ANTICANCER IMMUNOGENIC POTENTIAL OF ONCOLYTIC PEPTIDES: RECENT ADVANCES AND NEW PROSPECTS.

Oncolytic peptides are derived from natural host defense peptides/antimicrobial peptides produced in a wide variety of life forms. Over the past two decades, they have attracted much attention in both basic research and clinical applications. Oncolytic peptides were expected to act primarily on tumor cells and also trigger the immunogenic cell death. Their ability in the tumor microenvironment remodeling and potentiating the anticancer immunity has long been ignored. Despite the promising results, clinical application of oncolytic peptides is still hindered by their unsatisfactory bioactivity and toxicity to normal cells. To ensure safer therapy, various approaches are being developed. The idea of the Ukrainian research group was to equip peptide molecules with a "molecular photoswitch" - a diarylethene fragment capable of photoisomerization, allowing for the localized photoactivation of peptides within tumors reducing side effects. Such oncolytic peptides that may induce the membrane lysis-mediated cancer cell death and subsequent anticancer immune responses in combination with the low toxicity to normal cells have provided a new paradigm for cancer therapy. This review gives an overview of the broad effects and perspectives of oncolytic peptides in anticancer immunity highlighting the potential issues related to the use of oncolytic peptides in cancer immunotherapy. We summarize the current status of research on peptide-based tumor immunotherapy in combination with other therapies including immune checkpoint inhibitors, chemotherapy, and targeted therapy.

[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.

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.

DENDRITIC CELLS IN GLIOBLASTOMA TREATMENT: A MODERN VIEW OF THE PROBLEM AND OWN EXPERIENCE.

Glioblastoma (GBM) is the most aggressive primary malignant brain tumor in adults. The improvement of the efficacy of GBM treatment is an urgent problem encouraging the development of novel therapeutic strategies, in particular, immunotherapeutic modalities. With more understanding of the intimate interrelationships between the immune system and the mechanisms involved in cancer origin and progression, the skepticism related to the relevance of the immunotherapeutic approaches in the treatment of brain tumors is gradually decreasing. The review discloses the modern concepts on the association between CNS and the immune system. For a long time, CNS was considered as the immunoprivileged site that prevents the effects of immunotherapy in the treatment of brain tumors. Nowadays, these views are reconsidered, which opens the way to the use of immunotherapeutic approaches in GBM treatment. The results of the recent clinical trials on immunotherapy as a supplement to the conventional GBM treatment are considered. Vaccines based on the dendritic cell (DC) technology are regarded as the most promising for this purpose. The preliminary results of the Ukrainian clinical study are also presented and discussed. The results of the international clinical trials as well as our own experience give evidence of the relevance for using DC vaccines in the complex treatment of GBM, which is supported by the increased survival of patients and the safety of vaccine application. It is of high importance that GBM patients with the most unfavorable prognosis can benefit from DC vaccines as a component of the complex treatment. The prospects for immunotherapy in neurooncology are discussed.

[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.

EXPRESSION OF GENES INVOLVED IN P53 PATHWAY REGULATION IN NEUROBLASTOMA: A SHORT REVIEW.

The search for new prognostic and stratification genetic and epigenetic markers in neuroblastoma is an urgent problem in pediatric oncology. The review summarizes recent progress in studying the expression of genes involved in p53 pathway regulation in neuroblastoma. Several markers associated with recurrence risk and poor outcome are considered. Among them are MYCN amplification, high MDM2 and GSTP1 expression and homozygous mutant allele variant of GSTP1 gene A313G polymorphism. Prognostic criteria for neuroblastoma based on the analysis of miR-34a, miR-137, miR-380-5p, and miR-885-5p expression involved in regulating p53-mediated pathway are also considered. The authors' research data on the role of the above markers in regulation of this pathway in neuroblastoma are presented. The study of alterations in expression of microRNAs and genes involved in p53 pathway regulation will not only expand our understanding of the mechanisms of neuroblastoma pathogenesis but could substantiate new approaches for delineating risk groups and risk stratification of neuroblastoma patients as well as treatment optimization based on the genetic characteristics of the tumor.

Magnetically sensitive nanocomplex enhances antitumor efficacy of dendritic cell-based immunotherapy.

BACKGROUND: One of the major factors restricting in vivo efficacy of dendritic cells (DCs) based immunotherapy is the inefficient migration of these cells to the lymphoid tissue, wherein DCs activate antigen-specific T cells. A fundamentally new approach for the possibility of enhancing the antitumor effects of DC-based immunotherapy may be the use of magnetically sensitive nanocomplexes to increase the target delivery of DCs to the lymph nodes of the recipient. AIM: To study the antitumor and immunomodulatory effects of the DC-nanovaccine with magnetosensitive properties and its influence on the immunosuppressive tumor microenvironment in mice with sarcoma 37. MATERIALS AND METHODS: The antitumor, antimetastatic and immunomodulatory effects of DCs loaded with magnetic nanocomplex under magnetic field (MF) control in mice with sarcoma 37 have been investigated. RESULTS: Combined therapy contributed to a significant reduction in tumor volume and weight compared to the control group of mice and mice that received the DC vaccine without MF. Therapy with magnetically sensitive DC nanovaccine with and without the addition of the MF was accompanied by a significant down-regulation of the level of FoxP3, transforming growth factor ß, interleukin (IL)-10 and vascular endothelial growth factors, mRNA expression in tumor tissues. A significant increase in interferon-γ and IL-4 mRNA expression was found in mice treated with the magnetically sensitive DC nanovaccine under MF control. CONCLUSION: A significant increase in the antitumor efficacy of the DC vaccine can be achieved using magnetosensitive nanocarriers of tumor antigens under MF control.

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.

Prevalence of two BRCA1 mutations, 5382insC and 300T > G, in ovarian cancer patients from Ukraine.

Ovarian cancer is the seventh most common cancer in women worldwide and the leading cause of gynecological malignant diseases-related deaths in women. The most significant risk factor for ovarian cancer is an inherited genetic mutation in one of two genes: breast cancer gene 1 (BRCA1) or breast cancer gene 2 (BRCA2). The germline mutation c.5266dupC (also known as 5382insC or 5385insC) is the most common mutation among Slavic patients with breast and/or ovarian cancer. Missense mutation c.181T > G (also known as 300T > G or p.C61G) is regarded as the founder change in many Central European countries. We screened 306 ovarian cancer patients diagnosed at different ages by mutagenically separated polymerase chain reaction (PCR) and real-time PCR. A total of 25 BRCA1 mutations were detected (18 cases of 5382insC and 7 cases of 300 T > G). The frequency of the BRCA1 5382insC mutation is similar in breast and ovarian cancer patients from Ukraine, but the frequency of 300T > G was estimated in Ukraine at first time.

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.

Involvement of human beta-defensin-2 in regulation of malignant potential of cultured human melanoma cells.

BACKGROUND AND AIM: Human beta-defensin-2 (hBD-2) is an antimicrobial cationic peptide capable to control human carcinoma cell growth via cell cycle regulation. The present study was aimed on determination of hBD-2 influence on the growth patterns and malignant potential of cultured human melanoma cells. METHODS: The study was performed on cultured human melanoma cells of mel Z and mel Is lines treated with recombinant hBD-2 (rec-hBD-2); cell viability, proliferation, cell cycle distribution, and anchorage-independent growth were analyzed using MTT test, direct cell counting, flow cytometry, and colony forming assay respectively. Expression and/or phosphorylation levels of proteins involved in cell cycle control were evaluated by Western blotting. RESULTS: The treatment of mel Z and mel Is cells with rec-hBD-2 in a concentration range of 100-1000 nM resulted in a concentration-dependent suppression of cell proliferation, viability, and colony forming activity. It has been shown that rec-hBD-2 exerts its growth suppression effects via significant downregulation of B-Raf expression, activation of pRB and upregulation of p21(WAF1) expression, downregulation of cyclin D1 and cyclin E resulting in cell cycle arrest at G1/S checkpoint. CONCLUSION: According to obtained results, hBD-2 exerts its growth suppression effect toward human melanoma cells via downregulation of B-Raf, cyclin D1 and cyclin E expression, upregulation of p21(WAF1) expression and activation of pRB.

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.