Hippocampal astrocytic neogenin regulating glutamate uptake, a critical pathway for preventing epileptic response.

Epilepsy, a common neurological disorder, is featured with recurrent seizures. Its underlying pathological mechanisms remain elusive. Here, we provide evidence for loss of neogenin (NEO1), a coreceptor for multiple ligands, including netrins and bone morphological proteins, in the development of epilepsy. NEO1 is reduced in hippocampi from patients with epilepsy based on transcriptome and proteomic analyses. Neo1 knocking out (KO) in mouse brains displays elevated epileptiform spikes and seizure susceptibility. These phenotypes were undetectable in mice, with selectively depleted NEO1 in excitatory (NeuroD6-Cre+) or inhibitory (parvalbumin+) neurons, but present in mice with specific hippocampal astrocytic Neo1 KO. Additionally, neurons in hippocampal dentate gyrus, a vulnerable region in epilepsy, in mice with astrocyte-specific Neo1 KO show reductions in inhibitory synaptic vesicles and the frequency of miniature inhibitory postsynaptic current(mIPSC), but increase of the duration of miniature excitatory postsynaptic current and tonic NMDA receptor currents, suggesting impairments in both GABAergic transmission and extracellular glutamate clearance. Further proteomic and cell biological analyses of cell-surface proteins identified GLAST, a glutamate-aspartate transporter that is marked reduced in Neo1 KO astrocytes and the hippocampus. NEO1 interacts with GLAST and promotes GLAST surface distribution in astrocytes. Expressing NEO1 or GLAST in Neo1 KO astrocytes in the hippocampus abolishes the epileptic phenotype. Taken together, these results uncover an unrecognized pathway of NEO1-GLAST in hippocampal GFAP+ astrocytes, which is critical for GLAST surface distribution and function, and GABAergic transmission, unveiling NEO1 as a valuable therapeutic target to protect the brain from epilepsy.

HOXC10 indicates poor survival outcome in gastric cancer and promotes G1/S cell cycle transition through transcriptional repression of p21.

Homeobox (HOX) genes encode proteins that function as transcription factors during embryogenesis and tumorigenesis. We have previously reported upregulation of HOXC10 in gastric cancer (GC) tissues using cDNA microarray analysis. Though the functional role of HOXC10 in GC has been briefly reported, its specific mechanism is not fully understood. We analyzed the expression of HOXC10 in GC tissues, as well as its correlation with the survival outcome. By in vitro and in vivo assays, we further investigated the role of HOXC10 on cell cycle control and proliferation. Finally, we screened potential downstream targets of HOXC10 by cDNA microarray and explored the role of HOXC10 in p21 transcriptional repression through a dual luciferase reporter and chromatin immunoprecipitation. We illustrated the upregulation of HOXC10 in GC tissues and high HOXC10 expression related to poor survival outcome. Multivariable COX regression analysis showed that HOXC10 was an independent predictor of survival (HR=1.863; 95% CI: 1.076-3.225). Functionally, HOXC10 could promote GC cell proliferation and tumor growth in nude mice. Overexpression of HOXC10 accelerated G1/S cell cycle transition, whereas knocking down HOXC10 induced cell cycle arrest at the G1 phase. Critical factors of G1/S cell cycle transition including p21, CDK2, and c-Myc, were regulated by HOXC10. Importantly, an inverse correlation between p21 and HOXC10 expression in GC cell lines and tissues was observed. HOXC10 could directly bind to the promoter region of p21 and repress its transcriptional activity. Collectively, we identified HOXC10 as a predictor of poor prognosis in GC patients, and a novel transcriptional regulator of p21 in the G1/S cell cycle transition.

A novel heterozygous variant in PANX1 is associated with oocyte death and female infertility.

PURPOSE: Oocyte death is a severe clinical phenotype that causes female infertility and recurrent in vitro fertilization and intracytoplasmic sperm injection failure. We aimed to identify pathogenic variants in a female infertility patient with oocyte death phenotype. METHODS: Sanger sequencing was performed to screen PANX1 variants in the affected patient. Western blot analysis was used to check the effect of the variant on PANX1 glycosylation pattern in vitro. RESULTS: We identified a novel PANX1 variant (NM_015368.4 c.86G > A, (p. Arg29Gln)) associated with the phenotype of oocyte death in a non-consanguineous family. This variant displayed an autosomal dominant inheritance pattern with reduced penetrance. Western blot analysis confirmed that the missense mutation of PANX1 (c.86G > A) altered the glycosylation pattern in HeLa cells. Moreover, the mutation effects on the function of PANX1 were weaker than recently reported variants. CONCLUSION: Our findings expand the inheritance pattern of PANX1 variants to an autosomal dominant mode with reduced penetrance and enrich the variational spectrum of PANX1. These results help us to better understand the genetic basis of female infertility with oocyte death.

Mild and efficient copper-catalyzed oxidative cyclization of oximes with 2-aminobenzyl alcohols at room temperature: synthesis of polysubstituted quinolines.

A simple and efficient ligand-free Cu-catalyzed protocol for the synthesis of polysubstituted quinolines via oxidative cyclization of oxime acetates with 2-aminobenzyl alcohols at room temperature has been developed. The presented approach provides a new synthetic pathway leading to polysubstituted quinolines with good functional group tolerance under mild conditions. Moreover, this transformation can be applied for the preparation of quinolines on a gram scale. Oxime acetates serve as the internal oxidants in the reactions, thus making this method very attractive.

C-kit signaling promotes human pre-implantation 3PN embryonic development and blastocyst formation.

BACKGROUND: Although in vitro culture system has been optimized in the past few decades, the problem of few or no high quality embryos has been still not completely solved. Accordingly, fully understanding the regulatory mechanism of pre-implantation embryonic development would be beneficial to further optimize the in vitro embryo culture system. Recent studies have found the expression of c-kit in mouse embryo and its promotion effects on mouse embryonic development. However, it is unclear the expression, the role and the related molecular regulatory mechanism of c-kit in human pre-implantation embryo development. Therefore, the present study is to determine whether c-kit is expressed in human pre-implantation embryos, and to investigate the possible regulatory mechanism of c-kit signaling in the process of embryonic development. METHODS: The present study includes human immature oocytes and three pronucleus (3PN) embryos collected from 768 women (28-32 ages) undergoing IVF, and normal 2PN embryos collected from ICR mice. Samples were distributed randomly into three different experimental groups: SCF group: G-1™ (medium for culture of embryos from the pro-nucleate stage to day 3) or G-2™ (medium for culture of embryos from day3 to blastocyst stage) + HSA (Human serum album) solution + rhSCF; SCF + imanitib (c-kit inhibitor) group: G-1™ or G-2™ + HSA solution + rhSCF + imanitib; SCF + U0126 (MEK/ERK inhibitor) group: G-1™ or G-2™ + HSA solution + rhSCF + U0126; Control group: G-1™ or G-2™ + HSA solution + PBS; The rate of good quality embryos at day 3, blastulation at day 6 and good quality blastulation at day 6 were analysis. RT-PCR, western blot and immunofluorescence staining were applied to detect the target genes and proteins in samples collected from human or mice, respectively. RESULTS: c-kit was expressed ubiquitously in all human immature oocytes, 3PN embryos and 3PN blastocysts. In the experiment of human 3PN embryos, compared with other groups, SCF group showed obviously higher rate of good quality at day 3, better rate of blastocyst formation at day 6 and higher rate of good quality blastocyst formation at day 6. Furthermore, we observed a higher ETV5 expression in SCF group than that in other groups. Similar results were also found in animal experiment. Interestingly, we also found a higher phosphorylation level of MEK/ERK signal molecule in mice embryos from SCF group than those from other groups. Moreover, inhibition of MEK/ERK signaling would remarkably impeded the mice embryonic development, which might be due to the reduced ETV5 expression. CONCLUSIONS: The present study firstly revealed that c-kit signaling might promote the human pre-implantation embryonic development and blastocyst formation by up-regulating the expression of ETV5 via MEK/ERK pathway. Our findings provide a new idea for optimizing the in vitro embryo culture condition during ART program, which is beneficial to obtain high quality embryos for infertile patients.

[Advances in the roles and mechanisms of nonsense-mediated mRNA decay in embryonic development].

Nonsense-mediated mRNA decay (NMD) is originally identified as a widespread mRNA surveillance machinery in degrading 'aberrant' mRNA species with premature termination codons (PTCs) rapidly, which protects the cells from the accumulation of truncated proteins. Recent studies show that NMD can also regulate the degradation of normal gene transcripts, which execute important cellular and physiological functions. Therefore, NMD is considered as a highly conserved post-transcriptional regulatory mechanism in eukaryotes. NMD modulates 3% to 20% of the transcriptome from yeast to human directly or indirectly, which is essential for various physiological processes, such as cell homeostasis, stress response, proliferation, and differentiation. NMD can regulate the level of transcripts that involves in development, and single knockout of most NMD factors has an embryonic lethal effect. NMD plays an important role in the self-renewal, differentiation of embryonic stem cells and is critical during embryonic development. In this review, we summarized the latest advances in the roles and mechanisms of NMD in embryonic development, in order to provide new ideas for the research on embryonic development and the treatment of embryonic development related diseases.

The roles and mechanisms of the Hippo/YAP signaling pathway in the nervous system.

The Hippo signaling pathway, consisting of a highly conserved kinase cascade and downstream transcription co-activators YAP (Yes-associated protein)/TAZ (transcriptional coactivator with PDZ-binding motif), plays a key role in tissue homeostasis and organ size control by regulating the proliferation, differentiation and apoptosis of cells. During normal development, the precise control of neural cell numbers and spatial distributions of these neural cells is important for brain development. Recent studies have shown that the Hippo/YAP signaling pathway is actively involved in the self-renewal of neural stem cells, proliferation of neural progenitor cells, differentiation and activation of glial cells, and myelination of glial cells as well as in the development of neurological diseases. Due to its prominent role in the nervous system, it is necessary to further study on this pathway. In this review, we summarize the recent studies and focus on the roles and mechanisms of the Hippo/YAP signaling pathway in the nervous system, and provide insights for neural development and neural injury diseases.

Brain-derived Neurotrophic Factor Promotes the Migration of Olfactory Ensheathing Cells Through TRPC Channels.

Olfactory ensheathing cells (OECs) are a unique type of glial cells with axonal growth-promoting properties in the olfactory system. Organized migration of OECs is essential for neural regeneration and olfactory development. However, the molecular mechanism of OEC migration remains unclear. In the present study, we examined the effects of brain-derived neurotrophic factor (BDNF) on OEC migration. Initially, the "scratch" migration assay, the inverted coverslip and Boyden chamber migration assays showed that BDNF could promote the migration of primary cultured OECs. Furthermore, BDNF gradient attracted the migration of OECs in single-cell migration assays. Mechanistically, TrkB receptor expressed in OECs mediated BDNF-induced OEC migration, and BDNF triggered calcium signals in OECs. Finally, transient receptor potential cation channels (TRPCs) highly expressed in OECs were responsible for BDNF-induced calcium signals, and required for BDNF-induced OEC migration. Taken together, these results demonstrate that BDNF promotes the migration of cultured OECs and an unexpected finding is that TRPCs are required for BDNF-induced OEC migration. GLIA 2016;64:2154-2165.

High-Performance Monovalent Selective Cation Exchange Membranes with Ionically Cross-Linkable Side Chains: Effect of the Acidic Groups.

Inspired by the charge-governed protein channels located in the cell membrane, a series of polyether ether ketone-based polymers with side chains containing ionically cross-linkable quaternary ammonium groups and acidic groups have been designed and synthesized to prepare monovalent cation-selective membranes (MCEMs). Three acidic groups (sulfonic acid, carboxylic acid, and phenolic hydroxyl) with different acid dissociation constant (pKa) were selected to form the ionic cross-linking structure with quaternary ammonium groups in the membranes. The ionic cross-linking induced the nanophase separation and constructed ionic channels, which resulted in excellent mechanical performance and high cation fluxes. Interesting, the cation flux of membranes increased as the ionization of acidic groups increase, but the selectivity of MCEMs did not follow the same trend, which was mainly dependent on the affinity between the functional groups and the cations. Carboxyl group-containing MCEMs exhibited the best selectivity (9.01 for Li+/Mg2+), which was higher than that of the commercial monovalent cation-selective CIMS membrane. Therefore, it is possible to prepare stable MCEMs through a simple process using ionically cross-linkable polymers, and tuning acidic groups in the membranes provided an attractive approach to improving the cation flux and selectivity of MCEMs.

Repulsive migration of Schwann cells induced by Slit-2 through Ca2+-dependent RhoA-myosin signaling.

Schwann cells migrate along axons before initiating myelination during development and their migration facilitates peripheral nerve regeneration after injury. Axon guidance molecule Slit-2 is highly expressed during peripheral development and nerve regeneration; however, whether Slit-2 regulates the migration of Schwann cells remains a mystery. Here we show that Slit-2 receptor Robo-1 and Robo-2 were highly expressed in Schwann cells in vitro and in vivo. Using three distinct migration assays, we found that Slit-2 repelled the migration of cultured Schwann cells. Furthermore, frontal application of a Slit-2 gradient to migrating Schwann cells first caused the collapse of leading front, and then reversed soma translocation of Schwann cells. The repulsive effects of Slit-2 on Schwann cell migration depended on a Ca(2+) signaling release from internal stores. Interestingly, in response to Slit-2 stimulation, the collapse of leading front required the loss of F-actin and focal adhesion, whereas the subsequent reversal of soma translocation depended on RhoA-Rock-Myosin signaling pathways. Taken together, we demonstrate that Slit-2 repels the migration of cultured Schwann cells through RhoA-Myosin signaling pathways in a Ca(2+)-dependent manner.

Slit-2 repels the migration of olfactory ensheathing cells by triggering Ca2+-dependent cofilin activation and RhoA inhibition.

Olfactory ensheathing cells (OECs) migrate from the olfactory epithelium towards the olfactory bulb during development. However, the guidance mechanism for OEC migration remains a mystery. Here we show that migrating OECs expressed the receptor of the repulsive guidance factor Slit-2. A gradient of Slit-2 in front of cultured OECs first caused the collapse of the leading front, then the reversal of cell migration. These Slit-2 effects depended on the Ca(2+) release from internal stores through inositol (1,4,5)-triphosphate receptor channels. Interestingly, in response to Slit-2 stimulation, collapse of the leading front required the activation of the F-actin severing protein cofilin in a Ca(2+)-dependent manner, whereas the subsequent reversal of the soma migration depended on the reversal of RhoA activity across the cell. Finally, the Slit-2-induced repulsion of cell migration was fully mimicked by co-application of inhibitors of F-actin polymerization and RhoA kinase. Our findings revealed Slit-2 as a repulsive guidance factor for OEC migration and an unexpected link between Ca(2+) and cofilin signaling during Slit-2-triggered repulsion.

Morphological phenotypes of olfactory ensheathing cells display different migratory responses upon Slit-2.

Olfactory ensheathing cells (OECs) are a type of glial cells with morphological plasticity in the olfactory system. Cultured OECs display the process-bearing and flattened shape. Our previous studies have shown that the frontal application of Slit-2 gradient induced the collapse of leading front, and reversed the soma translocation of process-bearing OECs. However, the migratory properties of flattened OECs upon Slit-2 gradient remain elusive. Here, we found that Slit-2 gradient induced the collapse of their plasma membrane, and inhibited migration of flattened OECs. Upon to Slit-2 gradient, the leading front of flattened type 1 OECs firstly showed collapse and retraction, then gradually re-grew a new lamellipodia, finally, showed collapse again (this phenomenon was called as adaptation), while flattened type 2 OECs only showed collapse of plasma membrane. These different migratory responses upon Slit-2 stimulation were possibly due to their different sub-cellular distribution of Robo receptor. Furthermore, F-actin at the peripheral region of leading front was more sensitive to the Slit-2 stimulation than microtubules and the loss of F-actin might be implicated in initiating the collapse of flattened OECs. Finally, the adaptation of flattened type 1 OECs induced by Slit-2 was independent on protein synthesis. Taken together, these results demonstrate that morphological phenotypes of OECs display different migratory properties upon Slit-2 and an unexpected finding that the protein synthesis-independent adaptation in OECs induced by Slit-2.

[Migratory properties of vascular smooth muscle cells on extracellular matrix: a study on inverted coverslip migration assay].

Migration of vascular smooth muscle cells (VSMCs) is involved in vascular development and various vascular diseases; however, the molecular mechanisms of VSMC migration remain unclear. In this study, we established an inverted coverslip migration assay to study the migratory properties of cultured VSMCs on extracellular matrix. Pulmonary arterial smooth muscle cells (PASMCs) from rats were cultured and identified by immunocytochemistry. Each coverslip with a confluent monolayer of PASMCs was inverted to a larger coverslip which was coated with phosphate buffered saline (PBS, as a control), poly-D-lysine hydrobromide (PDL), laminin or Matrigel. After 24 h of migration over the larger coverslip, PASMCs were fixed, and reliably quantified. The roles and mechanisms of extracellular matrix in PASMC migration were further studied by wound-healing assay and immunocytochemistry. The results showed that: (1) The purity of the cultured PASMCs was (97 ± 3)%. (2) The number of PASMCs on laminin or Matrigel migrating out from the inverted coverslip was significantly increased compared with that on PBS or PDL, and the migratory distance of PASMCs on laminin or Matrigel was significantly farther than that on PBS or PDL. (3) The motility of PASMCs on laminin or Matrigel was significantly higher than that on PBS at 8 h, 12 h and 24 h after wounding, respectively. (4) F-actin staining showed that F-actin was congregated along the brim of the migrating cells from the inverted coverslip, and vinculin (a cell marker of focal adhesion) staining showed that the distribution of vinculin in PASMCs plated on laminin or Matrigel was significantly lower than that on PBS or PDL. These results suggest that the inverted coverslip migration assay is suitable to study VSMC migration, and laminin and Matrigel substrates may promote VSMC migration through inhibiting the formation of focal adhesion and regulating the cytoskeletal proteins.

A Y-shape connection device for pediatric patients with an indwelling catheter (Dia = 8Fr) during urodynamic studies, especially for filling phase measurements: a single-center prospective study for safety and effectiveness.

Introduction: This prospective study aimed to assess the effectiveness of a Y-shape connection device in reducing pain and bleeding in pediatric patients with indwelling catheters during urodynamic studies (UDS), while also obtaining effective results in the filling phase. Methods: A total of 45 pediatric patients with a mean age of 13 years were included, all of whom underwent both a UDS with the Y-shape connection device (Method A) and a standard UDS procedure (Method B). Results: The Y-shape connection device demonstrated similar overall urodynamic parameters compared to the standard procedure, while also resulting in significantly less bleeding (P = 0.006) and lower VAS scores during (1.12 ± 0.58 vs. 3.88 ± 1.01, P = 0.001) and after (0.12 ± 0.08 vs 2.91 ± 0.89, P = 0.001) the procedure. No adverse events were reported at the 1-month follow-up. Discussion: These findings suggest that the Y-shape connection device can effectively reduce pain and bleeding during and after UDS in pediatric patients with indwelling catheters (Dia = 8Fr), while also obtaining effective results in the filling phase. Therefore, this Y-shape connection device has a more significant value for children who require urodynamic studies and place more emphasis on filling phase parameters. Clinical trial registration: ChiCTR2300068280.

TFEB: a double-edged sword for tumor metastasis.

Transcription factor EB, a member of the microphthalmia-associated transcription factor (MiTF/TFE) family, is a master regulator of autophagy, lysosome biogenesis, and TAMs. Metastasis is one of the main reasons for the failure of tumor therapy. Studies on the relationship between TFEB and tumor metastasis are contradictory. On the positive side, TFEB mainly affects tumor cell metastasis via five aspects, including autophagy, epithelial-mesenchymal transition (EMT), lysosomal biogenesis, lipid metabolism, and oncogenic signaling pathways; on the negative side, TFEB mainly affects tumor cell metastasis in two aspects, including tumor-associated macrophages (TAMs) and EMT. In this review, we described the detailed mechanism of TFEB-mediated regulation of metastasis. In addition, we also described the activation and inactivation of TFEB in several aspects, including the mTORC1 and Rag GTPase systems, ERK2, and AKT. However, the exact process by which TFEB regulates tumor metastasis remains unclear in some pathways, which requires further studies.

RhoA-ROCK-Myosin pathway regulates morphological plasticity of cultured olfactory ensheathing cells.

Olfactory ensheathing cells (OECs) are glial cells in the olfactory system with morphological and functional plasticity. Cultured OECs have the flattened and process-bearing shape. Reversible changes have been found between these two morphological phenotypes. However, the molecular mechanism underlying the regulation of their morphological plasticity remains elusive. Using RhoA FRET biosensor, we found that the active RhoA signal mainly distributed in the lamellipodia and/or filopodia of OECs. Local disruption of these active RhoA distributions led to the morphological change from the flattened into process-bearing shape and promoted process outgrowth. Furthermore, RhoA pathway inhibitors, Toxin-B, C3, Y-27632 or over-expression of DN-RhoA blocked serum-induced morphological change of OECs from the process-bearing into flattened shape, whereas the activation of RhoA pathway by lysophosphatidic acid (LPA) promoted the morphological change from the process-bearing into flattened shape. Finally, ROCK-Myosin-F-actin as a downstream of RhoA pathway was involved in morphological plasticity of OECs. Taken together, these results suggest that RhoA-ROCK-Myosin pathway mediates the morphological plasticity of cultured OECs in response to extracellular cues.

2,2'-((1R,3R,4S)-4-methyl-4-vinylcyclohexane-1,3-diyl) bis(prop-2-en-1-amine), a bisamino derivative of ß-Elemene, inhibits glioblastoma growth through downregulation of YAP signaling.

ß-Elemene, a compound extracted from Chinese herb Curcuma wenyujin, has been demonstrated with antitumor effects in various cancers, including glioblastoma (GBM), a primary brain tumor with high morbidity and mortality. In this study, we reported a bisamino derivative of ß-Elemene, 2, 2'-((1R, 3R, 4S)-4-methyl-4-vinylcyclohexane-1, 3-diyl) bis(prop-2-en-1-amine) (compound 1), displayed a better anti-GBM effect than ß-Elemene with lower concentration. GBM cell lines (C6 and U87) were treated with compound 1 and subsequently analyzed by several assays. Compound 1 significantly inhibited the migration of C6 and U87 cells based on wound healing assay, transwell assay and inverted migration assay. Furthermore, colony formation assay, immunostaining and flow cytometry assays revealed that compound 1 significantly inhibited the proliferation of GBM cells. In addition, compound 1 induced the apoptosis of GBM cells. Mechanistically, we found Yes-associated protein (YAP) was down-regulated in compound 1-treated GBM cells, and the overexpression of YAP partially rescued the anti-GBM effects of compound 1. Finally, compound 1 suppresses the GBM growth in xenograft model through inactivation YAP signaling. Taken together, these results reveal that a novel derivative of ß-Elemene, compound 1, exhibits more potent anti-GBM activity than ß-Elemene through inactivating YAP signaling pathway, which will provide novel strategies for the treatment of GBM.

[cAMP mediates the morphological change of cultured olfactory ensheathing cells induced by serum].

Olfactory ensheathing cells (OECs) are a unique type of glia with common properties of astrocyte and Schwann cells. Cultured OECs have two morphological phenotypes, astrocyte-like OECs and Schwann cell-like OECs. Reversible changes have been found between these two morphological phenotypes. However, the molecular mechanism underlying the regulation of these reversible changes is still unknown. The aim of this paper is to establish a method for the morphology plasticity of cultured OECs, and investigate the underlying mechanism. Using the primary culture of OECs and immunocytochemistry, the morphology of OECs was observed under serum, serum free media or dB-cAMP drug treatment. Statistical analysis was performed to test differences among the percentages of OEC subtypes under these conditions. The results showed that under serum free media, (95.2±3.7)% of OECs showed Schwann cell-like morphology, and (4.8±3.7)% of OECs showed astrocyte-like morphology; however, under 10% serum media, (42.5±10.4)% of OECs exhibited Schwann cell-like morphology, and (57.5±10.4)% of OECs exhibited astrocyte-like morphology. When media was changed back to serum free media for 24 h, (94.8±5.0)% of OECs showed Schwann cell-like morphology, and (5.2±5.0)% of OECs showed astrocyte-like morphology. Furthermore, culture condition with or without serum did not affect the expression of OEC cell marker, p-75 and S-100. Finally, dB-cAMP, an analog of cAMP, through inhibiting the formation of F-actin stress fibers and focal adhesion, induced the morphology switch from astrocyte-like to Schwann cell-like morphology under serum condition, promoted the branches and the growth of processes. These results suggest that serum induces the morphology plasticity of cultured OECs, which is mediated by cytoplasmic cAMP level through regulating the formation of F-actin stress fibers and focal adhesion.

A combination index and glycoproteomics-based approach revealed synergistic anticancer effects of curcuminoids of turmeric against prostate cancer PC3 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Herbal medicines (HMs) often exert integration effects, including synergistic, additive and antagonistic effects, in such ways that they act on multiple targets and multiple pathways on account of their multiple components. Turmeric, made from the rhizome of Curcuma longa L., is a well-known HM prescribed in the polyherbal formulas for cancer treatment in traditional Chinese medicines (TCMs). However, neither the multiple anticancer compounds of turmeric nor the integration effects of these components are fully known. AIM OF THE STUDY: This work aims to develop a systematic approach to reveal the integration effect mechanisms of multiple anticancer compounds in turmeric against prostate cancer PC3 cells. MATERIALS AND METHODS: Combination index and omics technologies were applied to profile the integration effect mechanisms of bioactive compounds in proportions naturally found in turmeric. PC3 cell line (a prostate cancer cell line) fishing and high resolution mass spectrometry were employed to screen and identify the anticancer compounds from turmeric. The combinations which contain different cell-bound compounds in natural proportions were prepared for further evaluation of anti-cancer activity by using cell viability assays, and assessment of cell apoptosis and cell cycle analysis. Combination index analysis was applied to study the integration effects of the anticancer compounds in their natural proportions. Finally, quantitative glycoproteomics/proteomics and Western blot were implemented to reveal the potential synergistic effect mechanisms of the anticancer compounds based on their natural proportions in turmeric. RESULTS: Three curcuminoids (curcumin, CUR; demethoxycurcumin, DMC; bisdemethoxycurcumin, BDMC) in turmeric were discovered and shown to possess significant synergistic anticancer activities. Combination index analysis revealed an additive effect of CUR combined with DMC or BDMC and a slight synergistic effect of DMC combined with BDMC in natural proportions in turmeric, while a combination of all three curcuminoids (CUR, DMC and BDMC) at a ratio of 1:1:1 yielded superior synergistic effects. Interestingly, the presence of BDMC and DMC are essential for synergistic effect. Glycoproteomics and proteomics demonstrated that different curcuminoids regulate various protein pathways, such as ribosome, glycolysis/gluconeogenesis, biosynthesis of amino acids, and combination of CUR + DMC + BDMC showed the most powerful effects on down-regulation of protein expression. CONCLUSIONS: Our analytical approach provides a systematic understanding of the holistic activity and integration effects of the anti-cancer compounds in turmeric and three curcuminoids of turmeric showed a synergistic effect on PC3 cells.

Ginkgetin derived from Ginkgo biloba leaves enhances the therapeutic effect of cisplatin via ferroptosis-mediated disruption of the Nrf2/HO-1 axis in EGFR wild-type non-small-cell lung cancer.

BACKGROUND: Cisplatin (DDP) is the first-in-class drug for advanced and non-targetable non-small-cell lung cancer (NSCLC). A recent study indicated that DDP could slightly induce non-apoptotic cell death ferroptosis, and the cytotoxicity was promoted by ferroptosis inducer. The agents enhancing the ferroptosis may therefore increase the anticancer effect of DDP. Several lines of evidence supporting the use of phytochemicals in NSCLC therapy. Ginkgetin, a bioflavonoid derived from Ginkgo biloba leaves, showed anticancer effects on NSCLC by triggering autophagy. Ferroptosis can be triggered by autophagy, which regulates redox homeostasis. Thus, we aimed to elucidate the possible role of ferroptosis involved in the synergistic effect of ginkgetin and DDP in cancer therapy. METHODS: The promotion of DDP-induced anticancer effects by ginkgetin was examined via a cytotoxicity assay and western blot. Ferroptosis triggered by ginkgetin in DDP-treated NSCLC was observed via a lipid peroxidation assay, a labile iron pool assay, western blot, and qPCR. With ferroptosis blocking, the contribution of ferroptosis to ginkgetin + DDP-induced cytotoxicity, the Nrf2/HO-1 axis, and apoptosis were determined via a luciferase assay, immunostaining, chromatin immunoprecipitation (CHIP), and flow cytometry. The role of ferroptosis in ginkgetin + DDP-treated NSCLC cells was illustrated by the application of ferroptosis inhibitors, which was further demonstrated in a xenograft nude mouse model. RESULTS: Ginkgetin synergized with DDP to increase cytotoxicity in NSCLC cells, which was concomitant with increased labile iron pool and lipid peroxidation. Both these processes were key characteristics of ferroptosis. The induction of ferroptosis mediated by ginkgetin was further confirmed by the decreased expression of SLC7A11 and GPX4, and a decreased GSH/GSSG ratio. Simultaneously, ginkgetin disrupted redox hemostasis in DDP-treated cells, as demonstrated by the enhanced ROS formation and inactivation of the Nrf2/HO-1 axis. Ginkgetin also enhanced DDP-induced mitochondrial membrane potential (MMP) loss and apoptosis in cultured NSCLC cells. Furthermore, blocking ferroptosis reversed the ginkgetin-induced inactivation of Nrf2/HO-1 as well as the elevation of ROS formation, MMP loss, and apoptosis in DDP-treated NSCLC cells. CONCLUSION: This study is the first to report that ginkgetin derived from Ginkgo biloba leaves promotes DDP-induced anticancer effects, which can be due to the induction of ferroptosis.