Leonilla Elbling

Green tea extract and (–)-epigallocatechin-3-gallate, the major tea catechin, exert oxidant but lack antioxidant activities

Green tea is the most widely consumed beverage. It has attained high reputation as a health‐promoting dietary component ascribed to the antioxidant activity of (−)‐epigallocatechin‐3‐gallate (EGCG), its main polyphenolic constituent. Evidence is increasing that tea constituents can be cell damaging and pro‐oxidant themselves. These effects were suggested to be due to spontaneous H2O2 generation by polyphenols in solution. In the present study, we investigated the oxidant and antioxidant properties of green tea extracts (GTE) and of EGCG by means of the rodent macrophage‐like RAW 264.7 and human promyelocytic leukemic HL60 cell lines. The results obtained show that both under cell‐free conditions and in the presence of cells the oxidant activities of GTE and EGCG exceeded those of spontaneously generated H2O2 (FOX assay). Increase of intracellular oxidative stress was indicated by 2′,7′‐dichlorofluorescin probing, and the enhanced genotoxicity was demonstrated by the alkaline comet assay and by the micronucleus assay (cytokinesis block). Time‐ and dose‐dependent induction of cell death was monitored by trypan blue exclusion, MTT assay, and Hoechst staining. Furthermore, in our systems in vitro, EGCG neither directly scavenges H2O2 nor mediates other antioxidant activities but rather increased H2O2‐induced oxidative stress and DNA damage. In conclusion, our data suggest that detailed mechanistic studies on the effects of GTE and EGCG should be performed in vivo before excessive intake and/or topical application of green tea products can be recommended to healthy and/or diseased persons.

Resistance against novel anticancer metal compounds: Differences and similarities

The platinum antitumor drugs cisplatin, carboplatin and oxaliplatin are widely used components of modern cancer chemotherapy. However, their success is limited by severe adverse effects and because of the impact of intrinsic and acquired resistance mechanisms on tumor responses. Consequently, intense efforts have been made to develop new metal compounds that either display enhanced tumor specificity or are less prone to the development of resistance. Despite the synthesis of thousands of compounds during the last decades only very few novel metal drugs have successfully reached clinical development and/or approval so far. In this review we summarize the current knowledge on drug resistance mechanisms against novel metal compounds (including platinum, arsenic, ruthenium, gallium, titanium, copper, and lanthanum drugs), and address the question whether there might exist a general metal-drug resistance phenotype.

Multidrug resistance markers P-glycoprotein, multidrug resistance protein 1, and lung resistance protein in non-small cell lung cancer : prognostic implications

PurposeThe aim of this retrospective study was to comparatively investigate the expression of the three drug-resistance genes P-glycoprotein (P-gp), multidrug-resistance protein 1 (MRP1), and lung resistance protein (LRP), in non-small cell lung cancer (NSCLC) tissues, and to assess possible associations with clinicopathologic features.MethodsTumor specimens from 126 patients were analyzed by immunohistochemistry and, in selected cases, by reverse transcriptase polymerase chain reaction (RT-PCR), and data were statistically analyzed by SPSS.ResultsThe mean expression levels of tumor tissues in the case of P-gp and LRP did not exceed the one of normal epithelia, while MRP1 was significantly enhanced in NSCLC. A weak association was observed between higher grading and P-glycoprotein expression ( p <0.08) as well as lower grading and MRP1 expression in the case of adenocarcinoma ( p <0.05). MRP1 levels were highest in TNM stage I and declined with advanced stage ( p <0.03). A significant association was found between high MRP1 levels and longer overall survival ( N =115, p <0.04), which was highly significant in the patient group never treated with chemotherapy ( N =77; p <0.007). P-gp expression was enhanced in those patients who had received chemotherapy before surgery ( p <0.05).ConclusionsOur data point towards a major role of MRP1 in the intrinsic treatment resistance of NSCLC and suggest, in addition, a significant activation of P-gp expression during chemotherapy.

Expression of the major vault protein LRP in human non‐small‐cell lung cancer cells: Activation by short‐term exposure to antineoplastic drugs

Non‐small‐cell lung cancer (NSCLC) cells are characterised by resistance to the toxic impact of antineoplastic drugs both in vivo and in vitro. The lung resistance‐related protein (LRP), identical with the human major vault protein, is over‐expressed in a variety of tumour cells characterised by intrinsic or acquired chemoresistance. We investigated the expression and cellular localisation of LRP in 16 unselected NSCLC cell lines, immortalised bronchial epithelial cells and embryonic lung fibroblasts. All cell lines analysed expressed LRP mRNA, while protein expression ranged from undetectable up to high levels. Cell fractionation and immunofluorescence staining in selected cell lines localised LRP almost exclusively to the cytoplasm. LRP was contained in the 100,000 g pellet and absent in the soluble, cytosolic fraction and nuclei. A small proportion of LRP, however, was shown to be loosely associated with the outside of the nuclei. Sucrose gradient equilibrium centrifugation revealed presence of LRP exclusively in the fraction known to accumulate purified vault particles. Short‐term exposure (16 hr) to subtoxic daunomycin (DM)‐, and bleomycin (BM)‐concentrations significantly (up to 4‐fold) enhanced LRP expression in 2/4 cell lines tested. Cisplatin (CDDP) had a minor effect while vinblastine (VBL) was ineffective. At cytotoxic conditions all drugs rather decreased than increased LRP expression. When basic LRP expression was compared with chemosensitivity, a significant correlation was detected for resistance to CDDP but not DM, doxorubicin (DOX), etoposide (VP‐16), VBL and BM. Summing up, our data suggest a role of vaults both in basic CDDP resistance and, additionally, in an short‐term defensive response of NSCLC cells against several other drugs. Int. J. Cancer 88:293–300, 2000.

Fibroblast growth factor receptor-mediated signals contribute to the malignant phenotype of non-small cell lung cancer cells: therapeutic implications and synergism with epidermal growth factor receptor inhibition

Fibroblast growth factors (FGF) and their high-affinity receptors (FGFR) represent an extensive cellular growth and survival system. Aim of this study was to evaluate the contribution of FGF/FGFR-mediated signals to the malignant growth of non-small cell lung cancer (NSCLC) and to assess their potential as targets for therapeutic interventions. Multiple FGFR mRNA splice variants were coexpressed in NSCLC cells (n = 16) with predominance of FGFR1. Accordingly, both expression of a dominant-negative FGFR1 (dnFGFR1) IIIc-green fluorescent protein fusion protein and application of FGFR small-molecule inhibitors (SU5402 and PD166866) significantly reduced growth, survival, clonogenicity, and migratory potential of the majority of NSCLC cell lines. Moreover, dnFGFR1 expression completely blocked or at least significantly attenuated s.c. tumor formation of NSCLC cells in severe combined immunodeficient mice. Xenograft tumors expressing dnFGFR1 exhibited significantly reduced size and mitosis rate, enhanced cell death, and decreased tissue invasion. When FGFR inhibitors were combined with chemotherapy, antagonistic to synergistic in vitro anticancer activities were obtained depending on the application schedule. In contrast, simultaneous blockage of FGFR- and epidermal growth factor receptor-mediated signals exerted synergistic effects. In summary, FGFR-mediated signals in cooperation with those transmitted by epidermal growth factor receptor are involved in growth and survival of human NSCLC cells and should be considered as targets for combined therapeutic approaches. [Mol Cancer Ther 2008;7(10):3408–19]

Expression and functional activity of the ABC-transporter proteins P-glycoprotein and multidrug-resistance protein 1 in human brain tumor cells and astrocytes

The poor prognosis of glioma patients is partly based on the minor success obtained from chemotherapeutic treatments. Resistance mechanisms at the tumor cell level may be, in addition to the blood–brain barrier, involved in the intrinsic chemo-insensitivity of brain tumors. We investigated the expression of the drug-transporter proteins P-glycoprotein (P-gp) and multidrug-resistance protein 1 (MRP1) in cell lines (N=24) and primary cell cultures (N=36) from neuroectodermal tumors, as well as in brain tumor extracts (N=18) and normal human astrocytes (N=1). We found that a considerable expression of P-gp was relatively rare in glioma cells, in contrast to MRP1, which was constitutively overexpressed in cells derived from astrocytomas as well as glioblastomas. Also, normal astrocytes cultured in vitro expressed high amounts of MRP1 but no detectable P-gp. Meningioma cells frequently co-expressed P-gp and MRP1, while, most of the neuroblastoma cell lines express higher P-gp but lower MRP1 levels as compared to the other tumor types. Both, a drug-exporting and a chemoprotective function of P-gp as well as MRP1 could be demonstrated in selected tumor cells by a significant upregulation of cellular 3H-daunomycin accumulation and daunomycin cytotoxicity via administration of transporter antagonists. Summing up, our data suggest that P-gp contributes to cellular resistance merely in a small subgroup of gliomas, but frequently in neuroblastomas and meningiomas. In contrast, MRP1 is demonstrated to play a constitutive role in the intrinsic chemoresistance of gliomas and their normal cell counterpart.

Cellular Functions of Vaults and their Involvement in Multidrug Resistance

Vaults are evolutionary highly conserved ribonucleoprotein (RNP) particles with a hollow barrel-like structure. They are 41 x 73 nm in size and are composed of multiple copies of three proteins and small untranslated RNA (vRNA). The main component of vaults represents the 110 kDa major vault protein (MVP), whereas the two minor vault proteins comprise the 193 kDa vault poly(ADP-ribose) polymerase (VPARP) and the 240 kDa telomerase-associated protein-1 (TEP1). Vaults are abundantly present in the cytoplasm of eukaryotic cells and they were found to be associated with cytoskeletal elements as well as occasionally with the nuclear envelope. Vaults and MVP have been associated with several cellular processes which are also involved in cancer development like cell motility and differentiation. Due to the over-expression of MVP (also termed lung resistance-related protein or LRP) in several P-glycoprotein (P-gp)-negative chemoresistant cancer cell lines, vaults have been linked to multidrug resistance (MDR). Accordingly, high levels of MVP were found in tissues chronically exposed to xenobiotics. In addition, the expression of MVP correlated with the degree of malignancy in certain cancer types, suggesting a direct involvement in tumor development and/or progression. Based on the finding that MVP binds several phosphatases and kinases including PTEN, SHP-2 as well as Erk, evidence is accumulating that MVP might be involved in the regulation of important cell signalling pathways including the PI3K/Akt and the MAPK pathways. In this review we summarize the current knowledge concerning the vault particle and discuss its possible cellular functions, focusing on the role of vaults in chemotherapy resistance.

Expression of the multidrug resistance-associated protein (MRP) and chemoresistance of human non-small-cell lung cancer cells

Human non‐small‐cell lung cancer (NSCLC) is considered to be a chemotherapy‐refractory malignancy. The underlying mechanisms remain rather obscure. The multidrug resistance‐associated protein (MRP), mediating a multidrug resistance (MDR) phenotype, has been reported to be overexpressed in several drug‐selected lung cancer cell lines. A few previous studies have described intrinsic MRP expression in both NSCLC and normal lung tissues. However, the drug‐transporting activity as well as the correlation with chemoresistance is unclear. Using 15 unselected cell lines, we show that MRP (mRNA and protein as detected by reverse transcriptase polymerase chain reaction and immunoblot) is frequently expressed intrinsically, with markedly varying intensity, in NSCLC. Two cell lines expressed high MRP levels, one comparable to the drug‐selected controls (GLC4/ADR, HL‐60/AR) without, however, amplification of the MRP gene (Southern hybridization). Using 3H‐daunomycin (3H‐DM) and calcein as MRP substrates and probenecid (PRO), genistein (GEN), benzbromarone (BB), N‐ethylmaleimide (NEM) and verapamil (VP) as MRP modulators, drug accumulation studies revealed a transporting activity of MRP that correlated significantly with the gene expression data. Moreover, a significant correlation between MRP expression and chemoresistance against daunomycin (DM), doxorubicin (DOX), etoposide (VP‐16) and vinblastine (VBL), but not cisplatin (CDDP) and bleomycin (Bleo) (MTT‐based survival assay), was detected. Correlations mainly rested on the pronounced chemoresistance of 2 highly MRP‐expressing cell lines and did not reach significance when these cell lines were excluded. Int. J. Cancer 73:84–93, 1997.

The major vault protein is responsive to and interferes with interferon-γ-mediated STAT1 signals

The major vault protein (MVP) is the main component of vaults, large ribonucleoprotein particles implicated in the regulation of cellular signaling cascades and multidrug resistance. Here, we identify MVP as an interferon γ (IFN-γ)-inducible protein. Treatment with IFN-γ resulted in a significant upregulation of MVP promoter activity as well as mRNA and protein levels. Activation of MVP expression by IFN-γ involved transcriptional upregulation through the JAK/STAT pathway based on an interaction of STAT1 with an interferon-γ-activated site (GAS) within the proximal MVP promoter. Mutation of this site distinctly reduced basal as well as IFN-γ-stimulated MVP transcription. IFN-γ also significantly enhanced the translation rate of MVP. Ectopic MVP overexpression in the MVP-negative lung cancer cell model H65 led to a downregulation of three known IFN-γ-regulated genes, namely ICAM-1, CD13 and CD36. Additionally, presence of MVP in H65 cells blocked both basal and IFN-γ-induced ICAM-1 expression whereas downmodulation of endogenous MVP levels by shRNA enhanced IFN-γ-induced ICAM-1 expression in U373 glioblastoma cells. MVP-mediated IFN-γ insensitivity was accompanied by significantly reduced STAT1 phosphorylation at Y701 and diminished translocation of STAT1 into the nucleus. Summarizing, we identify MVP as an IFN-γ-responsive gene interfering with IFN-γ-activated JAK/STAT signals. These data further substantiate that the vault particle functions as a general interaction platform for cellular signaling cascades.

Overexpression of the human major vault protein in astrocytic brain tumor cells

Evidence has shown that the major human vault protein (MVP), which is identical to lung resistance‐related protein (LRP), may be causally involved in a special type of multidrug resistance (MDR). The purpose of this study was to investigate the expression and cellular localization of MVP in cells derived from brain tumors and other tumors of neuroectodermal origin. Using both established cell lines (n = 22) and primary explants (n = 30), we show that a distinct overexpression of the MVP gene at the mRNA (RT‐PCR) and protein (Western blot) levels is a characteristic feature of cells derived from astrocytic brain tumors. Primary cultures obtained from meningioma specimens also expressed high MVP levels, in contrast to neuroblastoma and medulloblastoma cells, which rarely contained detectable amounts of MVP. Normal human astrocytes cultured in vitro expressed MVP, although at low amounts compared with most malignant cell types. Basal MVP expression correlated with resistance against diverse antineoplastic drugs including anthracyclins, cisplatin and etoposide. By Western blot, MVP was also detected in all tumor samples taken from 7 glioma and 3 meningioma patients. Taken together, these data suggest overexpression of MVP as one explanation for the low efficacy of chemotherapeutic treatment of astrocytic brain tumors.