Both protein kinase C (PKC) and reactive oxygen species (ROS) are well-known signaling messengers cross-talking with each other to activate mitogen-activated protein kinases (MAPKs) for progression of hepatocellular carcinoma (HCC). However, the underlying mechanisms are not well elucidated. Especially, whether mitochondrial ROS (mtROS) is involved and how it triggers MAPK signaling are intriguing. In this study, we found mtROS generation and phosphorylation of MAPKs were mediated by PKCδ in HCCs treated with the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Heat shock protein 60 (HSP60), one of the chaperones in mitochondria was the major protein oxidized in TPA-treated HCCs. Moreover, depletion of HSP60 or expression of HSP60 cysteine mutant prevented TPA-induced phosphorylation of MAPKs. To delineate how HSP60 mediated MAPK activation, the role of Raf kinase inhibitor protein (RKIP), a negative regulator of MAPK, was investigated. TPA dissociated RKIP from HSP60 in both mitochondria and cytosol, concurrently with translocation of HSP60 and MAPK from mitochondria to cytosol, which was associated with robust phosphorylation of MAPKs in the cytosol. Moreover, TPA induced opposite phenotypical changes of HCCs, G1 cell cycle arrest, and cell migration, which were prevented by mtROS scavengers and depletion of PKCδ and HSP60. Consistently, TPA increased the migration-related genes, hydrogen peroxide inducible clone5, matrix metalloproteinase-1/3, lamininγ2, and suppressed the cell cycle regulator cyclin E1 (CCNE1) via PKCδ/mtROS/HSP60/MAPK-axis. Finally, c-jun and c-fos were required for TPA-induced expression of the migration-related genes and a novel microRNA, miR-6134, was responsible for TPA-induced suppression of CCNE1. In conclusion, PKCδ cross-talked with mtROS to trigger HSP60 oxidation for release of RKIP to activate MAPK, regulating gene expression for migration, and G1 cell cycle arrest in HCC. Targeted therapy aiming at key players like PKCδ, RKIP, and HSP60 is promising for preventing HCC progression.

Although butylidenephthalide (BP) is an efficient anticancer drug, its poor bioavailability renders it ineffective for treating drug-resistant brain tumors. However, this problem is overcome through the use of noninvasive delivery systems, including intranasal administration. Herein, the bioavailability, drug stability, and encapsulation efficiency (EE, up to 95%) of BP were improved by using cyclodextrin-encapsulated BP in liposomal formulations (CDD1). The physical properties and EE of the CDD1 system were investigated via dynamic light scattering, transmission electron microscopy, UV–Vis spectroscopy, and nuclear magnetic resonance spectroscopy. The cytotoxicity was examined via MTT assay, and the cellular uptake was observed using fluorescence microscopy. The CDD1 system persisted for over 8 h in tumor cells, which was a considerable improvement in the retention of the BP-containing cyclodextrin or the BP-containing liposomes, thereby indicating a higher BP content in CDD1. Nanoscale CDD1 formulations were administered intranasally to nude mice that had been intracranially implanted with temozolomide-resistant glioblastoma multiforme cells, resulting in increased median survival time. Liquid chromatography–mass spectrometry revealed that drug biodistribution via intranasal delivery increased the accumulation of BP 10-fold compared to oral delivery methods. Therefore, BP/cyclodextrin/liposomal formulations have potential clinical applications for treating drug-resistant brain tumors.

Presenilin-1 (PSEN1) is a crucial subunit within the γ-secretase complex and regulates β-amyloid (Aβ) production. Accumulated evidence indicates that n-butylidenephthalide (BP) acts effectively to reduce Aβ levels in neuronal cells that are derived from trisomy 21 (Ts21) induced pluripotent stem cells (iPSCs). However, the mechanism underlying this effect remains unclear. This article aims to investigate the possible mechanisms through which BP ameliorates the development of Alzheimer’s disease (AD) and verify the effectiveness of BP through animal experiments. Results from RNA microarray analysis showed that BP treatment in Ts21 iPSC-derived neuronal cells reduced long noncoding RNA (lncRNA) CYP3A43-2 levels and increased microRNA (miR)-29b-2-5p levels. Bioinformatics tool prediction analysis, biotin-labeled miR-29b-2-5p pull-down assay, and dual-luciferase reporter assay confirmed a direct negative regulatory effect for miRNA29b-2-5p on lnc-RNA-CYP3A43-2 and PSEN1. Moreover, BP administration improved short-term memory and significantly reduced Aβ accumulation in the hippocampus and cortex of 3xTg-AD mice but failed in miR-29b-2-5p mutant mice generated by CRISP/Cas9 technology. In addition, analysis of brain samples from patients with AD showed a decrease in microRNA-29b-2-5p expression in the frontal cortex region. Our results provide evidence that the LncCYP3A43-2/miR29-2-5p/PSEN1 network might be involved in the molecular mechanisms underlying BP-induced Aβ reduction.

The global rise of multidrug-resistant Enterobacter cloacae strains, especially those that are resistant to carbapenems and produce metallo-β-lactamases, poses a critical challenge in clinical settings owing to limited treatment options. While bacteriophages show promise in treating these infections, their use is hindered by scarce resources and insufficient genomic data. In this study, we isolated ECLFM1, a novel E. cloacae phage, from sewage water using a carbapenem-resistant clinical strain as the host. ECLFM1 exhibited rapid adsorption and a 15-min latent period, with a burst size of approximately 75 PFU/infected cell. Its genome, spanning 172,036 bp, was characterized and identified as a member of Karamvirus. In therapeutic applications, owing to a high multiplicity of infection, ECLFM1 showed increased survival in zebrafish infected with E. cloacae. This study highlights ECLFM1’s potential as a candidate for controlling clinical E. cloacae infections, which would help address challenges in treating multidrug-resistant strains and contribute to the development of alternative treatments.

Background: We explored the mechanisms of cell death induced by isochaihulactone treatment in LNCaP cells.Methods: LNCaP cells were treated with isochaihulactone and growth inhibition was assessed. Cell cycle profiles after isochaihulactone treatment were determined by flow cytometry. Expression levels of cell cycle regulatory proteins, caspase 9, caspase 3, and PARP were determined after isochaihulactone treatment. Signaling pathway was verified by inhibitors pre-treatment. Expression levels of early growth response gene 1 (EGR-1) and nonsteroidal anti-inflammatory drug-activated gene 1 (NAG-1) were determined to investigate their role in LNCaP cell death. NAG-1 expression was knocked down by si-NAG-1 siRNA transfection. Rate of cell death and proliferation were obtained by MTT assay.Results: Isochaihulactone caused cell cycle arrest at G2/M phase in LNCaP cells, which was correlated with an increase of p53 and p21 levels and downregulation of the checkpoint proteins cdc25c, cyclin B1, and cdc2. Bcl-2 phosphorylation and caspase activation were also observed. Isochaihulactone induced phosphorylation of c-Jun-N-terminal kinase (JNK), and JNK inhibitor partially reduced isochaihulactone-induced cell death. Isochaihulactone also induced the expressions of EGR-1 and NAG-1. Expression of NAG-1 was reduced by JNK inhibitor, and knocking down of NAG-1 inhibited isochaihulactone-induced cell death.Conclusions: Isochaihulactone apparently induces G2/M cell cycle arrest via downregulation of cyclin B1 and cdc2, and induces cellular death by upregulation of NAG-1 via JNK activation in LNCaP cells.