A comprehensive understanding of thioTEPA metabolism in the mouse using UPLC-ESI-QTOFMS-based metabolomics

ThioTEPA, an alkylating agent with anti-tumor activity, has been used as an effective anticancer drug since the 1950s. However, a complete understanding of how its alkylating activity relates to clinical efficacy has not been achieved, the total urinary excretion of thioTEPA and its metabolites is not resolved, and the mechanism of formation of the potentially toxic metabolites S-carboxymethylcysteine (SCMC) and thiodiglycolic acid (TDGA) remains unclear. In this study, the metabolism of thioTEPA in a mouse model was comprehensively investigated using ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC-ESI-QTOFMS) based-metabolomics. The nine metabolites identified in mouse urine suggest that thioTEPA underwent ring-opening, N-dechloroethylation, and conjugation reactions in vivo. SCMC and TDGA, two downstream thioTEPA metabolites, were produced from thioTEPA from two novel metabolites 1,2,3-trichloroTEPA (VII) and dechloroethyltrichloroTEPA (VIII). SCMC and TDGA excretion were increased about 4-fold and 2-fold, respectively, in urine following the thioTEPA treatment. The main mouse metabolites of thioTEPA in vivo were TEPA (II), monochloroTEPA (III) and thioTEPA-mercapturate (IV). In addition, five thioTEPA metabolites were detected in serum and all shared similar disposition. Although thioTEPA has a unique chemical structure which is not maintained in the majority of its metabolites, metabolomic analysis of its biotransformation greatly contributed to the investigation of thioTEPA metabolism in vivo, and provides useful information to understand comprehensively the pharmacological activity and potential toxicity of thioTEPA in the clinic.

Targeting PI3KC2β impairs proliferation and survival in acute leukemia, brain tumours and neuroendocrine tumours

Eight human catalytic phosphoinositide 3-kinase (PI3K) isoforms exist which are subdivided into three classes. While class I isoforms have been well-studied in cancer, little is known about the functions of class II PI3Ks.

Calpain-mediated cleavage of Atg5 switches autophagy to apoptosis

Autophagy-related gene (Atg) 5 is a gene product required for the formation of autophagosomes. Here, we report that Atg5, in addition to the promotion of autophagy, enhances susceptibility towards apoptotic stimuli. Enforced expression of Atg5-sensitized tumour cells to anticancer drug treatment both in vitro and in vivo. In contrast, silencing the Atg5 gene with short interfering RNA (siRNA) resulted in partial resistance to chemotherapy. Apoptosis was associated with calpain-mediated Atg5 cleavage, resulting in an amino-terminal cleavage product with a relative molecular mass of 24,000 (Mr 24K). Atg5 cleavage was observed independent of the cell type and the apoptotic stimulus, suggesting that calpain activation and Atg5 cleavage are general phenomena in apoptotic cells. Truncated Atg5 translocated from the cytosol to mitochondria, associated with the anti-apoptotic molecule Bcl-xL and triggered cytochrome c release and caspase activation. Taken together, calpain-mediated Atg5 cleavage provokes apoptotic cell death, therefore, represents a molecular link between autophagy and apoptosis--a finding with potential importance for clinical anticancer therapies.

Ex vivo assessment of chemotherapy-induced apoptosis and associated molecular changes in patient tumor samples

BACKGROUND: There are inherent conceptual problems in investigating the pharmacodynamics of cancer drugs in vivo. One of the few possible approaches is serial biopsies in patients. However, this type of research is severely limited by methodological and ethical constraints. MATERIALS AND METHODS: A modified 3-dimensional tissue culture technique was used to culture human tumor samples, which had been collected during routine cancer operations. Twenty tumor samples of patients with non-small cell lung cancer (NSCLC) were cultured ex vivo for 120 h and treated with mitomycin C, taxotere and cisplatin. The cytotoxic activity of the anticancer agents was quantified by assessing the metabolic activity of treated tumor cultures and various assays of apoptosis and gene expression were performed. RESULTS: The proliferative activity of the tissue was maintained in culture as assessed by Ki-67 staining. Mitomycin C, cisplatin and taxotere reduced the metabolic activity of the tumor tissue cultures by 51%, 29% and 20%, respectively, at 120 h. The decrease in metabolic activity corresponded to the induction of apoptosis as demonstrated by the typical morphological changes, such as chromatin condensation and nuclear fragmentation. In addition, activated caspase-3 could be verified in apoptotic cells by immunohistochemistry. To verify functional aspects of apoptosis, the induction of chemotherapy-induced cell death was inhibited with the caspase inhibitor z-VAD.fmk. RNA was extracted from the tissue cultures after 120 h of ex vivo drug treatment and was of sufficient quality to allow quantitative PCR. CONCLUSION: The 3-dimensional ex vivo culture technique is a useful method to assess the molecular effects of pharmacological interventions in human cancer samples in vitro. This culture technique could become an important tool for drug development and for the prediction of in vivo drug efficacy.

[Molecular targets in colon cancer]

Colorectal cancer is the second leading cause of cancer death in Switzerland. The nihilism that dominated the treatment of these patients for decades has been replaced by a measure of enthusiasm, given recent therapeutic advances. New anticancer drugs such as irinotecan and oxaliplatin have changed the standard chemotherapy treatment of metastatic colorectal cancer. However, the real hype has come from molecular targeted therapy. Identification of cellular processes characteristic of colon cancer has permitted therapeutic targeting with favorable therapeutic index. Inhibition of the epidermal growth factor receptor in the clinic has provided proof of principle that interruption of signal transduction cascades in patients has therapeutic potential. Angiogenesis, especially the vascular endothelial growth factor pathway, has been proven to be another highly successful molecular target. In this article, we will review molecular targets, which are under active clinical investigation in colon cancer.

Cardiac glycosides initiate Apo2L/TRAIL-induced apoptosis in non-small cell lung cancer cells by up-regulation of death receptors 4 and 5

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (Apo2L/TRAIL) belongs to the TNF family known to transduce their death signals via cell membrane receptors. Because it has been shown that Apo2L/TRAIL induces apoptosis in tumor cells without or little toxicity to normal cells, this cytokine became of special interest for cancer research. Unfortunately, cancer cells are often resistant to Apo2L/TRAIL-induced apoptosis; however, this can be at least partially negotiated by parallel treatment with other substances, such as chemotherapeutic agents. Here, we report that cardiac glycosides, which have been used for the treatment of cardiac failure for many years, sensitize lung cancer cells but not normal human peripheral blood mononuclear cells to Apo2L/TRAIL-induced apoptosis. Sensitization to Apo2L/TRAIL mediated by cardiac glycosides was accompanied by up-regulation of death receptors 4 (DR4) and 5 (DR5) on both RNA and protein levels. The use of small interfering RNA revealed that up-regulation of death receptors is essential for the demonstrated augmentation of apoptosis. Blocking of up-regulation of DR4 and DR5 alone significantly reduced cell death after combined treatment with cardiac glycosides and Apo2L/TRAIL. Combined silencing of DR4 and DR5 abrogated the ability of cardiac glycosides and Apo2L/TRAIL to induce apoptosis in an additive manner. To our knowledge, this is the first demonstration that glycosides up-regulate DR4 and DR5, thereby reverting the resistance of lung cancer cells to Apo2/TRAIL-induced apoptosis. Our data suggest that the combination of Apo2L/TRAIL and cardiac glycosides may be a new interesting anticancer treatment strategy.

Chemosensitization of carcinoma cells using epithelial cell adhesion molecule-targeted liposomal antisense against bcl-2/bcl-xL

Nanoscale drug delivery systems, such as sterically stabilized immunoliposomes binding to internalizing tumor-associated antigens, can increase therapeutic efficacy and reduce toxicity to normal tissues compared with nontargeted liposomes. The epithelial cell adhesion molecule (EpCAM) is of interest as a ligand for targeted drug delivery because it is abundantly expressed in solid tumors but shows limited distribution in normal tissues. To generate EpCAM-specific immunoliposomes for targeted cancer therapy, the humanized single-chain Fv antibody fragment 4D5MOCB was covalently linked to the exterior of coated cationic liposomes. As anticancer agent, we encapsulated the previously described antisense oligonucleotide 4625 specific for both bcl-2 and bcl-xL. The EpCAM-targeted immunoliposomes (SIL25) showed specific binding to EpCAM-overexpressing tumor cells, with a 10- to 20-fold increase in binding compared with nontargeted control liposomes. No enhanced binding was observed on EpCAM-negative control cells. On cell binding, SIL25 was efficiently internalized by receptor-mediated endocytosis, ultimately leading to down-regulation of both bcl-2 and bcl-xL expression on both the mRNA and protein level, which resulted in enhanced tumor cell apoptosis. In combination experiments, the use of SIL25 led to a 2- to 5-fold sensitization of EpCAM-positive tumor cells of diverse origin to death induction by doxorubicin. Our data show the promise of EpCAM-specific drug delivery systems, such as antisense-loaded immunoliposomes, for targeted cancer therapy.

Antiproliferative effects of mistletoe (Viscum album L.) extract in urinary bladder carcinoma cell lines

BACKGROUND: The aim of the study was to evaluate the antiproliferative potency of Viscum album extract (VA-E) in human bladder carcinoma cell lines with regard to its possible use for intravesical therapy of superficial bladder cancer. MATERIALS AND METHODS: Proliferation (MTT-test or 3H-thymidine incorporation), necrotic disintegration (3H-thymidine release of prelabelled cells) and portions of apoptotic and/or necrotic cells (Annexin-V binding, propidium iodide (PI) labelling and DNA-fluorescence profiles by flow cytometry) were measured in four different human bladder carcinoma cell lines (T24, TCCSUP, J82 and UM-UC3) cultured in vitro. RESULTS: Antiproliferative effects of VA-E were observed in the four bladder carcinoma cell lines tested. Metabolic activity could also be completely abrogated by short-time contact of the cells with VA-E. Apoptosis and necrosis, as underlying mechanisms of action, were differentially expressed by the different cell lines. CONCLUSION: VA-E and cytotoxic proteins, i.e., mistletoe lectins (ML) and viscotoxins (VT), were able to block the growth of bladder carcinoma cells. Together with the immunomodulating properties of VA-E, the observed antiproliferative potency might give a rationale for the topical intravesical application of VA-E for the treatment of superficial bladder cancer.

Antitumor activity of an epithelial cell adhesion molecule targeted nanovesicular drug delivery system

Site-specific delivery of anticancer agents to tumors represents a promising therapeutic strategy because it increases efficacy and reduces toxicity to normal tissues compared with untargeted drugs. Sterically stabilized immunoliposomes (SIL), guided by antibodies that specifically bind to well internalizing antigens on the tumor cell surface, are effective nanoscale delivery systems capable of accumulating large quantities of anticancer agents at the tumor site. The epithelial cell adhesion molecule (EpCAM) holds major promise as a target for antibody-based cancer therapy due to its abundant expression in many solid tumors and its limited distribution in normal tissues. We generated EpCAM-directed immunoliposomes by covalently coupling the humanized single-chain Fv antibody fragment 4D5MOCB to the surface of sterically stabilized liposomes loaded with the anticancer agent doxorubicin. In vitro, the doxorubicin-loaded immunoliposomes (SIL-Dox) showed efficient cell binding and internalization and were significantly more cytotoxic against EpCAM-positive tumor cells than nontargeted liposomes (SL-Dox). In athymic mice bearing established human tumor xenografts, pharmacokinetic and biodistribution analysis of SIL-Dox revealed long circulation times in the blood with a half-life of 11 h and effective time-dependent tumor localization, resulting in up to 15% injected dose per gram tissue. These favorable pharmacokinetic properties translated into potent antitumor activity, which resulted in significant growth inhibition (compared with control mice), and was more pronounced than that of doxorubicin alone and nontargeted SL-Dox at low, nontoxic doses. Our data show the promise of EpCAM-directed nanovesicular drug delivery for targeted therapy of solid tumors.

TRAIL-induced survival and proliferation of SCLC cells is mediated by ERK and dependent on TRAIL-R2/DR5 expression in the absence of caspase-8

Small cell lung cancer (SCLC) is characterized by an aggressive phenotype and acquired resistance to a broad spectrum of anticancer agents. TNF-related apoptosis-inducing ligand (TRAIL) has been considered as a promising candidate for safe and selective induction of tumor cell apoptosis without toxicity to normal tissues. Here we report that TRAIL failed to induce apoptosis in SCLC cells and instead resulted in an up to 40% increase in proliferation. TRAIL-induced SCLC cell proliferation was mediated by extracellular signal-regulated kinase 1 and 2, and dependent on the expression of surface TRAIL-receptor 2 (TRAIL-R2) and lack of caspase-8, which is frequent in SCLC. Treatment of SCLC cells with interferon-gamma (IFN-gamma) restored caspase-8 expression and facilitated TRAIL-induced apoptosis. The overall loss of cell proliferation/viability upon treatment with the IFN-gamma-TRAIL combination was 70% compared to TRAIL-only treated cells and more than 30% compared to untreated cells. Similar results were obtained by transfection of cells with a caspase-8 gene construct. Altogether, our data suggest that TRAIL-R2 expression in the absence of caspase-8 is a negative determinant for the outcome of TRAIL-based cancer therapy, and provides the rationale for using IFN-gamma or other strategies able to restore caspase-8 expression to convert TRAIL from a pro-survival into a death ligand.

Prognosis in pediatric hematologic malignancies is associated with serum concentration of mannose-binding lectin-associated serine protease-2 (MASP-2)

BACKGROUND: Mannose-binding lectin (MBL) and MBL-associated serine protease-2 (MASP-2) are key components of the lectin pathway of complement activation. Their serum concentrations show a wide interindividual variability. This study investigated whether the concentration of MBL and MASP-2 is associated with prognosis in pediatric patients with cancer. METHODS: In this retrospective multicenter study, MBL and MASP-2 were measured by commercially available ELISA in frozen remnants of serum taken at diagnosis. Associations of overall survival (OS) and event-free survival (EFS) with MBL and MASP-2 were assessed by multivariate Cox regression accounting for prognostically relevant clinical variables. RESULTS: In the 372 patients studied, median serum concentration of MBL was 2,808 microg/L (range, 2-10,060) and 391 microg/L (46-2,771) for MASP-2. The estimated 4-year EFS was 0.60 (OS, 0.78). In the entire, heterogeneous sample, MBL and MASP-2 were not significantly associated with OS or EFS. In patients with hematologic malignancies, however, higher MASP-2 was associated with better EFS in a significant and clinically relevant way (hazard ratio per tenfold increase (HR), 0.22; 95% CI, 0.09-0.54; P = 0.001). This was due to patients with lymphoma (HR, 0.11; 95% CI, 0.03-0.47; P = 0.003), but less for those with acute leukemia (HR, 0.35; 95% CI, 0.11-1.15; P = 0.083). CONCLUSION: In this study, higher MASP-2 was associated with better EFS in pediatric patients with hematologic malignancies, especially lymphoma. Whether MASP-2 is an independent prognostic factor affecting risk stratification and anticancer therapy needs to be assessed in prospective, disease-specific studies.

Taurolidine-citrate lock solution (TauroLock) significantly reduces CVAD-associated grampositive infections in pediatric cancer patients

BACKGROUND: Taurolidin/Citrate (TauroLock), a lock solution with broad spectrum antimicrobial activity, may prevent bloodstream infection (BSI) due to coagulase-negative staphylococci (CoNS or 'MRSE' in case of methicillin-resistant isolates) in pediatric cancer patients with a long term central venous access device (CVAD, Port- or/Broviac-/Hickman-catheter type). METHODS: In a single center prospective 48-months cohort study we compared all patients receiving anticancer chemotherapy from April 2003 to March 2005 (group 1, heparin lock with 200 IU/ml sterile normal saline 0.9%; Canusal Wockhardt UK Ltd, Wrexham, Wales) and all patients from April 2005 to March 2007 (group 2; taurolidine 1.35%/Sodium Citrate 4%; TauroLock, Tauropharm, Waldbüttelbrunn, Germany). RESULTS: In group 1 (heparin), 90 patients had 98 CVAD in use during the surveillance period. 14 of 30 (47%) BSI were 'primary Gram positive BSI due to CoNS (n = 4) or MRSE (n = 10)' [incidence density (ID); 2.30 per 1000 inpatient CVAD-utilization days].In group 2 (TauroLock), 89 patients had 95 CVAD in use during the surveillance period. 3 of 25 (12%) BSI were caused by CoNS. (ID, 0.45). The difference in the ID between the two groups was statistically significant (P = 0.004). CONCLUSION: The use of Taurolidin/Citrate (TauroLock) significantly reduced the number and incidence density of primary catheter-associated BSI due to CoNS and MRSE in pediatric cancer patients.

FUNCTIONALIZATION OF CARBON NANOTUBES FOR MESENCHYMAL STEM CELL-ASSISTED PHOTOTHERMAL THERAPY

Carbon nanotubes were first cut and functionalized with a newly developed reaction involving autoclaving and sonication in hydrogen peroxide. The functionalized nanotubes were characterized and evaluated for aqueous solubility. Studies which relate reaction conditions to final carbon nanotube length were conducted. Hydroxyl groups present on the carbon nanotubes served as a platform for a series of addition reactions, with the objective of conjugating streptavidin and fluorescent markers onto the carbon nanotubes. The modified nanotubes were attached onto the surface of biotinylated mesenchymal stem cells, creating a novel, tumor-homing delivery system for photothermal anticancer agents.

Epothilones as lead structures for the synthesis-based discovery of new chemotypes for microtubule stabilization

Epothilones are macrocyclic bacterial natural products with potent microtubule-stabilizing and antiproliferative activity. They have served as successful lead structures for the development of several clinical candidates for anticancer therapy. However, the structural diversity of this group of clinical compounds is rather limited, as their structures show little divergence from the original natural product leads. Our own research has explored the question of whether epothilones can serve as a basis for the development of new structural scaffolds, or chemotypes, for microtubule stabilization that might serve as a basis for the discovery of new generations of anticancer drugs. We have elaborated a series of epothilone-derived macrolactones whose overall structural features significantly deviate from those of the natural epothilone scaffold and thus define new structural families of microtubule-stabilizing agents. Key elements of our hypermodification strategy are the change of the natural epoxide geometry from cis to trans, the incorporation of a conformationally constrained side chain, the removal of the C3-hydroxyl group, and the replacement of C12 with nitrogen. So far, this approach has yielded analogs 30 and 40 that are the most advanced, the most rigorously modified, structures, both of which are potent antiproliferative agents with low nanomolar activity against several human cancer cell lines in vitro. The synthesis was achieved through a macrolactone-based strategy or a high-yielding RCM reaction. The 12-aza-epothilone ("azathilone" 40) may be considered a "non-natural" natural product that still retains most of the overall structural characteristics of a true natural product but is structurally unique, because it lies outside of the general scope of Nature's biosynthetic machinery for polyketide synthesis. Like natural epothilones, both 30 and 40 promote tubulin polymerization in vitro and at the cellular level induce cell cycle arrest in mitosis. These facts indicate that cancer cell growth inhibition by these compounds is based on the same mechanistic underpinnings as those for natural epothilones. Interestingly, the 9,10-dehydro analog of 40 is significantly less active than the saturated parent compound, which is contrary to observations for natural epothilones B or D. This may point to differences in the bioactive conformations of N-acyl-12-aza-epothilones like 40 and natural epothilones. In light of their distinct structural features, combined with an epothilone-like (and taxol-like) in vitro biological profile, 30 and 40 can be considered as representative examples of new chemotypes for microtubule stabilization. As such, they may offer the same potential for pharmacological differentiation from the original epothilone leads as various newly discovered microtubule-stabilizing natural products with macrolactone structures, such as laulimalide, peloruside, or dictyostatin.

Combined approach to hepatocellular carcinoma: a new treatment concept for nonresectable disease

Depending on tumor burden, hepatic function and patients' performance status, hepatocellular carcinoma is treated by surgery, local procedures, systemic therapy or palliation. The majority of patients are diagnosed at a stage where local therapy is the treatment of choice. Recently, the multikinase inhibitor sorafenib was found to improve the survival of patients with advanced hepatocellular carcinoma and conserved liver function. In this manuscript, we summarize the experimental evidence supporting the combination of a systemic targeted therapy with a local therapy. We also discuss the pros and cons of different schedules of combining such treatments. We conclude that there is enough of a theoretical argument to design clinical trials testing this strategy.