Difeng Zhu

The PI3K/AKT/MTOR signalling pathway plays an important role in the growth and proliferation of tumour cells. N-((3S,4S)-4-(3,4-Difluorophenyl)piperidin-3-yl)-2-fluoro-4-(1-methyl-1H-pyrazol-5-yl)benzamide (Hu7691) is a new-generation selective AKT inhibitor developed at Zhejiang University. In this study, we developed an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) for the measurement of Hu7691 in dog plasma. Plasma was precipitated with acetonitrile and then separated on a trifunctionally bonded alkyl column. Excellent separation efficiency and selectivity were achieved by adjusting the mobile phase ratio, with a total running time of only 5 min. The linear dynamic range of the calibration curve was 5–1000 ng/mL. The method was fully validated, and all performance metrics met the criteria. The validated method was used for the pharmacokinetic monitoring and bioavailability assessment of Hu7691 in dogs. The results showed that the area under the curve and peak plasma concentration of Hu7691 increased with increasing dose (oral 5, 10, 20 mg/kg, intravenous 10 mg/kg), and oral bioavailabilities were 86.7%, 50.8%, and 50.5%, respectively, indicating a high bioavailability of Hu7691 in dogs. This provides a test basis for the clinical application of the compound.

Forchlorfenuron (CPPU) is a plant growth regulator extensively used in agriculture. However, studies on CPPU pharmacokinetics are lacking. We established and validated a rapid, sensitive, and accurate liquid chromatography–mass spectrometry method for CPPU detection in rat plasma. CPPU pharmacokinetics was evaluated in adult and juvenile rats orally treated with 10, 30, and 90 mg/kg of the compound. The area under the plasma drug concentration–time curve from 0 to 24 h (AUC), at the final time point sampled (AUC_0–t), and the maximum drug concentration of CPPU increased in a dose-dependent manner. The pharmacokinetic parameters AUC_0–t and absolute bioavailability were higher in the juvenile rats than in adult rats. The mean residence time and AUC_0–t of juvenile rats in the gavage groups, except for the 10 mg/kg dose, were significantly higher in comparison to those observed for adult rats (p < 0.001). The plasma clearance of CPPU in juvenile rats was slightly lower than that in the adult rats. Taken together, juvenile rats were more sensitive to CPPU than adult rats, which indicates potential safety risks of CPPU in minors.

Dalbavancin is a novel semisynthetic glycopeptide antibiotic that comprises multiple homologs and isomers of similar polarities. However, pharmacokinetic studies have only analyzed the primary components of dalbavancin, namely B₀ and B₁. In this study, an ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed to simultaneously determinate and investigate the five homologous components of dalbavancin, namely, A₀, A₁, B₀, B₁, and B₂, in rat plasma. In this method, methanol was used to precipitate plasma, and a triple-bonded alkyl chromatographic column was used for molecule separation, using 0.1% formic acid-acetonitrile as the mobile phase for gradient elution. Targeted homologs were analyzed by a triple quadrupole mass spectrometer using positive electrospray ionization in multiple reaction monitoring mode. The linearity range was 50–2500 ng/mL with a high correlation coefficient (r² > 0.998). This method was successfully applied in the pharmacokinetic analysis of dalbavancin hydrochloride to investigate dalbavancin components in rats.

Ferroptosis is a newly characterized form of regulated cell death mediated by iron-dependent accumulation of lipid reactive oxygen species and holds great potential for cancer therapy. However, the molecular mechanisms underlying ferroptosis remain largely elusive. In this study, we define an integrative role of DJ-1 in ferroptosis. Inhibition of DJ-1 potently enhances the sensitivity of tumor cells to ferroptosis inducers both in vitro and in vivo. Metabolic analysis and metabolite rescue assay reveal that DJ-1 depletion inhibits the transsulfuration pathway by disrupting the formation of the S-adenosyl homocysteine hydrolase tetramer and impairing its activity. Consequently, more ferroptosis is induced when homocysteine generation is decreased, which might be the only source of glutathione biosynthesis when cystine uptake is blocked. Thus, our findings show that DJ-1 determines the response of cancer cells to ferroptosis, and highlight a candidate therapeutic target to potentially improve the effect of ferroptosis-based antitumor therapy.

Low progesterone level is always linked with pre-term birth. Therefore, maintaining of progesterone level is vital during pregnancy. Aldo-keto reductase family one member C1 (AKR1C1) catalyzes the reduction of progesterone to its inactive form of 20-alpha-hydroxy-progesterone and thus limits the biological effect of progesterone. In our effort to identify the natural compound that would specifically inhibit AKR1C1, liquiritin was found to be a selective and potent inhibitor of AKR1C1. Kinetic analyses in the S-(+)-1,2,3,4-tetrahydro-1-naphthol (s-tetralol) catalyzed by AKR1C1 in the presence of the inhibitors suggest that liquiritin is a competitive inhibitor by targeting the residues Ala-27, Val-29, Ala-25, and Asn-56 of AKR1C1. In HEC-1-B cells, treatment with liquiritin results in 85.00% of reduction in progesterone metabolism, which is mediated by AKR1C1 enzymatic activity. Overall, our study not only identify liquiritin as an inhibitor against AKR1C1, but also reveal that liquiritin may be served as a potential intervention strategy for preventing pre-term birth caused by low progesterone level.

The purpose of this study is to prove the lipid-regulating effects of neohesperidin (NHP) and explore the potential mechanisms related to fibroblast growth factor 21 (FGF21) and AMP-activated protein kinase (AMPK). Free fatty acids (FFAs)-induced lipid-accumulated HepG₂ cells, acutely egg yolk-induced dyslipidemia and chronically diet-induced obese (DIO) model mice were treated with NHP. Biochemical analyses were carried out to determine the lipid profiles. Western blotting and real-time PCR were employed to analyze FGF21, AMPK and the related proteins or mRNA expressions. Body weight and food intake were measured in DIO mice. siRNA or inhibitors of FGF21 or AMPK were utilized in further study. NHP showed potent hypolipidemic effect in HepG₂ cells loaded with FFAs and reversed the pathological changes of lipid in the acute or chronic dyslipidemia mouse model. It obviously improved the lipid profiles in plasma, liver and gastrocnemius muscles in DIO mice, and led to a significant body weight loss. Simultaneously, FGF21 protein expression or secretion, and AMPK/sirtuin type 1 (SIRT1)/peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α) axis or related molecules, was improved by NHP in HepG₂ cells and/or DIO mice. Furthermore, the siRNA or inhibitor targeting FGF21 or AMPK rejected the triglyceride-lowering effect of NHP. In conclusion, NHP regulates lipid metabolism in vivo and in vitro via FGF21 and AMPK/SIRT1/PGC-1α signaling axis.

Osteosarcoma is the most common bone cancer, and chemotherapy is currently indispensable for its treatment. Adriamycin has been claimed to be the most effective agent for osteosarcoma, however, the outcome of adriamycin chemotherapy remains unsatisfactory. Here, we reported a potent combination therapy that bortezomib, a proteasome inhibitor, enhances adriamycin‐induced apoptosis to eliminate osteosarcoma cells and we revealed that the activation of p‐eIF2α/ATF4/CHOP axis is the underlying associated mechanisms. First, we observed that bortezomib enhances adriamycin‐mediated inhibition of cell proliferation and enhances the apoptosis in osteosarcoma cell lines. Moreover, this drug combination produced more potent tumor‐growth inhibitory effects in human osteosarcoma cell line KHOS/NP xenografts. Our study showed that reactive oxygen species (ROS) plays an important role in apoptosis induced by adriamycin plus bortezomib, whereas ROS scavenger NAC could almost completely block the apoptosis induced by the combination treatment. Meanwhile, p‐eIF2α is remarkably elevated in the combination group. As a result, ATF4 exhibits strong activation which consequently induces the activation of CHOP and leads to the cell death. Finally, 13 primary osteosarcoma cells demonstrated potent response to the combination treatment. In a human osteosarcoma patient‐derived xenograft (PDX) model, our finding suggests that when combined with bortezomib, a relatively low dose of adriamycin produced more potent tumor‐growth inhibitory effects without increased toxicity. Thus, our findings not only provide a promising combination strategy to overcome osteosarcoma but also shed new light on the strategy of combining increased ROS and inhibited proteasome to open up new opportunities for the clinical development of chemotherapy regimens.