Inactivation of BAD by IKK inhibits TNFα-induced apoptosis independently of NF-κB activation.

The IκB kinase complex (IKK) is a key regulator of immune responses, inflammation, cell survival, and tumorigenesis. The prosurvival function of IKK centers on activation of the transcription factor NF-κB, whose target gene products inhibit caspases and prevent prolonged JNK activation. Here, we report that inactivation of the BH3-only protein BAD by IKK independently of NF-κB activation suppresses TNFα-induced apoptosis. TNFα-treated Ikkß(-/-) mouse embryonic fibroblasts (MEFs) undergo apoptosis significantly faster than MEFs deficient in both RelA and cRel due to lack of inhibition of BAD by IKK. IKK phosphorylates BAD at serine-26 (Ser26) and primes it for inactivation. Elimination of Ser26 phosphorylation promotes BAD proapoptotic activity, thereby accelerating TNFα-induced apoptosis in cultured cells and increasing mortality in animals. Our results reveal that IKK inhibits TNFα-induced apoptosis through two distinct but cooperative mechanisms: activation of the survival factor NF-κB and inactivation of the proapoptotic BH3-only BAD protein.

The BAD-BAX-Caspase-3 Cascade Modulates Synaptic Vesicle Pools via Autophagy.

The BAD-BAX-caspase-3 cascade is a canonical apoptosis pathway. Macroautophagy ("autophagy" hereinafter) is a process by which organelles and aggregated proteins are delivered to lysosomes for degradation. Here, we report a new function of the BAD-BAX-caspase-3 cascade and autophagy in the control of synaptic vesicle pools. We found that, in hippocampal neurons of male mice, the BAD-BAX-caspase-3 pathway regulates autophagy, which in turn limits the size of synaptic vesicle pools and influences the kinetics of activity-induced depletion and recovery of synaptic vesicle pools. Moreover, the caspase-autophagy pathway is engaged by fear conditioning to facilitate associative fear learning and memory. This work identifies a new mechanism for controlling synaptic vesicle pools, and a novel, nonapoptotic, presynaptic function of the BAD-BAX-caspase-3 cascade.SIGNIFICANCE STATEMENT Despite the importance of synaptic vesicles for neurons, little is known about how the size of synaptic vesicle pools is maintained under basal conditions and regulated by neural activity. This study identifies a new mechanism for the control of synaptic vesicle pools, and a new, nonapoptotic function of the BAD-BAX-caspase-3 pathway in presynaptic terminals. Additionally, it indicates that autophagy is not only a homeostatic mechanism to maintain the integrity of cells and tissues, but also a process engaged by neural activity to regulate synaptic vesicle pools for optimal synaptic responses, learning, and memory.

BH3-only sensors Bad, Noxa and Puma are Key Regulators of Tacaribe virus-induced Apoptosis.

Pathogenicity often differs dramatically among even closely related arenavirus species. For instance, Junín virus (JUNV), the causative agent of Argentine hemorrhagic fever (AHF), is closely related to Tacaribe virus (TCRV), which is normally avirulent in humans. While little is known about how host cell pathways are regulated in response to arenavirus infection, or how this contributes to virulence, these two viruses have been found to differ markedly in their ability to induce apoptosis. However, details of the mechanism(s) governing the apoptotic response to arenavirus infections are unknown. Here we confirm that TCRV-induced apoptosis is mitochondria-regulated, with associated canonical hallmarks of the intrinsic apoptotic pathway, and go on to identify the pro- and anti-apoptotic Bcl-2 factors responsible for regulating this process. In particular, levels of the pro-apoptotic BH3-only proteins Noxa and Puma, as well as their canonical transcription factor p53, were strongly increased. Interestingly, TCRV infection also led to the accumulation of the inactive phosphorylated form of another pro-apoptotic BH3-only protein, Bad (i.e. as phospho-Bad). Knockout of Noxa or Puma suppressed apoptosis in response to TCRV infection, whereas silencing of Bad increased apoptosis, confirming that these factors are key regulators of apoptosis induction in response to TCRV infection. Further, we found that while the highly pathogenic JUNV does not induce caspase activation, it still activated upstream pro-apoptotic factors, consistent with current models suggesting that JUNV evades apoptosis by interfering with caspase activation through a nucleoprotein-mediated decoy function. This new mechanistic insight into the role that individual BH3-only proteins and their regulation play in controlling apoptotic fate in arenavirus-infected cells provides an important experimental framework for future studies aimed at dissecting differences in the apoptotic responses between arenaviruses, their connection to other cell signaling events and ultimately the relationship of these processes to pathogenesis.

The Effect of NeuroAid (MLC901) on Cholestasis-Induced Spatial Memory Impairment with Respect to the Expression of BAX, BCL-2, BAD, PGC-1α and TFAM Genes in the Hippocampus of Male Wistar Rats.

Cholestasis is a bile flow reduction that is induced following Bile Duct Ligation (BDL). Cholestasis impairs memory and induces apoptosis. Apoptosis consists of two pathways: intrinsic and extrinsic. The intrinsic pathway is modulated by BCL-2 (B cell lymphoma-2) family proteins. BCL-2 (a pro-survival BCL-2 protein) has anti-apoptotic effect, while BAD (BCL-2-associated death) and BAX (BCL-2-associated X), the other members of BCL-2 family have pro-apoptotic effect. Furthermore, TFAM (mitochondrial transcriptional factor A) is involved in transcription and maintenance of mitochondrial DNA and PGC-1α (peroxisome proliferator-activated receptor γ coactivator-1α) is a master regulator of mitochondrial biogenesis. On the other hand, NeuroAid is a Traditional Chinese Medicine with neuroprotective and anti-apoptosis effects. In this study, we evaluated the effect of cholestasis on spatial memory and expression of BCL-2, BAD, BAX, TFAM, and PGC-1α in the hippocampus of rats. Additionally, we assessed the effect of NeuroAid on cholestasis-induced cognitive and genetic alterations. Cholestasis was induced by BDL surgery and NeuroAid was injected intraperitoneal at the dose of 0.4 mg/kg. Furthermore, spatial memory was evaluated using Morris Water Maze (MWM) apparatus. The results showed cholestasis impaired spatial memory, increased the expression of BAD and BAX, decreased the expression of TFAM and PGC-1α, and did not alter the expression of BCL-2. Also, NeuroAid decreased the expression of BAD and BAX and increased the expression of TFAM, PGC-1α, and BCL-2. In conclusion, cholestasis impaired spatial memory and increased the expression of pro-apoptotic genes. Also, cholestasis decreased the expression of TFAM and PGC-1α. Interestingly, NeuroAid restored the effects of cholestasis.

Chlorothalonil induces the intestinal epithelial barrier dysfunction in Caco-2 cell-based in vitro monolayer model by activating MAPK pathway.

The widespread use of chlorothalonil (CTL) has caused environmental residues and food contamination. Although the intestinal epithelial barrier (IEB) is directly involved in the metabolism and transportation of various exogenous compounds, there are few studies on the toxic effects of these compounds on the structure and function of IEB. The disassembly of tight junction (TJ) is a major cause of intestinal barrier dysfunction under exogenous compounds intake, but the precise mechanisms are not well understood. Here, we used Caco-2 cell monolayers as an in vitro model of human IEB to evaluate the toxicity of CTL exposure on the structure and function of IEB. Results showed that CTL exposure increased the paracellular permeability of the monolayers and downregulated mRNA levels of the TJ genes (ZO-1, OCLN, and CLDN1), polarity marker gene (SI), and anti-apoptosis gene (BCL-2) but upregulated the mRNA levels of apoptosis-related genes, including BAD, BAX, CASP3, and CASP8. Western blot analysis and immunofluorescence assay results showed the decreased levels and disrupted distribution of TJ protein network, including ZO-1 and CLDN1 in CTL-exposed IEB. In addition, the accumulation of intracellular reactive oxygen species, decreased mitochondrial membrane potential, and increased active CASP3 expression were observed in treated IEB. The result of TUNEL assay further confirmed the occurrence of cell apoptosis after CTL exposure. In addition, the phosphorylation of mitogen-activated protein kinases, including ERK, JNK and p38, was increased in CTL-exposed IEB. In summary, our results demonstrated that CTL exposure induced IEB dysfunction in Caco-2 cell monolayers by activating the mitogen-activated protein kinase pathway.

Proapoptotic Bad Involved in Brain Development, When Severely Defected, Induces Dramatic Malformation in Zebrafish.

The BH3-only molecule Bad regulates cell death via its differential protein phosphorylation, but very few studies address its effect on early embryonic development in vertebrate systems. In this work, we examined the novel role of zebrafish Bad in the initial programmed cell death (PCD) for brain morphogenesis through reducing environmental stress and cell death signaling. Bad was considered to be a material factor that because of the knockdown of Bad by morpholino oligonucleotides, PCD was increased and the reactive oxygen species (ROS) level was enhanced, which correlated to trigger a p53/caspase-8 involving cell death signaling. This Bad knockdown-mediated environmental stress and enhanced cell dying can delay normal cell migration in the formation of the three germ layers, especially the ectoderm, for further brain development. Furthermore, Bad defects involved in three-germ-layers development at 8 hpf were identified by in situ hybridization approach on cyp26, rtla, and Sox17 pattern expression markers. Finally, the Bad knockdown-induced severely defected brain was examined by tissue section from 24 to 48 h postfertilization (hpf), which correlated to induce dramatic malformation in the hindbrain. Our data suggest that the BH3-only molecule Bad regulates brain development via controlling programmed cell death on overcoming environmental stress for reducing secondary cell death signaling, which suggests that correlates to brain developmental and neurological disorders in this model system.

Assessment of Betulinic Acid Cytotoxicity and Mitochondrial Metabolism Impairment in a Human Melanoma Cell Line.

Melanoma represents one of the most aggressive and drug resistant skin cancers with poor prognosis in its advanced stages. Despite the increasing number of targeted therapies, novel approaches are needed to counteract both therapeutic resistance and the side effects of classic therapy. Betulinic acid (BA) is a bioactive phytocompound that has been reported to induce apoptosis in several types of cancers including melanomas; however, its effects on mitochondrial bioenergetics are less investigated. The present study performed in A375 human melanoma cells was aimed to characterize the effects of BA on mitochondrial bioenergetics and cellular behavior. BA demonstrated a dose-dependent inhibitory effect in both mitochondrial respiration and glycolysis in A375 melanoma cells and at sub-toxic concentrations (10 µM) induced mitochondrial dysfunction by eliciting a decrease in the mitochondrial membrane potential and changes in mitochondria morphology and localization. In addition, BA triggered a dose-dependent cytotoxic effect characterized by apoptotic features: morphological alterations (nuclear fragmentation, apoptotic bodies) and the upregulation of pro-apoptotic markers mRNA expression (Bax, Bad and Bak). BA represents a viable therapeutic option via a complex modulatory effect on mitochondrial metabolism that might be useful in advanced melanoma or as reliable strategy to counteract resistance to standard therapy.

Methylation of Promoters of Apoptosis-Related Genes in Blood Lymphocytes of Workers Exposed to Occupational External Irradiation.

We studied the effect of technogenic radiation on the degree of promoter methylation in genes involved in apoptosis in blood lymphocytes of workers exposed to long-term γ-radiation during their professional activities. Blood samples for the analysis were obtained from 11 conventionally healthy men aged from 54 to 71 years (mean 66 years), workers of the Siberian Group of Chemical Enterprises working experience from 27 to 40 years (mean 30 years); the external exposure dose was 175.88 mSv (158.20-207.81 mSv). In all examined subjects, the degree of methylation of the promoters of apoptosis-related genes ranged from 0.22 to 50.00%. A correlation was found between the degree of methylation of BCLAF1 promoters (p=0.035) with the age of workers, BAX promoters (p=0.0289) with high content of aberrant cells, and APAF1 promoters (p=0.0152) with increased number of dicentric chromosomes. A relationship was found between the dose of external irradiation and the degree of methylation of gene promoters of BAD (p=0.0388), BID (р=0.0426), and HRK (р=0.0101) genes.

Temporal DNA methylation pattern and targeted therapy in colitis-associated cancer.

DNA methylation plays a crucial role in the pathogenesis of various diseases, including colorectal cancer (CRC). However, the global and temporal DNA methylation pattern during initiation and progression of colitis-associated cancer (CAC) are still unknown, including the potential therapeutic strategy of targeting methylation for CAC. In the present study, the global DNA methylation pattern was determined at different time points during CAC using DNA methylation sequencing, followed by the Starburst plot integrating alterations and potential functional prediction analysis. After demonstrating the regulatory role of DNA methyltransferases (DNMTs) on the expression of hub-genes in CRC cells, DNMT inhibitors were administered to treat CAC mice. Our results indicated that 811 genes were hypermethylated at different time points during initiation and progression of CAC. Genes that were downregulated and hypermethylated during CAC, including hub-genes BAD and inositol polyphosphate phosphatase-like 1 (INPPL1), were involved in MAPK signaling pathways, kit receptor signaling pathways, apoptosis and EGF/EGFR signaling pathways. Upregulated DNMTs (DNMT1, DNMT3A and DNMT3B) mediated downregulation and hypermethylation of BAD and INPPL1 in CAC and CRC cells. Low doses of DNMT inhibitors (decitabine (DAC) and azacitidine (AZA)) exerted efficient antitumor effects in CAC, accompanied with upregulation of BAD and INPPL1 expression, and apoptosis induction. In summary, the present study demonstrates the temporal DNA methylation pattern during CAC and provides a novel therapeutic strategy for treating this disease.

Ribosomal protein small subunit 15A (RPS15A) inhibits the apoptosis of breast cancer MDA-MB-231 cells via upregulating phosphorylated ERK1/2, Bad, and Chk1.

AIM: To detect the expression and identify the role of Ribosomal protein S15A (RPS15A) in human breast cancer (BC). METHODS: Immunohistochemistry (IHC) was carried out for detecting the levels of RPS15A protein. Quantitative PCR was used to evaluate the mRNA level of RPS15A in one normal breast and three BC cell lines. Lentivirus-mediated shRNA targeting RPS15A was designed to investigate the impact of silencing RPS15A in MDA-MB-231 cell. RESULTS: Higher RPS15A expression was detected in tumor tissues than in para-cancer tissues, and higher RPS15A expression was related to larger tumor size and higher TNM stage. Also, RPS15A mRNA expression in all three BC cell lines was higher than that in normal breast cell (all P < .005). Further, RPS15A knockdown significantly suppressed MDA-MB-231 cell proliferation and induced apoptosis. Moreover, RPS15A knockdown increased the caspase-3/-7 activity, and suppressed the phosphorylated levels of ERK1/2, Bad, and Chk1 (all P < .01). CONCLUSION: RPS15A inhibits apoptosis via upregulating phosphorylated ERK1/2, Bad, and Chk1 in MDA-MB-231 cell line.

Induction of apoptosis in hematological cancer cells by dorsomorphin correlates with BAD upregulation.

AMPK is generally a tumor suppressor. However, once cancer arises, AMPK becomes a tumor promoter instead, driving cancer development. For such AMPK-driven cancers, AMPK blockade may be a valuable therapeutic strategy. Here we show that AMPK is upregulated in a variety of hematological cancers and plays key roles in maintaining viability of tumor cells. Blockade of AMPK signaling by dorsomorphin markedly induces apoptosis in Jurkat, K562 cell lines as well as primary cancerous B cells. Mechanistically, dorsomorphin significantly upregulates the expression of BAD, a pro-apoptotic member of the Bcl-2 gene family involved in initiating apoptosis. Reduction of BAD expression by RNA interference prevents apoptosis in response to AMPK inhibition. Thus, our data found BAD integrates the pro-apoptotic effects of dorsomorphin and provided novel insights into the mechanisms by which AMPK facilitates survival signaling in hematologic tumor cells.

Taurine inhibits neuron apoptosis in hippocampus of diabetic rats and high glucose exposed HT-22 cells via the NGF-Akt/Bad pathway.

Type 2 Diabetes causes learning and memory deficits that might be mediated by hippocampus neuron apoptosis. Studies found that taurine might improve cognitive deficits under diabetic condition because of its ability to prevent hippocampus neuron apoptosis. However, the effect and mechanism is not clear. In this study, we explore the effect and mechanism of taurine on inhibiting hippocampus neuron apoptosis. Sixty male Sprague-Dawley rats were randomly divided into control, T2D, taurine treatment (giving 0.5%, 1%, and 2% taurine in drinking water) groups. Streptozotocin was used to establish the diabetes model. HT-22 cell (hippocampus neurons line) was used for in vitro experiments. Morris Water Maze test was used to check the learning and memory ability, TUNEL assay was used to measure apoptosis and nerve growth factor (NGF); Akt/Bad pathway relevant protein was detected by western blot. Taurine improved learning and memory ability and significantly decreased apoptosis of the hippocampus neurons in T2D rats. Moreover, taurine supplement also inhibited high glucose-induced apoptosis in HT-22 cell in vitro. Mechanistically, taurine increased the expression of NGF, phosphorylation of Trka, Akt, and Bad, as well as reduced cytochrome c release from mitochondria to cytosol. However, beneficial effects of taurine were blocked in the presence of anti-NGF antibody or Akt inhibitor. Taurine could inhibit hippocampus neuron apoptosis via NGF-Akt/Bad pathway. These results provide some clues that taurine might be efficient and feasible candidate for improvement of learning and memory ability in T2D rats.

Design, expression, purification and crystallization of human 14-3-3ζ protein chimera with phosphopeptide from proapoptotic protein BAD.

14-3-3 protein isoforms regulate multiple processes in eukaryotes, including apoptosis and cell division. 14-3-3 proteins preferentially recognize phosphorylated unstructured motifs, justifying the protein-peptide binding approach to study 14-3-3/phosphotarget complexes. Tethering of human 14-3-3σ with partner phosphopeptides via a short linker has provided structural information equivalent to the use of synthetic phosphopeptides, simultaneously facilitating purification and crystallization. Nevertheless, the broader applicability to other 14-3-3 isoforms and phosphopeptides was unclear. Here, we designed a novel 14-3-3ζ chimera with a conserved phosphopeptide from BAD, whose complex with 14-3-3 is a gatekeeper of apoptosis regulation. The chimera could be bacterially expressed and purified without affinity tags. Co-expressed PKA efficiently phosphorylates BAD within the chimera and blocks its interaction with a known 14-3-3 phosphotarget, suggesting occupation of the 14-3-3 grooves by the tethered BAD phosphopeptide. Efficient crystallization of the engineered protein suggests suitability of the "chimeric" approach for studies of other relevant 14-3-3 complexes.

Differences in the mechanisms of proapoptotic BH3 proteins binding to Bcl-XL and Bcl-2 quantified in live MCF-7 cells.

Overexpression of antiapoptotic proteins including Bcl-XL and/or Bcl-2 contributes to tumor initiation, progression, and resistance to therapy by direct interactions with proapoptotic BH3 proteins. Release of BH3 proteins from antiapoptotic proteins kills some cancer cells and sensitizes others to chemotherapy. Binding of Bcl-XL and Bcl-2 to the BH3 proteins Bad, Bid, and the three major isoforms of Bim was measured for fluorescent protein fusions in live cells using fluorescence lifetime imaging microscopy and fluorescence resonance energy transfer. In cells the binding of the proteins at mitochondria is similar to the results from in vitro measurements. However, mutations in the BH3 region of Bim known to inhibit binding to Bcl-XL and Bcl-2 in vitro had much less effect in MCF-7 cells. Moreover, the BH3 mimetic ABT-737 inhibited Bad and Bid but not Bim binding to Bcl-XL and Bcl-2. Thus, the selectivity of ABT-737 also differs markedly from predictions made from in vitro measurements.

Piperine Inhibits Cell Proliferation and Induces Apoptosis of Human Gastric Cancer Cells by Downregulating Phosphatidylinositol 3-Kinase (PI3K)/Akt Pathway.

BACKGROUND Piperine has been reported to inhibit proliferation and induce apoptosis in various cancer cells. This study aimed to explore the efficacy and underlying mechanism of piperine in human gastric cancer. MATERIAL AND METHODS MTT assay was performed to examine the effect of piperine (concentrations of 0-300 µM) on the proliferation of human gastric cancer SNU-16 cells and normal human gastric epithelial GES-1 cells. Flow cytometry and Western blot were used to determine cell apoptosis and the expression level of protein (Cyto C, cleaved PARP, cleaved caspase-3, Bax, Bcl-2, Bad, Bcl-xl, PI3K, pPI3K, Akt, and pAkt), respectively. To further investigate the anti-tumor mechanism of piperine in SNU-16 cells, we used a small-molecule Akt activator SC79 in this study. The in vivo mechanism of piperine against gastric cancer was evaluated using a xenograft tumor model. RESULTS The results showed that piperine inhibited proliferation and induced apoptosis of SNU-16 cells. Piperine upregulated the protein expression of Bax, Bad, Cyto C, cleaved PARP, and cleaved caspase-3, but downregulated the protein expression of Bcl-2, Bcl-xl, pPI3k, and pAkt. However, SC79 reversed the function of piperine on the apoptosis-related proteins. An in vivo study revealed that, compared with the control group, the tumor volume of mice treated with piperine was significantly reduced. Piperine enhanced cleaved caspase-3 expression but decreased Ki-67 expression in a dose-dependent manner. Moreover, the nontoxicity effect of piperine was confirmed by H&E staining analysis in kidney and heart tissues of mice. CONCLUSIONS Our findings suggest that piperine inhibits proliferation and induces apoptosis of human gastric cancer cells through inhibition of the PI3K/Akt signaling pathway.

The RhoJ-BAD signaling network: An Achilles' heel for BRAF mutant melanomas.

Genes and pathways that allow cells to cope with oncogene-induced stress represent selective cancer therapeutic targets that remain largely undiscovered. In this study, we identify a RhoJ signaling pathway that is a selective therapeutic target for BRAF mutant cells. RhoJ deletion in BRAF mutant melanocytes modulates the expression of the pro-apoptotic protein BAD as well as genes involved in cellular metabolism, impairing nevus formation, cellular transformation, and metastasis. Short-term treatment of nascent melanoma tumors with PAK inhibitors that block RhoJ signaling halts the growth of BRAF mutant melanoma tumors in vivo and induces apoptosis in melanoma cells in vitro via a BAD-dependent mechanism. As up to 50% of BRAF mutant human melanomas express high levels of RhoJ, these studies nominate the RhoJ-BAD signaling network as a therapeutic vulnerability for fledgling BRAF mutant human tumors.

Torin2 overcomes sorafenib resistance via suppressing mTORC2-AKT-BAD pathway in hepatocellular carcinoma cells.

BACKGROUND: Sorafenib is an oral multi-kinase inhibitor that was approved by the US Food and Drug Administration for the treatment of patients with advanced hepatocellular carcinoma (HCC). However, resistance to sorafenib is an urgent problem to be resolved to improve the therapeutic efficacy of sorafenib. As the activation of AKT/mTOR played a pivotal role in sorafenib resistance, we evaluated the effect of a dual mTOR complex 1/2 inhibitor Torin2 on overcoming the sorafenib resistance in HCC cells. METHODS: The sorafenib-resistant Huh7 and Hep3B cell lines were established from their parental cell lines. The synergistic effect of sorafenib and Torin2 on these cells was measured by cell viability assay and quantified using the Chou-Talalay method. Apoptosis induced by the combination of sorafenib and Torin2 and the alteration in the specific signaling pathways of interest were detected by Western blotting. RESULTS: Sorafenib treatment inversely inhibited AKT in parental but activated AKT in sorafenib-resistant Huh7 and Hep3B HCC cells, which underscores the significance of AKT activation. Torin2 and sorafenib synergistically suppressed the viability of sorafenib-resistant cells via apoptosis induction. Torin2 successfully suppressed the sorafenib-activated mTORC2-AKT axis, leading to the dephosphorylation of Ser136 in BAD protein, and increased the expression of total BAD, which contributed to the apoptosis in sorafenib-resistant HCC cells. CONCLUSIONS: In this study, Torin2 and sorafenib showed synergistic cytostatic capacity in sorafenib-resistant HCC cells, via the suppression of mTORC2-AKT-BAD pathway. Our results suggest a novel strategy of drug combination for overcoming sorafenib resistance in HCC.

Chemical Standardization and Anti-Proliferative Activity of Ardisia elliptica Fruit against the HCT116 Human Colon Cancer Cell Line.

The present study is intended to carry out the chemical standardization and evaluation of the anti-proliferative activity of A. elliptica fruit extract. A. elliptica fruit powder was extracted with ethanol. The obtained extract was assessed for total phenolic content using the Folin-Ciocalteu method. Moreover, a simple, accurate, and precise reversed phase high-performance liquid chromatographic method was developed and validated to determine the embelin content of A. elliptica fruit extract. Then, the extract and embelin were investigated for their anti-proliferative effect against HCT-116 cells. Finally, the mechanisms of inhibition of the extract and embelin on the mRNA expression of pro-apoptotic genes Bad, Bax, and Caspase-8 and anti-apoptotic genes c-IAP1, Mcl-1, and XIAP were determined by real-time qRT-PCR. The phenolic content and embelin content of the extract were 5.20 ± 0.01 g of gallic acid equivalent per 100 g of dried fruit (g% GAE) and 5.57 ± 0.56 mg/g of extract, respectively. The extract and embelin showed strong anti-proliferative effects on HCT-116 cells with 50% inhibition concentration (IC50) values of 19.16 ± 1.09 µg/mL and 25.93 ± 1.75 µg/mL, respectively. The A. elliptica extract exhibited a significant increase in the mRNA level of Bad, Bax, and Caspase-8 and a significant decrease in c-IAP1, Mcl-1, and XIAP. Embelin showed a significant decrease in Mcl-1 and XIAP.

Paeoniflorin protects spiral ganglion neurons from cisplatin-induced ototoxicity: Possible relation to PINK1/BAD pathway.

The objective of this study was to elucidate whether paeoniflorin (PF) exerted an effect on cisplatin-induced spiral ganglion neuron (SGN) damage, with special attention given to the role of PINK1/BAD pathway in this process. Middle cochlear turn culture and C57BL/6 mice were utilized to identify the character of PF in vitro and in vivo. We found that cisplatin treatment led to SGN damage, in which reactive oxygen species (ROS) generation increased, PINK1 expression decreased, BAD accumulation on mitochondria raised and mitochondrial apoptotic pathway activated. Conversely, we demonstrated that PF pre-treatment obviously mitigated cisplatin-induced SGN damage. Mechanistic studies showed that PF could reduce ROS levels, increase PINK1 expression, decrease the BAD accumulation on mitochondria and, thus, alleviate the activated mitochondrial apoptosis in SGNs caused by cisplatin. Overall, the findings from this work reveal the important role of PF and provide another strategy against cisplatin-induced ototoxicity.

Genome-wide CRISPR-Cas9 viability screen reveals genes involved in TNF-α-induced apoptosis of human umbilical vein endothelial cells.

Tumor necrosis factor α (TNF-α), a pivotal cytokine in sepsis, protects the host against pathogens by promoting an inflammatory response while simultaneously inducing apoptosis of the vascular endothelium. Unfortunately, inhibitors targeting certain components of the TNF-α signaling pathway to reduce cellular apoptosis have failed to translate into clinical applications, partly due to the adverse effects of excessive immunosuppression. In an attempt to discover potential targets in the TNF-α signaling pathway to modulate moderate inflammation and apoptosis during the development of sepsis, we performed a pooled genome-wide CRISPR/Cas9 knockout screen in human umbilical vein endothelial cells (HUVECs). Tumor necrosis factor receptor superfamily member 1A (TNFRSF1A), B-cell lymphoma 2 (BCL2), Bcl2-associated death promoter (BAD), and NLR family member X1 (NLRX1) deficiencies were identified as the effective genetic suppressors of TNF-α cytotoxicity on a list of candidate regulators. CRISPR-mediated NLRX1 knockout conferred cellular resistance to challenge with TNF-α, and NLRX1 could be induced to colocalize with mitochondria following TNF-α stimulation. Thus, our work demonstrates the advantage of genome-scale screening with Cas9 and validates NLRX1 as a potential modulator of TNF-α-induced vascular endothelial apoptosis during sepsis.