Malgorzata Czyz

Parthenolide, a sesquiterpene lactone from the medical herb feverfew, shows anticancer activity against human melanoma cells in vitro.

Metastatic melanoma is a highly life-threatening disease. The lack of response to radiotherapy and chemotherapy highlights the critical need for novel treatments. Parthenolide, an active component of feverfew (Tanacetum parthenium), inhibits proliferation and kills various cancer cells mainly by inducing apoptosis. The aim of the study was to examine anticancer effects of parthenolide in melanoma cells in vitro. The cytotoxicity of parthenolide was tested in melanoma cell lines and melanocytes, as well as melanoma cells directly derived from a surgical excision. Adherent cell proliferation was measured by tetrazolium derivative reduction assay. Loss of the plasma membrane integrity, hypodiploid events, reactive oxygen species generation, mitochondrial membrane potential dissipation, and caspase-3 activity were assessed by flow cytometric analysis. Microscopy was used to observe morphological changes and cell detachment. Parthenolide reduced the number of viable adherent cells in melanoma cultures. Half maximal inhibitory concentration values around 4 μmol/l were determined. Cell death accompanied by mitochondrial membrane depolarization and caspase-3 activation was observed as the result of parthenolide application. Interestingly, the melanoma cells from vertical growth phase and melanocytes were less susceptible to parthenolide-induced cell death than metastatic cells when drug concentration was at least 6 μmol/l. Reactive oxygen species level was not significantly increased in melanoma cells. However, preincubation of parthenolide with the thiol nucleophile N-acetyl-cysteine protected melanoma cells from parthenolide-induced cell death suggesting the reaction with intracellular thiols as the mechanism responsible for parthenolide activity. In conclusion, the observed anticancer activity makes parthenolide an attractive drug candidate for further testing in melanoma therapy.

MITF in melanoma: mechanisms behind its expression and activity

MITF (microphthalmia-associated transcription factor) represents a melanocytic lineage-specific transcription factor whose role is profoundly extended in malignant melanoma. Over the last few years, the function of MITF has been tightly connected to plasticity of melanoma cells. MITF participates in executing diverse melanoma phenotypes defined by distinct gene expression profiles. Mutation-dependent alterations in MITF expression and activity have been found in a relatively small subset of melanomas. MITF activity is rather modulated by its upstream activators and suppressors operating on transcriptional, post-transcriptional and post-translational levels. These regulatory mechanisms also include epigenetic and microenvironmental signals. Several transcription factors and signaling pathways involved in the regulation of MITF expression and/or activity such as the Wnt/β-catenin pathway are broadly utilized by various types of tumors, whereas others, e.g., BRAFV600E/ERK1/2 are more specific for melanoma. Furthermore, the MITF activity can be affected by the availability of transcriptional co-partners that are often redirected by MITF from their own canonical signaling pathways. In this review, we discuss the complexity of a multilevel regulation of MITF expression and activity that underlies distinct context-related phenotypes of melanoma and might explain diverse responses of melanoma patients to currently used therapeutics.

Anti-apoptotic proteins on guard of melanoma cell survival

Apoptosis plays a pivotal role in sustaining proper tissue development and homeostasis. Evading apoptosis by cancer cells is a part of their adaption to microenvironment and therapies. Cellular integrity is predominantly maintained by pro-survival members of Bcl-2 family and IAPs. Melanoma cells are characterized by a labile and stage-dependent phenotype. Pro-survival molecules can protect melanoma cells from apoptosis and mediate other processes, thus enhancing aggressive phenotype. The essential role of Bcl-2, Mcl-1, Bcl-X(L), livin, survivin and XIAP was implicated for melanoma, often in a tumor stage-dependent fashion. In this review, the current knowledge of pro-survival machinery in melanoma is discussed.

MiRNA in melanoma-derived exosomes.

Proteins, RNAs and viruses can be spread through exosomes, therefore transport utilizing these nanovesicles is of the great interest. MiRNAs are common exosomal constituents capable of influencing expression of a variety of target genes. MiRNA signatures of exosomes are unique in cancer patients and differ from those in normal controls. The knowledge about miRNA profiles of tumor-derived exosomes may contribute to better diagnosis, determination of tumor progression and response to treatment, as well as to the development of targeted therapies. We summarize the current knowledge with regard to miRNAs that are found in exosomes derived from tumors, particularly from melanoma.

Parthenolide reduces the frequency of ABCB5-positive cells and clonogenic capacity of melanoma cells from anchorage independent melanospheres

Growing evidence suggests that the cancer stem cell phenotype in melanoma is dynamically regulated. Therefore, effective therapies have to target simultaneously bulk tumor cells and melanoma stem-like cells. The aim of the present study was to investigate the effects of parthenolide on heterogeneous cancer cell populations from anchorage-independent melanospheres. Cells derived from nodular melanoma specimens were grown under serum-free sphere-forming conditions. The effects of parthenolide on cellular viability, immunophenotype and self-renewing capacity were assessed with cells from dissociated melanospheres. Its penetration capacity was evaluated with intact melanospheres. In melanoma cells that survived treatment with parthenolide, a different immunophenotype than that in untreated control was found. The frequency of cells expressing the ABCB5 transporter was markedly reduced. Most importantly, melanoma cells that survived parthenolide treatment lost their self-renewing capacity. Significantly lower influence of drug on cellular viability and frequency of ABCB5-positive cells was observed in intact melanospheres. The potential clinical significance of our findings is based on the ability of parthenolide to affect both bulk and melanoma stem-like cells with clonogenic capacity and high expression of the ABCB5 transporter. Its low penetration capacity, however, may limit its action to easily accessible melanoma cells, either circulating in the blood or those in the vicinity to blood vessels within the tumor. Because of limited penetration capacity of parthenolide, this drug should be further explored as a part of multimodal therapies rather than as a stand-alone therapeutic agent.

Sphere formation and self-renewal capacity of melanoma cells is affected by the microenvironment.

Melanomas contain subsets of cancer stem-like cells with tumor-initiating capacity. The frequency of these cells in the tumor is still a topic of debate. We investigated the phenotypic plasticity of cancer cells grown as melanospheres to elucidate the influence of the microenvironment on some features of melanoma stem-like cells. Cells from surgical specimens of nodular melanoma were grown as anchorage-independent melanospheres in a stem cell medium and as adherent monolayer cultures in the presence of serum. Proliferation and viability were measured by cell counting and an acid phosphatase assay; surface marker expression was evaluated by flow cytometry, and the clonogenic potential of single cells was assessed by growth in soft agar. Patient-derived melanoma cells could be maintained in cell culture for more than 16 months when grown as melanospheres. In the presence of serum, melanospheres completely changed their growth characteristics and formed adherent monolayers. The transition from melanospheres to monolayers was accompanied by an apparent loss of clonogenic potential, an increased proliferation rate, and altered expressions of cell surface markers ABCB5, CD133, and CD49f. These changes, however, were reversible. Compared with adherent monolayer cultures, melanospheres are enriched in cells with clonogenic potential, reflecting the self-renewing capacity of cancer stem-like cells. This clonogenic potential can be lost and regained depending on the growth conditions. Our results demonstrate how easily melanoma cells change their function upon exposure to external stimuli and suggest that the frequency of melanoma stem-like cells strongly depends on the microenvironment.

Targeting NF-κB and HIF-1 Pathways for the Treatment of Cancer: Part I

The process of chronic inflammation is a common link which connects different kinds of environmental pollutants and infections with tumorigenesis. Transcription factor NF-κB is a common final target for many inflammatory and cell proliferation pathways, independent of the source of stimuli (e.g., cytokines, growth factors, environmental carcinogens, radiation, hypoxia, bacteria, and viruses). Over-activation of NF-κB has been confirmed in many tumors, resulting in worse prognosis for patient survival. Therefore, inhibition of cellular pathways for NF-κB activation is nowadays considered as a promising anti-cancer therapy and is extensively studied in clinical trials, or even has been adopted as an approved therapy in some kinds of cancer.

Pro-Survival Role of MITF in Melanoma

Melanoma is a therapy-resistant skin cancer due to numerous mechanisms supporting cell survival. Although components of melanoma cytoprotective mechanisms are overexpressed in many types of tumors, some of their regulators are characteristic for melanoma. Several genes mediating pro-survival functions have been identified as direct targets of microphthalmia-associated transcription factor (MITF), a melanocyte-specific modulator also recognized as a lineage addiction oncogene in melanoma. BRAF(V600E) and other proteins deregulated in melanoma influence MITF expression and activity, or they are the partners of MITF in melanoma response to radiotherapy and chemotherapeutics. In this review, the pro-survival activity of MITF is discussed.

Natural Compounds' Activity against Cancer Stem-Like or Fast-Cycling Melanoma Cells

Background Accumulating evidence supports the concept that melanoma is highly heterogeneous and sustained by a small subpopulation of melanoma stem-like cells. Those cells are considered as responsible for tumor resistance to therapies. Moreover, melanoma cells are characterized by their high phenotypic plasticity. Consequently, both melanoma stem-like cells and their more differentiated progeny must be eradicated to achieve durable cure. By reevaluating compounds in heterogeneous melanoma populations, it might be possible to select compounds with activity not only against fast-cycling cells but also against cancer stem-like cells. Natural compounds were the focus of the present study. Methods We analyzed 120 compounds from The Natural Products Set II to identify compounds active against melanoma populations grown in an anchorage-independent manner and enriched with cells exerting self-renewing capacity. Cell viability, cell cycle arrest, apoptosis, gene expression, clonogenic survival and label-retention were analyzed. Findings Several compounds efficiently eradicated cells with clonogenic capacity and nanaomycin A, streptonigrin and toyocamycin were effective at 0.1 µM. Other anti-clonogenic but not highly cytotoxic compounds such as bryostatin 1, siomycin A, illudin M, michellamine B and pentoxifylline markedly reduced the frequency of ABCB5 (ATP-binding cassette, sub-family B, member 5)-positive cells. On the contrary, treatment with maytansine and colchicine selected for cells expressing this transporter. Maytansine, streptonigrin, toyocamycin and colchicine, even if highly cytotoxic, left a small subpopulation of slow-dividing cells unaffected. Compounds selected in the present study differentially altered the expression of melanocyte/melanoma specific microphthalmia-associated transcription factor (MITF) and proto-oncogene c-MYC. Conclusion Selected anti-clonogenic compounds might be further investigated as potential adjuvants targeting melanoma stem-like cells in the combined anti-melanoma therapy, whereas selected cytotoxic but not anti-clonogenic compounds, which increased the frequency of ABCB5-positive cells and remained slow-cycling cells unaffected, might be considered as a tool to enrich cultures with cells exhibiting melanoma stem cell characteristics.

Proteomics in human cancer research

Proteomics is now widely employed in the study of cancer. Many laboratories are applying the rapidly emerging technologies to elucidate the underlying mechanisms associated with cancer development, progression, and severity in addition to developing drugs and identifying patients who will benefit most from molecular targeted compounds. Various proteomic approaches are now available for protein separation and identification, and for characterization of the function and structure of candidate proteins. In spite of significant challenges that still exist, proteomics has rapidly expanded to include the discovery of novel biomarkers for early detection, diagnosis and prognostication (clinical application), and for the identification of novel drug targets (pharmaceutical application). To achieve these goals, several innovative technologies including 2‐D‐difference gel electrophoresis, SELDI, multidimensional protein identification technology, isotope‐coded affinity tag, solid‐state and suspension protein array technologies, X‐ray crystallography, NMR spectroscopy, and computational methods such as comparative and de novo structure prediction and molecular dynamics simulation have evolved, and are being used in different combinations. This review provides an overview of the field of proteomics and discusses the key proteomic technologies available to researchers. It also describes some of the important challenges and highlights the current pharmaceutical and clinical applications of proteomics in human cancer research.