Salvianolic Acid B Significantly Suppresses the Migration of Melanoma Cells via Direct Interaction with ß-Actin.

Melanoma is the most aggressive and difficult to treat of all skin cancers. Despite advances in the treatment of melanoma, the prognosis for melanoma patients remains poor, and the recurrence rate remains high. There is substantial evidence that Chinese herbals effectively prevent and treat melanoma. The bioactive ingredient Salvianolic acid B (SAB) found in Salvia miltiorrhiza, a well-known Chinese herbal with various biological functions, exhibits inhibitory activity against various cancers. A375 and mouse B16 cell lines were used to evaluate the main targets and mechanisms of SAB in inhibiting melanoma migration. Online bioinformatics analysis, Western blotting, immunofluorescence, molecular fishing, dot blot, and molecular docking assays were carried out to clarify the potential molecular mechanism. We found that SAB prevents the migration and invasion of melanoma cells by inhibiting the epithelial-mesenchymal transition (EMT) process of melanoma cells. As well as interacting directly with the N-terminal domain of ß-actin, SAB enhanced its compactness and stability, thereby inhibiting the migration of cells. Taken together, SAB could significantly suppress the migration of melanoma cells via direct binding with ß-actin, suggesting that SAB could be a helpful supplement that may enhance chemotherapeutic outcomes and benefit melanoma patients.

Identification of small molecule inhibitors of G3BP-driven stress granule formation.

Stress granule formation is triggered by the release of mRNAs from polysomes and is promoted by the action of the RNA-binding proteins G3BP1/2. Stress granules have been implicated in several disease states, including cancer and neurodegeneration. Consequently, compounds that limit stress granule formation or promote their dissolution have potential as both experimental tools and novel therapeutics. Herein, we describe two small molecules, G3BP inhibitor a and b (G3Ia and G3Ib), designed to bind to a specific pocket in G3BP1/2 that is targeted by viral inhibitors of G3BP1/2 function. In addition to disrupting the co-condensation of RNA, G3BP1, and caprin 1 in vitro, these compounds inhibit stress granule formation in cells treated prior to or concurrent with stress and dissolve pre-existing stress granules. These effects are consistent across multiple cell types and a variety of initiating stressors. Thus, these compounds represent powerful tools to probe the biology of stress granules and hold promise for therapeutic interventions designed to modulate stress granule formation.

Luteolin exhibits synergistic therapeutic efficacy with erastin to induce ferroptosis in colon cancer cells through the HIC1-mediated inhibition of GPX4 expression.

Colon cancer continues to be a prevalent gastrointestinal malignancy with a bleak prognosis. The induction of ferroptosis, a new form of regulated cell death, has emerged as a potentially effective strategy for the treatment of colon cancer. However, numerous colon cancer cells display resistance to ferroptosis induced by erastin, a well-established ferroptosis inducer. Finding drugs that can enhance the susceptibility of colon cancer cells to erastin is of utmost importance. This study aimed to examine the synergistic therapeutic impact of combining erastin with a bioactive flavonoid compound luteolin on the ferroptosis-mediated suppression of colon cancer. Human colon cancer HCT116 and SW480 cells were used for the in vitro studies and a xenograft of colon cancer model in BALB/c nude mice was established for the in vivo experiments. The results showed that combinative treatment of luteolin and erastin effectively inhibited the viability and proliferation of colon cancer cells. Luteolin and erastin cotreatment synergistically induced ferroptosis, concomitant with a reduction in glutathione and an elevation in lipid peroxides. In vivo, combinative treatment of luteolin and erastin exhibited a pronounced therapeutic effect on xenografts of colon cancer, characterized by a significant induction of ferroptosis. Mechanistically, luteolin in combination with erastin synergistically reduced the expression of glutathione peroxidase 4 (GPX4), an antioxidase overexpressed in colon cancer cells. Furthermore, luteolin and erastin cotreatment significantly upregulated the expression of hypermethylated in cancer 1 gene (HIC1), a transcriptional repressor also recognized as a tumor suppressor. HIC1 overexpression notably augmented the suppression of GPX4 expression and facilitated ferroptotic cell death. In contrast, HIC1 silencing attenuated the inhibition of GPX4 expression and eliminated the ferroptosis. Conclusively, these results clearly demonstrated that luteolin acts synergistically with erastin and renders colon cancer cells vulnerable to ferroptosis through the HIC1-mediated inhibition of GPX4 expression, which may act as a promising therapeutic strategy.

Identification of small molecule inhibitors of G3BP-driven stress granule formation.

Stress granule formation is triggered by the release of mRNAs from polysomes and is promoted by the action of the paralogs G3BP1 and G3BP2. G3BP1/2 proteins bind mRNAs and thereby promote the condensation of mRNPs into stress granules. Stress granules have been implicated in several disease states, including cancer and neurodegeneration. Consequently, compounds that limit stress granule formation or promote their dissolution have potential as both experimental tools and novel therapeutics. Herein, we describe two small molecules, referred to as G3BP inhibitor a and b (G3Ia and G3Ib), designed to bind to a specific pocket in G3BP1/2 that is known to be targeted by viral inhibitors of G3BP1/2 function. In addition to disrupting co-condensation of RNA, G3BP1, and caprin 1 in vitro, these compounds inhibit stress granule formation in cells treated prior to or concurrent with stress, and dissolve pre-existing stress granules when added to cells after stress granule formation. These effects are consistent across multiple cell types and a variety of initiating stressors. Thus, these compounds represent ideal tools to probe the biology of stress granules and hold promise for therapeutic interventions designed to modulate stress granule formation.

ACSM3 suppresses proliferation and induces apoptosis and cell cycle arrest in acute myeloid leukemia cells via the regulation of IGF2BP2.

Acyl-CoA medium-chain synthetase-3 (ACSM3) has been reported to be involved in the malignant progression of multiple types of human cancer. Nevertheless, the role of ACSM3 in acute myeloid leukemia (AML) and its exact mechanism of action are as yet undefined. In the present study, the expression levels of ACSM3 and IGF2 mRNA-binding protein 2 (IGF2BP2) were evaluated using the Gene Expression Profiling Interactive Analysis database and AML cells. The Cell Counting Kit-8 assay and 5-ethynyl-2'-deoxyuridine staining were employed for the estimation of the cell proliferative activity. Induction of apoptosis and the assessment of the cell cycle were measured using flow cytometry and western blotting, respectively. The interaction of ACSM3 with IGF2BP2 was confirmed using an RNA immunoprecipitation assay. mRNA stabilization of ACSM3 following actinomycin D treatment was evaluated using reverse transcription-quantitative PCR analysis. The data indicated that the expression levels of ACSM3 were significantly downregulated, whereas those of IGF2BP2 were upregulated in tissues and AML cells. Downregulation of ACSM3 expression was closely associated with poor overall survival of patients with AML. ACSM3 overexpression repressed cell proliferative activity and induced apoptosis and cell cycle arrest. IGF2BP2 downregulated ACSM3 expression by reducing the stability of ACSM3 mRNA. In addition, IGF2BP2 overexpression counteracted the effects of ACSM3 overexpression noted on proliferation, induction of apoptosis and cell cycle arrest of HL-60 cells. In conclusion, ACSM3 repressed the cell proliferative activity and facilitated induction of apoptosis and cell cycle arrest in AML cells by modulating the expression of IGF2BP2.

Uncovering the effects and molecular mechanism of Astragalus membranaceus (Fisch.) Bunge and its bioactive ingredients formononetin and calycosin against colon cancer: An integrated approach based on network pharmacology analysis coupled with experimental validation and molecular docking.

Colon cancer is a highly malignant cancer with poor prognosis. Astragalus membranaceus (Fisch.) Bunge (Huang Qi in Chinese, HQ), a well-known Chinese herbal medicine and a popular food additive, possesses various biological functions and has been frequently used for clinical treatment of colon cancer. However, the underlying mechanism is not fully understood. Isoflavonoids, including formononetin (FMNT) and calycosin (CS), are the main bioactive ingredients isolated from HQ. Thus, this study aimed to explore the inhibitory effects and mechanism of HQ, FMNT and CS against colon cancer by using network pharmacology coupled with experimental validation and molecular docking. The network pharmacology analysis revealed that FMNT and CS exerted their anticarcinogenic actions against colon cancer by regulating multiple signaling molecules and pathways, including MAPK and PI3K-Akt signaling pathways. The experimental validation data showed that HQ, FMNT and CS significantly suppressed the viability and proliferation, and promoted the apoptosis in colon cancer Caco2 and HT-29 cells. HQ, FMNT and CS also markedly inhibited the migration of Caco2 and HT-29 cells, accompanied by a marked increase in E-cadherin expression, and a notable decrease in N-cadherin and Vimentin expression. In addition, HQ, FMNT and CS strikingly decreased the expression of ERK1/2 phosphorylation (p-ERK1/2) without marked change in total ERK1/2 expression. They also slightly downregulated the p-Akt expression without significant alteration in total Akt expression. Pearson correlation analysis showed a significant positive correlation between the inactivation of ERK1/2 signaling pathway and the HQ, FMNT and CS-induced suppression of colon cancer. The molecular docking results indicated that FMNT and CS had a strong binding affinity for the key molecules of ERK1/2 signaling pathway. Conclusively, HQ, FMNT and CS exerted good therapeutic effects against colon cancer by mainly inhibiting the ERK1/2 signaling pathway, suggesting that HQ, FMNT and CS could be useful supplements that may enhance chemotherapeutic outcomes and benefit colon cancer patients.

Two cardenolide glycosides from the seed fairs of Asclepias curassavica and their cytotoxic activities.

Two cardenolide glycosides, corotoxigenin 3-O-[ß-D-glucopyranosyl-(1→4)-6-deoxy-ß-D-glucopyranoside] (1) and coroglaucigenin 3-O-[ß-D-glucopyranosyl-(1→4)-6-deoxy-ß-D-glucopyranoside] (2), were isolated from the seed fairs of Asclepias curassavica. The structures of 1-2 were determined based on the combination of the analysis of their MS, NMR spectroscopic data and acid hydrolysis. The inhibitory effects of compounds 1 and 2 on human colorectal carcinoma cells (HCT116), non-small cell lung carcinoma cells (A549) and hepatic cancer cells (SMMC-7721) were evaluated. The results showed that both compounds 1 and 2 significantly inhibited the viability, proliferation, and migration of A549, HCT116 and SMMC-7721 cells, suggesting that compounds 1 and 2 can be applied in the treatment of lung, colon and liver cancers in clinical practice. This study may not only provide a scientific basis for clarifying the active ingredients in A. curassavica, but also help to understand its antitumor activity, which can promote the application of A. curassavica in clinical treatment of various cancers.

Malic enzyme 2 maintains protein stability of mutant p53 through 2-hydroxyglutarate.

Many types of cancer feature TP53 mutations with oncogenic properties. However, whether the oncogenic activity of mutant p53 is affected by the cellular metabolic state is unknown. Here we show that cancer-associated mutant p53 protein is stabilized by 2-hydroxyglutarate generated by malic enzyme 2. Mechanistically, malic enzyme 2 promotes the production of 2-hydroxyglutarate by adjusting glutaminolysis, as well as through a reaction that requires pyruvate and NADPH. Malic enzyme 2 depletion decreases cellular 2-hydroxyglutarate levels in vitro and in vivo, whereas elevated malic enzyme 2 expression increases 2-hydroxyglutarate production. We further show that 2-hydroxyglutarate binds directly to mutant p53, which reduces Mdm2-mediated mutant p53 ubiquitination and degradation. 2-Hydroxyglutarate supplementation is sufficient for maintaining mutant p53 protein stability in malic enzyme 2-depleted cells, and restores tumour growth of malic enzyme 2-ablated cells, but not of cells that lack mutant p53. Our findings reveal the previously unrecognized versatility of malic enzyme 2 catalytic functions, and uncover a role for mutant p53 in sensing cellular 2-hydroxyglutarate levels, which contribute to the stabilization of mutant p53 and tumour growth.

G3BP2 regulated by the lncRNA LINC01554 facilitates esophageal squamous cell carcinoma metastasis through stabilizing HDGF transcript.

Metastasis is the leading cause of death of patients with esophageal squamous cell carcinoma (ESCC). Although an increasing number of studies have demonstrated the involvement of G3BP2 in several human cancers, how G3BP2 interacts with long noncoding RNAs and regulates mRNA transcripts in mediating ESCC metastasis remains unclear. In this study, we uncovered that G3BP2 was upregulated in ESCC. Further analysis revealed that upregulation of G3BP2 was significantly correlated with lymph node metastasis, depth of tumor invasion and unfavorable outcomes in ESCC patients. Both in vitro and in vivo functional assays demonstrated that G3BP2 dramatically enhanced ESCC cell migration and invasion. Mechanistically, LINC01554 maintained the high G3BP2 expression in ESCC by protecting G3BP2 from degradation through ubiquitination and the interaction domains within LINC01554 and G3BP2 were identified. In addition, RNA-seq revealed that HDGF was regulated by G3BP2. G3BP2 bound to HDGF mRNA transcript to stabilize its expression. Ectopic expression of HDGF effectively abolished the G3BP2 depletion-mediated inhibitory effect on tumor cell migration. Intriguingly, introduction of compound C108 which can inhibit G3BP2 remarkedly suppressed ESCC cell metastasis in vitro and in vivo. Collectively, this study describes a newly discovered regulatory axis, LINC01554/G3BP2/HDGF, that facilitates ESCC metastasis and will provide novel therapeutic strategies for ESCC.

Chinese herbal medicines for prevention and treatment of colorectal cancer: From molecular mechanisms to potential clinical applications.

Worldwide, colorectal cancer (CRC) is one of the most common malignant tumors, leading to immense social and economic burdens. Currently, the main treatments for CRC include surgery, chemotherapy, radiotherapy and immunotherapy. Despite advances in the diagnosis and treatment of CRC, the prognosis for CRC patients remains poor. Furthermore, the occurrence of side effects and toxicities severely limits the clinical use of these therapies. Therefore, alternative medications with high efficacy but few side effects are needed. An increasing number of modern pharmacological studies and clinical trials have supported the effectiveness of Chinese herbal medicines (CHMs) for the prevention and treatment of CRC. CHMs may be able to effectively reduce the risk of CRC, alleviate the adverse reactions caused by chemotherapy, and prolong the survival time of patients with advanced CRC. Studies of molecular mechanisms have provided deeper insight into the roles of molecules from CHMs in treating CRC. This paper summarizes the current understanding of the use of CHMs for the prevention and treatment of CRC, the main molecular mechanisms involved in these processes, the role of CHMs in modulating chemotherapy-induced adverse reactions, and CHM's potential role in epigenetic regulation of CRC. The current study provides beneficial information on the use of CHMs for the prevention and treatment of CRC in the clinic, and suggests novel directions for new drug discovery against CRC.

G3BP1 Is a Tunable Switch that Triggers Phase Separation to Assemble Stress Granules.

The mechanisms underlying ribonucleoprotein (RNP) granule assembly, including the basis for establishing and maintaining RNP granules with distinct composition, are unknown. One prominent type of RNP granule is the stress granule (SG), a dynamic and reversible cytoplasmic assembly formed in eukaryotic cells in response to stress. Here, we show that SGs assemble through liquid-liquid phase separation (LLPS) arising from interactions distributed unevenly across a core protein-RNA interaction network. The central node of this network is G3BP1, which functions as a molecular switch that triggers RNA-dependent LLPS in response to a rise in intracellular free RNA concentrations. Moreover, we show that interplay between three distinct intrinsically disordered regions (IDRs) in G3BP1 regulates its intrinsic propensity for LLPS, and this is fine-tuned by phosphorylation within the IDRs. Further regulation of SG assembly arises through positive or negative cooperativity by extrinsic G3BP1-binding factors that strengthen or weaken, respectively, the core SG network.

A Genome-Editing Nanomachine Constructed with a Clustered Regularly Interspaced Short Palindromic Repeats System and Activated by Near-Infrared Illumination.

The RNA-guided CRISPR/Cas9 system is a powerful genome-editing technology with broad applications. Improving delivery efficiency and controllable activity of the CRISPR/Cas9 system is an area of intense research. We report the design, construction, and application of a CRISPR/Cas9 nanomachine (LACM), activated by a near-infrared (NIR) laser, which enables efficient delivery of single-guide RNA (sgRNA) into living cells and achieves controlled release of the sgRNA for the CRISPR/Cas9 activity. The LACM was constructed using a gold nanorod (AuNR) as a carrier that was decorated with dozens of protector DNAs stably hybridizing with the target binding domain of sgRNA. The DNA assembly on the AuNR protected the sgRNA. Irradiation with a NIR laser generated heat on the AuNR, resulting in controlled release of sgRNA, which guided the CRISPR/Cas9 genome editing. Successful editing of the EGFP and EMX1 genes in A549 and HEK293T cells, as well as knocking down of the PLK1 gene to induce apoptosis of the target cells, highlights the promising potential of the LACM for diverse applications.

Integrating Network Pharmacology and Experimental Validation to Investigate the Effects and Mechanism of Astragalus Flavonoids Against Hepatic Fibrosis.

Hepatic fibrosis (HF) represents the excessive wound healing where an excess amount of connective tissues is formed within the liver, finally resulting in cirrhosis or even hepatocellular carcinoma (HCC). Therefore, it is significant to discover the efficient agents and components to treat HF, thus restraining the further progression of hepatopathy. Astragalus membranaceus (Fisch.) Bunge [also called Astragali Radix (AR)] is a famous herb in traditional Chinese medicine (TCM), which possesses a variety of biological activities and exerts good therapeutic effects in the treatment of HF. Flavonoids account for the major active ingredients related to the AR pharmacological effects. Total AR flavonoids have been proved to exert inhibitory effects on hepatic fibrosis. This study aimed to further undertake network pharmacology analysis coupled with experimental validation and molecular docking to investigate the effects and mechanism of multiple flavonoid components from AR against liver fibrosis. The results of the network pharmacology analysis showed that the flavonoids from AR exerted their pharmacological effects against liver fibrosis by modulating multiple targets and pathways. The experimental validation data showed that the flavonoids from AR were able to suppress transforming growth factor beta 1 (TGF-ß1)-mediated activation of hepatic stellate cells (HSCs) and reduce extracellular matrix deposition in HSC-T6 cells via regulating the nuclear factor kappa B (NF-κB) signal transduction pathway. The results of the molecular docking study further showed that the flavonoids had a strong binding affinity for IκB kinase (IKKß) after docking into the crystal structure. The above results indicated that, flavonoids possibly exerted the anti-inflammatory effect on treating HF by mediating inflammatory signaling pathways. The potential mechanism of these flavonoids against liver fibrosis may be related to suppression of the NF-κB pathway through effective inhibition of IKKß. This study not only provides a scientific basis for clarifying the effects and mechanism of AR flavonoids against liver fibrosis but also suggests a novel promising therapeutic strategy for the treatment of liver fibrosis.

Synthesis and Biological Evaluation of Genistein-IR783 Conjugate: Cancer Cell Targeted Delivery in MCF-7 for Superior Anti-Cancer Therapy.

The flavonoid-based natural product genistein is a biologically active compound possessing promising anti-oxidant and anti-cancer properties. Poor pharmacokinetics along with low potency limit however the therapeutic application of genistein in cancer therapy. In order to overcome those limitations and to expand its therapeutic window of efficacy, we sought to covalently attach genistein with a heptamethine cyanine dye-IR 783-for cancer cell targeting and enhanced delivery to tumors. Herein we report the synthesis, a selective detailed characterization and preliminary in vitro/in vivo biological evaluation of genistein-IR 783 conjugate 4. The conjugate 4 displayed improved potency against human breast cancer MCF-7 cells (10.4 ± 1.0 µM) as compared with the parent genistein (24.8 ± 0.5 µM) or IR 783 (25.7 ± 0.7 µM) and exhibited selective high uptake in MCF-7 as against the normal mammary gland MCF-10A cells in various assays. In the cell viability assay, conjugate 4 exhibited over threefold lower potency against MCF-10A cells (32.1 ± 1.1 µM) suggesting that the anti-cancer profile of parent genistein is significantly improved upon conjugation with the dye IR783. Furthermore, the genistein-IR783 conjugate 4 was shown to be especially accumulated in MCF-7 cancer cells by fluorescent intensity measurements and inverted fluorescence microscopy in fixed cells as well as in live cells with time via live cell confocal fluorescence imaging. The mechanism-based uptake inhibition of conjugate 4 was observed with OATPs inhibitor BSP and in part with amiloride, as a macropinocytosis inhibitor. For the first time we have shown amiloride inhibited uptake of cyanine dye by about ~40%. Finally, genistein-IR 783 conjugate 4 was shown to be localized in MCF-7 tumor xenografts of mice breast cancer model via in vivo near infrared fluorescence (NIRF) imaging. In conclusion, conjugation of genistein with cyanine dye IR783 indeed improved its pharmacological profile by cancer cell selective uptake and targeting and therefore warrants further investigations as a new anti-cancer therapeutics derived from natural product genistein.

Evolution of Inosine-Specific Endonuclease V from Bacterial DNase to Eukaryotic RNase.

Endonuclease V (EndoV) cleaves the second phosphodiester bond 3' to a deaminated adenosine (inosine). Although highly conserved, EndoV homologs change substrate preference from DNA in bacteria to RNA in eukaryotes. We have characterized EndoV from six different species and determined crystal structures of human EndoV and three EndoV homologs from bacteria to mouse in complex with inosine-containing DNA/RNA hybrid or double-stranded RNA (dsRNA). Inosine recognition is conserved, but changes in several connecting loops in eukaryotic EndoV confer recognition of 3 ribonucleotides upstream and 7 or 8 bp of dsRNA downstream of the cleavage site, and bacterial EndoV binds only 2 or 3 nt flanking the scissile phosphate. In addition to the two canonical metal ions in the active site, a third Mn2+ that coordinates the nucleophilic water appears necessary for product formation. Comparison of EndoV with its homologs RNase H1 and Argonaute reveals the principles by which these enzymes recognize RNA versus DNA.

Houttuynia cordata Thunb. and its bioactive compound 2-undecanone significantly suppress benzo(a)pyrene-induced lung tumorigenesis by activating the Nrf2-HO-1/NQO-1 signaling pathway.

BACKGROUND: Lung cancer remains the most common cause of cancer-related deaths, with a high incidence and mortality in both sexes worldwide. Chemoprevention has been the most effective strategy for lung cancer prevention. Thus, exploring novel and effective candidate agents with low toxicity for chemoprevention is essential and urgent. Houttuynia cordata Thunb. (Saururaceae) (H. cordata), which is a widely used herbal medicine and is also popularly consumed as a healthy vegetable, exhibits anti-inflammatory, antioxidant and antitumor activity. However, the chemopreventive effect of H. cordata against benzo(a)pyrene (B[a]P)-initiated lung tumorigenesis and the underlying mechanism remain unclear. METHODS: A B[a]P-stimulated lung adenocarcinoma animal model in A/J mice in vivo and a normal lung cell model (BEAS.2B) in vitro were established to investigate the chemopreventive effects of H. cordata and its bioactive compound 2-undecanone against lung tumorigenesis and to clarify the underlying mechanisms. RESULTS: H. cordata and 2-undecanone significantly suppressed B[a]P-induced lung tumorigenesis without causing obvious systemic toxicity in mice in vivo. Moreover, H. cordata and 2-undecanone effectively decreased B[a]P-induced intracellular reactive oxygen species (ROS) overproduction and further notably protected BEAS.2B cells from B[a]P-induced DNA damage and inflammation by significantly inhibiting phosphorylated H2A.X overexpression and interleukin-1ß secretion. In addition, H. cordata and 2-undecanone markedly activated the Nrf2 pathway to induce the expression of the antioxidative enzymes heme oxygenase-1 (HO-1) and NAD(P)H: quinone oxidoreductase 1 (NQO-1). Nrf2 silencing by transfection with Nrf2 siRNA markedly decreased the expression of HO-1 and NQO-1 to diminish the reductions in B[a]P-induced ROS overproduction, DNA damage and inflammation mediated by H. cordata and 2-undecanone. CONCLUSIONS: H. cordata and 2-undecanone could effectively activate the Nrf2-HO-1/NQO-1 signaling pathway to counteract intracellular ROS generation, thereby attenuating DNA damage and inflammation induced by B[a]P stimulation and playing a role in the chemoprevention of B[a]P-induced lung tumorigenesis. These findings provide new insight into the pharmacological action of H. cordata and indicate that H. cordata is a novel candidate agent for the chemoprevention of lung cancer.

p53 regulation of ammonia metabolism through urea cycle controls polyamine biosynthesis.

Cancer cells exhibit altered and usually increased metabolic processes to meet their high biogenetic demands1,2. Under these conditions, ammonia is concomitantly produced by the increased metabolic processing. However, it is unclear how tumour cells dispose of excess ammonia and what outcomes might be caused by the accumulation of ammonia. Here we report that the tumour suppressor p53, the most frequently mutated gene in human tumours, regulates ammonia metabolism by repressing the urea cycle. Through transcriptional downregulation of CPS1, OTC and ARG1, p53 suppresses ureagenesis and elimination of ammonia in vitro and in vivo, leading to the inhibition of tumour growth. Conversely, downregulation of these genes reciprocally activates p53 by MDM2-mediated mechanism(s). Furthermore, the accumulation of ammonia causes a significant decline in mRNA translation of the polyamine biosynthetic rate-limiting enzyme ODC, thereby inhibiting the biosynthesis of polyamine and cell proliferation. Together, these findings link p53 to ureagenesis and ammonia metabolism, and further reveal a role for ammonia in controlling polyamine biosynthesis and cell proliferation.

A Systematic Review of Phytochemistry, Pharmacology and Pharmacokinetics on Astragali Radix: Implications for Astragali Radix as a Personalized Medicine.

Astragali radix (AR) is one of the most widely used traditional Chinese herbal medicines. Modern pharmacological studies and clinical practices indicate that AR possesses various biological functions, including potent immunomodulation, antioxidant, anti-inflammation and antitumor activities. To date, more than 200 chemical constituents have been isolated and identified from AR. Among them, isoflavonoids, saponins and polysaccharides are the three main types of beneficial compounds responsible for its pharmacological activities and therapeutic efficacy. After ingestion of AR, the metabolism and biotransformation of the bioactive compounds were extensive in vivo. The isoflavonoids and saponins and their metabolites are the major type of constituents absorbed in plasma. The bioavailability barrier (BB), which is mainly composed of efflux transporters and conjugating enzymes, is expected to have a significant impact on the bioavailability of AR. This review summarizes studies on the phytochemistry, pharmacology and pharmacokinetics on AR. Additionally, the use of AR as a personalized medicine based on the BB is also discussed, which may provide beneficial information to achieve a better and more accurate therapeutic response of AR in clinical practice.

Astragali radix and its main bioactive compounds activate the Nrf2-mediated signaling pathway to induce P-glycoprotein and breast cancer resistance protein.

ETHNOPHARMACOLOGICAL RELEVANCE: Astragali radix (Huang Qi, HQ), a well-known Chinese herbal medicine, is widely coadministered with many other drugs for treating diseases. The potential herb-drug interactions (HDIs) possibly occur during the combination therapy. P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) are the crucial targets that mediate the production of HDIs. We previously observed that HQ and its three main bioactive compounds, including Astragaloside IV (AS-IV), calycosin (CS) and formononetin (FMNT), could significantly induce the expression of P-gp and BCRP in HepG2 cells in vitro. However, their modulations on the function of P-gp and BCRP remain unknown; their impact on these two proteins expression in vivo is not clear; the exact regulatory mechanism has also not yet been explored. AIM OF THE STUDY: This study aimed to investigate the impact of HQ, AS-IV, CS and FMNT on P-gp and BCRP in vivo, and the exact regulatory mechanism involved. The effects of HQ and these compounds on the function of P-gp and BCRP were also studied. MATERIALS AND METHODS: Wild-type C57BL/6 mice and nuclear factor E2-related factor-2 knockout (Nrf2-/-) C57BL/6 mice were orally treated with HQ, AS-IV, CS or FMNT. The protein levels of P-gp and BCRP in the liver of mice were measured by using Western blot and immunohistochemistry. The mRNA levels were measured by using real-time PCR. The activation of the drugs on the antioxidant response element (ARE)-luciferin activity was studied by using reporter assay in a stably transfected HepG2-C8 cells. The efflux activity of P-gp and BCRP in HepG2 cells were tested by using flow cytometer with typical probes. RESULTS: HQ, AS-IV, CS and FMNT significantly upregulated the P-gp and BCRP expression in the liver of wild-type mice. The induction was significantly reversed in the Nrf2-/- mice. HQ and these compounds significantly increased the Nrf2 expression in wild-type mice. HQ and these compounds also markedly enhanced the ARE-luciferin activity and promoted the nuclear translocation of Nrf2 in cells. Besides, HQ and these compounds significantly enhanced the efflux activity of P-gp and BCRP, and increased the intracellular ATP levels. CONCLUSIONS: Our results proved that HQ and its main bioactive compounds could induce the P-gp and BCRP expression through the activation of the Nrf2-mediated signaling pathway. HQ and these compounds also significantly enhanced the efflux activity of P-gp and BCRP, and the increased intracellular ATP levels were likely involved in the increased P-gp and BCRP function. These results suggested that potentially HDIs likely occurred when HQ was used concomitantly with other drugs that are substrates of P-gp and BCRP.

Identification of a Novel Small RNA srvg23535 in Vibrio alginolyticus ZJ-T and Its Characterization With Phenotype MicroArray Technology.

Small non-coding RNAs (sRNAs) are important modulators of gene expression and are involved in the pathogenesis and survival of prokaryotes. However, few studies have been conducted with Vibrio alginolyticus, which limits our ability to probe the regulation of virulence and environmental adaptation by sRNAs in this opportunistic pathogen. In this study, the sRNA candidate srvg23535 was identified in V. alginolyticus ZJ-T. The precise transcript end, secondary structure, and sequence conservation were determined. A srvg23535 null mutant was constructed and characterized by using Phenotype MicroArray (PM) technology. In silico target prediction was conducted by IntaRNA and TargetRNA2. Subsequently, a 107 nt transcript was validated with a sigma70 promoter at the 5' end and a Rho-independent terminator at the 3' end. The sRNA srvg23535 had four stem-loop structures and was conserved among Vibrio harveyi, Vibrio parahaemolyticus, and Vibrio splendidus. Deletion of srvg23535 in V. alginolyticus ZJ-T led to a weaker utilization of D-mannose, D-melibiose, lactulose, and inosine as carbon sources but stronger utilization of L-cysteine as nitrogen source. Moreover, the srvg2353 mutant showed stronger resistance to osmotic stress but weaker resistance to pH stress. Additionally, a total of 22 common targets were identified and several were related to the observed phenotype of the mutant. This study indicated that the novel sRNA, srvg23535, is conserved and restricted to Vibrio spp., affecting the utilization of several carbon and nitrogen sources and the response to osmotic and pH stress. These results extend our understanding of sRNA regulation in V. alginolyticus and provide a significant resource for the further study of the precise target mRNAs of srvg23535, which may provide targets for antibacterial therapeutic or attenuated vaccines against Vibrio spp.