillum magneticum triggers apoptotic pathways in human breast cancer cells

The use of bacteria in cancer immunotherapy has the potential to bypass many shortcomings of conventional treatments. The ability of anaerobic bacteria to preferentially accumulate and replicate in hypoxic regions of solid tumors, as a consequence of bacterial metabolic needs, is particularly advantageous and key to boosting their immunostimulatory therapeutic actions in situ. While several of these bacterial traits are well-studied, little is known about their competition for nutrients and its effect on cancer cells which could serve as another potent and innate antineoplastic action. Here, we explored the consequences of the iron-scavenging abilities of a particular species of bacteria, Magnetospirillum magneticum, which has been studied as a potential new class of bacteria for magnetically targeted bacterial cancer therapy. We investigated their influence in hypoxic regions of solid tumors by studying the consequential metabolic effects exerted on cancer cells. To do so, we established an in vitro co-culture system consisting of the bacterial strain AMB-1 incubated under hypoxic conditions with human breast cancer cells MDA-MB-231. We first quantified the number of viable cells after incubation with magnetotactic bacteria demonstrating a lower rate of cellular proliferation that correlated with increasing bacteria-to-cancer cells ratio. Further experiments showed increasing populations of apoptotic cells when cancer cells were incubated with AMB-1 over a period of 24 h. Analysis of the metabolic effects induced by bacteria suggest an increase in the activation of executioner caspases as well as changes in levels of apoptosis-related proteins. Finally, the level of several human apoptosis-related proteins was investigated, confirming a bacteria-dependent triggering of apoptotic pathways in breast cancer cells. Overall, our findings support that magnetotactic bacteria could act as self-replicating iron-chelating agents and indicate that they interfere with proliferation and lead to increased apoptosis of cancer cells. This bacterial feature could serve as an additional antineoplastic mechanism to reinforce current bacterial cancer therapies.

Inducing Cytotoxicity in Colon Cancer Cells and Suppressing Cancer Stem Cells by Dolasetron and Ketoprofen through Inhibition of RNA Binding Protein PUM1.

Clinical trials of new drugs often face a high failure rate of approximately 45 percent due to safety and toxicity concerns. Repurposing drugs with well-established safety profiles becomes crucial in addressing this challenge. Colon cancer ranks as the third most prevalent cancer and the second leading cause of cancer related mortality worldwide. This study focuses on the RNA-binding protein pumilio1 (PUM1), a member of the PUF family involved in post-transcriptional gene expression regulation. By utilizing molecular docking techniques and FDA-approved drugs, potential inhibitors against PUM1 were identified. Notably, dolasetron and ketoprofen demonstrated promising results, exhibiting strong binding affinity, hydrophobic interactions, and favorable chemical reactivity according to Conceptual-DFT calculations. Both compounds effectively reduced cell viability, with IC50 values of 150 µM and 175 µM, respectively and shows long term inhibitory effects as seen by reduced in number of colonies. Moreover, they exhibited inhibitory effects on colon cancer stem cells, as indicated by reduced colonospheroid size and numbers. Apoptosis is induced by these compounds and has triggered activation of executioner caspase 3/7 in HCT116 cells which is evident through a caspase 3/7 assay and AO/EB staining, while the non-toxic effect of these compounds was evident from viability against non-cancerous cell line and hemolysis assay. Additionally, the treatment group showed a significant decrease in PUM1 and cancer stem cell markers expression compared to the control group. In conclusion, this study highlights the potential of targeting PUM1 as a novel approach to colon cancer treatment. Dolasetron and ketoprofen demonstrate promise as effective anti-cancer and anti-cancer stem cell drugs, inducing apoptosis in colon cancer cells through inhibition of PUM1.

Anastasis enhances metastasis and chemoresistance of colorectal cancer cells through upregulating cIAP2/NFκB signaling

Chemotherapy is a common strategy to treat cancer. However, acquired resistance and metastasis are the major obstacles to successful treatment. Anastasis is a process by which cells survive executioner caspase activation when facing apoptotic stress. Here we demonstrate that colorectal cancer cells can undergo anastasis after transient exposure to chemotherapeutic drugs. Using a lineage tracing system to label and isolate cells that have experienced executioner caspase activation in response to drug treatment, we show that anastasis grants colorectal cancer cells enhanced migration, metastasis, and chemoresistance. Mechanistically, treatment with chemotherapeutic drugs induces upregulated expression of cIAP2 and activation of NFκB, which are required for cells to survive executioner caspase activation. The elevated cIAP2/NFκB signaling persists in anastatic cancer cells to promote migration and chemoresistance. Our study unveils that cIAP2/NFκB-dependent anastasis promotes acquired resistance and metastasis after chemotherapy.

Chemotherapy-induced executioner caspase activation increases breast cancer malignancy through epigenetic de-repression of CDH12

Cancer relapse and metastasis are major obstacles for effective treatment. One important mechanism to eliminate cancer cells is to induce apoptosis. Activation of executioner caspases is the key step in apoptosis and was considered “a point of no return”. However, in recent years, accumulating evidence has demonstrated that cells can survive executioner caspase activation in response to apoptotic stimuli through a process named anastasis. Here we show that breast cancer cells that have survived through anastasis (anastatic cells) after exposure to chemotherapeutic drugs acquire enhanced proliferation and migration. Mechanistically, cadherin 12 (CDH12) is persistently upregulated in anastatic cells and promotes breast cancer malignancy via activation of ERK and CREB. Moreover, we demonstrate that executioner caspase activation induced by chemotherapeutic drugs results in loss of DNA methylation and repressive histone modifications in the CDH12 promoter region, leading to increased CDH12 expression. Our work unveils the mechanism underlying anastasis-induced enhancement in breast cancer malignancy, offering new therapeutic targets for preventing post-chemotherapy cancer relapse and metastasis.

Potential therapeutic action of tauroursodeoxycholic acid against cholestatic liver injury via hepatic Fxr/Nrf2 and CHOP-DR5-caspase-8 pathway.

Cholestasis is a pathophysiologic syndrome with limited therapeutic options. Tauroursodeoxycholic acid (TUDCA) has been employed to treat hepatobiliary disorders and is as effective as UDCA in alleviating cholestatic liver disease in clinical trials. Until now, TUDCA's mechanism of action toward cholestasis remains unclear. In the present study, cholestasis was induced with a cholic acid (CA)-supplemented diet or α-naphthyl isothiocyanate (ANIT) gavage in wild-type and Farnesoid X Receptor (FXR) deficient mice, using obeticholic acid (OCA) as control. The effects of TUDCA on liver histological changes, transaminase level, bile acid composition, hepatocyte death, expression of Fxr and nuclear factor erythroid 2-related factor 2 (Nrf2) and target genes, as well as apoptotic signaling pathways, were investigated. Treating CA-fed mice with TUDCA markedly alleviated liver injury, attenuated bile acids retention in liver and plasma, increased Fxr and Nrf2 nuclear levels and modulated the expression of targets regulating synthesis and transportation of bile acids, including BSEP, MRP2, NTCP and CYP7A1. TUDCA, but not OCA, activated Nrf2 signaling and exerted protective effects against cholestatic liver injury in Fxr-/- mice fed with CA. Furthermore, in both mice with CA- and ANIT-induced cholestasis, TUDCA decreased expression of GRP78 and CCAAT/enhancer-binding protein homologous protein (CHOP), reduced death receptor 5 (DR5) transcription, caspase-8 activation, and BID cleavage, and subsequently inhibited activation of executioner caspases and apoptosis in liver. We confirmed that TUDCA protected against cholestatic liver injury by alleviating BAs burden of dually activating hepatic Fxr and Nrf2. Moreover, inhibiting CHOP-DR5-caspase-8 pathway contributed to the anti-apoptotic effect of TUDCA in cholestasis.

Anastasis confers ovarian cancer cells increased malignancy through elevated p38 MAPK activation.

Activation of executioner caspases was once considered as a point of no return in apoptosis. However, in recent years, accumulating evidence has demonstrated that cells can survive executioner caspase activation in response to apoptotic stimuli through a process called anastasis. In this study, we developed a reporter system, mCasExpress, to track mammalian cells that survive executioner caspase activation. We demonstrate that anastatic ovarian cancer cells acquire enhanced migration following their transient exposure to apoptotic stimulus TRAIL or Paclitaxel. Moreover, anastatic cancer cells secrete more pro-angiogenic factors that enable tumor angiogenesis, growth and metastasis. Mechanistically, we demonstrate that activation of p38 MAPK, which occurs in a caspase-dependent manner in response to apoptotic stress to promote anastasis, persists at a higher level in anastatic cancer cells even after removal of apoptotic stimuli. Importantly, p38 is essential for the elevated migratory and angiogenic capacity in the anastatic cells. Our work unveils anastasis as a potential driver of tumor angiogenesis and metastasis.

Yamogenin-Induced Cell Cycle Arrest, Oxidative Stress, and Apoptosis in Human Ovarian Cancer Cell Line

Steroidal saponins are a group of compounds with complex structures and biological activities. They have anti-inflammatory, antimicrobial, fungicidal, and antitumor properties. Yamogenin is one of the spirostane saponins and occurs in Trigonella foenum-graecum, Asparagus officinalis, and Dioscorea collettii. It is a stereoisomer of diosgenin-a well-known compound whose activity and mechanisms of action in cancer cells are determined. However, the antitumor effect of yamogenin is still little known, and the mechanism of action has not been determined. In this study, we evaluated the effect of yamogenin on human ovarian cancer SKOV-3 cells in vitro by determining the cellular factors that trigger cell death. The viability of the cells was assessed with a Real-Time xCELLigence system and the cell cycle arrest with flow cytometry. The activity of initiator and executioner caspases (-8, -9, and -3/7) was estimated with luminometry and flow cytometry, respectively. The mitochondrial membrane depolarization, the level of oxidative stress, and DNA damage in the yamogenin-treated cells were also evaluated by flow cytometry. Genes expression analysis at the mRNA level was conducted with Real-Time PCR. Bid activation and chromatin condensation were estimated with fluorescent microscopy. The obtained results indicate that yamogenin has cytotoxic activity in SKOV-3 cells with an IC50 value of 23.90 ± 1.48 µg/mL and strongly inhibits the cell cycle in the sub-G1 phase. The compound also triggers cell death with a significant decrease in mitochondrial membrane potential, an increase in the level of oxidative stress (over two times higher in comparison to the control), and activation of caspase-8, -9, -3/7, as well as Bid. The results of genes expression indicate that the Tumor Necrosis Factor (TNF) Receptor Superfamily Members (TNF, TNFRSF10, TNFRSF10B, TNFRSF1B, and TNFRSF25), Fas Associated via Death Domain (FADD), and Death Effector Domain Containing 2 (DEDD2) were significantly upregulated and their relative expression was at least two times higher than in the control. Our work shows that yamogenin induces apoptosis in ovarian cancer cells, and both the extrinsic and mitochondrial-intrinsic pathways are involved in this process.

Ethanolic extracts of Pakistani euphorbiaceous plants induce apoptosis in breast cancer cells through induction of DNA damage and caspase-dependent pathway

BackgroundNumerous plants of Euphorbiaceae, thespurgefamily are traditionally used for the treatment of different diseases and recent studies also reported anti-oxidant, anti-inflammatory, and anti-tumor activities of these plants. However, the medicinal potential of several indigenous euphorbiaceous plants of Pakistan is not described yet. Therefore, we intended to evaluate the in vitro anti-breast cancer potential of 10 euphorbiaceous plants of Pakistan. MethodsCytotoxic screening of ethanolic extracts of selected plants was performed by MTT assay. The qualitative phytochemical analysis was performed to find the major groups of chemicals responsible for cytotoxic activity. To determine the genotoxic effect of plant extracts, microscopic analysis was carried out. Flow cytometry and fluorescent microscopic analysis were done to detect apoptosis. To find out the expression analysis of cell cycle and cell death regulatory genes, quantitative real-time polymerase reaction (qRT-PCR) was performed. ResultsAmong the 10 tested plants, ethanolic extracts of Croton tiglium (CTL) and Euphorbia royleana (ERA) were found to possess the highest anti-proliferative activity against breast cancer cells (MDA-MB-231, MCF-7), with IC50 values 100 and 80 µg/mL respectively. The phytochemical analysis confirmed the presence of phenols, flavonoids, and steroids in both plant extracts, whereas, glycosides and saponins were found only in CTL and ERA, respectively. The cellular aberrations and nuclear morphologies with a distinct DNA laddering pattern substantiated the genotoxic effects. Furthermore, our data showed that CTL and ERA induce cell cycle arrest at the G1/S phase by down-regulating the CDK4 and Cyclin D1 expression followed by caspase-dependent induction of apoptosis in both MCF-7 and MDA-MB-231 cells. However, based on the activation of initiator and executioner caspases, two distinct types of apoptotic pathways are proposed for these plants. The CTL prompted extrinsic while ERA triggered the intrinsic pathways of apoptosis. ConclusionOur data demonstrate the strong anti-proliferative and caspase-dependent apoptotic potential of CTL and ERA against breast cancer cells. Further studies are suggested to find clinical implications of these plants in breast cancer therapeutic.

An atlas of inter- and intra-tumor heterogeneity of apoptosis competency in colorectal cancer tissue at single-cell resolution

Cancer cells’ ability to inhibit apoptosis is key to malignant transformation and limits response to therapy. Here, we performed multiplexed immunofluorescence analysis on tissue microarrays with 373 cores from 168 patients, segmentation of 2.4 million individual cells, and quantification of 18 cell lineage and apoptosis proteins. We identified an enrichment for BCL2 in immune, and BAK, SMAC, and XIAP in cancer cells. Ordinary differential equation-based modeling of apoptosis sensitivity at single-cell resolution was conducted and an atlas of inter- and intra-tumor heterogeneity in apoptosis susceptibility generated. Systems modeling at single-cell resolution identified an enhanced sensitivity of cancer cells to mitochondrial permeabilization and executioner caspase activation compared to immune and stromal cells, but showed significant inter- and intra-tumor heterogeneity.

The proteolytic landscape of cells exposed to non-lethal stresses is shaped by executioner caspases

Cells are in constant adaptation to environmental changes to insure their proper functioning. When exposed to stresses, cells activate specific pathways to elicit adaptive modifications. Those changes can be mediated by selective modulation of gene and protein expression as well as by post-translational modifications, such as phosphorylation and proteolytic processing. Protein cleavage, as a controlled and limited post-translational modification, is involved in diverse physiological processes such as the maintenance of protein homeostasis, activation of repair pathways, apoptosis and the regulation of proliferation. Here we assessed by quantitative proteomics the proteolytic landscape in two cell lines subjected to low cisplatin concentrations used as a mild non-lethal stress paradigm. This landscape was compared to the one obtained in the same cells stimulated with cisplatin concentrations inducing apoptosis. These analyses were performed in wild-type cells and in cells lacking the two main executioner caspases: caspase-3 and caspase-7. Ninety-two proteins were found to be cleaved at one or a few sites (discrete cleavage) in low stress conditions compared to four hundred and fifty-three in apoptotic cells. Many of the cleaved proteins in stressed cells were also found to be cleaved in apoptotic conditions. As expected, ~90% of the cleavage events were dependent on caspase-3/caspase-7 in apoptotic cells. Strikingly, upon exposure to non-lethal stresses, no discrete cleavage was detected in cells lacking caspase-3 and caspase-7. This indicates that the proteolytic landscape in stressed viable cells fully depends on the activity of executioner caspases. These results suggest that the so-called executioner caspases fulfill important stress adaptive responses distinct from their role in apoptosis. Mass spectrometry data are available via ProteomeXchange with identifier PXD023488.

The actin nucleation factors JMY and WHAMM enable a rapid Arp2/3 complex-mediated intrinsic pathway of apoptosis.

The actin cytoskeleton is a well-known player in most vital cellular processes, but comparably little is understood about how the actin assembly machinery impacts programmed cell death pathways. In the current study, we explored roles for the human Wiskott-Aldrich Syndrome Protein (WASP) family of actin nucleation factors in DNA damage-induced apoptosis. Inactivation of each WASP-family gene revealed that two of them, JMY and WHAMM, are necessary for rapid apoptotic responses. JMY and WHAMM participate in a p53-dependent cell death pathway by enhancing mitochondrial permeabilization, initiator caspase cleavage, and executioner caspase activation. JMY-mediated apoptosis requires actin nucleation via the Arp2/3 complex, and actin filaments are assembled in cytoplasmic territories containing clusters of cytochrome c and active caspase-3. The loss of JMY additionally results in significant changes in gene expression, including upregulation of the WHAMM-interacting G-protein RhoD. Depletion or deletion of RHOD increases cell death, suggesting that RhoD normally contributes to cell survival. These results give rise to a model in which JMY and WHAMM promote intrinsic cell death responses that can be opposed by RhoD.

Apoptosis-inducing effects of Terminalia phanerophlebia leaf extracts on human renal cells

The study investigated the toxicogenic effects and molecular mechanisms of Terminalia phanerophlebia on human renal cells. The methylthiazol tetrazolium (MTT) assay was used to assess the cytotoxicity of a crude leaf extract of Terminalia phanerophlebia in Hek293 cells. Luminometry was used to determine caspase activity, phosphatidylserine externalization and necrosis. The comet assay evaluated DNA fragmentation and the lactate dehydrogenase (LDH) assay was used to assess membrane integrity. Western blotting measured the expression of bcl-2-like protein 4 (Bax), inhibitors of apoptosis proteins (IAP), cleaved poly (ADP-ribose) polymerase (cPARP), p53 and nuclear factor kappa B (NF-κB) in treated Hek293 cells. A reduction in cell viability was observed with a half maximal inhibitory concentration (IC50) of 1.36 mg/ml. At this concentration, Terminalia phanerophlebia decreased initiator and executioner caspase activity. Phosphatidylserine validated apoptosis in IC25-treated cells, while necrosis was observed in all treated cells. Comet formation indicated that there was DNA fragmentation. High levels of p53 resulted in upregulation of Bax in IC25-treated cells, while the opposite was noted in IC50-treated cells. Upregulation of NF-κB is associated with decreased caspase-3 and cPARP and further supported by an increase in IAP indicating that Hek293 cell death was mainly through apoptosis and necrosis. The findings suggest that while Terminalia phanerophlebia is a good extract for tuberculosis therapeutic intervention, it may exert toxic effects on kidney cells via the apoptotic pathway.

Akt1 and dCIZ1 promote cell survival from apoptotic caspase activation during regeneration and oncogenic overgrowth

Apoptosis is an ancient and evolutionarily conserved cell suicide program. During apoptosis, executioner caspase enzyme activation has been considered a point of no return. However, emerging evidence suggests that some cells can survive caspase activation following exposure to apoptosis-inducing stresses, raising questions as to the physiological significance and underlying molecular mechanisms of this unexpected phenomenon. Here, we show that, following severe tissue injury, Drosophila wing disc cells that survive executioner caspase activation contribute to tissue regeneration. Through RNAi screening, we identify akt1 and a previously uncharacterized Drosophila gene CG8108, which is homologous to the human gene CIZ1, as essential for survival from the executioner caspase activation. We also show that cells expressing activated oncogenes experience apoptotic caspase activation, and that Akt1 and dCIZ1 are required for their survival and overgrowth. Thus, survival following executioner caspase activation is a normal tissue repair mechanism usurped to promote oncogene-driven overgrowth.

Ex vivo Induction of Apoptotic Mesenchymal Stem Cell by High Hydrostatic Pressure

Among promising solutions for tissue repair and wound healing, mesenchymal stem (or stromal) cells (MSCs) have been a focus of attention and have become the most clinically studied experimental cell therapy. Recent studies reported the importance of apoptosis in MSC-mediated immunomodulation, in which apoptotic MSCs (apoMSCs) were shown to be superior to living MSCs. Nowadays, high hydrostatic pressure (HHP), a physical technique that uses only fluid pressure, has been developed and applied in various bioscience fields, including biotechnology, biomaterials, and regenerative medicine, as its safe and simply operation. In the current study, we investigated the impact of HHP treatment on human bone marrow-MSC survival and proliferation. Based on the detection of executioner caspase activation, phosphatidylserine exposure, DNA fragmentation (TUNEL) and irrefutable ultrastructural morphological changes on transmission electron microscopy (TEM), our data revealed that HHP treatment induced complete apoptosis in MSCs. Notably, this technique might provide manipulated products for use in cell-based therapies as manufacturing capability expands. We hope that our findings will contribute to the improvement of MSCs or EVs in translational research development.Graphical

Reversion of Pneumolysin-Induced Executioner Caspase Activation Redirects Cells to Survival.

Apoptosis is an indispensable mechanism for eliminating infected cells and activation of executioner caspases is considered to be a point of no return. Streptococcus pneumoniae, the most common bacterial pathogen causing community-acquired pneumonia, induces apoptosis via its pore-forming toxin pneumolysin, leading to rapid influxes of mitochondrial calcium [Ca2+]m as well as fragmentation, and loss of motility and membrane potential, which is accompanied by caspase-3/7 activation. Using machine-learning and quantitative live-cell microscopy, we identified a significant number of alveolar epithelial cells surviving such executioner caspase activation after pneumolysin attack. Precise single-cell analysis revealed the [Ca2+]m amplitude and efflux rate as decisive parameters for survival and death, which was verified by pharmacological inhibition of [Ca2+]m efflux shifting the surviving cells towards the dying fraction. Taken together, we identified the regulation of [Ca2+]m as critical for controlling the cellular fate under pneumolysin attack, which might be useful for therapeutic intervention during pneumococcal infection.

Hydroxypropyl-β-cyclodextrin causes massive damage to the developing auditory and vestibular system

2-hydroxypropyl-β-cyclodextrin (HPβCD), a cholesterol chelator used to treat Niemann-Pick C1 (NPC1) lysosomal storage disease, causes hearing loss in mammals by preferentially destroying outer hair cells. Because cholesterol plays an important role in early neural development, we hypothesized that HPβCD would cause more extensive damage to postnatal cochlear and vestibular structures in than adult rats. This hypothesis was tested by administering HPβCD to adult rats and postnatal day 3 (P3) cochlear and vestibular organ cultures. Adult rats treated with HPβCD developed hearing impairment and outer hair cell loss 3-day post-treatment; damage increased with dose from the high frequency base toward the low-frequency apex. The HPβCD-induced histopathologies were more severe and widespread in cochlear and vestibular cultures at P3 than in adults. HPβCD destroyed both outer and inner hair cells, auditory nerve fibers and spiral ganglion neurons as well as type I and type II vestibular hair cells and vestibular ganglion neurons. The early stage of HPβCD damage involved disruption of hair cell mechanotransduction and destruction of stereocilia. HPβCD-mediated apoptosis in P3 cultures was most-strongly initiated by activation of the extrinsic caspase-8 cell death pathway in cochlear and vestibular hair cells and neurons followed by activation of executioner caspase-3. Thus, HPβCD is toxic to all types of postnatal cochlear and vestibular hair cells and neurons in vitro whereas in vivo it only appears to destroy outer hair cells in adult cochleae. The more severe HPβCD-induced damage in postnatal cultures could be due to greater drug bioavailability in vitro and/or greater vulnerability of the developing inner ear.

In vitro analysis reveals necroptotic signaling does not provoke DNA damage or HPRT mutations

Most anticancer drugs provoke apoptotic signaling by damaging DNA or other means. Genotoxic therapies may enhance a patient’s risk of developing “therapy-related cancers” due to the accumulation of oncogenic mutations that may occur in noncancerous cells. Mutations can also form upon apoptotic signaling due to sublethal caspase activity, implying that apoptosis activating drugs may also be oncogenic. Necroptosis is a different way of killing cancer cells: this version of caspase-independent cell death is characterized by receptor-interacting protein kinase-3 (RIPK3) and mixed lineage kinase-like domain protein (MLKL) activation, leading to cell membrane rupture and controlled cell lysis. The mutagenic potential of sublethal necroptotic signaling has not yet been directly investigated. Smac mimetics drugs, which activate apoptotic or necroptotic cell death, do not induce mutations but the mechanistic basis for this lack of mutagenic activity has not been determined. In this study, we compared the mutagenic potential of these two cell death pathways by engineering cells to activate either apoptotic or necroptotic signaling by exposing them to Smac mimetics with or without TNFα, and/or enforcing or preventing expression of apoptotic or necroptotic regulators. We discovered that sublethal concentrations of Smac mimetics in contexts that activated apoptotic signaling provoked DNA damage and mutations in surviving cells. Mutagenesis was dependent on executioner caspase activation of the nuclease CAD. In contrast, RIPK3- and MLKL-dependent necroptotic signaling following Smac mimetic treatment was not mutagenic. Likewise, DNA damage was not provoked in cells expressing a lethal constitutively active MLKL mutant. These data reveal that cells surviving sublethal necroptotic signaling do not sustain genomic damage and provide hope for a reduced risk of therapy-related malignancies in patients treated with necroptosis-inducing drugs.