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.

Nrf2-Dependent Expression of Glutathione Antioxidant System Genes and Redox Status in Cells of In Vivo Drug-Resistant Murine P388 Leukemia Strains.

We measured the content of ROS and malondialdehyde in cells of in vivo drug-resistant murine P388 leukemia strains. It was found that the strains did not differ by malondialdehyde concentration, but intracellular concentration of ROS in cells of the cyclophosphamide-resistant strain (P388/CP) was higher than in cells of the original (P388) and other studied strains (P388/Rub, P388/cPt). Nuclear localization of the transcription factor Nrf2 in cells of strain P388/CP attested to its constitutive activation. Enhanced relative expression of the GCLM gene was found in all studied drug-resistant strains; the expression of the GSR and GPX1 genes was increased only in cells of the cyclophosphamide-resistant strain. These findings suggest that the mechanism of resistance of strain P388/CP is associated with increased activity of glutathione metabolism that developed as a result of activation of the antioxidant response transcription factor Nrf2 against the background of high intracellular concentration of ROS.

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.

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.

[Cyclic hydroxamic acids as chemosensitizers in cytostatic therapy].

In experimental animals with tumors it was studied antitumor activity of spirocyclic hydroxamic acids which could be classified as targeted agents as their target was enzyme histonedeacetylase, which was involved in the neoplastic process. The results showed that the hydroxamic acids were chemosensitizers for anticancer agents increasing their efficacy and enabling the researchers to reduce significantly the therapeutic dose. Also it was showed that hydroxamic acid, containing nitrogen mustard, was effective in the action on tumors with phenotype and genotype of multidrug resistance.

[Generalized mycoplasma infection in patients and carriers].

AIM: Study of possibility of generalization of mycoplasma infection in patients with urogenital pathology. MATERIALS AND METHODS: Among the examined patients 5 males characterized by risky sexual behavior with pronounced symptoms of infection or without those were selected. Patients were examined by a complex of methods for the presence of mycoplasma infection by culture, PCR, DFA, PHA, AHR and by detection of specific immune complexes in blood sera. Scrapes from urogenital tract, blood sera samples, urine, saliva, prostatic fluid were materials for the study. RESULTS: In blood of all patients in ELISA antibodies against Mycoplasma hominis were detected; in PHA they were detected only in 2 individuals. In all the patients in blood CIC were detected including antigens and DNA of one or several mycoplasma species. Sperm of 3 individuals was infected by Ureaplasma spp., 2--M. genitalium. In saliva of 2 individuals M. hominis was detected, 3--U. urealyticum. CONCLUSION: In all the examined patients the infection was shown to have generalized character. This phenomenon presents itself as quite significant because mycoplasma may cause anti-apoptotic and oncogenic effect.

[Antileukemic activity and development of resistance to cisplatin (CPT) and platinum (IV)-nitroxyl complex VS118].

Treatment with low doses (1/10 of LD50) of cisplatin and platinum (IV)-nitroxyl complex VS118 [e-ammin-d-(4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl)-a,f-bi s(acetate)-b,c-dichlorplatinum (IV)] was followed by a synergistic therapeutic effect (a 100% cure of animals) as compared with monotherapy with either drug. There was no synergistic increase in toxicity. The rates of resistance development decreased in the following order: P388/cPt+VS118, P388/cPt, P388/VS118. Resistant strains P388/cPt+VS18 and P388/VS118 were highly sensitive to doxorubicin, etoposide and cyclophoshamide. Further research in cPt+VS 118 combinations should be continued.

[The atypical forms of mycoplasmas persisting in the organism of mycoplasma's infected persons].

The antigens, DNA and RNA of mycoplasmas are preset in the blood serum of persons infected with urogenital mycoplasmas. The planting of patients' tests of blood serum containing antigen M. hominis on the artificial growth mediums resulted in the growth of mini-colonies of mini-cells (20-50 nm). The colonies subcultured hardly but sometimes formed solid bacterial lawn though never acquired "fried-egg" classical mycoplasma form. The proofs of identity of these colonies to M. hominis are presented. The mini-cells possessed infectiousness and ability to persist on a long-run in the internal organs of experimentally infected mice. Apparently, mini-cells are formed under impact of stress factors of the host immune defense and they are one of forms of mycoplasma's persistence in human organism.

Effect of nitric oxide donor, a modulator of tumor drug resistance, on cell death and p53 protein expression.

Exogenous NO donor 3,3-bis-(nitroxymethyl)oxetane (NMO) was synthesized at the Institute for Problems of Chemical Physics (Russian Academy of Sciences). This compound was shown to inhibit cell death (apoptosis and necrosis) in cyclophosphamide-sensitive and cyclophosphamide-resistant P388 murine tumor. p53 protein was expressed in both lines of tumor cells. NO donor NMO had little effect on p53 protein expression in cells of both stains. Our results suggest that the proapoptotic effect of NMO is mediated by the p53-independent molecular mechanisms.

Effect of drug resistance modulator, NO donor, on membrane structure and function of membrane-bound Ca2+-activated Mg2+-dependent ATPase.

Exogenous NO donor 3,3-bis-(nitroxymethyl)oxetane (NMO) was synthesized at the Institute for Problems of Chemical Physics (Russian Academy of Sciences). This compound was shown to inhibit Ca2+-ATPase isolated from normal muscular cells and tumor cells. Both hydrolytic and transport functions of the enzyme were inhibited under these conditions. These changes were probably related to changes in membrane structure caused by NO donor. Our results suggest that changes in intracellular Ca2+ concentration can modulate the formation of tumor drug resistance.

[Nitric oxide donor increases the effectiveness of cytostatic therapy and inhibits the development of drug resistance]. / Donor oksida azota povyshaet éffektivnost' tsitostaticheskoi terapii i zaderzhivaet razvitie lekarstvennoi rezistentnosti.

The investigation has established a potential of low-dosage chemotherapy with cytostatics when used in combination with nitric oxide (NO) donor. Such regimen resulted in more animals being cured of leukemias P388 and L1210 and longer survival. Similar effect was reported with transplantable intracerebral leukemia P388 in which case mean survival after cyclophosphamide plus NO-donor was three times as high as that after cyclophosphamide alone. Combination therapy also promoted animetastatic effect: melanoma B16 inhibition by cyclophosphamide alone was 50% vs. 80% after cyclophosphamide plus NO-donor. NO-donor inhibited development of drug resistance to cyclophosphamide.

Nitric oxide donor increases the efficiency of cytostatic therapy and retards the development of drug resistance.

The potentiality to increase the chemotherapeutic effectiveness of some cytostatics in low, subtherapeutic doses in combination with nitric oxide (NO) donor has been shown. This type of combined therapy results in significant increase in life span and number of survivors among mice bearing leukemias P388 and L-1210. A similar effect was observed for intracerebral leukemia P388 transplantation. In this case the life span of mice treated with cyclophosphamide and NO donor increased by three times in comparison to therapy with cyclophosphamide alone. The coinjection of nitric oxide donor and cytostatics improved the antimetastatic activity of the cytostatics: the index of melanoma B16 metastasis inhibition at the cyclophosphamide monotherapy is 50%; on addition of NO donor the index is over 80%. Comparative studies of NO donor (organic nitrate) and a similar compound in which ONO(2) moieties were replaced by OH groups demonstrated that the presence of NO(2) is required for adjuvant activity of compounds and confirmed that nitric oxide modifies the antitumor effects of cytostatics. It is shown also that nitric oxide donor retards the development of drug resistance to cyclophosphamide.

Nitrotriazole AK-2123 enhances mitomycin C activity in mice bearing multidrug-resistant tumors.

The often observed cross resistance of multidrug-resistant (MDR) tumors to mitomycin C (MMC) is surprising, as these tumors are, as a rule, sensitive to alkylating drugs, and the mechanism of MMC activity is connected to alkylation of DNA. This study shows that nitrotriazole AK-2123 significantly enhances the sensitivity of MDR-strains of P388 mouse leukemia (developed and characterized by authors previously) to mitomycin C. The modulating effect is dependent on the initial sensitivity of resistant tumors to MMC which is correlated with the existence or absence of sorcin (cytosole Ca2+-binding protein) gene coamplification in MDR-amplicon. In agreement with authors' previous data about AK-2123 influence on active Ca2+-transport, it is supposed that the modulatory effect of radiosensitizer is at least partially dependent on this capacity. AK-2123 has no own antitumor effect on investigated tumors and cannot modify the sensitivity of the parent tumor P388 to MMC.

[The radiosensitizer AK-2123 enhances sensitivity of MDR-tumors to Mitomycin C]. / Povyshenie chuvstvitel'nosti lekarstvenno-ustoichivykh opukholei k mitomitsinu C s pomoshch'iu radiosensibilizatora AK-2123.

It was demonstrated that radiosensitizer AK-2123 of the triazole group significantly enhanced the sensitivity of MDR-strains of P388 murine leukemia (reported by the authors earlier) to mitomycin C (MMC). There was a direct correlation between the modulating effect of AK-2123 and dose increase from 1 to 10 mg/kg. The effect depended on the initial sensitivity of the MMC-resistant strain which in turn correlated with the absence or presence of sorcin (cytosole low-molecular Ca(2+)-binding protein) gene coamplification in the mdr-amplicon. Since AK-2123 was reported earlier by us to disrupt active Ca(2+)-transport, it is suggested that the modulating effect of the radiosensitizer was at least partially due to said disruption. AK-2123 exerted no antitumor action of its own whatsoever. It could neither modify the sensitivity of parent strain P388 to MMC, nor overcome the cross resistance of one of the MDR-tumor strains under study to such drugs as etoposide and adriablastin.

[Microorganism persistence in hematopoietic tissue: means for detecting it and its significance]. / Persistentsiia mirkoorganizmov v krovetvornoi tkani: puti vyiavleniia i znachimost'.

Many microorganisms are capable of prolonged persistence in the marrow. In this study, carried out by the method of negative selection based on the treatment of mouse marrow cells with specific antimicrobial sera and complement, Mycoplasma arthritidis and L-forms of group B streptococci were found to be capable of persisting in the marrow in close association with the late category of clonogenic precursor cells, CFU-7, as well as, to a lesser extent, with late erythroid precursors, CFUe. Early colony-forming cells, CFUs-12 and PFUe, as well as granulocyto-macrophagal precursors, CFUgm, did not practically express antigens to the given infective agents on their surface.

Decreased sensitivity of multidrug-resistant tumor cells to cisplatin is correlated with sorcin gene co-amplification.

A set of multidrug resistant (MDR) murine leukemia P388 sublines processing 30-50-fold mdr1 gene amplification was obtained as a result of experimental chemotherapy with rubomycin, ruboxyl, vinblastine, vincristine, or combination of rubomycin and vincristine. Significant differences of developed MDR sublines in response to treatment with cisplatin, tiophosphamide, sarcolysin, and dopad were found. Strong correlation between drug sensitivity and a copy number of gene coding for 19-22 kDa calcium-binding sorcin gene co-amplification were hypersensitive to cisplatin and alkylating agents, the cell sublines showing amplification of sorcin DNA sequences did not possess such collateral sensitivity and even acquired cross-resistance. The dependence of sensitivity to cisplatin on sorcin gene co-amplification was confirmed by analysis of Djungarian hamster DM15 cell sublines that selected for MDR in vitro by colchicine.

[An immunological analysis of bacterial persistence in the bone marrow]. / Immunologicheskii analiz bakterial'noi persistentsii v kostnom mozge.

A method for the evaluation of bacterial persistence in the bone marrow in association with particular clonogenic target cells was developed. The method was based on the negative selection of cells expressing microbial antigens after treatment with hyperimmune antiserum specific to a given infective agent and the subsequent quantitation of target cells thus eliminated in appropriate assays. Using this approach, we demonstrated that Mycoplasma arthritidis and L-forms of Streptococcus strain L-406 were capable of persisting in murine bone marrow in close association with CFUs-7 (a subpopulation of hematopoietic stem cells) for at least several months after experimental infection. Francisella tularensis was also found to be capable to express on the CFUs-7 membranes. Persisting microorganisms enhanced both proliferation and migration of CFUs-7.

Phosphorus-containing metabolites in anthracycline-resistant murine leukemia P388 cells.

The method of 31P nuclear magnetic resonance was used to study in vivo the level of phosphorus-containing metabolites in P388 leukemia cells sensitive or resistant to rubomycin (daunomycin) and its nitroxyl analog-emoxyl. It was shown that decreased content of phosphomonoesters (PME) is characteristic of the resistant strains in comparison with the parent cells. Rubomycin and emoxyl were established not to affect practically the pool of phosphorus-containing metabolites in the cells of the resistant strains, but caused considerable increase of PME level in the cells of the parent strain.