Liver injury in septic mice were suppressed by a camptothecin-bile acid conjugate via inhibiting NF-κB signaling pathway.

BACKGROUND AND OBJECTIVES: Sepsis is a life-threatening organ dysfunction syndrome arising from uncontrolled inflammatory responses. Liver injury is a crucial factor for the prognosis of sepsis. Camptothecins (CPTs) have been reported to suppress the inflammatory response induced by sepsis. G2, a CPT-bile acid conjugate, has been demonstrated the property of liver targeting in our previous research. This study aimed to research the effects of G2 on liver injury induced by cecal ligation and puncture (CLP). METHODS: C57BL/6 mice were subjected to CLP surgery, and effects of G2 on liver damage and survival rates of CLP-induced mice were evaluated. To detect the related markers of hepatic injury or neutrophil infiltration, inflammatory cytokines and protein levels, hematoxylin-eosin staining assay, corresponding Detection Kits assay, ELISA and Western blot analysis were performed. RESULTS: Intraperitoneal administration of G2 reduced liver injury and enhanced the survival rates in mice with sepsis. Treatment with G2 decreased the levels of hepatic injury markers aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the serum of mice induced by CLP. The hepatic level of neutrophil infiltration marker myeloperoxidase (MPO) was reduced in G2 administration group. And the levels of serum inflammatory cytokines, including Tumor Necrosis Factor-α (TNFα), Interleukin-6 (IL-6) and IL-1ß, were decreased by G2. Furthermore, the results of Western blot analysis indicated that G2 suppressed the up-regulation of NF-κB p-P65 and p-IκBα. It suggested that G2 suppressed the activation of NF-κB signaling pathway. CONCLUSION: G2 alleviated sepsis-induced liver injury via inhibiting the NF-κB signaling pathway.

Digital gene expression analysis in the liver of ScpB-vaccinated and Streptococcus agalactiae-challenged Nile tilapia.

In recent years, streptococcal diseases have severely threatened the development of tilapia aquaculture, but effective prevention and control methods have not yet been established. To understand the immune responses of vaccinated Nile tilapia (Oreochromis niloticus), digital gene expression (DGE) technology was applied in this study to detect the gene expression profile of the Nile tilapia (O. niloticus) liver in response to ScpB (Streptococcal C5a peptidase from group B Streptococcus, ScpB) vaccination and a Streptococcus agalactiae-challenge. The control and the ScpB-vaccinated Nile tilapia yielded a total of 25,788,734 and 27,088,598 clean reads, respectively. A total of 1234 significant differentially expressed unigenes were detected (P < 0.05), of which 236 were significantly up-regulated, and 269 were significantly down-regulated (P < 0.05, |fold|>2, FDR<0.05). Of the differentially expressed gene, the identified genes which were enriched using databases of GO and KEGG could be categorized into a total of 67 functional groups and were mapped to 153 signaling pathways including 15 immune-related pathways. The differentially expressed genes (TLR1, TLR2, TLR3, TLR5, TLR9, MyD88, C3, IL-1ß, IL-10) were detected in the expression profiles, and this was subsequently verified via quantitative real-time PCR (qPCR). The results of this study can serve as a basis for future research not only on the molecular mechanism of S. agalactiae invasion, but also on the anti-S. agalactiae mechanism in targeted tissues of Nile tilapia.

Microwave-Assisted Degradation of Polysaccharide from Polygonatum sibiricum and Antioxidant Activity.

Four polysaccharide fractions (P-1: 71.40%, P-2: 1.95%, P-3: 1.14%, P-4: 1.64%) were isolated from crude Polygonatum sibiricum polysaccharide (PSP), processed by water extraction, ethanol precipitation, and further separated with diethylaminoethyl cellulose-52 anion-exchange chromatography. Their molecular weights and monosaccharide compositions were characterized by high performance gel chromatography with evaporative light scattering detector and ultraviolet-visible detector. The antioxidant activity of four polysaccharides fractions were assessed by the electron transfer menchanism (DPPH, ferric reducing power, and ABST assays) and chelation of transition metals (Fe2+ and Cu2+ chelation ability). The highest content fraction P-1 exhibited the lowest antioxidant activity, and the ranking of antioxidant capacity was P-4 > P-3 > P-2 > PSP > P-1. After processed by microwave-assisted degradation, the molecular weight of P-1 was decreased from 2.99 × 105 to 2.33 × 103 Da, while the antioxidant activity of degraded P-1 was about eightfold higher than natural P-1. These results indicated that the proposed microwave-assisted degradation approach was an efficacious methodology to improve their bioactivity by lower the molecular weight of polysaccharides. PRACTICAL APPLICATION: This study provided an environmentally friendly, convenient and efficient microwave-assisted degradation technology to process the neutral polysaccharides from Polygonatum sibiricum. The results could be used for the development and utilization of various plant polysaccharides as a kind of food supplement in our daily life.

Synthesis and biological activity of some bile acid-based camptothecin analogues.

In an effort to decrease the toxicity of camptothecin (CPT) and improve selectivity for hepatoma and colon cancer cells, bile acid groups were introduced into the CPT 20 or 10 positions, resulting in the preparation of sixteen novel CPT-bile acid analogues. The compounds in which a bile acid group was introduced at the 20-hydroxyl group of CPT showed better cytotoxic selectivity for human hepatoma and colon cancer cells than for human breast cancer cells. Fluorescence microscopy analysis demonstrated that one compound (E2) entered human hepatoma cells more effectively than it did human breast cancer cells. Compound G4 exhibited the best anti-tumour activity in vivo. These results suggested that introduction of a bile acid group at the 20-position of CPT could decrease toxicity in vivo and improve selectivity for hepatoma cells.

Coumarins from Edgeworthia chrysantha.

A new coumarin, edgeworic acid (1), was isolated from the flower buds of Edgeworthia chrysantha, together with the five known coumarins umbelliferone (2), 5,7-dimethoxycoumarin (3), daphnoretin (4), edgeworoside C (5), and edgeworoside A (6). Their structures were established on the basis of spectral data, particularly by the use of 1D NMR and several 2D shift-correlated NMR pulse sequences (1H-1H COSY, HSQC and HMBC), in combination with acetylation reactions.

Quantification of CPT13 in rat plasma using LC-MS/MS for a pharmacokinetic study.

A new, simple, sensitive and specific reversed-phase high performance liquid chromatographic (HPLC) method using tandem mass spectrometry detection was initially developed and validated for the analysis of 10-(2-pyrazolyl-ethoxy)-(20S)-camptothecin (CPT13) in rat plasma. Pretreatment of the sample obtained from plasma involved a single protein precipitation step with using acetonitrile containing 0.1% formic acid. An aliquot of 20 µl was injected into a C-18 column. The chromatographic separation was achieved using the mobile phase consisting of acetonitrile:water (35:65) at a flow rate of 1.0 mL/min. The total run time for each sample was 10 min, and camptothecin (CPT, IS) and CPT13 were well separated with retention times of 5.1 min and 5.6 min, respectively. Detection was performed using a triple quadrupole tandem mass spectrometer in multiple reaction monitoring (MRM) mode via an electrospray ionization (ESI) source. The calibration curve was linear (r² = 0.9998) over the concentration range of 1-1000 ng/mL, with a LLOQ of 1 ng/mL for CPT13. The inter- and intra-day precision (%R.S.D.) were <2.58% and 6.28%, respectively, and the accuracies (%) were within the range of 97.34-110.67%. CPT13 in rat plasma was stable when stored at -20 °C or 4 °C for three freeze-thaw cycles, The method was employed for the first time during pharmacokinetic studies of CPT13 in rats following a single intravenous dose (0.1 mg/kg) and three different oral doses (50 mg/kg, 30 mg/kg, and 10 mg/kg). This fully validated method was successfully applied to a pharmacokinetic study of CPT13 in rats.

[A fluorescent-probe detecting CYP2C9 * 3 DNA].

The mismatched CYP2C9 * 3 DNA was detected by a exciplex fluorescent-probe system. The exciplex fluorescent-probe model system comprises of two 12-mer oligonucleotides fluorescent-probes, complementary to neighbouring sites of a 24 (47) -mer DNA target and plasmid target, each equipped with moieties able to form an exciplex on correct, contiguous hybridization. Very similar results were obtained between the 24-mer target and the 47-mer target when WT and MT systems were detected by the exciplex fluorescent-probe. Exciplex bands at 505 nm were found for both 24(47)-mer WT and 24(47)-mer MT-targets at 5 degrees C, but were not distinctive enough to distinguish 24(47)-mer WT-target and 24(47)-mer MT-target. However the experiments were carried out at Tm, the exciplex band disappeared almost completely for 24(47)-mer MT-target system like control system, and there was still a strong exciplex band for the 24(47)-mer WT target system. Exciplex peaks at 505 nm were seen for the WT circular plasmids system, but not for MT circular plasmids. Therefore, mismatches of CYP2C9 * 3 DNA can be effectively detected by this exciplex construct, giving potential for single nucleotide polymorphism detection.

Cytotoxicity and topo I targeting activity of substituted 10--nitrogenous heterocyclic aromatic group derivatives of SN-38.

A series of 10-position substituted nitrogenous heterocyclic aromatic group derivatives of SN-38 were prepared. Most of these compounds possessed lower cytotoxicities than CPT. Compound 13 revealed potent cytotoxicity similar to CPT, and compounds 17, 18, and 19 showed similar cytotoxic activity to topotecan. All of the pyridine salt derivatives (7-16) revealed comparable or superior topo I inhibitory activity in relation to CPT. Ethyl in the 7-position of these compounds can increase the cytotoxicity and inhibitory activity to topo I compared with corresponding pyridine salts CPT derivatives (7a-13a) and simultaneously maintain good water solubility. This result is consistent with the SAR of CPT.

[A new class of exciplex-formed probe detect of specific sequence DNA].

The present research was to develop the exciplex-based fluorescence detection of DNA. A SNP-containing region of cytochrome P450 2C9 DNA systems was evaluated to define some of the structural and associated requirement of this new class of exciplex-formed probe, and a 24-base target was selected which contains single-nucleotide polymorphisms (SNP) in genes coding for cytochrome P450. The two probes were all 12-base to give coverage of a 24-base target region to ensure specificity within the human genome. Exciplex partners used in this study were prepared using analogous phosphoramide attachment to the 3'- or 5'-phosphate group of the appropriate oligonucleotide probes. The target effectively assembled its own detector by hybridization from components which were non-fluorescent at the detection wavelength, leading to the huge improvement in terms of decreased background. This research provides details of the effects of different partner, position of partners and different excitation wavelengths for the split-oligonucleotide probe system for exciplex-based fluorescence detection of DNA. This study demonstrates that the emission intensity of the excimer formed by new pyrene derivative is the highest in these excimer and exciplex, and the excimer is easy to be formed and not sensitive to the position of partners. However the exciplex formed by the new pyrene derivative and naphthalene emitted strongly at -505 nm with large Stokes shifts (120-130 nm), and the monomer emission at 390 and 410 nm is nearly zero. Excitation wavelength of 400 nm is the best for I(e505)/I(m410) (exciplex emission at 505 nm/monomer emission at 410 nm) of the exciplex. This method features lower background and high sensitivity. Moreover the exciplex is sensitive to the steric factor, different position of partners and microenvironment, so this exciplex system is promising and could be tried to identify the SNP genes.

Anti-proliferative and pro-apoptotic effect of CPT13, a novel camptothecin analog, on human colon cancer HCT8 cell line.

10-(2-pyrazolyl-ethoxy)-(20S)-camptothecin (CPT13) is a novel semi-synthetic analogue of camptothecin, our previous report had shown that it possessed higher in vitro cytoxicity activity towards human colon cancer HCT8 cell line than topotecan. In this study, the anti-proliferative effect of CPT13 on HCT8 cell line in vitro was analyzed. In order to further explore the underlying mechanism of cell growth inhibition of CPT13 towards HCT8 cell line, the cell cycle distribution, apoptosis proportion, the nuclei morphological changes and caspase-8 and caspase-3 activities were measured. Additionally the changes of mitochondrial morphology and membrane potential (DeltaPsim) were analyzed by atomic force microscopy (AFM) and flow cytometry, respectively. The results showed that CPT13 inhibited HCT8 cell growth by causing cell cycle arrest at G2/M transition and induced apoptosis, as evidenced by the typical apoptotic morphology such as condensation and fragmentation of nuclei and formation of apoptotic bodies. The changes of mitochondrial morphology, dose-dependently decrease in DeltaPsim and the enhancement of caspase-8 and caspase-3 activities were observed in different concentrations of drug treatment group. Our results suggest that CPT13 induces apoptosis by alternations of mitochondrial transmembrane depolarization, activation of caspase-8 and caspase-3. Therefore, CPT13 appears to be a potent drug against human colon cancer via induction of apoptosis and may be used as an alternative drug to therapy cancer.

Review camptothecin: current perspectives.

The review provides a detailed discussion of recent advances in the medicinal chemistry of camptothecin, a potent antitumor agent that targets topoisomerase I. Thousands of CPT derivatives have been synthesized. Two of them, Topotecan and Irinotecan, are commercially approved for use in clinic as antitumor agents while more are still in clinic trials. This review summarizes the current status of the modern synthetic approaches to CPT, the mechanism of action of CPT, the structure-activity relationship(SAR), a number of novel CPT analogs and their biologic activity. There is a systematic evaluation of A-, B- and E-ring- modified camptothecins reported recently.

[Synthesis of camptothecin glycosides by phase transfer-catalysis and its inhibitory activity against topo I].

AIM: To find new anticancer drug based on the structure of 10-hydroxy camptothecin. METHODS: Six camptothecin glycosides (7-12) were synthesized by phase transfer catalysis. The structures of all compounds synthesized were determined by 1H NMR, IR and MS. Their antitumor activity was evaluated on cancer cells in vitro, and inhibitory activity against Topo I was evaluated by molecular biologic method. RESULTS AND CONCLUSION: The result indicated that the yield of camptothecin glycosides by phase transfer catalysis is much higher than by the method from literature, camptothecin glycosides have much lower cytotoxicities on cancer cell in vitro, but have better inhibitory activity of topo I.

[Synthesis and antitumor activities of 10-hydroxy camptothecin derivatives].

AIM: To find new anticancer drug based on the structure of 10-hydroxy camptothecin. METHODS: Seven camptothecin derivatives (3 -9) were synthesized and the antitumor activities of these derivatives were evaluated. RESULTS: Structures of seven new compounds were determined by 1HNMR, IR, MS. Seven compounds showed inhibitory effects on Hela, BEL-7402, 7901 cell lines in vitro. Especially, compound 4 showed high bioactivities to all of the tumor cells in vitro, its anticancer activity against human cervical carcinoma Hela was much higher than that of 10-hydroxy camptothecin. CONCLUSION: Some compounds are worth further studying.

Synthesis and cytotoxicity of water soluble quaternary salt derivatives of camptothecin.

In an effort to improve the water solubility of camptothecin, 16 water soluble 10-substituted quaternary ammonium salt derivatives of camptothecin were prepared. Their antitumor activity was evaluated on cancer cells in vitro. All of these salts possess lower cytotoxicities than CPT in comparison. The camptothecin salts 16, 20 showed similar cytotoxic activity to topotecan. Especially the salts 21 showed similar cytotoxic activity to CPT in vitro.