Structural insights into the unusual core photocomplex from a triply extremophilic purple bacterium, Halorhodospira halochloris.

Halorhodospira (Hlr.) halochloris is a triply extremophilic phototrophic purple sulfur bacterium, as it is thermophilic, alkaliphilic, and extremely halophilic. The light-harvesting-reaction center (LH1-RC) core complex of this bacterium displays an LH1-Qy transition at 1,016 nm, which is the lowest-energy wavelength absorption among all known phototrophs. Here we report the cryo-EM structure of the LH1-RC at 2.42 Å resolution. The LH1 complex forms a tricyclic ring structure composed of 16 αßγ-polypeptides and one αß-heterodimer around the RC. From the cryo-EM density map, two previously unrecognized integral membrane proteins, referred to as protein G and protein Q, were identified. Both of these proteins are single transmembrane-spanning helices located between the LH1 ring and the RC L-subunit and are absent from the LH1-RC complexes of all other purple bacteria of which the structures have been determined so far. Besides bacteriochlorophyll b molecules (B1020) located on the periplasmic side of the Hlr. halochloris membrane, there are also two arrays of bacteriochlorophyll b molecules (B800 and B820) located on the cytoplasmic side. Only a single copy of a carotenoid (lycopene) was resolved in the Hlr. halochloris LH1-α3ß3 and this was positioned within the complex. The potential quinone channel should be the space between the LH1-α3ß3 that accommodates the single lycopene but does not contain a γ-polypeptide, B800 and B820. Our results provide a structural explanation for the unusual Qy red shift and carotenoid absorption in the Hlr. halochloris spectrum and reveal new insights into photosynthetic mechanisms employed by a species that thrives under the harshest conditions of any phototrophic microorganism known.

New insights on the photocomplex of Roseiflexus castenholzii revealed from comparisons of native and carotenoid-depleted complexes.

In wild-type phototrophic organisms, carotenoids (Crts) are primarily packed into specific pigment-protein complexes along with (Bacterio)chlorophylls and play important roles in the photosynthesis. Diphenylamine (DPA) inhibits carotenogenesis but not phototrophic growth of anoxygenic phototrophs and eliminates virtually all Crts from photocomplexes. To investigate the effect of Crts on assembly of the reaction center-light-harvesting (RC-LH) complex from the filamentous anoxygenic phototroph Roseiflexus (Rfl.) castenholzii, we generated carotenoidless (Crt-less) RC-LH complexes by growing cells in the presence of DPA. Here, we present cryo-EM structures of the Rfl. castenholzii native and Crt-less RC-LH complexes with resolutions of 2.86 Å and 2.85 Å, respectively. From the high-quality map obtained, several important but previously unresolved details in the Rfl. castenholzii RC-LH structure were determined unambiguously including the assignment and likely function of three small polypeptides, and the content and spatial arrangement of Crts with bacteriochlorophyll molecules. The overall structures of Crt-containing and Crt-less complexes are similar. However, structural comparisons showed that only five Crts remain in complexes from DPA-treated cells and that the subunit X (TMx) flanked on the N-terminal helix of the Cyt-subunit is missing. Based on these results, the function of Crts in the assembly of the Rfl. castenholzii RC-LH complex and the molecular mechanism of quinone exchange is discussed. These structural details provide a fresh look at the photosynthetic apparatus of an evolutionary ancient phototroph as well as new insights into the importance of Crts for proper assembly and functioning of the RC-LH complex.

Multifunctional Protein Hybrid Nanoplatform for Synergetic Photodynamic-Chemotherapy of Malignant Carcinoma by Homologous Targeting Combined with Oxygen Transport.

Photodynamic therapy (PDT) under hypoxic conditions and drug resistance in chemotherapy are perplexing problems in anti-tumor treatment. In addition, central nervous system neoplasm-targeted nanoplatforms are urgently required. To address these issues, a new multi-functional protein hybrid nanoplatform is designed, consisting of transferrin (TFR) as the multicategory solid tumor recognizer and hemoglobin for oxygen supply (ODP-TH). This protein hybrid framework encapsulates the photosensitizer protoporphyrin IX (PpIX) and chemotherapeutic agent doxorubicin (Dox), which are attached by a glutathione-responsive disulfide bond. Mechanistically, ODP-TH crosses the blood-brain barrier (BBB) and specifically aggregated in hypoxic tumors via protein homology recognition. Oxygen and encapsulated drugs ultimately promote a therapeutic effect by down-regulating the abundance of multidrug resistance gene 1 (MDR1) and hypoxia-inducible factor-1-α (HIF-1α). The results reveal that ODP-TH achieves oxygen transport and protein homology recognition in the hypoxic tumor occupation. Indeed, compared with traditional photodynamic chemotherapy, ODP-TH achieves a more efficient tumor-inhibiting effect. This study not only overcomes the hypoxia-related inhibition in combination therapy by targeted oxygen transport but also achieves an effective treatment of multiple tumors, such as breast cancer and glioma, providing a new concept for the construction of a promising multi-functional targeted and intensive anti-tumor nanoplatform.

Design, Synthesis and Antitumor Activity of Novel link-bridge and B-Ring Modified Combretastatin A-4 (CA-4) Analogues as Potent Antitubulin Agents.

A series of 12 novel acylhydrazone, chalcone and amide-bridged analogues of combretastatin A-4 were designed and synthesized toward tubulin. All these compounds were determined by elemental analysis, (1)H NMR, and MS. Among them, compound 7 with acylhydrazone-bridge, bearing a benzyl at the indole-N position, was identified as a potent antiproliferative agent against a panel of cancer cell lines with IC50 values ranging from 0.08 to 35.6 µM. In contrast, its cytotoxic effects on three normal human cells were minimal. Cellular studies have revealed that the induction of apoptosis by compound 7 was associated with a collapse of mitochondrial membrane potential, accumulation of reactive oxygen species, alterations in the expression of some cell cycle-related proteins (Cyclin B1, Cdc25c, Cdc2, P21) and some apoptosis-related proteins (Bax, PARP, Bcl-2, Caspase3). The docking mode showed the binding posture of CA-4 and compound 7 are similar in the colchicine-binding pocket of tubulin, as confirmed by colchicine-tubulin competitive binding assay, tubulin polymerization inhibitory activity, extracellular protein expression determination assay and confocal immunofluorescence microscopy. In vivo study, compound 7 effectively inhibited A549 xenograft tumor growth without causing significant loss of body weight suggesting that compound 7 is a promising new antimitotic agent with clinical potential.

Synthesis, physicochemical characterization and pharmacological investigation of indolacin-5-fluorouracil-1-ylmethyl ester.

The objective of this work is to synthesize indolacin-5-fluorouracil-1-ylmethyl ester and the structure was confirmed by means of UV, IR, 1H-NMR, 13C-NMR and mass spectrometry. The physicochemical parameters of melting point, solubility, apparent partition coefficient were investigated. S180 sarcoma, H22 hapatitic cancer and Lewis-transplanted mice were used to evaluate the anti-tumor activity of indolacini-5-fluorouracil-1-ylmethyl ester compared with 5-fluorouracil in vivo. Anti-inflammatory and analgesic activities were evaluated in mice. The inhibitory ratio of indolacini- 5-fluorouracil-1-ylmethyl ester is comparative to that of 5-fluorouracil. This study indicates that 5-fluorouracil-1-ylmethyl ester may represent a new anticancer predrug of 5-fluorouracil to produce a combined effect of indolacin and 5-fluorouracil for cancer therapy.

E Platinum, a newly synthesized platinum compound, induces autophagy via inhibiting phosphorylation of mTOR in gastric carcinoma BGC-823 cells.

A tightly regulated catabolic process named autophagy involves the degradation of intracellular components via lysosomes. Here we investigate the antitumor effect of E Platinum, a newly synthesized derivative of oxaliplatin, in vivo and in vitro. E Platinum exhibits growth inhibition of various tumor cells in a dose-dependent manner, but the mechanism underlying it is unclear. Based on theory introducing autophagy, we preliminarily investigate whether autophagy could contribute to the antitumor activity of E Platinum. Our results showed that autophagy induced by 12.5 µM E Platinum in gastric carcinoma BGC-823 cells was significantly characterized by the FITC-fluorescent microtubule associated protein 1 light chain 3 (MAP-LC3), lysosomal-rich/acidic compartments visualized with Lysotracker red (LTR-red) and an accumulation of numerous large autophagic vesicles within the cytoplasm, but not in the control cells. Meanwhile treatment of cells with 12.5 µM E Platinum resulted in conversion of water soluble LC3 (LC3-I) to lipidated and autophagosome-associated form (LC3-II) as well as increasing expression of autophagy protein Beclin 1. Activation of predominant lysosomal aspartic protease, LAMP-1 and cathepsin D, was demonstrated. Moreover, RNA interference targeting Beclin 1, inhibition of autophagy by 3-methyladenine (3-MA) and chloroquine significantly suppressed the above process as well as the BGC-823 cells growth inhibition triggered by 12.5 µM E Platinum. Studies of mechanism revealed that E Platinum suppressed activation of mTOR and p70S6K by decreasing phosphorylation of Akt, ERK1/2, JNK and p38 involved in mitogen-activated protein kinase signaling. We supported new evidences for E Platinum as a promising antitumor agent, involving with autophagy induction.

Effect of the timing of food intake on the absorption and bioavailability of carbamazepine immediate-release tablets in beagle dogs.

The effect of dosing time on the bioavailability of carbamazepine immediate-release (IR) tablets was investigated when administered to beagle dogs who were fasting, with co-administration of food (Co-food), and 0.5 h before food and 2 h after food. The study was conducted using a single dose of 200 mg (tablets/solution) with a 2 week washout period in a crossover design. Food intake significantly increased the rate and extent of tablet absorption. The C(max) (µg·ml⁻¹, 8.13/3.65) and t(max) (h, 1.83/0.92) were increased more than two-fold and the AUC0₋24 (µg·h·ml⁻¹, 20.09/8.19) was 2.5 times that of the values obtained under fasting conditions. The bioavailability of the tablets under fasting conditions was 91.2%, but increased to 223.5%, 182.8% and 148.4% in the Co-food, 0.5 h before food and 2 h after food groups, respectively (p < 0.05). Although there was no significant difference in the C(max) or AUC0₋24 between the treatments with food, the absorption appeared to be reduced to some extent when the tablets were given 2 h after food. The oral bioavailability of CBZ IR tablets was significantly affected by the timing of the food intake. This is maybe favored by the fluctuations in the level of bile salts with the timing of food intake. To obtain acute therapy for a drug with narrow therapeutic window, attention should be given to the dosing time and food intake interactions.

Reactive oxygen species-mitochondria pathway involved in FV-429-induced apoptosis in human hepatocellular carcinoma HepG2 cells.

FV-429 is a newly synthesized flavonoid with a bis(2-hydroxyethyl) amino propoxy substitution. In this study, we investigate the anticancer effect of FV-429 both in vivo and in vitro. These data have shown that FV-429 could significantly inhibit tumor growth in mice inoculated with Heps hepatoma cells without evident toxicity. After the treatment of FV-429 (40 mg/kg), the inhibitory rate of tumor weight was 52.12%. Then, we performed diamidinophenylindole staining and annexin V/propidium iodide double-staining assay to investigate the apoptosis induced by FV-429 in HepG2 cells. Further research revealed that FV-429 induced apoptosis through the mitochondrial apoptotic pathway, as indicated by a change in Bax/Bcl-2 ratios, collapse of mitochondrial membrane potential, the transposition of apoptotic-inducing factor and cytochrome c, caspase-3 and caspase-9 activation, and degradation of poly (ADP-ribose) polymerase. The accumulation of reactive oxygen species induced by FV-429 in HepG2 cells was also observed. Moreover, the mitogen-activated protein kinases, the downstream effect of reactive oxygen species accumulation including c-Jun N-terminal kinase and p38 mitogen-activated protein kinases, could be activated by FV-429. Taken together, our results provided a mechanistic framework for further exploration of FV-429 as a novel chemotherapy for human tumors.

Rice ABI5-Like1 regulates abscisic acid and auxin responses by affecting the expression of ABRE-containing genes.

Abscisic acid (ABA) regulates plant development and is crucial for plant responses to biotic and abiotic stresses. Studies have identified the key components of ABA signaling in Arabidopsis (Arabidopsis thaliana), some of which regulate ABA responses by the transcriptional regulation of downstream genes. Here, we report the functional identification of rice (Oryza sativa) ABI5-Like1 (ABL1), which is a basic region/leucine zipper motif transcription factor. ABL1 is expressed in various tissues and is induced by the hormones ABA and indole-3-acetic acid and stress conditions including salinity, drought, and osmotic pressure. The ABL1 deficiency mutant, abl1, shows suppressed ABA responses, and ABL1 expression in the Arabidopsis abi5 mutant rescued the ABA sensitivity. The ABL1 protein is localized to the nucleus and can directly bind ABA-responsive elements (ABREs; G-box) in vitro. A gene expression analysis by DNA chip hybridization confirms that a large proportion of down-regulated genes of abl1 are involved in stress responses, consistent with the transcriptional activating effects of ABL1. Further studies indicate that ABL1 regulates the plant stress responses by regulating a series of ABRE-containing WRKY family genes. In addition, the abl1 mutant is hypersensitive to exogenous indole-3-acetic acid, and some ABRE-containing genes related to auxin metabolism or signaling are altered under ABL1 deficiency, suggesting that ABL1 modulates ABA and auxin responses by directly regulating the ABRE-containing genes.

Effect of diallyl trisulfide on the pharmacokinetics of nifedipine in rats.

This study aimed to evaluate the effect of diallyl trisulfide (DATS), a major component derived from garlic, on the pharmacokinetics of nifedipine. Pharmacokinetic parameters of nifedipine were determined in rats following an oral gavage (3 mg/kg) or intravenous administration (0.75 mg/kg) of nifedipine with co-administration of DATS (20 mg/kg) and long-term pretreatment of DATS (20 mg/kg/d for 15 consecutive days). Compared to the control groups, higher C(max) and AUC(0-24 h) were observed for oral gavage of nifedipine after short-term and long-term pretreatment of DATS, whereas those for intravenous nifedipine were little changed. The oral bioavailabilities of nifedipine were remarkably enhanced via the concomitant use of DATS. In conclusion, DATS increased the oral exposure of nifedipine in rats likely by the modification of intestinal metabolism of nifedipine, indicating that combined use of DATS or DATS-containing supplement with nifedipine may require caution because high plasma concentrations may lead to an undesired toxicity of this agent. Practical Application: Patients suffering from cardiovascular disease should take caution in combined use of DATS or DATS-rich garlic supplement with nifedipine because long-term treatment of DATS could lead high plasma concentrations of nifedipine.

Gambogic acid-induced degradation of mutant p53 is mediated by proteasome and related to CHIP.

As an oncoprotein, mutant p53 is a potential tumor-specific target for cancer therapy. Most mutated forms of the protein are largely accumulated in cancer cells due to their increased stability. In the present study, we demonstrate that mutant p53 protein stability is regulated by gambogic acid (GA). Following GA exposure, protein levels of mutant p53 decreased while the mRNA levels were not affected in MDA-MB-435 cells, which indicate that GA down-regulates mutant p53 at post-transcription level. Co-treatment with GA and cycloheximide, a protein synthesis inhibitor, induced a decrease of half-life of mutant p53 protein. These findings indicated that the reduction of mutant p53 by GA was due to the destabilization and degradation of the protein. Furthermore, inhibition of proteasome activity by MG132 blocked GA-induced down-regulation of mutant p53, causing mutant p53 accumulation in detergent-insoluble cellular fractions. Further studies revealed that mutant p53 was ubiquitinated and it was chaperones related ubiquitin ligase carboxy terminus of Hsp70-interacting protein (CHIP) rather than MDM2 involved in the degradation of mutant p53. In addition, GA prevented Hsp90/mutant p53 complex formation and enhanced interaction of mutant p53 with Hsp70. Depletion of CHIP stabilized mutant p53 in GA treated cells. In conclusion, mutant p53 may be down-regulated by GA through chaperones-assisted ubiquitin/proteasome degradation pathway in cancer cells.

DHF-18, a new synthetic flavonoid, induced a mitochondrial-mediated apoptosis of hepatocarcinoma cells in vivo and in vitro.

A new synthetic flavonoid DHF-18, synthesized with a piperazine substitution, has been recently found to show potent anti-tumor activities both in vivo and in vitro. In this study, we demonstrated that DHF-18 significantly inhibited tumor growth in mice inoculated with Heps hepatoma cells without evident toxicity. After the treatment of 40mg/kg DHF-18, the inhibitory rate of tumor weight was 53.69%. To investigate whether apoptosis induction contributed to the anti-tumor effects of DHF-18, DAPI (diamidino-phenyl-indole) staining and Annexin V/PI (Propidium iodide) double staining were performed in our tests. The data showed that DHF-18 could induce the apoptosis cell death in HepG2 cells. Moreover, the apparent increase of intracellular reactive oxygen species levels and the reduction of mitochondria ΔΨm were both observed in HepG2 cells after DHF-18 treatment. Meanwhile, the transposition of apoptotic inducing factor (AIF) from mitochondria to nuclei, the release of cytochrome c from mitochondria and the activation of caspase-3, -9 were also detected, indicating that DHF-18 may induce apoptosis through a mitochondrial-mediated pathway. Additionally, DHF-18 decreased the expression of Bcl-2 protein, whereas the levels of Bax and Bak were found to increase after DHF-18 treatment. Moreover, the activation of caspase-8, the increase of TNF-R1 (Tumor necrosis factor receptor) and Bid were found. Taken together, our results suggested that DHF-18 may induce HeG2 cells apoptosis through a mitochondrial-dependent and independent pathway.

Wogonin potentiates the antitumor effects of low dose 5-fluorouracil against gastric cancer through induction of apoptosis by down-regulation of NF-kappaB and regulation of its metabolism.

Traditional Chinese medicines have been recognized as a new source of anticancer drugs or chemotherapy adjuvant to enhance the efficacy of chemotherapy and to ameliorate the side effects. Wogonin (WOG) has a potential for therapeutic use in the treatment of antitumor and chemoprophylaxis. 5-Fluorouracil (5-FU) is a key systemic chemotherapy drug and widely use in the treatment of solid tumors. In this study, we found that combination of WOG and 5-FU inhibited the viability of MGC-803 cells in a concentration-dependent manner and exhibited a synergistic anticancer effect (CI<1) when 5-FU was used at relatively low concentrations. The pro-apoptotic activity of two-drug combination was much stronger than single. Furthermore, WOG could decrease the mRNA levels of dihydropyrimidine dehydrogenase (DPD), the metabolic enzymes of 5-FU. WOG could inhibit the NF-kappaB nuclear translocation and I-kappaB phosphorylation. Moreover, combined treatment caused significantly growth inhibition of human tumor xenografts. In addition, WOG markedly enhanced the antitumor activity of low dose 5-FU (i.p. 10mg/kg/day), however there is no toxicity and influence on diet consumption in experimental animals. Taken together, our data's showed that WOG increased 5-FU retention for a prolonged catabolism by modulating 5-FU metabolic enzymes and sensitized the MGC-803 cells to 5-FU induced apoptosis by inhibiting the NF-kappaB nuclear translocation. The anti-gastric cancer effect of two-drug combination was much stronger than that of WOG or 5-FU alone. These results may be relevant to design new clinical therapeutic strategies against gastric cancer in future.

Reactive oxygen species-mitochondria pathway involved in LYG-202-induced apoptosis in human hepatocellular carcinoma HepG(2) cells.

Previously, we demonstrated that LYG-202, a newly synthesized flavonoid with a piperazine substitution, exhibited obvious antitumor activity in vivo and in vitro. The exact mechanism of this new compound remains unclear. In the present study, we examined the effects of LYG-202 on reactive oxygen species (ROS) production and the downstream signaling pathway in the apoptosis of human hepatocellular carcinoma HepG(2) cells. Pretreatment with NAC (N-acetylcysteine), a ROS production inhibitor, partly inhibited the apoptosis induced by LYG-202 via blocking the ROS generation. Further data revealed that LYG-202 induced ROS accumulation followed by a decrease in mitochondrial membrane potential (MMP), release of cytochrome c (Cyt c) and apoptosis-inducing factor (AIF) to cytosol, which induced apoptosis of the cells. Moreover, the mitogen-activated protein kinases (MAPK), the downstream effect of ROS accumulation including c-Jun N-terminal kinase (JNK) and p38 MAPK, could be activated by LYG-202. Taken together, the generation of ROS might play an important role in LYG-202-induced mitochondrial apoptosis pathway, which provided further support for LYG-202 as a novel anticancer therapeutic candidate.

Colon-specific drug delivery systems based on cyclodextrin prodrugs: in vivo evaluation of 5-aminosalicylic acid from its cyclodextrin conjugates.

AIM: To investigate the release of cyclodextrin-5-aminosalicylic acid (CyD-5-ASA) in cecum and colon. METHODS: An anti-inflammatory drug 5-ASA was conjugated onto the hydroxyl groups of alpha-, beta- and gamma-cyclodextrins (CyDs) through an ester linkage, and the in vivo drug release behavior of these prodrugs in rat's gastrointestinal tract after the oral administration was investigated. RESULTS: The 5-ASA concentration in the rat's stomach and small intestine after the oral administration of CyD-5-ASA conjugate was much lower than that after the oral administration of 5-ASA alone. The lower concentration was attributable to the passage of the conjugate through the stomach and small intestine without significant degradation or absorption, followed by the degradation of the conjugate site-specific in the cecum and colon. The oral administration of CyD-5-ASA resulted in lower plasma and urine concentration of 5-ASA than that of 5-ASA alone. CONCLUSION: CyD-5-ASA conjugates may be used as prodrugs for colon-specific drug delivery system.

Time- and pH-dependent colon-specific drug delivery for orally administered diclofenac sodium and 5-aminosalicylic acid.

AIM: To investigate Time- and pH-dependent colon-specific drug delivery systems (CDDS) for orally administered diclofenac sodium (DS) and 5-aminosalicylic acid (5-ASA), respectively. METHODS: DS tablets and 5-ASA pellets were coated by ethylcellulose (EC) and methacrylic acid copolymers (Eudragit L100 and S100), respectively. The in vitro release behavior of the DS coated tablets and 5-ASA coated pellets were examined, and then in vivo absorption kinetics of DS coated tablets in dogs were further studied. RESULTS: Release profile of time-dependent DS coated tablets was not influenced by pH of the dissolution medium, but the lag time of DS release was primarily controlled by the thickness of the coating layer. The thicker the coating layer, the longer the lag time of DS release is. On the contrary, in view of the pH-dependent 5-ASA coated pellets, 5-ASA release was significantly governed by pH. Moreover, the 5-ASA release features from the coated pellets depended upon both the combination ratio of the Eudragit L100 and S100 pH-sensitive copolymers in the coating formulation and the thickness of the coating layer. The absorption kinetic studies of the DS coated tablets in dogs demonstrated that in vivo lag time of absorption was in a good agreement with in vitro lag time of release. CONCLUSION: Two types of CDDS, prepared herein by means of the regular coating technique, are able to achieve site-specific drug delivery targeting at colon following oral administration, and provide a promising strategy to control drug release targeting the desired lower gastrointestinal region.

Multidrug resistance P-glycoprotein: crucial significance in drug disposition and interaction.

This article reviews recent advances in our understanding of the structure, drug interaction mechanism, and substrate molecular requirements of P-glycoprotein (P-gp) and its emerging crucial role in drug disposition and the modulation of drug interaction. In view of its wide localization in normal tissues, the broad variety of structurally and functionally unrelated substrates of P-gp, and its ATP-dependent outward-oriented transport, P-gp actively participates in intestinal secretion, blood-tissue barriers, and biliary and renal excretions for many exogenous substrates, and also performs a protective role to prevent entry of xenobiotics. Moreover, the importance of P-gp-mediated drug interactions in clinical practice can hardly be underestimated, since it may result in severe side effects, such as digitalis drug interaction. Polymorphism or single nucleotide polymorphism (SNP) associated with P-gp may exert a significant effect on the pharmacokinetic behavior of its substrates, a fact which has major clinical implications and suggests careful dose adjustment for individual treatment. Moreover, dietary components and pharmaceutical excipients may modulate P-gp activity, and as a result affect in vivo drug disposition and therapeutic efficacy; examples include grapefruit juice, Pluronic P85, PEG 300, etc. In summary, it should be emphasized that P-gp is an integral component in the process of drug discovery, development strategy,