Expression of STAT3 and hypoxia markers in long-term surviving malignant glioma patients.

BACKGROUND: Glioblastoma is a malignant and aggressive type of central nevous system malignancy characterized by many distinct biological features including extensive hypoxia. Hypoxia in glioblatoma associates with complex signaling patterns including activation of several pathways such as MAPK, PI3K-AKT/mTOR and IL-6/JAK/STAT3 with the master regulator HIF-1, which in turn drive particular tumor behaviors determining, in the end, treatment outcomes and patients fate. Thus, the present study was designed to investigate the expression of selected hypoxia related factors including STAT3 in a small set of long-term surviving glioma patients. METHODS: The expression of selected hypoxia related factors including STAT3 was evaluated in a time series of formalin fixed paraffin embedded and cryopreserved glioma samples from repeatedly resected patients. In addition, comparative studies were also conducted on primary glioma cells derived from original patient samples, stabilized glioma cell lines and tumor-xenograft mice model. Obtained data were correlated with clinical findings too. RESULTS: Glioblastoma samples of the analyzed patients displayed heterogeneity in the expression of hypoxia- related and EMT markers with most interesting trend being observed in pSTAT3. This heterogeneity was subsequently confirmed in other employed models (primocultures derived from glioblastoma tissue resections, cryopreserved tumor specimens, stabilized glioblastoma cell line in vitro and in vivo) and concerned, in particular, STAT3 expression which remained stable. In addition, subsequent studies on the role of STAT3 in the context of glioblastoma hypoxia demonstrated opposing effects of its deletion on cell viability as well as the expression of hypoxia and EMT markers. CONCLUSIONS: Our results suport the importance of STAT3 expression and activity in the context of hypoxia in malignant glioblastoma long-term surviving glioma patients while emphasizing heterogeneity of biological outcomes in varying employed tumor models.

Comparative Analysis of miRNA and EMT Markers in Metastatic Colorectal Cancer.

Colorectal cancer (CRC) is the fourth most commonly diagnosed malignant condition in the world. Micro RNAs (miRNAs) as well as epithelial to mesenchymal transition (EMT) play an important role in the pathogenesis of CRC. We performed a comparative analysis of the expression of selected miRNA genes and EMT markers in bioptic samples from patients (n = 45) with primary CRC or metastatic (m)CRC to the regional lymph node using reverse transcription-quantitative PCR and IHC staining. Results: Out of all miRNA analyzed, the miR-17 expression was most significantly different and associated with lower risk of CRC spread to the lymph node. In addition, significant relationships were found between the tumor side localization and several miRNAs expressions (miR-9, miR-29b, miR-19a, miR-19b, miR-21, miR-106a, miR-20a and miR-17). In addition, of the examined EMT markers, only VEGFA expression correlated with tumor progression (tumor grade G2). In the examined set of patient samples and their matched healthy tissue, several specific molecular markers (miRNAs associated with EMT and tumor progression) were identified with a promising prognostic potential. Their further examination in larger patient cohorts is planned to validate the present data.

Flubendazole exhibits anti-glioblastoma effect by inhibiting STAT3 and promoting cell cycle arrest.

Glioblastoma multiforme (GBM) belongs to most aggressive and invasive primary brain tumor in adults whose prognosis and survival remains poor. Potential new treatment modalities include targeting the cytoskeleton. In our study, we demonstrated that repurposed drug flubendazole (FLU) significantly inhibits proliferation and survival of GBM cells. FLU exerted its effect by affecting microtubule structure and our results also suggest that FLU influences tubulins expression to a certain degree. Moreover, FLU effects decreased activation of STAT3 and also partially inhibited its expression, leading to upregulation of p53 signaling pathway and subsequent cell cycle arrest at G2/M phase as well as caspase-dependent cell death in GBM cells. These results suggest FLU as a promising agent to be used in GBM treatment and prompting further testing of its effects on GBM.

Role of N-Cadherin in Epithelial-to-Mesenchymal Transition and Chemosensitivity of Colon Carcinoma Cells.

(1) Background: N-cadherin expression, epithelial-to-mesenchymal transition (EMT) and aggressive biological phenotype of tumor cells are linked although the underlying mechanisms are not entirely clear. (2) Methods: In this study, we used two different in vitro cell models with varying N-cadherin expression (stabilized lines and primocultures) and investigated their select biological features including the degree of their chemoresistance both in vitro as well as in vivo. (3) Results: We report that although enforced N-cadherin expression changes select morphological and behavioral characteristics of exposed cells, it fails to successfully reprogram cells to the aggressive, chemoresistant phenotype both in vitro as well as in vivo as verified by implantation of those cells into athymic mice. Conversely, primocultures of patient-colonic cells with naturally high levels of N-cadherin expression show fully aggressive and chemoresistant phenotype pertinent to EMT (in vitro and in vivo), with a potential to develop new mutations and in the presence of dysregulated regulatory pathways as represented by investigated miRNA profiles. (4) Conclusions: The presented results bring new facts concerning the functional axis of N-cadherin expression and related biological features of colon cancer cells and highlight colon cancer primocultures as a useful model for such studies.

Evaluating the Use of TiO2 Nanoparticles for Toxicity Testing in Pulmonary A549 Cells.

Purpose: Titanium dioxide nanoparticles, 25 nm in size of crystallites (TiO2 P25), are among the most produced nanomaterials worldwide. The broad use of TiO2 P25 in material science has implied a request to evaluate their biological effects, especially in the lungs. Hence, the pulmonary A549 cell line has been used to estimate the effects of TiO2 P25. However, the reports have provided dissimilar results on caused toxicity. Surprisingly, the physicochemical factors influencing TiO2 P25 action in biological models have not been evaluated in most reports. Thus, the objective of the present study is to characterize the preparation of TiO2 P25 for biological testing in A549 cells and to evaluate their biological effects. Methods: We determined the size and crystallinity of TiO2 P25. We used four techniques for TiO2 P25 dispersion. We estimated the colloid stability of TiO2 P25 in distilled water, isotonic NaCl solution, and cell culture medium. We applied the optimal dispersion conditions for testing the biological effects of TiO2 P25 (0-100 µg.mL-1) in A549 cells using biochemical assays (dehydrogenase activity, glutathione levels) and microscopy. Results: We found that the use of fetal bovine serum in culture medium is essential to maintain sufficient colloid stability of dispersed TiO2 P25. Under these conditions, TiO2 P25 were unable to induce a significant impairment of A549 cells according to the results of biochemical and microscopy evaluations. When the defined parameters for the use of TiO2 P25 in A549 cells were met, similar results on the biological effects of TiO2 P25 were obtained in two independent cell laboratories. Conclusion: We optimized the experimental conditions of TiO2 P25 preparation for toxicity testing in A549 cells. The results presented here on TiO2 P25-induced cellular effects are reproducible. Therefore, our results can be helpful for other researchers using TiO2 P25 as a reference material.

Induced Zinc Loss Produces Heterogenous Biological Responses in Melanoma Cells.

Zinc levels in serum and/or tissue are reported to be altered in melanoma with unknown effects on melanoma development and biology. The purpose of this study was to examine the effects of acute chelation of free intracellular zinc pools in melanoma cell lines Bowes and A375, as well as selected melanoma tissue explants with high or low intracellular free zinc. Zinc chelating agent TPEN at the concentration of 25 µM was employed during 48 h, which significantly reduced intracellular free zinc while decreasing melanoma cell proliferation, inducing G1/S arrest and cell damage leading to mitochondrial, caspase-dependent apoptosis. Chelation of free zinc was also associated with increased generation of superoxide in cell lines but not marked lysosomal membrane damage. Conversely, melanoma explant cultures mostly displayed time-dependent loss of lysosomal membrane integrity in the presence of slowly growing superoxide levels. Loss of free zinc-dependent p53 activity was similarly disparate in individual melanoma models. Surviving melanoma cells were arrested in the cell cycle, and varying proportions of them exhibited features characteristic of premature senescence, which increased in time despite zinc reloading. The present results show that melanoma cells with varying free zinc levels respond to its acute loss in a number of individual ways, reflecting activated mechanisms including oxidative stress, lysosomal damage, and p53 activity leading to heterogenous outcomes including cell death, transient, and/or permanent cell cycle arrest and premature senescence.

The Effect of Chronic Exposure of Graphene Nanoplates on the Viability and Motility of A549 Cells.

Graphene and its derivatives are popular nanomaterials used worldwide in many technical fields and biomedical applications. Due to such massive use, their anticipated accumulation in the environment is inevitable, with a largely unknown chronic influence on living organisms. Although repeatedly tested in chronic in vivo studies, long-term cell culture experiments that explain the biological response to these nanomaterials are still scarce. In this study, we sought to evaluate the biological responses of established model A549 tumor cells exposed to a non-toxic dose of pristine graphene for eight weeks. Our results demonstrate that the viability of the A549 cells exposed to the tested graphene did not change as well as the rate of their growth and proliferation despite nanoplatelet accumulation inside the cells. In addition, while the enzymatic activity of mitochondrial dehydrogenases moderately increased in exposed cells, their overall mitochondrial damage along with energy production changes was also not detected. Conversely, chronic accumulation of graphene nanoplates in exposed cells was detected, as evidenced by electron microscopy associated with impaired cellular motility.

Silencing of E-cadherin expression leads to increased chemosensitivity to irinotecan and oxaliplatin in colorectal cancer cell lines.

Colorectal carcinoma (CRC) is a leading malignant disease in most developed countries. In advanced stages it presents with metastatic dissemination and significant chemoresistance. Despite intensive studies, no convincing evidence has been published concerning the association of cadherins and epithelial-mesenchymal transition (EMT) as a direct cause of acquired chemoresistance in CRC. The present study was designed to investigate the role of E-cadherin in EMT and its associated chemosensitivity/chemoresistance in four immortalized CRC cell lines representing various stages of CRC development (i.e. HT29 and Caco-2-early, SW480 and SW620 late). The expression of E-cadherin gene CDH1 was downregulated by the specific siRNA. Cell proliferation and chemosensitivity to irinotecan (IT) and oxaliplatin (OPT) were detected using WST-1 and x-CELLigence Real Time analysis. Expression of selected EMT markers were tested and compared using RT-PCR and western blot analysis in both variants (E-cadherin silenced and non-silenced) of each cell line. We have discovered that downregulation of E-cadherin expression has a diverse effect on both cell proliferation as well as the expression of EMT markers in individual tested CRC cell lines, with Caco-2 cells being the most responsive. On the other hand, reduced E-cadherin expression resulted in increased sensitivity of all cell lines to IT and mostly to OPT which might be related to changes in intracellular metabolism of these drugs. These results suggest dichotomy of E-cadherin involvement in the phenotypic EMT spectrum of CRC and warrants further mechanistic studies.

Complex Interplay of Genes Underlies Invasiveness in Fibrosarcoma Progression Model.

Sarcomas are a heterogeneous group of mesenchymal tumours, with a great variability in their clinical behaviour. While our knowledge of sarcoma initiation has advanced rapidly in recent years, relatively little is known about mechanisms of sarcoma progression. JUN-murine fibrosarcoma progression series consists of four sarcoma cell lines, JUN-1, JUN-2, JUN-2fos-3, and JUN-3. JUN-1 and -2 were established from a single tumour initiated in a H2K/v-jun transgenic mouse, JUN-3 originates from a different tumour in the same animal, and JUN-2fos-3 results from a targeted in vitro transformation of the JUN-2 cell line. The JUN-1, -2, and -3 cell lines represent a linear progression from the least transformed JUN-2 to the most transformed JUN-3, with regard to all the transformation characteristics studied, while the JUN-2fos-3 cell line exhibits a unique transformation mode, with little deregulation of cell growth and proliferation, but pronounced motility and invasiveness. The invasive sarcoma sublines JUN-2fos-3 and JUN-3 show complex metabolic profiles, with activation of both mitochondrial oxidative phosphorylation and glycolysis and a significant increase in spared respiratory capacity. The specific transcriptomic profile of invasive sublines features very complex biological relationships across the identified genes and proteins, with accentuated autocrine control of motility and angiogenesis. Pharmacologic inhibition of one of the autocrine motility factors identified, Ccl8, significantly diminished both motility and invasiveness of the highly transformed fibrosarcoma cell. This progression series could be greatly valuable for deciphering crucial aspects of sarcoma progression and defining new prognostic markers and potential therapeutic targets.

Acute Increases in Intracellular Zinc Lead to an Increased Lysosomal and Mitochondrial Autophagy and Subsequent Cell Demise in Malignant Melanoma.

Changes in zinc content and dysregulated zinc homeostatic mechanisms have been recognized in several solid malignancies such as prostate cancer, breast cancer, or pancreatic cancer. Moreover, it has been shown that zinc serum and/or tissue levels are altered in melanoma with varying effects on melanoma development and biology. This study was conducted to explore the effects of acute increases of intracellular zinc in a set of melanoma tissue explants obtained from clinical samples. Measurements of their zinc content showed an extant heterogeneity in total and free intracellular zinc pools associated with varying biological behavior of individual cells, e.g., autophagy levels and propensity to cell death. Use of zinc pyrithione elevated intracellular zinc in a short time frame which resulted in marked changes in mitochondrial activity and lysosomes. These alterations were accompanied by significantly enhanced autophagy flux and subsequent cell demise in the absence of typical apoptotic cell death markers. The present results show for the first time that acutely increased intracellular zinc in melanoma cells specifically enhances their autophagic activity via mitochondria and lysosomes which leads to autophagic cell death. While biologically relevant, this discovery may contribute to our understanding and exploration of zinc in relation to autophagy as a means of controlling melanoma growth and survival.

Biology of Glioblastoma Multiforme-Exploration of Mitotic Catastrophe as a Potential Treatment Modality.

Glioblastoma multiforme (GBM) represents approximately 60% of all brain tumors in adults. This malignancy shows a high biological and genetic heterogeneity associated with exceptional aggressiveness, leading to a poor survival of patients. This review provides a summary of the basic biology of GBM cells with emphasis on cell cycle and cytoskeletal apparatus of these cells, in particular microtubules. Their involvement in the important oncosuppressive process called mitotic catastrophe will next be discussed along with select examples of microtubule-targeting agents, which are currently explored in this respect such as benzimidazole carbamate compounds. Select microtubule-targeting agents, in particular benzimidazole carbamates, induce G2/M cell cycle arrest and mitotic catastrophe in tumor cells including GBM, resulting in phenotypically variable cell fates such as mitotic death or mitotic slippage with subsequent cell demise or permanent arrest leading to senescence. Their effect is coupled with low toxicity in normal cells and not developed chemoresistance. Given the lack of efficient cytostatics or modern molecular target-specific compounds in the treatment of GBM, drugs inducing mitotic catastrophe might offer a new, efficient alternative to the existing clinical management of this at present incurable malignancy.

The Evaluation of Glioblastoma Cell Dissociation and Its Influence on Its Behavior.

PURPOSE: Primary cell lines are a valuable tool for evaluation of tumor behavior or sensitivity to anticancer treatment and appropriate dissociation of cells could preserve genomic profile of the original tissue. The main aim of our study was to compare the influence of two methods of glioblastoma multiforme (GBM) cell derivation (mechanic-MD; enzymatic-ED) on basic biological properties of thus derived cells and correlate them to the ones obtained from stabilized GBM cell line A-172. METHODS: Cell proliferation and migration (xCELLigence Real-Time Cell Analysis), expression of microRNAs and protein markers (RT-PCR and Western blotting), morphology (phase contrast and fluorescent microscopy), and accumulation of temozolomide (TMZ) and its metabolite 5-aminoimidazole-4-carboxamide (AIC) inside the cells (LC-MS analysis) were carried out in five different samples of GBM (GBM1, GBM2, GBM32, GBM33, GBM34), with each of them processed by MD and ED types of isolations. The same analyses were done in the A-172 cell line too. RESULTS: Primary GBM cells obtained by ED or MD approaches significantly differ in biological behavior and properties of these cells. Unlike in primary MD GBM cells, higher proliferation, as well as migration, was observed in primary ED GBM cells, which were also associated with the acquired mesenchymal phenotype and higher sensitivity to TMZ. Finally, the same analyses of stabilized GBM cell line A-172 revealed several important differences in measured parameters. CONCLUSIONS: GBM cells obtained by MD and ED dissociation show considerable heterogeneity, but based on our results, MD approach should be the preferred method of primary GBM cell isolation.

Cardiac Troponins are Among Targets of Doxorubicin-Induced Cardiotoxicity in hiPCS-CMs.

Modern diagnostic strategies for early recognition of cancer therapeutics-related cardiac dysfunction involve cardiac troponins measurement. Still, the role of other markers of cardiotoxicity is still unclear. The present study was designed to investigate dynamics of response of human cardiomyocytes derived from induced pluripotent stem cells (hiPCS-CMs) to doxorubicin with the special emphasis on their morphological changes in relation to expression and organization of troponins. The hiPCS-CMs were treated with doxorubicin concentrations (1 and 0.3 µM) for 48 h and followed for next up to 6 days. Exposure of hiPCS-CMs to 1 µM doxorubicininduced suppression of both cardiac troponin T (cTnT) and cardiac troponin I (cTnI) gene expression. Conversely, lower 0.3 µM doxorubicin concentration produced no significant changes in the expression of aforementioned genes. However, the intracellular topography, arrangement, and abundance of cardiac troponin proteins markedly changed after both doxorubicin concentrations. In particular, at 48 h of treatment, both cTnT and cTnI bundles started to reorganize, with some of them forming compacted shapes extending outwards and protruding outside the cells. At later intervals (72 h and onwards), the whole troponin network collapsed and became highly disorganized following, to some degree, overall changes in the cellular shape. Moreover, membrane permeability of cardiomyocytes was increased, and intracellular mitochondrial network rearranged and hypofunctional. Together, our results demonstrate complex effects of clinically relevant doxorubicin concentrations on hiPCS-CM cells including changes in cTnT and cTnI, but also in other cellular compartments contributing to the overall cytotoxicity of this class of cytostatics.

Selected Aspects of Chemoresistance Mechanisms in Colorectal Carcinoma-A Focus on Epithelial-to-Mesenchymal Transition, Autophagy, and Apoptosis.

Chemoresistance has been found in all malignant tumors including colorectal carcinoma (CRC). Nowadays chemoresistance is understood as a major reason for therapy failure, with consequent tumor growth and spreading leading ultimately to the patient's premature death. The chemotherapy-related resistance of malignant colonocytes may be manifested in diverse mechanisms that may exist both prior to the onset of the therapy or after it. The ultimate function of this chemoresistance is to ensure the survival of malignant cells through continuing adaptation within an organism, therefore, the nature and spectrum of cell-survival strategies in CRC represent a highly significant target of scientific inquiry. Among these survival strategies employed by CRC cells, three unique but significantly linked phenomena stand out-epithelial-to-mesenchymal transition (EMT), autophagy, and cell death. In this mini-review, current knowledge concerning all three mechanisms including their emergence, timeline, regulation, and mutual relationships will be presented and discussed.

The effect of sodium butyrate and cisplatin on expression of EMT markers.

Histone modifications play a key role in the epigenetic regulation of gene transcription in cancer cells. Histone acetylations are regulated by two classes of enzymes, histone acetyltransferases (HATs) and histone deacetylases (HDACs). HDACs are increased in ovarian carcinomas and they are involved in carcinogenesis and resistance to chemotherapeutic agents. In our study we investigated anticancer effect of HDAC inhibitor sodium butyrate (NaBu) on cisplatin-sensitive and cisplatin-resistant ovarian cell lines A2780 and A2780cis. A2780 and A2780cis were treated with NaBu alone or in combination with cisplatin (CP). NaBu inhibited the growth of both cell lines and enhanced cytotoxic effect of CP. Exposure to NaBu for 24 h induced cell cycle arrest. The expressions of EMT-related genes and proteins were further investigated by qPCR and western blot analysis. Loss of E-cadherin has been shown to be crucial in ovarian cancer development. We found that NaBu dramatically induce expression of E-cadherin gene (CDH1) and protein levels in A2780 and A2780cis. We investigated correlation between transcription and methylation of CDH1gene. Methylation level analysis in 32 CpG sites in CDH1 gene (promoter/exon1 regions) was performed using bisulfite NGS (Next Generation Sequencing). We found that cisplatin-resistant cell line A2780cis cells differ from their cisplatin-sensitive counterparts in the CDH1 methylation. Methylation in A2780cis cells is elevated compared to A2780. However, NaBu-induced expression of CDH1 was not accompanied by CDH1 demethylation. NaBu treatment induced changes in expression of EMT-related genes and proteins. Interestingly E-cadherin zinc finger transcriptional repressor SNAIL1 was upregulated in both cell lines. Mesenchymal marker vimentin was downregulated. Matrix metalloproteases (MMPs) are necessary for pericellular proteolysis and facilitate migration and invasion of tumour cells. NaBu induced mRNA expression of MMPs, mild changes in activities of gelatinases MMP2 and MMP9 were detected. Our data demonstrate that NaBu sensitizes cisplatin-resistant ovarian cancer cells, re-established E-cadherin expression, but it was not able to reverse the EMT phenotype completely.

Inositol hexaphosphate limits the migration and the invasiveness of colorectal carcinoma cells in vitro.

Inositol hexaphosphate (IP6), also known as phytic acid, has been shown to exhibit anticancer effects in a number of preclinical tumor models. IP6 decreases proliferation by arresting cells in the G0/G1 phase, inhibits iron-mediated oxidative reactions, enhances differentiation and stimulates apoptosis. The present study attempted to characterize the effect of IP6 on the migration and adhesion of colon cancer SW620 cells. IP6 was assessed at concentrations of 0.2 and 1 mM during 12, 24 and 48 h of exposure. Migration ability was measured with the real-time xCELLigence Real-Time Cell Analyzer Dual Purpose system. The expression of mRNA and proteins involved in migration and cancer progression [epithelial cell adhesion molecule, intercellular adhesion molecule-1, ß-catenin, N-cadherin, E-cadherin, matrix metalloproteinase (MMP)-2 and MMP-9] was determined by reverse transcription-quantitative polymerase chain reaction and western blot analysis. The changes in the expression and subcellular localization of E-cadherin were determined by indirect immunofluorescence. IP6 induced a decrease in the migration ability of the tested SW620 cell line. IP6-treated cells also showed decreased expression of N-cadherin, increased levels of E-cadherin and decreased expression of MMP-2 and MMP-9. These results indicated that IP6 has potential to modulate the migration ability and expression of markers associated with invasion in SW620 cells; however, further analysis is necessary to obtain a detailed understanding of the mechanism of action.

Flubendazole and mebendazole impair migration and epithelial to mesenchymal transition in oral cell lines.

Benzimidazole anthelmintics flubendazole and mebendazole are microtubule-targeting drugs that showed considerable anti-cancer activity in different preclinical models. In this study, the effects of flubendazole and mebendazole on proliferation, migration and cadherin switching were studied in a panel of oral cell lines in vitro. Both compounds reduced the viability of the PE/CA-PJ15 and H376 oral squamous carcinoma cells and of the premalignant oral keratinocytes DOK with the IC50 values in the range of 0.19-0.26 µM. Normal oral keratinocytes and normal gingival fibroblasts were less sensitive to the treatment. Flubendazole and mebendazole also reduced the migration of the PE/CA-PJ15 cell in concentrations that had no anti-migratory effects on the normal gingival fibroblasts. Levels of the focal adhesion kinase FAK, Rho-A and Rac1 GTPases and the Rho guanine nucleotide exchange factor GEF-H1 were decreased in both PE/CA-PJ15 cells and gingival fibroblasts following treatment. Both drugs also interfered with cadherin switching in the model of TGF-ß-induced epithelial to mesenchymal transition (EMT) in the DOK cell line. Levels of N-cadherin were reduced in the TGF-ß induced cells co-treated with flubendazol and mebendazole in very low concentration (50 nM). These results suggest direct effects of both benzimidazoles on selected processes of EMT in oral cell lines such as cadherin switching as well as cellular migration.

Oxaliplatin and irinotecan induce heterogenous changes in the EMT markers of metastasizing colorectal carcinoma cells.

In patients with advanced colorectal cancer (CRC), surgery is complemented with systemic therapy - chemotherapy and radiochemotherapy. Although the patients' overall survival has been significantly improved, tumor resistance is still a frequent cause of chemotherapy failure. Several factors contribute to chemoresistance of tumor cells including changes related with epithelial-mesenchymal transition (EMT). The present study was designed to verify the presence of EMT markers in paired CRC primary cell lines obtained from primary tumor sites and lymph node metastases of three patients and to investigate the effect of irinotecan and oxaliplatin treatment on these markers as well. The samples of the higher stage of CRC and positive for angioinvasion were selected and qPCR, western blot analysis, migration assay, cytotoxicity testing was performed. Results confirmed the increased expression of several markers characteristic of EMT and invasiveness in lymph node metastatic cells, with a significant variability between individual samples. Irinotecan and oxaliplatin decreased migration activity of the cells and to the varying degree affected the expression of EMT and invasiveness markers. In conclusion, in CRC EMT is present in metastatic cells over a phenotypic continuum whose expression is altered heterogeneously upon irinotecan and oxaliplatin treatment.

Increases in Intracellular Zinc Enhance Proliferative Signaling as well as Mitochondrial and Endolysosomal Activity in Human Melanocytes.

BACKGROUND/AIMS: Zinc (Zn) is an important microelement required by skin cells for a variety of biological processes. The role of Zn in melanocyte proliferation and homeostasis has to date not been investigated. METHODS: Human dermal melanocytes were isolated from patients and their proliferative activity determined along with both total and labile Zn content. Subsequently, changes in proliferation as well as in Zn content were determined upon exposure of the dermal melanocytes to external Zn. Further in-depth analyses were undertaken aimed at measuring the expression of proliferation-related proteins (determined by immunoblotting and densitometry), as well as changes in mitochondrial biogenesis and membrane potential (assessed by fluorescence-based cellometry) along with endolysosomal activity (determined by spectrofluorimetrically-measured elevation in fluorescence of lysosomal-aimed non-fuorescent substrate). RESULTS: Human skin melanocytes accumulate externally added Zn, a process which dose-dependently enhances their injury or proliferative activity. Enhanced proliferation is accompanied by an increased expression of the proteins AKT3, ERK1/2, c-MYC and CYCD. In addition, Zn-enriched melanocytes exhibit enhanced mitochondrial biogenesis, with individual mitochondria possessing stabilized mitochondrial membrane potential as well as showing elevated ATP and superoxide levels. Moreover, upon external exposure, Zn enters lysosomes/melanosomes, the activity of which is stimulated along with the process of autophagy. CONCLUSION: The determination of the unique Zn-dependent stimulation of melanocytes and in particular the enhancement of the cells' mitochondrial as well as lysosomal/melanosomal activities may prove important in tracing the sequence of steps in the process of melanomagenesis.