WNT-Conditioned Mechanism of Exit from Postchemotherapy Shock of Differentiated Tumour Cells.

BACKGROUND: the present study aims to prove or disprove the hypothesis that the state of copy number aberration (CNA) activation of WNT signalling pathway genes accounts for the ability of differentiated tumour cells to emerge from postchemotherapy shock. METHODS: In the first step, the CNA genetic landscape of breast cancer cell lines BT-474, BT-549, MDA-MB-231, MDA-MD-468, MCF7, SK-BR-3 and T47D, which were obtained from ATCC, was examined to rank cell cultures according to the degree of ectopic activation of the WNT signalling pathway. Then two lines of T47D with ectopic activation and BT-474 without activation were selected. The differentiated EpCAM+CD44-CD24-/+ cells of these lines were subjected to IL6 de-differentiation with formation of mammospheres on the background of cisplatin and WNT signalling inhibitor ICG-001. RESULTS: it was found that T47D cells with ectopic WNT signalling activation after cisplatin exposure were dedifferentiated to form mammospheres while BT-474 cells without ectopic WNT-signalling activation did not form mammospheres. The dedifferentiation of T47D cells after cisplatin exposure was completely suppressed by the WNT signalling inhibitor ICG-001. Separately, ICG-001 reduced, but did not abolish, the ability to dedifferentiate in both cell lines. CONCLUSIONS: these data support the hypothesis that the emergence of differentiated tumour cells from postchemotherapy shock after chemotherapy is due to ectopic activation of WNT signalling pathway genes.

Transcriptomic Analysis of CRISPR/Cas9-Mediated PARP1-Knockout Cells under the Influence of Topotecan and TDP1 Inhibitor.

Topoisomerase 1 (TOP1) is an enzyme that regulates DNA topology and is essential for replication, recombination, and other processes. The normal TOP1 catalytic cycle involves the formation of a short-lived covalent complex with the 3' end of DNA (TOP1 cleavage complex, TOP1cc), which can be stabilized, resulting in cell death. This fact substantiates the effectiveness of anticancer drugs-TOP1 poisons, such as topotecan, that block the relegation of DNA and fix TOP1cc. Tyrosyl-DNA phosphodiesterase 1 (TDP1) is able to eliminate TOP1cc. Thus, TDP1 interferes with the action of topotecan. Poly(ADP-ribose) polymerase 1 (PARP1) is a key regulator of many processes in the cell, such as maintaining the integrity of the genome, regulation of the cell cycle, cell death, and others. PARP1 also controls the repair of TOP1cc. We performed a transcriptomic analysis of wild type and PARP1 knockout HEK293A cells treated with topotecan and TDP1 inhibitor OL9-119 alone and in combination. The largest number of differentially expressed genes (DEGs, about 4000 both up- and down-regulated genes) was found in knockout cells. Topotecan and OL9-119 treatment elicited significantly fewer DEGs in WT cells and negligible DEGs in PARP1-KO cells. A significant part of the changes caused by PARP1-KO affected the synthesis and processing of proteins. Differences under the action of treatment with TOP1 or TDP1 inhibitors alone were found in the signaling pathways for the development of cancer, DNA repair, and the proteasome. The drug combination resulted in DEGs in the ribosome, proteasome, spliceosome, and oxidative phosphorylation pathways.

Human Blood Extracellular Vesicles Activate Transcription of NF-kB-Dependent Genes in A549 Lung Adenocarcinoma Cells.

Extracellular vesicles (EVs) produced by various cell types are heterogeneous in size and composition. Changes in the RNA sets of EVs in biological fluids are considered the basis for the development of new approaches to minimally invasive diagnostics and the therapy of human diseases. In this study, EVs were obtained from the blood of healthy donors by centrifugation, followed by ultracentrifugation. It was shown that EVs consist of several populations including small exosome-like vesicles and larger microvesicle-like particles. The composition of EVs' RNAs was determined. A549 lung adenocarcinoma cells were incubated with EV and the NGS analysis of differentially expressed genes was performed. During the incubation of A549 cells with EVs, the levels of mRNA encoding components for the NF-kB signaling pathway increased, as well as the expression of genes controlled by the NF-kB transcription factor. Overall, our results suggest that components of EVs trigger the NF-kB signaling cascade in A549 cells, leading to the transcription of genes including cytokines, adhesion molecules, cell cycle regulators, and cell survival factors. Our data provide insight into the interaction between blood EVs and human cells and can be used for designing new tools for the diagnosis and treatment of human diseases.

Cytotoxic and Antitumor Activity of Lactaptin in Combination with Autophagy Inducers and Inhibitors.

Autophagy is a degradative process in which cellular organelles and proteins are recycled to restore homeostasis and cellular metabolism. Autophagy can be either a prosurvival or a prodeath process and remains one of the most fundamental processes for cell vitality. Thus autophagy modulation is an important approach for reinforcement anticancer therapeutics. Earlier we have demonstrated that recombinant analog of human milk protein lactaptin (RL2) induced apoptosis of various cultured cancer cells and activated lipidation of microtubule-associated protein 1 light chain 3 (LC3). In this study we investigated whether autophagy inhibitors-chloroquine (CQ), Ku55933 (Ku), and 3-methyladenine (3MA)-or inducer-rapamycin (Rap)-can enhance cytotoxic activity of lactaptin analog in cancer cells and its anticancer activity in the mice model. Western Blot analysis revealed that RL2 induced short-term autophagy in MDA-MB-231 and MCF-7 cells at early stages of incubation and that these data were confirmed by the transmission electron microscopy of autophagosome/autophagolysosome formation. RL2 stimulates reactive oxygen species (ROS) production, autophagosomes accumulation, upregulation of ATG5 with processing of LC3I to LC3II, and downregulation of p62/sequestosome 1 (p62). We have shown that autophagy modulators, CQ, Ku, and Rap, synergistically increased cytotoxicity of RL2, and RL2 with CQ induced autophagic cell death. In addition, CQ, Ku, and Rap in combination with RL2 decreased activity of lysosomal protease Cathepsin D. More importantly, combining RL2 with CQ, we improved antitumor effect in mice. Detected synergistic cytotoxic effects of both types of autophagy regulators, inhibitors, and inducers with RL2 against cancer cells allow us to believe that these combinations can be a basis for the new anticancer approach. Finally, we suppose that CQ and Rap promoting of short-term RL2-induced autophagy interlinks with final autophagic cell death.

Establishment of primary human breast cancer cell lines using "pulsed hypoxia" method and development of metastatic tumor model in immunodeficient mice.

BACKGROUND: Among breast cancer (BC) patients the outcomes of anticancer therapy vary dramatically due to the highly heterogeneous molecular characteristics of BC. Therefore, an extended panel of BC cell lines are required for in vitro and in vivo studies to find out new characteristic of carcinogenesis and metastasis. The purpose of this study was to develop patient-derived BC cell cultures and metastatic tumor models representing a tool for personal therapy and translational research. METHODS: Breast cancer cells were prepared by optimizing technique from tumor samples. We used real-time RT-PCR, flow cytometry, western blotting, cytotoxicity assay, karyotyping and fluorescent and electron microscopy analyses to characterize the established cell lines. BC xenografts in scid mice were used for in vivo tumorigenicity studies. RESULTS: The technique of preparing primary cells was optimized and this resulted in a high output of viable and active proliferated cells of nine patient-derived breast cancer cell lines and one breast non-malignant cell line. High E-cadherine and EpCAM expression correlated positively with epithelial phenotype while high expression of N-cadherine and Vimentin were shown in cells with mesenchymal phenotype. All mesenchymal-like cell lines were high HER3-positive-up to 90%. More interesting than that, is that two cell lines under specific culturing conditions (pulsed hypoxia and conditioned media) progressively transformed from mesenchymal to epithelial phenotypes displaying the expression of respective molecular markers proving that the mesenchymal-to-epithelial transition occurred. Becoming epithelial, these cells have lost HER3 and decreased HER2 membrane receptors. Three of the established epithelial cancer cell lines were tumorigenic in SCID mice and the generated tumors exhibited lobules-like structures. Ultrastructure analysis revealed low-differentiate phenotype of tumorigenic cell lines. These cells were in near-triploid range with multiple chromosome rearrangements. Tumorigenic BrCCh4e cells, originated from the patient of four-course chemotherapy, initiated metastasis when they were grafted subcutaneous with colonization of mediastinum lymph nodes. CONCLUSIONS: The developed BC cells metastasizing to mediastinum lymph nodes are a relevant model for downstream applications. Moreover, our findings demonstrate that pulsed hypoxia induces transformation of primary fibroblastoid breast cancer cells to epithelial-like cells and both of these cultures-induced and original-don't show tumor initiating capacity.

Characterization of primary normal and malignant breast cancer cell and their response to chemotherapy and immunostimulatory agents.

BACKGROUND: The phenomenon of chemotherapy-resistant cancers has necessitated the development of new therapeutics as well as the identification of specific prognostic markers to predict the response to novel drugs. Primary cancer cells provide a model to study the multiplicity of tumourigenic transformation, to investigate alterations of the cellular response to various molecular stimuli, and to test therapeutics for cancer treatment. METHODS: Here, we developed primary cultures of human breast tissue - normal cells (BN1), cancer cells (BC5), and cells from a chemotherapy-treated tumour (BrCCh1) to compare their response to conventional chemotherapeutics and to innate immunity stimulators with that of the immortalized breast cells MCF7, MDA-MB-231, and MCF10A. Expression of the progesterone receptor (PGR), oestrogen receptor (ER) α and ß, human epidermal growth factor receptor (HER) 2 and 3 and aromatase CYP19, as well as expression of interferon-induced protein with tetratricopeptide repeats 3 (IFIT3) mRNA in human breast cells were characterized. RESULTS: We revealed that BC5 carcinoma cells were PGRlow/ERbhigh/ERa-/Cyp19+, the BrCCh1 cells that originated from the recurrent tumour were PGR-/ERb+/ERa-/Cyp19+, and normal BN cells were PGR-/ERb+/ERa-/Cyp19high. The treatment of primary culture cells with antitumour therapeutics revealed that BrCCh1 cells were doxorubicine-resistant and sensitive to cisplatin. BC5 cells exhibited low sensitivity to tamoxifen and cisplatin. The innate immunity activators interferon-α and an artificial small nucleolar RNA analogue increased expression of IFIT3 at different levels in primary cells and in the immortalized breast cells MCF7, MDA-MB-231, and MCF10A. The relative level of activation of IFIT3 expression was inversely correlated with the baseline level of IFIT3 mRNA expression in breast cell lines. CONCLUSION: Our data demonstrated that primary cancer cells are a useful model for the development of novel cancer treatments. Our findings suggest that expression of IFIT3 mRNA can be used as a prognostic marker of breast cancer cell sensitivity to immunostimulating therapeutics.

Artificial Analogues of Circulating Box C/D RNAs Induce Strong Innate Immune Response and MicroRNA Activation in Human Adenocarcinoma Cells.

Fragments of small nucleolar RNAs (snoRNAs) were found among various non-coding RNAs (ncRNAs) circulating in human blood. Currently, the function of such cell-free sno-derived-RNAs is not clearly defined. This work is aimed at identifying regulatory pathways controlled by extracellular snoRNAs. In order to determine the molecular targets and pathways affected by artificial snoRNAs, we performed Illumina array analysis of MCF-7 human adenocarcinoma cells transfected with box C/D RNAs. The genes related to the innate immune response and apoptotic cascades were found to be activated in transfected cells compared with control cells. Intriguingly, the transfection of MCF-7 cells with artificial box C/D snoRNAs also increased the transcription of several microRNAs, such as mir-574, mir-599 and mir-21. Our data demonstrated that extracellular snoRNAs introduced into human cells may function as gene expression modulators, with activation of microRNA genes being one of the regulatory mechanisms.

Tumor-Specific Peptide, Selected from a Phage Peptide Library, Enhances Antitumor Activity of Lactaptin.

A recombinant analogue of lactaptin (RL2), a new potential anticancer molecule, induces apoptosis in cultured tumor cells. The tumor suppression efficacy of RL2 was shown against mouse hepatoma-1 cells and MDA-MB-231 human breast adenocarcinoma cells. The RL2-based therapeutic drug lactaptin is distributed evenly throughout the organism, which reduces its antitumor efficacy. In the current study, we obtained a genetic construct that allows production of the recombinant fusion protein T3-RL2, consisting of RL2 and T3 peptide (YTYDPWLIFPAN), in E. coli cells. T3 peptide was selected from a phage peptide library as a result of two screenings: in vitro using MDA-MB-231 cell culture and in vivo using a mouse xenograft model of breast cancer MDA-MB-231. It was shown that the displayed peptide T3 provides binding and internalization of phage particles by MDA-MB-231 cells and their specific accumulation in MDA-MB-231 tumor tissue. In addition, based on the nucleotide sequences coding RL2 and the known tumor-targeting peptide iRGD, we obtained genetic constructs that provide synthesis of fusion proteins RL2-iRGD and RL-iRGD-His. We studied the cytotoxic activity of fusion proteins T3-RL2, RL2-iRGD and RL-iRGD-His in vitro using MDA-MB-231 and MCF-7 human adenocarcinoma cells. The in vitro results showed that the fusion proteins inhibit proliferation of both cell cultures, and their cytotoxic activity is higher than that of RL2. In vivo experiments on the study of the antitumor efficacy of the obtained fusion proteins demonstrated that T3-RL2 protein significantly inhibits MDA-MB-231 tumor growth in a xenograft model compared with RL2, while the antitumor effect of RL2-iRGD and RL-iRGD-His proteins is comparable to the effect of RL2.

Sensitivity of endometrial cancer cells from primary human tumor samples to new potential anticancer peptide lactaptin.

PURPOSE: Endometrial carcinoma is the most common gynecologic malignancy which is associated with a poor prognosis when diagnosed at an advanced stage; therefore, the discovery of efficacious new drugs is required to reinforce conventional chemotherapy. Short-term cultures of primary cells from endometrial tumors could be used for testing new anticancer therapeutics as well as for the development of personalized cancer therapy strategy. Here, the antitumor effect of a recombinant analogue of lactaptin (RL2), a new potential anticancer molecule, was examined against primary human endometrial cancer cells. MATERIALS AND METHODS: Primary cell cultures of malignant and normal human endometrium were performed by enzymatic digestion of endometrial tissue from biopsy material. Real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) was performed to determine the messenger ribonucleic acid (mRNA) state of estrogen (ERs) and progesterone (PRs) hormone receptors and aromatase (Cyp 19) in cell cultures. Dynamic monitoring of cell adhesion and proliferation was made using the iCELLigence system (ASEA Biosciences). The sensitivity of cell cultures to conventional anticancer drugs and the lactaptin analog was estimated by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, flow cytometry, and the iCELLligence system. RESULTS: Established short-term primary cultures of endometrial cancer cells were ERα/ERß/PR-positive and sensitive for RL2. The IC 50 values of doxorubicin and cisplatin were determined for all of the primary cultures designed. KE normal cells displaying low Cyp19 mRNA levels and high ERß and PR mRNA levels were more resistant to RL2 treatment as well as to cisplatin and doxorubicin. CONCLUSIONS: Our results indicate that the recombinant analog of lactaptin, RL2, exerts cytotoxic effects against primary hormone-dependent endometrial tumor cells in vitro with features of apoptosis.

Lactaptin induces p53-independent cell death associated with features of apoptosis and autophagy and delays growth of breast cancer cells in mouse xenografts.

Lactaptin, the proteolytic fragment of human milk kappa-casein, induces the death of various cultured cancer cells. The mechanisms leading to cell death after lactaptin treatment have not been well characterized. In this study the in vivo and in vitro effects of a recombinant analogue of lactaptin (RL2) were examined. Following treatment with the recombinant analogue of lactaptin strong caspase -3, -7 activation was detected. As a consequence of caspase activation we observed the appearance of a sub-G1 population of cells with subdiploid DNA content. Dynamic changes in the mRNA and protein levels of apoptosis-related genes were estimated. No statistically reliable differences in p53 mRNA level or protein level were found between control and RL2-treated cells. We observed that RL2 constitutively suppressed bcl-2 mRNA expression and down regulated Bcl-2 protein expression in MDA-MB-231 cells. We demonstrated that RL2 penetrates cancer and non-transformed cells. Identification of the cellular targets of the lactaptin analogue revealed that α/ß-tubulin and α-actinin-1 were RL2-bound proteins. As the alteration in cellular viability in response to protein stimulus can be realized not only by way of apoptosis but also by autophagy, we examined the implications of autophagy in RL2-dependent cell death. We also found that RL2 treatment induces LC3-processing, which is a hallmark of autophagy. The autophagy inhibitor chloroquine enhanced RL2 cytotoxicity to MDA-MB-231 cells, indicating the pro-survival effect of RL2-dependent autophagy. The antitumour potential of RL2 was investigated in vivo in mouse xenografts bearing MDA-MB-231 cells. We demonstrated that the recombinant analogue of lactaptin significantly suppressed the growth of solid tumours. Our results indicate that lactaptin could be a new molecule for the development of anticancer drugs.