Zsuzsanna Szabó

Expression of miRNA-21 and miRNA-221 in clear cell renal cell carcinoma (ccRCC) and their possible role in the development of ccRCC

AIM Clear cell renal cell carcinoma (ccRCC) is the third most common urological cancer after prostate and bladder cancer but has the highest rate of mortality affecting over 40% of patients. microRNAs (miRNAs) are small noncoding RNAs that have become potential biomarkers and molecular targets for cancer treatment. Molecular markers such as miRNAs may have a role in the diagnosis of ccRCC. In this study, we examined the expressions of miRNA-21 and miRNA-221 in renal cancer patients׳ tumor and adjacent paired normal tissues investigating the possible role of these miRNAs in the development of ccRCC. MATERIALS AND METHODS Renal tumors (n = 24) and paired normal renal tissue (n = 24) samples, obtained from the Department of Urology, University of Debrecen, were analyzed for miRNA-21 and miRNA-221 expressions with quantitative real-time polymerase chain reaction. RESULTS miRNA-21 and miRNA-221 expressions were significantly up-regulated in tumor specimens compared to normal tissue (P<0.05). miRNA-21 and miRNA-221 showed coexpression pattern in 19 (79.2%) cases of tumor samples and 8 (33.3%) cases of paired normal renal tissues. Increased miRNA pattern showed a positive correlation with pathological status of the patients. CONCLUSIONS Expression of oncogenic miRNA-21 and miRNA-221 in human ccRCC tumor tissue samples compared to adjacent nontumorous tissues might suggest that these miRNAs are involved in the development of ccRCC.

Airborne LiDAR point cloud in mapping of fluvial forms: a case study of a Hungarian floodplain

The aim of this paper was to analyze the ground and low vegetation points of a Light Detection and Ranging (LiDAR) point cloud from the aspect of the generated digital terrain model (DTM). We determined the height difference between the surveyed surface and the DTM and the level of interspersion of ground and low vegetation points in a floodplain. Finally, we performed a supervised classification with topographic (elevation, slope and aspect) variables and an Normalized Difference Vegetation Index (NDVI) layer to identify swales and point bars as floodplain forms. Cross sections of field surveys provided reference data to express the magnitude of the bias on the DTM caused by the vegetation, and we proved that the bias can reach the 60% of the relative height and depth of the floodplain forms (mean error was 0.15 ± 0.12 m). A landscape metric, the Aggregation Index, provided an appropriate tool to analyze and quantify the interspersion of the ground and vegetation points: indicating a high level of interspersion of the classified points, i.e. proved that vegetation points where the last echoes reflected from the vegetation became ground points. Floodplain classification performed best with the common use of DTM, slope, aspect and NDVI coverages, with 71% overall accuracy.

IN VITRO ACTIVITY OF FLUCONAZOLE AND AMPHOTERICIN B AGAINST CANDIDA INCONSPICUA CLINICAL ISOLATES AS DETERMINED BY THE TIME-KILL METHOD

Candida inconspicua is an emerging pathogen in immunocompromised patients possessing inherently decreased susceptibility to fluconazole. We determined the MICs and killing activity of fluconazole and amphotericin B against C. inconspicua clinical isolates as well as reference strain C. inconspicua ATCC 16783 for comparison. MICs were determined using the standard broth microdilution method. Killing rates were determined using time-kill methodology at 0.5-16 x MIC fluconazole and amphotericin B concentrations. Fluconazole and amphotericin B MIC values varied between 16-128 mg/l and 0.5-1 mg/l, respectively. In time kill-assays fluconazole showed fungistatic effect at 1-16 x MIC concentrations against all tested strains after 24 h-incubation, but became fungicidal after 48 h at 4-16 x MIC concentrations. The time necessary to achieve fungicidal endpoint at 1 mg/l amphotericin B concentration ranged from 2 to 24 h. Our in vitro results confirm the data that fluconazole is ineffective against C. inconspicua at the fluconazole serum concentration attainable in humans. Amphotericin B due to its rapid killing activity seems to be a good alternative for the treatment of infections caused by C. inconspicua.

Characterization of luteinizing hormone-releasing hormone receptor type I (LH-RH-I) as a potential molecular target in OCM-1 and OCM-3 human uveal melanoma cell lines

Introduction Uveal melanoma (UM) is the most common primary intraocular malignancy with very poor prognosis. Conventional chemotherapy only rarely prolongs the survival, therefore patients require novel treatment modalities. The discovery of specific receptors for hypothalamic hormones on cancer cells has led to the development of radiolabeled and cytotoxic hormone analogs. Materials and methods In the present study, our aim was to investigate the expression of mRNA for receptors of luteinizing hormone-releasing hormone type I (LH-RH-I) and LH-RH ligand in OCM-1 and OCM-3 human uveal melanoma cell lines. The presence and binding characteristics of LH-RH-I receptor protein was further studied by Western blot, immunocytochemistry and ligand competition assay. The expression of mRNA and protein for LH-RH-I receptors has been also studied using tumor samples originating from nude mice xenografted with OCM-1 or OCM-3 cells. Results The mRNA for LH-RH-I receptor has been detected in OCM-1 and OCM-3 cell lines and was found markedly higher in OCM-3 cells. The mRNA for LH-RH-I receptors was also observed in both UM xenograft models in vivo with higher levels in OCM-3. The presence of LH-RH-I receptor protein was found in both cell lines in vitro by immunocytochemistry and Western blot, and also in tumor tissue samples grown in nude mice by Western blot. Both human uveal melanoma models investigated showed specific high affinity receptors for LH-RH-I using ligand competition assay. The mRNA for LH-RH ligand has also been detected in OCM-1 and OCM-3 cell lines and cancer tissues. Conclusion The demonstration of the expression of LH-RH-I receptors in OCM-1 and OCM-3 human UM cell lines suggests that they could serve as potential molecular target for therapy. Our findings support the development of new therapeutic approaches based on cytotoxic LH-RH analogs or modern powerful antagonistic analogs of LH-RH targeting LH-RH-I receptors in UM.

Somatostatin Receptors as Molecular Targets in Human Uveal Melanoma

Uveal melanoma (UM) is the most common primary intraocular malignancy in adults, with an incidence of 4–5 cases per million. The prognosis of UM is very poor. In the present study, our aim was to investigate the expression of mRNA and protein for somatostatin receptor types-1, -2, -3, -4, -5 (SSTR-1–5) in human UM tissue samples and in OCM-1 and OCM-3 human UM cell lines by qRT-PCR, western blot and ligand competition assay. The mRNA for SSTR-2 showed markedly higher expression in UM tissues than SSTR-5. The presence of SSTRs was demonstrated in 70% of UM specimens using ligand competition assay and both human UM models displayed specific high affinity SSTRs. Among the five SSTRs, the mRNA investigated for SSTR-2 and SSTR-5 receptors was strongly expressed in both human UM cell lines, SSTR-5 showing the highest expression. The presence of the SSTR-2 and SSTR-5 receptor proteins was confirmed in both cell lines by western blot. In summary, the expression of somatostatin receptors in human UM specimens and in OCM-1 and OCM-3 human UM cell lines suggests that they could serve as a potential molecular target for therapy of UM using modern powerful cytotoxic SST analogs targeting SSTR-2 and SSTR-5 receptors.

Experimental therapy of doxorubicin resistant human uveal melanoma with targeted cytotoxic luteinizing hormone-releasing hormone analog (AN-152).

Background: Cytotoxic analogs of LHRH (luteinizing hormone‐releasing hormone) can be successfully used for the treatment of hormone‐dependent cancers such as prostatic, ovarian, endometrial, but our knowledge about their effect on hormone‐independent cancers such as human uveal melanoma (UM) is limited. Previously, we have demonstrated that 46% of UM express full‐length LHRH receptors. This finding has led us to further examine the mechanism of action of LHRH receptor based targeted therapies in this malignancy. Aims: In the present study we investigated the cellular uptake of doxorubicin (DOX) and cytotoxic LHRH analog AN‐152 (AEZS‐108, zoptarelin doxorubicin) on human UM cell lines (OCM3) and its DOX resistant form OCM3DOX320 by confocal laser scanning microscopy. The LHRH receptor expression was characterized by RT‐PCR and immunocytochemistry. Results: We were able to establish a new, stable and DOX resistant human UM cell line OCM3DOX320. Our results demonstrated the expression of splice variants and isoforms of receptor for LHRH in OCM3 UM cell line and its doxorubicin resistant form OCM3DOX320. It has been revealed by MTT assay that AN‐152 inhibited cell proliferation in a dose dependent manner in OCM3DOX320 cells. Furthermore, receptor‐mediated uptake of AN‐152 was demonstrated using confocal laser scanning microscopy in both cell line. Conclusions: Our results suggest that the antiproliferative effect of AN‐152 can be detected even if only LHRH receptor isoforms are expressed. Our study also demonstrates the LHRH receptor‐mediated uptake of AN‐152 in DOX resistant OCM3DOX320 cells. Our experiments provide new insights into a potential targeted therapy of UM and give further details about the accumulation of AN‐152 in hormone‐independent DOX‐resistant cells expressing splice variants of the LHRH receptors. Graphical abstract Figure. No Caption available.

Synthesis and in vitro biochemical evaluation of oxime bond-linked daunorubicin–GnRH-III conjugates developed for targeted drug delivery

Gonadotropin releasing hormone-III (GnRH-III), a native isoform of the human GnRH isolated from sea lamprey, specifically binds to GnRH receptors on cancer cells enabling its application as targeting moieties for anticancer drugs. Recently, we reported on the identification of a novel daunorubicin–GnRH-III conjugate (GnRH-III–[4Lys(Bu), 8Lys(Dau=Aoa)] with efficient in vitro and in vivo antitumor activity. To get a deeper insight into the mechanism of action of our lead compound, the cellular uptake was followed by confocal laser scanning microscopy. Hereby, the drug daunorubicin could be visualized in different subcellular compartments by following the localization of the drug in a time-dependent manner. Colocalization studies were carried out to prove the presence of the drug in lysosomes (early stage) and on its site of action (nuclei after 10 min). Additional flow cytometry studies demonstrated that the cellular uptake of the bioconjugate was inhibited in the presence of the competitive ligand triptorelin indicating a receptor-mediated pathway. For comparative purpose, six novel daunorubicin–GnRH-III bioconjugates have been synthesized and biochemically characterized in which 6Asp was replaced by D-Asp, D-Glu and D-Trp. In addition to the analysis of the in vitro cytostatic effect and cellular uptake, receptor binding studies with 125I-triptorelin as radiotracer and degradation of the GnRH-III conjugates in the presence of rat liver lysosomal homogenate have been performed. All derivatives showed high binding affinities to GnRH receptors and displayed in vitro cytostatic effects on HT-29 and MCF-7 cancer cells with IC50 values in a low micromolar range. Moreover, we found that the release of the active drug metabolite and the cellular uptake of the bioconjugates were strongly affected by the amino acid exchange which in turn had an impact on the antitumor activity of the bioconjugates.

Effect of preventive and curative fungicide treatment on Fusarium proliferatum infected maize — a field trial

There are extensive data on effects of antifungal agents on the plant pathogens, especially on Fusariums spp. species. However, investigations on the interaction of chemicals and the treated cultivars are rare. The aim of the study was to test two types of fungicide mixtures, azoxystrobin-propiconazole, and prothioconazole-tebuconazole, which are applied in wheat cultivars intensively, on six fodder maize hybrids that were infected with Fusarium proliferatum in the R1 growth stage in a field trial. The effect of the fungicide treatment was tested on the starch content and antifungal, antioxidant polyphenols of the kernels in the R3–R4 and R6 stage of the cultivars. The level of the fungal presence and the fumonisin concentration of the kernels were increased significantly under the artificial infection. The fumonisin concentration was variable at the R6 stage of the hybrid maize kernels. The treatment with prothioconazole and tebuconazole was found to be suitable when it was done before flowering, while the azoxystrobin-propiconazole treatments were equally successful before and after maize flowering considering the decreasing fumonisin concentration of the kernels. Both fungicide mixtures, when they were applied after maize flowering, affected the starch biosynthesis to the R3–R4 stage significantly. Meanwhile, azoxystrobin-propiconazole also significantly affected the antioxidant flavone/flavanol contents from the R3–R4 stage to the R6 stage.