Redox-Active Compounds in the Therapy of Drug-Resistant Murine Leukemia P388 Strains.

The efficiency of combinations of cytostatics cisplatin and adriamycin with antioxidant sodium 3-(3'-tert-butyl-4-hydroxyphenyl)propyl thiosulfate (TS-13), and nitric oxide (NO) donor NaNO2 was evaluated on two drug-resistant strains of leukemia P388 with changed redox-status of cells. Simultaneous use of both NO donor and TS-13 in combinations with the cytostatics did not increase the efficiency of therapy. In addition, antioxidant activity of TS-13, NaNO2, and their combinations was studied by the method of luminol-dependent chemiluminescence on the model systems with the use of the homogenized cells of sensitive strain and two drug-resistant strains of leukemia P388. It was shown that TS-13 and NO donor produced opposite effects: TS-13 decreased, while NO donor increased the content of free radicals in the model system. Combinations of antioxidant TS-13 and NO donor should be used with consideration for the redox-status of tumor treated.

Nrf2-Regulated Antioxidant System in Cells of In Vivo Drug-Resistant P388 Murine Leukemia Strains.

We studied the expression of Nrf2 transcription factor and antioxidant system proteins in drug-resistant murine leukemia strains P388 in vivo, as well as the redox status of cells under conditions of induced oxidative stress. Immunoblotting and real-time PCR showed that the cyclophosphamide-resistant strain P388 (P388/CP) exhibits Nrf2-mediated drug resistance. Cells of the P388/CP strain are characterized by high expression of Nrf2, which leads to a significant increase in the expression of ARE genes and antioxidant system proteins, as well as to the effective maintenance of redox homeostasis under conditions of induced oxidative stress. Taking into account the important role of Nrf2 overexpression in reducing the effectiveness of chemotherapy in patients with different leukemias, the P388/CP strain can be of great interest as a model in the development of new drugs for the treatment of malignant neoplasms.

Cladoic Acid, an Anti-Trypanosoma cruzi Polyketide from Cladosporium sp. TP-F2020.

A new polyketide, cladoic acid, was isolated from a fungus of the genus Cladosporium. The structure of the highly oxygenated trans-decalin ring with an all-E triene side chain was elucidated by extensive spectroscopic analysis. The unique chair/twist-boat conformation of the trans-decalin core and the flexibility of the B-ring were demonstrated by computer-aided conformational analysis. Cladoic acid was active against Trypanosoma cruzi and inhibited the proliferation of amastigotes and epimastigotes with IC50 values of 27 and 46 µM, respectively, but it did not show any appreciable activity against P388 murine leukemia cells, bacteria, or fungi, indicating it is a potential candidate for drug development against Chagas disease.

Structure revision of tricholomenyn B, an antimitotic geranylcyclohexenone rediscovered as a bitter and antibacterial substance, from a basidiomycete Tricholoma japonicum (Shiro-shimeji).

Tricholomenyn B, an antimitotic geranylcyclohexenone originally discovered from a basidiomycete Tricholoma acerbum, was isolated as a bitter and antibacterial constituent from fruiting bodies of T. japonicum. Careful comparison of NMR, MS, and other physicochemical properties of the isolated substance with the literature values revised a previously proposed macrolide structure 1 to a macrodiolide 2. Compound 2 was perceived bitter at a minimum dose of 37.5 µg, showed weak antimicrobial activity against Kocuria rhizophila and Staphylococcus aureus, and was marginally cytotoxic (IC50 2.6 µM) against P388 murine leukemia cells.

The Increase of Antileukemic Efficiency of Doxorubicin and Other Cytostatics Combined with Platinum(IV)-Nitroxyl Complex ВС118.

Combined treatment of murine leukemia P388 with doxorubicin and platinum(IV)-nitroxyl complex ВС118 administered in low doses improved efficiency of treatment (cure of 83% of animals) without increasing toxicity.

Small-Scale Preparation of Fluorescently Labeled Chemical Probes from Marine Cyclic Peptides, Kapakahines A and F.

A number of bioactive marine natural products have been isolated so far, but it is still difficult to disclose their modes of action. In this study, we prepared fluorescently labeled chemical probes from the cytotoxic marine cyclic peptides kapakahines A (1) and F (2) to visualize their localization as the first step of the study of their modes of action. We used fluorescent dyes 3a or 3a/b (a 1:1 mixture of 3a and 3b) whose terminal N-hydroxysuccinimide (NHS) group can react with the free amino groups of kapakahines. The fluorescently labeled kapakahine A (Kap A-5-FL, 5a) stained P388 murine leukemia cells and HeLa human cervical cancer cells, while cells treated with fluorescently labeled kapakahine F (Kap F-5-FL, 6a) only weakly stained them. Further analysis of the confocal images of the stained cells with higher magnification (×100) indicated the localization of Kap A-5-FL (5a) in the cells. In this paper, we report the small-scale preparation and a new delivery method of fluorescent probes, as well as the application of these procedures to cell staining.

Preclinical Study of Pharmacological Properties of Doxorubicin-NPh.

Preclinical study of therapeutic properties of an innovative drug Doxorubicin-NPh (doxorubicin in the form of ultrafine suspension of phospholipid liposomes) in comparison with free doxorubicin (Doxorubicin-Teva) and protected doxorubicin (Caelyx) was performed on transplanted murine tumor models. All these drugs were efficient in Ca755 breast carcinoma model (tumor growth inhibition ≈100%, increase in lifespan 90.6-114.3%). In P388 lymphocytic leukemia and LLC lung carcinoma, advantages of the protected doxorubicin by the benefit/risk ratio (width of therapeutic interval) were demonstrated: Caelyx>Doxorubicin-NPh>Doxorubicin-Teva. Doxorubicin-NPh and Caelyx exhibited similar therapeutic activity in the LLC model, especially when administered 3 times with 3-day intervals; for Doxorubicin-Teva, the optimal interval between the injections was 7 days.

Causes and possibilities to circumvent cyclophosphamide toxicity.

Cyclophosphamide is an inert prodrug converted into 4-hydroxycyclophosphamide (OHCP) by hepatic hydroxylation. OHCP is in equilibrium with its tautomeric aldophosphamide (ALDO). From ALDO, the cytotoxic active metabolites are formed enzymatically by phosphodiesterases; these are the alkylating metabolite phosphoramide mustard (PAM) and the proapoptotic aldehyde 3-hydroxypropanal (HPA). PAM damages the DNA by alkylation; HPA amplifies the thereby induced apoptosis. The generally accepted view that acrolein, which is believed to be formed in the formation of PAM by ß-elimination from ALDO would be mainly responsible for the toxicity of cyclophosphamide, has to be revised because no acrolein is formed in the systemic circulation of patients after cyclophosphamide administration. It is shown that not acrolein, but OHCP itself is the true toxic metabolite of cyclophosphamide. Toxicity tests with OHCP and PAM were carried out, which demonstrated that OHCP unfolds its toxicity, not as a carrier of PAM but is toxic itself by reacting with nucleophilic groups of macromolecules, for example, thiol groups of membrane proteins. Further experiments demonstrate that the toxicity of oxazaphosphorine cytostatics may be drastically reduced if the formation of the pharmacologically active metabolite ALDO bypasses the formation of OHCP. Toxicity experiments in mice with S-ethanol-cyclophosphamide (SECP) that hydrolyzes to OHCP show that SECP is as toxic as OHCP, whereas the thiazolidine of ALDO, which hydrolyzes to ALDO bypassing OHCP is 7-9 times less toxic without loss of antitumor activity.

Complete NMR assignment and absolute configuration of k4610422, a norditerpenoid inhibitor of testosterone-5α-reductase originally from Streptosporangium: rediscovery from a thermophilic Actinomadura.

A norditerpenoid k4610422 (1), an inhibitor of testosterone-5α-reductase originally discovered from a mesophilic rare actinomycete of the genus Streptosporangium, was isolated from the culture extract of a thermophilic actinomycete Actinomadura sp. The complete 1H and 13C NMR assignment and absolute configuration of 1 were addressed by spectroscopic measurements including NOESY and CD spectra coupled with ECD calculation, which allowed to establish the (5 R,9 S,10 R,13 S)-configuration. Compound 1 was moderately cytotoxic against P388 murine leukemia cells with IC50 30 µM.

Activity of Antioxidant Enzymes and Content of Reduced Glutathione in Cells of Drug-Resistant Murine Leukemia P388 Strains.

Activities of superoxide dismutase and catalase and content of reduced glutathione in cells of drug-resistant murine leukemia P388 strains were studied without or after administration of antitumor compounds. In the absence of chemotherapeutic agents, no significant differences in activities of the studied enzymes in cells of the initial strain and strains resistant to cyclophosphamide, cisplatin, and rubomycin were observed. Compounds to which resistance was developed did not significantly affect activity of enzymes in cells of drug-resistant strains, while the use of compounds that were not resistance inductors was accompanied by a significant decrease in enzyme activity in cells resistant to cisplatin and rubomycin. In cells of strains resistant to cisplatin and cyclophosphamide, the content of reduced glutathione significantly differed from that in the initial strain. In addition, the concentration of reduced glutathione in cells of cyclophosphamide-resistant strain considerably decreased upon addition of the drug producing a therapeutic effect. Our findings suggest that the mechanism of resistance of in vivo derived cyclophosphamide resistant cell strain is related to increased level of reduced glutathione and activity of its metabolism.

Labrenzbactin from a coral-associated bacterium Labrenzia sp.

A new catecholate-containing siderophore, labrenzbactin (1), was isolated from the fermentation broth of a coral-associated bacterium Labrenzia sp. The structure and absolute configuration of 1 was determined by spectroscopic methods and Marfey's analysis. Overall, 1 showed antimicrobial activity against Ralstonia solanacearum SUPP1541 and Micrococcus luteus ATCC9341 with MIC values of 25 and 50 µg ml-1, respectively, and cytotoxicity against P388 murine leukemia cells with an IC50 of 13 µM.

Comparative Analysis of Bioactivity of the Russian-Made Antitumor Substances of the Nitrosourea Group.

We performed an in vivo comparative study of activity of three substances of the nitrosourea group produced in Russia. All substances demonstrated high antitumor activity against various solid and leukemic tumors. Aranosa significantly enhanced life duration in mice with leukemia (by 65-194%) and inhibited the growth of solid tumors (by 49-99.6%). Lisomustine and ormustine showed higher activity than aranose. Single administration of lisomustine increased life span of mice (by 22-114%) and resulted in cure of all animals in four models: lymphoblastic leukemia L-1210, lymphocytic leukemia P-388, Lewis lung carcinoma, and cervical cancer RShM-5. After ormustine treatment, full recovery was observed only in groups with lymphocytic leukemia P-388 and cervical cancer RShM-5. These findings attest to higher activity of lisomustine in the studied models.

Catenulobactins A and B, Heterocyclic Peptides from Culturing Catenuloplanes sp. with a Mycolic Acid-Containing Bacterium.

The production of two new heterocyclic peptide isomers, catenulobactins A (1) and B (2), in cultures of Catenuloplanes sp. RD067331 was significantly increased when it was cocultured with a mycolic acid-containing bacterium. The planar structures and absolute configurations of the catenulobactins were determined based on NMR/MS and chiral-phase GC-MS analyses. Catenulobactin B (2) displayed Fe(III)-chelating activity and moderate cytotoxicity against P388 murine leukemia cells.

Umezawamides, new bioactive polycyclic tetramate macrolactams isolated from a combined-culture of Umezawaea sp. and mycolic acid-containing bacterium.

New polycyclic tetramate macrolactams, Umezawamides A (1) and B (2) were isolated from a combined-culture of Umezawaea sp. RD066910 and mycolic-acid containing bacterium Tsukamurella pulmonis TP-B0596. Their planar structures and partial stereochemistries were determined based on the spectroscopic analysis, MMFF conformational search, and ECD calculations. Umezawamides are the first secondary metabolites isolated from the genus Umezawaea and they exhibited cytotoxicities to P388 murine leukemia cells. Furthermore, umezawamide A (1) showed growth inhibitory activity against Candida albicans.

The Effectiveness of Cyclic Hydroxamic Acid CHA-5 against Drug-Resistant P388 Leukemia Strains.

We studied the effectiveness of cyclic hydroxamic acid CHA-5 against drug-resistant and multidrug-resistant murine P388 leukemia strains. More than 60% mice receiving transplantation of rubomycin-resistant leukemia P388 strain survived after CHA-5 monotherapy; combined therapy with CHA-5 and cisplatin was also highly effective. Vincristine-resistant tumor was highly sensitive to combined treatment with CHA-5 and cyclophosphamide. It should be emphasized that standard antitumor agents were used in very low doses in combination therapy and CHA-5 significantly potentiated their effect.

Total Syntheses of Lepadiformine Marine Alkaloids with Enantiodivergency, Utilizing Hg(OTf)2 -Catalyzed Cycloisomerization Reaction and their Cytotoxic Activities.

The enantioselective total syntheses of lepadiformine marine alkaloids, azatricyclic natural products isolated from marine tunicates, were completed. These alkaloids have a unique chemical structure characterized by the trans-1-azadecalin (AB ring system) fused with the spirocyclic ring (AC ring system). Here we found that a cycloisomerization reaction from functionalized linear substrates to a 1-azaspiro[4.5]decane framework corresponding to the AC ring in lepadiformines is promoted by a catalytic amount of mercury(II) triflate (Hg(OTf)2 ). The total syntheses of (-)-lepadiformines A and B were achieved in 28 % and 21 % overall yields, respectively, through the novel cycloisomerization reaction. The syntheses of (+)- and (-)-lepadiformine C hydrochloride salts also enabled us to determine the absolute configuration of natural lepadiformine C. It has been found that a phenomenon of enantiodivergence occurs in lepadiformine alkaloids from a single species of marine tunicate, Clavelina moluccensis. The cytotoxic activities of synthesized lepadiformine hydrochloride salts and their synthetic intermediates were evaluated.

Chemical analyses and in vitro and in vivo toxicity of fruit methanol extract of Sechium edule var. nigrum spinosum.

CONTEXT: Sechium edule (Jacq.) Sw. (Cucurbitaceae) is used in ethnomedicine, but the diversity of the varietal groups of this species has not often been considered. This is important because we previously reported that different variety of species exhibit different activities across different tumor cell lines. OBJECTIVE: This study investigates the chemical composition and biological activities of extracts obtained from S. edule var. nigrum spinosum. MATERIALS AND METHODS: The leukemia P388 cell line and mononuclear bone marrow cells (MNCBMs) were treated with the extract at a concentration ranging from 40 to 2370 µg/mL for cytotoxicity and viability assays. CD-1 mice were treated with 8-5000 mg/kg extract and monitored every hour for the first 24 h and subsequently for seven days for signs of toxicity (LD50). In addition, the chromatographic profile of the extract was determined by HPLC. RESULTS: The extract inhibits the proliferation of both P388 cells and MNCBMs, with IC50 values of 927 and 1911 µg/mL, respectively, but reduced the viability and induced the apoptosis of only leukemia cells. The LD50 was higher than 5000 mg/kg, and this concentration did not alter the blood chemistry or cell count but doubled the mitotic index in the bone marrow. The HPLC showed the presence of cucurbitacins, phloridzin, naringenin, phloretin, apigenin, and gallic, chlorogenic, vanillic, p-hydroxybenzoic, caffeic, and p-coumaric acids. DISCUSSION AND CONCLUSION: Sechium edule var. nigrum spinosum contains bioactive compounds that explain the antiproliferative and nutraceutical activities, and its lack of physiological side effects constitutes an added value to a widely consumed vegetable.

Identification of Pyridinium with Three Indole Moieties as an Antimicrobial Agent.

A novel pyridinium with three indole moieties, tricepyridinium, was obtained from the culture of an Escherichia coli clone incorporating metagenomic libraries from the marine sponge Discodermia calyx. For the important structural elements of tricepyridinium to be investigated for antibacterial activity, tricepyridinium and its analogues were chemically synthesized. Tricepyridinium had antimicrobial activity, but not against E. coli, and cytotoxicity against P388 cells. Additional bioassays with its synthetic analogues revealed that the intriguing combination of the indole moieties, most likely derived from three tryptamines, as well as the pyridinium moiety were chiefly responsible for its potent biological activities.

Cytototoxic constituents from the bark of Chisocheton cumingianus (Meliaceae).

A new lanostane-type triterpenoid, 3ß-hydroxy-25-ethyl-lanost-9(11),24(24')-diene (1), along with 3ß-hydroxy-lanost-7-ene (2) and ß-sitosterol-3-O-acetate (3) was isolated from the stem bark of C. cumingianus. The chemical structure of the new compound was elucidated on the basis of spectroscopic data. All of the compounds were evaluated for their cytotoxic effects against P-388 murine leukemia cells. Compounds 1-3 showed cytotoxicity against P-388 murine leukemia cells with IC50 values of 28.8 ± 0.10, 4.29 ± 0.03, and 100.18 ± 0.16 µg/ml, respectively.

Sulfoureido Lipopeptides from the Marine Sponge Discodermia kiiensis.

New N-sulfoureidylated lipopeptides, sulfolipodiscamides A-C (1-3), were isolated by gel filtration chromatography of the n-butanol fraction of the marine sponge Discodermia kiiensis. By extensive NMR analyses and high-resolution mass spectrometry, the structures of 1-3 were elucidated as having an unprecedented N-sulfoureidyl group on the d-citrulline residue, a distinct feature that was not found in the structurally related lipodiscamides A-C (4-6), derived from the ether fraction of the same sponge. Furthermore, the absolute configurations of 1-3 were confirmed by comparisons of the HPLC retention times of the hydrolytic products and the corresponding authentic lipodiscamides. Interestingly, sulfolipodiscamide A displayed a 2.3-fold increase in cytotoxicity against murine leukemia (P388) cells, compared to the unconjugated parent compound.