Extrinsic Apoptosis Research Articles

Evaluation of CuO nanoparticle toxicity on 3D bioprinted human iPSC-derived cardiac tissues

Particulate matter has been identified as a significant environmental threat to human health. As one of its components, copper oxide nanoparticles (CuO NP) have been found highly potent in cytotoxicity. However, the elucidation of its mechanism is still limited. This study investigated the toxicity of CuO NP toward a cardiac tissue. To better recapitulate the species-specific tissue phenotype and toxin response, we developed a human induced pluripotent stem cells (iPSC)-derived cardiac micro-tissue. With the precise deposition of the cell and scaffold material enabled by rapid 3D bioprinting, the cardiac micro-tissue showed a mature phenotype and was incorporated with a force gauge to enable contraction measurement. We discovered an LD50 of 7.176 g/mL from the CuO NP treatment outcome of the micro-tissue with a downward trend in tissue force as toxicity increased. We also identified mitochondrial damage and activation of extrinsic apoptosis as a significant pathway to mediate the tissue toxicity.

Chemotherapeutic potential of matcha green tea (Camellia sinensis) polyphenols to induce cell-cycle arrest and apoptosis in WERI-Rb-1 retinoblastoma cells

IntroductionCamellia sinensis has been used as a traditional medicine in Southeast Asia to treat various diseases for centuries. However, their efficacy against retinoblastoma has not been thoroughly investigated and characterized. This study aims to determine the cytotoxicity, cell cycle, and apoptotic induction by matcha green tea extract (MGTE) on retinoblastoma cancer cell lines. MethodsThe cytotoxic activity of MGTE was evaluated using MTT assay, and the morphology of cells was observed by fluorescence microscopy. Apoptosis and cell-cycle distribution were determined by propidium iodide (PI) and Annexin V staining, further analyzed through flow cytometry. The effect of MGTE on gene expression of MGTE-treated WERI-Rb-1 cells was investigated using quantitative polymerase chain reaction (qPCR) analysis. ResultsThe results showed that MGTE possessed high cytotoxic activity on WERI-Rb-1 human retinoblastoma cells with IC50 13.3 ± 1.40 µg/mL at 72 h. Flow cytometry results also proved that MGTE induced apoptotic cell death in the sub G0 phase. It was also observed that the chemotherapeutic effect of MGTE occurred via extrinsic and intrinsic apoptosis pathway that was mediated by the activation of caspase 3, caspase 8, caspase 9, Bad, and Bax, which culminated in the apoptosis of retinoblastoma cancer cells. ConclusionThis study suggests that matcha green tea could be used as an apoptosis-inducing anticancer agent for retinoblastoma cancer treatment, with further rigorous studies. The promising baseline information on the chemotherapeutic potential of MGTE provides a potential strategy for shedding light on retinoblastoma treatment.

Caspase 3 Expressions in Children with Acute Lymphoblastic Leukemia During Induction Phase Chemotherapy

Introduction: Caspase-3 is a crucial mediator of the extrinsic apoptosis pathway. The role of caspase-3 for extrinsic apoptosis signalling is still a challenge and should be exploited in childhood ALL. This study aimed to compare the caspase-3 expression in the patient’s bone marrow before and after the induction phase of chemotherapy in childhood ALL. It will also to correlate the mean difference in caspase-3 expression between ALL standard-risk and ALL high-risk patients. Methods: Seventeen newly diagnosed ALL subjects were enrolled in this study. Caspase-3 expression in bone marrow was assessed using flow cytometry and monoclonal antibodies. A T-test and a paired T-test were used to compare between groups. The correlation coefficient between ALL groups was evaluated using Spearman’s test and linear regression with a significant p-value of 0.05. Results: The caspase-3 expression is higher after induction therapy. However, it showed an insignificant difference (16.56+12.91% vs 27.71+12.33%; p = 0.08, p > 0.05). The mean difference of caspase-3 in ALL high-risk groups was significantly higher than in ALL standard-risk groups with a positive correlation (p = 0.007, r = 0.756). Conclusion: The caspase-3 expression after induction phase chemotherapy was increased in all standard-risk and high-risk patients; other lymphoblast apoptosis markers need to be confirmed alongside caspase-3.

Synthesis, in silico modelling, and in vitro biological evaluation of substituted pyrazole derivatives as potential anti-skin cancer, anti-tyrosinase, and antioxidant agents

Twenty-five azole compounds (P1–P25) were synthesised using regioselective base-metal catalysed and microwave-assisted approaches, fully characterised by high-resolution mass spectrometry (HRMS), nuclear magnetic resonance (NMR), and infrared spectra (IR) analyses, and evaluated for anticancer, anti-tyrosinase, and anti-oxidant activities in silico and in vitro. P25 exhibited potent anticancer activity against cells of four skin cancer (SC) lines, with selectivity for melanoma (A375, SK-Mel-28) or non-melanoma (A431, SCC-12) SC cells over non-cancerous HaCaT-keratinocytes. Clonogenic, scratch-wound, and immunoblotting assay data were consistent with anti-proliferative results, expression profiling therewith implicating intrinsic and extrinsic apoptosis activation. In a mushroom tyrosinase inhibition assay, P14 was most potent among the compounds (half-maximal inhibitory concentration where 50% of cells are dead, IC50 15.9 μM), with activity greater than arbutin and kojic acid. Also, P6 exhibited noteworthy free radical-scavenging activity. Furthermore, in silico docking and absorption, distribution, metabolism, excretion, and toxicity (ADMET) simulations predicted prominent-phenotypic actives to engage diverse cancer/hyperpigmentation-related targets with relatively high affinities. Altogether, promising early-stage hits were identified – some with multiple activities – warranting further hit-to-lead optimisation chemistry with further biological evaluations, towards identifying new skin-cancer and skin-pigmentation renormalising agents.

The anticancer effect of Salvia pisidica essential oil through promotion intrinsic and extrinsic apoptosis pathways in human cancer cell lines

IntroductionSpecies of the Salvia genus are aromatic herbs and traditionally used for many medicinal purposes in Anatolia. The genus has produced an extraordinarily large number of beneficial secondary metabolites belonging to numerous chemical groups. Salvia pisidica is an endemic plant of southwest Anatolia, the dried parts of which are used as herbal tea and local materia medica. MethodsThe composition of endemic S. pisidica essential oil was determined through GC-MS studies. In vitro cytotoxicity activity and apoptosis analysis were investigated by MTT assay, q-PCR and Annexin V staining experiments. ResultsOur results showed that 1,8 cineole (19.15 %) camphor (18.12 %) and γ-gurjunene (8.58 %) were the major constituents of the essential oil. It showed cytotoxic effects on the human breast cancer MCF-7, colon carcinoma Caco-2, hepatocyte carcinoma HepG2, and prostate carcinoma LnCap. The results of q-PCR and image-cytometer analysis showed that the essential oil caused induction of apoptosis by intrinsic and extrinsic pathways in all cancer cell lines. ConclusionS. pisidica essential oil demonstrates considerable cytotoxic activity due to an apoptosis-related mechanism, suggesting that their bioactive components could be used as natural anticancer substances.

Selenium deficiency causes oxidative stress and activates inflammation, apoptosis, and necroptosis in the intestine of weaned calves.

Selenium performs a variety of biological functions in organisms, including antioxidant and anti-inflammatory effects. This study investigated how selenium deficiency affects weaned calves' intestines. According to Inductively coupled plasma mass spectrometry (ICP-MS) analysis of intestinal selenium concentrations in calves, the Se-D group had a significantly lower concentration of selenium. Hematoxylin-eosin staining showed that the intestinal epithelial cells were detached, the goblet cells were lost, and the intestinal villi were fragmented and loosely arranged in the Se-D group, along with hyperemia and inflammatory infiltration. Of the 22 selenoprotein genes, 9 were downregulated in response to selenium deficiency in Reverse transcription-PCR (RT-PCR), whereas 6 genes were upregulated. In the Se-D group, oxidative stress was detected by measuring redox levels in the intestines. Furthermore, TdT-mediated dUTP Nick-End Labeling (TUNEL) staining, RT-PCR, and Western blotting (WB) results indicated that both intrinsic and extrinsic apoptosis pathways are activated in the intestine during selenium deficiency. Selenium deficiency also induced necroptosis in the intestine through upregulation of MLKL, RIPK1, and RIPK3 mRNA levels. In addition, according to hematoxylin-eosin staining and ELISA, selenium-deficient calves had severe inflammation in their intestines. As a result of RT-PCR and WB analyses, we found that selenium deficiency was associated with nuclear factor kappa-B (NF-κB) and mitogen-activated protein kinase (MAPK) pathways. Our study suggested that weaned calves' intestines are affected by selenium deficiency, which causes oxidative stress, inflammation, apoptosis, and necroptosis.

HAMLET effect on cell death and mitochondrial respiration in colorectal cancer cell lines with KRAS/BRAF mutations

PurposeTreatment of advanced colorectal cancer (CRC) depends on the correct selection of personalized strategies. HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) is a natural proteolipid milk compound that might serve as a novel cancer prevention and therapy candidate. Our purpose was to investigate HAMLET effect on viability, death pathway and mitochondrial bioenergetics of CRC cells with different KRAS/BRAF mutational status in vitro.MethodsWe treated three cell lines (Caco-2, LoVo, WiDr) with HAMLET to evaluate cell metabolic activity and viability, flow cytometry of apoptotic and necrotic cells, pro- and anti-apoptotic genes, and protein expressions. Mitochondrial respiration (oxygen consumption) rate was recorded by high-resolution respirometry system Oxygraph-2 k.ResultsThe HAMLET complex was cytotoxic to all investigated CRC cell lines and this effect is irreversible. Flow cytometry revealed that HAMLET induces necrotic cell death with a slight increase in an apoptotic cell population. WiDr cell metabolism, clonogenicity, necrosis/apoptosis level, and mitochondrial respiration were affected significantly less than other cells.ConclusionHAMLET exhibits irreversible cytotoxicity on human CRC cells in a dose-dependent manner, leading to necrotic cell death and inhibiting the extrinsic apoptosis pathway. BRAF-mutant cell line is more resistant than other type lines. HAMLET decreased mitochondrial respiration and ATP synthesis in CaCo-2 and LoVo cell lines but did not affect WiDr cells’ respiration. Pretreatment of cancer cells with HAMLET has no impact on mitochondrial outer and inner membrane permeability.

IKK-mediated TRAF6 and RIPK1 interaction stifles cell death complex assembly leading to the suppression of TNF-α-induced cell death.

Tumor necrosis factor α (TNF-α) is a pro-inflammatory cytokine capable of inducing extrinsic apoptosis and necroptosis. Tumor necrosis factor receptor-associated factor 6 (TRAF6), an E3 ligase, is a member of the TRAF family of proteins, which mediates inflammatory signals by activating nuclear factor kappa B (NFкB) and mitogen-activated protein kinase (MAPK). Although the functions of TRAF6 have been identified, its role in TNF-α-induced cell death remains poorly understood. Here, we report that TRAF6 is a negative modulator of TNF-α-induced cell death but does not affect TNF-α-induced NFκB activation. TRAF6 deficiency accelerates both TNF-α-induced apoptosis and necroptosis; however, the acceleration can be reversed by reconstituting TRAF6 or TRAF6C70A, suggesting that E3 ligase activity is not required for this activity. Mechanistically, TRAF6 directly interacts with RIPK1 during TNF-α-induced cell death signaling, which prevents RIPK1 from interacting with components of the cell death complex such as itself, FADD or RIPK3. These processes suppress the assembly of the death complex. Notably, IKK was required for TRAF6 to interact with RIPK1. In vivo, Traf6-/- embryos exhibited higher levels of cell death in the liver but could be rescued by the simultaneous knockout of Tnf. Finally, TRAF6 knockdown xenografts were highly sensitive to necroptotic stimuli. We concluded that TRAF6 suppresses TNF-α-induced cell death in coordination with IKK complexes in vivo and in vitro by suppressing the assembly of cell death complex.

In Vitro Assessment of the Impact of Ultraviolet B Radiation on Oral Healthy and Tumor Cells

Ultraviolet radiation (UVR) is generally considered a primary tumorigenic agent. While UVR exposure has been studied, especially at the skin level, the impact of UV exposure on internal tissues and its effect on the appearance and the development of tumors has not yet been fully examined. Although there are maximum limits for UVR exposure on external tissues, other internal tissues, such as oral tissue, can be exposed to UVR as well. Over the course of diagnosis and treatment, oral cells may be exposed to ultraviolet radiation; however, there has not been an established limit for UV radiation exposure. Therefore, the aim of the current study was to examine the effects of ultraviolet-B (UVB) radiation at two doses (2.5 and 5 J/cm2) on tumor cells (pharyngeal carcinoma and tongue carcinoma) and healthy cells (gingival fibroblasts). The viability of the cells and their morphology, actin filaments, and nuclei structures; the expression of anti-apoptotic (Bcl-2) and pro-apoptotic (Bax) genes; and the roles of caspases-3/7, 8, and 9 were determined after the cells had been exposed to UVB. The experiments revealed that both types of cell lines showed reductions in viability, especially at a dose of 5 J/cm2. Additionally, apoptotic-like changes (rounding of the cells, the condensation of the nuclei, the re-organization of the actin filaments) were observed in all analyzed cells. The expression of anti-apoptotic (Bcl-2) and pro-apoptotic (Bax) genes revealed that UVB (5 J/cm2) may induce apoptosis in both oral tumor and healthy cells. Moreover, an analysis of caspases-3/7, 8, and 9 showed that UVB exposure enhanced their activity, suggesting that cell death could be caused by both intrinsic and extrinsic apoptosis. Accordingly, UVB exposure at the maximum doses used in dental practices (5 J/cm2) induced nonselective apoptotic changes, thereby reducing both tumor and healthy cell viability.

Abstract 6146: Tumor suppressor p73 primes extrinsic apoptosis via transcriptional activation of DR5 and c-FLIP

Abstract The tumor suppressor p73 is a member of p53 family. p73 transcriptionally activates p53-targeted genes involved in cell cycle regulation and apoptosis. It is well known that p73 induces cellular apoptosis via the intrinsic apoptotic pathway upon cellular stresses. However, the role of p73 in extrinsic apoptosis is poorly studied. We observed cell cycle arrested at the G1 phase in cells infected with a p73-expressing recombinant adenovirus (Ad-p73). We observed that overexpression of p73 upregulated c-FLIP at the protein level, in addition to DR5 and p21. c-FLIP is an anti-apoptotic factor that inhibits caspase 8/10 activation, thus blocking extrinsic pathway apoptosis. We demonstrated that c-FLIP is a transcriptional target of p73 based on our observations that: 1) p73 increases c-FLIP at mRNA level, 2) p73 binds to the c-FLIP promoter and 3) p73 enhances c-FLIP promoter-driven luciferase expression. We sought to explore whether the upregulation of c-FLIP prevents extrinsic apoptosis in cancer cells following p73 activation. To address this question, p73 was overexpressed in cells by adenovirus infection and c-FLIP was knocked down by siRNA. Overexpression of p73 induced cellular apoptosis in the cells with knockdown of c-FLIP and this effect was blocked by knockdown of caspase 8. Our results suggest that p73 induces extrinsic apoptosis when c-FLIP is depleted in cancer cells. We further found that the knockdown of DR5 inhibited p73-induced apoptosis in c-FLIP-deficient cancer cells. By contrast, PUMA knockdown did not block p73-induced apoptosis. Our results suggest that DR5 is required for cell extrinsic apoptosis in c-FLIP-deficient cancer cells upon p73 activation. p21 is the cyclin dependent kinase inhibitor that leads to cell cycle arrest at the G1/S phase. Knockdown of p21 enhanced cleaved-caspase 8 and cleaved-PARP in c-FLIP-depleted cancer cells treated with Ad-p73. Our results, taken together, suggest that the p73 primes extrinsic apoptosis via DR5 and the priming level appears to be titrated at the level of c-FLIP. Upregulation of c-FLIP decreases the priming level, resulting in cells converting to cell cycle arrest and survival via p21 whereas reduction of c-FLIP leads the primed cancer cells to undergo extrinsic apoptosis upon p73 treatment in cancer cells. Our discovery of p73 transcriptional upregulation of c-FLIP provides a rationale for depleting c-FLIP to improve antitumor efficacy of p73-targeting cancer therapy. Citation Format: Shengliang Zhang, Wafik S. El-Deiry. Tumor suppressor p73 primes extrinsic apoptosis via transcriptional activation of DR5 and c-FLIP [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6146.

Abstract 393: Determinants of birinapant efficacy in triple negative breast cancer

Abstract Background: Triple-Negative Breast Cancer (TNBC) is an aggressive subtype of breast cancer with few targeted therapies, limiting patients to harsh systemic treatments and resulting in the lowest 5-year survival rate of all breast cancer subtypes. This survival rate plummets after metastasis to another site, making it crucial to identify new therapeutic options for TNBC patients to prevent recurrence and metastasis. The small molecule therapy birinapant, a secondary mitochondrial activator of caspases (SMAC) mimetic, represents a potential new avenue for TNBC treatment. Birinapant initiates apoptotic signaling through targeting of Inhibitor of Apoptosis (IAP) proteins in the Tumor Necrosis Factor Receptor (TNFR) extrinsic apoptosis signaling pathway, leading to the formation of a death complex, caspase activity, and apoptosis. We have found that approximately 25% of TNBC patient-derived xenograft (PDX) and xenograft-derived organoid (PDxO) models are exquisitely sensitive to birinapant treatment. However, the majority of our TNBC patient-derived lines remain resistant through unknown means. We seek to understand the parameters for birinapant response and the mechanism of inherent resistance in TNBC using our patient-derived TNBC models. Approach and Results: Preliminary Western blot experiments revealed that treatment of birinapant-sensitive and resistant PDX tumors in vivo and PDxOs in vitro with birinapant causes degradation of cIAP1 and cIAP2, but only causes apoptosis in birinapant-sensitive lines. Therefore, there may be a disruption of necessary downstream apoptotic signaling in the birinapant-resistant lines. Through follow-up Western blot experiments we determined that at baseline, birinapant-resistant PDxOs do not lack the TNFR extrinsic apoptosis proteins required for apoptosis signaling after IAP degradation, nor is there an abundance of these proteins in the birinapant-sensitive PDxOs. Furthermore, to determine if TNFR extrinsic apoptosis signaling is impaired in birinapant-resistant PDxOs, we treated birinapant-resistant and -sensitive PDxOs with the TNF-a cytokine, which is known to enhance extrinsic apoptosis signaling in the presence of birinapant. Treatment with TNF-a and birinapant did not induce apoptosis in the birinapant-resistant PDxOs, suggesting that the extrinsic apoptosis signaling pathway may be the culprit of resistance in these patient-derived lines. Discussion: Follow-up experiments will address whether an inability to execute extrinsic apoptosis through other death receptor pathways explains birinapant resistance in these models. This work will allow us to uncover what predisposes TNBC patients to be resistant or sensitive to birinapant, and determine how this may be harnessed therapeutically to improve outcomes for TNBC patients. This project was funded in part with federal funds under HHSN261201500003I and U54CA224076. Citation Format: Elisabeth A. Brown, Chieh-Hsiang Yang, Emilio Cortes-Sanchez, Zhengtao Chu, Jeevan Govindharajulu, Ralph Parchment, James Doroshow, Apurva Srivastava, Alana Welm. Determinants of birinapant efficacy in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 393.

Dentatin triggers ROS-mediated apoptosis, G0/G1 cell cycle arrest and release of Th1-related cytokines in colorectal carcinoma cells

Naturally occurring carbazole alkaloid, Dentatin (DTN), is known to inhibit human cancer cell lines. However, it’s inhibitory and immunomodulatory activities in colorectal carcinoma HCT-116 cells remain obscure. This study compares DTN’s inhibitory effect against 5-FU in HCT-116 cells using in vitro anticancer assays including cell cytotoxicity, migration, colony formation, cell cycle and apoptosis, while assessing its immunomodulatory activity via Th1/Th2/Th17 inflammatory cytokines assay. DTN triggered the intrinsic and extrinsic apoptosis pathways by actuating caspases-8, -9 and -3 while inhibiting cell migration and colonies formation, and inducing cell cycle arrest at the G0/G1 phase. Accumulation of iROS with loss of MMP and DNA damage was observed with elevation of several Th1 cytokines, the TNF-α and IFN-γ with lower pro-inflammatory IL-6 compared to 5-fluorouracil. In conclusion, DTN triggered ROS-mediated apoptosis with release of inflammatory cytokines, potentially evoking anticancer immunity in CRC. Further validation of DTN’s mechanism in vivo is warranted.

Antrodin C Isolated from Antrodia Cinnamomea Induced Apoptosis through ROS/AKT/ERK/P38 Signaling Pathway and Epigenetic Histone Acetylation of TNFα in Colorectal Cancer Cells.

Antrodin C, a maleimide derivative compound isolated from the ethanol extract of the mycelium of Antrodia cinnamomea, is an endemic fungus of Taiwan and a potential chemoprotective agent. However, the molecular mechanisms underlying the mode of action of antrodin C on cancer cells, especially in human colorectal cancer (CRC), remain unclear. The cell death and ROS of the antrodin-C-treated HCT-116 cells were measured by annexin V-FITC/propidium iodide staining, DCFDA, and Fluo-3 fluorescence staining assays. Moreover, signaling molecules regulating TNFα cell death pathways and ROS/AKT/ERK/P38 pathways were also detected in cells treated with antrodin C by Western blotting and chromatin immunoprecipitation. The effects of antrodin C were determined in HCT-116 cell xenograft animal models in terms of tumor volumes and histopathological evaluation. Treatment with antrodin C triggered the activation of extrinsic apoptosis pathways (TNFα, Bax, caspase-3, and -9), and also suppressed the expression of anti-apoptotic molecules Bcl-2 in HCT-116 cells in a time-dependent manner. Antrodin C also decreased cell proliferation and growth through the inactivation of cyclin D1/cyclin for the arrest of the cell cycle at the G1 phase. The activation of the ROS/AKT/ERK/P38 pathways was involved in antrodin-C-induced transcriptional activation, which implicates the role of the histone H3K9K14ac (Acetyl Lys9/Lys14) of the TNFα promoters. Immunohistochemical analyses revealed that antrodin C treatment significantly induced TNFα levels, whereas it decreased the levels of PCNA, cyclin D1, cyclin E, and MMP-9 in an in vivo xenograft mouse model. Thus, antrodin C induces cell apoptosis via the activation of the ROS/AKT/ERK/P38 signaling modules, indicating a new mechanism for antrodin C to treat CRC in vitro and in vivo.

Patients with hepatocellular carcinoma that die during the first year of liver transplantation have high blood sFasL concentrations.

Fas ligand (FasL) is one ligand that activates extrinsic apoptosis pathway. High expression in lymphocytes of FasL have been found in patients with acute rejection of liver transplantation (LT). No high blood concentrations of soluble FasL (sFasL) have been found in patients with acute LT rejection; however, the samples size of those studies was small. To determine whether patients with hepatocellular carcinoma (HCC) that dead during the first year of LT have higher blood sFasL concentrations previously to LT that those who that remain alive in a study of higher sample size. Patients underwent LT due to HCC were included in this retrospective study. Serum sFasL levels prior to LT were measured and one-year LT mortality was registered. Non-surviving patients (n = 14) showed higher serum sFasL levels [477 (269-496) vs 85 (44-382) pg/mL; P < 0.001] than surviving patients (n = 113). Serum sFasL levels (pg/mL) were associated with mortality (OR = 1.006; 95%CI = 1.003-1.010; P = 0.001) independently of age of LT donor in the logistic regression analysis. We report for the first time that HCC patients who die within the first year of HT have higher blood sFasL concentrations prior to HT than those who remain alive.

Toxic effects of zinc oxide nanoparticles as a food additive in goat mammary epithelial cells

Zinc oxide nanoparticles (ZnO NPs) have recently been used as food preservatives and additives because of their good antibacterial and nutritional functions. This study performed RNA-seq analyses to evaluate the potential toxicity of ZnO NPs on goat mammary epithelial cells (GMECs) in vitro. Our results suggested that the ZnO NP treatment significantly reduced GMEC viability in a time- and dose-dependent manner. Transcriptomic analysis showed that ZnO NP exposure changed the expression levels of more than 500 genes in GMECs, including various biological pathways. We observed that decreased mitochondrial membrane potential caused mitochondrial dysfunction. Further study indicated that the treatment of cells with ZnO NPs resulted in the accumulation of reactive oxygen species (ROS), which led to oxidative stress. Meanwhile, the expression of genes (TNFα, TNFR1, FADD, Caspase 8 and Caspase 6) associated with the death receptor pathway was upregulated, which indicated the death receptor-mediated extrinsic apoptosis pathway was activated. Moreover, the expression levels of Bax, Cytc, Caspase 3 and Caspase 9 were upregulated, while the expression levels of Bcl2 were downregulated, which indicated mitochondria-mediated intrinsic apoptosis pathway was activated. More notably, ZnO NP exposure increased the expression levels of ER stress-related genes (PERK, ATF4, eIF2α and CHOP) and proteins (p-PERK, p-eIF2α, PERK and CHOP). Furthermore, gene ontology (GO) terms and genes related to autophagy were altered, suggesting that exposure to ZnO NPs might activate autophagy in GMECs. In summary, our findings showed that ZnO NPs could exert significant toxic effects on GMECs through multiple mechanisms. These pathways are related to each other and influence each other to participate in ZnO NPs-induced the damage of GMECs. Thus, their safe use in the feed and food industry should be considered. Meanwhile, RNA-seq might represent a new method of assessing the toxicity mechanisms of nanomaterials.

TUBB4B is a novel therapeutic target in non-alcoholic fatty liver disease-associated hepatocellular carcinoma.

Non-alcoholic fatty liver disease-associated hepatocellular carcinoma (NAFLD-HCC) is an emerging malignancy due to the rising prevalence of NAFLD. However, no drug is available to target NAFLD-HCC. In this study, we aim to unravel novel therapeutic targets of NAFLD-HCC utilizing a high-throughput CRISPR/Cas9 screening strategy. We utilized the Epi-drug CRISPR/Cas9 library consisting of single-guide RNAs (sgRNAs) targeting over 1,000 genes representing the FDA-approved drug targets and epigenetic regulators to perform loss-of-function screening in two NAFLD-HCC cell lines (HKCI2 and HKCI10). CRISPR/Cas9 library screening unraveled TUBB4B as an essential gene for NAFLD-HCC cell growth. TUBB4B was overexpressed in NAFLD-HCC tumors compared with adjacent normal tissues (N = 17) and was associated with poor survival (p < 0.01). RNA-sequencing and functional assays revealed that TUBB4B knockout in NAFLD-HCC promoted cell apoptosis, cell cycle arrest, and cellular senescence, leading to suppressed NAFLD-HCC growth in vitro and in vivo. We identified that TUBB4B inhibitor mebendazole (MBZ), an FDA-approved drug, inhibited NAFLD-HCC growth by inducing apoptosis and cellular senescence. Since protein expression of pro-survival Bcl-xL was induced in TUBB4B knockout NAFLD-HCC cells, we examined combination of TUBB4B inhibition with navitoclax, a Bcl-xL inhibitor that selectively targets senescent cells. Consistent with our hypothesis, either TUBB4B knockout or MBZ synergized with navitoclax to inhibit NAFLD-HCC cell growth via the induction of intrinsic and extrinsic apoptosis pathways. In summary, TUBB4B is a novel therapeutic target in NAFLD-HCC. Inhibition of TUBB4B with MBZ in combination with navitoclax synergistically inhibited NAFLD-HCC cell growth, representing a promising strategy for the treatment of NAFLD-HCC. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Elevated soluble fas blood concentrations in patients dying from spontaneous intracerebral hemorrhage.

Several studies of spontaneous intracerebral hemorrhage (SICH) patients have shown apoptotic changes in brain samples after hematoma evacuation. However, there have been no data on the association between blood concentrations of soluble fas (sFas) (the main surface death receptor of the extrinsic apoptosis pathway) and the prognosis of spontaneous intracranial hypotension (SIH) patients. To determine whether there is an association between blood sFas concentrations and SICH patient mortality. We included patients with severe and supratentorial SIH. Severe was defined as having Glasgow Coma Scale < 9. We determined serum sFas concentrations at the time of severe SICH diagnosis. We found that non-surviving patients (n = 36) compared to surviving patients (n = 39) had higher ICH score (P = 0.001), higher midline shift (P = 0.004), higher serum sFas concentrations (P < 0.001), and lower rate of early hematoma evacuation (P = 0.04). Multiple logistic regression analysis showed an association between serum sFas concentrations and 30-d mortality (odds ratio = 1.070; 95% confidence interval = 1.014-1.129; P = 0.01) controlling for ICH score, midline shift, and early hematoma evacuation. The association of blood sFas concentrations and SICH patient mortality is a novel finding in our study.

Primary and Secondary microRNA Modulation of the Extrinsic Pathway Apoptosis in Hepatocellular Carcinoma

One of the most common malignant liver diseases is hepatocellular carcinoma, which has a high recurrence rate and a low five-year survival rate. It is very heterogeneous both in structure and between patients, which complicates diagnosis, prognosis and response to treatment. In this regard, an individualized, patient-centered approach becomes important, in which the use of mimetics and hsa-miRNA inhibitors involved in the pathogenesis of the disease may be determinative. From this point of view hsa-miRNAs are of interest, their aberrant expression is associated with poor prognosis for patients and is associated with tumor progression due to dysregulation of programmed cell death (apoptosis). However, the effect of hsa-miRNA on tumor development depends not only on its direct effect on expression of genes – primary targets, but also on secondary targets mediated by regulatory pathways. And while the former are actively studied, the role of secondary targets of these hsa-miRNAs in modulating apoptosis is still unclear. The present work summarizes data on hsa-miRNAs whose primary targets are key genes of the extrinsic pathway of apoptosis. Their aberrant expression is associated with early disease relapse and poor patient outcome. For these hsa-miRNAs, using the software package ANDSystem, we reconstructed the regulation of the expression of secondary targets and analyzed their impact on the activity of the extrinsic pathway of apoptosis. The potential effect of hsa-miRNAs mediated by the action on secondary targets is shown to negatively correlate with the number of their primary targets. It is also shown that hsa-miR-373, hsa-miR-106b and hsa-miR-96 have the highest priority as the markers of hepatocellular carcinoma, whose action on the secondary targets enhances their anti-apoptotic effect.

Utilizing mixture design response surface methodology to determine effective combinations of plant derived compounds as prostate cancer treatments.

Prostate cancer (PC) is estimated to cause 13.1% of all new cancer cases in the United States in 2021. Natural bioactive compounds have drawn the interest of researchers worldwide in their efforts to find novel treatments for PC. Many of these bioactive compounds have been identified from traditional Chinese medicine (TCM) remedies often containing multiple bioactive compounds. However, in vitro studies frequently focus on the compounds in isolation. We used mixture design response surface methodology (MDRSM) to assess changes in PC cell viability after 48 h of treatment to identify the optimal mixture of all 35 three-compound combinations of seven bioactive compounds from TCM. We used berberine, wogonin, shikonin, curcumin, triptolide, emodin, and silybin to treat PC3 and LNCaP human PC cells at their IC50 concentrations that we calculated. These compounds modulate many chemotherapeutic pathways including intrinsic and extrinsic apoptosis, increasing reactive oxygen species, decreasing metastatic pathways, inhibiting cell cycle progression. We hypothesize that because these compounds bind to unique molecular targets to activate different chemotherapeutic pathways, they will act synergistically to decrease tumor cell viability. Results from MDRSM showed that two-way combinations were more effective than three-way or single compounds. Most notably wogonin, silybin, emodin and berberine responded well in two-compound combinations with each other in PC3 and LNCaP cells. We then conducted cell viability tests combining two bioactive compound ratios with docetaxel (Doc) and found significant results within the LNCaP cell line. In particular, mixtures of berberine and wogonin, berberine and silybin, emodin and berberine, and emodin and silybin reduced LNCaP cell viability up to an average of 90.02%. The two-compound combinations were significantly better than docetaxel treatment of LNCaP cells. Within the PC3 cells, we show that a combination of berberine, wogonin and docetaxel is just as effective as docetaxel alone. Thus, we provide new combination treatments that are highly effective in vitro for treating androgen-dependent and androgen-independent PC.

CXCR4 enhances the inhibitory effects of bone mesenchymal stem cells on lung cell apoptosis in a rat model of smoking-induced COPD.

Chronic obstructive pulmonary disease is the 3rd leading cause of death worldwide, and the available treatments are unsatisfactory, resulting in a major economic burden. As cellular therapy is commonly used for lung disease, we investigated a treatment with CXCR4-overexpressing BMSCs in a COPD model. We extracted and purified Bone marrow mesenchymal stem cells (BMSCs) from SD rats. COPD apoptosis model was established by cigarette smoke exposure. BMSCs (1 × 106 cells per injection)were transplanted in vivo twice a month during model establishment, and alveolar rupture in the lung was assessed. Lung cell apoptosis was assessed by terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) analysis, and the concentrations of apoptotic proteins in the lungs were detected by Western blotting. We successfully isolated BMSCs and established CXCR4-overexpressing BMSCs. qRT‒PCR and Western blotting detection both reveal that CXCR4 mRNA level and protein both significantly higher expression in CXCR4-BMSCs than the pBABE-BMSCs. Continuous cigarette smoke exposure caused alveolar septal rupture: In the model group, the alveolar mean linear intercept in the first month was significantly lower than that in the third month (p < 0.05). In the third month, the alveolar mean linear intercept values of the control and CXCR4-BMSC groups were lower than those of the model group (control group p < 0.01, CXCR4-BMSC group p < 0.05), and TUNEL staining revealed that the apoptosis rates of the control and CXCR4-BMSC groups were significantly lower than those of the model group (p < 0.01). Furthermore, the levels of the apoptotic proteins cleaved caspase-8, cleaved caspase-3 and cleaved PARP-1 were higher in the model group than in the control group (p < 0.05) and significantly lower in the CXCR4-BMSC group than in the model group (p < 0.05). The transplantation of CXCR4-overexpressing BMSCs during COPD model generation significantly inhibited apoptosis via the extrinsic apoptosis pathway. CXCR4 enhances the inhibitory effects of bone mesenchymal stem cells on lung cell apoptosis in a rat model of smoking-induced COPD.