Regulator of Ribosome Synthesis 1 (RRS1) Stabilizes GRP78 and Promotes Breast Cancer Progression.

Regulator of ribosome synthesis 1 (RRS1), a crucial regulatory factor in ribosome biogenesis, exerts a remarkable impact on the progression of breast cancer (BC). However, the exact mechanisms and pathways have not yet been fully elucidated. To investigate the impact of RRS1 on BC growth and metastasis, along with its underlying mechanisms. We discovered that RRS1 is overexpressed in BC tissues and cell lines. This study aims to regulate the level of RRS1 through lentiviral transfection technology to explore its potential function in BC cells. Knockdown of RRS1 resulted in the inhibition of cell proliferation, invasion, and migration, whereas overexpression had the opposite effects. We firstly identified the interaction between RRS1 and Glucose-Regulated Protein 78 (GRP78) using Co-immunoprecipitation (Co-IP) combined with mass spectrometry analysis, providing evidences of co-localization and positive regulation between RRS1 and GRP78. We observed that RRS1 inhibited the degradation of GRP78 through the ubiquitin-proteasome pathway, resulting in the stabilization of GRP78. In addition, our findings suggested that RRS1 promoted BC progression by activating the GRP78-mediated phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway. In conclusion, this newly discovered RRS1/GRP78 signaling axis provides a molecular and theoretical basis for further exploring the mechanisms of breast cancer invasion and metastasis.

Ribosome Biogenesis Regulator 1 Homolog (RRS1) Promotes Cisplatin Resistance by Regulating AEG-1 Abundance in Breast Cancer Cells.

Many ribosomal proteins are highly expressed in tumors and are closely related to their diagnosis, prognosis and pathological characteristics. However, few studies are available on the correlation between ribosomal proteins and chemoresistance. RRS1 (human regulator of ribosome synthesis 1), a critical nuclear protein involved in ribosome biogenesis, also plays a key role in the genesis and development of breast cancer by protecting cancer cells from apoptosis. Given that apoptosis resistance is one of the causes of the cisplatin resistance of tumor cells, our aim was to determine the relationship between RRS1 and cisplatin resistance in breast cancer cells. Here, we report that RRS1 is associated with cisplatin resistance in breast cancer cells. RRS1 silencing increased the sensitivity of MCF-7/DDP cells to cisplatin and inhibited cancer cell proliferation by blocking cell cycle distribution and enhancing apoptosis. AEG-1 (astrocyte elevated gene-1) promotes drug resistance by interfering with the ubiquitination and proteasomal degradation of MDR1 (multidrug resistance gene 1), thereby enhancing drug efflux. We found that RRS1 binds to and stabilizes AEG-1 by inhibiting ubiquitination and subsequent proteasomal degradation, which then promotes drug efflux by upregulating MDR1. Furthermore, RRS1 also induces apoptosis resistance in breast cancer cells through the ERK/Bcl-2/BAX signaling pathway. Our study is the first to show that RRS1 sensitizes breast cancer cells to cisplatin by binding to AEG-1, and it provides a theoretical basis to improve the efficacy of cisplatin-based chemotherapy.

The Large Molecular Weight Polysaccharide from Wild Cordyceps and Its Antitumor Activity on H22 Tumor-Bearing Mice.

Cordyceps has anti-cancer effects; however, the bioactive substance and its effect are still unclear. Polysaccharides extracted from Cordyceps sinensis, the fugus of Cordyceps, have been reported to have anti-cancer properties. Thus, we speculated that polysaccharides might be the key anti-tumor active ingredients of Cordyceps because of their larger molecular weight than that of polysaccharides in Cordyceps sinensis. In this study, we aimed to investigate the effects of wild Cordyceps polysaccharides on H22 liver cancer and the underlying mechanism. The structural characteristics of the polysaccharides of WCP were analyzed by high-performance liquid chromatography, high-performance gel-permeation chromatography, Fourier transform infrared spectrophotometry, and scanning electron microscopy. Additionally, H22 tumor-bearing BALB/c mice were used to explore the anti-tumor effect of WCP (100 and 300 mg/kg/d). The mechanism by WCP inhibited H22 tumors was uncovered by the TUNEL assay, flow cytometry, hematoxylin-eosin staining, quantitative reverse transcription-polymerase chain reaction, and Western blotting. Here, our results showed that WCP presented high purity with an average molecular weight of 2.1 × 106 Da and 2.19 × 104 Da. WCP was determined to be composed of mannose, glucose, and galactose. Notably, WCP could inhibit the proliferation of H22 tumors not only by improving immune function, but also by promoting the apoptosis of tumor cells, likely through the IL-10/STAT3/Bcl2 and Cyto-c/Caspase8/3 signaling pathways, in H22 tumor-bearing mice. Particularly, WCP had essentially no side effects compared to 5-FU, a common drug used in the treatment of liver cancer. In conclusion, WCP could be a potential anti-tumor product with strong regulatory effects in H22 liver cancer.

Polysaccharides extracted from mulberry fruits (Morus nigra L.): antioxidant effect of ameliorating H2O2-induced liver injury in HepG2 cells.

BACKGROUND: Mori Fructus is an economical and readily available traditional Chinese medicine and food. Polysaccharides in Mori Fructus have clear antioxidant activity and have been found to alleviate oxidative stress (OS)-induced liver damage in experimental studies. The mechanism of regulation of cellular antioxidant activity by mulberry polysaccharides has been suggested to be Nrf2, but it is not clear whether the Nrf2 pathway is mediated by activation of other targets, and the exact process of effects in hepatocytes has yet to be elucidated. METHODS: In this study, the basic characterization of total polysaccharides extracted from mulberry fruits (Morus nigra Linn.) was analyzed. A model of oxidative damage induced by H2O2 in HepG2 cells was established. The levels of cellular oxidation-related markers, including ROS, SOD and Gpx, were then examined. Furthermore, Q-PCR and Western-blot were used to detect the expression of genes and proteins related to the PI3K/Akt-mediated Nrf2 signaling pathway. RESULTS: The results showed that a total mulberry polysaccharides (TMP) has a molecular weight of 57.5 kDa with a pyranose ring mainly composed of glucose (48.81%), galactose (22.79%) and mannose (18.2%). TMP reduced the accumulation of ROS in HepG2 cells after H2O2 treatment and modulated the activity of SOD and Gpx. Q-PCR and Western-blot showed that TMP could up-regulate the expression of p-PI3K, p-AKT, Nrf2, NQO1 and HO-1. CONCLUSIONS: This study demonstrates that TMP can reduce ROS accumulation in H2O2-treated HepG2 cells and restore cell viability by activating the PI3K/AKT-mediated Nrf2 pathway. TMP may be a potent antioxidant agent that could slow down oxidative damage to the liver.

Ferulic Acid Alleviates Hepatic Lipid Accumulation and Inflammation by Improving Proximal and Distal Intestinal Barriers in NAFLD Mice.

Non-alcoholic fatty liver disease (NAFLD) is closely associated with low-grade chronic inflammation which is usually induced by intestinal dysbiosis. As ferulic acid (FA) has been proven effective at improving the intestinal integrity, we aimed to determine the effect of dietary FA on NAFLD development in high-fat dieted (HFD) mice, a well-established model of NAFLD. Male C57BL/6J mice were fed either a normal chow diet (ND) or HFD with or without FA (40 mg/kg) orally for 6 weeks. FA significantly alleviated lipid metabolism disorder and reduced liver inflammation in HFD mice (P < 0.05). As expected, FA improved the ileal intestinal integrity likely via the Nuclear factor E2-related factor 2 (Nrf2)/Heme oxygenase-1 (HO-1) signaling pathway. Importantly, we found that FA also relieved HFD-induced gut microbiota dysbiosis by inhibiting the growth of harmful bacteria such as Helicobacter and increased the abundance of many short-chain fatty acids (SCFAs)-producing bacteria (P < 0.05). Our data indicated that FA not only increased the colonic levels of SCFAs, but also maintained the colonic barrier integrity by up-regulating the expression of the epithelial tight junction protein. These data indicated that FA alleviated NAFLD by reducing circulating lipopolysaccharide levels. These effects may be due to improved proximal and distal intestinal barriers, which presumably mediated through the interaction of FA with the gut microbiota.

Cordyceps proteins alleviate lupus nephritis through modulation of the STAT3/mTOR/NF-кB signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Cordyceps is a parasitic edible fungus, which is a unique Chinese medicinal material. It has been reported to have immunomodulatory effects and use in kidney disease. Especially, Cordyceps has been used in the treatment of lupus nephritis (LN). AIM OF STUDY: Cordyceps proteins (CP) have a favorable bidirectional immunomodulatory functions and may have therapeutic potential for LN. However, the underlying molecular mechanism remains unknown. So this study aimed to examine the activities of CP in LN and possible mechanism. MATERIALS AND METHODS: So proteomics was performed to detect proteins components of Cordyceps, and analysis it. In addition, MRL/lpr mice were used to study the progression of LN. The MRL/lpr mice were fed either CP (i.g, 0.5, 1.0, 1.5 g/kg/d), prednisolone acetate (PA, i.g, 6 mg/kg/d), or Bailing capsule (BC, i.g, 0.75 g/kg/d) for 8 weeks. Hematoxylin-eosin (H&E), Periodic Acid Schif (PAS) and Masson's stainings, Immunofluorescence, and Immunohistochemistry were performed to verify the therapeutic effect of CP on MRL/lpr mice. The mechanism by CP alimerated LN was uncovered by Western blotting (WB) and Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) methods. RESULTS: Our results revealed that CP blocked proteinuria production and renal inflammatory infiltratation in MRL/lpr mice to reduce the renal fibrosis. In addition, CP worked better than BC which is artificial Cordyceps fungus powder in regulating proteinuria to urine creatinine ratio and interleukin-4(IL-4) protein amount. Especially, CP modulated the STAT3/mTOR/NF-кB signaling pathway in LN mice and brought a more pronounced lowering effect on the contents of IL-6 and IL-1ß than the PA. CONCLUSION: CP could be a potential anti-inflammatory immune product with strong regulatory effects and potency than BC and PA in nephritis therapeutics.

The role of DDX46 in breast cancer proliferation and invasiveness: A potential therapeutic target.

DDX46, a member of DEAD-box (DDX) proteins, is associated with various cancers, while its involvement in the pathogenesis of breast cancer hasn't been reported so far. The study demonstrated the overexpression of DDX46 in human breast cancer cells and tissue samples, and correlated with high histological grade and lymph node metastasis. Downregulation of DDX46 in the breast cancer cell lines inhibited their proliferation and invasiveness in vitro. Furthermore, the growth of MDA-MB-231 xenografts was suppressed in nude mice by DDX46 knockingdown. Taken together, our findings suggest that DDX46 is an oncogenic factor in human breast cancer, and a potential therapeutic target.

Regulation of the intestinal flora: A potential mechanism of natural medicines in the treatment of type 2 diabetes mellitus.

Diabetes mellitus comprises a group of heterogeneous disorders, which are usually subdivided into type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM). Both genetic and environmental factors have been implicated in the onset of diabetes. Type 1 diabetes primarily involves autoimmune insulin deficiency. In comparison, type 2 diabetes is contributed by the pathological state of insulin deficiency and insulin resistance. In recent years, significant differences were found in the abundance of microflora, intestinal barrier, and intestinal metabolites in diabetic subjects when compared to normal subjects. To further understand the relationship between diabetes mellitus and intestinal flora, this paper summarizes the interaction mechanism between diabetes mellitus and intestinal flora. Furthermore, the natural compounds found to treat diabetes through intestinal flora were classified and summarized. This review is expected to provide a valuable resource for the development of new diabetic drugs and the applications of natural compounds.

Downregulated RRS1 inhibits invasion and metastasis of BT549 through RPL11­c­Myc­SNAIL axis.

Regulator of ribosome synthesis 1 (RRS1) is a key factor in ribosome biosynthesis and other cellular functions. High level of RRS1 in breast cancer cell lines is associated with increased cell proliferation, invasion and migration. RRS1 controls the assembly of the 60s subunit and maturation of 25S rRNA during ribosome biosynthesis. In this study, lentiviral transfection of sh­RNA was used to knock down the level of RRS1, to detect the effect of RRS1 on cell function and to explore the specific mechanism of RRS1 affecting cell invasion and metastasis by COIP and dual­luciferase reporter gene assays. The present study found that RRS1 knockdown reduced the accumulation of ribosome protein L11 (RPL11) in the nucleolus, which then migrated to the nucleoplasm and bound to c­Myc. This inhibited trans­activation of SNAIL by c­Myc and eventually decreased the invasion and metastasis capacity of the human breast cancer cell line BT549. Taken together, RRS1 regulates invasion and metastasis of human breast cancer cells through the RPL11­c­Myc­SNAIL axis. The findings are of great significance for exploring the mechanism of breast cancer invasion and metastasis and the corresponding regulatory factors.

Advances in the Relationship Between Regulator of Ribosome Synthesis 1 (RRS1) and Diseases.

A regulator of ribosome synthesis 1 (RRS1) was discovered in yeast and is mainly localized in the nucleolus and endoplasmic reticulum. It regulates ribosomal protein, RNA biosynthesis, and protein secretion and is closely involved in cellular senescence, cell cycle regulation, transcription, translation, oncogenic transformation etc., Mutations in the RRS1 gene are associated with the occurrence and development of Huntington's disease and cancer, and overexpression of RRS1 promotes tumor growth and metastasis. In this review, the structure, function, and mechanisms of RRS1 in various diseases are discussed.

MYCN­amplified neuroblastoma cell­derived exosomal miR­17­5p promotes proliferation and migration of non­MYCN amplified cells.

Neuroblastoma (NB) is considered a highly prevalent extracranial solid tumor in young children, and the upregulation of N­myc proto­oncogene (MYCN) is closely associated with the late stages of NB and poor prognostic outcomes. The current study was designed to evaluate the effects of exosomal microRNA (miRNA/miR)­17­5p from MYCN­amplified NB cells on the proliferative and migratory potential of non­MYCN amplified NB cells. miR­17­5p was found to activate the PI3K/Akt signaling cascade by targeting PTEN, and the overexpression of miR­17­5p was found to promote cellular migration and proliferation in vitro. Further experimentation revealed that the elevated expression of miR­17­5p in SK­N­BE(2) cell­derived exosomes significantly promoted the proliferative and migratory capacities of SH­SY5Y cells by inhibiting PTEN. Collectively, these findings demonstrated that miR­17­5p derived from MYCN­amplified NB cell exosomes promoted the migration and proliferation of non­MYCN amplified cells, highlighting an exosome­associated malignant role for miR­17­5p.

TMEM166 inhibits cell proliferation, migration and invasion in hepatocellular carcinoma via upregulating TP53.

Transmembrane protein 166 (TMEM166), an endoplasmic reticulum-associated protein, functions in many diseases via regulating autophagy and/or apoptosis. However, the role of TMEM166 in hepatocellular carcinoma (HCC) remains largely unknown. In this study, we detected the expression of TMEM166 in HCC by real-time fluorescent quantitative PCR (RT-qPCR), immunohistochemistry and western blot. To investigate its biological function and underlying mechanism in HCC, TMEM166 was overexpressed in HCC cell lines and assessed its effects on cell proliferation, migration, invasion, apoptosis and cell cycle by MTT assay, wound healing assay, Transwell assay, Annexin V-FITC/PI assay, JC-1 staining and flow cytometry assay, respectively. Results demonstrated that the expression of TMEM166 was significantly decreased in HCC and was associated with advanced TNM clinical stage and poor clinical outcome of HCC patients. TMEM166 overexpression inhibited HCC cells proliferation, migration and invasion. Furthermore, TMEM166 inhibited cell proliferation by inducing apoptosis and cell cycle arrest via upregulating anti-oncogene TP53 and TP53 knockdown significantly alleviated the anti-tumor effects of TMEM166 on HCC cells. This study provides the first comprehensive analysis the role of TMEM166 in HCC. TMEM166 displays a fine anti-tumor activity on HCC cells involving a mechanism of upregulating TP53. This study suggests TMEM166 is a potential target for the treatment of HCC.

Isatin inhibits the invasion and metastasis of SH­SY5Y neuroblastoma cells in vitro and in vivo.

Indoline­2,3­dione or indole­1H­2,3­dione, commonly known as isatin, is found in plants of genus Isatin and in Couropita guianancis aubl, and inhibits tumor cell proliferation through its antioxidant effects. The present study analyzed the effect of isatin on the malignant phenotype of neuroblastoma cells, and reported that isatin significantly inhibited neuroblastoma cell proliferation, invasion and migration in vitro in a dose­dependent manner, and distant metastasis in tumor­bearing mice. Mechanistically, isatin inhibited lysine­specific histone demethylase (LSD)1 and reversed the blockade on p53, thereby activating the apoptotic pathway. The inhibitory effect of isatin on LSD1 may be mediated via direct binding and molecular docking or indirectly through the TGFß/ERK/NF­κB signaling pathway. Isatin also alleviated the renal and hepatic toxicity of cyclophosphamide in the tumor­bearing mice, indicating its potential as a candidate drug as well as an adjuvant for treating metastatic neuroblastoma.

TIM-3 blockade combined with bispecific antibody MT110 enhances the anti-tumor effect of γδ T cells.

As ideal cells that can be used for adoptive cell therapy, γδ T cells are a group of homogeneous cells with high proliferative and tumor killing ability. However, γδ T cells are apt to apoptosis and show decreased cytotoxicity under persistent stimulation in vitro and cannot aggregate at tumor sites efficiently in vivo, both of which are two main obstacles to tumor adoptive immunotherapy. In this study, we found that the immune checkpoint T-cell immunoglobulin domain and mucin domain 3 (TIM-3) were up-regulated significantly on γδ T cells during their ex vivo expansion and this up-regulation contributed to the dysfunction of γδ T cells. Although the killing ability of γδ T cells against breast cancer cells which exhibited a high level of epithelial cell adhesion molecule (EpCAM) was enhanced, the level of TIM-3 on γδ T cells was also further up-regulated under the application of the bispecific antibody MT110 (anti-CD3 × anti-EpCAM) which can redirect T cells to target cells. Besides, these γδ T cells with up-regulated TIM-3 exhibited an increased susceptibility to apoptosis. By reinvigorating dysfunctional γδ T cells and promoting them to accumulate at tumor sites, the combined use of TIM-3 inhibitor and MT110 could further enhance the anti-tumor effect of the adoptively transfused γδ T cells. These results may have clinical implications for the design of new translational anti-tumor regimens aimed at combining checkpoint blockade and immune cell redirection.

Isatin inhibits SH-SY5Y neuroblastoma cell invasion and metastasis through PTEN signaling.

OBJECTIVE: Isatin has gained attention in recent years owing to its anticancer properties and is thought to offer medical benefits. Isatin is an endogenous oxidized indole with various behavioral and metabolic properties and is commonly found in mammalian tissues and fluids. It has several plausible applications in biomedical research and has also been investigated as a potential anticancer agent. However, its effects on neuroblastoma (NB) cells are unclear. Here, we evaluate the effects of isatin on neuroblastoma cell metastasis and invasion and reveal the underlying mechanism. METHODS: NB cell viability was evaluated with the cell counting kit (CCK)-8 assay. NB cell invasion and migration abilities were tested with transwell and wound healing experiments. The relative mRNA expression of associated molecules was detected with real-time polymerase chain reaction (RT-PCR) and quantitative PCR. The expression level of related proteins was detected with western blotting. RESULTS: Isatin inhibited the proliferation, invasion, and migration of neuroblastoma cells in a dose-dependent manner. Isatin increased the expression level of H3K4m1 and phosphatase and tensin homolog (PTEN) and decreased the phosphorylation level of PTEN downstream proteins phosphoinositide 3-kinase, protein kinase B, mammalian target of rapamycin, focal adhesion kinase, and SHC. Together, these results support the potential anti-metastatic effects of isatin on NB cells.

Functional role of RRS1 in breast cancer cell proliferation.

RRS1 (human regulator of ribosome synthesis 1), an essential nuclear protein involved in ribosome biogenesis, is overexpressed in some human cancers, yet its role in breast cancer remains unclear. Here, we report a functional analysis of RRS1 in breast cancer and its likely mechanism. Immunohistochemistry (IHC) and RT-qPCR analyses indicated that RRS1 was commonly overexpressed in breast cancer tissues. The copy numbers of RRS1 were higher in tumours compared with those for normal tissues. And there was a significant correlation between copy number and mRNA expression. In addition, RRS1 overexpression was significantly correlated with lymph node metastasis and poor survival. RRS1 mRNA and protein levels were also significantly increased in a panel of human breast cancer cell lines. RRS1 knockdown inhibited proliferation and induced apoptosis and cell cycle arrest in all three cell lines. Furthermore, RRS1 knockdown suppressed the tumour formation and growth of MDA-MB-231 cells in nude mice. Additionally, RRS1 knockdown activated p53 and p21 in MCF-7 cells. A marked increase in the quantity of ribosome-free RPL11 was detected by Western blot. Moreover, co-immunoprecipitation (CoIP) experiments showed that RRS1 knockdown activated p53 by facilitating the direct contact of MDM2 and RPL11/RPL5. Taken together, our results suggest that RRS1 may contribute to breast cancer proliferation through RPL11/MDM2-mediated p53 activation. Therefore, RRS1 may be a promising target for breast cancer therapy.

Evaluation of medicated feeds with antiparasitical and immune-enhanced Chinese herbal medicines against Ichthyophthirius multifiliis in grass carp (Ctenopharyngodon idellus).

Since malachite green was banned for using in food fish due to its carcinogenic and teratogenic effects on human, the search of alternative drug to treat Ichthyophthirius multifiliis becomes urgent. This study aimed to (1) evaluate the ethanol extracts of medicinal plants Cynanchum atratum, Zingiber officinale, Cynanchum paniculatum, immunostimulant (A), and immunostimulant (B) for their efficacy against I. multifiliis, and (2) determine effects of medicated feeds with C. atratum, Z. officinale, C. paniculatum, and immunostimulant (A) to treat I. multifiliis in grass carp. The results in this study showed that the minimum concentrations of C. atratum, Z. officinale, and C. paniculatum extracts for killing all theronts were 16, 8, and 16 mg/L, respectively. In vivo experiments, fish fed with medicated feeds of C. atratum for 10 days, or Z. officinale for 3 days, or combination of three plants for 10 days resulted in a significant reduction in the I. multifiliis infective intensity on grass carp after theronts exposure. Grass carp fed with medicated feeds of immunostimulant (A) for 21 days showed no infection and 100 % of survival 15 days post theronts exposure. Therefore, immunostimulant (A) is a promising feed supplement to treated I. multifiliis with good antiparasitic efficacy.