Silencing CCT3 induces ferroptosis through the NOD1-NF-κB signaling pathway in bladder cancer.

Bladder cancer (BCa) is a lethal malignancy of the urinary system and exhibits a poor prognosis. Chaperonin-containing tailless complex polypeptide 1 subunit 3 (CCT3) acts as an oncogene in various tumors, whereas its effect on BCa remains unknown. We identified the ferroptosis-associated differentially expressed genes through bioinformatic analysis and selected CCT3 for further verification. The levels of cell viability, apoptosis, migration, invasion, and proliferation were measured to clarify the effect of silencing CCT3 on BCa cells. Then we evaluated the role of CCT3 knockdown in vivo. Ferroptosis was assessed by the expression detection of the ferroptosis-related proteins. The underlying mechanism was predicted by RNA sequencing and verified by an agonist for nucleotide-binding and oligomerization domain 1 (NOD1). Western blotting was conducted to detect the protein expression of NOD1, nuclear factor kappa B (NF-κB) inhibitor alpha (IκBα), and phospho-IκBα (p-IκBα). In vitro, down-regulation of CCT3 suppressed the cell viability, migration, invasion, and proliferation, as well as induced apoptosis of BCa cells. In vivo, silencing CCT3 elevated the body weight of mice and suppressed the BCa progression. In addition, CCT3 knockdown could induce ferroptosis in vitro and in vivo. CCT3 knockdown suppressed the expression of NOD1 and p-IκBα/IκBα and the NOD1 agonist could reverse the effect of CCT3 suppression on BCa in vitro and in vivo. In summary, our findings demonstrate that silencing CCT3 inhibits BCa via induction of ferroptosis and suppression of the NOD1-NF-κB pathway.

Alcohol consumption and liver phenotype of individuals with alpha-1 antitrypsin deficiency.

BACKGROUND AND AIMS: Alpha-1 antitrypsin deficiency is an inherited disorder caused by alpha-1 antitrypsin (AAT) mutations. We analysed the association between alcohol intake and liver-related parameters in individuals with the heterozygous/homozygous Pi*Z AAT variant (Pi*MZ/Pi*ZZ genotype) found in the United Kingdom Biobank and the European Alpha1 liver consortium. METHODS: Reported alcohol consumption was evaluated in two cohorts: (i) the community-based United Kingdom Biobank (17 145 Pi*MZ, 141 Pi*ZZ subjects, and 425 002 non-carriers [Pi*MM]); and (ii) the European Alpha1 liver consortium (561 Pi*ZZ individuals). Cohort (ii) included measurements of carbohydrate-deficient transferrin (CDT). RESULTS: In both cohorts, no/low alcohol intake was reported by >80% of individuals, while harmful consumption was rare (~1%). Among Pi*MM and Pi*MZ individuals from cohort (i), moderate alcohol consumption resulted in a <30% increased rate of elevated transaminases and ~50% increase in elevated gamma-glutamyl transferase values, while harmful alcohol intake led to an at least twofold increase in the abnormal levels. In Pi*ZZ individuals from both cohorts, moderate alcohol consumption had no marked impact on serum transaminase levels. Among Pi*ZZ subjects from cohort (ii) who reported no/low alcohol consumption, those with increased CDT levels more often had signs of advanced liver disease. CONCLUSIONS: Pi*MZ/Pi*ZZ genotype does not seem to markedly aggravate the hepatic toxicity of moderate alcohol consumption. CDT values might be helpful to detect alcohol consumption in those with advanced fibrosis. More data are needed to evaluate the impact of harmful alcohol consumption.

Microgels as Platforms for Antibody-Mediated Cytokine Scavenging.

Therapeutic antibodies are the key treatment option for various cytokine-mediated diseases, such as rheumatoid arthritis, psoriasis, and inflammatory bowel disease. However, systemic injection of these antibodies can cause side effects and suppress the immune system. Moreover, clearance of therapeutic antibodies from the blood is limiting their efficacy. Here, water-swollen microgels are produced with a size of 25 µm using droplet-based microfluidics. The microgels are functionalized with TNFα antibodies to locally scavenge the pro-inflammatory cytokine TNFα. Homogeneous distribution of TNFα-antibodies is shown throughout the microgel network and demonstrates specific antibody-antigen binding using confocal microscopy and FLIM-FRET measurements. Due to the large internal accessibility of the microgel network, its capacity to bind TNFα is extremely high. At a TNFα concentration of 2.5 µg mL-1 , the microgels are able to scavenge 88% of the cytokine. Cell culture experiments reveal the therapeutic potential of these microgels by protecting HT29 colorectal adenocarcinoma cells from TNFα toxicity and resulting in a significant reduction of COX II and IL8 production of the cells. When the microgels are incubated with stimulated human macrophages, to mimic the in vivo situation of inflammatory bowel disease, the microgels scavenge almost all TNFα that is produced by the cells.

Dual proteotoxic stress accelerates liver injury via activation of p62-Nrf2.

Protein accumulation is the hallmark of various neuronal, muscular, and other human disorders. It is also often seen in the liver as a major protein-secretory organ. For example, aggregation of mutated alpha1-antitrypsin (AAT), referred to as PiZ, is a characteristic feature of AAT deficiency, whereas retention of hepatitis B surface protein (HBs) is found in chronic hepatitis B (CHB) infection. We investigated the interaction of both proteotoxic stresses in humans and mice. Animals overexpressing both PiZ and HBs (HBs-PiZ mice) had greater liver injury, steatosis, and fibrosis. Later they exhibited higher hepatocellular carcinoma load and a more aggressive tumor subtype. Although PiZ and HBs displayed differing solubility properties and distinct distribution patterns, HBs-PiZ animals manifested retention of AAT/HBs in the degradatory pathway and a marked accumulation of the autophagy adaptor p62. Isolation of p62-containing particles revealed retained HBs/AAT and the lipophagy adapter perilipin-2. p62 build-up led to activation of the p62-Nrf2 axis and emergence of reactive oxygen species. Our results demonstrate that the simultaneous presence of two prevalent proteotoxic stresses promotes the development of liver injury due to protein retention and activation of the p62-Nrf2 axis. In humans, the PiZ variant was over-represented in CHB patients with advanced liver fibrosis (unadjusted odds ratio = 9.92 [1.15-85.39]). Current siRNA approaches targeting HBs/AAT should be considered for these individuals. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

miR-143 Induces the Apoptosis of Prostate Cancer LNCap Cells by Suppressing Bcl-2 Expression.

BACKGROUND Prostate cancer has become a serious threat to the life of patients. microRNAs are small non-coding RNA molecules that regulate the growth and apoptosis of cells. We aimed to investigate the regulation and mechanism of microRNA (miR-143) in the proliferation and apoptosis of prostate cancer LNCap cells. MATERIAL AND METHODS miR-143 and control scramble miRNA were synthesized and respectively transfected into LNCap cells. The proliferation and apoptosis were detected by MTT assay, flow cytometry, and caspase-3 activity assay. The intracellular expression of Bcl-2 was determined by Western blot. Further, LNCap cells were transfected with small interfering RNA (siRNA) targeting Bcl-2 (siBcl-2) or plasmid expressing Bcl-2, followed by transfection of miR-143 or control miRNA. Bcl-2 expression was detected by Western blot, and cell apoptosis was measured by caspase-3 activity assay. RESULTS Transfection of miR-143 significantly inhibited the proliferation of LNCap cells (P=0.0073), increased the percentage of externalized phosphatidylserine (P=0.0042), activated the caspase-3 (P=0.0012), and decreased the expression of Bcl-2 (P=0.012) when compared with the control miRNA group. The expression of Bcl-2 was significantly reduced after siBcl-2 transfection. The apoptosis in the siBcl-2+miR-143 group was significantly increased compared with that in the miR-143 group (P=0.036), whereas there was no significant difference in the apoptosis between the siBcl-2+miRNA and miRNA groups. The expression of Bcl-2 was obviously higher after the transfection of Bcl-2-expressing plasmid. The apoptosis in Bcl-2+miR-143 group was significantly reduced compared with the miR-143 group (P=0.031), whereas no significant difference in the apoptosis was detected between the miRNA and Bcl-2+miRNA groups. CONCLUSIONS Transfection of miR-143 induces the apoptosis of prostate cancer LNCap cells by down-regulating Bcl-2 expression, suggesting that Bcl-2 might be a potential therapeutic target for prostate cancer.

[Expression of androgen receptor in genital tissue of patients with congenital hypospadias and simple chordee].

OBJECTIVE: To investigate the localization and expression characteristics of androgen receptor (AR) in genital tissue of patients with congenital hypospadias and simple chordee. METHODS: Between August 2005 and January 2007, dorsal prepuce, ventral perimeatal skin, and urethral plate were harvested from 25 patients with congenital hypospadias (aged from 1 year and 11 months to 19 years with an average of 3 years and 7 months) and 4 patients with simple chordee (aged from 3 years and 6 months to 16 years with an average of 7 years and 1 month). Prepuce by circumcision from 18 patients was used as control. The expression intensity and distribution of AR were assessed with immunohistochemistry. RESULTS: AR was expressed in prepuce tissues from congentital hypospadias, simple chordee, and control. The AR positive cell rates were 62.94% +/- 5.40% and 62.87% +/- 5.33% in dorsal and ventral prepuce of control patients respectively, and were 59.00% +/- 3.75%, 58.46% +/- 4.14%, and 52.30% +/- 3.53% in dorsal prepuce, ventral perimeatal skin, and urethral plate of patients with congenital hypospadias respectively. AR positive cell rate was significantly lower in patients with congenital hypospadias than in control patients (P < 0.05), and in urethral plate than in dorsal prepuce and ventral perimeatal skin of patients with congenital hypospadias (P < 0.05), and no significant difference was detected between dorsal and ventral specimens (P > 0.05). Stratified analysis showed a similar expression mode in severe hypospadias group and severe chordee group (P < 0.05). In mild to moderate hypospadias group and mild to moderate chordee group, no significant difference was shown when dorsal and ventral skin specimens were compared to that in normal control (P > 0.05), with AR expression diminished in urethral plate (P < 0.05), and AR decrease was relative to severity of chordee (P < 0.05). The AR positive cell rates were 59.69% +/- 2.73%, 55.71% +/- 1.67%, and 51.92% +/- 1.87% in dorsal, ventral skin, and urethral plate of patients with chordee respectively. Reducing tendency of AR expression was observed. CONCLUSION: AR expression decreases in penile skin of patients with congenital hypospadias and simple chordee, especially in urethral plate.