Anticancer Effect of Cathelicidin LL-37, Protegrin PG-1, Nerve Growth Factor NGF, and Temozolomide: Impact on the Mitochondrial Metabolism, Clonogenic Potential, and Migration of Human U251 Glioma Cells.

Glioblastoma (GBM) is one of the most aggressive and lethal malignancy of the central nervous system. Temozolomide is the standard of care for gliomas, frequently results in resistance to drug and tumor recurrence. Therefore, further research is required for the development of effective drugs in order to guarantee specific treatments to succeed. The aim of current study was to investigate the effects of nerve growth factor (NGF), human cathelicidin (LL-37), protegrin-1 (PG-1), and temozolomide on bioenergetic function of mitochondria, clonogenicity, and migration of human U251 glioma cells. Colony formation assay was used to test the ability of the glioma cells to form colonies in vitro. The U251 glioma cells migration was evaluated using wound-healing assay. To study the mitochondrial metabolism in glioma cells we measured oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) using a Seahorse XF cell Mito stress test kit and Seahorse XF cell Glycolysis stress kit, respectively. We revealed that LL-37, NGF, and TMZ show strong anti-tumorigenic activity on GMB. LL-37 (4 µM), TMZ (155 µM), and NGF (7.55 × 10-3 µM) inhibited 43.9%-60.3%, 73.5%-81.3%, 66.2% the clonogenicity of glioma U251 cells for 1-2 days, respectively. LL-37 (4 µM), and NGF (7.55 × 10-3 µM) inhibited the migration of U251 glioma cells on the third and fourth days. TMZ also inhibited the migration of human glioma U251 cells over 1-3 days. In contrast, PG-1 (16 µM) stimulated the migration of U251 glioma cells on the second, fourth, and sixth days. Anti-mitogenic and anti-migration activities of NGF, LL-37, and TMZ maybe are relation to their capacity to reduce the basal OCR, ATP-synthetase, and maximal respiration of mitochondria in human glioma U251 cells. Glycolysis, glycolytic capacity and glycolytic spare in glioma U251 cells haven`t been changed under the effect of NGF, LL-37, PG-1, and TMZ in regard to control level. Thus, LL-37 and NGF inhibit migration and clonogenicity of U251 glioma cells, which may indicate that these compounds have anti-mitogenic and anti-migration effects on human glioma cells. The study of the mechanisms of these effects may contribute in the future to the use of NGF and LL-37 as therapeutic agents for gliomas.

In Vitro Evaluation of the Cytotoxic Effect of Streptococcus pyogenes Strains, Protegrin PG-1, Cathelicidin LL-37, Nerve Growth Factor and Chemotherapy on the C6 Glioma Cell Line.

Brain cancer treatment, where glioblastoma represents up to 50% of all CNS malignancies, is one of the most challenging calls for neurooncologists. The major driver of this study was a search for new approaches for the treatment of glioblastoma. We tested live S. pyogenes, cathelicidin family peptides and NGF, assessing the oncolytic activity of these compounds as monotherapy or in combination with chemotherapeutics. For cytotoxicity evaluation, we used the MTT assay, trypan blue assay and the xCELLigence system. To evaluate the safety of the studied therapeutic approaches, we performed experiments on normal human fibroblasts. Streptococci and peptides demonstrated high antitumor efficiency against glioma C6 cells in all assays applied, surpassing the effect of chemotherapeutics (doxorubicin, carboplatin, cisplatin, etoposide). A real-time cytotoxicity analysis showed that the cell viability index dropped to 21% 2-5 h after S. pyogenes strain exposure. It was shown that LL-37, PG-1 and NGF also exhibited strong antitumor effects on C6 glioma cells when applied at less than 10-4 M. Synergistic effects for combinations of PG-1 with carboplatin and LL-37 with etoposide were shown. Combinations of S. pyogenes strain #7 with NGF or LL-37 demonstrated a cytotoxic effect (56.7% and 57.3%, accordingly) on C6 glioma cells after 3 h of exposure.

The Phenomenon of Multidrug Resistance in Glioblastomas.

The most common and aggressive brain tumor in the adult population is glioblastoma (GBM). The lifespan of patients does not exceed 22 months. One of the reasons for the low effectiveness of GBM treatment is its radioresistance and chemoresistance. In the current review, we discuss the phenomenon of multidrug resistance of GBM in the context of the expression of ABC family transporter proteins and the mechanisms of proliferation, angiogenesis, and recurrence. We focused on the search of molecular targets among growth factors, receptors, signal transduction proteins, microRNAs, transcription factors, proto-oncogenes, tumor suppressor genes, and their single-nucleotide polymorphisms.

Related expression of TRKA and P75 receptors and the changing copy number of MYC-oncogenes determine the sensitivity of brain tumor cells to the treatment of the nerve growth factor in combination with cisplatin and temozolomide.

Objectives Oncological diseases are an urgent medical and social problem. The chemotherapy induces not only the death of the tumor cells but also contributes to the development of their multidrug resistance and death of the healthy cells and tissues. In this regard, the search for the new pharmacological substances with anticancer activity against drug-resistant tumors is of utmost importance. In the present study we primarily investigated the correlation between the expression of TrkA and p75 receptors with the nerve growth factor (NGF) and cisplatin or temozolomide sensitivity of anaplastic astrocytoma (AA), glioblastoma (GB) and medulloblastoma (MB) cell cultures. We then evaluated the changing of copy numbers of MYCC and MYCN and its correlation with cytotoxicity index (CI) in MB cells under NGF exposition. Methods The primary cell cultures were obtained from the tumor biopsy samples of the patients with AA (n=5), GB (n=7) or MB (n=25) prior to radiotherapy and chemotherapy. The cytotoxicity effect of NGF and its combinations with cisplatin or temozolomide, the relative expression of TrkA and p75 receptors, its correlations with CI in AA, GB and MB primary cell cultures were studied by trypan blue cytotoxicity assay and immunofluorescence staining respectively. The effect of NGF on MYCC and MYCN copy numbers in MB cell cultures was studied by fluorescence in situ hybridization. Results We found that the expression of TrkA and p75 receptors (p=0.03) and its ratio (p=0.0004) depends on the sensitivity of AA and GB cells to treatment with NGF and its combinations with cisplatin or temozolomide. NGF reduces (p<0.05) the quantity of MB cells with six or eight copies of MYCN and three or eight copies of MYCC. Besides, NGF increases (p<0.05) the quantity of MB cells containing two copies of both oncogenes. The negative correlation (r=-0.65, p<0.0001) is established between MYCC average copy numbers and CI of NGF in MB cells. Conclusions The relative expression of NGF receptors (TrkA/p75) and its correlation with CI of NGF and its combinations in AA and GB cells point to the mechanism involving a cell death signaling pathway. NGF downregulates (p<0.05) some increased copy numbers of MYCC and MYCN in the human MB cell cultures, and upregulates normal two copies of both oncogenes (p<0.05).

Related expression of TRKA and P75 receptors and the changing copy number of MYC-oncogenes determine the sensitivity of brain tumor cells to the treatment of the nerve growth factor in combination with cisplatin and temozolomide.

OBJECTIVES: Oncological diseases are an urgent medical and social problem. The chemotherapy induces not only the death of the tumor cells but also contributes to the development of their multidrug resistance and death of the healthy cells and tissues. In this regard, the search for the new pharmacological substances with anticancer activity against drug-resistant tumors is of utmost importance. In the present study we primarily investigated the correlation between the expression of TrkA and p75 receptors with the nerve growth factor (NGF) and cisplatin or temozolomide sensitivity of anaplastic astrocytoma (AA), glioblastoma (GB) and medulloblastoma (MB) cell cultures. We then evaluated the changing of copy numbers of MYCC and MYCN and its correlation with cytotoxicity index (CI) in MB cells under NGF exposition. METHODS: The primary cell cultures were obtained from the tumor biopsy samples of the patients with AA (n=5), GB (n=7) or MB (n=25) prior to radiotherapy and chemotherapy. The cytotoxicity effect of NGF and its combinations with cisplatin or temozolomide, the relative expression of TrkA and p75 receptors, its correlations with CI in AA, GB and MB primary cell cultures were studied by trypan blue cytotoxicity assay and immunofluorescence staining respectively. The effect of NGF on MYCC and MYCN copy numbers in MB cell cultures was studied by fluorescence in situ hybridization. RESULTS: We found that the expression of TrkA and p75 receptors (p=0.03) and its ratio (p=0.0004) depends on the sensitivity of AA and GB cells to treatment with NGF and its combinations with cisplatin or temozolomide. NGF reduces (p<0.05) the quantity of MB cells with six or eight copies of MYCN and three or eight copies of MYCC. Besides, NGF increases (p<0.05) the quantity of MB cells containing two copies of both oncogenes. The negative correlation (r=-0.65, p<0.0001) is established between MYCC average copy numbers and CI of NGF in MB cells. CONCLUSIONS: The relative expression of NGF receptors (TrkA/p75) and its correlation with CI of NGF and its combinations in AA and GB cells point to the mechanism involving a cell death signaling pathway. NGF downregulates (p<0.05) some increased copy numbers of MYCC and MYCN in the human MB cell cultures, and upregulates normal two copies of both oncogenes (p<0.05).

The spectrum of pathogenic variants of the ATP7B gene in Wilson disease in the Russian Federation.

BACKGROUND: Wilson's disease (WD) is a rare inherited disorder caused by mutations in the ATP7B gene resulting in copper accumulation in different organs. However, data on ATP7B mutation spectrum in Russia and worldwide are insufficient and contradictory. The objective of the present study was estimation of the frequency of ATP7B gene mutations in the Russian population of WD patients. MATERIALS AND METHODS: 75 WDpatients were examined by next-generation sequencing (NGS). A targeted panel NimbleGen SeqCap EZ Choice: 151012_HG38_CysFib_EZ_HX3 (ROCHE)was designed for analysis of ATP7B gene and possible modifier genes. Retrospective assessment of a diagnostic WD score (Leipzig, 2001) was also performed. RESULTS: 31 mutations in ATP7B gene were detected. Two most frequent mutations were c.3207C > A (51,85% of alleles) and c.3190 G > A (8,64% of alleles). Single rare mutations were detected in 29% of cases. In 96% cases mutations of both copies of the ATP7B were revealed. We also observed 3 novel potentially pathogenic variants which were not previously described (c.1870-8A > G, c.3655A > T (p.Ile1219Phe), c.3036dupC (p.Lys1013fs). For 25% of patients at the time of the manifestation the diagnosis WD could not be established using the earlier proposed diagnostic score. There was a remarkable delay in diagnosis for the majority of patients. Only 33% of patients WD was diagnosed in three months after the first symptoms, 29%patients - in 3-12 months, 30% - in 1-10 years, in 8% - more than 10 years. Generally, clinical appearance of WD may be rather variable at manifestation and genetic profiling at this step is the only way to confirm the presence of WD.

X-linked and autosomal dominant forms of the ichthyosis in coinheritance.

According to modern classification, there are two forms of inherited ichthyoses: syndromic and non-syndromic, each of them consists of more than ten different nosologies. The commonest types of the ichthyosis are X-linked recessive (prevalence 1/2000-6000 in men) and autosomal dominant, or ichthyosis vulgaris with incomplete penetrance (1/250-1000). The X-linked form is associated with mutations in steroid sulfatase STS gene, it is noteworthy that there is a full deletion of the gene in 90% of cases. Ichthyosis vulgaris is caused by heterozygous mutations in the FLG gene encoding filaggrin. It is important to note that the clinical forms of these diseases are indistinguishable. The aim of this study was to search for pathogenic or likely pathogenic mutations which are associated with various forms of the inherited ichthyosis such as other inherited diseases with similar phenotypic signs. The sequencing was done on a HiSeq 4000 sequencer (Illumina) by paired-end reading (2 × 150 bp). The identified mutation p.Arg2037Ter in the heterozygous condition has been described before in databases as being pathogenic. Also, our patient has a full deletion of the STS gene and it was found that our patient carries two pathogenic mutations which are related to different forms of the inherited ichthyosis.