Esculin induces endoplasmic reticulum stress and drives apoptosis and ferroptosis in colorectal cancer via PERK regulating eIF2α/CHOP and Nrf2/HO-1 cascades.

ETHNOPHARMACOLOGICAL RELEVANCE: Cortex fraxini (also known as Qinpi), the bark of Fraxinus rhynchophylla Hance and Fraxinus stylosa Lingelsh, constitutes a crucial component in several traditional Chinese formulas (e.g., Baitouweng Tang, Jinxiao Formula, etc.) and has demonstrated efficacy in alleviating intestinal carbuncle and managing diarrhea. Cortex fraxini has demonstrated commendable anticancer activity in the realm of Chinese ethnopharmacology; nevertheless, the underlying mechanisms against colorectal cancer (CRC) remain elusive. AIM OF THE STUDY: Esculin, an essential bioactive compound derived from cortex fraxini, has recently garnered attention for its ability to impede viability and induce apoptosis in cancer cells. This investigation aims to assess the therapeutic potential of esculin in treating CRC and elucidate the underlying mechanisms. MATERIALS AND METHODS: The impact of esculin on CRC cell viability was assessed using CCK-8 assay, Annexin V/PI staining, and Western blotting. Various cell death inhibitors, along with DCFH-DA, ELISA, biochemical analysis, and Western blotting, were employed to delineate the modes through which esculin induces HCT116 cells death. Inhibitors and siRNA knockdown were utilized to analyze the signaling pathways influenced by esculin. Additionally, an azomethane/dextran sulfate sodium (AOM/DSS)-induced in vivo CRC mouse model was employed to validate esculin's potential in inhibiting tumorigenesis and to elucidate its underlying mechanisms. RESULTS: Esculin significantly suppressed the viability of various CRC cell lines, particularly HCT116 cells. Investigation with diverse cell death inhibitors revealed that esculin-induced cell death was associated with both apoptosis and ferroptosis. Furthermore, esculin treatment triggered cellular lipid peroxidation, as evidenced by elevated levels of malondialdehyde (MDA) and decreased levels of glutathione (GSH), indicative of its propensity to induce ferroptosis in HCT116 cells. Enhanced protein levels of protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) and p-eIF2α suggested that esculin induced cellular endoplasmic reticulum (ER) stress, subsequently activating the Nrf2/ARE signaling pathway and initiating the transcriptional expression of heme oxygenase (HO)-1. Esculin-induced excessive expression of HO-1 could potentially lead to iron overload in HCT116 cells. Knockdown of Ho-1 significantly attenuated esculin-induced ferroptosis, underscoring HO-1 as a critical mediator of esculin-induced ferroptosis in HCT116 cells. Furthermore, utilizing an AOM/DSS-induced colorectal cancer mouse model, we validated that esculin potentially inhibits the onset and progression of colon cancer by inducing apoptosis and ferroptosis in vivo. CONCLUSIONS: These findings provide comprehensive insights into the dual induction of apoptosis and ferroptosis in HCT116 cells by esculin. The activation of the PERK signaling pathway, along with modulation of downstream eIF2α/CHOP and Nrf2/HO-1 cascades, underscores the mechanistic basis supporting the clinical application of esculin on CRC treatment.

Metabolic heterogeneity protects metastatic mucosal melanomas cells from ferroptosis.

Cancer heterogeneity has been proposed to be one of the main causes of metastatic dissemination and therapy failure. However, the underlying mechanisms of this phenomenon remain poorly understood. Melanoma is an aggressive malignancy with a high heterogeneity and metastatic potential. Therefore, the present study investigated the possible association between cancer heterogeneity and metastasis in melanoma. In total, two novel Chinese oral mucosal melanoma (COMM) cell lines, namely COMM­1 and COMM­2, were established for exploring methods into preventing the loss of cellular heterogeneity caused by long­term cell culture. Each cell line was grown under two different models of culture, which yielded two subtypes, one exhibited an adhesive morphology (COMM­AD), whereas the other was grown in suspension (COMM­SUS). Compared with the COMM­AD cells, the COMM­SUS cells exhibited higher metastatic capacities and autofluorescence. Further investigations indicated that the COMM­SUS cells exhibited metabolic reprogramming by taking up lactate produced by COMM­AD cells at increased levels to accumulate NADH through monocarboxylate transporter 1, whilst also increasing NADPH levels through the pentose phosphate pathway (PPP). Additionally, increased NADH and NADPH levels in the COMM­SUS cells, coupled with the upregulation of the anti­ferroptotic proteins, glutathione peroxidase 4 and ferrop-tosis suppressor protein 1, enabled them to resist ferroptotic cell death induced by oxidative stress during hematogenous dissemination. The inhibition of ferroptosis was found to substantially increase the metastatic capacity of COMM­AD cells. Furthermore, suppressing lactate uptake and impairing PPP activation significantly decreased the metastatic potential of the COMM­SUS cells. Thus, the present study on metabolic heterogeneity in COMM cells potentially provides a novel perspective for exploring this mechanism underlying cancer metastasis.

Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro.

In order to construct an effective and realistic model for tongue squamous cell carcinoma (TSCC) in vitro, the methods were created to produce decellularized tongue extracellular matrix (TEM) which provides functional scaffolds for TSCC construction. TEM provides an in vitro niche for cell growth, differentiation, and cell migration. The microstructures of native extracellular matrix (ECM) and biochemical compositions retained in the decellularized matrix provide tissue-specific niches for anchoring cells. The fabrication of TEM can be realized by deoxyribonuclease (DNase) digestion accompanied with a serious of organic or inorganic pretreatment. This protocol is easy to operate and ensures high efficiency for the decellularization. The TEM showed favorable cytocompatibility for TSCC cells under static or stirred culture conditions, which enables the construction of the TSCC model. A self-made bioreactor was also used for the persistent stirred condition for cell culture. Reconstructed TSCC using TEM showed the characteristics and properties resembling clinical TSCC histopathology, suggesting the potential in TSCC research.

The n-type Ge photodetectors with gold nanoparticles deposited to enhance the responsivity.

Gold nanoparticles (AuNPs) have been deposited on n-type Ge photodetectors to improve the responsivity. Two different coverage ratios, including 10.5 and 30.3% of AuNPs have been prepared, and the fabricated photodetectors are compared with the control sample. The 1,310-nm responsivities at -2 V of the control, 10.5% AuNPs, and 30.3% AuNPs samples are 465, 556, and 623 mA/W, respectively. The AuNPs could increase the responsivities due to the plasmon resonance. The reflectance spectra of these samples have been measured to verify that plasmon resonance contributes to the forward scattering of incident light. The reflectance decreases with AuNP deposition, and a denser coverage results in a smaller reflectance. The smaller reflectance indicates more light could penetrate into the Ge active layer, and it results in a larger responsivity.

Antigenic sites of H1N1 influenza virus hemagglutinin revealed by natural isolates and inhibition assays.

The antigenic sites of hemagglutinin (HA) are crucial for understanding antigenic drift and vaccine strain selection for influenza viruses. In 1982, 32 epitope residues (called laboratory epitope residues) were proposed for antigenic sites of H1N1 HA based on the monoclonal antibody-selected variants. Interestingly, these laboratory epitope residues only cover 28% (23/83) mutation positions for 9 H1N1 vaccine strain comparisons (from 1977 to 2009). Here, we propose the entropy and likelihood ratio to model amino acid diversity and antigenic variant score for inferring 41 H1N1 HA epitope residues (called natural epitope residues) with statistically significant scores according to 1572 HA sequences and 197 pairs of HA sequences with hemagglutination inhibition (HI) assays of natural isolates. By combining both natural and laboratory epitope residues, we identified 62 (11 overlapped) residues clustered into five antigenic sites (i.e., A-E) which are highly correlated to the antigenic sites of H3N2 HA. Our method recognizes sites A, B and C as critical sites for escaping from neutralizing antibodies in H1N1 virus. Experimental results show that the accuracies of our models are 81.2% and 82.2% using 41 and 62 epitope residues, respectively, for predicting antigenic variants on 197 paring HA sequences. In addition, our model can detect the emergence of epidemic strains and reflect the genetic diversity and antigenic variant between the vaccine and circulating strains. Finally, our model is theoretically consistent with the evolution rates of H3N2 and H1N1 viruses and is often consistent to WHO vaccine strain selections. We believe that our models and the inferred antigenic sites of HA are useful for understanding the antigenic drift and evolution of influenza A H1N1 virus.

SH2B1beta enhances fibroblast growth factor 1 (FGF1)-induced neurite outgrowth through MEK-ERK1/2-STAT3-Egr1 pathway.

Genetic studies have established the crucial roles of FGF signaling, FGF-induced gene expression and morphogenesis during embryogenesis. In this study, we showed that overexpressing a signaling adaptor protein, SH2B1beta, enhanced FGF1-induced neurite outgrowth in PC12 cells. SH2B1beta has previously been shown to promote nerve growth factor (NGF) and glial cell line-derived neurotrophic factor (GDNF)-induced neurite outgrowth, in part, through prolonging NGF and GDNF-induced signaling. To delineate how SH2B1beta promotes FGF1-induced neurite outgrowth, we examined its role in FGF1-dependent signaling. Our data suggest that SH2B1beta enhances and prolongs FGF1-induced MEK-ERK1/2 and PI3K-AKT pathways. We also provided the first evidence that FGF1 induces the phosphorylation of signal transducer and activator of transcription 3 (STAT3) at serine 727 [pSTAT3(S727)] in PC12 cells. SH2B1beta enhances this phosphorylation and the expression of the immediate early gene, Egr1. Through inhibitor assays, we have further shown that MEK-ERK1/2 is required for FGF1-induced neurite outgrowth, pSTAT3(S727) and Egr1 expression. Moreover, inhibiting Rho kinase, ROCK, enhances FGF1-induced neurite outgrowth through pSTAT3(S727)-independent manner. Taken together, our results demonstrate, for the first time, that SH2B1beta enhances FGF1-induced neurite outgrowth in PC12 cells mainly through MEK-ERK1/2-STAT3-Egr1 pathway.

An endothelial-cell-enriched primary culture system to study vascular endothelial growth factor (VEGF A) expression in a teleost, the Japanese eel (Anguilla japonica).

A partial gene for eel (Anguilla japonica) vascular endothelial growth factor (VEGF) has been cloned and an endothelial-cell-enriched primary culture derived from rete mirabile established to study regulation of the expression of the eel VEGF gene. Cells were cultured in M199 medium containing 0.1% fetal calf serum (FCS) and serum-free M199 medium for long-and short-term experiments, respectively. Cells were separately treated with cobalt ions (Co2+), basic fibroblast growth factor (bFGF), and estradiol (E2), which have been demonstrated to stimulate mammalian VEGF A expression, followed by quantification of the VEGF mRNA levels by real-time reverse transcription polymerase chain reaction. Our results show that: (1) the deduced eel VEGF protein encoded by the cloned gene is about 130 amino acids in length, and is closely related to a zebrafish (Danio rerio) VEGF A; (2) the endothelial-cell-enriched rete mirabile primary culture containing mainly (over 70%) the capillary endothelial cells; (3) the expression levels of the eel VEGF transcript were increased by Co2+, bFGF, and E2 treatments in a dose-and time-dependent manner. Our data demonstrate that an eel partial VEGF gene has been cloned and its regulation of expression in endothelial-cell-enriched rete mirabile cell culture is similar to that in higher vertebrates.