Tolga Eichhorn

P-glycoprotein and its inhibition in tumors by phytochemicals derived from Chinese herbs.

P-glycoprotein belongs to the family of ATP-binding cassette (ABC) transporters. It functions in cellular detoxification, pumping a wide range of xenobiotic compounds, including anticancer drugs out of the cell. In cancerous cells, P-glycoprotein confers resistance to a broad spectrum of anticancer agents, a phenomenon termed multidrug resistance. An attractive strategy for overcoming multidrug resistance is to block the transport function of P-glycoprotein and thus increase intracellular concentrations of anticancer drugs to lethal levels. Efforts to identify P-glycoprotein inhibitors have led to numerous candidates, none of which have passed clinical trials with cancer patients due to their high toxicity. The search for naturally inhibitory products from traditional Chinese medicine may be more promising because natural products are frequently less toxic than chemically synthesized substances. In this review, we give an overview of molecular and clinical aspects of P-glycoprotein and multidrug resistance in the context of cancer as well as Chinese herbs and phytochemicals showing inhibitory activity towards P-glycoprotein.

Molecular interaction of artemisinin with translationally controlled tumor protein (TCTP) of Plasmodium falciparum

Malaria causes millions of death cases per year. Since Plasmodium falciparum rapidly develops drug resistance, it is of high importance to investigate potential drug targets which may lead to novel rational therapy approaches. Here we report on the interaction of translationally controlled tumor protein of P. falciparum (PfTCTP) with the anti-malarial drug artemisinin. Furthermore, we investigated the crystal structure of PfTCTP. Using mass spectrometry, bioinformatic approaches and surface plasmon resonance spectroscopy, we identified novel binding sites of artemisinin which are in direct neighborhood to amino acids 19-46, 108-134 and 140-163. The regions covered by these residues are known to be functionally important for TCTP function. We conclude that interaction of artemisinin with TCTP may be at least in part explain the antimalarial activity of artemisinin.

Shikonin Directly Targets Mitochondria and Causes Mitochondrial Dysfunction in Cancer Cells

Chemotherapy is a mainstay of cancer treatment. Due to increased drug resistance and the severe side effects of currently used therapeutics, new candidate compounds are required for improvement of therapy success. Shikonin, a natural naphthoquinone, was used in traditional Chinese medicine for the treatment of different inflammatory diseases and recent studies revealed the anticancer activities of shikonin. We found that shikonin has strong cytotoxic effects on 15 cancer cell lines, including multidrug-resistant cell lines. Transcriptome-wide mRNA expression studies showed that shikonin induced genetic pathways regulating cell cycle, mitochondrial function, levels of reactive oxygen species, and cytoskeletal formation. Taking advantage of the inherent fluorescence of shikonin, we analyzed its uptake and distribution in live cells with high spatial and temporal resolution using flow cytometry and confocal microscopy. Shikonin was specifically accumulated in the mitochondria, and this accumulation was associated with a shikonin-dependent deregulation of cellular Ca2+ and ROS levels. This deregulation led to a breakdown of the mitochondrial membrane potential, dysfunction of microtubules, cell-cycle arrest, and ultimately induction of apoptosis. Seeing as both the metabolism and the structure of mitochondria show marked differences between cancer cells and normal cells, shikonin is a promising candidate for the next generation of chemotherapy.

Pharmacogenomic Identification of c-Myc/Max-Regulated Genes Associated with Cytotoxicity of Artesunate towards Human Colon, Ovarian and Lung Cancer Cell Lines

Development of novel therapy strategies is one of the major pressing topics of clinical oncology to overcome drug resistance of tumors. Artesunate (ART) is an anti-malarial drug, which also exerts profound cytotoxic activity towards cancer cells. We applied a gene-hunting approach using microarray-based transcriptome-wide mRNA expression profiling and COMPARE analyses. We identified a set of genes, whose expression was associated either with high IC50 values or low IC50 values for ART. Therefore, these genes may function as resistance or sensitivity factors for response of tumor cells towards ART. This viewpoint is conceivable for genes involved in ribosomal activity, drug transport, cellular antioxidant defense, apoptosis, cell proliferation, cell cycle progression etc. An investigation of underlying signal transduction by pathway analysis suggested a role of the signaling pathways related to tumor necrosis factor (TNF) and the tumor suppressor p53. On the other hand, there were genes without obvious functional link to cellular response to ART, such as genes involved in the survival of cochlear outer and inner hair cells etc. We proved the hypothesis that ART influences the activity of transcription factors regulating downstream genes involved or not involved in response of cancer cells towards ART. This would explain the identification of genes with and without obvious relation to the cytotoxic activity of ART by microarray and COMPARE analyses. By analysis of the binding motifs for the transcription factors c-Myc and Max, we indeed found that 53 of 56 genes contained one or more binding sites for c-Myc/Max upstream of the gene-location. We conclude that c-Myc and Max-mediated transcriptional control of gene expression might contribute to the therapeutic effects of ART in cancer cells, but may also confer unwanted side effects by affecting therapy-unrelated genes.

Factors determining sensitivity or resistance of tumor cell lines towards artesunate

Clinical oncology is still challenged by the development of drug resistance of tumors that result in poor prognosis for patients. There is an urgent necessity to understand the molecular mechanisms of resistance and to develop novel therapy strategies. Artesunate (ART) is an anti-malarial drug, which also exerts profound cytotoxic activity towards cancer cells. We first applied a gene-hunting approach using cluster and COMPARE analyses of microarray-based transcriptome-wide mRNA expression profiles. Among the genes identified by this approach were genes from diverse functional groups such as structural constituents of ribosomes (RPL6, RPL7, RPS12, RPS15A), kinases (CABC1, CCT2, RPL41), transcriptional and translational regulators (SFRS2, TUFM, ZBTB4), signal transducers (FLNA), control of cell growth and proliferation (RPS6), angiogenesis promoting factors (ITGB1), and others (SLC25A19, NCKAP1, BST1, DBH, FZD7, NACA, MTHFD2). Furthermore, we applied a candidate gene approach and tested the role of resistance mechanisms towards established anti-cancer drugs for ART resistance. By using transfected or knockout cell models we found that the tumor suppressor p16(INK4A) and the anti-oxidant protein, catalase, conferred resistance towards ART, while the oncogene HPV-E6 conferred sensitivity towards ART. The tumor suppressor p53 and its downstream protein, p21, as well as the anti-oxidant manganese-dependent superoxide dismutase did not affect cellular response to ART. In conclusion, our pharmacogenomic approach revealed that response of tumor cells towards ART is multi-factorial and is determined by gene expression associated with either ART sensitivity or resistance. At least some of the functional groups of genes (e.g. angiogenesis promoting factors, cell growth and proliferation-associated genes signal transducers and kinases) are also implicated in clinical responsiveness of tumors towards chemotherapy. It merits further investigation, whether ART is responsive in clinically refractory tumors and whether the genes identified in the present study also determine clinical responsiveness towards ART.

Anticancer activity of Salvia officinalis essential oil against HNSCC cell line (UMSCC1)

BACKGROUND Every year there are several hundred thousand new cases of oral cancer worldwide. Clinical oncology is still challenged by toxicity and side effects of multimodal therapy strategies in which is associated with poor prognosis for patients. There is an urgent necessity to develop novel therapy strategies. As the majority of anticancer drugs are of natural origin, natural products represent a valuable source for the identification and development of novel treatment options for cancer. The aim of this investigation was to study the cytotoxicity of Salvia officinalis L. (sage) essential oil. METHODS Salvia officinalis essential oil was gained by aqueous extraction from plant material and subsequently analyzed by gas chromatography. The cytotoxicity of the essential oil on the squamous human cell carcinoma cell line of the oral cavity (UMSCC1) was assessed with the XTT assay. These experiments revealed the half maximal inhibitory concentration (IC(50)) of the essential oil. It was used in the microarray-based analysis of gene expression of UMSSC1 cells. The results were submitted to a signaling pathway analysis. RESULTS The main constituents of Salvia officinalis essential oil include the monoterpenes thujone, β-pinene, and 1,8-cineol. Low concentrations of the essential oil increased vitality of the UMSCC1 cells. Beyond the concentration of the IC(50) of 135 µg/ml, sage essential oil reduced UMSSC1 cells viability to a minimum. In the microarray gene expression analysis, genes involved in cancer, cellular growth and proliferation, cell death, cell morphology, cell cycle, gene expression, and DNA repair were the most prominent. The three most significantly regulated pathways by sage were aryl hydrocarbon receptor signaling, cell cycle (G1/S checkpoint) regulation, and p53 signaling. CONCLUSION To the best of our knowledge, this study suggests for the first time the ability of Salvia officinalis essential oil to inhibit human HNSCC cell growth. The therapeutic potential of sage essential oil might exceed that of its common use in otorhinolaryngology.

Polyhydroxylated Steroidal Glycosides from Paris polyphylla

Three new steroidal saponins, parisyunnanosides G-I (1-3), one new C(21) steroidal glycoside, parisyunnanoside J (4), and three known compounds, padelaoside B (5), pinnatasterone (6), and 20-hydroxyecdyson (7), were isolated from the rhizomes of Paris polyphylla Smith var. yunnanensis. Compounds 1 and 3 have unique trisdesmoside structures that include a C-21 β-d-galactopyranose moiety. All compounds were evaluated for their cytotoxicity against human CCRF leukemia cells.

Self-medication with nutritional supplements and herbal over-thecounter products

In recent years, the popularity increased for nutritional supplements and herbal products. Prescription drugs, but not herbal therapies are paid by health insurances. They are sold over-the-counter (OTC) on the patients’ own expense. However, there are potential risks of self-medication, e.g. incorrect self-diagnosis, severe adverse reactions, dangerous drug interactions, risk of addiction etc. They are often used by patients at their own discretion without knowledge of and control by their physicians. Certain users are at risk of intoxication. Multiple medications taken by older patients increase the risk for adverse drug reactions, drug-drug interactions, and compliance problems for this age group (polypharmacy). Herbals should be discontinued prior to operations to avoid interactions with anesthetics or anticoagulants. Herbal preparations may also be carcinogenic or interfere with cancer treatments. Pregnant women use various OTC preparations. However, in many cases, it is unclear whether their use is safe for mother or baby. Self-medication with herbals is also largely distributed among anxious and depressive patients, and patients with other conditions and symptoms. The popularity of herbal products has also brought concerns on quality, efficacy and safety. Cases of botanical misidentification, contaminations with heavy metals, pesticides, radioactivity, organic solvents, microbials as well as adulteration with chemical drugs necessitate the establishment of international quality control standards. Hepatotoxic effects have been reported for more than 300 plant species, and some commonly used herbs have been demonstrated to interact with Western medication. Health care professionals have a critical responsibility assessing the self-care ability of their patients. Databases are available for pharmacists with information on action, side effects and toxicities as well as herb-drug interactions. There is a need for established guidelines regarding the correct use of nutritional supplements and herbal OTC preparations (phytovigilance). Physicians, pharmacists, and other health care professionals have to counsel patients and the general public on the benefits and risks associated with herbal drugs. Information centers for consumers and general practitioners are needed, and convincing evidence on safety and efficacy of herbal products has to be demonstrated in placebo-controlled, double blind and randomized clinical trials.

Krebshemmende Wirkung des ätherischen Salbei-Öls auf eine Plattenepithelzellkarzinom-Zelllinie der Mundhöhle (UMSCC1)

BACKGROUND Every year there are several hundred thousand new cases of oral cancer worldwide. Clinical oncology is still challenged by toxicity and side effects of multimodal therapy strategies in which is associated with poor prognosis for patients. There is an urgent necessity to develop novel therapy strategies. As the majority of anticancer drugs are of natural origin, natural products represent a valuable source for the identification and development of novel treatment options for cancer. The aim of this investigation was to study the cytotoxicity of Salvia officinalis L. (sage) essential oil. METHODS Salvia officinalis essential oil was gained by aqueous extraction from plant material and subsequently analyzed by gas chromatography. The cytotoxicity of the essential oil on the squamous human cell carcinoma cell line of the oral cavity (UMSCC1) was assessed with the XTT assay. These experiments revealed the half maximal inhibitory concentration (IC(50)) of the essential oil. It was used in the microarray-based analysis of gene expression of UMSSC1 cells. The results were submitted to a signaling pathway analysis. RESULTS The main constituents of Salvia officinalis essential oil include the monoterpenes thujone, β-pinene, and 1,8-cineol. Low concentrations of the essential oil increased vitality of the UMSCC1 cells. Beyond the concentration of the IC(50) of 135 µg/ml, sage essential oil reduced UMSSC1 cells viability to a minimum. In the microarray gene expression analysis, genes involved in cancer, cellular growth and proliferation, cell death, cell morphology, cell cycle, gene expression, and DNA repair were the most prominent. The three most significantly regulated pathways by sage were aryl hydrocarbon receptor signaling, cell cycle (G1/S checkpoint) regulation, and p53 signaling. CONCLUSION To the best of our knowledge, this study suggests for the first time the ability of Salvia officinalis essential oil to inhibit human HNSCC cell growth. The therapeutic potential of sage essential oil might exceed that of its common use in otorhinolaryngology.

Bioinformatic and experimental fishing for artemisinin-interacting proteins from human nasopharyngeal cancer cells

Determining interacting cellular partners of drugs by chemical proteomic techniques is complex and tedious. Most approaches rely on activity-based probe profiling and compound-centric chemical proteomics. The anti-malarial artemisinin also exerts profound anti-cancer activity, but the mechanisms of action are incompletely understood. In the present investigation, we present a novel approach to identify artemisinin-interacting target proteins. Our approach overcomes usual problems in traditional fishing procedures, because the drug was attached to a surface without further chemical modification. The proteins identified effect among others, cell cycle arrest, apoptosis, inhibition of angiogenesis, disruption of cell migration, and modulation of nuclear receptor responsiveness. Furthermore, a bioinformatic approach confirmed experimentally identified proteins and suggested a large number of other interacting proteins. Theoretically predicted interaction partners may serve as a starting point to complete the whole set of proteins binding artemisinin.