Methadone-mediated sensitization of glioblastoma cells is drug and cell line dependent.

PURPOSE: D,L-methadone (MET), an analgesic drug used for pain treatment and opiate addiction, has achieved attention from oncologists and social media as possible chemoensitizing agent in cancer therapy, notably brain cancer (glioblastoma multiforme, GBM). MET has been reported to enhance doxorubicin-induced cytotoxicity in GBM cells via activation of the µ-opioid receptor (MOR). Here, we extended this work and quantified the toxic effect of MET in comparison to other opioids alone and in combination with doxorubicin and the clinically more relevant alkylating drug temozolomide (TMZ), using a set of GBM cell lines and primary GBM cells. METHODS: MOR expression in GBM cells was investigated by immunofluorescence and immunoblotting. Resistance to drugs alone and in combination with anticancer drugs was assessed by MTT assays. Concentration effect curves were fitted by nonlinear regression analysis and IC50 values were calculated. Apoptosis and necrosis rates were determined by annexin V/propidium iodide (PI)-flow cytometry. RESULTS: MET alone was cytotoxic in all GBM cell lines and primary GBM cells at high micromolar concentrations (IC50 ~ 60-130 µM), observed both in the metabolic MTT assay and by quantifying apoptosis and necrosis, while morphine and oxycodone were not cytotoxic in this concentration range. Naloxone was not able to block MET-induced cytotoxicity, indicating that cell death-inducing effects of MET are not MOR-dependent. We recorded doxorubicin and TMZ concentration- response curves in combination with fixed MET concentrations. MET enhanced doxorubicin-induced cytotoxicity in only one cell line, and in primary cells it was observed only in a particular MET concentration range. In all assays, MET was not effective in sensitizing cells to TMZ. In two cell lines, MET even decreased the cell's sensitivity to TMZ. CONCLUSION: MET was found to be cytotoxic in GBM cells in vitro only at high, clinically not relevant concentrations, where it was effective in inducing apoptosis and necrosis. Sensitizing effects were only observed in combination with doxorubicin, but not with TMZ, and are dependent on cell line and the applied drug concentration. Therefore, our findings do not support the use of MET in the treatment of GBM in combination with TMZ, as no sensitizing effect of MET was observed.

Liquid Chromatography-High-Resolution Mass Spectrometry-Based In Vitro Toxicometabolomics of the Synthetic Cathinones 4-MPD and 4-MEAP in Pooled Human Liver Microsomes.

Synthetic cathinones belong to the most often seized new psychoactive substances on an international level. This study investigated the toxicometabolomics, particularly the in vitro metabolism of 2-(methylamino)-1-(4-methylphenyl)-1-pentanone (4-MPD) and 2-(ethylamino)-1-(4-methylphenyl)-1-pentanone (4-MEAP) in pooled human liver microsomes (pHLM) using untargeted metabolomics techniques. Incubations were performed with the substrates in concentrations ranging from 0, 12.5, and 25 µM. Analysis was done by means of high-performance liquid chromatography coupled to high-resolution mass spectrometry (HPLC-HRMS/MS) in full scan only and the obtained data was evaluated using XCMS Online and MetaboAnalyst. Significant features were putatively identified using a separate parallel reaction monitoring method. Statistical analysis was performed using Kruskal-Wallis test for prefiltering significant features and subsequent hierarchical clustering, as well as principal component analysis (PCA). Hierarchical clustering or PCA showed a distinct clustering of all concentrations with most of the features z-scores rising with the concentration of the investigated substances. Identification of significant features left many of them unidentified but revealed metabolites of both 4-MPD and 4-MEAP. Both substances formed carboxylic acids, were hydroxylated at the alkyl chain, and formed metabolites after combined hydroxylation and reduction of the cathinone oxo group. 4-MPD additionally formed a dihydroxy metabolite and a hydroxylamine. 4-MEAP was additionally found reduced at the cathinone oxo group, N-dealkylated, and formed an oxo metabolite. These findings are the first to describe the metabolic pathways of 4-MPD and to extend our knowledge about the metabolism of 4-MEAP. Findings, particularly the MS data of the metabolites, are essential for setting up metabolite-based toxicological (urine) screening procedures.

Thioredoxin Confers Intrinsic Resistance to Cytostatic Drugs in Human Glioma Cells.

Thioredoxin (Trx) overexpression is known to be a cause of chemotherapy resistance in various tumor entities. However, Trx effects on resistance are complex and depend strictly on tissue type. In the present study, we analyzed the impact of the Trx system on intrinsic chemoresistance of human glioblastoma multiforme (GBM) cells to cytostatic drugs. Resistance of GBM cell lines and primary cells to drugs and signaling inhibitors was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Impact of Trx inhibition on apoptosis was investigated by proteome profiling of a subset of proteins and annexin V apoptosis assays. Trx-interacting protein (TXNIP) was overexpressed by transfection and protein expression was determined by immunoblotting. Pharmacological inhibition of Trx by 1-methyl-2-imidazolyl-disulfide (PX-12) reduced viability of three GBM cell lines, induced expression of active caspase-3, and reduced phosphorylation of AKT-kinase and expression of ß-catenin. Sensitivity to cisplatin could be restored by both PX-12 and recombinant expression of the upstream Trx inhibitor TXNIP, respectively. In addition, PX-12 also sensitized primary human GBM cells to temozolomide. Combined inhibition of Trx and the phosphatidylinositide 3-kinase (PI3K) pathway resulted in massive cell death. We conclude that the Trx system and the PI3K pathway act as a sequential cascade and could potentially present a new drug target.

Inhibition of the PI3K but not the MEK/ERK pathway sensitizes human glioma cells to alkylating drugs.

BACKGROUND: Intrinsic chemoresistance of glioblastoma (GBM) is frequently owed to activation of the PI3K and MEK/ERK pathways. These signaling cascades are tightly interconnected however the quantitative contribution of both to intrinsic resistance is still not clear. Here, we aimed at determining the activation status of these pathways in human GBM biopsies and cells and investigating the quantitative impact of both pathways to chemoresistance. METHODS: Receptor tyrosine kinase (RTK) pathways in temozolomide (TMZ) treatment naive or TMZ resistant human GBM biopsies and GBM cells were investigated by proteome profiling and immunoblotting of a subset of proteins. Resistance to drugs and RTK pathway inhibitors was assessed by MTT assays. Apoptotic rates were determined by Annexin V staining and DNA damage with comet assays and immunoblotting. RESULTS: We analyzed activation of RTK pathways by proteome profiling of tumor samples of patients which were diagnosed a secondary GBM and underwent surgery and patients which underwent a second surgery after TMZ treatment due to recurrence of the tumor. We observed substantial activation of the PI3K and MEK/ERK pathways in both groups. However, AKT and CREB phosphorylation was reduced in biopsies of resistant tumors while ERK phosphorylation remained unchanged. Subsequent proteome profiling revealed that multiple RTKs and downstream targets are also activated in three GBM cell lines. We then systematically describe a mechanism of resistance of GBM cell lines and human primary GBM cells to the alkylating drugs TMZ and cisplatin. No specific inhibitor of the upstream RTKs sensitized cells to drug treatment. In contrast, we were able to restore sensitivity to TMZ and cisplatin by inhibiting PI3K in all cell lines and in human primary GBM cells. Interestingly, an opposite effect was observed when we inhibited the MEK/ERK signaling cascade with two different inhibitors. CONCLUSIONS: Temozolomide treatment naive and TMZ resistant GBM biopsies show a distinct activation pattern of the MEK/ERK and PI3K signaling cascades indicating a role of these pathways in resistance development. Both pathways are also activated in GBM cell lines, however, only the PI3K pathway seems to play a crucial role in resistance to alkylating agents and might serve as drug target for chemosensitization.

Is sunitinib a Narrow Therapeutic Index Drug? - A systematic review and in vitro toxicology-analysis of Sunitinib vs. Imatinib in cells from different tissues.

Narrow Therapeutic Index Drugs (NTIDs) are characterized by a small range between therapeutic and toxicological effect. Missing international harmonized definition for NTIDs the EMA does not even have a definition of NTIDs in contrast to the U.S. FDA, Health Canada, and the Japanese NIHS. Sunitinib, a tyrosine kinase inhibitor (TKI), indicated for the treatment of certain cancer types, will be running off-patent soon. Falling into the category of NTID would have a major impact on regulatory requirements for generic applications. Our analyses of metadata revealed numerous arguments in favor of a NTID designation. We used in vitro experiments to also give initial experimental answers. Five cell types of different tissue origin were examined for determination of IC50-values in cell viability assays. For comparison, the first-in-class TKI Imatinib was used as reference non-NTID drug. In addition, apoptotic proteins were investigated with respect to their expression and phosphorylation status. These in vitro experiments showed systematically higher toxicity of Sunitinib compared to Imatinib and a different expression and phosphorylation pattern of apoptotic proteins. In vitro data can only give preliminary results and further experiments with clinical blood samples and tumor biopsies are needed to finally clarify NTID status of Sunitinib.

Systemic therapy of Cushing's syndrome.

Cushing's disease (CD) in a stricter sense derives from pathologic adrenocorticotropic hormone (ACTH) secretion usually triggered by micro- or macroadenoma of the pituitary gland. It is, thus, a form of secondary hypercortisolism. In contrast, Cushing's syndrome (CS) describes the complexity of clinical consequences triggered by excessive cortisol blood levels over extended periods of time irrespective of their origin. CS is a rare disease according to the European orphan regulation affecting not more than 5/10,000 persons in Europe. CD most commonly affects adults aged 20-50 years with a marked female preponderance (1:5 ratio of male vs. female). Patient presentation and clinical symptoms substantially vary depending on duration and plasma levels of cortisol. In 80% of cases CS is ACTH-dependent and in 20% of cases it is ACTH-independent, respectively. Endogenous CS usually is a result of a pituitary tumor. Clinical manifestation of CS, apart from corticotropin-releasing hormone (CRH-), ACTH-, and cortisol-producing (malign and benign) tumors may also be by exogenous glucocorticoid intake. Diagnosis of hypercortisolism (irrespective of its origin) comprises the following: Complete blood count including serum electrolytes, blood sugar etc., urinary free cortisol (UFC) from 24 h-urine sampling and circadian profile of plasma cortisol, plasma ACTH, dehydroepiandrosterone, testosterone itself, and urine steroid profile, Low-Dose-Dexamethasone-Test, High-Dose-Dexamethasone-Test, after endocrine diagnostic tests: magnetic resonance imaging (MRI), ultra-sound, computer tomography (CT) and other localization diagnostics. First-line therapy is trans-sphenoidal surgery (TSS) of the pituitary adenoma (in case of ACTH-producing tumors). In patients not amenable for surgery radiotherapy remains an option. Pharmacological therapy applies when these two options are not amenable or refused. In cases when pharmacological therapy becomes necessary, Pasireotide should be used in first-line in CD. CS patients are at an overall 4-fold higher mortality rate than age- and gender-matched subjects in the general population. The following article describes the most prominent substances used for clinical management of CS and gives a systematic overview of safety profiles, pharmacokinetic (PK)-parameters, and regulatory framework.

Clinical pharmacology and regulatory consequences of GnRH analogues in prostate cancer.

INTRODUCTION: GnRH (gonadotropin-releasing hormone) analogues are long-term known to be safe and effective in the clinical management of hormone-dependent advanced prostate cancer. However, their unusual mechanism of action of de-sensitizing pituitary receptors makes generic market entry challenging. In addition, safety aspects like initial flare-up, breakthrough escape, and miniflares render planning and organization of clinical registration trials a complex project. REGULATORY REQUIREMENTS THERAPEUTIC EQUIVALENCE: Regulatory requirements are high as these medicines are compared to bilateral surgical castration with a 100% success rate. GnRH analogues will be used probably even wider in the near future due to demographic development and extension of indications. However, they are challenged by their antagonistic counterparts, which are avoiding flare-up phenomena. The following article deals with regulatory requirements of GnRH analogues in regard to their clinical characteristics.

Clinical pharmacology of tyrosine kinase inhibitors becoming generic drugs: the regulatory perspective.

Over the last decades, billions have been spent and huge efforts have been taken in basic and clinical cancer research [CA Cancer J Clin63:11-30]. About a decade ago, the arms race between drugs and cancer cells reached a new level by introduction of tyrosine kinase inhibitors (TKI) into pharmacological anti-cancer therapy. According to their molecular mechanism of action, TKI in contrast to so-called "classic" or "conventional" cytostatics belong to the group of targeted cancer medicines, characterized by accurately fitting with biological structures (i.e. active centers of kinases). Numerous (partly orphan) indications are covered by this new class of substances. Approximately ten years after the first substances of this class of medicines were authorized, patent protection will end within the next years. The following article covers clinical meaning and regulatory status of anti-cancer TKI and gives an outlook to what is expected from the introduction of generic anti-cancer TKI.

Targeting adipose tissue.

Two different types of adipose tissues can be found in humans enabling them to respond to starvation and cold: white adipose tissue (WAT) is generally known and stores excess energy in the form of triacylglycerol (TG), insulates against cold, and serves as a mechanical cushion. Brown adipose tissue (BAT) helps newborns to cope with cold. BAT has the capacity to uncouple the mitochondrial respiratory chain, thereby generating heat rather than adenosine triphosphate (ATP). The previously widely held view was that BAT disappears rapidly after birth and is no longer present in adult humans. Using positron emission tomography (PET), however, it was recently shown that metabolically active BAT occurs in defined regions and scattered in WAT of the adult and possibly has an influence on whole-body energy homeostasis. In obese individuals adipose tissue is at the center of metabolic syndrome. Targeting of WAT by thiazolidinediones (TZDs), activators of peroxisome proliferator-activated receptor γ (PPARγ) a 'master' regulator of fat cell biology, is a current therapy for the treatment of type 2 diabetes. Since its unique capacity to increase energy consumption of the body and to dissipate surplus energy as heat, BAT offers new perspectives as a therapeutic target for the treatment of obesity and associated diseases such as type 2 diabetes and metabolic syndrome. Recent discoveries of new signaling pathways of BAT development give rise to new therapeutic possibilities in order to influence BAT content and activity.

Platinum resistance in breast and ovarian cancer cell lines.

Breast and ovarian cancers are among the 10 leading cancer types in females with mortalities of 15% and 6%, respectively. Despite tremendous efforts to conquer malignant diseases, the war on cancer declared by Richard Nixon four decades ago seems to be lost. Approximately 21,800 women in the US will be diagnosed with ovarian cancer in 2011. Therefore, its incidence is relatively low compared to breast cancer with 207.090 prognosed cases in 2011. However, overall survival unmasks ovarian cancer as the most deadly gynecological neoplasia. Platinum-based chemotherapy is emerging as an upcoming treatment modality especially in triple negative breast cancer. However, in ovarian cancer Platinum-complexes for a long time are established as first line treatment. Emergence of a resistant phenotype is a major hurdle in curative cancer therapy approaches and many scientists around the world are focussing on this issue. This review covers new findings in this field during the past decade.

Overcoming cisplatin resistance of ovarian cancer cells by targeted liposomes in vitro.

The clinical application of cisplatin to treat solid tumours is often limited by the development of tumour cell resistance against this cytostatic agent. Although liposomal carriers of cisplatin are currently in clinical development, approaches to functionally overcome cisplatin resistance by liposomes have hardly been reported. We prepared PEGylated cisplatin-containing liposomes with diameters of about 110 nm and targetability to transferrin receptors (TfR) to correlate cisplatin cell uptake with cytotoxicity in sensitive and cisplatin resistant ovarian cancer cells A2780 compared to the free drug. Whereas the cell entry of free cisplatin was reduced by factor 4 after 24h in resistant cells, liposomal uptake was similar in both cell lines and not affected by resistance. Cytotoxicity was clearly related to intracellular platinum levels, which were even higher for liposomal vs. free cisplatin in the resistant cells after 24, 48, and 72 h and slightly lower in the sensitive cells. However, TfR targeting was of less impact on activity in comparison to non-targeted liposomes. Detection of cellular ATP levels within 24h allowed postulations on the intracellular fate of the liposomes. Altogether, this study strongly supports approaches to overcome cisplatin resistance by a liposomal application of the drug.

NF546 [4,4'-(carbonylbis(imino-3,1-phenylene-carbonylimino-3,1-(4-methyl-phenylene)-carbonylimino))-bis(1,3-xylene-alpha,alpha'-diphosphonic acid) tetrasodium salt] is a non-nucleotide P2Y11 agonist and stimulates release of interleukin-8 from human monocyte-derived dendritic cells.

The G protein-coupled P2Y(11) receptor is involved in immune system modulation. In-depth physiological evaluation is hampered, however, by a lack of selective and potent ligands. By screening a library of sulfonic and phosphonic acid derivatives at P2Y(11) receptors recombinantly expressed in human 1321N1 astrocytoma cells (calcium and cAMP assays), the selective non-nucleotide P2Y(11) agonist NF546 [4,4'-(carbonylbis(imino-3,1-phenylene-carbonylimino-3,1-(4-methyl-phenylene)carbonylimino))-bis(1,3-xylene-alpha,alpha'-diphosphonic acid) tetrasodium salt] was identified. NF546 had a pEC(50) of 6.27 and is relatively selective for P2Y(11) over P2Y(1), P2Y(2), P2Y(4), P2Y(6), P2Y(12), P2X(1), P2X(2), and P2X(2)-X(3). Adenosine-5'-O-(3-thio)triphosphate (ATPgammaS), a nonhydrolyzable analog of the physiological P2Y(11) agonist ATP, and NF546 use a common binding site as suggested by molecular modeling studies and their competitive behavior toward the nanomolar potency antagonist NF340 [4,4'-(carbonylbis(imino-3,1-(4-methyl-phenylene)carbonylimino))bis(naphthalene-2,6-disulfonic acid) tetrasodium salt] in Schild analysis. The pA(2) of NF340 was 8.02 against ATPgammaS and 8.04 against NF546 (calcium assays). NF546 was further tested for P2Y(11)-mediated effects in monocyte-derived dendritic cells. Similarly to ATPgammaS, NF546 led to thrombospondin-1 secretion and inhibition of lipopolysaccharide-stimulated interleukin-12 release, whereas NF340 inhibited these effects. Further, for the first time, it was shown that ATPgammaS or NF546 stimulation promotes interleukin 8 (IL-8) release from dendritic cells, which could be inhibited by NF340. In conclusion, we have described the first selective, non-nucleotide agonist NF546 for P2Y(11) receptors in both recombinant and physiological expression systems and could show a P2Y(11)-stimulated IL-8 release, further supporting the immunomodulatory role of P2Y(11) receptors.

Y-box-binding protein YB-1 identifies high-risk patients with primary breast cancer benefiting from rapidly cycled tandem high-dose adjuvant chemotherapy.

PURPOSE: To investigate the potential of Y-box-binding protein YB-1, a multifunctional protein linked to tumor aggressiveness and multidrug resistance, to identify patients with breast cancer likely to benefit from dose-intensified chemotherapy regimens. PATIENTS AND METHODS: YB-1 was immunohistochemically determined in 211 primary tumors from the prospective, randomized West German Study Group WSG-AM-01 trial in high-risk (> or = 10 involved lymph-nodes) breast cancer (HRBC). Predictive impact of YB-1 was assessed by multivariate survival analysis, including time-varying factor-therapy interactions. RESULTS: At median follow-up of 61.7 months, patients receiving rapidly cycled tandem high-dose therapy (HD; two cycles [2x] epirubicin 90 mg/m(2) and cyclophosphamide 600 mg/m(2) every 14 days, followed by 2x epirubicin 90 mg/m(2), cyclophosphamide 3,000 mg/m(2), and thiotepa 400 mg/m(2) every 21 days) had better disease-free survival (DFS; hazard ratio [HR] = 0.62; 95% CI, 0.44 to 0.89) and overall survival (OS; HR = 0.59; 95% CI, 0.4 to 0.89) than those receiving conventional dose-dense chemotherapy (DD; 4x epirubicin 90 mg/m(2) and cyclophosphamide 600 mg/m(2), followed by 3x cyclophosphamide 600 mg/m(2), methotrexate 40 mg/m(2), and fluorouracil 600 mg/m(2) every 14 days). High YB-1 was associated with aggressive tumor phenotype (negative steroid hormone receptor status, positive human epidermal growth factor receptor 2 and p53 status, high MIB-1, unfavorable tumor grade) and poor OS (median 78 v 97 months; P = .01). In patients with high YB-1, HD yielded a 63-month median DFS (P = .001) and a 46-month median OS advantage (P = .002) versus DD. In multivariate models, patients with high B-1 receiving HD (v DD) had one third the hazard rate after 20 months for DFS and one sixth after 40 months for OS. CONCLUSION: In a randomized prospective cancer therapy trial, for the first time, a strong predictive impact of YB-1 on survival has been demonstrated: enhanced benefit from HD (v DD) therapy occurs in HRBC with high YB-1. Future trials could therefore address optimal chemotherapeutic strategies,taking YB-1 into account.

Hyperactivation of the insulin-like growth factor receptor I signaling pathway is an essential event for cisplatin resistance of ovarian cancer cells.

Platinum plays a central role in the therapy of ovarian cancer, and the emergence of platinum resistance is a major obstacle for clinical management of the disease. We treated A2780 ovarian cancer cells by weekly cycles of cisplatin over a period of 6 months and unveiled that enhanced insulin-like growth factor I receptor (IGF-IR) expression and autocrine IGF-I are associated with hyperactivation of the IGF-IR and phosphatidylinositol-3-OH kinase (PI3K) pathways in cisplatin-resistant cells. IGF-IR expression levels increased during treatment cycles and correlated with cisplatin resistance. Purified IGF-I induced cisplatin resistance in diverse ovarian cancer cell lines, and small molecule inhibitors proved that IGF-IR and PI3K are essential for cisplatin resistance. Similar results were obtained with BG-1 ovarian cancer cells. Cytogenetic and array comparative genomic hybridization analyses revealed selection and de novo formation of chromosomal alterations during resistance development. An analysis of gene expression profiles of primary ovarian carcinomas identified the regulatory subunit PIK3R2 of PI3-kinase as a significant negative prognosis factor for ovarian cancer. We conclude that targeting the IGF-IR and the PI3K pathways is a promising new strategy to treat cisplatin-resistant ovarian carcinomas.

Acquired cisplatin resistance in the head-neck cancer cell line Cal27 is associated with decreased DKK1 expression and can partially be reversed by overexpression of DKK1.

Head and neck cancers are treated by a combination of surgery, radiotherapy and/or chemotherapy. The clinical success of cisplatin-based chemotherapy, mostly in combination with 5-FU or a taxane, is however limited by multifactorial intrinsic or acquired resistance. So far, known genes involved in cisplatin resistance do not sufficiently allow the prediction of cancer chemosensitivity. Thus, the purpose of this study was to search for further genes involved in cisplatin resistance by differential gene expression analysis of the parental tongue cancer cell line Cal27 and its 10-fold more resistant sub-cell line Cal27cis, which was obtained by treating Cal27 with increasing concentrations of cisplatin. As found by the suppression subtractive hybridization, expression of DKK1, an inhibitor of canonical WNT signaling, was decreased in Cal27cis. Microarray analysis, qPCR and ELISA confirmed the approximately 2-fold difference in expression. Cisplatin treatment and serum starvation increased by 2-fold the secretion of DKK1 in Cal27 and Cal27cis, thus rendering DKK1-levels significantly different in both cell lines under basal and stress conditions. Recombinant overexpression of DKK1 in Cal27 and Cal27cis resulted in clonal cell lines, which were both 2.2- to 3-fold more sensitive toward cisplatin in cell viability (MTT) and in proliferation (BrdU) assays. In conclusion, acquired (10-fold) resistance of Cal27 against cisplatin is associated with decreased DKK1 expression and could partially be reversed by DKK1 overexpression, thus suggesting DKK1 and the WNT signaling pathway as a marker and target for cisplatin chemosensitivity.

Epidermal growth factor receptor pathway analysis identifies amphiregulin as a key factor for cisplatin resistance of human breast cancer cells.

The use of platinum complexes for the therapy of breast cancer is an emerging new treatment modality. To gain insight into the mechanisms underlying cisplatin resistance in breast cancer, we used estrogen receptor-positive MCF-7 cells as a model system. We generated cisplatin-resistant MCF-7 cells and determined the functional status of epidermal growth factor receptor (EGFR), MAPK, and AKT signaling pathways by phosphoreceptor tyrosine kinase and phospho-MAPK arrays. The cisplatin-resistant MCF-7 cells are characterized by increased EGFR phosphorylation, high levels of AKT1 kinase activity, and ERK1 phosphorylation. In contrast, the JNK and p38 MAPK modules of the MAPK signaling pathway were inactive. These conditions were associated with inactivation of the p53 pathway and increased BCL-2 expression. We investigated the expression of genes encoding the ligands for the ERBB signaling cascade and found a selective up-regulation of amphiregulin expression, which occurred at later stages of cisplatin resistance development. Amphiregulin is a specific ligand of the EGFR (ERBB1) and a potent mitogen for epithelial cells. After exposure to cisplatin, the resistant MCF-7 cells secreted amphiregulin protein over extended periods of time, and knockdown of amphiregulin expression by specific short interfering RNA resulted in a nearly complete reversion of the resistant phenotype. To demonstrate the generality and importance of our findings, we examined amphiregulin expression and cisplatin resistance in a variety of human breast cancer cell lines and found a highly significant correlation. In contrast, amphiregulin levels did not significantly correlate with cisplatin resistance in a panel of lung cancer cell lines. We have thus identified a novel function of amphiregulin for cisplatin resistance in human breast cancer cells.

The Y-box binding protein YB-1 is associated with progressive disease and mediates survival and drug resistance in multiple myeloma.

Current knowledge about molecular mechanisms underlying disease progression and drug resistance in multiple myeloma (MM) is still limited. Here, we analyzed the potential pathogenetic role of the Y-box binding protein YB-1 in MM. YB-1 is a member of the cold-shock domain protein superfamily and involved in various cellular functions such as proliferation. Immunohistochemical analyses revealed that neither normal bone marrow (BM) plasma cells (PCs), premalignant PCs of patients with monoclonal gammopathy of unknown significance (MGUS), nor MM cells with a mature morphology showed expression of YB-1 in situ. In contrast, YB-1 was strongly expressed in situ in normal PC precursor blasts as well as in a MM subset and in vitro in all of the evaluated MM cell lines. The YB-1-expressing MM cells were characterized by an immature morphology and a highly proliferative phenotype as defined by Ki 67 expression. We observed that siRNA-mediated knockdown of YB-1 decreased proliferation and induced apoptosis in MM cells even in the presence of BM stromal cells. Furthermore, we found that overexpression of YB-1 mediated resistance toward doxorubicin-induced apoptosis in MM cells. Thus, YB-1 contributes to disease progression, survival, and drug resistance in MM and might therefore provide an attractive therapeutic target.