SCFFBXW5-mediated degradation of AQP3 suppresses autophagic cell death through the PDPK1-AKT-MTOR axis in hepatocellular carcinoma cells.

AQP3 (aquaporin 3 (Gill blood group)), a member of the AQP family, is an aquaglyceroporin which transports water, glycerol and small solutes across the plasma membrane. Beyond its role in fluid transport, AQP3 plays a significant role in regulating various aspects of tumor cell behavior, including cell proliferation, migration, and invasion. Nevertheless, the underlying regulatory mechanism of AQP3 in tumors remains unclear. Here, for the first time, we report that AQP3 is a direct target for ubiquitination by the SCFFBXW5 complex. In addition, we revealed that downregulation of FBXW5 significantly induced AQP3 expression to prompt macroautophagic/autophagic cell death in hepatocellular carcinoma (HCC) cells. Mechanistically, AQP3 accumulation induced by FBXW5 knockdown led to the degradation of PDPK1/PDK1 in a lysosomal-dependent manner, thus inactivating the AKT-MTOR pathway and inducing autophagic death in HCC. Taken together, our findings revealed a previously undiscovered regulatory mechanism through which FBXW5 degraded AQP3 to suppress autophagic cell death via the PDPK1-AKT-MTOR axis in HCC cells.Abbreviation: BafA1: bafilomycin A1; CQ: chloroquine; CRL: CUL-Ring E3 ubiquitin ligases; FBXW5: F-box and WD repeat domain containing 5; HCC: hepatocellular carcinoma; HSPA8/HSC70: heat shock protein family A (Hsp70) member 8; 3-MA: 3-methyladenine; PDPK1/PDK1: 3-phosphoinositide dependent protein kinase 1; RBX1/ROC1: ring-box 1; SKP1: S-phase kinase associated protein 1; SCF: SKP1-CUL1-F-box protein.

Daurisoline Inhibits ESCC by Inducing G1 Cell Cycle Arrest and Activating ER Stress to Trigger Noxa-Dependent Intrinsic and CHOP-DR5-Dependent Extrinsic Apoptosis via p-eIF2α-ATF4 Axis.

Esophageal squamous cell carcinoma (ESCC), one of the most malignant human cancers in clinic, requires novel treatment. Daurisoline (DAS) is a component of traditional Chinese herb, which exhibits anti-cancer effects by autophagy inhibition and metastasis suppression. However, the effect and mechanism of DAS on ESCC remain unclear. Here, we found that DAS inhibited cell proliferation and colony formation in both human ESCC cell lines EC1 and ECA109. Mechanistically, DAS induced p21-/p27-dependent G1 phase cell cycle arrest and apoptosis in a dose-dependent manner. The induction of apoptosis by DAS was largely dependent on the activation of the transcription factor ATF4 and its downstream NOXA-dependent intrinsic and CHOP-DR5-dependent extrinsic apoptotic pathway. ATF4 activation induced by DAS was due to the generation of excessive reactive oxygen species (ROS) and the subsequent activation of endoplasmic reticulum (ER) stress through the p-eIF2α-ATF4 signal pathway, which can be largely abrogated by N-acetylcysteine (NAC), a scavenger of ROS. Moreover, DAS treatment significantly inhibited tumor growth and reduced tumor weight in the tumor xenograft mouse model by up-regulating key proteins related to cell cycle arrest and apoptotic pathway. Taken together, these findings identified DAS as a novel candidate for the treatment of ESCC.

Andrographolide, a diterpene lactone from the Traditional Chinese Medicine Andrographis paniculate, induces senescence in human lung adenocarcinoma via p53/p21 and Skp2/p27.

BACKGROUND: Senescence leads to permanent cell-cycle arrest and is a potential target for cancer therapy. Andrographolide (AD) is a diterpene lactone isolated from Traditional Chinese Medicine (TCM) Andrographis paniculate, which has been used as an anti-inflammatory drug in clinical practice with the potential to target senescence in recalcitrant lung cancer. PURPOSE: To determine whether AD can induce senescence in human lung adenocarcinoma in vitro and in vivo and to elucidate the underlying mechanisms. METHODS: SA-ß-Gal staining was used to detect the expression of senescence-associated ß-galactosidase (SA-ß-Gal) in human lung adenocarcinoma cells A549 and NCI-H1795. DNA damage was examined by the detection of γH2AX foci. Cell cycle was analyzed by flow cytometry. Cancer cell proliferation was determined by ATPlite assay and clonogenic survival assay in vitro. Tumor growth was determined in a mouse model of A549. The expression level of proteins and mRNA was estimated by Western blotting and Quantitative RT-PCR, respectively. Small interfering RNA (siRNA) was used to knock down p21, p27 and p53 to explore the potential mechanism of AD-induced senescence in human lung adenocarcinoma cells. RESULTS: AD-induced A549 and NCI-H1795 cell senescence determined by increased cell size, flattened morphology, DNA damage, cell cycle arrest as well as the increased expression of ß-galactosidase. AD inhibited cell proliferation in lung cells in vitro and lung cells xenograft growth in nude mice. p21 and p27, the major cell cycle regulators and mediators of senescence, were upregulated at the protein level in AD-treated A549 lung adenocarcinoma in vitro and in vivo. Further studies demonstrated that AD induced cell senescence via p53/p21 and Skp2/p27. CONCLUSION: In the present study, we found that the primary anti-inflammatory drug AD could have a potential antitumor effect in lung cancer. We demonstrated that AD induced lung adenocarcinoma senescence in vitro and in vivo via p53/p21 and Skp2/p27 for the first time. AD is therefore a promising senescence-inducing therapeutic for recalcitrant human lung adenocarcinoma.

Cullin3-TNFAIP1 E3 Ligase Controls Inflammatory Response in Hepatocellular Carcinoma Cells via Ubiquitination of RhoB.

Rho family GTPase RhoB is the critical signaling component controlling the inflammatory response elicited by pro-inflammatory cytokines. However, the underlying mechanisms of RhoB degradation in inflammatory response remain unclear. In this study, for the first time, we identified that TNFAIP1, an adaptor protein of Cullin3 E3 ubiquitin ligases, coordinated with Cullin3 to mediate RhoB degradation through ubiquitin proteasome system. In addition, we demonstrated that downregulation of TNFAIP1 induced the expression of pro-inflammatory cytokines IL-6 and IL-8 in TNFα-stimulated hepatocellular carcinoma cells through the activation of p38/JNK MAPK pathway via blocking RhoB degradation. Our findings revealed a novel mechanism of RhoB degradation and provided a potential strategy for anti-inflammatory intervention of tumors by targeting TNFAIP1-RhoB axis.

An anticancer agent-loaded PLGA nanomedicine with glutathione-response and targeted delivery for the treatment of lung cancer.

Stimuli response or controlled release is a new research hotspot in nanomedicine; however, there is scarce research on organic nanomedicines with stimuli responses, which limits their practical biological applications. In addition, homoharringtonine (HHT) has been used as an effective anticancer agent, but reducing its toxicity and side effects is an urgent problem to be solved. Herein, an EGFR (epidermal growth factor receptor) aptamer-modified HHT-loaded PLGA-SS-PEG nanomedicine was developed. The nanomaterial possesses spherical morphology and admirable biocompatibility. After targeted endocytosis in tumour cells via the selective recognition between EGFR and its aptamer, the PLGA nanomedicine is triggered by a high GSH level and releases its cargo in lung cancer cells. The in vitro and in vivo results reveal that the PLGA nanomedicine not only inhibited the proliferation and promoted the apoptosis of lung cancer cells, but also possessed better therapeutic efficacy and less toxic side effects compared with the free anticancer agent. Consequently, this study provides a novel approach to construct a biodegradable nanomedicine with targeted recognition and stimuli response. Moreover, it inhibited the proliferation of lung cancer cells with high efficiency and low toxicity. Importantly, the PLGA nanomedicine demonstrates encouraging potential as a multifunctional nano-system applicable for cancer therapy.

Smart and dual-targeted BSA nanomedicine with controllable release by high autolysosome levels.

Targeting modifications and smart responsiveness of nanomedicines can enable anticancer drugs to be selectively delivered to and controllably released in tumour cells or tissues, which can reduce the treatment's toxicity and side effects. Good biocompatibility is crucial for the clinical application of any nanomedicine. In this study, a double-targeting molecule, an RGD peptide- and 4-(2-aminoethyl) morpholine-modified, doxorubicin (DOX)-loaded bovine serum albumin (BSA) nanomedicine, that can be controllably released by the high levels of autophagic lysosomes in tumour cells was developed. The size of the spherical BSA nanoparticles is approximately 60 nm. In vitro experiments indicated that the RGD peptide- and 4-(2-aminoethyl) morpholine-modified, DOX-loaded BSA nanomedicine has a better therapeutic effect than free DOX. In vivo experiments suggested that the BSA nanomedicine can successfully suppress the progression of PC9 xenograft tumours. This phenomenon may be attributable to the endocytosis of a relatively large amount of nanomedicine and the effective release of the loaded chemotherapeutic agent, as induced by high levels of autolysosomes. Collectively, the results of this study provide a smart approach for increasing therapeutic efficacy using a double-targeting molecule-modified BSA nanomedicine.

The difference between blood-associated and water-associated herbs of Danggui-Shaoyao San in theory of TCM, based on serum pharmacochemistry.

Danggui-Shaoyao San (DSS) is a famous Chinese formula for activating blood circulation and promoting urination. This study was to investigate the difference of material basis between a blood-associated herbs group and a water-associated herbs group. According to the theory of traditional Chinese medicine, the formula can be divided into a blood-associated herbs group (Angelica sinensis, Paeonia lactiflora and Ligusticum chuanxiong) and a water-associated herbs group (Atractylodes macrocephala, Alisma orientale and Poria cocos). The HPLC fingerprint of the formula was established for quality control. Serum samples from rats, orally administrated DSS, and the decomposed recipes of DSS, were analyzed by HPLC-DAD and the transitional blood components of DSS were identified. Twenty-one common peaks were identified in the fingerprint of DSS. Contents of paeoniflorin, albiflorin, ferulic acid and alisol B 23-acetate in co-decoction were significantly higher than those in individual decoction. Eleven peaks belonged to the blood-associated herbs group (four metabolites and seven prototype components; paeoniflorin and ferulic acid appeared in prototype components), whereas six peaks belonged to the water-associated herbs group (three metabolites and three prototype components). It was concluded that the serum pharmacochemistry is a meaningful approach for clarifying the difference between blood-associated and water-associated herbs in chemical composition.

Synergistic inhibition of autophagy and neddylation pathways as a novel therapeutic approach for targeting liver cancer.

Liver cancer is the second-most frequent cause of cancer death in the world and is highly treatment resistant. We reported previously that inhibition of neddylation pathway with specific NAE inhibitor MLN4924, suppressed the malignant phenotypes of liver cancer. However, during the process, MLN4924 induces pro-survival autophagy as a mechanism of drug resistance. Here, we report that blockage of autophagy with clinically-available autophagy inhibitors (e.g. chloroquine) significantly enhanced the efficacy of MLN4924 on liver cancer cells by triggering apoptosis. Mechanistically, chloroquine enhanced MLN4924-induced up-regulation of pro-apoptotic proteins (e.g. NOXA) and down-regulation of anti-apoptotic proteins. Importantly, the down-regulation of NOXA expression via siRNA silencing substantially attenuated apoptosis of liver cancer cells. Further mechanistic studies revealed that blockage of autophagy augmented MLN4924-induced DNA damage and reactive oxygen species (ROS) generation. The elimination of DNA damage or blockage of ROS production significantly reduced the expression of NOXA, and thereby attenuated apoptosis and reduced growth inhibition of liver cancer cells. Moreover, blockage of autophagy enhanced the efficacy of MLN4924 in an orthotopic model of human liver cancer, with induction of NOXA and apoptosis in tumor tissues. These findings provide important preclinical evidence for clinical investigation of synergistic inhibition of neddylation and autophagy in liver cancer.

Targeting protein neddylation with an NEDD8-activating enzyme inhibitor MLN4924 induced apoptosis or senescence in human lymphoma cells.

Recent studies indicate that post-translational protein neddylation is required for the maintenance of cell viability in several lymphoma cell lines, while inhibition of the neddylation pathway with an NEDD8-activating enzyme (NAE) inhibitor MLN4924 induces apoptosis in lymphoma cells. However, the mechanism by which neddylation inhibition induces apoptosis in lymphoma cells has not been fully elucidated. Moreover, it is unknown whether neddylation inhibition triggers non-apoptotic cell-killing responses, such as cell senescence, in lymphoma cells. Here, we report that MLN4924 specifically inhibited protein neddylation, inactivated cullin-RING E3 ligase (CRL), the best-known neddylation substrate, and induced the accumulation of tumor-suppressive CRL substrates in lymphoma cells. Moreover, MLN4924 potently suppressed the growth of lymphoma cells by inducing G2 cell-cycle arrest, followed by apoptosis or senescence in a cell line-dependent manner. MLN4924-induced apoptosis was mediated by intrinsic apoptotic signaling with substantial up-regulation of pro-apoptotic Bik and Noxa as well as down-regulation of anti-apoptotic XIAP, c-IAP1 and c-IAP2, while senescence induction upon neddylation inhibition seemed dependent on the expression of tumor suppressor p21/p27. Together, these findings expand our understanding on how lymphoma cells respond to neddylation inhibition and support the development of neddylation inhibitors (e.g. MLN4924) for the treatment of lymphoma.

[Mechanism Study on Danggui Shaoyao San and Guizhi Fuling Wan For Treating Primary Dysmenorrheal Based on Biological Network].

OBJECTIVE: To investigate the molecular mechanisms of Danggui Shaoyao San and Guizhi Fuling Wan for treating primary dysmenorrhea of liver depression and blood stasis syndrome by biological network method. METHODS: Targets of the formula were collected from. PubMed database, and targets of primary dysmenorrhea were searched from Gene Card database. Then, the "formula-syndromes-disease" network was constructed and analyzed with Cytoscape software. Molecular docking approach was used to verify estrogen-like effect of ingredients. RESULTS: The "formula-syndromes-disease" shared multiple targets, which involves a variety of important nodes, such as IL and PGF2α, and played the effects of anti-inflammatory and analgesic. Danggui Shaoyao San could influence the function of hormones, such as corticotropin releasing hormone, and inhibit hyperactivity of adrenal axis. Guizhi Fuling Wan mainly affected a series of inflammation caused by cyclooxygenase and had the effects of blood coagulation. Estrogen-like effect of Danggui Shaoyao Sanwas stronger than that of Guizhi Fuling Wan. CONCLUSION: The novel approach will present an effective strategy for theory study of Danggui Shaoyao San and Guizhi Fuling Wan for treating primary dysmenorrhea.

Cantharidin combined with chemotherapy for Chinese patients with metastatic colorectal cancer.

BACKGROUND: This systematic analysis was conducted to evaluate the efficacy and safety of cantharidin combined with chemotherapy in treating Chinese patients with metastatic colorectal cancer. METHODS: Clinical studies evaluating the efficacy and safety of cantharidin combined with chemotherapy on response and safety for Chinese patients with colorectal cancer were identified using a predefined search strategy. Pooled response rate (RR) of treatment were calculated. RESULTS: When cantharidin combined with chemotherapy, 4 clinical studies which included 155 patients with advanced colorectal cancer were considered eligible for inclusion. The systematic analysis suggested that, in all patients, pooled RR was 46.5% (72/155) in cantharidin combined regimens. Major adverse effects were neutropenia, leukopenia, fatigue, and anemia with cantharidin combined treatment; no treatment related deaths occurred. CONCLUSION: This systematic analysis suggests that cantharidin combined regimens are associated with high response rate and accepted toxicity in treating Chinese patients with metastatic colorectal cancer suggesting that randomized clinical trials are now warranted.

Transcriptome and proteome exploration to provide a resource for the study of Agrocybe aegerita.

BACKGROUND: Agrocybe aegerita, the black poplar mushroom, has been highly valued as a functional food for its medicinal and nutritional benefits. Several bioactive extracts from A. aegerita have been found to exhibit antitumor and antioxidant activities. However, limited genetic resources for A. aegerita have hindered exploration of this species. METHODOLOGY/PRINCIPAL FINDINGS: To facilitate the research on A. aegerita, we established a deep survey of the transcriptome and proteome of this mushroom. We applied high-throughput sequencing technology (Illumina) to sequence A. aegerita transcriptomes from mycelium and fruiting body. The raw clean reads were de novo assembled into a total of 36,134 expressed sequences tags (ESTs) with an average length of 663 bp. These ESTs were annotated and classified according to Gene Ontology (GO), Clusters of Orthologous Groups (COG), and Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathways. Gene expression profile analysis showed that 18,474 ESTs were differentially expressed, with 10,131 up-regulated in mycelium and 8,343 up-regulated in fruiting body. Putative genes involved in polysaccharide and steroid biosynthesis were identified from A. aegerita transcriptome, and these genes were differentially expressed at the two stages of A. aegerita. Based on one-dimensional gel electrophoresis (1-DGE) coupled with electrospray ionization liquid chromatography tandem MS (LC-ESI-MS/MS), we identified a total of 309 non-redundant proteins. And many metabolic enzymes involved in glycolysis were identified in the protein database. CONCLUSIONS/SIGNIFICANCE: This is the first study on transcriptome and proteome analyses of A. aegerita. The data in this study serve as a resource of A. aegerita transcripts and proteins, and offer clues to the applications of this mushroom in nutrition, pharmacy and industry.

Induction of cell senescence by targeting to Cullin-RING Ligases (CRLs) for effective cancer therapy.

Cullin-RING ligases (CRLs) are the biggest family of multiunit ubiquitin E3 ligases, controlling many biological processes by promoting the degradation of a broad spectrum of proteins associated with cell cycle, signal transduction and cell growth. The dysfunction of CRLs causes a lot of diseases including cancer, which meanwhile offers us a promising approach to cancer therapy by targeting to CRLs. Recent studies have demonstrated that genetic or pharmaceutical inactivation of CRLs often leads to cancer cell death by activating multiple cell-killing pathways including senescence, an emerging anticancer mechanism of therapeutic agents. Here, we summarize the induction of cellular senescence and its mechanism of action, triggered by targeting to specific subunits of CRLs via multiple approaches including siRNA silencing, genetic knockout as well as small molecule inhibitor, exhibiting anticancer effect in vitro and in vivo.

Inactivation of the Cullin (CUL)-RING E3 ligase by the NEDD8-activating enzyme inhibitor MLN4924 triggers protective autophagy in cancer cells.

The multiunit Cullin (CUL)-RING E3 ligase (CRL) controls diverse biological processes by targeting a mass of substrates for ubiquitination and degradation, whereas its dysfunction causes carcinogenesis. Post-translational neddylation of CUL, a process triggered by the NEDD8-activating enzyme E1 subunit 1 (NAE1), is required for CRL activation. Recently, MLN4924 was discovered via a high-throughput screen as a specific NAE1 inhibitor and first-in-class anticancer drug. By blocking CUL neddylation, MLN4924 inactivates CRL and causes the accumulation of CRL substrates that trigger cell cycle arrest, senescence and/or apoptosis to suppress the growth of cancer cells in vitro and in vivo. Recently, we found that MLN4924 also triggers protective autophagy in response to CRL inactivation. MLN4924-induced autophagy is attributed partially to the inhibition of mechanistic target of rapamycin (also known as mammalian target of rapamycin, MTOR) activity by the accumulation of the MTOR inhibitory protein DEPTOR, as well as reactive oxygen species (ROS)-induced stress. Moreover, the blockage of autophagy response enhances apoptosis in MLN4924-treated cells. Together, our findings not only reveal autophagy as a novel cellular response to CRL inactivation by MLN4924, but also provide a piece of proof-of-concept evidence for the combination of MLN4924 with autophagy inhibitors to enhance therapeutic efficacy.

The Nedd8-activating enzyme inhibitor MLN4924 induces autophagy and apoptosis to suppress liver cancer cell growth.

Posttranslational neddylation of cullins in the Cullin-Ring E3 ligase (CRL) complexes is needed for proteolytic degradation of CRL substrates, whose accumulation induces cell-cycle arrest, apoptosis, and senescence. The Nedd8-activating enzyme (NAE) is critical for neddylation of CRL complexes and their growth-promoting function. Recently, the anticancer small molecule MLN4924 currently in phase I trials was determined to be an inhibitor of NAE that blocks cullin neddylation and inactivates CRL, triggering an accumulation of CRL substrates that trigger cell-cycle arrest, apoptosis, and senescence in cancer cells. Here, we report that MLN4924 also triggers autophagy in response to CRL inactivation and that this effect is important for the ability of MLN4924 to suppress the outgrowth of liver cancer cells in vitro and in vivo. MLN4924-induced autophagy was attributed partially to inhibition of mTOR activity, due to accumulation of the mTOR inhibitory protein Deptor, as well as to induction of reactive oxygen species stress. Inhibiting autophagy enhanced MLN4924-induced apoptosis, suggesting that autophagy is a survival signal triggered in response to CRL inactivation. In a xenograft model of human liver cancer, MLN4924 was well-tolerated and displayed a significant antitumor effect characterized by CRL inactivation and induction of autophagy and apoptosis in liver cancer cells. Together, our findings support the clinical investigation of MLN4924 for liver cancer treatment and provide a preclinical proof-of-concept for combination therapy with an autophagy inhibitor to enhance therapeutic efficacy.

A novel lectin from Agrocybe aegerita shows high binding selectivity for terminal N-acetylglucosamine.

A novel lectin was isolated from the mushroom Agrocybe aegerita (designated AAL-2) by affinity chromatography with GlcNAc (N-acetylglucosamine)-coupled Sepharose 6B after ammonium sulfate precipitation. The AAL-2 coding sequence (1224 bp) was identified by performing a homologous search of the five tryptic peptides identified by MS against the translated transcriptome of A. aegerita. The molecular mass of AAL-2 was calculated to be 43.175 kDa from MS, which was consistent with the data calculated from the amino acid sequence. To analyse the carbohydrate-binding properties of AAL-2, a glycan array composed of 465 glycan candidates was employed, and the result showed that AAL-2 bound with high selectivity to terminal non-reducing GlcNAc residues, and further analysis revealed that AAL-2 bound to terminal non-reducing GlcNAc residues with higher affinity than previously well-known GlcNAc-binding lectins such as WGA (wheatgerm agglutinin) and GSL-II (Griffonia simplicifolia lectin-II). ITC (isothermal titration calorimetry) showed further that GlcNAc bound to AAL-2 in a sequential manner with moderate affinity. In the present study, we also evaluated the anti-tumour activity of AAL-2. The results showed that AAL-2 could bind to the surface of hepatoma cells, leading to induced cell apoptosis in vitro. Furthermore, AAL-2 exerted an anti-hepatoma effect via inhibition of tumour growth and prolongation of survival time of tumour-bearing mice in vivo.