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Tumor markers play an important role in the identification of human malignancies. It has been shown that the carcinoembryonic antigen (CEA, CEACAM5) is a promoter of metastasis in epithelial cancers that is widely used as a clinical marker. The aim of this study is to elucidate the network of genes that are involved in the CEA-induced liver metastasis. Previously, we have shown that CEA is accumulated in the lungs and livers of rats by interacting with their macrophages. We identified and cloned a new gene (CEAR) for the CEA-binding protein, which is located on the surface of fixed liver macrophages, Kupffer cells (Bajenova et al, 2001). It has been shown that the interaction of CEA and CEAR proteins increases the production of IL-1, IL-10, IL-6, TNF-α cytokines (Thomas et al, 2011). This interaction changes the expression of liver adhesion molecules that enhances the survival of cancer cells to the liver. We also suggested that CEA synthesis by cancer cells may influence the E-cadherin adhesion junction complexes and have shown that CEA production violates the functional relationship between Ecadherin and its partners α-, β- and p120 catenin. A new type of interaction was discovered between the CEA and β-catenin and the increased amount of β-catenin in the nuclei of CEA producing cells. The data show that CEA production can cause the dissociation of cancer cells and trigger cancer progression. The CEA synthesis also alters splicing of p120 catenin protein and causes the release of soluble E-cadherin. Previously, CEA and epithelial E-cadherin were considered as independent tumor markers. Our data explain the correlation between the elevated levels of CEA and the increase in soluble E-cadherin in the progression of colorectal cancer (Bajenova et al, 2014).

We carried out a comparative transcriptome analysis of CEA-producing cell lines. The RNA transcriptome libraries were obtained and sequenced. By pairwise comparisons of CEA producing and non-producing cell lines using Cummerband program, we selected the set of genes (90 total genes) whose expression have been changed in the CEA-producing cell lines (overexpressed or downregulated). The biological processes that are linked to this differential gene expression were identified by Gene Set Enrichment Analysis (GSEA). In total, 8 significantly enriched GO terms related to the cellular components and biological processes were identified. Using KEGG and GO databases, we also identified the signaling pathways involved in the response to CEA. These findings have direct medical application, since they allow not only to establish the relationships between the existing biomarkers but also to discover the new ones. These biomarkers can be used for diagnosis and monitoring of metastatic carcinomas and for the drug development.

Acute myelomonocytic leukemia (FAB M4) is one of the most common forms of acute myeloid leukemia (AML). This AML form is characterized by rapid accumulation transformed myeloblasts and monoblasts in bone marrow, with the rapid suppression of normal hematopoiesis. Bone marrow microenvironment is one of the main factors determining drug resistance of leukemic cells. It is known that the adhesion of leukemic cells to mesenchymal stem cell and bone marrow extracellular matrix (laminin, collagen) enhances their drug resistance. However, it remains unknown whether the emergence of drug resistance when cell–cell contacts are formed only between leukemia cells, without the involvement of bone marrow stromal elements. We studied the role of cell aggregation in drug resistance of leukemic cells. We used the bone marrow mononuclear cells (BMMC) isolated from the patients with acute myelomonocytic leukemia. For the formation of multicellular aggregates, BMMC were cultivated in 96-well plates coated with 1.5% agarose. We showed that resistance of BMMC to bortezomib, doxorubicin and fludarabine in multicellular aggregates was increased. In three-dimensional multicellular aggregates of BMMC index IC50 for bortezomib, doxorubicin and fludarabine was 7 ± 1 ng/ml, 1 ± 0.4 mkM and 0.8 ± 0.05 mkM, respectively. In control condition, index IC50 bortezomib, doxorubicin and fludarabine was significantly lower, 2 ± 0.5 ng/ml, 0.3 ± 0.05 mkM and 0.07 ± 0.001 mkM, respectively. In multicellular aggregates of BMMC number of mitotic cells and expression of Ki-67 protein were not significantly different from the control. It has also been shown that cells in multicellular aggregates increased expression antiapoptotic protein Bcl-2. Suppression of BMMC aggregation by culturing the cells in medium containing 0.9% methylcellulose resulted in decreased IC50 index for bortezomib, doxorubicin and fludarabine, 2 ± 0.7 ng/ml, 0.12 ± 0.004 mkM and 0.04 ± 0.005 mkM, respectively. Expression of the Bcl-2 protein was also decreased. This work demonstrates the involvement of cell aggregation in the formation of drug resistance phenotype in leukemic cells.

The work was supported by the Russian Foundation for Basic Research (Russia) (No. 14-04-32183, 14- 04-32191), the scholarship of the President of the Russian Federation (Russia) (No. SP-6867.2013.4, SP-1519.2015.4), and by the Government of the Russian Federation (Russia) (No.14.Z50.31.0028).

Background

Metformin is a antidiabetic drug with anticancer properties. However, the mechanism action by which metformin affects various cancer cells still unknown. It is known that tumor growth is accompanied by changes in the metabolic cascade that includes overproduction of lactate and adenosine. The adenosine is released into the extracellular environment and regulates differentiation, proliferation, and angiogenesis of tumor mass. We found that lactate is activator of key enzyme of adenosine metabolism – adenosine deaminase (ADA).

Aim

The aim of our study was to investigate the catabolism of adenosine in the tumor while taking metformin.

Materials and methods

In this study we investigated the level of adenosine, inosine, hypoxanthine and ADA activity in 15 women aged 46–76 years, with breast cancer (BC) T2-4N1M0 (cancer tissues) during treatment with metformin, 1000 mg per day for 3 months. Control group – 15 women aged 46–76 years, with stage T2-4N1M0 breast cancer (cancer tissues) without metformin therapy.

Statistical analysis was performed using the license package StatSoft. Statistica 12.0.

Results

ADA activity during treatment with metformin was 2-fold increased: 12.1 ± 2.49 nmol/min*mg in comparison with 4.77 ± 0.943 nmol/min*mg. Concentration of catabolic products of adenosine degradation was increased before metformin therapy. Inosine level was 0.121 ± 0.041 micro mol/g tissue (BC tissues from women without metformin 0.042 ± 0.015 micro mol/g tissue). Hypoxanthine 2.45 ± 0.428 micro mol/g tissue (in comparison with 0.711 ± 0.269 micro mol/g tissue). Whereas, adenosine level in BC after metformin therapy was 0.226 ± 0.148 micro mol/g tissue (in comparison with 0.186 ± 0.056 micro mol/g tissue), that were not significantly different.

Conclusion

Thus, we have found that metformin significantly increases the rate of catabolism of adenosine, and this in turn reduces the inhibitory effect on the tumor microenvironment cytotoxic cells. Therefore, our data for the first time provide novel evidence for a mechanism that the anticancer activities of metformin are due to adenosine metabolism regulation.