Intrinsic Apoptosis Research Articles

Concurrent inhibition of pBADS99 synergistically improves MEK inhibitor efficacy in KRASG12D-mutant pancreatic ductal adenocarcinoma

Therapeutic targeting of KRAS-mutant pancreatic ductal adenocarcinoma (PDAC) has remained a significant challenge in clinical oncology. Direct targeting of KRAS has proven difficult, and inhibition of the KRAS effectors have shown limited success due to compensatory activation of survival pathways. Being a core downstream effector of the KRAS-driven p44/42 MAPK and PI3K/AKT pathways governing intrinsic apoptosis, BAD phosphorylation emerges as a promising therapeutic target. Herein, a positive association of the pBADS99/BAD ratio with higher disease stage and worse overall survival of PDAC was observed. Homology-directed repair of BAD to BADS99A or small molecule inhibition of BADS99 phosphorylation by NCK significantly reduced PDAC cell viability by promoting cell cycle arrest and apoptosis. NCK also abrogated the growth of preformed colonies of PDAC cells in 3D culture. Furthermore, high-throughput screening with an oncology drug library to identify potential combinations revealed a strong synergistic effect between NCK and MEK inhibitors in PDAC cells harboring either wild-type or mutant-KRAS. Mechanistically, both mutant-KRAS and MEK inhibition increased the phosphorylation of BADS99 in PDAC cells, an effect abrogated by NCK. Combined pBADS99-MEK inhibition demonstrated strong synergy in reducing cell viability, enhancing apoptosis, and achieving xenograft stasis in KRAS-mutant PDAC. In conclusion, the inhibition of BADS99 phosphorylation enhances the efficacy of MEK inhibition, and their combined inhibition represents a mechanistically based and potentially effective therapeutic strategy for the treatment of KRAS-mutant PDAC.

Boolean modeling of breast cancer signaling pathways uncovers mechanisms of drug synergy.

Breast cancer is one of the most common types of cancer in females. While drug combinations have shown potential in breast cancer treatments, identifying new effective drug pairs is challenging due to the vast number of possible combinations among available compounds. Efforts have been made to accelerate the process with in silico predictions. Here, we developed a Boolean model of signaling pathways in breast cancer. The model was tailored to represent five breast cancer cell lines by integrating information about cell-line specific mutations, gene expression, and drug treatments. The models reproduced cell-line specific protein activities and drug-response behaviors in agreement with experimental data. Next, we proposed a calculation of protein synergy scores (PSSs), determining the effect of drug combinations on individual proteins' activities. The PSSs of selected proteins were used to investigate the synergistic effects of 150 drug combinations across five cancer cell lines. The comparison of the highest single agent (HSA) synergy scores between experiments and model predictions from the MDA-MB-231 cell line achieved the highest Pearson's correlation coefficient of 0.58 with a great balance among the classification metrics (AUC = 0.74, sensitivity = 0.63, and specificity = 0.64). Finally, we clustered drug pairs into groups based on the selected PSSs to gain further insights into the mechanisms underlying the observed synergistic effects of drug pairs. Clustering analysis allowed us to identify distinct patterns in the protein activities that correspond to five different modes of synergy: 1) synergistic activation of FADD and BID (extrinsic apoptosis pathway), 2) synergistic inhibition of BCL2 (intrinsic apoptosis pathway), 3) synergistic inhibition of MTORC1, 4) synergistic inhibition of ESR1, and 5) synergistic inhibition of CYCLIN D. Our approach offers a mechanistic understanding of the efficacy of drug combinations and provides direction for selecting potential drug pairs worthy of further laboratory investigation.

Inhibition of TNBC Cell Growth by Paroxetine: Induction of Apoptosis and Blockage of Autophagy Flux.

The strategy of drug repurposing has gained traction in the field of cancer therapy as a means of discovering novel therapeutic uses for established pharmaceuticals. Paroxetine (PX), a selective serotonin reuptake inhibitor typically utilized in the treatment of depression, has demonstrated promise as an agent for combating cancer. Nevertheless, the specific functions and mechanisms by which PX operates in the context of triple-negative breast cancer (TNBC) remain ambiguous. This study aimed to examine the impact of PX on TNBC cells in vitro as both a standalone treatment and in conjunction with other pharmaceutical agents. Cell viability was measured using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, apoptosis was assessed through flow cytometry, and the effects on signaling pathways were analyzed using RNA sequencing and Western blot techniques. Furthermore, a subcutaneous tumor model was utilized to assess the in vivo efficacy of combination therapy on tumor growth. The results of our study suggest that PX may activate the Ca2+-dependent mitochondria-mediated intrinsic apoptosis pathway in TNBC by potentially influencing the PI3K/AKT/mTOR pathway as well as by inducing cytoprotective autophagy. Additionally, the combination of PX and chemotherapeutic agents demonstrated moderate inhibitory effects on 4T1 tumor growth in an in vivo model. These findings indicate that PX may exert its effects on TNBC through modulation of critical molecular pathways, offering important implications for improving chemosensitivity and identifying potential therapeutic combinations for clinical use.

Natural 2',4-Dihydroxy-4',6'-dimethoxy Chalcone Isolated from Chromolaena tacotana Inhibits Breast Cancer Cell Growth through Autophagy and Mitochondrial Apoptosis.

Breast cancer (BC) is one of the most common cancers among women. Effective treatment requires precise tailoring to the genetic makeup of the cancer for improved efficacy. Numerous research studies have concentrated on natural compounds and their anti-breast cancer properties to improve the existing treatment options. Chromolaena tacotana (Klatt) R.M. King and H. Rob (Ch. tacotana) is a notable source of bioactive hydroxy-methylated flavonoids. However, the specific anti-BC mechanisms of these flavonoids, particularly those present in the plant's inflorescences, remain partly undefined. This study focuses on assessing a chalcone derivative extracted from Ch. tacotana inflorescences for its potential to concurrently activate regulated autophagy and intrinsic apoptosis in luminal A and triple-negative BC cells. We determined the chemical composition of the chalcone using ultraviolet (UV) and nuclear magnetic resonance (NMR) spectroscopy. Its selective cytotoxicity against BC cell lines was assessed using the MTT assay. Flow cytometry and Western blot analysis were employed to examine the modulation of proteins governing autophagy and the intrinsic apoptosis pathway. Additionally, in silico simulations were conducted to predict interactions between chalcone and various anti-apoptotic proteins, including the mTOR protein. Chalcone was identified as 2',4-dihydroxy-4',6'-dimethoxy-chalcone (DDC). This compound demonstrated a selective inhibition of BC cell proliferation and triggered autophagy and intrinsic apoptosis. It induced cell cycle arrest in the G0/G1 phase and altered mitochondrial outer membrane potential (∆ψm). The study detected the activation of autophagic LC3-II and mitochondrial pro-apoptotic proteins in both BC cell lines. The regulation of Bcl-XL and Bcl-2 proteins varied according to the BC subtype, yet they showed promising molecular interactions with DDC. Among the examined pro-survival proteins, mTOR and Mcl-1 exhibited the most favorable binding energies and were downregulated in BC cell lines. Further research is needed to fully understand the molecular dynamics involved in the activation and interaction of autophagy and apoptosis pathways in cancer cells in response to potential anticancer agents, like the hydroxy-methylated flavonoids from Ch. tacotana.

Bisphenol A regulates bladder cells responses via control of G2/M-phase cell cycle, apoptotic signaling, MAPK pathway, and transcription factor-associated MMP modulation.

Bisphenol A (BPA), an exogenous endocrine-disrupting chemical,is widely used to produce polycarbonate plastics. The widely used BPA has been detected in human urine samples, raising public anxiety about the detrimental effects of BPA on the bladder. In this study, we explored regulatory mechanisms for the adverse effects of BPA in human bladder BdFCand T24 cells. BPA induced extrinsic and intrinsic apoptosis and G2/M cell cycle arrest caused by the ATM-CHK1/CHK2-CDC25c-CDC2 signaling, which ultimately inhibited the growth of human bladder cells. We also found that BPA decreased the binding activity of AP-1 and NF-κB transcription factors in human bladder cells, which inhibited migration and invasion through matrix metallopeptidase-2 and -9 inactivation. Phosphorylation of MAPKs was implicated with BPA-mediated detrimental effects in human bladder cells. Collectively, our results provide a novel explanation for the underlying molecular mechanisms that BPA induces cytotoxicity in human bladder cells.

Intranasal Delivery of Anti-Apoptotic siRNA Complexed with Fas-Signaling Blocking Peptides Attenuates Cellular Apoptosis in Brain Ischemia.

Ischemic stroke-induced neuronal cell death leads to the permanent impairment of brain function. The Fas-mediating extrinsic apoptosis pathway and the cytochrome c-mediating intrinsic apoptosis pathway are two major molecular mechanisms contributing to neuronal injury in ischemic stroke. In this study, we employed a Fas-blocking peptide (FBP) coupled with a positively charged nona-arginine peptide (9R) to form a complex with negatively charged siRNA targeting Bax (FBP9R/siBax). This complex is specifically designed to deliver siRNA to Fas-expressing ischemic brain cells. This complex enables the targeted inhibition of Fas-mediating extrinsic apoptosis pathways and cytochrome c-mediating intrinsic apoptosis pathways. Specifically, the FBP targets the Fas/Fas ligand signaling, while siBax targets Bax involved in mitochondria disruption in the intrinsic pathway. The FBP9R carrier system enables the delivery of functional siRNA to hypoxic cells expressing the Fas receptor on their surface-a finding validated through qPCR and confocal microscopy analyses. Through intranasal (IN) administration of FBP9R/siCy5 to middle cerebral artery occlusion (MCAO) ischemic rat models, brain imaging revealed the complex specifically localized to the Fas-expressing infarcted region but did not localize in the non-infarcted region of the brain. A single IN administration of FBP9R/siBax demonstrated a significant reduction in neuronal cell death by effectively inhibiting Fas signaling and preventing the release of cytochrome c. The targeted delivery of FBP9R/siBax represents a promising alternative strategy for the treatment of brain ischemia.

Research and Development Progression of Oridonin for Hematological Malignancies Therapy.

Oridonin is a tetracyclic diterpenoid compound extracted from the medicinal herb Isodon and related species. Since 1976, studies have reported the significant anti-tumor activity of oridonin in vivo. Recently, an increasing number of studies have confirmed the anti-tumor effects of oridonin in various types of cancers, and its effect on hematological malignancies stands out. Herein, we have systematically reviewed the anti-- tumor effects of oridonin and its specific mechanisms in hematological malignancies, including the regulation of cancer proteins, activation of intrinsic and extrinsic apoptosis signaling pathways, accumulation of reactive oxygen species (ROS), modulation of chaperone proteins and miRNA expression, combination therapy with chemotherapeutic drugs, and the development of its derivatives. Taken together, oridonin exhibits multiple anti-tumor activities and serves as a multi-target agent, making it worthy of further investigation.

Cobalt oxide nanoparticles functionalized with glucose and conjugated to Ellagic acid (Co3O4@Glu-Ellagic acid NPs) induce apoptotic genes in HepG2, liver cancer cell line

Surface modification of metal nanoparticles can be a solution to obtain more efficient and biocompatible drugs. In this study, the anticancer mechanism of cobalt oxide nanoparticles (NPs) functionalized with glucose and conjugated with Ellagic acid (Co3O4@Glu-Ellagic acid NPs) in a liver cancer cell line was investigated. Physicochemical characteristics of NPs were investigated by Fourier-transform infrared spectroscopy (FT-IR), Transmission electron microscopy (TEM), (Scanning Electron Microscopy) SEM, X-Ray diffraction analysis (XRD), Energy Dispersive Spectroscopy mapping (EDS-mapping), Dynamic light scattering (DLS), and zeta potential. Cytotoxicity of Co3O4@Glu-Ellagic acid NPs in normal (THLE2) and cancer (HepG2) hepatocyte cell lines was characterized by MTT and flow cytometry assays. Relative expression of the CASP8, CASP9, and p53 genes was assessed by Real-Time PCR. The synthesized nanoparticles were spherical, without considerable agglomeration, and their size was in the range of 20 to 60 nm. Surface charge and DLS size were − 30.1 mV and 280 nm, respectively. Treatment of the studied cells with the nanoparticles displayed their antiproliferative potential; however, liver cancer cells were considerably more sensitive to the nanoparticles than normal hepatocyte cells. The 50 % inhibitory concentration of Co3O4@Glu-Ellagic acid NPs for normal and cancer hepatocyte cells were 201 and 97 μg/mL, respectively. Treating HepG2 cells with Co3O4@Glu-Ellagic acid NPs led to a significant increase in the percentage of apoptotic cells (from 0.66 % to 50.8 %) and expression of the CASP8, CASP9, and p53 genes by 2.88, 2.11, and 2.0 folds that suggests apoptogenic property of the nanoparticles through activation of the intrinsic and extrinsic apoptosis pathways. Measuring Reactive Oxygen Species (ROS) level showed that in treated cancer cells with Co3O4@Glu-Ellagic acid nanoparticles the ROS level increased. Moreover, Hoechst staining indicated the anti-tumor effect of Co3O4@Glu-Ellagic acid NP by appearance of apoptotic bodies in HepG2 cells. This work revealed that Co3O4@Glu-Ellagic acid NPs are potent apoptogenic agent against liver cancer cells.

Synergistic effects of Bacillus coagulans and Newcastle disease virus on human colorectal adenocarcinoma cell proliferation.

Colorectal cancer (CRC) is a common type of cancer that has a high death rate and is becoming more common in developed countries. Currently, there are several treatment options available for CRC patients, and clinical trials are being conducted to improve conventional therapies. This study investigates the combined impact of Bacillus coagulans (B.C) and Newcastle disease virus (NDV) on the growth of human colorectal adenocarcinoma cells (HT29 cell line). The HT29 cell line was cultured under controlled laboratory conditions. They were treated with Fluorouracil (5-FU), NDV, and B.C., after which various assessments were conducted to determine the effects of these treatments. These assessments included MTT assay for cytotoxicity, evaluation of cell viability, and measurement of caspase 8 and 9 activity levels. The significance of the data was determined at a threshold of P<0.05 following analysis. The usage of NDV and B.C significantly increased cell death and reduced cell growth in the HT29 cell line, when compared to the control group. Moreover, the combined application of NDV and B.C along with 5-FU exhibited a synergistic effect in decreasing the proliferation of HT29 cells. Additionally, the results indicated that intrinsic apoptosis pathway was activated by B.C and NDV. It appears that utilizing oncolytic viruses (OV) and bacteria in conjunction with chemotherapy drugs could potentially aid in reducing the growth of colorectal cancer cells. However, further research is necessary, including animal studies, to confirm the efficacy of this treatment method.

Chemical profile and biological properties of the Piper corcovadense C.DC. essential oil

The essential oil fromPiper corcovadense D.DC. (EOPc), an important plant belonging to the Piperaceae family, which is commonly found in the northern region of Brazil and poorly explored scientifically, was used in this study. Thus, the EOPc was characterized chemically by Gas Chromatography/Mass Spectrometry (GC/MS) and the antioxidant and antimicrobial activities and their potential effects on cutaneous melanoma (SK-MEL-28) and healthy peripheral blood mononuclear (PBMC) cells were determined. The major compounds identified in the EOPc were: trans-sesquisabinene hydrate, trans-caryophyllene, β-pinene, trans-β-farnesene, 14-hydroxycaryophyllene, limonene and p-cymene. The EOPc demonstrated antioxidant activity as evaluated by Folin-Ciocalteu reagent (FC) reducing capacity, DPPH, and ABTS methods. The values found were respectively 5.41 ± 0.17 mg GAE mL−1 (GAE: Gallic acid equivalent), 2.88 ± 0.17 µmol TE mL−1 (TE: Trolox equivalent) and 6.26 ± 0.02 µmol TE mL−1. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined for different bacterial strains. The EOPc at a concentration of 2.61 µg mL−1 exhibited both bactericidal and bacteriostatic properties against Escherichia coli. The EOPc showed potential antitumor activity as it reduced the cell viability of human cutaneous melanoma cells SK-MEL-28. Besides, the EOPc did not exhibit cytotoxic activity against healthy PBMCs, indicating that it does not harm healthy cells at the tested concentrations. The EOPc increased the levels of ROS at concentrations of 250 µg mL−1. The EOPc also did not stimulate the mobilization of endogenous antioxidant defenses, as assessed by total thiol (PSH) and non-protein thiols (NPSH). Thus, the study suggests that the EOPc has antioxidant and antimicrobial properties due to the presence of specific compounds. It also exhibits antitumor potential against cutaneous melanoma cells while showing no cytotoxicity to healthy PBMCs. It directly influenced ROS levels at the highest tested concentration in the cells, suggesting an antitumor effect related to the intrinsic apoptosis pathway. Nevertheless, while the study has initial findings, the results are promising and indicate an attractive biological potential of P. corcovadense, mainly in human cutaneous melanoma cells.

Abstract A074: Targeting dormant disseminated tumor cells and their permissive niche by pro-resolving mediators derived from resolution-phase macrophages

Abstract Metastatic breast cancer can recur years after primary tumor resection and adjuvant therapy and appears to arise from quiescent disseminated tumor cells (QDTC) that persist at distant sites and resist conventional therapies. To date there are no treatments to target QDTCs. Previously, the fibrotic-like microenvironment enriched with Type I collagen (Col-I) was shown to be required for the switch of QDTC to metastatic outgrowth in lungs of mice. Here, we examined whether soluble mediators secreted by ex-vivo generated pro-resolving CD11blow macrophages (CM-Mres) will reinstate resolution of inflammation thus inhibiting the establishment of the fibrotic-like-niche and in turn prevent the emergence of QDTC to metastatic outgrowth. Our findings indicate that CM-Mres promoted immune silencing of alveolar macrophages at the metastatic site where dormant DTCs reside. Immune silencing is one of the hallmarks of resolution of inflammation. Furthermore, CM-Mres inhibited the establishment of a fibrotic-like niche resulting in the inhibition of the metastatic outgrowth in vitro and in vivo. This was due to inhibition of fibroblasts differentiation to myofibroblasts independent of TGFbeta1 canonical signaling and abolishment of Col-I expression. Furthermore, CM-Mres deactivated myofibroblasts as part of the resolution process by inducing an increase in reactive oxygen species (ROS) via activation of NADPH oxidase. This in turn induced DNA damage leading to Go/G1 arrest and intrinsic apoptosis. Similarly, apoptosis mediated by oxidative stress was induced by CM-Mres in dormant and outbreaking QDTCs. Taken together our results demonstrate for the first time that by reinstating resolution of inflammation via physiological mediators, secreted by pro-resolving macrophages, can prevent metastatic outgrowth of QDTCs at their permissive site. Since conventional therapies fail to eradicate dormant DTCs and their transition to clinical metastasis, our findings hold great promise in the quest to identify novel strategies to prevent or treat recurrent metastatic disease. Citation Format: Odelya Gilon, Keren Weidenfeld, Hadell Samara, Yonatan Feuermann, Sagie Schif-Zuck, Sergei Butenko, Edmond Sabo, Amiram Ariel, Dalit Barkan. Targeting dormant disseminated tumor cells and their permissive niche by pro-resolving mediators derived from resolution-phase macrophages [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A074.

Formation of reactive species via high power microwave induced DNA damage and promoted intrinsic pathway-mediated apoptosis in lung cancer cells: An in vitro investigation

Lung cancer continues to be the second most common cancer diagnosed and the main cause of cancer-related death globally, which requires novel and effective treatment strategies. When considering treatment options, non-small cell lung cancer (NSCLC) remained a challenge, seeking new therapeutic strategies. High-power microwave (HPM) progressions have facilitated the advancement of new technologies as well as improvements to those already in use. The impact of HPM on NSCLC has not been investigated before. In this work, we uncovered the effect of pulsed HPM on NSCLC (H460 and A549) for the first time and the most likely underlying mechanisms. Two NSCLC (H460 and A549) cells and lung normal MRC5 were exposed to HPM (15, 30, 45, and 60) pulses (2.1 mJ/pulse). After exposure, the effects were observed at 12, 24, 48, and 72 h. HPM primarily increases the level of intracellular reactive species by a strong electric field of ∼27 kV/cm, which altered NSCLC viability, mitochondrial activity, and death rates. A model for the production of intracellular reactive species by HPM was also presented. NSCLC is found to be affected by HPM through DNA damage (upregulation of ATR/ATM, Chk1/Chk2, and P53) and increased expression of apoptotic markers. NAC scavenger and CPTIO-inhibitor confirm that the reactive species are mainly accountable for cellular effects. In order to ensure suitability for real-world usage, the skin depth was calculated as 30 mm. ROS played a main role in inducing cellular effects, with NO species possibly playing a contributing role. These findings clarify the cellular mechanisms underlying HPM-induced cell death, potentially advancing therapeutic approaches for treating NSCLC, and a useful first step for future investigations in this area. Moreover, this technique has the potential to serve as an adjunct to non-surgical methods in cancer therapy.

Caspase-dependent apoptosis in Ribloflavin transporter deficiency iPSCs and derived motor neurons

Riboflavin Transporter Deficiency (RTD) is a rare genetic, childhood-onset disease. This pathology has a relevant neurological involvement, being characterized by motor symptoms, ponto-bulbar paralysis and sensorineural deafness. Such clinical presentation is associated with muscle weakness and motor neuron (MN) degeneration, so that RTD is considered part of the MN disease spectrum. Based on previous findings demonstrating energy dysmetabolism and mitochondrial impairment in RTD induced Pluripotent Stem cells (iPSCs) and iPSC-derived MNs, here we address the involvement of intrinsic apoptotic pathways in disease pathogenesis using these patient-specific in vitro models by combined ultrastructural and confocal analyses. We show impaired neuronal survival of RTD iPSCs and MNs. Focused Ion Beam/Scanning Electron Microscopy (FIB/SEM) documents severe alterations in patients’ cells, including deranged mitochondrial ultrastructure, and altered plasma membrane and nuclear organization. Occurrence of aberrantly activated apoptosis is confirmed by immunofluorescence and TUNEL assays. Overall, our work provides evidence of a role played by mitochondrial dysfunction in RTD, and identifies neuronal apoptosis as a contributing event in disease pathogenesis, indicating intrinsic apoptosis pathways as possible relevant targets for more effective therapeutical approaches.

The E3 ubiquitin ligase Itch regulates death receptor and cholesterol trafficking to affect TRAIL-mediated apoptosis

The activation of apoptosis signalling by TRAIL (TNF-related apoptosis-inducing ligand) through receptor binding is a fundamental mechanism of cell death induction and is often perturbed in cancer cells to enhance their cell survival and treatment resistance. Ubiquitination plays an important role in the regulation of TRAIL-mediated apoptosis, and here we investigate the role of the E3 ubiquitin ligase Itch in TRAIL-mediated apoptosis in oesophageal cancer cells. Knockdown of Itch expression results in resistance to TRAIL-induced apoptosis, caspase-8 activation, Bid cleavage and also promotes cisplatin resistance. Whilst the assembly of the death-inducing signalling complex (DISC) at the plasma membrane is not perturbed relative to the control, TRAIL-R2 is mis-localised in the Itch-knockdown cells. Further, we observe significant changes to mitochondrial morphology alongside an increased cholesterol content. Mitochondrial cholesterol is recognised as an important anti-apoptotic agent in cancer. Cells treated with a drug that increases mitochondrial cholesterol levels, U18666A, shows a protection from TRAIL-induced apoptosis, reduced caspase-8 activation, Bid cleavage and cisplatin resistance. We demonstrate that Itch knockdown cells are less sensitive to a Bcl-2 inhibitor, show impaired activation of Bax, cytochrome c release and an enhanced stability of the cholesterol transfer protein STARD1. We identify a novel protein complex composed of Itch, the mitochondrial protein VDAC2 and STARD1. We propose a mechanism where Itch regulates the stability of STARD1. An increase in STARD1 expression enhances cholesterol import to mitochondria, which inhibits Bax activation and cytochrome c release. Many cancer types display high mitochondrial cholesterol levels, and oesophageal adenocarcinoma tumours show a correlation between chemotherapy resistance and STARD1 expression which is supported by our findings. This establishes an important role for Itch in regulation of extrinsic and intrinsic apoptosis, mitochondrial cholesterol levels and provides insight to mechanisms that contribute to TRAIL, Bcl-2 inhibitor and cisplatin resistance in cancer cells.

NF‐κB1 promotes miR‐27a‐5p upregulation in acrylamide‐induced toxicity in rats

AbstractAcrylamide (AA) is a neurotoxic and potentially carcinogenic contaminant, usually generated during food processing. MicroRNAs are a class of noncoding small RNAs, which serve essential pathogenic roles in linking exposure to toxic contaminants with their pathological outcomes. Our previous research identified miR‐27a‐5p as a key regulator in AA toxicity, as it induces mitochondria‐dependent apoptosis by targeting Btf3 in rats. However, the mechanisms by which miR‐27a‐5p is regulated to exert its toxic effects remain unclear. Here, the transcription factors that bind to the promoter of miR‐27a‐5p and their potential regulatory functions are investigated in cell lines and rats. The results showed that nuclear factor kappa‐B 1 (NF‐κB1) is a promising transcription factor from bioinformatic analysis. The luciferase reporter assay demonstrated that NF‐κB1 enhances the transcriptional activity of the miR‐27a‐5p promoter, whereas the cleavage under targets and tagmentation assay successfully verified the specific binding sites at −305/−298 bp. Furthermore, the inhibition of NF‐κB1 resulted in the suppression of miR‐27a‐5p expression and its associated target pathways, with additional validation provided by in vivo experimentation. Consequently, the upregulation of miR‐27a‐5p induced by AA and its subsequent intrinsic apoptosis can be attributed to the binding of NF‐κB1 to the miR‐27a‐5p promoter. This binding event may serve as the initial step in the molecular network regulated by miR‐27a‐5p that underlies AA toxicity.

Molecular cloning and characterization of BNIP3 and NIX1/2 and their role in DHA-induced mitophagy and apoptosis in grass carp (Ctenopharyngodon idellus) adipocytes

B-cell lymphoma-2 and adenovirus E1B 19-kDa-interacting protein 3 (BNIP3) and BNIP3 like (BNIP3L or NIX) play a vital role in regulating mitophagy and the intrinsic apoptosis in mammals, but their gene characterizations remain unclear in fish. Herein, bnip3, nix1 and nix2 were isolated and characterized from grass carp (Ctenopharyngodon idellus), which encode peptides of 194, 233 and 222 amino acids, respectively. As typical BH3-only proteins, grass carp BNIP3, NIX1 and NIX2 proteins contain BH3 and C-terminal transmembrane domains for inducing apoptosis. Moreover, the LC3-interacting region motif of BNIP3, NIX1 and NIX2 is also conserved in grass carp. Phylogenetic analyses also demonstrated that nix1 and nix2 may have originated from the genome duplication event. Expression pattern analysis indicated that bnip3, nix1 and nix2 were highest expressed in brain, followed by eye (bnip3) and liver (nix1 and nix2). BNIP3, NIX1 and NIX2 localized to the nucleus and the cytoplasm, with a predominant localization to mitochondria within the cytoplasm. In the present study, we found that 200 μM DHA impaired the mitochondrial function, manifested as the decreased antioxidant ability, cellular ATP content and mitochondrial membrane potential in grass carp adipocytes. In addition, the gene expression and enzyme activities of caspase family were significantly increased in 200 μM DHA group, indicating that adipocyte apoptosis was induced. Meanwhile, DHA increased the gene expression of bnip3, nix1 and nix2 in a dose-dependent manner in grass carp adipocytes. The colocalization of mitochondria and lysosomes was promoted by 200 μM DHA treatment, implying that BNIP3/NIX-related mitophagy was activated in adipocytes. Based on these findings, it can be inferred that BNIP3/NIX-related mitophagy may be involved in the adipocyte apoptosis induced by DHA in grass carp.

IKAROS and AIOLOS directly regulate AP-1 transcriptional complexes and are essential for NK cell development.

Ikaros transcription factors are essential for adaptive lymphocyte function, yet their role in innate lymphopoiesis is unknown. Using conditional genetic inactivation, we show that Ikzf1/Ikaros is essential for normal natural killer (NK) cell lymphopoiesis and IKZF1 directly represses Cish, a negative regulator of interleukin-15 receptor resulting in impaired interleukin-15 receptor signaling. Both Bcl2l11 and BIM levels, and intrinsic apoptosis were increased in Ikzf1-null NK cells, which in part accounts for NK lymphopenia as both were restored to normal levels when Ikzf1 and Bcl2l11 were co-deleted. Ikzf1-null NK cells presented extensive transcriptional alterations with reduced AP-1 transcriptional complex expression and increased expression of Ikzf2/Helios and Ikzf3/Aiolos. IKZF1 and IKZF3 directly bound AP-1 family members and deletion of both Ikzf1 and Ikzf3 in NK cells resulted in further reductions in Jun/Fos expression and complete loss of peripheral NK cells. Collectively, we show that Ikaros family members are important regulators of apoptosis, cytokine responsiveness and AP-1 transcriptional activity.

Phytol and (−)-α-bisabolol Synergistically trigger intrinsic apoptosis through redox and Ca2+ imbalance in non-small cell lung cancer

This study aimed to identify a combination of natural compounds that could enhance lung cancer treatment by targeting multiple pathways and reducing toxicity. The Chow-Talay method's CI value of 0.75 demonstrated that the synergistic combination of phytol and (−)-α-bisabolol (Phy + Bis) exhibited a significant anti-proliferative effect on A549 cells. After 24 h of treatment, Phy + Bis significantly altered the morphology of A549 cells and activated apoptosis. Increased levels of reactive oxygen species (ROS) are evidence that the combination treatments induced oxidative stress. Consequently, there was an increase in intracellular calcium concentration ([Ca2+]i), leading to perturbations in the membrane potential (ΔΨm). Intrinsic apoptosis was triggered by these effects, which resulted in depolarization of the mitochondrial membrane, activation of Cas 9 and Cas 3, upregulation of Bax and Cyt-C, and downregulation of Bcl-2. Phy + Bis also caused G0/G1 phase arrest and cell senescence, with senescent cells expressing more p53. The combined therapy prevented the migration of A549 cells, possibly by interfering with the polymerization of the actin cytoskeleton. Importantly, Phy + Bis did not have any harmful effects on human lung L-132 cells, red blood cells, peripheral blood mononuclear cells, and an in vivo artemia model. Overall, phytol and (−)-α-bisabolol appear to be a promising combination as a safe and effective therapy choice for non-small cell lung cancer.

B4 suppresses lymphoma progression by inhibiting fibroblast growth factor binding protein 1 through intrinsic apoptosis.

Lymphoma positions as the fifth most common cancer, in the world, reporting remarkable deaths every year. Several promising strategies to counter this disease recently include utilizing small molecules that specifically target the lymphoma cellular proteins to overwhelm its progression. FGFBP1 is a soluble intracellular protein that progresses cancer cell proliferation and is upregulated in several cancers. Therefore, inhibiting FGFBP1 could significantly slow down lymphoma progression through triggering apoptosis. Thus, in this study, a flavonoid B4, isolated from Cajanus cajan, has been investigated for its effects of B4 on lymphoma, specifically as an FGFBP1 inhibitor. B4 could selectively hinder the growth of lymphoma cells by inducing caspase-dependent intrinsic apoptosis through G1/S transition phase cell cycle arrest. RNA sequencing analysis revealed that B4 regulates the genes involved in B-cell proliferation and DNA replication by inhibiting FGFBP1 in vitro. B4 increases the survival rate of lymphoma mice. B4 also represses the growth of patient-derived primary lymphoma cells through FGFBP1 inhibition. Drug affinity responsive target stability experimentations authorize that B4 powerfully binds to FGFBP1. The overexpression of FGFBP1 raises the pharmacological sensitivity of B4, supplementing its specific action on lymphoma cells. This study pioneers the estimation of B4 as a possible anticancer agent for lymphoma treatment. These outcomes highlight its selective inhibitory effects on lymphoma cell growth by downregulating FGFBP1 expression through intrinsic apoptosis, causing mitochondrial and DNA damage, ultimately leading to the inhibition of lymphoma progression. These suggest B4 may be a novel FGFBP1 inhibitor for the lymphoma treatment.

Propranolol Treatment Reduces A549-Derived Lung Cancer Spheroids via Intrinsic Apoptosis

Propranolol Treatment Reduces A549-Derived Lung Cancer Spheroids via Intrinsic Apoptosis