RAB31 in glioma-derived endothelial cells promotes glioma cell invasion via extracellular vesicle-mediated enrichment of MYO1C.

Extracellular vesicles (EV), important messengers in intercellular communication, can load and transport various bioactive components and participate in different biological processes. We previously isolated glioma human endothelial cells (GhECs) and found that GhECs, rather than normal human brain endothelial cells (NhECs), exhibit specific enrichment of MYO1C into EVs and promote the migration of glioma cells. In this study, we explored the mechanism by which MYO1C is secreted into EVs. We report that such secretion is dependent on RAB31, RAB27B, and FAS. When expression of RAB31 increases, MYO1C is enriched in secretory EVs. Finally, we identified an EV export mechanism for MYO1C that promotes glioma cell invasion and is dependent on RAB31 in GhECs. In summary, our data indicate that the knockdown of RAB31 can reduce enrichment of MYO1C in extracellular vesicles, thereby attenuating the promotion of glioma cell invasion by GhEC-EVs.

The tumor-enriched small molecule gambogic amide suppresses glioma by targeting WDR1-dependent cytoskeleton remodeling.

Glioma is the most prevalent brain tumor, presenting with limited treatment options, while patients with malignant glioma and glioblastoma (GBM) have poor prognoses. The physical obstacle to drug delivery imposed by the blood‒brain barrier (BBB) and glioma stem cells (GSCs), which are widely recognized as crucial elements contributing to the unsatisfactory clinical outcomes. In this study, we found a small molecule, gambogic amide (GA-amide), exhibited the ability to effectively penetrate the blood-brain barrier (BBB) and displayed a notable enrichment within the tumor region. Moreover, GA-amide exhibited significant efficacy in inhibiting tumor growth across various in vivo glioma models, encompassing transgenic and primary patient-derived xenograft (PDX) models. We further performed a genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) knockout screen to determine the druggable target of GA-amide. By the combination of the cellular thermal shift assay (CETSA), the drug affinity responsive target stability (DARTS) approach, molecular docking simulation and surface plasmon resonance (SPR) analysis, WD repeat domain 1 (WDR1) was identified as the direct binding target of GA-amide. Through direct interaction with WDR1, GA-amide promoted the formation of a complex involving WDR1, MYH9 and Cofilin, which accelerate the depolymerization of F-actin to inhibit the invasion of patient-derived glioma cells (PDCs) and induce PDC apoptosis via the mitochondrial apoptotic pathway. In conclusion, our study not only identified GA-amide as an effective and safe agent for treating glioma but also shed light on the underlying mechanisms of GA-amide from the perspective of cytoskeletal homeostasis.

Gambogic amide inhibits angiogenesis by suppressing VEGF/VEGFR2 in endothelial cells in a TrkA-independent manner.

CONTEXT: Gambogic amide (GA-amide) is a non-peptide molecule that has high affinity for tropomyosin receptor kinase A (TrkA) and possesses robust neurotrophic activity, but its effect on angiogenesis is unclear. OBJECTIVE: The study investigates the antiangiogenic effect of GA-amide on endothelial cells (ECs). MATERIALS AND METHODS: The viability of endothelial cells (ECs) treated with 0.1, 0.15, 0.2, 0.3, 0.4, and 0.5 µM GA-amide for 48 h was detected by MTS assay. Wound healing and angiogenesis assays were performed on cells treated with 0.2 µM GA-amide. Chicken eggs at day 7 post-fertilization were divided into the dimethyl sulfoxide (DMSO), bevacizumab (40 µg), and GA-amide (18.8 and 62.8 ng) groups to assess the antiangiogenic effect for 3 days. mRNA and protein expression in cells treated with 0.1, 0.2, 0.4, 0.8, and 1.2 µM GA-amide for 6 h was detected by qRT-PCR and Western blots, respectively. RESULTS: GA-amide inhibited HUVEC (IC50 = 0.1269 µM) and NhEC (IC50 = 0.1740 µM) proliferation, induced cell apoptosis, and inhibited the migration and angiogenesis at a relatively safe dose (0.2 µM) in vitro. GA-amide reduced the number of capillaries from 56 ± 14.67 (DMSO) to 20.3 ± 5.12 (62.8 ng) in chick chorioallantoic membrane (CAM) assay. However, inactivation of TrkA couldn't reverse the antiangiogenic effect of GA-amide. Moreover, GA-amide suppressed the expression of VEGF and VEGFR2, and decreased activation of the AKT/mTOR and PLCγ/Erk1/2 pathways. CONCLUSIONS: Considering the antiangiogenic effect of GA-amide, it might be developed as a useful agent for use in clinical combination therapies.

PIWIL2 interacting with IKK to regulate autophagy and apoptosis in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is one of the most common malignancies and cause of death from cancer in China. Previous studies showed that autophagy and apoptosis inhibition are critical for the survival of ESCC cells. However, the underlying mechanisms remain to be clarified. Recently, we found that PIWIL2, a novel cancer testis protein, is highly expressed in ESCC and associated with high T-stage and poor 5-year survival rate in patients. Our further study showed that PIWIL2 can directly bind to IKK and promote its phosphorylation, leading to phosphorylation of IκB and subsequently nuclear translocation of NF-κB for apoptosis inhibition. Meanwhile, PIWIL2 competitively inhibits binding of IKK to TSC1, and thus deactivate mTORC1 pathway which suppresses ULK1 phosphorylation and initiation of autophagy. The mouse xenograft model suggested that PIWIL2 can promote ESCC growth in an IKK-dependent manner. This present work firstly revealed that PIWIL2 can play a role in regulating autophagy and apoptosis, and is associated with poor prognosis in ESCC patients, providing novel insights into the roles of PIWIL2 in tumorigenesis.

Contamination profiles and health impact of benzothiazole and its derivatives in PM2.5 in typical Chinese cities.

Although benzothiazole and its derivatives (BTHs) are considered emerging contaminants in diverse environments and organisms, little information is available about their contamination profiles and health impact in ambient particles. In this study, an optimized method of ultrasound-assisted extraction coupled with the selected reaction monitoring (SRM) mode of GC-EI-MS/MS was applied to characterize and analyze PM2.5-bound BTHs from three cities of China (Guangzhou, Shanghai, and Taiyuan) during the winter of 2018. The total BTH concentration (ΣBTHs) in PM2.5 samples from the three cities decreased in the order of Guangzhou > Shanghai > Taiyuan, independently of the PM2.5 concentration. Despite the large variation in concentration of ΣBTHs in PM2.5, 2-hydroxybenzothiazole (OTH) was always the predominant compound among the PM2.5-bound BTHs and accounted for 50-80% of total BTHs in the three regions. Results from human exposure assessment and toxicity screening indicated that the outdoor exposure risk of PM2.5-bound BTHs in toddlers was much higher than in adults, especially for OTH. The developmental and reproduction toxicity of OTH was further explored in vivo and in vitro. Exposure of mouse embryonic stem cells (mESCs) to OTH for 48 h significantly increased the intracellular reactive oxygen species (ROS) and induced DNA damage and apoptosis via the functionally activating p53 expression. In addition, the growth and development of zebrafish embryos were found to be severely affected after OTH treatment. An overall metabolomics study was conducted on the exposed zebrafish larvae. The results indicated that exposure to OTH inhibited the phenylalanine hydroxylation reaction, which further increased the accumulation of toxic phenylpyruvate and acetylphenylalanine in zebrafish. These findings provide important insights into the contamination profiles of PM2.5-bound BTHs and emphasize the health risk of OTH.

PIWIL2 stabilizes ß-catenin to promote cell cycle and proliferation in tumor cells.

PIWIL2 belongs to the PIWI protein subfamily and is widely expressed in a variety of tumors. Previous studies have shown that PIWIL2 has the characteristics of oncogene. Recently we reported that PIWIL2 suppresses GSK3ß activity to regulate circadian rhythms through SRC-PI3K-AKT pathway. As GSK3ß is a key part of the ß-catenin destruction complex, it plays a vital role in regulating the degradation of ß-catenin. Besides, the activated ß-catenin/CyclinD1 pathway is involved in the proliferation of tumor cells. It is intriguing to investigate whether PIWIL2 regulates ß-catenin and downstream pathway. In this study, we found that PIWIL2 suppressed GSK3ß induced phosphorylation and ubiquitination of ß-catenin, and thus increased ß-catenin accumulation in the nucleus. By up-regulating ß-catenin and CyclinD1, PIWIL2 can promote cell cycle and proliferation in tumor cells. Taken together, our results revealed a novel function of PIWIL2 in regulating ß-catenin/CyclinD1 pathway in tumor cells, providing a new perspective for PIWIL2 as an oncogene.

RAE1 promotes BMAL1 shuttling and regulates degradation and activity of CLOCK: BMAL1 heterodimer.

Circadian rhythm is an autoregulatory rhythm, which is sustained by various mechanisms. The nucleocytoplasmic shuttling of BMAL1 is essential for CLOCK translocation between cytoplasm and nucleus and maintenance of the correct pace of the circadian clock. Here we showed that RAE1 and NUP98 can promote the degradation of BMAL1 and CLOCK. Knockdown of RAE1 and NUP98 suppressed BMAL1 shuttling, leading to cytoplasm accumulation of CLOCK. Furthermore, Chip assay showed that knockdown of RAE1 and NUP98 can enhance the interaction between CLOCK: BMAL1 and E-box region in the promoters of Per2 and Cry1 while reducing its transcription activation activity. Our present study firstly revealed that RAE1 and NUP98 are critical regulators for BMAL1 shuttling.