Anticancer Activity of Continentalic Acid in B-Cell Lymphoma

Aralia continentalis has been used in Korea as a folk remedy for arthralgia, rheumatism, and inflammation. However, its anti-lymphoma effect remains uncharacterized. Here, we demonstrate that A. continentalis extract and its three diterpenes efficiently kill B-lymphoma cells. Our in vitro and in vivo results suggest that the cytotoxic activities of continentalic acid, a major diterpene from A. continentalis extract, are specific towards cancer cells while leaving normal murine cells and tissues unharmed. Mechanistically, continentalic acid represses the expression of pro-survival Bcl-2 family members, such as Mcl-1 and Bcl-xL. It dissociates the mitochondrial membrane potential, leading to the stimulation of effector caspase 3/7 activities and, ultimately, cell death. Intriguingly, this agent therapeutically synergizes with roflumilast, a pan-PDE4 inhibitor that has been successfully repurposed for the treatment of aggressive B-cell malignancies in recent clinical tests. Our findings unveiled that A. continentalis extract and three of the plant’s diterpenes exhibit anti-cancer activities. We also demonstrate the synergistic inhibitory effect of continentalic acid on the survival of B-lymphoma cells when combined with roflumilast. Taken in conjunction, continentalic acid may hold significant potential for the treatment of B-cell lymphoma.

Global Reprogramming of Apoptosis-Related Genes during Brain Development

To enable long-term survival, mammalian adult neurons exhibit unique apoptosis competence. Questions remain as to whether and how neurons globally reprogram the expression of apoptotic genes during development. We systematically examined the in vivo expression of 1923 apoptosis-related genes and associated histone modifications at eight developmental ages of mouse brains. Most apoptotic genes displayed consistent temporal patterns across the forebrain, midbrain, and hindbrain, suggesting ubiquitous robust developmental reprogramming. Although both anti- and pro-apoptotic genes can be up- or downregulated, half the regulatory events in the classical apoptosis pathway are downregulation of pro-apoptotic genes. Reduced expression in initiator caspases, apoptosome, and pro-apoptotic Bcl-2 family members restrains effector caspase activation and attenuates neuronal apoptosis. The developmental downregulation of apoptotic genes is attributed to decreasing histone-3-lysine-4-trimethylation (H3K4me3) signals at promoters, where histone-3-lysine-27-trimethylation (H3K27me3) rarely changes. By contrast, repressive H3K27me3 marks are lost in the upregulated gene groups, for which developmental H3K4me3 changes are not predictive. Hence, developing brains remove epigenetic H3K4me3 and H3K27me3 marks on different apoptotic gene groups, contributing to their downregulation and upregulation, respectively. As such, neurons drastically alter global apoptotic gene expression during development to transform apoptosis controls. Research into neuronal cell death should consider maturation stages as a biological variable.

Apoptosis Quantification in Tissue: Development of a Semi-Automatic Protocol and Assessment of Critical Steps of Image Processing

Apoptosis is associated with numerous phenotypical characteristics, and is thus studied with many tools. In this study, we compared two broadly used apoptotic assays: TUNEL and staining with an antibody targeting the activated form of an effector caspase. To compare them, we developed a protocol based on commonly used tools such as image filtering, z-projection, and thresholding. Even though it is commonly used in image-processing protocols, thresholding remains a recurring problem. Here, we analyzed the impact of processing parameters and readout choice on the accuracy of apoptotic signal quantification. Our results show that TUNEL is quite robust, even if image processing parameters may not always allow to detect subtle differences of the apoptotic rate. On the contrary, images from anti-cleaved caspase staining are more sensitive to handle and necessitate being processed more carefully. We then developed an open-source Fiji macro automatizing most steps of the image processing and quantification protocol. It is noteworthy that the field of application of this macro is wider than apoptosis and it can be used to treat and quantify other kind of images.

Deletion of TLR4 reduces apoptosis and improves histology in a murine kidney transplant model

Acute kidney injury (AKI) after transplantation of human deceased donor kidneys is associated with upregulation of tubular toll like receptor 4 (TLR4), but whether TLR4 is required for AKI is unknown. We hypothesized that TLR4 knockout mice (TLR4KO) subjected to cold ischemia followed by kidney transplant (CI + Txp) would be protected from AKI. C57Bl/6J wild type or TLR4KO kidneys were subjected to CI + Txp into wild type recipients. Tubular cell apoptosis, tubular injury and cast formation were significantly improved in recipients of TLR4KO kidneys. TLR4KO kidneys also demonstrated significantly decreased expression of the effector caspase 8. Brush border injury scores and serum creatinine were not different in recipients of TLR4KO versus wild type kidneys. Phosphorylated RIP3 and MLKL through which TLR4 signals programmed necrosis were expressed in both recipient groups. In addition, TNF-α and TNFR1 expression were significantly increased in recipient serum and TLR4KO kidneys respectively after CI + Txp, suggesting continued activation of programmed necrosis despite TLR4 deletion. Our results suggest that TLR4 deletion decreases apoptosis via inhibition of the death receptor pathway and decreases tubular injury and cast formation.

Apoptosis Quantification in Tissue: Development of a Semi- automatic Protocol and Assessment of Critical Steps of Image Processing

Apoptosis is associated with numerous phenotypical characteristics, and is thus studied with many tools. In this study, we compared two broadly used apoptotic assays: TUNEL and staining with an antibody targeting the activated form of an effector caspase. To compare them, we developed a protocol based on commonly used tools such as filters, zprojection and thresholding. Even though it is commonly used in imageprocessing protocols, thresholding remains a recurring problem. Here we analyzed the impact of processing parameters and readout choice on the accuracy of apoptotic signal quantification. Our results show that TUNEL is quite robust, even if image processing parameters can allow or not to detect subtle differences of the apoptotic rate. On the contrary, images from anticleaved caspase staining are more sensitive to handle and proved to necessitate to be processed more carefully. We then developed an open source Fiji macro automatizing most steps of the image processing and quantification protocol. It is noteworthy that the field of application of this macro is wider than apoptosis as it can perfectly be used to treat and quantify other kind of images.

Drice restrains Diap2-mediated inflammatory signalling and intestinal inflammation

The Drosophila IAP protein, Diap2, is a key mediator of NF-κB signalling and innate immune responses. Diap2 is required for both local immune activation, taking place in the epithelial cells of the gut and trachea, and for mounting systemic immune responses in the cells of the fat body. We have found that transgenic expression of Diap2 leads to a spontaneous induction of NF-κB target genes, inducing chronic inflammation in the Drosophila midgut, but not in the fat body. Drice is a Drosophila effector caspase known to interact and form a stable complex with Diap2. We have found that this complex formation induces its subsequent degradation, thereby regulating the amount of Diap2 driving NF-κB signalling in the intestine. Concordantly, loss of Drice activity leads to accumulation of Diap2 and to chronic intestinal inflammation. Interestingly, Drice does not interfere with pathogen-induced signalling, suggesting that it protects from immune responses induced by resident microbes. Accordingly, no inflammation was detected in transgenic Diap2 flies and Drice-mutant flies reared in axenic conditions. Hence, we show that Drice, by restraining Diap2, halts unwanted inflammatory signalling in the intestine.

GO-Y078, a Curcumin Analog, Induces Both Apoptotic Pathways in Human Osteosarcoma Cells via Activation of JNK and p38 Signaling

Osteosarcoma is the most common primary bone malignancy in teenagers and continues to confer a generally poor prognosis due to its highly metastatic potential. Poor solubility in water and instability of curcumin limits its bioavailability for use in the adjuvant situation to improve the prognosis and the long-term survival of patients with osteosarcoma. To further obtain information regarding the apoptosis induced by a new curcumin analog, GO-Y078, in human osteosarcoma cells, flow cytometric analysis, annexin V-FITC/PI apoptosis staining assay, human apoptosis array, and Western blotting were employed. GO-Y078 dose-dependently decreased viabilities of human osteosarcoma U2OS, MG-63, 143B, and Saos-2 cells and induced sub-G1 fraction arrest and apoptosis in U2OS and 143B cells. In addition to the effector caspase 3 and poly adenosine diphosphate-ribose polymerase, GO-Y078 significantly activated both initiators of extrinsic caspase 8 and intrinsic caspase 9, whereas cellular inhibitors of apoptosis 1 (cIAP-1) and X-chromosome-linked IAP (XIAP) in U2OS and 143B cells were significantly repressed. Moreover, GO-Y078 increased phosphorylation of extracellular signal-regulated protein kinases (ERK)1/2, c-Jun N-terminal kinases (JNK)1/2, and p38 in U2OS and 143B cells. Using inhibitors of JNK (JNK-in-8) and p38 (SB203580), GO-Y078′s increases in cleaved caspases 8, 9, and 3 could be expectedly suppressed, but they could not be affected by co-treatment with the ERK inhibitor (U0126). Altogether, GO-Y078 simultaneously induces both apoptotic pathways and cell arrest in U2OS and 143B cells through activating JNK and p38 signaling and repressing IAPs. These findings contribute to a better understanding of the mechanisms responsible for GO-Y078′s apoptotic effects on human osteosarcoma cells.

An Integrative Apoptotic Reaction Model for extrinsic and intrinsic stimuli

Apoptosis, a form of programmed cell death central to all multicellular organisms, plays a key role during organism development and is often misregulated in cancer. Devising a single model applicable to distinct stimuli and conditions has been limited by lack of robust observables. Indeed, previous numerical models have been tailored to fit experimental datasets in restricted scenarios, failing to predict response to different stimuli. We quantified the activity of three caspases simultaneously upon intrinsic or extrinsic stimulation to assemble a comprehensive dataset. We measured and modeled the time between maximum activity of intrinsic, extrinsic and effector caspases, a robust observable of network dynamics, to create the first integrated Apoptotic Reaction Model (ARM). Observing how effector caspases reach maximum activity first irrespective of stimuli used, led us to identify and incorporate a missing feedback into a successful model for extrinsic stimulation. By simulating different recently performed experiments, we corroborated that ARM adequately describes them. This integrated model provides further insight into the indispensable feedback from effector caspase to initiator caspases.

Immunohistochemical Expression Patterns of Tight Junction Proteins, Pro-Apoptotic and Anti-Apoptotic Factors on Progression of Intestinal Mucositis of Onco-Hematological Patients under Epirubicin-Based Chemotherapy

Chemotherapy and radiation are often accompanied by complications such as intestinal mucositis. The aim of this study was to assess by immunohistochemical assay the consequences of epirubicin-based therapy applied to onco-hematological patients, on the mucosal cells that undergo apoptosis and on the tight junction proteins, immediately before and after a short time of chemotherapy administration. We assessed the protein expression and distribution of the pro-apoptotic Bax, anti-apoptotic Bcl-2 and effector Caspase-3 as key proteins in apoptosis pathways and the changes in immunopositivity of Claudin-1 and ZO-1 tight junction proteins. Results show that the Bcl-2 family is involved in intestinal damage via Caspase-3 dependent apoptosis of epithelial cells. Additionally, the intestinal mucositis activates other injurious pathways through a dramatic drop in Claudin-1 and ZO-1 expressions, contributing for a while to a structural and functional integrity disruption of the intestinal epithelium.

Cholecalciferol Mediates Apoptosis in Siha Cervical Cancer Line via Autocrine Mechanisms

Cervical cancer disproportionately affects low-resource countries and is a significant health burden in South Africa. Pre-clinical studies have demonstrated numerous anti-cancer actions of vitamin D metabolites. Here, the anti-cancer action of the vitamin D precursor, cholecalciferol, was investigated in a high-grade cervical cancer cell line, SiHa. SiHa cell cultures were treated with a range of cholecalciferol doses (26 nM, 104 nM, 260 nM and 2600 nM) for 72 hours. Cell count and viability were assessed by crystal violet and trypan blue assays, respectively. Apoptotic cell death was investigated by flow cytometry, which measured mitochondrial membrane potential (∆Ψm), phosphatidylserine (PS) externalisation, effector caspase activation and the expression of DNA damage markers. Additionally, brightfield microscopy and transmission electron microscopy (TEM) were respectively used to characterise morphological and ultrastructural features of apoptosis. Expression of the vitamin D metabolising system (VDMS) – consisting of cholecalciferol activating (CYP2R1 and CYP27A1), calcidiol activating (CYP27B1) and calcidiol inactivating (CYP24A1) enzymes, and the vitamin D receptor (VDR) – was assessed by qPCR and Western blots. Data were analysed using a one-way ANOVA and Bonferroni post-hoc tests and p < 0.05 was considered statistically significant. Significant decreases in cell count (p = 0.011) and cell viability (p < 0.0001) were identified in SiHa cells treated with 2600 nM cholecalciferol. Furthermore, biochemical markers at 2600 nM treatment were significant for apoptosis, and included decreased ∆Ψm (p = 0.0145); increased PS externalisation (p = 0.0439); terminal caspase activation (p = 0.0025); and nuclear damage (p = 0.004). Moreover, biochemical apoptosis was corroborated by classical apoptotic features observed by brightfield microscopy and TEM. Additionally, a significant increase in CYP2R1 gene (p < 0.0001) and protein (p = 0.021) expression, and a converse significant decrease in CYP27B1 gene (p = 0.003) and protein expression (p = 0.031) were observed at 2600 nM cholecalciferol treatment. Furthermore, significant increases in VDR gene (p = 0.033) and protein (p = 0.04) expression, and CYP24A1 gene (p < 0.0001) and protein (p = 0.0274) expression were observed at 2600 nM cholecalciferol. In summary, high-dose cholecalciferol treatment of SiHa cervical cancer cells inhibits cell growth, induces apoptosis, and furthermore, upregulates CYP2R1 and VDR expression. Taken together, these findings suggest that autocrine activation of cholecalciferol to calcidiol may mediate VDR signalling of cell growth inhibition, and apoptosis in SiHa experimental cultures.