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.

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.

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.

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.

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.

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.

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.

Fruit extract from a Sechium edule hybrid induce apoptosis in leukaemic cell lines but not in normal cells.

The antiproliferative potential of a crude extract from the chayote hybrid H-837-07-GISeM® and its potential for apoptosis induction were assessed in leukaemic cell lines and normal mouse bone marrow mononuclear cells (BM-MNCs). The extract strongly inhibited the proliferation of the P388, J774, and WEHI-3 cell lines (with an IC50 below 1.3 µg·mL(-1)), reduced cell viability, and induced apoptotic body production, phosphatidylserine translocation, and DNA fragmentation. However, the extract had no effect on BM-MNCs. We postulate that these properties make the extract a good candidate for an anti-tumour agent for clinical use.

[Assessment of Antitumor Effect of Submerged Culture of Ophiocordyceps sinensis and Cordyceps militaris].

Ophiocordyceps sinensis and Cordyceps militaris metabolites showed a high potential in the treatment of tumors as well as some other diseases. Antitumor properties of O. sinensis and C. militaris submerged mycelium were investigated. It was found that the O. sinensis dry biomass in a dose of 50 mg/kg administered once a day to the mice with subcutaneously inoculated P388 lympholeucosis lowered the tumor growth by 65% vs. 54% for the C. militaris dry biomass. The water extract of O. sinensis submerged culture however accelerated the growth of the P388 lympholeucosis tumor node in the mice almost two times, compared to the control. A greater caution in using this fungus as a source of biologically active substances is required since unwanted tumor-stimulating effects can arise.

[Antitumor Activity of Polysaccharides from Ganoderma lucidum Mycelium: in vivo Comparative Study].

Fractions of water soluble and alkali soluble polysaccharides, as well as fucogalactan, a water soluble polysaccharide, and xylomannan, an alkali soluble polysaccharide, were isolated from the Ganoderma lucidum submerged mycelium. When administered orally, the polysaccharides showed antitumor activity in vivo on murine models of solid tumors. Xylomannan and fucogalactan showed the highest antitumor activity. Sensitivity to xylomannan was more pronounced in adenocarcinoma Ca755 as compared to the T-cell lymphocytic leukemia P388. The antitumor activity of the water soluble polysaccharides total fractions from the mycelium and fruiting bodies of the G. lucidum strain was almost identical. The maximum antitumor effect of the mycelium water soluble polysaccharides total fraction was observed with the use of the daily dose of 2 mg/kg.

Numerosol A-D, new cembranoid diterpenes from the soft coral Sinularia numerosa.

Four new cembrane-type diterpenes; numerosol A-D (1-4); along with a known steroid; gibberoketosterol (5); were isolated from the Taiwanese soft coral Sinularia numerosa. The structures of these metabolites were determined by extensive analysis of spectroscopic data. Gibberoketosterol (5) exhibited cytotoxicity against P-388 (mouse lymphocytic leukemia) cell line with an ED50 of 6.9 µM.

Cytotoxicity and apoptosis induced by alfalfa (Medicago sativa) leaf extracts in sensitive and multidrug-resistant tumor cells.

Alfalfa (Medicago sativa) has been used to cure a wide variety of ailments. However, only a few studies have reported its anticancer effects. In this study, extracts were obtained from alfalfa leaves and their cytotoxic effects were assessed on several sensitive and multidrug-resistant tumor cells lines. Using the mouse leukaemia P388 cell line and its doxorubicin-resistant counterpart (P388/DOX), we showed that the inhibition of cell growth induced by alfalfa leaf extracts was mediated through the induction of apoptosis, as evidenced by DNA fragmentation analysis. The execution of programmed cell death was achieved via the activation of caspase-3, leading to PARP cleavage. Fractionation of toluene extract (To-1), the most active extract obtained from crude extract, led to the identification of 3 terpene derivatives and 5 flavonoids. Among them, (-)-medicarpin, (-)-melilotocarpan E, millepurpan, tricin, and chrysoeriol showed cytotoxic effects in P388 as well as P388/DOX cells. These results demonstrate that alfalfa leaf extract may have interesting potential in cancer chemoprevention and therapy.

Effect of doxorubicin/pluronic SP1049C on tumorigenicity, aggressiveness, DNA methylation and stem cell markers in murine leukemia.

PURPOSE: Pluronic block copolymers are potent sensitizers of multidrug resistant cancers. SP1049C, a Pluronic-based micellar formulation of doxorubicin (Dox) has completed Phase II clinical trial and demonstrated safety and efficacy in patients with advanced adenocarcinoma of the esophagus and gastroesophageal junction. This study elucidates the ability of SP1049C to deplete cancer stem cells (CSC) and decrease tumorigenicity of cancer cells in vivo. EXPERIMENTAL DESIGN: P388 murine leukemia ascitic tumor was grown in BDF1 mice. The animals were treated with: (a) saline, (b) Pluronics alone, (c) Dox or (d) SP1049C. The ascitic cancer cells were isolated at different passages and examined for 1) in vitro colony formation potential, 2) in vivo tumorigenicity and aggressiveness, 3) development of drug resistance and Wnt signaling activation 4) global DNA methylation profiles, and 5) expression of CSC markers. RESULTS: SP1049C treatment reduced tumor aggressiveness, in vivo tumor formation frequency and in vitro clonogenic potential of the ascitic cells compared to drug, saline and polymer controls. SP1049C also prevented overexpression of BCRP and activation of Wnt-ß-catenin signaling observed with Dox alone. Moreover, SP1049C significantly altered the DNA methylation profiles of the cells. Finally, SP1049C decreased CD133(+) P388 cells populations, which displayed CSC-like properties and were more tumorigenic compared to CD133(-) cells. CONCLUSIONS: SP1049C therapy effectively suppresses the tumorigenicity and aggressiveness of P388 cells in a mouse model. This may be due to enhanced activity of SP1049C against CSC and/or altered epigenetic regulation restricting appearance of malignant cancer cell phenotype.

Crystal structure of SsfS6, the putative C-glycosyltransferase involved in SF2575 biosynthesis.

The molecule known as SF2575 from Streptomyces sp. is a tetracycline polyketide natural product that displays antitumor activity against murine leukemia P388 in vivo. In the SF2575 biosynthetic pathway, SsfS6 has been implicated as the crucial C-glycosyltransferase (C-GT) that forms the C-C glycosidic bond between the sugar and the SF2575 tetracycline-like scaffold. Here, we report the crystal structure of SsfS6 in the free form and in complex with TDP, both at 2.4 Å resolution. The structures reveal SsfS6 to adopt a GT-B fold wherein the TDP and docked putative aglycon are consistent with the overall C-glycosylation reaction. As one of only a few existing structures for C-glycosyltransferases, the structures described herein may serve as a guide to better understand and engineer C-glycosylation.

Steroidal esters of the aromatic nitrogen mustard 2-[4-N,N-bis(2-chloroethyl)amino-phenyl]butanoic acid (2-PHE-BU): synthesis and in-vivo biological evaluation.

On the basis of the results of in-silico predictions and in an effort to extend our structure-activity relationship studies, the aromatic nitrogen mustard 2-[4-N,N-bis(2-chloroethyl) amino-phenyl]butanoic acid (2-PHE-BU) was synthesized and conjugated with various steroidal alcohols. The resulting steroidal esters were evaluated for their in-vivo toxicity and antileukemic activity in P388-leukemia-bearing mice. The new derivatives showed significantly reduced toxicity and marginally improved antileukemic activity compared with free 2-PHE-BU. Nevertheless, they did not prove to be superior either to the template steroidal ester used for in-silico predictions or to previously synthesized steroidal esters of aromatic nitrogen mustards. The results obtained indicate that in-silico design predictions may guide the design and synthesis of new bioactive steroidal esters, but further parameters should be considered aiming at the discovery of compounds with optimum activity.

The antileukemic activity of modified fibrinogen-methotrexate conjugate.

BACKGROUND: The search for new, innovative methods to treat all types of diseases, especially cancer-related ones, is a challenge taken by pharmaceutical companies and academic institutions. The use of conjugates containing widely-known and widely-used bioactive substances is one of the ways to solve this problem. Research into drug binding with macromolecular carrier systems has joined the search for new therapeutic strategies. METHODS: The main goal of this paper is the potential offered by the use of fibrinogen derivatives as an antileukemic drug carrier. Physicochemical properties of the obtained conjugate were analyzed, characterizing alterations in relation to the starting carrier and analyzing biological implications. The intraperitoneally (i.p.) inoculated P388 mouse leukemia model for in vivo studies was used. RESULTS AND CONCLUSIONS: Conjugates consisting of a fibrinogen derivative with a covalently bound anticancer drug were developed. Carrier preparation and a conjugate synthesis in aqueous solution were formulated, as well as purification of the conjugate was performed. The study showed that the survival of leukemia mice treated with FH-MTX conjugate was indeed significantly longer than survival in both untreated animals (control) and mice treated with unbound MTX. A significant increase in the antileukemic activity of MTX conjugated with hydrolysed fibrinogen was observed as compared with the unconjugated drug. Reported data suggest that hydrolysed fibrinogen can serve as a carrier molecule for the MTX drug with the aim of enhancing its antileukemic activity. GENERAL SIGNIFICANCE: Conjugates consisting of a fibrinogen derivative with a covalently bound anticancer drug seem to be a promising anticancer drug.

Application of artificial neural networks for the prediction of antitumor activity of a series of acridinone derivatives.

Artificial neural networks (ANNs) have been applied for the quantitative structure-activity relationships (QSAR) studies of antitumor activity of acridinone derivatives. Molecular modeling studies were performed with the use of HyperChem and Dragon computer programs and molecular geometry optimization using MM+ molecular mechanics and semi-empirical AM1 method, and several molecular descriptors of agents were obtained. A high correlation resulted between the ANN predicted antitumor activity and that one from biological experiments for the data used in the testing set of acridinones was obtained with correlation coefficient on the level of 0.9484. Moreover, the sensitivity analysis indicated that molecular parameters describing geometrical properties as well as lipophilicity of acridinone derivative molecule are important for acridinones antitumor activity.

Overexpression of apoptosis-associated speck-like protein in P388D1 murine lymphoma cells affects metastatic properties.

Apoptosis-associated speck-like protein (ASC) is a bipartite adaptor molecule that participates in inflammation and apoptosis. ASC silencing has been observed in a significant proportion of human cancers. Here, we examined the role of ASC overexpression in the metastasis of the P388D1 murine lymphoma cell line to the liver, lung, spleen and kidney. First, we determined that the P388D1 cells express ASC. Then, ASC overexpression in P388D1 was achieved by transfecting pEGFP-ASC-C2 into the P388D1 cells. Furthermore, after the ASC-overexpressing P388D1 cells were injected into DBA/2 mice through the vena caudalis, their metastasis to the lung and the liver was significantly reduced in the pEGFP-ASC-C2-transfected group. These data indicate that ASC overexpression affects the in vivo metastatic properties of P388D1 cells.

Preclinical evaluation of amiodarone for the treatment of murine leukemia P388. In vivo and in vitro investigation.

PURPOSE: The purpose of the present study was the investigation of antileukemic effect of amiodarone in leukemia P388 BDF1 bearing mice and its genotoxic and cytostatic effect in cultured normal human lymphocytes. METHODS: Leukemia P388 was used in this study. BDF1 mice were used for chemotherapy evaluation in vivo. The antitumor activity was assessed by the oncostatic parameter T/C, representing the increase of life span of drug-treated animals vs. controls. Lymphocyte cultures were used to study the genotoxic and cytostatic effect in vitro, expressed by enhanced sister chromatid exchange (SCE) and reduced proliferation rate indices (PRIS). RESULTS: Amiodarone was found to exert antileukemic potency against leukemia P388 bearing mice at all three different treatment schedules used, yielding T/C values of 155%, 163% with one cure and 230%. In the in vitro cytogenic experiments, significant increase of SCE rates by amiodarone was observed at 0.2 µM, while at the same concentration significant suppression of PRIS was achieved. CONCLUSION: According to the National Cancer Institute (NCI), a compound is characterized as potential chemotherapeutic deserving further evaluation if it produces T/C values≥125%. On the other hand the SCE assay has predictive value as a clinical assay for drugs exhibiting a strong correlation between cell killing and induction of SCEs. Further studies are warranted to clarify the structure-activity relationship of amiodarone.