Unveiling the Molecular Mechanism of Trastuzumab Resistance in SKBR3 and BT474 Cell Lines for HER2 Positive Breast Cancer.

HER2-positive breast cancer is one of the most prevalent forms of cancer among women worldwide. Generally, the molecular characteristics of this breast cancer include activation of human epidermal growth factor receptor-2 (HER2) and hormone receptor activation. HER2-positive is associated with a higher death rate, which led to the development of a monoclonal antibody called trastuzumab, specifically targeting HER2. The success rate of HER2-positive breast cancer treatment has been increased; however, drug resistance remains a challenge. This fact motivated us to explore the underlying molecular mechanisms of trastuzumab resistance. For this purpose, a two-fold approach was taken by considering well-known breast cancer cell lines SKBR3 and BT474. In the first fold, trastuzumab treatment doses were optimized separately for both cell lines. This was done based on the proliferation rate of cells in response to a wide variety of medication dosages. Thereafter, each cell line was cultivated with a steady dosage of herceptin for several months. During this period, six time points were selected for further in vitro analysis, ranging from the untreated cell line at the beginning to a fully resistant cell line at the end of the experiment. In the second fold, nucleic acids were extracted for further high throughput-based microarray experiments of gene and microRNA expression. Such expression data were further analyzed in order to infer the molecular mechanisms involved in the underlying development of trastuzumab resistance. In the list of differentially expressed genes and miRNAs, multiple genes (e.g., BIRC5, E2F1, TFRC, and USP1) and miRNAs (e.g., hsa miR 574 3p, hsa miR 4530, and hsa miR 197 3p) responsible for trastuzumab resistance were found. Downstream analysis showed that TFRC, E2F1, and USP1 were also targeted by hsa-miR-8485. Moreover, it indicated that miR-4701-5p was highly expressed as compared to TFRC in the SKBR3 cell line. These results unveil key genes and miRNAs as molecular regulators for trastuzumab resistance.

Extracellular Vesicles as Signal Carriers in Malignant Thyroid Tumors?

Extracellular vesicles (EVs) are small, membranous structures involved in intercellular communication. Here, we analyzed the effects of thyroid cancer-derived EVs on the properties of normal thyroid cells and cells contributing to the tumor microenvironment. EVs isolated from thyroid cancer cell lines (CGTH, FTC-133, 8505c, TPC-1 and BcPAP) were used for treatment of normal thyroid cells (NTHY), as well as monocytes and endothelial cells (HUVEC). EVs' size/number were analyzed by flow cytometry and confocal microscopy. Gene expression, protein level and localization were investigated by qRT-PCR, WB and ICC/IF, respectively. Proliferation, migration and tube formation were analyzed. When compared with NTHY, CGTH and BcPAP secreted significantly more EVs. Treatment of NTHY with cancer-derived EVs changed the expression of tetraspanin genes, but did not affect proliferation and migration. Cancer-derived EVs suppressed tube formation by endothelial cells and did not affect the phagocytic index of monocytes. The number of 6 µm size fraction of cancer-derived EVs correlated negatively with the CD63 and CD81 expression in NTHY cells, as well as positively with angiogenesis in vitro. Thyroid cancer-derived EVs can affect the expression of tetraspanins in normal thyroid cells. It is possible that 6 µm EVs contribute to the regulation of NTHY gene expression and angiogenesis.

Analysis of the Role of FRMD5 in the Biology of Papillary Thyroid Carcinoma.

BACKGROUND: Thyroid carcinoma (TC) is the most common endocrine system malignancy, and papillary thyroid carcinoma (PTC) accounts for >80% of all TC cases. Nevertheless, PTC pathogenesis is still not fully understood. The aim of the study was to elucidate the role of the FRMD5 protein in the regulation of biological pathways associated with the development of PTC. We imply that the presence of certain genetic aberrations (e.g., BRAF V600E mutation) is associated with the activity of FRMD5. METHODS: The studies were conducted on TPC1 and BCPAP (BRAF V600E) model PTC-derived cells. Transfection with siRNA was used to deplete the expression of FRMD5. The mRNA expression and protein yield were evaluated using RT-qPCR and Western blot techniques. Proliferation, migration, invasiveness, adhesion, spheroid formation, and survival tests were performed. RNA sequencing and phospho-kinase proteome profiling were used to assess signaling pathways associated with the FRMD5 expressional status. RESULTS: The obtained data indicate that the expression of FRMD5 is significantly enhanced in BRAF V600E tumor specimens and cells. It was observed that a drop in intracellular yield of FRMD5 results in significant alternations in the migration, invasiveness, adhesion, and spheroid formation potential of PTC-derived cells. Importantly, significant divergences in the effect of FRMD5 depletion in both BRAF-wt and BRAF-mutated PTC cells were observed. It was also found that knockdown of FRMD5 significantly alters the expression of multidrug resistant genes. CONCLUSIONS: This is the first report highlighting the importance of the FRMD5 protein in the biology of PTCs. The results suggest that the FRMD5 protein can play an important role in controlling the metastatic potential and multidrug resistance of thyroid tumor cells.

Combinatory Treatment with miR-7-5p and Drug-Loaded Cubosomes Effectively Impairs Cancer Cells.

BACKGROUND: Multidrug resistance (MDR) is an emerging problem in the treatment of cancer. Therefore, there is a necessity for novel strategies that would sensitize tumor cells to the administered chemotherapeutics. One of the innovative approaches in fighting drug-resistant tumors is the treatment of cancer with microRNA (miRNA), or the use of cubosomes (lipid nanoparticles) loaded with drugs. Here, we present a study on a novel approach, which combines both tools. METHODS: Cubosomes loaded with miR-7-5p and chemotherapeutics were developed. The effects of drug- and miRNA-loaded vehicles on glioma- (A172, T98G), papillary thyroid- (TPC-1) and cervical carcinoma-derived (HeLa) cells were analyzed using molecular biology techniques, including quantitative real-time PCR, MTS-based cell proliferation test, flow cytometry and spheroids formation assay. RESULTS: The obtained data indicate that miR-7-5p increases the sensitivity of the tested cells to the drug, and that nanoparticles loaded with both miRNA and the drug produce a greater anti-tumor effect in comparison to the free drug treatment. It was found that an increased level of apoptosis in the drug/miRNA co-treated cells is accompanied by an alternation in the expression of the genes encoding for key MDR proteins of the ABC family. CONCLUSIONS: Overall, co-administration of miR-7-5p with a chemotherapeutic can be considered a promising strategy, leading to reduced MDR and the induction of apoptosis in cancer cells.

Identification of CD34+/PGDFRα+ Valve Interstitial Cells (VICs) in Human Aortic Valves: Association of Their Abundance, Morphology and Spatial Organization with Early Calcific Remodeling.

Aortic valve interstitial cells (VICs) constitute a heterogeneous population involved in the maintenance of unique valvular architecture, ensuring proper hemodynamic function but also engaged in valve degeneration. Recently, cells similar to telocytes/interstitial Cajal-like cells described in various organs were found in heart valves. The aim of this study was to examine the density, distribution, and spatial organization of a VIC subset co-expressing CD34 and PDGFRα in normal aortic valves and to investigate if these cells are associated with the occurrence of early signs of valve calcific remodeling. We examined 28 human aortic valves obtained upon autopsy. General valve morphology and the early signs of degeneration were assessed histochemically. The studied VICs were identified by immunofluorescence (CD34, PDGFRα, vimentin), and their number in standardized parts and layers of the valves was evaluated. In order to show the complex three-dimensional structure of CD34+/PDGFRα+ VICs, whole-mount specimens were imaged by confocal microscopy, and subsequently rendered using the Imaris (Bitplane AG, Zürich, Switzerland) software. CD34+/PDGFRα+ VICs were found in all examined valves, showing significant differences in the number, distribution within valve tissue, spatial organization, and morphology (spherical/oval without projections; numerous short projections; long, branching, occasionally moniliform projections). Such a complex morphology was associated with the younger age of the subjects, and these VICs were more frequent in the spongiosa layer of the valve. Both the number and percentage of CD34+/PDGFRα+ VICs were inversely correlated with the age of the subjects. Valves with histochemical signs of early calcification contained a lower number of CD34+/PDGFRα+ cells. They were less numerous in proximal parts of the cusps, i.e., areas prone to calcification. The results suggest that normal aortic valves contain a subpopulation of CD34+/PDGFRα+ VICs, which might be involved in the maintenance of local microenvironment resisting to pathologic remodeling. Their reduced number in older age could limit the self-regenerative properties of the valve stroma.

Combination of ERK2 inhibitor VX-11e and voreloxin synergistically enhances anti-proliferative and pro-apoptotic effects in leukemia cells.

ERK1/2 inhibitors are new promising anticancer drugs. The aim of this study was to investigate the effect of the combination of ERK2 inhibitor VX-11e and voreloxin on MOLM-14, K562, REH and MOLT-4 leukemia cell lines. We found that VX-11e alone and in combination with voreloxin significantly decreased ERK activation in all cell lines tested. To evaluate the interactions of the drugs, cells were treated for 24 h with VX-11e or voreloxin alone and in combination at fixed ratios based on IC50 values. The combinatorial effects of both drugs were synergistic over a wide range of concentrations in MOLM-14, REH and MOLT-4 cell lines. In K562 cells, three effects were found to be additive, one antagonistic and only one synergistic. The results showed that incubation with both VX-11e and voreloxin inhibited the growth of leukemia cells, affected cell cycle and induced apoptosis. Furthermore, the molecular mechanism of these effects might be attributed to an increased expression of p21 and a decreased expression of survivin and NF-κB in all cell lines tested except from K562 cells. In conclusion, combination of VX-11e and voreloxin can exert a synergistic anticancer effect in leukemia cells.

Transcription Factor Prospero Homeobox 1 (PROX1) as a Potential Angiogenic Regulator of Follicular Thyroid Cancer Dissemination.

It is well known that Prospero homeobox 1 (PROX1) is a crucial regulator of lymphangiogenesis, that reprograms blood endothelial cells to lymphatic phenotype. However, the role of PROX1 in tumor progression, especially in angiogenesis remains controversial. Herein, we studied the role of PROX1 in angiogenesis in cell lines derived from follicular thyroid cancer (FTC: FTC-133) and squamous cell carcinoma of the thyroid gland (SCT: CGTH-W-1) upon PROX1 knockdown. The genes involved in angiogenesis were selected by RNA-seq, and the impact of PROX1 on vascularization potential was investigated using human umbilical vein endothelial cells (HUVECs) cultured in conditioned medium collected from FTC- or SCT-derived cancer cell lines after PROX1 silencing. The angiogenic phenotype was examined in connection with the analysis of focal adhesion and correlated with fibroblast growth factor 2 (FGF2) levels. Additionally, the expression of selected genes involved in angiogenesis was detected in human FTC tissues. As a result, we demonstrated that PROX1 knockdown resulted in upregulation of factors associated with vascularization, such as metalloproteinases (MMP1 and 3), FGF2, vascular endothelial growth factors C (VEGFC), BAI1 associated protein 2 (BAIAP2), nudix hydrolase 6 (NUDT6), angiopoietin 1 (ANGPT1), and vascular endothelial growth factor receptor 2 (KDR). The observed molecular changes resulted in the enhanced formation of capillary-like structures by HUVECs and upregulated focal adhesion in FTC-133 and CGTH-W-1 cells. The signature of selected angiogenic genes' expression in a series of FTC specimens varied depending on the case. Interestingly, PROX1 and FGF2 showed opposing expression levels in FTC tissues and seven thyroid tumor-derived cell lines. In summary, our data revealed that PROX1 is involved in the spreading of thyroid cancer cells by regulation of angiogenesis.

New aspects of antiproliferative activity of 4-hydroxybenzyl isothiocyanate, a natural H2S-donor.

The effect of 4-hydroxybenzyl isothiocyanate (HBITC), a natural H2S-donor from white mustard seeds (Sinapis alba), on the proliferation of human neuroblastoma (SH-SY5Y) and glioblastoma (U87MG) cells was studied and some aspects of the mechanism of its activity were suggested. The inhibition of both SH-SY5Y and U87MG cell proliferation was associated with an increase in the thiosulfate level, the number of cells with the inactive form of Bcl-2 protein, and with a decrease of mitochondrial membrane potential. Interestingly, HBITC results in downregulation of p53 protein and upregulation of p21 protein levels in SH-SY5Y cells. In the presence of elevated levels of H2S and thiosulfate, the sulfhydryl groups of p53 protein as well as Bcl-2 protein could be modified via HBITC-induced S-sulfuration or by oxidative stress. It seems that the induction of p21 protein level is mediated in SH-SY5Y cells by p53-independent mechanisms. In addition, HBITC-treatment caused downregulation of the level of mitochondrial rhodanese and 3-mercaptopyruvate sulfurtransferase, and consequently increased the level of the reactive oxygen species in SH-SY5Y cells.

Combined orcein and martius scarlet blue (OMSB) staining for qualitative and quantitative analyses of atherosclerotic plaques in brachiocephalic arteries in apoE/LDLR-/- mice.

Numerous cellular and extracellular components should be analyzed in sections of atherosclerotic plaques to assess atherosclerosis progression and vulnerability. Here, we combined orcein (O) staining for elastic fibers and martius scarlet blue (MSB) polychrome to visualize various morphological contents of plaque in brachiocephalic arteries (BCA) of apoE/LDLR-/- mice. Elastic fibers (including broken elastic laminae and 'buried' fibrous caps) were stained purple and they could be easily distinguished from collagen fibers (blue). Orcein allowed clear identification of even the finest elastic fibers. Erythrocytes were stained yellow and they could easily be discerned from mature fibrin (red). Old fibrin tends to acquire blue color. The method of OMSB staining is simple, takes less than 1 h to perform and can be adapted to automatic stainers. Most importantly, the color separation is good enough to allow digital automatic segmentation of specific components in tissue section and quantitative analysis of the plaque constituents. OMSB was used to compare atherosclerotic plaques in proximal and distal regions of BCA in apoE/LDLR-/- mice. In conclusion, OMSB staining represents a novel staining that could be routinely used for qualitative and quantitative microscopic assessments of formaldehyde-fixed and paraffin-embedded sections of arteries with atherosclerotic lesions.

A possible mechanism of inhibition of U87MG and SH-SY5Y cancer cell proliferation by diallyl trisulfide and other aspects of its activity.

The study was conducted to elucidate the mechanism of antiproliferative and antioxidative action of diallyl trisulfide (DATS), a garlic-derived organosulfur compound. Changes in the L-cysteine desulfuration, and the levels of cystathionine and non-protein thiols in DATS-treated human glioblastoma (U87MG) and neuroblastoma (SH-SY5Y) cells were investigated. The inhibition of proliferation of the investigated cells by DATS was correlated with an increase in the inactivated form of Bcl-2. In U87MG cells, an increased level of sulfane sulfur and an increased activity of 3-mercaptopyruvate sulfurtransferase (MPST) and rhodanese, the enzymes involved in sulfane sulfur generation and transfer, suggest that DATS can function as a donor of sulfane sulfur atom, transferred by sulfurtransferases, to sulfhydryl groups of cysteine residues of Bcl-2 and in this way lower the level of active form of Bcl-2 by S-sulfuration. Diallyl trisulfide antioxidative effects result from an increased level of cystathionine, a precursor of cysteine, and an increased glutathione level. MPST and rhodanese, the level of which is increased in the presence of DATS, can serve as antioxidant proteins.

[Lysozyme--occurrence in nature, biological properties and possible applications]. / Lizozym--wystepowanie w przyrodzie, wlasciwosci biologiczne i mozliwosci zastosowan.

Lysozyme (LZ, muramidase, N-acetylmuramylhydrolase) is a protein occuring in animals, plants, bacteria and viruses. It can be found e.g. in granules of neutrophils, macrophages and in serum, saliva, milk, honey and hen egg white. The enzyme hydrolyzes the ß-1,4 glycosidic bonds between N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG) of cell wall peptidoglycan (PG) in Gram-positive and Gram-negative bacteria. In the animal kingdom, three muramidase types have been identified: the c-type (chicken type), the g-type (goose-type) and the i-type (invertebrates). The c-type LZ from hen egg white is a model for the study of protein structure and function. Muramidase shows bactericidal activity mainly against Gram-positive bacteria. Cytolytic activity against cells of Gram-negative bacteria has not been proved. Bacterial cells have developed defense mechanisms that allow them to avoid the action of LZ. They are based e.g. on the production of enzyme inhibitors or modification of the PG. LZ is one of the most studied enzymes and yet not all aspects characterizing this protein are fully understood. One of the most important unresolved issues concerning the biological function of LZ is the role of muramidase in the bactericidal action of serum against Gram-negative bacteria. In order to clarify the function of LZ, the enzyme is e.g. removed from the serum by adsorption onto bentonite (montmorillonite, MMT). By using X-ray diffraction techniques it has been shown that MMT after contact with the serum is delaminated. The problems associated with folding of muramidase and LZ participation in the development of amyloidoses also await explanation.

Current Non-Metal Nanoparticle-Based Therapeutic Approaches for Glioblastoma Treatment.

The increase in the variety of nano-based tools offers new possibilities to approach the therapy of poorly treatable tumors, which includes glioblastoma multiforme (GBM; a primary brain tumor). The available nanocomplexes exhibit great potential as vehicles for the targeted delivery of anti-GBM compounds, including chemotherapeutics, nucleic acids, and inhibitors. The main advantages of nanoparticles (NPs) include improved drug stability, increased penetration of the blood-brain barrier, and better precision of tumor targeting. Importantly, alongside their drug-delivery ability, NPs may also present theranostic properties, including applications for targeted imaging or photothermal therapy of malignant brain cells. The available NPs can be classified into two categories according to their core, which can be metal or non-metal based. Among non-metal NPs, the most studied in regard to GBM treatment are exosomes, liposomes, cubosomes, polymeric NPs, micelles, dendrimers, nanogels, carbon nanotubes, and silica- and selenium-based NPs. They are characterized by satisfactory stability and biocompatibility, limited toxicity, and high accumulation in the targeted tumor tissue. Moreover, they can be easily functionalized for the improved delivery of their cargo to GBM cells. Therefore, the non-metal NPs discussed here, offer a promising approach to improving the treatment outcomes of aggressive GBM tumors.

Enhancement of Temozolomide Stability and Anticancer Efficacy by Loading in Monopalmitolein-Based Cubic Phase Nanoparticles.

Temozolomide (TMZ) is a prodrug possessing a wide spectrum of anticancer activities. TMZ is pharmacologically inactive, but at a physiological pH, it is quickly converted to an active metabolite, 5-aminoimidazole-4-carboxamide, and a methyldiazonium cation. Due to its chemical nature, TMZ presents some capability of crossing the blood-brain barrier and therefore is used as a first-line agent in the treatment of gliomas. Here, we aimed to improve the anticancer effectiveness of TMZ by loading it into cubosomes, which are lipid nanoparticles recognized as efficient nano-based drug delivery systems. TMZ was incorporated into the monoolein (MO)- and monopalmitolein (MP)-derived cubic phases to improve its stability and half-life. It was considered that the drug release rate may vary between the MO and MP cubosomes, as the water channels of MP phases are larger than those of MO cubosomes. Therefore, we expected that due to the MPs' ability to entrap more drug molecules inside the mesophase, the concentration of TMZ available to cancer cells would be enhanced. This assumption was supported by biological analyses using the A-172 and drug-resistant T98G glioma-derived cell lines. The strongest reduction in viability was observed for A-172 cells treated with TMZ-loaded MP nanoparticles. Importantly, the TMZ-loaded MPs also caused a significant anticancer effect in the drug-resistant T98G glioma-derived cells. Both MO and MP empty cubic phases did not affect the survival of the tested cells. Concluding, TMZ-loaded cubosomes present strong anticancer properties. Encapsulating the drug within the lipid nanostructure helps to protect the drug from degradation and allows for greater accumulation of TMZ at the tumor site. Together with chemical-based features of mesophases related to increased cargo size and kinetic properties, we imply that MPs may be considered as a highly efficient nano-based drug delivery system to treat poorly curable tumors including gliomas.

Influence of mosquito-borne biological agents on health risks among soldiers and military personnel.

INTRODUCTION AND OBJECTIVE: Mosquitoes are the most important vector group for humans, and three genera - Aedes, Anopheles and Culex, are of greatest significance in the transmission of pathogens to humans and animals. The geographical expansion of vectors can lead to the spread diseases into new regions. Soldiers exercise in the field, participate in missions, or are stationed in Military Contingents located in different climatic conditions, which is directly related to exposure to mosquitoborne diseases. OBJECTIVE: The aim is to describe the role of mosquitoes in the transmission of selected pathogens of medical and epidemiological importance, which pose a new threat in Europe, pointing to soldiers and other military personnel as particularly vulnerable occupational groups. REVIEW METHODS: PubMed and other online resources and publications were searched to evaluate scientific relevance. BRIEF DESCRIPTION OF THE STATE OF KNOWLEDGE.: In recent years in Europe, attention has been drawn to emerging infectious diseases transmitted by mosquitoes, including malaria, Dengue fever, West Nile fever and Chikungunya fever. West Nile virus infections were recorded in many European countries, including Greece, Italy, Germany and Austria. Soldiers, due to their tasks, are particularly vulnerable to vector-borne diseases. In order to reduce the exposure of soldiers to mosquito-borne diseases various protection measures are used. SUMMARY: Some of vector-borne diseases belong to emerging infectious diseases and may pose a threat to public health. The burden on soldiers with these diseases can be significant, which is the reason why methods of surveillance and the control of vectors are being developed.

Resolving the role of podoplanin in the motility of papillary thyroid carcinoma-derived cells using RNA sequencing.

The intracellular level of podoplanin (PDPN), a transmembrane protein of still unclear function, is frequently altered in metastatic tumors. High expression of PDPN is frequently observed in papillary thyroid cancer (PTC) specimens. Similarly, PTC-derived cell lines (BCPAP and TPC1, harboring the BRAF V600E mutation and RET/PTC1 fusion, respectively), also present enhanced PDPN yield. We previously reported that depletion of PDPN impairs migration of TPC1 cells, but augments metastasis of BCPAP cells. Interestingly, this phenomenon stays in contrast to the migratory pattern observed for wild-type cells, where TPC1 exhibited higher motility than BCPAP cells. Here, we aimed to elucidate the potential role of PDPN in regulation of molecular mechanisms leading to the diverse metastatic features of the studied PTC-derived cells. We consider that this phenomenon may be caused by alternative regulation of signaling pathways due to the presence of the mutated BRAF allele or RET/PTC1 fusion. The high-throughput RNA sequencing (RNA-seq) technique was used to uncover the genes and signaling pathways affected in wild-type and PDPN-depleted TPC1 and BCPAP cells. We found that changes in the expression of various factors of signaling pathways, like RHOA and RAC1 GTPases and their regulators, are linked with both high PDPN levels and presence of the BRAF V600E mutation. We imply that the suppressed motility of wild-type BCPAP cells results from overactivation of RHOA through natively high PDPN expression. This process is accompanied by inhibition of the PI3K kinase and consequently RAC1, due to overactivation of RAS-mediated signaling and the PTEN regulator.

Cellular and molecular basis of thyroid autoimmunity.

Autoimmune thyroid disease (AITD) is the most common human autoimmune disease. The two major clinical manifestations of AITD are Graves' disease and Hashimoto's thyroiditis (HT). AITD is characterized by lymphocytic infiltration of the thyroid gland, leading either to follicular cell damage, thyroid gland destruction, and development of hypothyroidism (in HT) or thyroid hyperplasia, induced by thyroid antibodies which activate thyrotropin receptor (TSHR) on thyrocytes, leading to hyperthyroidism. The aim of this review is to present up-to-date picture of the molecular and cellular mechanisms that underlie the pathology of AITD. Based on studies involving patients, animal AITD models, and thyroid cell lines, we discuss the key events leading to the loss of immune tolerance to thyroid autoantigens as well as the signaling cascades leading to the destruction of thyroid gland. Special focus is given on the interplay between the environmental and genetic factors, as well as ncRNAs and microbiome contributing to AITD development. In particular, we describe mechanistic models by which SNPs in genes involved in immune regulation and thyroid function, such as CD40, TSHR, FLT3, and PTPN22, underlie AITD predisposition. The clinical significance of novel diagnostic and prognostic biomarkers based on ncRNAs and microbiome composition is also underscored. Finally, we discuss the possible significance of probiotic supplementation on thyroid function in AITD.

Simvastatin Coadministration Modulates the Electrostatically Driven Incorporation of Doxorubicin into Model Lipid and Cell Membranes.

Understanding the interactions between drugs and lipid membranes is a prerequisite for finding the optimal way to deliver drugs into cells. Coadministration of statins and anticancer agents has been reported to have a positive effect on anticancer therapy. In this study, we elucidate the mechanism by which simvastatin (SIM) improves the efficiency of biological membrane penetration by the chemotherapeutic agent doxorubicin (DOX) in neutral and slightly acidic solutions. The incorporation of DOX, SIM, or a combination of them (DOX:SIM) into selected single-component lipid membranes, zwitterionic unsaturated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), neutral cholesterol, and negatively charged 1,2-dimyristoyl-sn-glycero-3-phospho-l-serine (DMPS) was assessed using the Langmuir method. The penetration of neutral lipid monolayers by the codelivery of SIM and DOX was clearly facilitated at pH 5.5, which resembles the pH conditions of the environment of cancer cells. This effect was ascribed to partial neutralization of the DOX positive charge as the result of intermolecular interactions between DOX and SIM. On the other hand, the penetration of the negatively charged DMPS monolayer was most efficient in the case of the positively charged DOX. The efficiency of the drug delivery to the cell membranes was evaluated under in vitro conditions using a panel of cancer-derived cell lines (A172, T98G, and HeLa). MTS and trypan blue exclusion assays were performed, followed by confocal microscopy and spheroid culture tests. Cells were exposed to either free drugs or drugs encapsulated in lipid carriers termed cubosomes. We demonstrated that the viability of cancer cells exposed to DOX was significantly impaired in the presence of SIM, and this phenomenon was greatly magnified when DOX and SIM were coencapsulated in cubosomes. Overall, our results confirmed the utility of the DOX:SIM combination delivery, which enhances the interactions between neutral components of cell membranes and positively charged chemotherapeutic agents.

Sulfurtransferases and Cystathionine Beta-Synthase Expression in Different Human Leukemia Cell Lines.

The studies concerned the expression of sulfurtransferases and cystathionine beta-synthase in six human leukemia cell lines: B cell acute lymphoblastic leukemia-B-ALL (REH cells), T cell acute lymphoblastic leukemia-T-ALL (DND-41 and MOLT-4 cells), acute myeloid leukemia-AML (MV4-11 and MOLM-14 cells), and chronic myeloid leukemia-CML (K562 cells). Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis were performed to determine the expression of thiosulfate sulfurtransferase, 3-mercaptopyruvate sulfurtransferase, gamma-cystathionase, and cystathionine beta-synthase on the mRNA and protein level. Interestingly, we found significant differences in the mRNA and protein levels of sulfurtransferases and cystathionine beta-synthase in the studied leukemia cells. The obtained results may contribute to elucidating the significance of the differences between the studied cells in the field of sulfur compound metabolism and finding new promising ways to inhibit the proliferation of various types of leukemic cells by modulating the activity of sulfurtransferases, cystathionine beta-synthase, and, consequently, the change of intracellular level of sulfane sulfur as well as H2S and reactive oxygen species production.

The Role of miRNA-7 in the Biology of Cancer and Modulation of Drug Resistance.

MicroRNAs (miRNAs, miRs) are small non-coding RNA (ncRNA) molecules capable of regulating post-transcriptional gene expression. Imbalances in the miRNA network have been associated with the development of many pathological conditions and diseases, including cancer. Recently, miRNAs have also been linked to the phenomenon of multidrug resistance (MDR). MiR-7 is one of the extensively studied miRNAs and its role in cancer progression and MDR modulation has been highlighted. MiR-7 is engaged in multiple cellular pathways and acts as a tumor suppressor in the majority of human neoplasia. Its depletion limits the effectiveness of anti-cancer therapies, while its restoration sensitizes cells to the administered drugs. Therefore, miR-7 might be considered as a potential adjuvant agent, which can increase the efficiency of standard chemotherapeutics.

Targeting the MAPK/ERK and PI3K/AKT Signaling Pathways Affects NRF2, Trx and GSH Antioxidant Systems in Leukemia Cells.

The mitogen-activated protein kinase (MAPK)/extracellular signal kinase (ERK) and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signal transduction pathways have been implicated in the pathogenesis of leukemia. The aim of this study was to investigate the effect of the combination of ERK1/2 inhibitor AZD0364 and PI3K inhibitor ZSTK474 on acute lymphoblastic leukemia (ALL) REH, MOLT-4, acute myeloid leukemia (AML) MOLM-14, and chronic myeloid leukemia (CML) K562 cell lines. To evaluate the interactions of the drugs, cells were treated for 48 h with AZD0364 or ZSTK474 alone and in combination at fixed ratios. The combinatorial effects of both inhibitors were synergistic over a wide range of concentrations in REH, MOLT-4, and MOLM-14 cell lines. However, in K562 cells, the effects were found to be antagonistic. Furthermore, AZD0364 and ZSTK474 significantly decreased both ERK1/2 and AKT activation in REH, MOLT-4, and MOLM-14 cells. The results showed that incubation with both AZD0364 and ZSTK474 inhibited cell viability, increased reactive oxygen species (ROS) production, and induced apoptosis in leukemia cells. We observed that combined treatment with AZD0364 and ZSTK474 affected nuclear factor-κB (NF-κB) and antioxidant protein levels: NF-E2-related factor 2 (NRF2), heme oxygenase-1 (HO-1), thioredoxin (Trx), thioredoxin reductase (TrxR), and the reduced glutathione/oxidized glutathione (GSH/GSSG) ratio. These effects were accompanied with decreased antiapoptotic survivin protein level. However, distinct cell line dependent effects were observed. In conclusion, the combination of AZD0364 and ZSTK474 can exert a synergistic anticancer effect in ALL and AML cells, which is associated with the induction of oxidative stress and the involvement of cellular antioxidant defense mechanisms.