Development of bruneomycin and rubomycin resistance in mouse lymphadenosis tumor cells and Staphyloccus when these preparations are used separately or together

Two sublines of mouse lymphadenosis (L-5178) resistant to bruneomycin and rubomycin used alone, as well as a subline with induced resistance to the combination of these drugs were employed in the study. The studies showd that in separate use of rubomycin and bruneomycin the tumor cell resistance to the respective drug was evident at the 10th passage. After 30 passages neither bruneomycin nor rubomycin produced reliable inhibition of the tumor growth in the respective subline of lyphadenosis. When the antibiotics were used in combination, no significant decrease in sensitivity of the tumor cells to either of the drugs or their combination was observed. The experiment with Staph. aureus also showed that the rate of the resistance increase to the drug combination was lower than that to the drugs used alone. Therefore, it was shown that the combined use of bruneomycin and rubomycin provided a means for preventing to a significant extent of development of the resistance in lymphadenosis tumor cells and Staph. aureus. This may be considered as an indication for clinical trials of the above combination.     

Study using the probit-analysis method of the process of increasing resistance to dipin and bruneomycin in Fisher L-5178 lymphadenosis cells in experiments on animals]

The study of the process of resistance induction in the cells of lymphadenosis to dipin (subline L-5178/D) and bruneomycin (subline L-5178/B) showed that with increasing of the resistance relation between the drug dose and the antitumor effect gradually faded. It was found that only at the first stages of induction of the resistance increase may be found by comparison of values ED50 and ED70 of the lymphadenosis sensitive strain and its resistant sublines. The probit-analysis method provided registration of the resistance increase also at later stages. In this case the resistance level may be estimated by the slope of the dose-response curve.     

Plasmodium berghei: Development of resistance to clindamycin and minocycline in mice

Strains of Plasmodium berghei resistant to clindamycin or minocycline were selected by a procedure in which groups of infected mice were treated with increasing doses of drug during each of a series of subpassages. Groups of five mice, each infected by intravenous inoculation with 10 million parasitized erythrocytes, were treated orally with different doses of drug for four consecutive days beginning on the day of infection. Subpassages were routinely made by Day 7, using donor mice from the group that had been treated with the highest dose of drug that allowed for some development of parasitemia during the preceding passage. Drug doses were increased in each passage as dictated by the development of parasitemia during the previous treated passage.The rate of development of resistance to clindamycin or minocycline was much slower than to conventional antimalarials such as chloroquine, quinine, or pyrimethamine. P. berghei developed total resistance to the latter compounds in nine to 12 treated passages in mice over a period of 60 to 85 days. In contrast, development of total resistance to clindamycin required 42 treated passages over a period of 300 days. Total resistance to minocycline was not attained during 86 successive minocycline-treated passages in mice over a period of 600 days, but a sixfold increase in resistance to minocycline was observed.The clindamycin-resistant strain was normally sensitive to minocycline, chloroquine, quinine, and pyrimethamine. The strain partially resistant to minocycline was normally sensitive to clindamycin, chloroquine, quinine, and pyrimethamine. Resistance to clindamycin was stable during 51 drug-free passages in mice over a period of 1 year. Resistance to minocycline was unstable. During 16 drug-free passages in mice the strain reverted towards normal sensitivity to minocycline. Strains resistant to clindamycin or minocycline showed no difference in rate of development in mice as compared to the parent strain. Likewise, only minor morphological modifications were seen in Giemsa-stained blood smears between the two resistant strains and the parent strain.These results suggest that other species of malaria may develop resistance to clindamycin or minocycline. Should resistance to one of these compounds appear, however, it should not invalidate the use of the other in the treatment of malaria.     

Dynamics of the development of oval cell population induced in the liver by dipin and partial hepatectomy

It has been shown that dipin in combination with partial hepatectomy (model of dipin hepato-carcinogenesis) induces massive oval cell proliferation. The main stages of oval cells development examined by means of light microscopy of half-thin slices are the following: 1) appearance around portal tracts--1-3 weeks; 2) migration into parenchyma along terminal branches of portal vessels--3-8 weeks; 3) maximum development--8-10 weeks; 4) induction focuses of growth of new formed hepatocytes around portal tracts--8-10 weeks; 5) forcing of oval cells towards the centre of liver lobules and their elimination. There was close correlation between oval cells development, fibrosis and inflammation. Oval cells remained around portal tracts until the development of numerous hepatomas in 10 months after treatment.     

Myeloinhibitory effect of the action of some antitumor antibiotics and its comparative assessment]

General toxic and myeloinhibitory effects of some antitumor antibiotics, such as rubomycin, olivomycin, bruneomycin and karminomycin administered intraperitoneally in a single LD50 to mice were studied. It was found that the general toxicity of bruneomycin and karminomycin was almost the same and 5 to 8 times higher than that of rubomycin and olivomycin. The use of the above antibiotics resulted in definite shifts in the blood systems of healthy mice. The most significant suppression of hemopoesis accompanied by a pronounced depression of the number of the myelocariocytes was observed after the use of olivomycin. The effect of karminomycin was characterized by suppression of erythro-, myelo- and lymphopoesis and depression of the number of the granulocytes and lymphocytes of the blood. Bruneomycin and rubomycin had a short-time myeloinhibitory effect. The erythroid cords of the bone marrow proved to be most sensitive to the inhibitory effect of the antibiotics. However, inhibition of the erythropoesis accompanied by deep reticulocytopenia did not induce the respective depression of the erythrocyte number. The lymphoid cords was in the 2nd place by its sensitivity to the antibiotics and the myeloid and megocariocytal cords were in the 3rd and the 4th places respectively. Complete reduction of hemopoesis in the animals was observed by the 10th day of the drugs use.     

Pathomorphological picture of the liver in mice administered antitumor antibiotics intraperitoneally and its comparative assessment]

A single administration of carminomycin, ribomycin or olivomycin in LD50 or treatment of the experimental animals with these antibiotics for 10 days in the therapeutic doses equal to 10 per cent of the LD50 induced distrophic and necrobiotic changes in the liver. The use of bruneomycin in the equivalent doses induced sclerotic process in addition to the above doses resulted in a decrease in the colour intensity of DNA, RNA and protein as compared to the control, the content of glycogen and a marked increase in the amount of lipids in the hepatocyte cytoplasm. The most pronounced shifts were observed with the use of carminomycin, rubomycin and especially bruneomycin in single doses. With the use of olivomycin in a single dose the shifts were less pronounced. It should be noted that with the use of carminomycin and rubomycin the damages were of the same character by their intensity. The changes in the liver on the use of carminomycin, rubomycin and olivomycin in single doses or during the treatment course were reversible, while on the use of bruneomycin they preserved to the end of the experiment.     

Gold nanoparticles: Mutagen,antimutagen, or comutagen?

The effect of ultrasmall gold nanoparticles (GNPs), both direct and in combination with the alkylating mutagen dipin, on the genetic structures of male germ cells in mice were studied using the meiotic micronucleus assay. It was shown for the first time that GNPs can exhibit mutagenic, antimutagenic, and comutagenic activity depending on the experimental conditions.     

Response of the spermatogenic system to chemical mutagen dipin in SAMP1 senescence-accelerated mice

Specific features of spermatogenesis were studied in senescence-accelerated mice of the strain SAMP1 after one-time injection of the chemical mutagen dipin. Quantitative and histomorphological changes in the spermatogenic epithelium proved to develop gradually. Cell loss and disorganization of spermatogenesis reached the peak as late as on days 28 and 35 after the injection. Differentiating spermatogonia manifested increased sensitivity to dipin. In prophase I of meiosis, developing spermatocytes proved to be less sensitive to the cytotoxic action of dipin at the pachytene than at the preleptotene-leptotene stages. Spermatogenesis in most seminiferous tubules was restored by day 56 after dipin treatment. At the end of the experiment (day 100), both quantitative parameters and morphological pattern of spermatogenesis did not differ significantly from those in the control. Thus, the cytotoxic action of dipin does not lead to irreversible structural disorganization of the spermatogenic epithelium in SAMP1 mice. Radioautography revealed a large proportion of highly differentiated Sertoli cells with ³H-thymidine-labeled nuclei in experimental animals. In some cases, structures resembling embryonic seminiferous tubules were revealed in the vicinity of rete testis in histological sections of testes of experimental mice. These structures contained the cells morphologically similar to gonocytes and immature Sertoli cells.     

Combined effect of alkylating compounds on human chromosomes]

A combined effect of thiophosphamide and dipin on chromosomes of unstimulated human lymphocytes was studied in experiments carried out thrice according to the scheme of a complete two-factor experiment. The exposure to the mutagens lasted for an hour, the concentrations being from 3.17 to 22.19 . 10-5 M. After washing a fresh medium with PHA was added to lymphocytes and then they were cultivated during 60 hrs. Both at separate and combined action of tested chemicals the effect observed in all the cytogenetic tests was not subjected to a linear dependence on the concentration of mutagens. With the change in the proportion between thiophosphamide and dipin, given that the summed number of their molecules was constant, the cytogenetic effect was directly proportional to the part of each mutagen in a combined treatment. The analysis of variance and regression analysis of the results obtained as well as the analysis of types of appearing chromosome aberrations and the analysis of distribution of breaks in cells at the combined action showed that, taking into account that the concentration dependences were not linear. the combined effect of thiophosphamide and dipin was additive, without any interaction of individual effects.     

Exploiting the Synergy between Carboplatin and ABT-737 in the Treatment of Ovarian Carcinomas

Platinum drug-resistance in ovarian cancers mediated by anti-apoptotic proteins such as Bcl-xL is a major factor contributing to the chemotherapeutic resistance of recurrent disease. Consequently, concurrent inhibition of Bcl-xL in combination with chemotherapy may improve treatment outcomes for patients. Here, we develop a mathematical model to investigate the potential of combination therapy with ABT-737, a small molecule inhibitor of Bcl-xL, and carboplatin, a platinum-based drug, on a simulated tumor xenograft. The model is calibrated against in vivo experimental data, wherein xenografts established in mice were treated with ABT-737 and/or carboplatin on a fixed periodic schedule. The validated model is used to predict the minimum drug load that will achieve a predetermined level of tumor growth inhibition, thereby maximizing the synergy between the two drugs. Our simulations suggest that the infusion-duration of each carboplatin dose is a critical parameter, with an 8-hour infusion of carboplatin given weekly combined with a daily bolus dose of ABT-737 predicted to minimize residual disease. The potential of combination therapy to prevent or delay the onset of carboplatin-resistance is also investigated. When resistance is acquired as a result of aberrant DNA-damage repair in cells treated with carboplatin, drug delivery schedules that induce tumor remission with even low doses of combination therapy can be identified. Intrinsic resistance due to pre-existing cohorts of resistant cells precludes tumor regression, but dosing strategies that extend disease-free survival periods can still be identified. These results highlight the potential of our model to accelerate the development of novel therapeutics such as BH3 mimetics.     

Action of carminomycin on leukemic cell proliferation and some problems of its use in the combined therapy of leukemias

The effect of carminomycin on the mitotic cycle of the cells of the transplantable leukemia L-1210 and the therapeutic activity of other antitumor drugs, such as phopurine and cyclophosphane was studied on mice BDF1. It was found that the cells in phases S and G2 of the mitotic cycle were most sensitive to carminomycin. Transfer G1--S and phase G1 were characterized by resistance to the antibiotic effect. When carminomycin was used in combination with phopurine or cyclophosphane, clear dependence of the therapeutic efficacy on the treatment scheme was noted. Simultaneous administration of all the three drugs resulted in potentiation of the antileukemic effect. An analogous effect was observed when carminomycin was administered prior to phopurine or cyclophosphane. When the order of the drugs use was reverse, the efficacy of the combined therapy was significantly lower than the summation antileukemic effect of the drugs.     

Alkylating Damage by Dipin of Hematopoietic and Stromal Cells of the Bone Marrow

Effect of alkylating agent dipin was studied on hematopoietic (CFU-S) and stromal (CFU-F) progenitor cells. Single administration of dipin (0.06 mg/g) to adult (CBA × C57Bl/6) F1 hybrid mice induced a long-term (2 years) oscillations in the numbers of day 7 CFU-S and day 11 CFU-S in the bone marrow and spleen. Dipin also damaged the hematopoietic stroma as indicated by decreased numbers of CFU-F which remained low for at least a year. The capacity of stromal cells to form ectopic hematopoietic foci was considerably decreased and also remained low for 10 months. The obtained data suggest high dipin sensitivity of the earliest hematopoietic and stromal cells. The dynamics of CFU-S numbers in the hematopoietic organs supports their functioning on the basis of clonal succession (Kay, 1965).__________Translated from Izvestiya Akademii Nauk, Seriya Biologicheskaya, No. 3, 2005, pp. 267–272.Original Russian Text Copyright © 2005 by Domaratskaya, Bueverova, Payushina, Starostin.     

Effect of the anthracycline group antibiotics, mitomycin C and bruneomycin, on the transduction of drug resistance in staphylococci]

The effect of various concentrations of antitumor antibiotics, such as carminomycin, rubomycin, adriamycin, mitomycin C and bruneomycin on transduction of erythromycin resistance from the donor strain 8325 P II/de of Staph, aureus to the recipient strain 8325-I in different transduction systems was studied. It was shown that the above antibiotics inhibited the transduction in the systems with constant presence of the drugs. Preliminary treatment of the recipient cells with the drugs in the subbacteriostatic doses did not decrease the transfer frequency. The preliminary treatment of the donor cells resulted in an increase in the phase titer and the transfer frequency in the "preliminary-treated donor + recipient" system.     

Defective B cell tolerance induction in New Zealand black mice. I. Macrophage independence and comparison with other autoimmune strains

B cell unresponsiveness was examined in vitro by using spleen cells from autoimmune NZB, BXSB/Mp male, MRL/Mp-Ipr/Ipr (MRL/l), and control mice, and the tolerogen trinitrophenyl human gamma-globulin (TNP-HGG). The B cell subset responsive to TNP-Brucella abortus in each autoimmune and control strain that was tested was highly susceptible to tolerance induction with the use of high epitope density conjugates (TNP30HGG and TNP32HGG). When a tolerogen with a lower epitope density was used (TNP7HGG), several control strains were all rendered tolerant in a thymic-independent and hapten-specific manner. NZB B cells were resistant to all concentrations of TNP7HGG tested, whereas B cells from BXSB/Mp male and MRL/1 mice were resistant to low concentrations of this tolerogen. NZB mice were resistant in addition to tolerance induction with TNP9HGG, TNP10HGG, and TNP12.7HGG. Experiments were performed to determine whether splenic macrophages played a role in resistance to tolerance in NZB mice. The mixing of NZB and control DBA/2J T cell-depleted splenocytes revealed no modulatory effects by the accessory cells in culture. Moreover, B cells rigorously depleted of macrophages by double Sephadex G-10 column passage exhibited characteristic patterns of resistance or susceptibility in NZB and control strains, respectively. These findings support the conclusion that resistance to tolerance in NZB mice is determined at the B cell level and are consistent with the hypothesis that diverse immunoregulatory disturbances contribute in varying degrees to the development of systemic lupus erythematosus in different inbred strains of mice.     

The assessment of the level of genetic damages accumulated with age and induced in liver cells by the micronucleus production]

Latent genetic disturbances in aging liver cells can be registered during interphase by the appearance of micronuclei resulting from certain chromosomal aberrations. Micronuclei were also detected in postmitotic hepatocytes of mouse liver regenerating after partial resection of CCl4 poisoning. In 1.5- and 2-month-old mice, the proportion of micronuclei-containing cells was on average 0.59 and 0.89%, respectively. At the age of 4 and 7 months, the proportion of aberrant cells in hepatocyte population, including cells containing multiple micronuclei, increased to 5.93 and 11.7%, respectively. In order to evaluate parameters used to characterize "spontaneous" aging, experiments were performed in which genetic disturbances were induced by x-irradiation or treatment with dipin, an alkylating agent (individually or in combination); the effect was determined one and two months after the treatment. The yield of micronuclei under the conditions of a mild treatment (irradiation at a dose of 0.7 and 1.4 Gr or dipin at a dose of 30 mg/kg body weight) was similar to that observed during aging. The possible reasons for the increased (as compared to the published data) rate of genetic disturbances in arbitrary intact animals are discussed.     

Alternative Signaling Pathways as Potential Therapeutic Targets for Overcoming EGFR and c-Met Inhibitor Resistance in Non-Small Cell Lung Cancer

The use of tyrosine kinase inhibitors (TKIs) against EGFR/c-Met in non-small cell lung cancer (NSCLC) has been shown to be effective in increasing patient progression free survival (PFS), but their efficacy is limited due to the development of resistance and tumor recurrence. Therefore, understanding the molecular mechanisms underlying development of drug resistance in NSCLC is necessary for developing novel and effective therapeutic approaches to improve patient outcome. This study aims to understand the mechanism of EGFR/c-Met tyrosine kinase inhibitor (TKI) resistance in NSCLC. H2170 and H358 cell lines were made resistant to SU11274, a c-Met inhibitor, and erlotinib, an EGFR inhibitor, through step-wise increases in TKI exposure. The IC₅₀ concentrations of resistant lines exhibited a 4–5 and 11–22-fold increase for SU11274 and erlotinib, respectively, when compared to parental lines. Furthermore, mTOR and Wnt signaling was studied in both cell lines to determine their roles in mediating TKI resistance. We observed a 2–4-fold upregulation of mTOR signaling proteins and a 2- to 8-fold upregulation of Wnt signaling proteins in H2170 erlotinib and SU11274 resistant cells. H2170 and H358 cells were further treated with the mTOR inhibitor everolimus and the Wnt inhibitor XAV939. H358 resistant cells were inhibited by 95% by a triple combination of everolimus, erlotinib and SU11274 in comparison to 34% by a double combination of these drugs. Parental H2170 cells displayed no sensitivity to XAV939, while resistant cells were significantly inhibited (39%) by XAV939 as a single agent, as well as in combination with SU11274 and erlotinib. Similar results were obtained with H358 resistant cells. This study suggests a novel molecular mechanism of drug resistance in lung cancer.     

Hierarchy of in vitro sensitivity and resistance of tumor cells to cytotoxic effector cells,cytokines, drugs and toxins

Summary Drug resistance of tumor cells has led to the development of other therapeutic modalities including biological response modifiers, lymphokine-activated killer cells (LAK), and cytokines alone and in combination. The premise of these alternative modalities is that drug resistance can be overcome by other cytotoxic agents or cytotoxic effector cells. However, the relationship between tumor cell sensitivity to these different agents and the cytotoxicity caused by drugs is not known or well understood. Thus, understanding the relationship between these different systems of tumor cell cytotoxicity is essential for optimal therapeutic intervention. To this end, we compared the tumor cell cytotoxicity mediated by recombinant tumor necrosis factor (rTNF), cytotoxic effector cells (natural killer cells, monocytes, LAK cells), chemotherapeutic drugs, and microbial toxins. Human tumor cell lines sensitive and resistant to rTNF or drugs were used to evaluate the effectiveness of the other cytotoxic modalities. Sensitivity was considered as tumor cell cytotoxicity above 15% while resistance refers to that below 10%. Cell lines tested consisted of several histological types such as brain, lung, colon and ovarian tumors. In our experiments, cell lines made resistant to rTNF by coculture were also relatively resistant to unactivated monocytes and their supernatants. These lines were sensitive to all other methods tested including activated monocytes, natural killer and LAK cells, drugs, and toxins. The tumor lines naturally resistant to rTNF were found to have various degrees of sensitivity and resistance to these other systems. Upon the analysis of our data, a pattern emerged that suggested a hierarchy of sensitivity and resistance of the tumor cells to the cytotoxic mechanisms explored. From a majority of cell lines resistant to rTNF to a minority of lines resistant to LAK, we found an interesting gradation of sensitivity and/or resistance to the other cytotoxic modalities employed. The hypothesis of an underlying common mechanism of action within these systems is discussed.Supported in part by grant CA43 121 from the Department of Health and Human services, NIH, and NRSA clinical and fundamental immunology training grant A107 126, NIH (J. S.), and in part by a grant from the Concern Foundation, Los Angeles and a gift from the Boiron Foundation     

Estimation of the frequencies of induced mutations in spermatogenic cells of senescence-accelerated prone mice of the SAMP1 strain

The results obtained in this work demonstrate the dynamics of cytogenetic changes of spermatogenic cells in senescence-accelerated prone mice, strain SAMP1, after a single exposure to a chemical mutagen, dipin, at a genetically active dose of 30 mg/kg. In the time interval between days 3 and 28 the frequency of induced spermatogonial micronuclei does not significantly exceed the level of spontaneous mutagenesis. The lack of an experimental effect of micronuclei in this time interval is probably a consequence of mitotic delay and (or) of the death of a considerable part of genetically defective cells in the spermatogonial compartment. Different stages of meiosis exhibit different chemical sensibilities: the yield of round spermatids with micronuclei is maximum after treatment of early primary spermatocytes (preleptotene-leptotene stage) with dipin. The high sensibility of preleptotene and leptotene spermatocytes is confirmed by the sperm head shape abnormality assay. Chromosome damage caused by dipin in spermatogonial stem cells is irreversible, as evidenced by a sharp increase in the frequencies of spermatogonial and meiotic micronuclear aberrations within long periods after treatment. Increased genetic instability in the stem compartment does not lead to irreversible degradation of the system of development of male sex cells in senescence-accelerated SAMP1 mice.     

Experimental study on chemotherapeutic efficacy, acute toxic action and nephrotoxicity of combinations of eremomycin with tobramycin

Chemotherapeutic efficacy of eremomycin in combination with tobramycin was investigated on a model of experimental sepsis of albino mice caused by Staphylococcus aureus cultures resistant to methicillin. Eremomycin is a novel original antibiotic of the glycopeptide structure isolated in the USSR and tobramycin is an aminoglycoside. Acute toxicity of the combination with a wide range of the dose fixed proportions was studied on mice and the nephrotoxic action of the antibiotics and their combinations administered intravenously for 5 days was studied on albino rats. The experiments showed that the chemotherapeutic effect of eremomycin in combination with tobramycin was of synergistic nature. Acute toxicity of the combined drugs mainly summed up and somewhat increased when the proportion of tobramycin and eremomycin was 1:2.4 or 1:3.6. Eremomycin had a dose-depended nephrotoxicity. Summing up of the nephrotoxic action of the drugs on their combined use was observed.     

Influence of doxorubicin on the development of resistance of staphylococci to ceftriaxone

Effect of combined use of doxorubicin and ceftriaxone on 5 strains of Staphylococcus aureus (standard S. aureus ATCC 29213 and 4 isolated strains) was studied. The method of passages in meat-pepton broth with constant and increasing concentrations of ceftriaxone in presence of 1/2 and 1/4 minimum inhibitory concentration of doxorubicin was used. It has been shown that doxorubicin in such concentrations does not influence on the development of resistance of tested strains to ceftriaxone. The combination of doxorubicin as anti-tumor drug with intercalary action and ceftriaxone does not increase the risk of development of resistance of staphylococci to antibiotics, which has a matter during their combined use.