Inhibition Effect of Chloroquine and Integrin-Linked Kinase Knockdown on Translation in Melanoma Cells.

The twofold role of autophagy in cancer is often the therapeutic target. Numerous regulatory pathways are shared between autophagy and other molecular processes needed in tumorigenesis, such as translation or survival signaling. Thus, we have assumed that ILK knockdown should promote autophagy, and used together with chloroquine, an autophagy inhibitor, it could generate a better anticancer effect by dysregulation of common signaling pathways. Expression at the protein level was analyzed using Western Blot; siRNA transfection was done for ILK. Analysis of cell signaling pathways was monitored with phospho-specific antibodies. Melanoma cell proliferation was assessed with the crystal violet test, and migration was evaluated by scratch wound healing assays. Autophagy was monitored by the accumulation of its marker, LC3-II. Our data show that ILK knockdown by siRNA suppresses melanoma cell growth by inducing autophagy through AMPK activation, and simultaneously initiates apoptosis. We demonstrated that combinatorial treatment of melanoma cells with CQ and siILK has a stronger antitumor effect than monotherapy with either of these. It generates the synergistic antitumor effects by the decrease of translation of both global and oncogenic proteins synthesis. In our work, we point to the crosstalk between translation and autophagy regulation.

Expression of Alternative Splice Variants of 6-Phosphofructo-2-kinase/Fructose-2,6-bisphosphatase-4 in Normoxic and Hypoxic Melanoma Cells.

Cancer-specific isoenzyme of phosphofructokinase II (PFKFB4), as our previous research has shown, may be one of the most important enzymes contributing to the intensification of glycolysis in hypoxic malignant melanoma cells. Although the PFKFB4 gene seems to play a crucial role in the progression of melanoma, so far there are no complete data on the expression of PFKFB4 at the isoform level and the influence of hypoxia on alternative splicing. Using RT-qPCR and semi-quantitative RT-PCR, we presented the PFKFB4 gene expression profile at the level of six isoforms described in the OMIM NCBI database in normoxic and hypoxic melanoma cells. Additionally, using VMD software, we analyzed the structure of isoforms at the protein level, concluding about the catalytic activity of individual isoforms. Our research has shown that five isoforms of PFKFB4 are expressed in melanoma cells, of which the D and F isoforms are highly constitutive, while the canonical B isoform seems to be the main isoform induced in hypoxia. Our results also indicate that the expression profile at the level of the PFKFB4 gene does not reflect the expression at the level of individual isoforms. Our work clearly indicates that the PFKFB4 gene expression profile should be definitely analyzed at the level of individual isoforms. Moreover, the analysis at the protein level allowed the selection of those isoforms whose functional validation should be performed to fully understand the importance of PFKFB4 expression in the metabolic adaptation of malignant melanoma cells.

AFM-based Analysis of Wharton's Jelly Mesenchymal Stem Cells.

Wharton's jelly mesenchymal stem cells (WJ-MSCs) are multipotent stem cells that can be used in regenerative medicine. However, to reach the high therapeutic efficacy of WJ-MSCs, it is necessary to obtain a large amount of MSCs, which requires their extensive in vitro culturing. Numerous studies have shown that in vitro expansion of MSCs can lead to changes in cell behavior; cells lose their ability to proliferate, differentiate and migrate. One of the important measures of cells' migration potential is their elasticity, determined by atomic force microscopy (AFM) and quantified by Young's modulus. This work describes the elasticity of WJ-MSCs during in vitro cultivation. To identify the properties that enable transmigration, the deformability of WJ-MSCs that were able to migrate across the endothelial monolayer or Matrigel was analyzed by AFM. We showed that WJ-MSCs displayed differences in deformability during in vitro cultivation. This phenomenon seems to be strongly correlated with the organization of F-actin and reflects the changes characteristic for stem cell maturation. Furthermore, the results confirm the relationship between the deformability of WJ-MSCs and their migration potential and suggest the use of Young's modulus as one of the measures of competency of MSCs with respect to their possible use in therapy.

Integrin-linked kinase regulates cadherin switch in bladder cancer.

Cadherin switch is specific of epithelial-mesenchymal transition (EMT) and is closely related to tumor cell invasion. However, the molecular mechanism that promotes the phenotypic changes remains unclear and elusive. We found that integrin-linked kinase (ILK) is a key factor involved in cadherin switch. The expression and activity of ILK are elevated in a variety of cancers but its mechanisms are not exactly understood. In this report, we studied the role and mechanism of ILK in EMT of human bladder cancer. We showed that silencing of ILK expression by small interfering RNA (siRNA) significantly abolished the nuclear translocation or the presence of markers associated with EMT like Snail, Twist, Zeb, and beta-catenin. ILK knockdown by siRNA suppressed N-cadherin expression and increased re-expression of E-cadherin in bladder cancer cells. We suggest that ILK is a major signaling factor involved in EMT. It is essential to understand the molecular mechanism of EMT in aim to possibly use it in search for new therapeutic targets.

A comparative study of glycoproteomes in androgen-sensitive and -independent prostate cancer cell lines.

Prostate cancer is one of the most common malignancies in men and is predicted to be the second leading cause of cancer-related deaths. After 6-18 months, hormone ablation treatment results in androgen-independent growth of cancer cells, metastasis and progression. The mechanism of androgen-independent growth of prostatic carcinoma cells is still unknown. Identification of factors that facilitate the transition from androgen-dependent to independent states is crucial in designing future diagnostics and medication strategies. To understand the biochemical meaning of hormone dependency deprivation, glycoproteins enriched profiles were compared between DU145 (hormone non-responding) and LNCaP (hormone responding) prostate cancer cells. These results allow for anticipation on the important role of glycosylation in malignant transformation. Both Tn antigen and complex antennary N-oligosaccharides were recognized. Their occurrence might be involved in the development and progression of tumor, and failure of hormone ablation therapy. Among identified proteins in androgen-sensitive cells nucleolin (P19338) was found that is widely described as apoptosis inhibitor, and also transporter of molecules from the membrane to the cytoplasm or nucleus. In addition, 14-3-3 protein family (P27348, P31946, P61981, P63104, P62258, Q04917, and P31947) was investigated across available databases as it forms stable complexes with glycoproteins. Our studies indicate that isoforms: sigma and eta were found in androgen-dependent prostate cancer cells, while other isoforms were present in androgen non-responding cells. 14-3-3 binding partners are involved in cancer pathogenesis. These findings may contribute to a better understanding of prostate cancer tumorigenesis and to a more efficient prognosis and individual therapy in a future. However, it still remains to be revealed how important those changes are for androgen dependency loss in prostate cancer patients carried out on clinically relevant populations.

Tanespimycin and tipifarnib exhibit synergism in inducing apoptosis in melanoma cell lines from later stages of tumor progression.

Many anticancer strategies rely on efficient induction of apoptosis. The need for development of drug combinations with a strong pro-apoptotic activity is of particular interest in melanoma resistant to currently available chemotherapeutic regimes. We studied the pro-apoptotic properties of combination of tanespimycin+tipifarnib in five melanoma cell lines representing various stages of tumor progression. Our results show that in cells derived from vertical- and metastatic-phase the combination of tested drugs is strongly cytotoxic and efficient in inducing apoptosis, as evidenced by activation of caspase-9 and caspase-3 and enhanced fragmentation of DNA.

Cancer cell detection in tissue sections using AFM.

Currently, cancer diagnosis relies mostly on morphological examination of exfoliated, aspirated cells or surgically removed tissue. As long as standard diagnosis is concerned, this classical approach seems to be satisfactory. In the recent years, cancer progression has been shown to be accompanied by alterations in mechanical properties of cells. This offers the detection of otherwise unnoticed cancer cell disregarded by histological analysis due to insignificant manifestations. One of techniques, sensitive to changes in mechanical properties, is the atomic force microscopy, which detects cancer cells through their elastic properties. Such measurements were applied to tissue sections collected from patients suffering from various cancers. Despite of heterogeneity and complexity of cancer cell sections, the use of the Young's modulus as an indicator of cell elasticity allow for detection of cancer cells in tissue slices.

Effects of kinetin riboside on proliferation and proapoptotic activities in human normal and cancer cell lines.

Kinetin riboside (KR) is a N6-substituted derivative of adenosine. It is a natural compound which occurs in the milk of coconuts on the nanomole level. KR was initially shown to selectively inhibit proliferation of cancer cells and induce their apoptosis. We observed that KR inhibited growth (20-80%) of not only human cancer, but also normal cells and that this effect strongly depended on the type of cells. The anti-apoptotic Bcl-2 protein was downregulated, while proapoptotic Bax was upregulated in normal as well as in cancer cell lines, upon exposure to KR. Cytochrome c level increased in the cytosol upon treatment of cells with KR. The activity of caspases (ApoFluor®Green Caspase Activity Assay), as well as caspase-3 (caspase-3 activation assay) were increased mainly in cancer cells. The expression of procaspase 9 and its active form in the nucleus as well as in cytosol of KR-treated cells was elevated. In contrast, no effect of KR on caspase 8 expression was noted. The results indicated that non-malignant cells were less sensitive to KR then their cancer analogs and that KR most likely stimulated apoptosis mechanism of cancer cells through the intrinsic pathway.

Characterization of N-cadherin unbinding properties in non-malignant (HCV29) and malignant (T24) bladder cells.

The expression of N-cadherin, characteristic of various cancers, very often leads to changes in the cells' adhesive properties. Thus, we sought to find out if N-cadherin expressed in various, but cancer-related cells, differs in its functional properties that could contribute to variations in cells' phenotypes. In our work, measurements of an unbinding force of a single N-cadherin molecule, probed with the same antibody both on a surface of living non-malignant (HCV29) and malignant cells (T24) of bladder cancer, were carried out with the use of an atomic force microscopy. The results show the enhanced N-cadherin level in T24 malignant cells (8.7% vs. 3.6% obtained for non-malignant one), confirmed by the Western blot and the immunohistochemical staining. The effect was accompanied by changes in unbinding properties of an individual N-cadherin molecule. Lower unbinding force values (26.1 ± 7.1 pN) in non-malignant cells reveal less stable N-cadherin complexes, as compared to malignant cells (61.7 ± 14.6 pN). This suggests the cancer-related changes in a structure of the binding site of the antibody, located at the extracellular domain of N-cadherin.

[Beta-carotene regulates the expression of proapoptotic BAX and CAPN2 in HL-60, U-937 and TF-1 - human acute myeloid leukemia cell lines; microarray, RQ-PCR and Western Blot analysis]. / Wplyw beta-karotenu na ekspresje proapoptotycznych genów BAX i CAPN2 w komórkach linii ostrej bialaczki szpikowej HL-60, U-937 i TF-1; analiza technika mikromacierzy, ilosciowej PCR i Western Blot.

Beta carotene (BC) is a nutritional compound widespread in foods which can influence vital cellular functions--differentiation, proliferation and apoptosis of normal and cancer cells. However its role in the carcinogenesis remains controversial. We performed a microarray expression analysis in three human acute leukemia cell lines (HL-60, U937 and TF-1) exposed to 10mM BC and found that BC stimulated the apoptosis in all studied cell lines. This effect was most evident in the HL-60 cell line and correlated with increased expression of proapoptotic BAX and CAPN2 genes. The micro-array findings were replicated by the quantitative BAX and CAPN2 expression analysis using real-time PCR and by Western Blot on protein level. The biological tests (TUNEL method) for apoptosis showed consistent proapoptotic effects in all studied cell lines. In this paper the stimulatory effect of BC on apoptosis (enhanced expression of proapoptotic genes and proteins) in human acute myeloid leukemia cells was confirmed. The most potent activation of apoptosis in the HL-60 cells is in line with other investigators observations suggesting distinct molecular mechanism of apoptosis stimulation by BC in different human acute myeloid leukemia cells.

Recent Progress in Discovering the Role of Carotenoids and Their Metabolites in Prostatic Physiology and Pathology with a Focus on Prostate Cancer-A Review-Part I: Molecular Mechanisms of Carotenoid Action.

Among the vast variety of plant-derived phytochemicals, the group of carotenoids has continuously been investigated in order to optimize their potential application in the area of dietary intervention and medicine. One organ which has been especially targeted in many of these studies and clinical trials is the human prostate. Without doubt, carotenoids (and their endogenous derivatives-retinoids and other apo-carotenoids) are involved in intra- and intercellular signaling, cell growth and differentiation of prostate tissue. Due to the accumulation of new data on the role of different carotenoids such as lycopene (LC) and ß-carotene (BC) in prostatic physiology and pathology, the present review aims to cover the past ten years of research in this area. Data from experimental studies are presented in the first part of the review, while epidemiological studies are disclosed and discussed in the second part. The objective of this compilation is to emphasize the present state of knowledge regarding the most potent molecular targets of carotenoids and their main metabolites, as well as to propose promising carotenoid agents for the prevention and treatment of prostatic diseases.

Stiffening of DU145 prostate cancer cells driven by actin filaments - microtubule crosstalk conferring resistance to microtubule-targeting drugs.

The crucial role of microtubules in the mitotic-related segregation of chromosomes makes them an excellent target for anticancer microtubule targeting drugs (MTDs) such as vinflunine (VFL), colchicine (COL), and docetaxel (DTX). MTDs affect mitosis by directly perturbing the structural organisation of microtubules. By a direct assessment of the biomechanical properties of prostate cancer DU145 cells exposed to different MTDs using atomic force microscopy, we show that cell stiffening is a response to the application of all the studied MTDs (VFL, COL, DTX). Changes in cellular rigidity are typically attributed to remodelling of the actin filaments in the cytoskeleton. Here, we demonstrate that cell stiffening can be driven by crosstalk between actin filaments and microtubules in MTD-treated cells. Our findings improve the interpretation of biomechanical data obtained for living cells in studies of various physiological and pathological processes.

Integrin linked kinase regulates endosomal recycling of N-cadherin in melanoma cells.

Malignant transformation is characterized by a phenotype "switch" from E- to N-cadherin - a major hallmark of epithelial to mesenchymal transition (EMT). The increased expression of N-cadherin is commonly followed by a growing capacity for migration as well as resistance to apoptosis. Integrin Linked Kinase (ILK) is a key molecule involved in EMT and progression of cancer cells. ILK is known as a major signaling mediator involved in cadherin switch, but the specific mechanism through which ILK modulates N-cadherin expression is still not clear. Studies were carried out on human melanoma WM793 and 1205Lu cell lines. Expression of proteins was analyzed using PCR and Western Blot; siRNA transfection was done for ILK. Analysis of cell signaling pathways was monitored with phospho-specific antibodies. Subcellular localization of protein was studied using the ProteoExtract Subcellular Kit and Western blot analysis. Our data show that ILK knockdown by siRNA did suppress N-cadherin expression in melanoma, but only at the protein level. The ILK silencing-induced decrease of N-cadherin membranous expression in melanoma highlights the likely crucial role of ILK in the coordination of membrane trafficking through alteration of Rab expression. It is essential to understand the molecular mechanism of increased N-cadherin expression in cancer to possibly use it in the search of new therapeutic targets.

mTOR inhibitor everolimus reduces invasiveness of melanoma cells.

The mammalian target of rapamycin (mTOR) plays a key role in several cellular processes: proliferation, survival, invasion, and angiogenesis, and therefore, controls cell behavior both in health and in disease. Dysregulation of the mTOR signaling is involved in some of the cancer hallmarks, and thus the mTOR pathway is an important target for the development of a new anticancer therapy. The object of this study is recognition of the possible role of mTOR kinase inhibitors-everolimus single and in combination with selected downstream protein kinases inhibitors: LY294002 (PI3 K), U0126 (ERK1/2), GDC-0879 (B-RAF), AS-703026 (MEK), MK-2206 (AKT), PLX-4032 (B-RRAF) in cell invasion in malignant melanoma. Treatment of melanoma cells with everolimus led to a significant decrease in the level of both phosphorylated: mTOR (Ser2448) and mTOR (Ser2481) as well as their downstream effectors. The use of protein kinase inhibitors produced a significant decrease in metalloproteinases (MMPs) activity, as well as diminished invasion, especially when used in combination. The best results in the inhibition of both MMPs and cell invasiveness were obtained for the combination of an mTOR inhibitor- everolimus with a B-RAF inhibitor-PLX-4032. Slightly less profound reduction of invasiveness was obtained for the combinations of an mTOR inhibitor-everolimus with ERK1/2 inhibitor-U126 or MEK inhibitor-AS-703026 and in the case of MMPs activity decrease for PI3 K inhibitor-LY294002 and AKT inhibitor-MK-2206. The simultaneous use of everolimus or another new generation rapalog with selected inhibitors of crucial signaling kinases seems to be a promising concept in cancer treatment.

Probing the recognition specificity of αVß1 integrin and syndecan-4 using force spectroscopy.

The knowledge on how cells interact with microenvironment is particularly important in understanding the interaction of cancer cells with surrounding stroma, which affects cell migration, adhesion, and metastasis. The main cell surface receptors responsible for the interaction with extracellular matrix (ECM) are integrins, however, they are not the only ones. Integrins are accompanied to other molecules such as syndecans. The role of the latter has not yet been fully established. In our study, we would like to answer the question of whether integrins and syndecans, possessing similar functions, share also similar unbinding properties. By using single molecule force spectroscopy (SMFS), we conducted measurements of the unbinding properties of αVß1 and syndecan-4 in the interaction with vitronectin (VN), which, as each ECM protein, possesses two binding sites specific to integrins and syndecans. The unbinding force and the kinetic off rate constant derived from SMFS describe the stability of single molecular complex. Obtained data show one barrier transition for each complex. The proposed model shows that the unbinding of αVß1 from VN proceeds before the unbinding of SDC-4. However, despite different unbinding kinetics, the access to both receptors is needed for cell growth and proliferation.

The new face of nucleolin in human melanoma.

Nucleolin is multifunctional protein mainly present in nucleoli but also detected in cytoplasm and plasma membranes. Extranuclear nucleolin differs from the nuclear form by its glycosylation. Studies on expression of nucleolin in breast cancer suggest a possible association to the metastatic cascade. In the present study, Vicia villosa lectin (VVL) precipitation followed by subsequent polyacrylamide gel electrophoresis and mass spectrometry analysis demonstrates nucleolin as a VVL-positive glycoprotein expressed in melanoma. The presence of VVL-positive nucleolin in the melanoma cell membrane and cytoplasm was confirmed by confocal microscopy. Using bioinformatic peptide prediction programs, nucleolin was shown to contain multiple possible MHC class-I binding peptides in its sequence which makes nucleolin an interesting melanoma marker and target for immunodiagnostic and possibly therapeutic purposes.

[Effect of conjugated linoleic acid isomers on proliferation of mammary cancer cells]. / Wplyw izomerów sprzezonego kwasu linolowego na proliferacje komórek nowotworowych piersi.

Conjugated linoleic acid (CLA) is a collective term describing a mixture of positional and geometric isomers of linoleic acid with two conjugated double bonds. Among these compounds two isomers cis9, trans11 and trans10, cis12 have received considerable attention. They are present in milk and meat fat of ruminants and the cis9, trans11 (rumenic acid) is the most abundant with lesser amounts of the trans10, cis12 isomer. A considerable number of papers suggest anticarcinogenic properties of CLA, including their ability to suppress the growth of different cancer cell lines in vitro. It was also found that these isomers may act through different mechanisms to inhibit carcinogenesis. In view of the above, the objective of this paper was evaluation of isomer-specific effects of the natural CLA isomers i.e. cis9, trans11 and trans10, cis12 on proliferation of mammary cancer cell lines. Two mammary cancer cell lines were used: MCF-7 and T47D (ATCC Collection). The cells were incubated with both CLA isomers: cis9,trans11 or trans10, cis12 within the range of 5-200 microM for 24-120 h. There were no toxic effects of any of the isomers in the range of 5-100 microM, as indicated by the Cytotoxicity Detection Kit (Roche) and cells proliferation was determined in these experimental conditions. Proliferation of cells was determined using Cell Proliferation ELISA BrdU (5' bromo-2' deoxyuridine), based on incorporation of BrdU to DNA of growing cells. The results obtained indicate that both isomers suppress the proliferation of the studied mammary cancer cell lines i.e. MCF-7 and T47D, especially when treated with the CLA isomers for 48 h or longer. Of the studied lines the strongest growth-suppressing function approximately 65% was observed for the line T47D.

mTOR inhibitor Everolimus-induced apoptosis in melanoma cells.

Melanoma is the most aggressive, therapy-resistant skin cancer. The mammalian target of rapamycin (mTOR), the serine/threonine kinase which integrates both intracellular and extracellular signals, plays a crucial role in coordinating the balance between the growth and death of cells. The object of this study is a comparison of the influence of mTOR inhibitor everolimus in the concentration range between 20 nM and 10 µM, used individually and in combination with selected downstream protein kinases inhibitors: LY294002 (PI3K), U0126 (ERK1/2), AS-703026 (MEK) and MK-2206 (AKT) on the expression of pro-survival proteins: p-Bcl-2 (S70), p-Bcl-2 (T56), Bcl-2, Bcl-xL, Mcl-1, activity of caspase-3, proliferation and induction of apoptosis in melanoma cells. Current results clearly show that the nanomolar concentration of the mTOR inhibitor everolimus in combination with the inhibitor of MAP kinase (AS-703026) or AKT kinase (MK-2206) is effective in inducing apoptosis and reducing proliferation of melanoma cells. The herein research results confirm the hypothesis on the important role of mTOR signaling in cancer progression, and gives hope that implementation of successful combination of its inhibitors will find recognition and application in cancer treatment in the near future.