Contribution of reactive oxygen species to the anticancer activity of aminoalkanol derivatives of xanthone.

Reactive oxygen species (ROS) are critically involved in the action of anticancer agents. In this study, we investigated the role of ROS in the anticancer mechanism of new aminoalkanol derivatives of xanthone. Most xanthones used in the study displayed significant pro-oxidant effects similar to those of gambogic acid, one of the most active anticancer xanthones. The pro-oxidant activity of our xanthones was shown both directly (by determination of ROS induction, effects on the levels of intracellular antioxidants, and expression of antioxidant enzymes) and indirectly by demonstrating that the overexpression of manganese superoxide dismutase decreases ROS-mediated cell senescence. We also observed that mitochondrial dysfunction and cellular apoptosis enhancement correlated with xanthone-induced oxidative stress. Finally, we showed that the use of the antioxidant N-acetyl-L-cysteine partly reversed these effects of aminoalkanol xanthones. Our results demonstrated that novel aminoalkanol xanthones mediated their anticancer activity primarily through ROS elevation and enhanced oxidative stress, which led to mitochondrial cell death stimulation; this mechanism was similar to the activity of gambogic acid.

Cytotoxicity of etoposide in cancer cell lines in vitro after BCL-2 and C-RAF gene silencing with antisense oligonucleotides.

BCL-2 and C-RAF genes are overexpressed in most types of cancers. Although these genes are mediators in different molecular pathways their main characteristic is the antiapoptotic activity, thus cells that overexpress either BCL-2 or C-RAF lose their ability to undergo apoptotic death being resistant to chemotherapeutic agents and/or physiologic mediators of cell death (e.g., TNF-alpha). Both anti-C-RAF, and anti-BCL-2 oligonucleotides were tested as chemosensitizers in cancer therapy. The aim of the study was to investigate the effects of the combined use of antisense oligonucleotides (ASOs) targeting BCL-2 and C-RAF transcripts on the in vitro cancer cell cultures exposed to etoposide. Cells were transfected with phosphorothioate BCL-2 and C-RAF ASOs. To sustain high intracellular level of ASOs, 3-day transfection was used, and it was followed by a single treatment with 20 microM etoposide for 5 h. The following cancer cell lines were tested: A549, HeLa, and T24. Sequence-specific decrease in BCL-2, and C-RAF mRNA levels were confirmed by real-time RT-PCR: after 1-day treatment mRNA levels decreased by 9-42% of the normal expression in cells treated with 50-1200 nM ASOs. Also, the induction of cell death in all transfected cultures in a concentration-dependent manner was confirmed by MTT assay,microscopic analysis of cell morphology, and the measurement of histone H3 expression. Results also showed that both ASOs effectively potentiated etoposide-induced cytotoxicity; the strongest effects were obtained in A549 (lung cancer). This observation suggests that lower concentrations of both antisense oligonucleotides may be used, at least for this type of cancer, to obtain high efficiency of etoposide-induced cell death enhancement. Simultaneous use of two ASOs in 3-day treatment allows us to lower concentrations needed to obtain significant treatment results thus enabling to diminish sequence-unspecific toxicity.

Repetitive extragenic palindromic PCR (REP-PCR) as a method used for bulking process detection in activated sludge.

Bulking of activated sludge is a world-widely prevalent problem and can lead to loss of bio-oxidation, further deterioration of effluent quality, and even to a complete breakdown of the entire treatment process. Most common reasons of bulking are bacterial community changes, especially excessive growth of filamentous bacteria or excess of biopolymers on surface of non-filamentous microbes. Because of complex nature of the bulking phenomenon, the successful bulking control strategy finding is still a very important need awaiting new options and advices. The repetitive extragenic palindromic PCR (REP-PCR) fingerprinting method has been applied to distinguish bacterial community in non-bulking and bulking activated sludge. The characteristic REP-PCR fingerprinting patterns, using the Ward's clustering method, have been analyzed to determine homology/similarity relation between particular non-bulking and bulking sludge sampling. The received clustering results were in high concordance with activated sludge typing done based on physicochemical sludge analysis. The choice and application of molecular typing method in sludge analysis will depend upon the needs, skill level, and resources of the laboratory. The proposed REP-PCR method and statistical analysis of fingerprinting patterns seems to be simple, rapid, and effective methods to show differences between population in non-bulking and bulking activated sludge. It is easy to implement, and it may be useful for routinely activated sludge monitoring as well as may be helpful in early detection of bulking process.

Repetitive extragenic palindromic PCR (REP-PCR) as an alternative method for detection of bulking in activated sludge.

Bulking of activated sludge is a world-wide problem which negatively affects wastewater treatment efficiency. The most common reasons of bulking are bacterial community changes, especially excessive growth of filamentous bacteria (filamentous bulking) or excess of biopolymers on the surface of non-filamentous microbes (non-filamentous or Zoogleal bulking). Because of the complex nature of the bulking phenomenon finding a successful bulking control strategy remains a very important issue that awaits new options and advices. The REP-PCR fingerprinting method has been applied to distinguish a bacterial community in non-bulking and bulking activated sludge. The characteristic REP-PCR fingerprinting patterns were compared with each other in terms of the presence or absence of bands and in terms of measured integrated optical density (IOD) of the bands. The obtained fingerprinting patterns, using Ward's clustering method, have been analyzed to determine homology/similarity relations between specific non-bulking and bulking sludge sampling. The received clustering results were in high concordance with activated sludge typing which generally is done based on physicochemical sludge analysis. The proposed REP-PCR method and statistical analysis of fingerprinting patterns seems to be a simple, rapid and effective method revealing differences between populations in non-bulking and bulking activated sludge. It may be useful for routine activated sludge monitoring and may be helpful in the early detection of the bulking process.

Influence of interferon-alpha and ribavirin on the transcription of interferon-gamma and IFN-gamma receptor genes in Jurkat cells.

Interferon-alpha and ribavirin are currently the only drugs registered in the chronic hepatitis C therapy. Their actions are based on both direct antiviral activities, and their influence on genes expressions. In the presented study, using Jurkat cell line as an in vitro model for interferon-gamma synthesis, influence of interferon-alpha and ribavirin on the expressions of IFN-gamma and its receptor subunits (IFNgR1 and IFNgR2) were studied. Expressions of the studied genes were measured at the transcriptional level using Real-Time RT-PCR method. Results indicate that both drugs, IFN-alpha and ribavirin, induced changes in IFN-gamma and its receptor expressions. While IFN-alpha stimulated the expressions of the studied genes (IFN- gamma, IFNgR1, and IFNgR2), ribavirin showed the contradictory influence. The inhibitory effect of ribavirin dominated IFN-alpha action and was responsible for the decrease in the mRNA levels of IFN-gamma and the receptor of IFN-gamma. This phenomenon observed at in vitro model may be responsible for the IFN-gamma decrease during hepatitis C therapy with IFN-alpha and ribavirin which was suggested by some authors.

[Changes in etoposide-induced apoptosis of HeLa tumor cells transfected with antisense oligonucleotide for BCL-2 mRNA]. / Zmiany w indukcji apoptozy komórek linii nowotworowej HeLa za pomoca etopozydu w obecnosci oligonukleotydu antysensownego dla mRNA genu BCL-2.

UNLABELLED: Overexpression of genes involved in proliferation, including genes of BCL family, is often found in cells of most malignant tumors. Currently developed strategies of cancer treatment include trials combining classical chemotherapy with silencing of genes expression using the gene therapy. The aim of the study was to induce silencing of BCL-2 gene expression in HeLa tumor cell line using antisense oligonucleotides (ASOs) technique. MATERIAL AND METHODS: Studies were carried out on in vitro HeLa cell cultures treated with etoposide. Cells were transfected by lipofection with ASO targeting BCL-2 mRNA. Effects of BCL-2 silencing were determined by Real-Time RT-PCR, proliferation/cytotoxicity MTT [3-(4,5-dimethylthiazol-2-yl)-2-5-dipheryl tetrazolium bromide] test, and by microscopic detection of apoptotic cells. RESULTS: We showed a decrease in BCL-2 mRNA level in cells transfected with anti-BCL-2 ASO at concentration ranging from 50 to 1200 nM. Apoptotic cells were detected more frequently in transfected cultures compared with untreated controls. However, MTT tests did not display significant decrease of cell proliferation in the transfected cultures as compared with cultures treated with etoposide alone. CONCLUSIONS: 1. These results indicate that the studied ASO sequence-specifically decreases BCL-2 expression in HeLa cells in vitro, although its action is limited mostly by transfection efficiency. 2. The use of the anti-BCL-2 oligonucleotide in combination with etoposide results in significant deacrease of proliferation in cell cultures and this phenomenon is a result of synergy between used chemotherapy and gene therapy.

Analysis of swine leukocyte antigen class I gene profiles and porcine endogenous retrovirus viremia level in a transgenic porcine herd inbred for xenotransplantation research.

Molecular characterization of swine leukocyte antigen (SLA) genes is important for elucidating the immune responses between swine-donor and human-recipient in xenotransplantation. Examination of associations between alleles of SLA class I genes, type of pig genetic modification, porcine endogenous retrovirus (PERV) viral titer, and PERV subtypes may shed light on the nature of xenograft acceptance or rejection and the safety of xenotransplantation. No significant difference in PERV gag RNA level between transgenic and non-transgenic pigs was noted; likewise, the type of applied transgene had no impact on PERV viremia. SLA-1 gene profile type may correspond with PERV level in blood and thereby influence infectiveness. Screening of pigs should provide selection of animals with low PERV expression and exclusion of specimens with PERV-C in the genome due to possible recombination between A and C subtypes, which may lead to autoinfection. Presence of PERV-C integrated in the genome was detected in 31.25% of specimens, but statistically significant increased viremia in specimens with PERV-C was not observed. There is a need for multidirectional molecular characterization (SLA typing, viremia estimation, and PERV subtype screening) of animals intended for xenotransplantation research in the interest of xeno-recipient safety.

Reduced mobility of fibroblast growth factor (FGF)-deficient myoblasts might contribute to dystrophic changes in the musculature of FGF2/FGF6/mdx triple-mutant mice.

Development and regeneration of muscle tissue is a highly organized, multistep process that requires cell proliferation, migration, differentiation, and maturation. Previous data implicate fibroblast growth factors (FGFs) as critical regulators of these processes, although their precise role in vivo is still not clear. We have explored the consequences of the loss of multiple FGFs (FGF2 and FGF6 in particular) for muscle regeneration in mdx mice, which serve as a model for chronic muscle damage. We show that the combined loss of FGF2 and FGF6 leads to severe dystrophic changes in the musculature. We found that FGF6 mutant myoblasts had decreased migration ability in vivo, whereas wild-type myoblasts migrated normally in a FGF6 mutant environment after transplantation of genetically labeled myoblasts from FGF6 mutants in wild-type mice and vice versa. In addition, retrovirus-mediated expression of dominant-negative versions of Ras and Ral led to a reduced migration of transplanted myoblasts in vivo. We propose that FGFs are critical components of the muscle regeneration machinery that enhance skeletal muscle regeneration, probably by stimulation of muscle stem cell migration.

[Genital warts associated with HPV infection during II and III trimester of pregnancy--a case report and analysis of treatment options]. / Klykciny konczyste u ciezarnej w II i III trymestrze ciazy--opis sytuacji klinicznej i analiza zastosowanego leczenia.

More than 30 HPV types can infect the genital tract. Viral infection can be present in clinical, subclinical or latent form. A visible genital form of HPV infection are genital warts, which are commonly caused by HPV types 6 and 11, and appear on the vulva, cervix, vagina, urethra and anus. Oncogenic HPV types 16, 18, 31, 33 and 35 are also found in genital warts and are associated with vulval (VII), cervical (CIN) and anal (AIN) intraepithelial neoplasia. The general prevalence of HPV infection in the form of visible genital warts estimates to about 1% of sexually active adults. Approximately 15% of the infected group / of all adults have a subclinical or latent infection and at least 80% had been infected with one or more genital HPV types at some point in their lives. The highest rate of frequency of infections occurs in the group of adults, aged from 18 to 28. Over the last twenty years figures have shown a constant growth of the infection rate, which also includes pregnant women. Genital warts can proliferate during pregnancy due to altered immunity and increased blood supply. Cryotherapy, electrocautery, laser therapy, surgery or trichloroacetic acid may be used to remove the warts. In the paper a case report on genital warts associated with HPV infection during II and III trimester of pregnancy and analysis of treatment options has been presented.

[Expression patterns in isoforms of vascular endothelial growth-factors in tissue samples of vulval cancer T1 N2M0 stage]. / Wzory ekspresji izoform naczyniowo-sródblonkowego czynnika wzrostu (VEGF) w raku sromu T1N2M0.

OBJECTIVES: The aim of the study was estimating the expression of isoforms of mRNA vascular endothelial growth factor: VEGF121, VEGF145, VEGF165, VEGF183, VEGF189, VEGF206, in tissue samples of vulvar cancer, normal and inguinal lymph nodes metastases. MATERIALS AND METHODS: The material for this investigation of expression of mRNA selected genes was 4 tissue specimens from women with vulvar cancer T1N2M0. The method used for quantitative determination of the numbers of mRNA copies of selected genes was the QRT-PCR and QPCR technique, with the use of an ABI PRISM 7700 (TaqMan) sequence detector. The nucleotide sequence for starters and probes for quantitative RT-PCR was designed with the use of the Primer Express Version 1.0 ABI PRISM software. RESULTS: Expression of all mRNA isoforms VEGF was found in the samples. The number of mRNA copies obtained in 1 microgram total mRNA extract of the genes examined in tissue sections with various pathomorphology were clearly differentiated. CONCLUSIONS: Expression profile of aforementioned genes indicated a definitive relationship between vascular endothelial growth factor isoform and vulvar cancer. These findings provide evidence that the expression pattern of mRNA VEGF isoform should find broad applications in clinical and research settings.

Modulation of porcine endogenous retroviruses (PERVs) expression in vitro by shRNA in the presence of cyclosporine A and dexamethasone.

BACKGROUND: Despite the fact that the risk of Porcine Endogenous Retroviruses (PERV) infection and propagation in human recipients is extremely low, such an event cannot be completely ruled out, especially in immunosuppressed patients. Therefore, the aim of this study was to analyze the expression of PERVs in vitro in the presence of immunosuppression agents: cyclosporine A (CsA), and dexamethasone (DEX). We investigated the possible interactions between immunosuppression drugs, CsA and DEX, and the efficiency of anti-PERV RNAi. MATERIAL/METHODS: Plasmid-based vectors expressing shRNAs against all PERV genes were constructed and analyzed. PERVs expression in cultures transfected with anti-PERV RNAi constructions and treated with CsA or DEX was analyzed by Real-Time RT-PCR, Western blot, and by the measurement of RT activity. RESULTS: Both CsA and DEX inhibited PERVs expression in cell cultures in vitro. RNAi constructions efficiently knocked down PERV expression in Circe, and de novo PERV-infected HeLa and HEK-293 cell cultures. Pretreatment of Circe cultures with CsA or DEX increased PERVs knockdown by RNAi, but no specific interaction between the drugs and transfection efficiency was observed. CONCLUSIONS: Our results demonstrate that cyclosporine A and dexamethasone decrease expression of PERVs in vitro. We also proved that these drugs did not synergize or antagonize RNAi-mediated knockdown of PERVs. These observations may be beneficial in immunosuppressed xenograft recipients; however, due to the controversial literature data concerning influence of immune suppression on graft recipients, our results should be further analyzed.

Different extent of cardiac malfunction and resistance to oxidative stress in heterozygous and homozygous manganese-dependent superoxide dismutase-mutant mice.

AIMS: The mitochondrially expressed manganese-dependent superoxide dismutase (MnSOD, SOD2) is an essential antioxidative enzyme that is necessary for normal heart function. In this study, we investigated the heart function of mice that were exposed to increased oxidative stress for time periods of up to 6 months due to decreased MnSOD activity caused by heterozygous deletion of the MnSOD gene. METHODS AND RESULTS: We generated a mouse strain in which the gene encoding MnSOD was exchanged against a cassette containing the SOD cDNA under the control of the tetracycline response element. After breeding with mice carrying the tetracycline receptor, compound mice express MnSOD depending on the presence of tetracycline. Without tetracycline receptor the MnSOD gene is fully inactivated, and animals show an MnSOD-deficient phenotype. Using echocardiographic recordings, we found an impairment of left ventricular functions: MnSOD+/- mice displayed a decrease in fraction shortening and ejection fraction and an increase in left ventricular internal diameter in systole. Furthermore, MnSOD+/- mice developed heart hypertrophy with accompanying fibrosis and necrosis revealed by immunhistochemical analysis. Although we did not find an increase in apoptosis in MnSOD+/- hearts under normal conditions, we observed an increase of the number of apoptotic cells and vascular senescence after treatment with doxorubicin. CONCLUSION: Our study demonstrates that lifelong reduction of MnSOD activity has a negative effect on normal heart function. This animal model presents a valuable tool to investigate the mechanism of heart pathology reported in patients bearing different polymorphic variants of the MnSOD gene and to develop new therapeutic strategies through manipulation of the antioxidative defence system.