Development of an algorithm for biomedical image analysis of the spatial organization of collagen in breast cancer tissue of patients with different clinical status.

Collagen, the main component of the tumor microenvironment, plays a key role in the development of breast cancer (BCa); however, the specific changes in its spatial organization during tumor progression have not been definitively elucidated. The existing and available methods for assessing the morphometric parameters of the stroma's fibrous component are insufficient for a detailed description of the state of collagen fibers and for assessing its changes to evaluate the aggressiveness of the BCa course. The aim of the work was to develop an algorithm for microphoto analysis to assess the spatial organization of collagen in BCa tissue of patients with different clinical statuses. The study was conducted on 60 tissue samples of stage I-II BCa. The processed images were analyzed using the software packages CurveAlign v4.0 and imagej. We established that the increase in BCa stage and the decrease in tumor differentiation grade are associated with decreased length, width, and straightness of collagen fibers, as well as their increased density. The formation of an aggressive basal molecular BCa subtype was accompanied by an increase in tumor-stroma ratio. The obtained results indicate the possibility of practical application of the developed algorithm for evaluating the spatial organization of collagen in BCa tissue to predict the aggressiveness of the disease course.

Osteonectin (SPARC) prognostic value in prostate cancer.

BACKGROUND: Prostate cancer (PCa) is common malignancy among men worldwide. To date only few molecular markers are available to predict its course and outcome. SPARC is considered to be promising prognostic marker of PCa due to its involvement in various cancer processes. MATERIALS AND METHODS: study was conducted on PCa surgical primary tumor samples, obtained from 84 patients. Level of SPARC mRNA expression was estimated using RT-qPCR. To identify SPARC protein (osteonectin) in prostate tissue, immunohistochemical analysis was conducted. Bioinformatical analysis was performed on UALCAN and TNMplot resources. RESULTS: bioinformatical analysis demonstrated that SPARC mRNA levels are decreased in PCa samples, in comparison to normal tissue. In patients with lymph node metastases its levels are 1.26 times higher; p = 4.66E-02, than in N0 category. Ex vivo study demonstrated that SPARC expression was elevated on both mRNA and protein levels in PCa patients with lymph node metastases (by 2.34 and 1.91, respectively, p < 0.05). We established higher levels of SPARC mRNA and protein in PCa patients with T3 tumors, as well as high Gleason score. Estimation of survival rates demonstrated that PCa patients with a high level of SPARC mRNA and protein have decreased overall 2-year survival. CONCLUSIONS: SPARC protein was overexpressed on mRNA and protein levels in patients with presence of lymph node metastases and higher Gleason score of tumors. Also, both mRNA and protein upregulation were associated with worse survival rates. The current study has therefore provided further evidence that SPARC is indeed linked to the prognosis and aggressiveness of human PCa.

Does insulin make breast cancer cells resistant to doxorubicin toxicity?

The effects of insulin on the doxorubicin (Dox) sensitivity of breast cancer cell line MCF-7 and its Dox-resistant counterpart MCF-7/Dox were studied and glucose metabolism, content of essential minerals, and the expression of several microRNAs in these cells upon exposure to insulin and Dox were compared. Cell viability colorimetric assay, colorimetric enzymatic technique, flow cytometry, immunocytochemical techniques, inductively-coupled plasma atomic emission spectroscopy, and quantitative polymerase chain reaction were used in the study. We found that insulin in high concentration significantly suppressed Dox toxicity, especially in parental MCF-7 cell line. The increase in proliferative activity triggered by insulin in MCF-7 but not MCF-7/Dox cells occurred in the setting of the increased level of specific binding sites for insulin and increased glucose uptake. Insulin treatment of MCF-7 cells in low and high concentrations resulted in the increase of Mg, Ca, and Zn content while in DOX-resistant cells, only Mg content increased upon exposure to insulin. High concentration of insulin increased the expression of kinase Akt1, P-glycoprotein 1 (P-gp1) and DNA excision repair protein ERCC-1 in MCF-7 cells, while in MCF-7/Dox cells, Akt1 expression decreased, and cytoplasmic expression of P-gp1 increased. In addition, insulin treatment affected expression of miR-122-5p, miR-133a-3p, miR-200b-3p, and miR-320a-3p. The decreased manifestation of biological effects of insulin in Dox-resistant cells could be partly explained by the different patterns of energy metabolism in MCF-7 cells and their Dox-resistant counterpart.

Benign and malignant prostate neoplasms show different spatial organization of collagen.

AIM: To compare the indicators of the spatial organization of collagen and its regulating factors between benign and malignant prostate neoplasms. METHODS: The study involved tumor tissue samples from 40 patients with stage II-III prostate cancer (PCa) and 20 patients with benign prostatic hyperplasia (BPH). The localization of collagen was determined with a Masson trichrome stain. To establish quantitative indicators of the spatial organization of collagen, morphometric studies were carried out with the CurveAlign and ImageJ programs. RESULTS: PCa tissue had two times lower collagen density (P<0.0001) and 1.3 times lower levels of collagen alignment (P=0.018) compared with BPH tissue. In PCa tissue, collagen fibers were shorter (by 24.2%; P<0.001) and thicker (by 15.5%; P<0.001). PCa tissue samples showed significantly higher levels of metalloproteinase (MMP)-2 (by 2.4 times; P=0.001), MMP-8 (by 2.3 times; P=0.007), and MMP-13 (by 1.9 times; P=0.004). CONCLUSIONS: Collagen matrix spatial organization features, as well as its regulatory factors, could be potential biomarkers of malignant prostate neoplasms.

Mast cells as a tumor microenvironment factor associated with the aggressiveness of prostate cancer.

We aimed to investigate the relationship between the degree of mast cells' (MCs) infiltration and clinicopathological features of prostate cancer (PCa) malignancy and to find out the possible mechanisms of the involvement of these cells in the formation of the aggressive course of the PCa development. The study was conducted on the clinical material of 60 patients with PCa of stages II-III. MCs in the PCa tissue were determined by a histochemical method using toluidine blue. The expression of osteopontin (OPN) was studied by the immunohistochemical method. The expression of miRNA-21, -126, -146a, -181a, and -221 was investigated by quantitative real-time PCR. Statistical processing of the results was performed using the GraphPad Prism 8 program. Our results demonstrated that the increased level of infiltration and degranulation of MCs in the PCa tissue was associated with such indices of the malignancy of the tumor process as the Gleason score and the preoperative PSA level in the blood serum of patients. A high level of MCs infiltration of the PCa tissue was associated with a significant decrease in the two-year recurrence-free survival rates of the patients by 23.3% (р=0.0455). A high degree of MCs infiltration of the PCa tissue was associated with 1.2 times (p=0.0347) higher level of OPN expression and 1.7 (p=0.0051) and 1.65 (p=0.0087) times lower levels of miR-126 and miR-181a expression, respectively. The obtained results indicate the participation of MCs as a factor of the tumor microenvironment in the PCa progression.

Post-operative unadjuvanted therapeutic xenovaccination with chicken whole embryo vaccine suppresses distant micrometastases and prolongs survival in a murine Lewis lung carcinoma model.

Immunotherapy in the form of anticancer vaccination relies on the mobilization of the patient's immune system against specific cancer antigens. Instead of focusing on an autologous cell lysate, which is not always available in clinical practice, the present study investigates vaccines utilizing xenogeneic foetal tissue that are rich in oncofoetal antigens. Lewis lung carcinoma (LLC)-challenged C57BL/6 mice were treated with either a xenogeneic vaccine made from chicken whole embryo, or a xenogeneic vaccine made from rat embryonic brain tissue, supplemented with a Bacillus subtilis protein fraction as an adjuvant. Median and overall survival, size of metastatic foci in lung tissue and levels of circulating CD8a+ T cells were evaluated and compared with untreated control mice. Following primary tumour removal, a course of three subcutaneous vaccinations with xenogeneic chicken embryo vaccine led to significant increase in overall survival rate (100% after 70 days of follow-up vs. 40% in untreated control mice), significant increase in circulating CD8a+ T cells (18.18 vs. 12.6% in untreated control mice), and a significant decrease in the area and incidence of metastasis foci. The xenogeneic rat brain tissue-based vaccine did not improve any of the investigated parameters, despite promising reports in other models. We hypothesize that the proper selection of antigen source (tissue) can constitute an effective immunotherapeutic product.

Enhanced antitumor activity of surface-modified iron oxide nanoparticles and an α-tocopherol derivative in a rat model of mammary gland carcinosarcoma.

Maghemite (γ-Fe2O3) nanoparticles were obtained by coprecipitation of ferrous and ferric salts in an alkaline medium followed by oxidation; the nanoparticles were coated with poly(N,N-dimethylacrylamide) (PDMA) and characterized by transmission electron microscopy, attenuated total reflection (ATR) Fourier transform infrared (FTIR) spectroscopy, dynamic light scattering, thermogravimetric and elemental analyses, and magnetic measurements in terms of particle morphology, size, polydispersity, amount of coating, and magnetization, respectively. The effects of α-tocopherol (Toc) and its phenolic (Toc-6-OH) and acetate (Toc-6-Ac) derivatives on Fe2+ release from γ-Fe2O3@PDMA, as well as from γ-Fe2O3 and CuFe2O4 nanoparticles (controls), were examined in vitro using 1,10-phenanthroline. The presence of tocopherols enhanced spontaneous Fe2+ release from nanoparticles, with Toc-6-OH exhibiting more activity than neat Toc. All of the nanoparticles tested were found to initiate blood lipid oxidation in a concentration-dependent manner, as determined by analysis of 2-thiobarbituric acid reactive species. Wistar rats with Walker-256 carcinosarcoma (a model of mammary gland carcinosarcoma) received Toc-6-Ac, magnetic nanoparticles, or their combination per os, and the antitumor activity of each treatment was determined in vivo. γ-Fe2O3@PDMA nanoparticles exhibited increased antitumor activity compared to both commercial CuFe2O4 particles and the antitumor drug doxorubicin. Moreover, increased antitumor activity was observed after combined administration of γ-Fe2O3@PDMA nanoparticles and Toc-6-Ac; however, levels of bilirubin, aspartate aminotransferase, and white bloods normalized and did not differ from those of the intact controls. The antitumor activity of the γ-Fe2O3 nanoparticles strongly correlated with Fe2+ release from the nanoparticles but not with nanoparticle-initiated lipid peroxidation in vitro.

Bacterial ghosts as adjuvants in syngeneic tumour cell lysate-based anticancer vaccination in a murine lung carcinoma model.

Instead of relying on external anticancer factors for treatment, immunotherapy utilizes the host's own immune system and directs it against given tumour antigens. This study demonstrated that it is possible to overcome the documented immunosuppressive properties of tumour cell lysate by supplementing it with appropriate adjuvant. Lewis lung carcinoma (LLC)­challenged C57BL/6 mice were treated with LLC cryo­lysate mixed with either bacterial ghosts (BGs) generated from E. coli Nissle 1917 or B. subtilis 70 kDa protein as adjuvants. Median and overall survival, the size of metastatic foci in lung tissue and levels of circulating CD8a+ T cells were evaluated and compared to the untreated control mice or mice treated with LLC lysate alone. After primary tumour removal, a course of three subcutaneous vaccinations with LLC lysate supplemented with BGs led to a significant increase in overall survival (80% after 84 days of follow­up vs. 40% in untreated control mice), a significant increase in circulating CD8a+ T cells (16.57 vs. 12.6% in untreated control mice) and a significant decrease in metastasis foci area and incidence. LLC lysate supplemented with B. subtilis protein also improved the inspected parameters in the treated mice, when compared against the untreated control mice, but not to a significant degree. Therefore, whole cell lysate supplemented with BGs emerges as an immunostimulatory construct with potential clinical applications in cancer treatment.

Synthesis and antiproliferative activity of conformationally restricted 1,2,3-triazole analogues of combretastatins in the sea urchin embryo model and against human cancer cell lines.

A series of 1,5-diaryl- and 4,5-diaryl-1,2,3-triazole derivatives of combretastatin A4 were synthesized and evaluated as antimitotic microtubule destabilizing agents using the sea urchin embryo model. Structure-activity relationship studies identified compounds substituted with 3,4,5-trimethoxyphenyl and 3,4-methylenedioxy-5-methoxyphenyl ring A and 4-methoxyphenyl ring B as potent antiproliferative agents with high cytotoxicity against a panel of human cancer cell lines including multi-drug resistant cells. 4,5-Diaryl-1,2,3-triazoles (C-C geometry) were found to be considerably more active than the respective 1,5-diaryl-1,2,3-triazoles (N-C geometry). Compound 10ad' induced G2/M cell cycle arrest and apoptosis in human T-leukemia Jurkat cells via caspase 2/3/9 activation and downregulation of the antiapoptotic protein XIAP. A mitotic catastrophe has been evaluated as another possible cell death mode.

Microspectroscopic Study of Liposome-to-cell Interaction Revealed by Förster Resonance Energy Transfer.

We report the Förster resonance energy transfer (FRET)-labeling of liposomal vesicles as an effective approach to study in dynamics the interaction of liposomes with living cells of different types (rat hepatocytes, rat bone marrow, mouse fibroblast-like cells and human breast cancer cells) and cell organelles (hepatocyte nuclei). The in vitro experiments were performed using fluorescent microspectroscopic technique. Two fluorescent dyes (DiO as the energy donor and DiI as an acceptor) were preloaded in lipid bilayers of phosphatidylcholine liposomes that ensures the necessary distance between the dyes for effective FRET. The change in time of the donor and acceptor relative fluorescence intensities was used to visualize and trace the liposome-to-cell interaction. We show that FRET-labeling of liposome vesicles allows one to reveal the differences in efficiency and dynamics of these interactions, which are associated with composition, fluidity, and metabolic activity of cell plasma membranes.

Cancer education in Ukraine.

The main features of the system of 'higher medical education' in Ukraine are presented. The principles of undergraduate, specialist training, and postgraduate education on oncology are described in detail and discussed in terms of European standards of cancer education. It is underlined that the cancer education in the system of higher and postgraduate education should be continuous, multidisciplinary, and of high quality.

Iron metabolism disturbances in the MCF-7 human breast cancer cells with acquired resistance to doxorubicin and cisplatin.

The development of resistance of cancer cells to therapeutic agents is the major obstacle in the successful treatment of breast cancer and the main cause of breast cancer recurrence. The results of several studies have demonstrated an important role of altered cellular iron metabolism in the progression of breast cancer and suggested that iron metabolism may be involved in the acquisition of a cancer cell drug-resistant phenotype. In the present study, we show that human MCF-7 breast cancer cells with an acquired resistance to the chemotherapeutic drugs doxorubicin (MCF-7/DOX) and cisplatin (MCF-7/CDDP) exhibited substantial alterations in the intracellular iron content and levels of iron-regulatory proteins involved in the cellular uptake, storage and export of iron, especially in profoundly increased levels of ferritin light chain (FTL) protein. The increased levels of FTL in breast cancer indicate that FTL may be used as a diagnostic and prognostic marker for breast cancer. Additionally, we demonstrate that targeted downregulation of FTL protein by the microRNA miR-133a increases sensitivity of MCF-7/DOX and MCF-7/CDDP cells to doxorubicin and cisplatin. These results suggest that correction of iron metabolism abnormalities may substantially improve the efficiency of breast cancer treatment.

Molecular mechanisms of radiation resistance in doxorubicin-resistant breast adenocarcinoma cells.

A positive response to breast cancer treatment is largely dependent on the successful combination of anticancer treatment modalities, such as chemotherapy and radiation therapy. Unfortunately, chemotherapy resistance occurs frequently. Furthermore, drug­resistant tumors can become unresponsive to other antitumor therapies, and they often fail to respond to radiation therapy. The molecular structures underlying the radiation responses of chemoresistant cells and tumors are not well understood. We analyzed the effect of ionizing radiation on MCF-7 human breast adenocarcinoma cells and their doxorubicin­resistant variant, MCF-7/DOX. The results demonstrated that drug­resistant MCF-7/DOX cells were less susceptible to radiation-induced DNA damage and apoptosis. This was proven through gene expression profiling, lower levels of γH2AX foci upon irradiation, and altered levels of DNA repair proteins, including pATM, KU70 and RAD51. Additionally, MCF-7/DOX drug­resistant cells harbored DNA polymerases with significantly low fidelity. In summary, our study revealed that drug-resistant MCF-7/DOX cells have high DNA repair potential and low-fidelity DNA polymerases, seemingly sacrificing specificity and efficiency to gain higher survival potential. In the long run, this may lead to an increased probability of mutation accumulation and further the development of an even more pronounced resistance phenotype. Therefore, this study provides a roadmap for the analysis of the roles of the DNA repair function and effectiveness, and apoptosis in response to radiation, chemotherapy and combinations of both treatment modalities.

Involvement of microRNA-451 in resistance of the MCF-7 breast cancer cells to chemotherapeutic drug doxorubicin.

Many chemotherapy regiments are successfully used to treat breast cancer; however, often breast cancer cells develop drug resistance that usually leads to a relapse and worsening of prognosis. We have shown recently that epigenetic changes such as DNA methylation and histone modifications play an important role in breast cancer cell resistance to chemotherapeutic agents. Another mechanism of gene expression control is mediated via the function of small regulatory RNA, particularly microRNA (miRNA); its role in cancer cell drug resistance still remains unexplored. In the present study, we investigated the role of miRNA in the resistance of human MCF-7 breast adenocarcinoma cells to doxorubicin (DOX). Here, we for the first time show that DOX-resistant MCF-7 cells (MCF-7/DOX) exhibit a considerable dysregulation of the miRNAome profile and altered expression of miRNA processing enzymes Dicer and Argonaute 2. The mechanistic link of miRNAome deregulation and the multidrug-resistant phenotype of MCF-7/DOX cells was evidenced by a remarkable correlation between specific miRNA expression and corresponding changes in protein levels of their targets, specifically those ones that have a documented role in cancer drug resistance. Furthermore, we show that microRNA-451 regulates the expression of multidrug resistance 1 gene. More importantly, transfection of the MCF-7/DOX-resistant cells with microRNA-451 resulted in the increased sensitivity of cells to DOX, indicating that correction of altered expression of miRNA may have significant implications for therapeutic strategies aiming to overcome cancer cell resistance.

Epigenetic profiling of multidrug-resistant human MCF-7 breast adenocarcinoma cells reveals novel hyper- and hypomethylated targets.

The successful treatment of cancer requires a clear understanding of multiple interacting factors involved in the development of drug resistance. Presently, two hypotheses, genetic and epigenetic, have been proposed to explain mechanisms of acquired cancer drug resistance. In the present study, we examined the alterations in epigenetic mechanisms in the drug-resistant MCF-7 human breast cancer cells induced by doxorubicin (DOX) and cisplatin (cisDDP), two chemotherapeutic drugs with different modes of action. Despite this difference, both of the drug-resistant cell lines displayed similar pronounced changes in the global epigenetic landscape showing loss of global DNA methylation, loss of histone H4 lysine 20 trimethylation, increased phosporylation of histone H3 serine 10, and diminished expression of Suv4-20h2 histone methyltransferase compared with parental MCF-7 cells. In addition to global epigenetic changes, the MCF-7/DOX and MCF-7/cisDDP drug-resistant cells are characterized by extensive alterations in region-specific DNA methylation, as indicated by the appearance of the number of differentially methylated DNA genes. A detailed analysis of hypo- and hypermethylated DNA sequences revealed that the acquisition of drug-resistant phenotype of MCF-7 cells to DOX and cisDDP, in addition to specific alterations induced by a particular drug only, was characterized by three major common mechanisms: dysfunction of genes involved in estrogen metabolism (sulfatase 2 and estrogen receptor alpha), apoptosis (p73, alpha-tubulin, BCL2-antagonist of cell death, tissue transglutaminase 2 and forkhead box protein K1), and cell-cell contact (leptin, stromal cell-derived factor receptor 1, activin A receptor E-cadherin) and showed that two opposing hypo- and hypermethylation processes may enhance and complement each other in the disruption of these pathways. These results provided evidence that epigenetic changes are an important feature of cancer cells with acquired drug-resistant phenotype and may be a crucial contributing factor to its development. Finally, deregulation of similar pathways may explain the existence and provide mechanism of cross-resistance of cancer cells to different types of chemotherapeutic agents.

Signal transduction pathways in Burkitt's lymphoma cell lines BL41 and DG75 with different sensitivity to doxorubicin.

AIM: To understand the biochemical basis of cell sensitivity to cytotoxic effect of doxorubicine (DOX), we investigated signaling cascades mediated by c-Jun N-terminal protein kinases (JNK1/2), p38 mitogen-activated protein kinases (MAPK), extracellular signal-regulated protein kinase (ERK1/2) and protein kinase B/Akt in both DOX-sensitive BL41 and the DOX-resistant DG75 Burkitt's lymphoma (BL) cell lines. METHODS: To test the effect of DOX on different signaling cascades, BL41 and DG75 cells were treated with DOX for varying lengths of time. Cytotoxic effect of DOX was analyzed by Hoechst 33342 staining. Total amount of JNK1/2, ERK1/2, p38 MARK, Akt proteins, and also phosphorylated/activated forms of these enzymes were detected using Western blot analysis with specific antibodies. Immunophenotypic analysis of BL41 and DG75 cells was performed by indirect immunofluorescence staining. RESULTS: Our findings demonstrated that DOX treatment of the BL41 cells led to sustained activation of JNK1/2 and p38 MAPK. This activation/phosphorylation did not result from increased expression of either JNK1/2 or p38 MAPK since protein levels of JNK1/2 and p38 MAPK in DOX-treated and untreated cells were unaltered. Apoptotic signaling cascade induced by DOX in BL41 cell was accompanied by Akt dephosphorylation. The effect of DOX in drug-resistant cell line DG75 convoyed by dephosphorylation of JNK1/2, p38 MAPK and activation of Akt. Fate of BL cells did not depend from ERK activity. CONCLUSION: The outcome of cellular response to DOX in BL cell lines is determined by interference of at least three signaling pathways: JNK1/2, p38 MAPK and PKB/Akt. The balance between Akt/PKB and MAPK pathways is important in determining whether BL cells survive or undergo apoptosis in response to DOX treatment.

Role of polyamines in the function of nuclear transcription factor NF-kappaB in breast cancer cells.

UNLABELLED: The aim of the work was the investigation of the influence of polyamines on formation of nuclear transcription factor kappa B (NF-kappaB) complex with specific DNA-sequences nuclear response elements (NRE), and the study of content of p50 and p65 (NF-kappaB subunits) in breast tumor cells under polyamines metabolism modulation. METHODS: The model experiments in vitro were carried out on cultured MCF-7 cells, in vivo experiments -- on experimental Ca755 and Walker-256 tumors using surface plasmon resonance (SPR) and Western-blotting analysis. RESULTS: The data have demonstrated that polyamines falicitated p50 -- NRE binding. Under conditions of polyamine depletion, an increase of content of both p50 and p65 subunits of NF-kappaB in the nucleus has been registered (especially that of p50 subunits). Upon addition of alpha-DFMO + putrescine to the culture medium of MCF-7 cells, the decrease of the levels of p50 and p65 proteins in the nuclei has been observed. CONCLUSION: Polyamines influence the activity of NF-kappaB transcription factor in breast tumor cells.

Total proteolytic activity and content of the main proteinase inhibitors in blood plasma of rats bearing doxorubicin-sensitive and doxorubicin-resistant Guerin carcinoma.

UNLABELLED: The AIM of work was to evaluate the alteration of the total proteolytic activity (TPA) and the levels of alpha(1)-proteinase inhibitor (alpha1PI) and alpha(2)-macroglobuline (alpha2M) in blood plasma of rats bearing Guerin carcinoma upon the development of Doxorubicin (DOX) resistance. MATERIALS AND METHODS: TPA and alpha1PI and alpha2M content in the blood plasma of male Wistar rats bearing DOX-resistant and DOX-sensitive Guerin carcinoma were evaluated by standard biochemical methods. RESULTS: During growth of both DOX-sensitive and DOX-resistant Guerin carcinoma, TPA decrease in blood plasma and the increase of alpha1PI levels were registered; in DOX-resistant group this effect was more pronounced. Alpha2M content in blood plasma of animals from both experimental groups was considerably smaller than that of the control and was the lowest in DOX-resistant group. CONCLUSION: The growth of DOX-resistant Guerin carcinoma is accompanied by imbalance of proteolysis processes in the blood plasma, particularly, alteration of TPA and alpha1PI and alpha2M levels.