COMPARISON OF 2D AND 3D PRIMARY CELL CULTURES OBTAINED FROM EXPLANT OF HIGH-GRADE UROTHELIAL BLADDER CANCER.

Studying the biological characteristics of bladder cancer in primary culture can be an effective way for diagnostic and prognostic purposes, as well as choosing a scheme for personalized therapy. AIM: To characterize and compare 2D and 3D primary cell cultures obtained from the same tumor sample resected from a patient with high-grade bladder cancer. MATERIALS AND METHODS: 2D and 3D primary cell cultures were obtained from explants of resected bladder cancer. Glucose metabolism, lactate dehydrogenase (LDH) activity, and level of apoptosis were studied. RESULTS: Multicellular tumor spheroids (3D) differ from planar culture (2D) by more pronounced consumption of glucose from the culture medium (1.7 times higher than 2D on Day 3 of culture), increased lactate dehydrogenase activity (2.5 times higher on Day 3 vs. Day 1 of cultivation, while in 2D culture LDH activity is constant), stronger acidification of the extracellular environment (pH dropped by 1 in 3D and by 0.5 in 2D). Spheroids demonstrate enhanced resistance to apoptosis (1.4 times higher). CONCLUSION: This methodological technique can be used both for tumor characterization and for selection of optimal postoperative chemotherapeutic schemes.

Expression of TLR4 and major inflammatory cytokines in patients with bladder cancer of different grade and stage.

BACKGROUND: The bladder cancer is immunogenic, and neoantigens generated by tumor cells trigger a notable immune response in the host. On the other hand, multiple immune escape mechanisms allow for avoiding the recognition by the host immune system. Toll-like receptor type 4 and inflammatory cytokines play major role in the immune response to bladder cancer. AIM: To assess the expression of TLR4 and the genes of major inflammatory cytokines in tumor cells and in unaffected tissue of the bladder. MATERIALS AND METHODS: The pairs of samples from the urinary bladder tumor and unaffected adjacent tissue were obtained from 50 surgically treated patients with bladder cancer. The level of expression of TLR4, TGF-ß1, INF-γ, TNF-α genes was evaluated by real-time polymerase chain reaction. RESULTS: Bladder cancer cells are characterized by lower expression levels of TLR4, TGF-ß1, INF-γ, TNF-α as compared to unaffected tissue. In patients with recurrent cancer, expression of TLR-4 and cytokines does not change both in tumor and in unaffected tissue of the bladder. Expression of TLR4 is identically low both in low- and high-grade cancer. Expression levels of the INF-γ and TNF-α are remarkably low in muscle-invasive cancer compared to the unaffected bladder tissue. The level of TGF-ß1 in bladder cancer is comparable to the unaffected tissue of the bladder, while in the intact and metastatic lymph nodes it is significantly upregulated. CONCLUSION: Bladder cancer tissue differs from the unaffected part of the bladder wall in the level of TLR4, TGF-ß1, INF-γ, TNF-α expression.

Changes in expression of TLR-4, TGF-ß, INF-γ, TNF-α in cultured T24/83 cells of invasive bladder cancer treated with cisplatin and/or polyphenolic adjuvant melanin.

BACKGROUND: Toll-like receptor 4 (TLR4) is known to be involved in carcinogenesis and cancer progression. Changes in TLR4 expression are associated with changes in the expression of key cellular cytokines (transforming growth factor-ß (TGF-ß), tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ)), which affect cancer progression and metastasis. AIM: To study changes in the expression of TLR4, TGF-ß, TNF-α, IFN-γ genes, the level of apoptosis and cell cycle distribution in human invasive urothelial carcinoma T24/83 cells under the treatment with polyphenolic adjuvant compound of fungal origin melanin, cytotoxic drug cisplatin, and combination of both. MATERIALS AND METHODS: T24/83 cells were incubated with cisplatin (0.05 mM), melanin (5 µg/ml), or their combination. The expression level of TLR-4, TGF-ß, INF-γ, TNF-α was evaluated by the real time polymerase chain reaction. The flow cytometry was used to study cell cycle distribution, proliferative activity and level of apoptosis. Morphological analysis of the Т24/83 cells was performed as well. RESULTS: Melanin, cisplatin, and their combination downregulate TLR4 expression (2.67; 1.28; and 2.73-fold decrease, respectively) and TNF-α expression (6.5; 1.4; and 1.7-fold decrease, respectively). Melanin did not affect TGF-ß expression while cisplatin caused 13-fold downregulation of TGF-ß. The combined use of cisplatin and melanin decreased TGF-ß expression by 6.5 times. The upregulation of IFN-γ by melanin, cisplatin, and their combination was demonstrated (4.3; 6.7; and 2-fold increase, respectively). All treatment modalities increased the level of apoptosis in T24/83 cells. Melanin treatment increased significantly the proportion of fibroblast-like cells in T24/83 culture with decreased cell adhesion to the substrate. CONCLUSIONS: Melanin, cisplatin, and combination of both agents affect significantly TLR4, TNF-α, TGF-ß, INF-γ expression, cell cycle distribution and morphology in T24/83 cells suggesting their transition to less aggressive phenotype.

The effect of perioperative analgesic drugs omnopon and dexketoprofen on the functional activity of immune cells in murine model of tumor surgery.

We aimed to investigate the effect of perioperative analgesia with nonselective cyclooxygenase-2 inhibitor dexketoprofen and opioid drug omnopon on the functional activity of immune cells in tumor excision murine model. Lewis lung carcinoma cells were transplanted into hind paw of C57/black mice. On the 23th day tumor was removed. Analgesic drugs were injected 30 min before and once a day for 3 days after the surgery. Biological material was obtained a day before, 1 day and 3 days after the tumor removal. IFN-γ, IL-4, IL-10 and TGF-ß mRNA levels in splenic cells were assessed by quantitative real-time RT-PCR. Cytotoxic activity of splenocytes was estimated by flow cytometry. We found that in splenocytes of mice received opioid analgesia IL-10 mRNA level was increased 2.3 times on day one after the surgery compared to preoperative level (P < 0.05), while in dexketoprofen group this parameter did not change. IFN-γ gene expression level on day 3 after tumor removal was 40% higher in splenocytes of dexketoprofen treated mice as compared with omnopon treated animals (P < 0.05). Cytotoxic activity of splenocytes on day 3 postsurgery was (62.2 ± 2.4)% in dexketoprofen against (50.2 ± 3.3)% in omnopon group. In conclusion, perioperative analgesia with cyclooxygenase inhibitor dexketoprofen in contrast to opioid analgesia with omnopon preserves higher functional activity of murine immune cells in the experimental model of tumor surgery.

Prognostic significance of MDM2 gene expression in childhood neuroblastoma.

AIM: To investigate the association of MDM2 expression at the mRNA levels in neuroblastoma with clinical features and unfavorable disease factors to determine the possibility of it usage as a prognostic marker of neuroblastoma. MATERIALS AND METHODS: Total RNA and DNA were extracted from tumor tissue samples of total 91 neuroblastoma patients (mean age: 39.45 ± 4.81 months). MDM2 mRNA levels were detected with Q-PCR. TP53 gene deletion was detected with FISH method. MYCN amplification was detected with -Q-PCR analysis in fresh tumor samples and FISH in FFPE samples. RESULTS: We investigated the association of MDM2 mRNA expression with clinical outcome in neuroblastoma patients (n = 91). Kaplan - Meier curves showed a significant association of high MDM2 expression with poor event-free survival (p < 0.001). Clinical outcome of patients without MYCN amplification with low MDM2 expression was associated with better event-free survival than with high MDM2 expression (p < 0.001). Overexpression of MDM2 can be used as significant prognostic marker for patient stratification on risk groups and treatment optimization. CONCLUSION: Our results showed that the high expression of MDM2 at mRNA levels is an important factor of neuroblastoma prognosis. It may be a valuable additional molecular marker in guiding specific therapy in MYCN non-amplified NB patients without TP53 gene deletion.

Effect of Pd(II) and Ni(II) coordination compounds with 4-amino-3-mercapto-5-methyl-1,2,4-triazole on the mitochondrial dehydrogenases activity.

Pd(I) and Ni(II) complex compounds: [Pd(AMMT)2]Cl2 (1), [Pd(AMMT)4]Cl2 (2) and [Ni(AMMT)2(H2O)](NO3)2 (3) with 4-amino-3-mercapto-5-methyl-1,2,4-triazole (AMMT) have been synthesized. The spectral characteristics of 1, 2 were studied by 1H (13C) NMR and UV-Vis spectroscopy. X-ray diffraction studies established that all complexes contain the AMMT molecule, which are coordinated to the central metal ion in the thione tautomeric form. At the ratio M: L = 1:2 ligand is coordinated in bidentate chelate manner by the nitrogen of amino- and sulfur of mercapto group (compounds 1, 3). But the molar ratio M: L = 1:4 leads to monodentate coordination of AMMT molecules only by sulfur of mercaptogroup (complex 2). Vacant coordination sites of the metal ion are occupied by water molecules (complex 3). The screening of complexes 1-3 and starting compounds [AMMT, K2PdCl4 (4), Ni(NO3)2 · 6H2O (5)] by their mitochondrial dehydrogenase activity have been performed by us for the first time, resulting in established that the Pd(II) complexes (1, 2), Pd(II) salt (4) and AMMT normalize the activity of mitochondrial dehydrogenases of cancer HeLa cells, identified by MTT-test. In contrast, the Ni(II) complex (3) and Ni(II) salt (5) do not stimulate the activity of mitochondrial dehydrogenases. It has been found, that all investigated compounds do not affect on the cell cycle and the level of apoptotic cells as well as do not show a toxic effect. Thus, these results indicate that AMMT and Pd(II) complexes may be used as modifiers of mitochondrial respiration, which dysfunction is particularly evident in the tumor cells.

Biologic activities of recombinant human-beta-defensin-4 toward cultured human cancer cells.

AIM: The aim of the study was in vitro analysis of biological activity of recombinant human beta-defensin-4 (rec-hBD-4). METHODS: hBD-4 cDNA was cloned into pGEX-2T vector, and recombinant plasmid was transformed into E. coli BL21(DE3) cells. To purify soluble fusion GST-hBD-4 protein, affinity chromatography was applied. Rec-hBD-4 was cleaved from the fusion protein with thrombin, and purified by reverse phase chromatography on Sep-Pack C18. Effects of rec-hBD-4 on proliferation, viability, cell cycle distribution, substrate-independent growth, and mobility of cultured human cancer cells of A431, A549, and TPC-1 lines were analyzed by direct cell counting technique, MTT assay, flow cytofluorometry, colony forming assay in semi-soft medium, and wound healing assay. RESULTS: Rec-hBD-4 was expressed in bacterial cells as GST-hBD-4 fusion protein, and purified by routine 3-step procedure (affine chromatography on glutathione-agarose, cleavage of fusion protein by thrombin, and reverse phase chromatography). Analysis of in vitro activity of rec-hBD-4 toward three human cancer cell lines has demonstrated that the defensin is capable to affect cell behaviour in concentration-dependent manner. In 1-100 nM concentrations rec-hBD-4 significantly stimulates cancer cell proliferation and viability, and promotes cell cycle progression through G2/M checkpoint, greatly enhances colony-forming activity and mobility of the cells. Treatment of the cells with 500 nM of rec-hBD-4 resulted in opposite effects: significant suppression of cell proliferation and viability, blockage of cell cycle in G1/S checkpoint, significant inhibition of cell migration and colony forming activity. CONCLUSION: Recombinant human beta-defensin-4 is biologically active peptide capable to cause oppositely directed effects toward biologic features of cancer cells in vitro dependent on its concentration.

Immunological markers of anti-tumor dendritic cells vaccine efficiency in patients with non-small cell lung cancer.

AIM: To investigate the quantitative and functional status of peripheral blood lymphocytes in patients with non-small cell lung cancer during DC-vaccine therapy and identify the most informative immunological parameters which are associated with clinical outcome. MATERIALS AND METHODS: The study was conducted within the framework of randomized phase III clinical trial of DC-vaccine efficacy in patients with non-small cell lung cancer. Quantitative composition of peripheral blood lymphocytes was determined by flow cytometry. Cytokines mRNA expression level was estimated using real-time RT-PCR. RESULTS: In our study the most pronounced changes in the immune system have been defined after fourth DC-vaccine injection. Immunologic features such as reduction the MIP-1α mRNA expression level, increasing the RANTES mRNA expression level and NK-cells count, retention CD4/CD8 ratio at physiological level were associated with favorable clinical outcome after DC-immunotherapy. CONCLUSIONS: Immunological markers established in our investigation can be used for estimation of DC-immunotherapy efficiency. The results of our research are very promising, but these data should be confirmed in further studies with a large cohort of patients.

[About the impact of the dendritic cell autovaccine on the results of treatment of non-small-cell lung cancer patients].

In thoracic department of the National Cancer Institute studied the effectiveness of dendritic cell autovaccine in the postoperative period in non-small-cell lung cancer patients. The results, showing good tolerance dendritic cell autovaccine. Shows the formation of the expressed antigen immune response after repeated injections dendritic cell autovaccine, as manifested after 4 revaccination. Results of survival patients non-small-cell lung cancer who received postoperative dendritic cell autovaccines demonstrate the high efficiency of the method and its applicability with a minimum of side effects. Further study of survival of patients non-small-cell lung cancer who received immunotherapy treatment, monitoring of compliance with the best mode of repeated injections.

[Effect of dendritic cell autovaccine on the treatment outcome in patients with ovarian cancer].

During I/II phase clinical trial in ovarian cancer (OC) patients two types of autologous anticancer vaccines based on dendritic cells have been tested, and a comparative analysis of their effectiveness have been performed. It was shown that the anticancer vaccines based on DC, "loaded" with autologous tumor cell lysate obtained by treatment of tumor cells by cytotoxic lectins B. subtilis had higher efficiency, compared with the standard DC--autovaccine. The presence of antigen-specific immune response observed after at least four vaccinations. Obtained results open the prospects to improve the basic treatment of OC patients by this method. The results of immunological examinations create preconditions for individual optimization of the DC-vaccine therapy.