Use of antioxidant nanoliposomes for co-delivery of PTEN plasmids and plumbagin to induce apoptosis in hepatic cancer cells.

Hepatocellular carcinoma remains a challenging contributor to the global cancer and related mortality, and claims approximately 800,000 deaths each year. Dysregulation or loss of function mutations involving the tumor suppressor gene, phosphatase and tensin homolog deleted on chromosome ten (PTEN), has been well-characterized in various cancers to elicit anomalous cell proliferation and oncogenic transformation. However, the delivery and bioavailability of genes/drugs of interest to carcinomas remains a serious bottleneck behind the success of any anti-cancer formulation. In this study, we have engineered nanoliposomes containing PTEN plasmids, plumbagin, and antioxidant cerium oxide nanoparticles (Lipo-PTEN-Plum) to restore the PTEN expression and inhibit the AKT/PI3K pathway. The Lipo-PTEN-Plum was quasi-spherical in shape with ∼110 nm diameter and ∼64% plumbagin loading efficiency. The Lipo-PTEN-Plum was successfully internalized HepG2 cells, restore PTEN expression and inhibit PI3K/AKT pathway to induce death in cells grown in monolayer and in form of spheroids. Mechanistically, the formulation showed G2/M cell cycle arrest, DNA damage and apoptosis in hepatic cancer cells. Other cellular events such as Caspase-7 overexpression and PI3K (phosphoinositide 3-kinase), AKT (a serine/threonine protein kinase), PARP [Poly (ADP-ribose) polymerases], and mTOR (Mammalian target of rapamycin) inhibition led to the apoptosis in hepatic cancer cells. The mRNA expression profile of PTEN, PI3K, AKT3, Caspase-7, PARP and mTOR proteins, primarily controlling the cancer cell proliferation and apoptosis, suggest that exogenous supply of PTEN could regulate the expression of oncogenic proteins and thus cancer progression.

BV6 enhances apoptosis in Lung cancer cells by ameliorating caspase expressions through attenuation of XIAP, cIAP-1, and cIAP-2 proteins.

Objective: The present study aimed to investigate the inhibitory role of second mitochondria determined activator of caspases mimetic on inhibitor of apoptosis proteins (IAPs) and regulation of caspases in nonsmall cell lung cancer cell line. Materials and Methods: Dimethyl sulfoxide and 3-(4, 5-dimethyl thizol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay was done to determine the IC50 of BV6 using NCI-H23 cell line. The levels of mRNA of X-linked IAP (XIAP), cellular IAP (cIAP-1), cIAP-2, caspase-6, and caspase-7 in H23 cell line were evaluated by a quantitative real-time polymerase chain reaction, while their protein expressions were tested using western blotting. Results: Two doses of BV6 dependently downregulated the expression of mRNA of XIAP (P = 0.002, P= 0.0003 vs. untreated), cIAP-1 (P = 0.05, P = 0.005 vs. untreated), and cIAP-2 (P = 0.001, P = 0.0002 vs. untreated), respectively, while the compound upregulated the mRNA expression of caspase-6 (P = 0.001, P < 0.0001 vs. untreated) and caspase-7 (P = 0.001, P = 0.0004 vs. untreated), respectively. Dose dependent of BV6 treatment significantly decreased the protein level of XIAP (P = 0.003, P = 0.007 vs. untreated), cIAP-1 (P = 0.02, P = 0.01 vs. untreated), and cIAP-2 (P = 0.008,P = 0.008 vs. untreated), respectively. However, the compound increased the protein level of caspase-6 and caspase-7 when compared to untreated control (P = 0.006,P = 0.001) and (P = 0.01, P = 0.001), respectively. Conclusions: The result showed that BV6 treatment reduced the level of mRNA of XIAP, cIAP-1, and cIAP-2 and increased the gene expression of caspase-6 and caspase-7 in NCI-H23 cell line. Therefore, the study revealed that BV6 could be used in future as additional therapeutics in lung cancer.

Downregulation of SOX21-AS1 Alleviated Cisplatin Resistance in Cervical Cancer Through Epithelial-Mesenchymal Transition Inhibition.

Cisplatin is widely used in chemotherapies in cervical cancer (CC). Nevertheless, drug resistance in cancer patients poses a major threat to efficacy of treatment. To explore the underlying modulatory mechanism of SOX21-AS1 in cisplatin resistance in CC cell and mice models, Gepia database was referred for SOX21-AS1 expression in cancer tissues and normal ones. Reverse transcription quantitative real-time polymerase chain reaction was used to measure the differential expression of SOX21-AS1 in parental Siha cells and cisplatin-resistant Siha/DDP cells. Luciferase reporter gene assays were conducted to verify putative bindings between SOX21-AS1 and miR-9-3p. Western blot method was employed to evaluate the changes in cleaved-caspase 7 protein expression. Cisplatin resistance was evaluated in each transfected group using cell counting kit 8 method after cells were exposed to cisplatin (0, 7.5, 15, 30, 60, 120, and 240 µg/mL) for 24 hours. Flow cytometry method was used to measure the apoptosis rates. Cell migration and invasion were measured using Transwell assays. Immunofluorescence method was applied to observe epithelial to mesenchymal transition (EMT) markers, including E-cadherin, Snail, matrix metalloproteinase (MMP)3, and MMP9. Siha/DDP cell groups stably transfected with sh-NC and sh-SOX21-AS1 were injected through tail vein of Balb/C mice. Lung tissue sections were used for hematoxylin and eosin staining and immunohistochemistry analysis. SOX1-AS1 expression was higher in cancer tissues than normal ones and was also higher in Siha/DDP rather than Siha cells. SOX21-AS1 was targeted by miR-9-3p in CC cells. Downregulation of SOX21-AS1 or overexpression of miR-9-3p inhibited cisplatin resistance in Siha/DDP cells and reduced cell invasion and migration and attenuated EMT progression. In vivo, the SOX21-AS1 knockdown led to less severe lung metastasis. Downregulation of SOX21-AS1 alleviated cisplatin resistance in CC through EMT inhibition.

SALIS transcriptionally represses IGFBP3/Caspase-7-mediated apoptosis by associating with STAT5A to promote hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the most common subtype of liver cancer and the second most fatal cancer in the world despite the great therapeutic advances in the past two decades, which reminds us of the gap in fully understanding the oncogenic mechanism of HCC. To explore the key factors contributing to the progression of HCC, we identified a LncRNA, termed SALIS (Suppression of Apoptosis by LINC01186 Interacting with STAT5A), functions in promoting the proliferation, colony formation, migration and invasion while suppressing apoptosis in HCC cells. Mechanistic study indicated SALIS physically associates with transcription factor STAT5A and binds to the promoter regions of IGFBP3 and Caspase-7 to transcriptionally repress their expression and further inhibit apoptosis. Our findings identified SALIS as an oncogene to promote HCC by physically binding with STAT5A to inhibit the expression of pro-apoptotic IGFBP3 and Caspase-7, which suggests novel therapeutic targets for HCC treatments.

Executioner caspases 3 and 7 are dispensable for intestinal epithelium turnover and homeostasis at steady state.

Apoptosis is widely believed to be crucial for epithelial cell death and shedding in the intestine, thereby shaping the overall architecture of the gastrointestinal tract, but also regulating tolerance induction, pinpointing a role of apoptosis intestinal epithelial cell (IEC) turnover and maintenance of barrier function, and in maintaining immune homeostasis. To experimentally address this concept, we generated IEC-specific knockout mice that lack both executioner caspase-3 and caspase-7 (Casp3/7ΔIEC), which are the converging point of the extrinsic and intrinsic apoptotic pathway. Surprisingly, the overall architecture, cellular landscape, and proliferation rate remained unchanged in these mice. However, nonapoptotic cell extrusion was increased in Casp3/7ΔIEC mice, compensating apoptosis deficiency, maintaining the same physiological level of IEC shedding. Microbiome richness and composition stayed unaffected, bearing no sign of dysbiosis. Transcriptome and single-cell RNA sequencing analyses of IECs and immune cells revealed no differences in signaling pathways of differentiation and inflammation. These findings demonstrate that during homeostasis, apoptosis per se is dispensable for IEC turnover at the top of intestinal villi intestinal tissue dynamics, microbiome, and immune cell composition.

Knockout of caspase-7 gene improves the expression of recombinant protein in CHO cell line through the cell cycle arrest in G2/M phase.

BACKGROUND: Chinese hamster ovary cell line has been used routinely as a bioproduction factory of numerous biopharmaceuticals. So far, various engineering strategies have been recruited to improve the production efficiency of this cell line such as apoptosis engineering. Previously, it is reported that the caspase-7 deficiency in CHO cells reduces the cell proliferation rate. But the effect of this reduction on the CHO cell productivity remained unclear. Hence, in the study at hand the effect of caspase-7 deficiency was assessed on the cell growth, viability and protein expression. In addition, the enzymatic activity of caspase-3 was investigated in the absence of caspase-7. RESULTS: Findings showed that in the absence of caspase-7, both cell growth and cell viability were decreased. Cell cycle analysis illustrated that the CHO knockout (CHO-KO) cells experienced a cell cycle arrest in G2/M phase. This cell cycle arrest resulted in a 1.7-fold increase in the expression of luciferase in CHO-KO cells compared to parenteral cells. Furthermore, in the apoptotic situation the enzymatic activity of caspase-3 in CHO-KO cells was approximately 3 times more than CHO-K1 cells. CONCLUSIONS: These findings represented that; however, caspase-7 deficiency reduces the cell proliferation rate but the resulted cell cycle arrest leads to the enhancement of recombinant protein expression. Moreover, increasing in the caspase-3 enzymatic activity compensates the absence of caspase-7 in the caspase cascade of apoptosis.

Knockout of caspase-7 gene improves the expression of recombinant protein in CHO cell line through the cell cycle arrest in G2/M phase

BACKGROUND: Chinese hamster ovary cell line has been used routinely as a bioproduction factory of numerous biopharmaceuticals. So far, various engineering strategies have been recruited to improve the production efficiency of this cell line such as apoptosis engineering. Previously, it is reported that the caspase-7 deficiency in CHO cells reduces the cell proliferation rate. But the effect of this reduction on the CHO cell productivity remained unclear. Hence, in the study at hand the effect of caspase-7 deficiency was assessed on the cell growth, viability and protein expression. In addition, the enzymatic activity of caspase-3 was investigated in the absence of caspase-7. RESULTS: Findings showed that in the absence of caspase-7, both cell growth and cell viability were decreased. Cell cycle analysis illustrated that the CHO knockout (CHO-KO) cells experienced a cell cycle arrest in G2/M phase. This cell cycle arrest resulted in a 1.7-fold increase in the expression of luciferase in CHO-KO cells compared to parenteral cells. Furthermore, in the apoptotic situation the enzymatic activity of caspase-3 in CHO-KO cells was approximately 3 times more than CHO-K1 cells. CONCLUSIONS: These findings represented that; however, caspase-7 deficiency reduces the cell proliferation rate but the resulted cell cycle arrest leads to the enhancement of recombinant protein expression. Moreover, increasing in the caspase-3 enzymatic activity compensates the absence of caspase-7 in the caspase cascade of apoptosis.

The use of HRM shifts in qPCR to investigate a much neglected aspect of interference by intracellular nanoparticles.

Genetic molecular studies used to understand potential risks of engineered nanomaterials (ENMs) are incomplete. Intracellular residual ENMs present in biological samples may cause assay interference. This report applies the high-resolution melt (HRM) feature of RT-qPCR to detect shifts caused by the presence of gold nanoparticles (AuNPs). A universal RNA standard (untreated control) sample was spiked with known amounts of AuNPs and reverse transcribed, where 10 reference genes were amplified. The amplification plots, dissociation assay (melt) profiles, electrophoretic profiles and HRM difference curves were analysed and detected interference caused by AuNPs, which differed according to the amount of AuNP present (i.e. semi-quantitative). Whether or not the assay interference was specific to the reverse transcription or the PCR amplification step was tested. The study was extended to a target gene-of-interest (GOI), Caspase 7. Also, the effect on in vitro cellular samples was assessed (for reference genes and Caspase 7). This method can screen for the presence of AuNPs in RNA samples, which were isolated from biological material in contact with the nanomaterials, i.e., during exposure and risk assessment studies. This is an important quality control procedure to be implemented when quantifying the expression of a GOI from samples that have been in contact with various ENMs. It is recommended to further examine 18S, PPIA and TBP since these were the most reliable for detecting shifts in the difference curves, irrespective of the source of the RNA, or, the point at which the different AuNPs interacted with the assay.

Characterization of caspase-7 interaction with RNA.

Apoptosis is a regulated form of cell death essential to the removal of unwanted cells. At its core, a family of cysteine peptidases named caspases cleave key proteins allowing cell death to occur. To do so, each caspase catalytic pocket recognizes preferred amino acid sequences resulting in proteolysis, but some also use exosites to select and cleave important proteins efficaciously. Such exosites have been found in a few caspases, notably caspase-7 that has a lysine patch (K38KKK) that binds RNA, which acts as a bridge to RNA-binding proteins favoring proximity between the peptidase and its substrates resulting in swifter cleavage. Although caspase-7 interaction with RNA has been identified, in-depth characterization of this interaction is lacking. In this study, using in vitro cleavage assays, we determine that RNA concentration and length affect the cleavage of RNA-binding proteins. Additionally, using binding assays and RNA sequencing, we found that caspase-7 binds RNA molecules regardless of their type, sequence, or structure. Moreover, we demonstrate that the N-terminal peptide of caspase-7 reduces the affinity of the peptidase for RNA, which translates into slower cleavages of RNA-binding proteins. Finally, employing engineered heterodimers, we show that a caspase-7 dimer can use both exosites simultaneously to increase its affinity to RNA because a heterodimer with only one exosite has reduced affinity for RNA and cleavage efficacy. These findings shed light on a mechanism that furthers substrate recognition by caspases and provides potential insight into its regulation during apoptosis.

A large multiethnic GWAS meta-analysis of cataract identifies new risk loci and sex-specific effects.

Cataract is the leading cause of blindness among the elderly worldwide and cataract surgery is one of the most common operations performed in the United States. As the genetic etiology of cataract formation remains unclear, we conducted a multiethnic genome-wide association meta-analysis, combining results from the GERA and UK Biobank cohorts, and tested for replication in the 23andMe research cohort. We report 54 genome-wide significant loci, 37 of which were novel. Sex-stratified analyses identified CASP7 as an additional novel locus specific to women. We show that genes within or near 80% of the cataract-associated loci are significantly expressed and/or enriched-expressed in the mouse lens across various spatiotemporal stages as per iSyTE analysis. Furthermore, iSyTE shows 32 candidate genes in the associated loci have altered gene expression in 9 different gene perturbation mouse models of lens defects/cataract, suggesting their relevance to lens biology. Our work provides further insight into the complex genetic architecture of cataract susceptibility.

A single-cell analytical approach to quantify activated caspase-3/7 during osteoblast proliferation, differentiation, and apoptosis.

The protein heterogeneity at the single-cell level has been recognized to be vital for an understanding of various life processes during animal development. In addition, the knowledge of accurate quantity of relevant proteins at cellular level is essential for appropriate interpretation of diagnostic and therapeutic results. Some low-copy-number proteins are known to play a crucial role during cell proliferation, differentiation, and also in apoptosis. The fate decision is often based on the concentration of these proteins in the individual cells. This is likely to apply also for caspases, cysteine proteases traditionally associated with cell death via apoptosis but recently being discovered also as important factors in cell proliferation and differentiation. The hypothesis was tested in bone-related cells, where modulation of fate from apoptosis to proliferation/differentiation and vice versa is particularly challenging, e.g., towards anti-osteoporotic treatments and anti-cancer strategies. An ultrasensitive and highly selective method based on bioluminescence photon counting was used to quantify activated caspase-3/7 in order to demonstrate protein-level heterogeneity in individual cells within one population and to associate quantitative measurements with different cell fates (proliferation, differentiation, apoptosis). The results indicate a gradual increase of caspase-3/7 activation from the proliferative status to differentiation (more than three times) and towards apoptosis (more than six times). The findings clearly support one of the putative key mechanisms of non-apoptotic functions of pro-apoptotic caspases based on fine-tuning of their activation levels.

Evaluation of the effects of phosphorylation of synthetic peptide substrates on their cleavage by caspase-3 and -7.

Caspases are a family of enzymes that play roles in cell death and inflammation. It has been suggested that in the execution phase of the apoptotic pathway, caspase-3, -6 and -7 are involved. The substrate specificities of two proteases (caspases 3 and 7) are highly similar, which complicates the design of compounds that selectively interact with a single enzyme exclusively. The recognition of residues other than Asp in the P1 position of the substrate by caspase-3/-7 has been reported, promoting interest in the effects of phosphorylation of amino acids in the direct vicinity of the scissile bond. To evaluate conflicting reports on this subject, we synthesized a series of known caspase-3 and -7 substrates and phosphorylated analogs, performed enzyme kinetic assays and mapped the peptide cleavage sites using internally quenched fluorescent peptide substrates. Caspases 3 and 7 will tolerate pSer at the P1 position but only poorly at the P2' position. Our investigation demonstrates the importance of peptide length and composition in interpreting sequence/activity relationships. Based on the results, we conclude that the relationship between caspase-3/-7 and their substrates containing phosphorylated amino acids might depend on the steric conditions and not be directly connected with ionic interactions. Thus, the precise effect of phospho-amino acid residues located in the vicinity of the cleaved bond on the regulation of the substrate specificity of caspases remains difficult to predict. Our observations allow to predict that natural phosphorylated proteins may be cleaved by caspases, but only when extended substrate binding site interactions are satisfied.

The genotoxicity potential of luteolin is enhanced by CYP1A1 and CYP1A2 in human lymphoblastoid TK6 cells.

Luteolin (5,7,3',4'-tetrahydroxyflavone) belongs to the flavone subclass of flavonoids. Luteolin and its glycosides are present in many botanical families, including edible plants, fruits, and vegetables. While the beneficial properties of luteolin have been widely studied, fewer studies have investigated its toxicity. In the present study, using human lymphoblastoid TK6 cells and our newly developed TK6-derived cell lines that each stably express a single human cytochrome P450 (CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C18, 2C9, 2C19, 2D6, 2E1, 3A4, 3A5, and 3A7), we systematically evaluated luteolin-induced cytotoxicity and genotoxicity, and the role of specific CYPs in the bioactivation of luteolin. Treatments with luteolin for 4-24 h induced cytotoxicity, apoptosis, DNA damage, and chromosome damage in a concentration-dependent manner. Subsequently, we observed that luteolin-induced cytotoxicity and genotoxicity, measured by the high-throughput micronucleus assay, were significantly increased in TK6 cells transduced with CYP1A1 and 1A2. In addition, key apoptosis and DNA damage biomarkers, including cleaved PARP-1, cleaved caspase-3, and phosphorylated histone 2AX (γH2A.X), were all significantly increased in the CYP1A1- and 1A2-expressing cells compared with the empty vector controls. Analysis by LC-MS/MS revealed that TK6 cells biotransformed the majority of luteolin into diosmetin, a less toxic O-methylated flavone, after 24 h; the presence of CYP1A1 and 1A2 partially reversed this process. Altogether, these results indicate that metabolism by CYP1A1 and 1A2 enhanced the toxicity of luteolin in vitro. Our results further support the utility of our TK6 cell system for identification of the specific CYPs responsible for chemical bioactivation and toxicity potential.

Synthesis of novel phytol-derived γ-butyrolactones and evaluation of their biological activity.

The synthesis of phytol-derived γ-butyrolactones as well as their evaluation for deterrent activity towards peach-potato aphid Myzus persicae and antiproliferative activity against four selected cancer cell lines are reported. Products were obtained in good yields (19-96%) and their structures were fully characterized by spectroscopic data (NMR, HRMS). Four synthesized δ-halo-γ-lactones (4-7) are new and have not been previously described in the literature. In the choice test phytol (1) appeared deterrent to M. persicae, whereas modifications of its structure did not cause the avoidance of the treated leaves by the aphids. In contrast, aphids were attracted to the leaves treated with the new trans-δ-chloro-γ-lactone (6). Electrical Penetration Graph (EPG) technique applied to explore the aphid probing and feeding activity revealed that neither phytol nor lactone 6 affected aphid probing and the consumption of phloem sap, which means that both phytol and the lactone 6 might have acted as postingestive modifiers of aphid behavior. The results of in vitro antitumor assays showed that obtained phytol derivatives exhibit cytotoxic activity against studied cancer cell lines (leukemia, lung and colon carcinoma and its doxorubicin resistant subline). Halolactones 4-6 were identified as the compounds, which arrest cell cycle of leukemia cells mainly in G2/M and S phases.

Impaired mitochondrial quality control in Rett Syndrome.

Rett Syndrome (RTT) is a rare neurodevelopmental disorder caused in the 95% of cases by mutations in the X-linked MECP2 gene, affecting almost exclusively females. While the genetic basis of RTT is known, the exact pathogenic mechanisms that lead to the broad spectrum of symptoms still remain enigmatic. Alterations in the redox homeostasis have been proposed among the contributing factors to the development and progression of the syndrome. Mitochondria appears to play a central role in RTT oxidative damage and a plethora of mitochondrial defects has already been recognized. However, mitochondrial dynamics and mitophagy, which represent critical pathways in regulating mitochondrial quality control (QC), have not yet been investigated in RTT. The present work showed that RTT fibroblasts have networks of hyperfused mitochondria with morphological abnormalities and increased mitochondrial volume. Moreover, analysis of mitophagic flux revealed an impaired PINK1/Parkin-mediated mitochondrial removal associated with an increase of mitochondrial fusion proteins Mitofusins 1 and 2 (MFN1 and 2) and a decrease of fission mediators including Dynamin related protein 1 (DRP1) and Mitochondrial fission 1 protein (FIS1). Finally, challenging RTT fibroblasts with FCCP and 2,4-DNP did not trigger a proper apoptotic cell death due to a defective caspase 3/7 activation. Altogether, our findings shed light on new aspects of mitochondrial dysfunction in RTT that are represented by defective mitochondrial QC pathways, also providing new potential targets for a therapeutic intervention aimed at slowing down clinical course and manifestations in the affected patients.

Analysis of the lamprey genotype provides insights into caspase evolution and functional divergence.

The cysteine-containing aspartate specific proteinase (caspase) family plays important roles in apoptosis and the maintenance of homeostasis in lampreys. We conducted genomic and functional comparisons of six distinct lamprey caspase groups with human counterparts to determine how these expanded molecules evolved to adapt to the changing caspase-mediated signaling pathways. Our results showed that lineage-specific duplication and rearrangement were responsible for expanding lamprey caspases 3 and 7, whereas caspases 1, 6, 8, and 9 maintained a relatively stable genome and protein structure. Lamprey caspase family molecules displayed various expression patterns and were involved in the innate immune response. Caspase 1 and 7 functioned as a pattern recognition receptor with a broad-spectrum of microbial recognition and bactericidal effect. Additionally, caspases 1 and 7 may induce cell apoptosis in a time- and dose-dependent manner; however, apoptosis was inhibited by caspase inhibitors. Thus, these molecules may reflect the original state of the vertebrates caspase family. Our phylogenetic and functional data provide insights into the evolutionary history of caspases and illustrate their functional characteristics in primitive vertebrates.

Glutathione is a potential therapeutic target for acrolein toxicity in the cornea.

Toxic and volatile chemicals are widely used in household products and previously used as warfare agents, causing a public health threat worldwide. This study aimed to evaluate the extent of injury and mechanisms of acrolein toxicity in the cornea. Primary human corneal stromal fibroblasts cultures (hCSFs) from human donor cornea were cultured and exposed to acrolein toxicity with -/+ N-acetylcysteine (NAC) to study the mode of action in the presence of Buthionine sulphoximine (BSO). PrestoBlue and MTT assays were used to optimize acrolein, NAC, and BSO doses for hCSFs. Cell-based assays and qRT-PCR analyses were performed to understand the acrolein toxicity and mechanisms. Acrolein exposure leads to an increased reactive oxygen species (ROS), compromised glutathione (GSH) levels, and mitochondrial dysfunction. The TUNEL and caspase assays showed that acrolein caused cell death in hCSFs. These deleterious effects can be mitigated using NAC in hCSFs, suggesting that GSH can be a potential target for acrolein toxicity in the cornea.

Role of CASP7 polymorphisms in noise-induced hearing loss risk in Han Chinese population.

Genetic factors and gene-environment interaction may play an important role in the development of noise induced hearing loss (NIHL). 191 cases and 191 controls were selected by case-control study. Among them, case groups were screened from workers exposed to noise in binaural high-frequency hearing thresholds greater than 25 dB (A). Workers with hearing thresholds ≤ 25 dB (A) in any binaural frequency band were selected to the control group, based on matching factors such as age, exposure time to noise, and operating position. The blood samples from two groups of workers were subjected to DNA extraction and SNP sequencing of CASP3 and CASP7 genes using the polymerase chain reaction ligase detection reaction method. Conditional logistic regression correction was used to analyze the genetic variation associated with susceptibility to NIHL. There was an association between rs2227310 and rs4353229 of the CASP7 gene and the risk of NIHL. Compared with the GG genotype, the CC genotype of rs2227310 reduced the risk of NIHL. Compared with CC genotype, the TT genotype of rs4353229 reduced the risk of NIHL. Workers carrying the rs2227310GG and rs4353229CC genotype had an increased risk of NIHL compared to workers without any high-risk genotype. There were additive interaction and multiplication interaction between CASP7rs2227310 and CNE, and the same interaction between CASP7rs4353229 and CNE. The interaction between the CASP7 gene and CNE significantly increased the risk of NIHL. The genetic polymorphisms of CASP7rs2227310GG and CASP7rs4353229CC were associated with an increased risk of NIHL in Han Chinese population and have the potential to act as biomarkers for noise-exposed workers.

Substantial induction of non-apoptotic CD4 T-cell death during the early phase of HIV-1 infection in a humanized mouse model.

Several mechanisms underline induction of CD4 T-cell death by human immunodeficiency virus (HIV) infection. For a long time, apoptosis was considered central to cell death involved in the depletion of CD4 T cells during HIV infection. However, which types of cell death are induced during the early phase of HIV infection in vivo remains unclear. In this study, CD4 T-cell death induced in early HIV infection was characterized using humanized mice challenged with CCR5-tropic (R5) or CXCR4-tropic (X4) HIV-1. Results showed that CD4 T-cell death was induced in the spleen 3 days post-challenge with both R5 and X4 HIV-1. Although cell death without caspase-1 and caspase-3/7 activation was preferentially observed, caspase-1+ pyroptosis was also significantly induced within the memory subpopulation by R5 or X4 HIV-1 and the naïve subpopulation by X4 HIV-1. In contrast, caspase-3/7+ apoptosis was not enhanced by either R5 or X4 HIV-1. Furthermore, phosphorylated mixed lineage kinase domain-like protein+ necroptosis was induced by only X4 HIV-1. These findings indicate that various types of non-apoptotic CD4 T-cell death, such as pyroptosis and necroptosis, are induced during the early phase of HIV infection in vivo.

Combination treatment of Src inhibitor Saracatinib with GMI, a Ganoderma microsporum immunomodulatory protein, induce synthetic lethality via autophagy and apoptosis in lung cancer cells.

Saracatinib is an oral Src-kinase inhibitor and has been studied in preclinical models and clinical trials of cancer therapy. GMI, a fungal immunomodulatory protein from Ganoderma microsporum, possesses antitumor capacity. The aim of this study is to evaluate the cytotoxic effect of combination treatment with saracatinib and GMI on parental and pemetrexed-resistant lung cancer cells. Cotreatment with saracatinib and GMI induced synergistic and additive cytotoxic effect in A549 and A400 cells by annexin V/propidium iodide assay and combination index. Using western blot assay, saracatinib, and GMI combined treatment synergistically induced caspase-7 activation in A549 cells. Different from A549 cells, saracatinib and GMI cotreatment markedly increased LC3B-II in A400 cells. ATG5 silencing abolished the caspase-7 activation and reduced cell death in A549 cells after cotreatment. This is the first study to provide a novel strategy of treating lung cancer with or without drug resistance via combination treatment with GMI and saracatinib.