Induction Of Apoptosis In Response Research Articles

Comparative Toxicity of TiO2 and Sn-Doped TiO2 Nanoparticles in Zebrafish After Acute and Chronic Exposure.

This study was conducted to assess the toxicological potential of synthesized pure and Sn-doped TiO2 NPs (Sn-TiO2 NPs) in zebrafish after acute and chronic exposure. The pure TiO2 NPs, 4%, and 8% Sn-TiO2 NPs were synthesized and characterized using X-ray diffraction, Scanning Electron Microscope, diffuse reflectance spectra, dynamic light scattering, and zeta potential analyses. The pure TiO2 NPs, 4%, and 8% Sn-TiO2 NPs were spherical with average sizes of about 40, 28, and 21 nm, respectively, indicating significant size reduction of TiO2 NPs following Sn doping. According to our results, the LC50-96h increased in the order of 8% Sn-TiO2 NPs (45 mg L-1) < 4% Sn-TiO2 NPs (80.14 mg L-1) < pure TiO2 NPs (105.47 mg L-1), respectively. Exposure of fish to Sn-TiO2 NPs after 30 days resulted in more severe histopathological alterations in gills, liver, intestine, and kidneys than pure TiO2 NPs. Furthermore, Sn-doping significantly elevated malondialdehyde levels and micronuclei frequency, indicating increased oxidative stress and genotoxicity. Expression analysis revealed altered expression of various genes, including upregulation of pro-apoptotic Bax gene and downregulation of anti-apoptotic Bcl-2 gene, suggesting potential induction of apoptosis in response to Sn-doped NPs. Additionally, antioxidant genes (Gpx, Sod, Cat, and Ucp-2) and stress response gene (Hsp70) showed altered expression, suggesting complex cellular responses to mitigate the toxic effects. Overall, this study highlights the concerning impact of Sn-doping on the toxicity of TiO2 NPs in zebrafish and emphasizes the need for further research to elucidate the exact mechanisms underlying this enhanced toxicity.

The Cellular Impact, Immediate Targets and Mechanism of Action of NSD2 in t(4;14) Multiple Myeloma

INTRODUCTION: NSD2 is a potential therapeutic target in the 15% of cases of multiple myeloma that carry a t(4;14) translocation. As a consequence of the translocation, strong Ig enhancers are placed in proximity to NSD2 and FGFR3. FGFR3 is deleted at a subsequent time point in 25% of cases, while all cases retain NSD2 expression consistent with it likely being the key oncogenic driver. NSD2 is an H3K36 dimethyl transferase, a histone modification mark associated with active transcription, but the molecular targets and the mechanism by which NSD2 regulates its targets is unknown. The cellular function of NSD2 is also largely unknown, but previous shRNA knockdown studies have suggested it is involved in cell proliferation. The major limitation to understanding the role of NSD2 is the lack of an appropriate experimental system with which to investigate its molecular and cellular function. To address this deficit and to identify the primary direct targets of NSD2 as a means to elucidate its mechanism of action we have generated NSD2 dTAG and NSD2 KO cells together with NSD2 reconstitution systems. METHODS: We have utilized CRISPR gene editing to knock-in a dTAG in NSD2 in the t(4;14) KMS-11 cell line. This system enables the inducible and rapid degradation of NSD2 following the addition of a small-molecule degrader which is otherwise inert. We have also generated clonal NSD2 KO KMS-11 cells that allow us to observe and study the stable phenotypes of NSD2 KO. Because H3K36me2 deposited by NSD2 leads to induction of DNMT3A-mediated DNA methylation and suppression of PRC2-mediated H3K27me3, we have generated DNMT3A KO on top of NSD2dTAG/KO and have used two different PRC2 inhibitors to dissect the dependency of NSD2 function on such secondary modifications. To systematically dissect the dependency on NSD2 isoforms and its methyltransferase activity, we have reconstituted NSD2 in both NSD2 dTAG and NSD2 KO KMS-11 cells, including WT NSD2I WT, NSD2II WT, NSD2 Y1179A (LOF), NSD2 E1099K (GOF). To maximally match the timing of NSD2 perturbation, NSD2 reconstitution was generated in NSD2 dTAG and NSD2 KO cells in a Tet-ON and Tet-OFF expression system, respectively. Further we have generated KMS-11 cells overexpressing Dox-inducible H3.3K36M to investigate the consequences of abrogation of H3K36me2 by this dominant negative mutant of H3.3. We have done SLAM-seq to assess the transcriptional targets of NSD2 and will extend this with ATAC-seq and CUT&amp;Tag to identify the molecular targets of NSD2. RESULTS: We show that NSD2 is rapidly degraded within 1 h when a degrader is added and subsequently a significant reduction of H3K36me2 is seen by 24 hours although it takes more than 3 days to see its deep depletion. We observed a reciprocal increase of H3K27me3 as previously noted. Depletion of H3K36me2 and increase in H3K27me3 was also seen in the NSD2 KO KMS-11 cells. Paradoxically we didn't see an induction of apoptosis in response to the degradation of NSD2 or its KO. In sharp contrast to the previously published shRNA results only a mild inhibition of cell proliferation was observed in NSD2 KO cells and over an extended period of 3 weeks in the dTAG system. We didn't observe an effect of H3.3 K36M overexpression on cell proliferation although it abolished H3K36me2 very efficiently. These experiments demonstrate that NSD2 is not essential for viability of KMS-11 cells but is required for optimal cell growth, which is likely independent of its H3K36me2 methyltransferase activity. SLAMseq results show that NSD2 degradation in the dTAG system causes transcriptional changes of a specific set of newly synthesized mRNAs. ATAC-seq and CUT&amp;Tag experiments are being run and will be superimposed on the SLAM-seq data to identify immediate targets of NSD2. Subsequently, use of PRC2 inhibitors and DNMT3A KO cells in the dTAG cells and the NSD2 reconstitution experiments will uncover the mechanisms of how NSD2 controls the transcription of its targets. CONCLUSION: We show that NSD2 is not essential for viability of KMS-11 cells but is required for optimal cell growth and that NSD2 controls the transcription of a specific set of genes and does not exert a global impact on the transcriptome. We will further define the immediate targets of NSD2 by leveraging our SLAM-seq, ATAC-seq, and CUT&amp;Tag data in NSD2 dTAG/KO and NSD2-reconstituted cells and by blocking the crosstalk with other epigenetic marks (i.e. H3K27me3 and DNAme).

The epigenetic modifier HDAC2 and the checkpoint kinase ATM determine the responses of microsatellite instable colorectal cancer cells to 5-fluorouracil

The epigenetic modifier histone deacetylase-2 (HDAC2) is frequently dysregulated in colon cancer cells. Microsatellite instability (MSI), an unfaithful replication of DNA at nucleotide repeats, occurs in about 15% of human colon tumors. MSI promotes a genetic frameshift and consequently a loss of HDAC2 in up to 43% of these tumors. We show that long-term and short-term cultures of colorectal cancers with MSI contain subpopulations of cells lacking HDAC2. These can be isolated as single cell-derived, proliferating populations. Xenografted patient-derived colon cancer tissues with MSI also show variable patterns of HDAC2 expression in mice. HDAC2-positive and HDAC2-negative RKO cells respond similarly to pharmacological inhibitors of the class I HDACs HDAC1/HDAC2/HDAC3. In contrast to this similarity, HDAC2-negative and HDAC2-positive RKO cells undergo differential cell cycle arrest and apoptosis induction in response to the frequently used chemotherapeutic 5-fluorouracil, which becomes incorporated into and damages RNA and DNA. 5-fluorouracil causes an enrichment of HDAC2-negative RKO cells in vitro and in a subset of primary colorectal tumors in mice. 5-fluorouracil induces the phosphorylation of KAP1, a target of the checkpoint kinase ataxia-telangiectasia mutated (ATM), stronger in HDAC2-negative cells than in their HDAC2-positive counterparts. Pharmacological inhibition of ATM sensitizes RKO cells to cytotoxic effects of 5-fluorouracil. These findings demonstrate that HDAC2 and ATM modulate the responses of colorectal cancer cells towards 5-FU.Graphical abstract

Bisphenol A induces apoptosis in response to DNA damage through c-Abl/YAPY357/ p73 pathway in P19 embryonal carcinoma stem cells

Bisphenol A (2,2-bis(4′-hydroxyphenyl) propane, BPA) is a well-known endocrine-disrupting compound that is widely used in various daily products and exhibits embryonic development toxicity and genotoxicity. However, the affected signaling pathways involved in embryonic development especially the interactions of involved proteins remain unclear. In our previous study (Ge et al., 2021), BPA induces DNA damage and apoptosis in Xenopus embryos, resulting in multiple malformations of larvae. However, the signaling pathways induced for apoptosis response to DNA damage are still not well elucidated. Here, we systematically elucidated the enriched pathways affected by BPA and illustrated the interactions of involved proteins. Results indicated that BPA affected multiple embryonic development pathways including Hippo, TGF-β, Wnt, and Notch pathways. Furthermore, the protein-protein interaction network suggested that the c-Abl/YAPY357/p73 pathway may play a key role in apoptosis induction in response to DNA damage. P19 embryonal carcinoma stem cells, as a developmental toxicity model, were treated with different BPA concentrations to establish an in vitro model to verify the role of the c-Abl/YAPY357/p73 pathway in apoptosis. BPA triggered DNA damage and significantly upregulated the expression levels of c-Abl, phosphorylated YAPY357, phosphorylated p73Y99, and cleaved caspase-3 protein (p < 0.05), thus decreasing cell viability and transcriptionally activating the p73 target genes Bax and Puma. These data suggested that BPA activated the c-Abl/YAPY357/p73 pathway in response to DNA damage. Imatinib, an inhibitor of tyrosine kinase c-Abl, significantly downregulated the elevated expression levels of p-YAPY357, p-p73Y99 and cleaved caspase-3 (p < 0.05) caused by BPA and then ameliorated the cell index of P19 cells in the BPA-treated group. Therefore, this substance restrained the phosphokinase activity of c-Abl and suppressed the c-Abl/YAPY357/p73 pathway. Results showed that the c-Abl/YAPY357/p73 pathway served as a mechanism for caspase-3 activation that induced the apoptosis response to DNA damage stress.

Atovaquone Demonstrates Single Agent Activity and an Additive Effect in Combination with Cytotoxic Chemotherapy (Cytarabine and Daunorubicin) in AML

Background: To improve outcomes for pediatric acute myeloid leukemia (AML), identifying and targeting mechanisms of therapy resistance is critical. Recently, the antimicrobial drug atovaquone (AQ) was shown to reduce IL-6/STAT3 signaling in vitro and to improve relapse-free survival in adult AML patients who used AQ for prophylaxis after bone marrow transplant (Xiang et al, Blood, 2016). Clinically, this FDA approved drug is well tolerated, even in patients with organ dysfunction. It has established pediatric dosing, and plasma concentrations at standard doses are typically 40-80µM. We performed in vitro and in vivo experiments to evaluate the efficacy of AQ as a single agent and in combination with standard induction chemotherapy (cytarabine, daunorubicin, and etoposide). In order to ensure clinical relevance, all experiments examined cytotoxicity at clinically achievable doses.Methods: AML cell lines were incubated with vehicle control or increasing doses of AQ (1µM - 30µM) for 24, 48, 72, or 96 hours, and then apoptosis was quantified by Annexin-V staining. Primary pediatric AML cells were exposed to 30µM AQ for the same time periods. To reduce spontaneous apoptosis of primary AML cells over prolonged culture, samples were cultured in HS5 stromal cell-conditioned media (CM) or co-cultured with HS5 stromal cells. For in vivo studies, female NSG mice were injected with 5x105 THP-1 cells expressing firefly luciferase (THP-1.ffluc). Mice were randomly assigned to receive AQ 200 mg/kg daily (n=6) or vehicle (n=5) by oral gavage, starting on day 1, and disease burden was monitored by bioluminescent imaging 3 times a week. A cohort of male mice receiving VC or AQ starting the day of injection with THP-1.ffluc cells were followed for survival (n=7 for each group). For in vitro drug combination cytotoxicity assays, 103 THP-1.ffluc cells per well were plated in 96 well plates with media only, single drugs at increasing concentrations, and AQ+chemotherapy combinations (AQ 1-15uM, AraC 0.1-3uM, Dauno 0.03-0.6uM, VP-16 0.3-3uM). After 48 hours, viability was determined by luminescence. Background luminescence of media-only wells was subtracted from all values and means of 4 replicates were used for analysis. All values were normalized to the cells + vehicle wells, which were set to 0 to quantify the percent affected. The Calcusyn method was used to calculate the combination index (CI). An AQ+chemotherapy combination was considered synergistic if CI<0.8, additive if CI>0.8 and <1.2, or antagonistic if CI>1.2.Results: All 7 AML cell lines demonstrated time and dose-dependent induction of apoptosis in response to 1-30µM doses of AQ. IC50s were within the range of typical clinically achievable doses. For AML cell lines, the IC50s (in µM) after 72 hour incubation were: MOLM13 13.7, MV4-11 14.0, THP-1 15.7, NB4 16.9, Kasumi 1 18.6, HL60 33.7, KG1 98.9. AQ was generally less potent against the CML cell line K562 and the precursor B ALL cell line RS4 (72 hour IC50s were 116.6 and 81.4 respectively). All tested primary samples demonstrated AQ induced apoptosis that increased over time. After subtracting spontaneous apoptosis at each timepoint, the apoptosis induced by AQ at 72 hours for 5 patient samples supported by co-culture were 36, 37, 39, 67 and 87%. Annexin-V+% for the same patient samples supported by CM were 19, 13, 51, 47, and 24%. Bioluminescent imaging of female NSG mice xenografted with THP-1.ffluc cells and treated with single agent AQ daily starting on the day of cell injection demonstrated decreased disease burden compared to control mice (for example on day 21, the average radiance of VC treated mice vs AQ treated mice was 2 x 105 vs 6.3 x 104 p/s/cm²/sr; p=.0048 by ANOVA). Single agent AQ prolonged survival of male xenografted mice with all VC mice succumbing to disease at a median of 63 days vs 1/7 AQ treated mice by day 70 (p=.003 by log-rank test). Our drug combination assays found an antagonistic effect in combination with etoposide, and an additive effect with cytarabine and daunorubicin.Conclusions: Atovaquone induced dramatic levels of apoptosis of AML cells at clinically achievable doses in vitro ; reduced disease burden and improved survival in AML xenografts; and had an additive effect with standard chemotherapy drugs for pediatric AML. Our data support further evaluation of atovaquone for pediatric AML. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The synergy of BET inhibitors with aurora A kinase inhibitors in MYCN-amplified neuroblastoma is heightened with functional TP53

Amplification of MYCN is a poor prognostic feature in neuroblastoma (NBL) indicating aggressive disease. We and others have shown BET bromodomain inhibitors (BETi) target MYCN indirectly by downregulating its transcription. Here we sought to identify agents that synergize with BETi and to identify biomarkers of resistance. We previously performed a viability screen of ∼1,900 oncology-focused compounds combined with BET bromodomain inhibitors against MYCN-amplified NBL cell lines. Reanalysis of our screening results prominently identified inhibitors of aurora kinase A (AURKAi) to be highly synergistic with BETi. We confirmed the anti-proliferative effects of several BETi+AURKAi combinations in MYCN-amplified NBL cell lines. Compared to single agents, these combinations cooperated to decrease levels of N-myc. We treated both TP53-wild type and mutant, MYCN-amplified cell lines with the BETi JQ1 and the AURKAi Alisertib. The combination had improved efficacy in the TP53-WT context, notably driving apoptosis in both genetic backgrounds. JQ1+Alisertib combination treatment of a MYCN-amplified, TP53-null or TP53-restored genetically engineered mouse model of NBL prolonged survival better than either single agent. This was most profound with TP53 restored, with marked tumor shrinkage and apoptosis induction in response to combination JQ1+Alisertib. BETi+AURKAi in MYCN-amplified NBL, particularly in the context of functional TP53, provided anti-tumor benefits in preclinical models. This combination should be studied more closely in a pediatric clinical trial.

Integrin α2β1 inhibition attenuates prostate cancer cell proliferation by cell cycle arrest, promoting apoptosis and reducing epithelial-mesenchymal transition.

Integrin α2β1 plays an important role in cellular migration and metastasis processes associated with prostate cancer. The aim of this study was to assess whether selective inhibition of integrin α2β1 is an effective strategy to target metastatic prostate cancer cells.In this regard, we examined the effects of the inhibitor BTT-3033, which selectively interferes with the connection between integrin a2b1 and its ligand, on migration, epithelial-mesenchymal transition (EMT), cell cycle arrest, apoptosis, and specific intracellular signaling pathways using LNcap-FGC and DU-145 prostate cancer cell lines. Western blot analysis and immunocytochemistry assays showed that inhibition of integrin a2b1 inhibits EMT, through the increased expression of E-cadherin and decreased expression of N-cadherin and vimentin. Scratch wound healing assays revealed a direct effect on integrin α2β1 in the migration capacity of cells. In addition, treatment with BTT-3033 induced a reduction in cell viability and proliferation, as assessed by MTT and BrdU assays. In addition, the results show that BTT-3033 inhibits cell proliferation by inducing G1 cell cycle arrest. Moreover, inhibition of integrin α2β1 induces apoptosis through the activation of ROS, Bax protein upregulation, caspase-3 activation, and depletion of ΔΨm. Molecular signaling studies showed that integrin α2β1 was a positive regulator of MKK7 phosphorylation. In conclusion, our results reveal a critical role for integrin a2b1 in the proliferation of prostate cancer cells, as demonstrated by EMT inhibition, cell cycle arrest, and apoptosis induction in response to treatment with its specific inhibitor BT-3033.

Mitochondrial manganese superoxide dismutase knock-down increases oxidative stress and caspase-3 activity in the white shrimp Litopenaeus vannamei exposed to high temperature, hypoxia, and reoxygenation

Shrimp are increasingly exposed to warmer temperatures and lower oxygen concentrations in their habitat due to climate change. These conditions may lead to oxidative stress and apoptosis. We studied the effects of high temperature, hypoxia, reoxygenation, and the combination of these factors on lipid peroxidation, protein carbonylation, and caspase-3 activity in gills of white shrimp Litopenaeus vannamei. Silencing of mitochondrial manganese superoxide dismutase (mMnSOD) was used to determine the role of this enzyme in response to the abiotic stressors described above, to avoid oxidative damage and apoptosis. In addition, mMnSOD gene expression and mitochondrial SOD activity were evaluated to determine the efficiency of silencing this enzyme. The results showed that there was no effect of the abiotic stress conditions on the thiobarbituric acid reactive substances (TBARS), but protein carbonylation increased in all the oxidative stress treatments and caspase-3 activity decreased in hypoxia at 28 °C. On the other hand, mMnSOD-silenced shrimp experienced higher oxidative stress, since TBARS, carbonylated proteins and caspase-3 activity increased in some silenced treatments. Unexpectedly, mitochondrial SOD activity increased in some of the silenced treatments as well. Altogether, these results suggest that mMnSOD has a key role in shrimp for the prevention of oxidative damage development and induction of apoptosis in response to hypoxia, reoxygenation, high temperature, and their interactions, as conditions derived from climate change.

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.

Abstract 5030: Vitamin D exhibits therapeutic anti-tumor and anti-angiogenic potential by reducing VEGF levels and altering TIMP/MMP system in breast cancer

Abstract Background: Clinical and experimental observations suggest that Vitamin D deficiency associates with increased risk of breast cancer. Furthermore, studies concluded that Vitamin D precipitates in significant anti-neoplastic effects against breast, and inducing apoptosis and affecting angiogenesis in other types of cancer. However, the exact mechanism underlying this anti-angiogenic effect of Vitamin D is still elusive. This study aims to investigate the anti-cancer role of Vitamin D anti-cancer and its association with modulation of major factors involved in angiogenesis and metastasis in breast cancer cell lines. Experimental methods: The hormonally active form of vitamin D, 1α,25-dihydroxyvitamin D3 (calcitriol), was used to evaluate anti-proliferative effect, inhibition of cell cycle progression and apoptosis induction in two breast cancer cell lines, MCF-7 and MDA-MB-231. Furthermore, impact of calcitriol on expression and activity of tissue inhibitors of metalloproteinases (TIMP), major groups of the matrix metalloproteinases (MMPs), vascular endothelial growth factor (VEGF), and tumor growth factor (TGF-β1) among others involved in tumor progression and angiogenesis were also examined. Results and Discussions: Calcitriol treatment (10 µM) significantly inhibited cell proliferation in a timely dependent manner. Both cell lines showed G1 cell cycle arrest, decreased levels of the cell death inhibitor, survivin, and apoptosis induction in response to calcitriol treatment. Proteome Profiler array analysis revealed that calcitriol resulted in reducing the levels of major pro-angiogenesis regulators, especially VEGF, TGF-β1, Urokinase-type plasminogen activator (uPA), and Amphiregulin. Analysis of mRNA and proteins expression upon calcitriol exposure showed an increase of TIMP1, TIMP2 and a decrease in MMP 2, 3 and 9 levels. Finally, quantitative analysis using ELISA revealed that calcitriol treatment reduced MMP2 and MMP9 activities. Conclusion: Our study demonstrates potential therapeutic property of calcitriol through inhibiting of pro-angiogenetic markers in breast cancer. Citation Format: Khuloud Bajbouj, Jasmin Shafarin, Ahmad Sawan, Ahmed Shehada, Walaa Eldin Elkhalifa. Vitamin D exhibits therapeutic anti-tumor and anti-angiogenic potential by reducing VEGF levels and altering TIMP/MMP system in breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5030.

Induction of apoptosis in Ogg1-null mouse embryonic fibroblasts by GSH depletion is independent of DNA damage

Reactive oxygen species (ROS) within the cell are rapidly detoxified by antioxidants such as glutathione. Depletion of glutathione will therefore increase levels of intracellular ROS, which can lead to oxidative DNA damage and the induction of apoptosis. The working hypothesis was that Ogg1 null mouse embryonic fibroblasts (mOgg1−/− MEFs) would be more sensitive in response to GSH depletion due to their deficiency in the removal of the oxidative DNA modification, 8-oxo-7,8-dihydroguanine (8-oxoG). Following GSH depletion, an increase in intracellular ROS and a subsequent induction of apoptosis was measured in mOgg1−/− MEFs; as expected. Unexpectedly, an elevated basal level of ROS was identified in mOgg1-/- MEFs compared to wild type MEFs; which we suggest is partly due to the differential expression of key anti-oxidant genes. The elevated basal ROS levels in mOgg1−/− MEFs were not accompanied by a deficiency in ATP production or a large increase in 8-oxoG levels. Although 8-oxoG levels did increase following GSH depletion in mOgg1-/- MEFs; this increase was significantly lower than observed following treatment with a non-toxic dose of hydrogen peroxide. Reconstitution of Ogg1 into mOgg1−/− MEFs resulted in an increased viability following glutathione depletion, however this rescue did not differ between a repair-proficient and a repair-impaired variant of Ogg1. The data indicates that induction of apoptosis in response to oxidative stress in mOgg1−/− MEFs is independent of DNA damage and OGG1-initiated DNA repair.

The SCFSkp2 ubiquitin ligase complex modulates TRAIL-R2-induced apoptosis by regulating FLIP(L)

TRAIL-R2 (DR5) is a clinically-relevant therapeutic target and a key target for immune effector cells. Herein, we identify a novel interaction between TRAIL-R2 and the Skp1-Cullin-1-F-box (SCF) Cullin-Ring E3 Ubiquitin Ligase complex containing Skp2 (SCFSkp2). We find that SCFSkp2 can interact with both TRAIL-R2’s pre-ligand association complex (PLAC) and ligand-activated death-inducing signalling complex (DISC). Moreover, Cullin-1 interacts with TRAIL-R2 in its active NEDDylated form. Inhibiting Cullin-1’s DISC recruitment using the NEDDylation inhibitor MLN4924 (Pevonedistat) or siRNA increased apoptosis induction in response to TRAIL. This correlated with enhanced levels of the caspase-8 regulator FLIP at the TRAIL-R2 DISC, particularly the long splice form, FLIP(L). We subsequently found that FLIP(L) (but not FLIP(S), caspase-8, nor the other core DISC component FADD) interacts with Cullin-1 and Skp2. Importantly, this interaction is enhanced when FLIP(L) is in its DISC-associated, C-terminally truncated p43-form. Prevention of FLIP(L) processing to its p43-form stabilises the protein, suggesting that by enhancing its interaction with SCFSkp2, cleavage to the p43-form is a critical step in FLIP(L) turnover. In support of this, we found that silencing any of the components of the SCFSkp2 complex inhibits FLIP ubiquitination, while overexpressing Cullin-1/Skp2 enhances its ubiquitination in a NEDDylation-dependent manner. DISC recruitment of TRAF2, previously identified as an E3 ligase for caspase-8 at the DISC, was also enhanced when Cullin-1’s recruitment was inhibited, although its interaction with Cullin-1 was found to be mediated indirectly via FLIP(L). Notably, the interaction of p43-FLIP(L) with Cullin-1 disrupts its ability to interact with FADD, caspase-8 and TRAF2. Collectively, our results suggest that processing of FLIP(L) to p43-FLIP(L) at the TRAIL-R2 DISC enhances its interaction with co-localised SCFSkp2, leading to disruption of p43-FLIP(L)’s interactions with other DISC components and promoting its ubiquitination and degradation, thereby modulating TRAIL-R2-mediated apoptosis.

Isobaric tags for relative and absolute quantitation-based quantitative proteomic analysis of X-linked inhibitor of apoptosis and H2AX in etoposide-induced renal cell carcinoma apoptosis.

Background:X-linked inhibitor of apoptosis (XIAP) is a vital factor in the anti-apoptosis mechanism of tumors and is highly expressed in renal cell carcinoma (RCC). However, the mechanism through which XIAP regulates DNA damage repair is unknown. This study investigated the regulatory mechanism of XIAP in etoposide-induced apoptosis in two Caki-1 cell lines with high or low XIAP expression.Methods:The two cell lines were established using RNA interference technology. The differentially expressed proteins in the two cell lines were globally analyzed through an isobaric tags for relative and absolute quantitation-based quantitative proteomics approach. Proteomic analysis revealed 255, 375, 362, and 5 differentially expressed proteins after 0, 0.5, 3, and 12 h of drug stimulation, respectively, between the two cell lines. The identified differentially expressed proteins were involved in numerous biological processes. In addition, the expression of histone proteins (H1.4, H2AX, H3.1, H3.2, and H3.3) was drastically altered, and the effects of XIAP silencing were accompanied by the marked downregulation of H2AX. Protein-protein interactions were assessed and confirmed through immunofluorescence and Western blot analyses.Results:The results suggested that XIAP may act as a vital cell signal regulator that regulates the expression of DNA repair-related proteins, such as H2AX, and influences the DNA repair process.Conclusions:Given these functions, XIAP may be the decisive factor in determining the sensitivity of RCC cell apoptosis induction in response to chemotherapeutic agents.

Physical PEGylation Enhances The Cytotoxicity Of 5-Fluorouracil-Loaded PLGA And PCL Nanoparticles.

PurposeThe main goal of this study is to evaluate the impact of physical incorporation of polyethylene glycol (PEG) into 5-fluorouracil (5-FU)-loaded polymeric nanoparticles (NPs).MethodsThe 5-FU-loaded NPs were prepared utilizing a simple double emulsion method using polycaprolactone (PCL) and polylactic-co-glycolic acid (PLGA) with or without PEG 6000. The surface charge, particle size, and shape of NPs were evaluated by standard procedures. Both Fourier Transform Infrared Spectroscopy and X-ray diffraction spectra of the 5-FU loaded NPs were compared against the pure 5-FU. The in vitro release profile of 5-FU from the NPs was monitored by the dialysis tubing method. Cell death and apoptosis induction in response to 5-FU NP exposure were measured by MTT and Annexin–V/7-amino-actinomycin D (7-AAD) assays, respectively, in Daoy, HepG2, and HT-29 cancer cell lines.ResultsThe 5-FU loaded NPs were found to be spherical in shape with size ranging between 176±6.7 and 253.9±8.6 nm. The zeta potential varied between −7.13± 0.13 and −27.06±3.18 mV, and the entrapment efficiency was between 31.96% and 74.09%. The in vitro release of the drug followed a two-phase mode characterized by rapid release in the first 8 hrs followed by a period of slow release up to 72 hrs with composition-based variable extents. Cells exposed to NPs demonstrated a significant cell death which correlated with the ratio of PEG in the formulations in Daoy and HepG2 cells but not in HT-29 cells. Formulations (F1–F3) significantly induced early apoptosis in HT-29 cell lines.ConclusionThe physical PEGylation significantly enhanced the entrapment and loading efficiencies of 5-FU into NPs formulated with PLGA and PCL. It also fostered the in vitro cytotoxicity of 5-FU-loaded NPs in both Daoy and HepG2 cells. Induction of early apoptosis was confirmed for some of the formulations.

BOK promotes erythropoiesis in a mouse model of myelodysplastic syndrome.

Myelodysplastic syndromes are clonal hematopoietic stem cell disorders characterized by cytopenia and intramedullary apoptosis. BCL-2 Ovarian Killer (BOK) is a pro-apoptotic member of the BCL-2 family of proteins which, when stabilized from endoplasmic reticulum-associated degradation (ERAD), induces apoptosis in response to ER stress. Although ER stress appropriately activates the unfolded protein response (UPR) in BOK-disrupted cells, the downstream effector signaling that includes ATF4 is defective. We used Nup98-HoxD13 (NHD13) transgenic mice to evaluate the consequences of BOK loss on hematopoiesis and leukemogenesis. Acute myeloid leukemia developed in 36.7% of NHD13 mice with a Bok gene knockout between the age of 8 and 13months and presented a similar overall survival to the NHD13 mice. The loss of BOK exacerbated anemia in NHD13 mice, and NHD13/BOK-deficient mice exhibited significantly lower hemoglobin, lower mean cell hemoglobin concentration, and higher mean cell volume than NHD13 mice. Hematopoietic progenitor cell assays revealed a decreased amount of erythroid progenitor stem cells (BFU-E) in the bone marrow of NHD13-transgenic/BOK-deficient mice. RT-qPCR analysis demonstrated decreased mean value of ATF4 in the erythroid progenitors of NHD13 and NHD13/BOK-deficient mice. Our results suggest that in addition to induction of apoptosis in response to ER stress, BOK may regulate erythropoiesis when certain erythroid progenitors experience cell stress.

PS988 CHARACTERIZATION OF A NOVEL, POTENT SMALL MOLECULE MDM2 ANTAGONIST WHICH ACTIVATES WILD‐TYPE P53 AND INDUCES APOPTOSIS IN AML

Background:In the presence of various stress signals, p53 acts as a tumor suppressor by regulating the expression of a multitude of genes to elicit cellular responses such as cell cycle arrest and apoptosis. The activity of p53 is tightly regulated by MDM2, an E3 ubiquitin ligase that acts as a primary inhibitor of p53 function by, for example, targeting p53 for proteasomal degradation. Early studies have demonstrated that blocking the MDM2‐p53 interaction in tumors carrying wild‐type p53 prevents p53 degradation and reactivates it. Small molecule MDM2 antagonists that inhibit the MDM2‐p53 interaction, therefore, present a promising strategy for cancer therapy and a number of these compounds are in clinical development.Aims:Herein, we describe the characterization of a novel, potent small molecule MDM2 antagonist in AML in vitro and in vivo pre‐clinical models and in patient‐derived AML blast cells.Methods:A panel of p53 wild‐type AML cell lines was tested for reduction in cell proliferation using Alamar Blue assay following treatment with the compound. Induction of apoptosis was measured by flow cytometry using a fluorescent caspase‐3 substrate or Annexin V. Target engagement was analyzed by Western Blotting and TaqMan qRT‐PCR. The MV‐4‐11 mouse systemic model was used to test in vivo sensitivity to the compound. Primary AML blasts were isolated from patients using combinations of antibodies against CD34, CD33, CD45, and CD117.Results:We have applied structure‐based design to develop a novel, potent, orally bioavailable MDM2 antagonist. The compound exhibits EC50 &lt;1 nM against the full‐length MDM2 protein in a cell‐free ELISA and increases p53 levels in a wide range of p53 wild‐type cells (e.g. EC50 = 10 nM for p53 induction in SJSA‐1 osteosarcoma cells).When tested in a panel of p53 wild‐type AML cell lines, the compound exerted a strong anti‐proliferative effect with GI50 values of &lt;30 nM being observed in 9 out of 11 cell lines. In contrast, the compound had little effect on p53 mutant KG‐1 cells (GI50 &gt;10 mM). In addition, many of the p53 wild‐type AML cell lines showed a strong induction of apoptosis in response to treatment with the compound. Activation of p53 was evident by an increase in the expression of p53 and that of its well‐known transcriptional targets such as p21 and MDM2. Consistent with these findings, a detailed study of gene expression changes in MV‐4‐11 confirmed clear transcriptional activation of several p53 target genes (CDKN1A, MDM2, BBC3, FAS, GADD45, BAX) 2–6 hours after addition of the compound.In accordance with its potent activity in vitro, the compound displayed significant in vivo efficacy in the MV‐4‐11 mouse systemic model of AML. Here, QDx14 oral dosing at well tolerated doses demonstrated a clear reduction in tumor burden. Furthermore, p53 activation by the compound triggered apoptosis when tested in primary AML blast cells isolated from patients.Summary/Conclusion:Taken together, our findings demonstrate that the compound exhibits potent activity against AML cells that retain wild‐type p53, thus meriting further clinical investigations.

Mode of pP38 and Nf-kand946 Signalling Pathway on Induction of Apoptosis in Response to Flavonoids of Aloe Vera in Glioblastoma

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Studies on phytoconstituents, in vitro antioxidant, antibacterial, antiparasitic, antimicrobial, and anticancer potential of medicinal plant Lasiosiphon eriocephalus decne (Family: Thymelaeaceae)

Background: The present study has been undertaken with an objective to determine the antioxidant, antimicrobial, antiparasitic, and cytotoxic activity of endangered medicinal plant Lasiosiphon eriocephalus. Materials and Methods: Aqueous, methanol, and ethanol extracts of leaves, stem bark, and flowers of the selected medicinal plant were screened for the presence of phenolics, tannins, and flavonoids. In vitro antioxidant activity was evaluated by 1,1-diphenyl-2-picryl hydrazine and 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid) assay. The antiparasitic activity of extracts was tested against protozoan endoparasite Leishmania donovani, and antimicrobial activity plant extracts were determined against Klebsiella pneumonia, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. The anticancer properties of plant extracts were studied by cytotoxicity on cancer cells including HeLa and MCF-7 determined by MTT assay and DNA fragmentation assay. Results: The extracts screened for phytochemical analysis was found to contain phenolics, tannins, and flavonoids. The water and solvent extracts of leaves, and flowers had shown strong antioxidant activity in vitro. The leaves and flower extract showed the ability to inhibit the growth of both Gram-positive as well as Gram-negative bacteria. The results also showed increased antimicrobial and antiparasitic activities of ethanol extracts of flowers then leaves and followed by bark. The present study revealed that the treatment of crude extract of leaves as well as stem bark of L. eriocephalus exhibited strong cytotoxic activity against tested cancer cell lines. Conclusion: These results indicated that the possible secondary metabolites from the crude extract of L. eriocephalous plant have a potential as antioxidant, antiparasitic, antibacterial, and cytotoxic agent against cancer cells. However, further study needs to be carried out to understand the detailed molecular mechanism involved in the inhibition of cell proliferation and induction of apoptosis in response to phytoconstituents derived from L. eriocephalus.

Patulin Induces Autophagy-Dependent Apoptosis through Lysosomal-Mitochondrial Axis and Impaired Mitophagy in HepG2 Cells.

Patulin (PAT) is a compound produced by fungi including those of the Aspergillus, Penicillium, and Byssochlamys species. PAT has been linked with negative outcomes in certain microorganisms and animal species, but how it causes hepatotoxicity is poorly understood. In this study, we determined that, by treating HepG2 cells using PAT, these cells could be induced to rapidly undergo autophagy, and this was followed within 12 h of treatment by lysosomal membrane permeabilization (LMP) and cathepsin B release. We were able to block these outcomes if cells were treated with 3-methyladenine (3MA), an inhibitor of autophagy, prior to PAT treatment. Moreover, PAT-induced collapse of mitochondrial membrane potential (ΔΨm) depended both on cathepsin B and autophagy. 3MA was further able to reduce the induction of apoptosis in response to PAT, suggesting that autophagy is a driving mechanism for this apoptotic induction. Inhibiting cathepsin B using CA-074 Me further reduced PAT-induced collapses of ΔΨm, mitochondiral cytochrome c release, and apoptosis. We also found that extended treatment of HepG2 cells using PAT over a period of 24 h led to the impairment of mitophagy such that morphologically swollen mitochondria accumulated within cells, and PINK1 failed to colocalize with LC3. Together these data reveal that PAT treatment can promote the induction of apoptosis in HepG2 cells in a manner dependent upon autophagy that progresses via the lysosomal-mitochondrial axis. This study thereby affords new insights into the mechanisms by which PAT drives hepatotoxicity.

Radical change of apoptotic strategy following irradiation during later period of embryogenesis in medaka (Oryzias latipes)

Induction of apoptosis in response to various genotoxic stresses could block transmission of teratogenic mutations to progeny cells. The severity of biological effects following irradiation depends on the stage at which embryos are irradiated during embryogenesis. We reported previously that irradiation of medaka embryos 3 days post fertilization (dpf) with 10 Gy of gamma rays induced high incidence of apoptotic cells in the mid-brain, however, the embryos hatched normally and developed without apparent malformations. To determine the severity of biological effects following irradiation during a later period of embryogenesis, embryos of various developmental stages were irradiated with 15 Gy of gamma rays and examined for apoptotic induction at 24 h after irradiation in the brain, eyes and pharyngeal epithelium tissues, which are actively proliferating and sensitive to irradiation. Embryos irradiated at 3 dpf exhibited many apoptotic cells in these tissues, and all of them died due to severe malformations. In contrast, embryos irradiated at 5 dpf showed no apoptotic cells and subsequently hatched without apparent malformations. Embryos irradiated at 4 dpf had relatively low numbers of apoptotic cells compared to those irradiated at 3 dpf, thereafter most of them died within 1 week of hatching. In adult medaka, apoptotic cells were not found in these tissues following irradiation, suggesting that apoptosis occurs during a restricted time period of medaka embryogenesis throughout the life. No apoptotic cells were found in irradiated intestinal tissue, which is known to be susceptible to radiation damage in mammals, whereas many apoptotic cells were found in proliferating spermatogonial cells in the mature testis following irradiation. Taken together, with the exception of testicular tissue, the results suggest a limited period during medaka embryogenesis in which irradiation-induced apoptosis can occur.