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INTRODUCTION: Immunosuppressant drugs are increasingly being used in the reproductive years. Theoretically, such medications could affect fetal health either through changes in the sperm DNA or through fetal exposure caused by a presence in the seminal fluid. This systematic overview summarizes existing literature on the spermatotoxic and genotoxic potentials of methotrexate (MTX), a drug widely used to treat rheumatic and dermatologic diseases, and mycophenolate mofetil (MMF), which alone or supplemented with ganciclovir (GCV) may be crucial for the survival of organ transplants.MATERIAL AND METHODS: The systematic overview was performed in accordance with the PRISMA guidelines: A systematic literature search of the MEDLINE and Embase databases was done using a combination of relevant terms to search for studies on spermatotoxic or genotoxic changes related to treatment with MTX, GCV or MMF. The search was restricted to English language literature, and to in vivo animal studies (mammalian species) and clinical human studies.RESULTS: A total of 102 studies were identified, hereof 25 human and 77 animal studies. For MTX, human studies of immunosuppressive dosages show transient effect on sperm quality parameters, which return to reference values within 3 months. No human studies have investigated the sperm DNA damaging effect of MTX, but in other organs the genotoxic effects of immunosuppressive doses of MTX are fluctuating. In animals, immunosuppressive and cytotoxic doses of MTX adversely affect sperm quality parameters and show widespread genotoxic damages in various organs. Cytotoxic doses transiently change the DNA material in all cell stages of spermatogenesis in rodents. For GCV and MMF, data are limited and the results are indeterminate, for which reason spermatotoxic and genotoxic potentials cannot be excluded.CONCLUSIONS: Data from human and animal studies indicate transient spermatotoxic and genotoxic potentials of immunosuppressive and cytotoxic doses of MTX. There are a limited number of studies investigating GCV and MMF.

Summary With the aim of overcoming the well-known limitations of platinum-based antineoplastic drugs, recent efforts have focused on the development of new anticancer agents containing metals other than platinum. Among these agents, organopalladium compounds have received significant recent attention due to their generally high stability under physiological conditions. A significant number of these compounds have shown promising in vitro and in vivo antiproliferative activity toward several cisplatin-sensitive and cisplatin-resistant tumors and have sometimes exhibited a different mechanism of action compared to platinum-based drugs. In this review, recent advances in the field of organopalladium compounds as potential anticancer agents are discussed.

Vinorelbine (VLB) is a semi-synthetic Vinca alkaloid which is currently used in treatment of different cancer types mainly advanced breast cancer (ABC) and advanced/metastatic non-small cell lung cancer (NSCLC). However, its marketed formulation has been reported to have serious side effects, such as granulocytopenia, which is the major dose-limiting toxicity. Other unwanted effects include venous discoloration and phlebitis proximal to the site of injection, as well as localized rashes and urticaria, blistering, and skin sloughing. Our long-term aim in synthesizing a novel nanomicellar vinorelbine formulation is to reduce or even eliminate these side effects and increase drug activity by formulating the drug in a lipid-based system as a nanomedicine targeted to the site of action. To this end, the purpose of this study was to prepare, characterize, and determine the in vitro efficacy of vinorelbine-loaded sterically stabilized, biocompatible, and biodegradable phospholipid nanomicelles (SSM; size, similar to 15 nm). Our results indicated that vinorelbine incorporate at high quantities and within the interface between the core and palisade sections of the micelles. Incorporation ratio of drug within sterically stabilized micelles increased as the total amount of drug in the system increased, and no drug particles were formed at the highest drug concentrations tested. The nanomicellar formulation of vinorelbine was similar to 6.7-fold more potent than vinorelbine dissolved in DMSO on MCF-7 cell line. Collectively, these data indicate that vinorelbine-loaded SSM can be developed as a new, safe, stable, and effective nanomedicine for the treatment of breast and lung cancers.

Objectives: Palladium complexes are potent and less toxic molecules in comparison to other metal based agents. Here, we characterized two palladium(II) saccharinate complexes with terpyridine for their cell cycle specificity. Materials and methods: Cells were arrested at Gl, Gl/S boundary or mitosis using mimosine, double-Thymidine block, aphidicolin, nocodazole or colcemid, and evaluated based on morphology and flow cytometry. Synchronized cells were treated with the Pd(II) complexes, and viability was measured via MTT assay. Results: While treatment of arrested cells with the Pd(II) complexes resulted in no significant change in cell death in HCT-116 and MDA-MB-231 cells, HeLa cells were more sensitive in S/G1. The main form of cell death was found to be apoptosis. Conclusions: Pd(II) complexes appear to be cell-cycle non-specific, while cell line dependent differences may be observed. Cells die through apoptosis regardless of the cell cycle stage, which makes these complexes more promising as anti-cancer agents. İstanbul Üniversitesi - 36696

Clinical efficacy of anticancer chemotherapies is dramatically hampered by multidrug resistance (MDR) dependent on inherited traits, acquired defence against toxins, and adaptive mechanisms mounting in tumours. There is overwhelming evidence that molecular events leading to MDR are regulated by redox mechanisms. For example, chemotherapeutics which overrun the first obstacle of redox-regulated cellular uptake channels (MDR1, MDR2, and MDR3) induce a concerted action of phase I/II metabolic enzymes with a temporal redox-regulated axis. This results in rapid metabolic transformation and elimination of a toxin. This metabolic axis is tightly interconnected with the inducible Nrf2-linked pathway, a key switch-on mechanism for upregulation of endogenous antioxidant enzymes and detoxifying systems. As a result, chemotherapeutics and cytotoxic by-products of their metabolism (ROS, hydroperoxides, and aldehydes) are inactivated and MDR occurs. On the other hand, tumour cells are capable of mounting an adaptive antioxidant response against ROS produced by chemotherapeutics and host immune cells. The multiple redox-dependent mechanisms involved in MDR prompted suggesting redox-active drugs (antioxidants and prooxidants) or inhibitors of inducible antioxidant defence as a novel approach to diminish MDR. Pitfalls and progress in this direction are discussed.

International audience; The term “immunogenic cell death” (ICD) is commonly employed to indicate a peculiar instance of regulated cell death (RCD) that engages the adaptive arm of the immune system. The inoculation of cancer cells undergoing ICD into immunocompetent animals elicits a specific immune response associated with the establishment of immunological memory. Only a few agents are intrinsically endowed with the ability to trigger ICD. These include a few chemotherapeutics that are routinely employed in the clinic, like doxorubicin, mitoxantrone, oxaliplatin, and cyclophosphamide, as well as some agents that have not yet been approved for use in humans. Accumulating clinical data indicate that the activation of adaptive immune responses against dying cancer cells is associated with improved disease outcome in patients affected by various neoplasms. Thus, novel therapeutic regimens that trigger ICD are urgently awaited. Here, we discuss current combinatorial approaches to convert otherwise non-immunogenic instances of RCD into bona fide ICD.

Purpose In preclinical models, there is synergism between chemotherapy and recombinant human tumor necrosis factor (TNF) –related apoptosis-inducing ligand (TRAIL) on apoptosis induction in tumor cells. Therefore, the prognostic relevance was analyzed of the expression of TRAIL and its death receptors DR4 and DR5 on disease-free survival and overall survival in stage III colon cancer patients treated with adjuvant chemotherapy. Methods Tissue microarrays were constructed of primary tumor tissue from 376 stage III colon cancer patients treated in a randomized adjuvant chemotherapy study (fluorouracil/levamisole v fluorouracil/levamisole/leucovorin) and stained immunohistochemically for TRAIL, DR4, and DR5. Log-rank tests and Cox proportional hazard analysis, with adjustment for treatment arm, sex, age, N stage, microsatellite instability status, and p53 mutation status, were performed. Results The majority of tumors showed high expression of TRAIL (83%), DR4 (92%), and DR5 (87%). Median follow-up was 43 months. High DR4 expression was associated with worse disease-free survival (odds ratio [OR] = 2.19; 95% CI, 1.06 to 4.53; P = .03), worse overall survival (OR = 2.22; 95% CI,1.03 to 4.81; P = .04) and shorter time to recurrence (P = .02) compared with those with low DR4 expression. TRAIL or DR5 expression had no prognostic value. Conclusion High DR4 expression is associated with worse disease-free and overall survival in stage III adjuvant-treated colon cancer patients. Evaluation of DR4 expression in stage III colon cancer patients may identify a subset requiring more aggressive adjuvant treatment.

Doxorubicin (DOX) and ifosfamide (IFO) are the most active single agents in soft tissue sarcomas (STS). Tumour necrosis factor-alpha (TNF-alpha) is used for STS in the setting of isolated limb perfusions. Like TNF-alpha, TNF-related apoptosis-inducing ligand (TRAIL) induces apoptosis. In contrast to TNF-a preliminary studies suggest that TRAIL lacks systemic side effects. The effects of TRAIL alone and in combination with DOX or 4-hydroxy-IFO were evaluated in the TNF-alpha sensitive rhabdomyosarcoma cell line KYM-1, its 5-fold TNF-alpha sensitive subline KD4 and its >150-fold TNF-a resistant subline 37138R. Membrane expression of TRAIL-receptors DR4 (death receptor 4), DR5 (pro-apoptotic), DcR1 (decoy receptor 1), DcR2 (anti-apoptotic) was assessed by flow cytometry. Cytotoxicity was determined by microculture tetrazolium assays. Apoptosis assays were performed with acridine orange. DOX (doxorubicin) and 4-OH-IFO decreased survival in all cell lines; a 2-fold resistance was observed for both drugs in 37138R. All cell lines expressed DR4 and DR5, but hardly any DcR1 or DcR2. TRAIL was cytotoxic in KYM-1, even more in KD4 and induced massive apoptosis; 37138R was >500-fold resistant to TRAIL and little apoptosis could be observed. TRAIL plus DOX showed synergistic cytotoxicity in KYM-1 and 37138R. TRAIL plus 4-OH-IFO showed addition in all three cell lines. DOX plus TRAIL-induced more cytotoxicity and apoptosis in all cell lines compared to TRAIL alone. In 37138R, DOX overcame resistance to TRAIL. In KYM-1, KD4 and 37138R, sensitivity and resistance to TNF-a and TRAIL parallels. TRAIL-resistance was independent from expression of TRAIL-receptors. DOX with TRAIL could overcome TRAIL-resistance in 37138R cells.

Doxorubicin (DOX) and ifosfamide (IFO) are the most active single agents in soft tissue sarcomas (STS). Tumour necrosis factor-alpha (TNF-alpha) is used for STS in the setting of isolated limb perfusions. Like TNF-alpha, TNF-related apoptosis-inducing ligand (TRAIL) induces apoptosis. In contrast to TNF-a preliminary studies suggest that TRAIL lacks systemic side effects. The effects of TRAIL alone and in combination with DOX or 4-hydroxy-IFO were evaluated in the TNF-alpha sensitive rhabdomyosarcoma cell line KYM-1, its 5-fold TNF-alpha sensitive subline KD4 and its >150-fold TNF-a resistant subline 37138R. Membrane expression of TRAIL-receptors DR4 (death receptor 4), DR5 (pro-apoptotic), DcR1 (decoy receptor 1), DcR2 (anti-apoptotic) was assessed by flow cytometry. Cytotoxicity was determined by microculture tetrazolium assays. Apoptosis assays were performed with acridine orange. DOX (doxorubicin) and 4-OH-IFO decreased survival in all cell lines; a 2-fold resistance was observed for both drugs in 37138R. All cell lines expressed DR4 and DR5, but hardly any DcR1 or DcR2. TRAIL was cytotoxic in KYM-1, even more in KD4 and induced massive apoptosis; 37138R was >500-fold resistant to TRAIL and little apoptosis could be observed. TRAIL plus DOX showed synergistic cytotoxicity in KYM-1 and 37138R. TRAIL plus 4-OH-IFO showed addition in all three cell lines. DOX plus TRAIL-induced more cytotoxicity and apoptosis in all cell lines compared to TRAIL alone. In 37138R, DOX overcame resistance to TRAIL. In KYM-1, KD4 and 37138R, sensitivity and resistance to TNF-a and TRAIL parallels. TRAIL-resistance was independent from expression of TRAIL-receptors. DOX with TRAIL could overcome TRAIL-resistance in 37138R cells.

Recombinant human (rh) TNF-related apoptosis-inducing ligand (TRAIL) harbors potential as an anticancer agent. RhTRAIL induces apoptosis via the TRAIL receptors TRAIL-R1 and TRAIL-R2 in tumors and is non-toxic to nonhuman primates. Because limited data are available about TRAIL receptor distribution, we performed an immunohistochemical (IHC) analysis of the expression of TRAIL-R1, TRAIL-R2, the anti-apoptotic TRAIL receptor TRAIL-R3, and TRAIL in normal human and chimpanzee tissues. In humans, hepatocytes stained positive for TRAIL and TRAIL receptors and bile duct epithelium for TRAIL, TRAIL-R1, and TRAIL-R3. In brains, neurons expressed TRAIL-R1, TRAIL-R2, TRAIL-R3 but no TRAIL. In kidneys, TRAIL-R3 was negative, tubuli contorti expressed TRAIL-R1, TRAIL-R2, and TRAIL, and cells in Henle's loop expressed only TRAIL-R2. Heart myocytes showed positivity for all proteins studied. In colon, TRAIL-R1, TRAIL-R2, and TRAIL were present. Germ and Leydig cells were positive for all proteins studied. Endothelium in liver, heart, kidney, and testis lacked TRAIL-R1 and TRAIL-R2. In alveolar septa and bronchial epithelium TRAIL-R2 was expressed, brain vascular endothelium expressed TRAIL-R2 and TRAIL-R3, and in heart vascular endothelium only TRAIL-R3 was present. Only a few differences were observed between human and chimpanzee liver, brain, and kidney. In contrast to human, chimpanzee bile duct epithelium lacked TRAIL, TRAIL-R1, and TRAIL-R3, lung and colon showed no TRAIL or its receptors, TRAIL-R3 was absent in germ and Leydig cells, and vascular endothelium showed only TRAIL-R2 expression in the brain. In conclusion, comparable expression of TRAIL and TRAIL receptors was observed in human and chimpanzee tissues. Lack of liver toxicity in chimpanzees after rhTRAIL administration despite TRAIL-R1 and TRAIL-R2 expression is reassuring for rhTRAIL application in humans. (J Histochem Cytochem 52:821–831, 2004)

PURPOSE: Several in vitro studies have shown that non-small cell lung cancer (NSCLC) cell lines are sensitive to apoptosis induction by the recombinant human (rh) tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) death ligand, indicating that rhTRAIL might become an attractive molecule for treatment of NSCLCs. To investigate the therapeutic potential of rhTRAIL, the expression of TRAIL and its apoptosis-inducing receptors DR4 and DR5 was evaluated in tumors of stage III NSCLC patients.EXPERIMENTAL DESIGN: Before treatment, tumor biopsies from locally advanced NSCLC patients were obtained by bronchoscopy. DR4, DR5, and TRAIL expression were determined immunohistochemically in 87 tumors. Patients were randomized for treatment with 60 Gy radiotherapy with or without carboplatin as radiosensitizer.RESULTS: DR4, DR5, and TRAIL were expressed in 99%, 82%, and 91% of the tumors, respectively. Seventeen percent of the samples expressed only DR4 and no DR5. In NSCLCs with squamous cell differentiation, a typical staining pattern for DR4 and DR5 was observed. Cells from the basal layer were strongly positive, and the more mature cells were less positive or negative. An inverse staining pattern was observed for TRAIL. Poorly differentiated areas showed strong staining intensity for DR4, DR5, and TRAIL. DR5-positive staining was associated with increased risk of death (odds ratio, 5.76; 95% confidence interval, 1.04-31.93; P = 0.045).CONCLUSIONS: The majority of the locally irresectable stage III NSCLCs expressed at least one of the two death receptors for TRAIL. Therefore, these death receptors may provide a target for the use of rhTRAIL as a new adjunct in the treatment of stage III NSCLC.

PURPOSE: Several in vitro studies have shown that non-small cell lung cancer (NSCLC) cell lines are sensitive to apoptosis induction by the recombinant human (rh) tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) death ligand, indicating that rhTRAIL might become an attractive molecule for treatment of NSCLCs. To investigate the therapeutic potential of rhTRAIL, the expression of TRAIL and its apoptosis-inducing receptors DR4 and DR5 was evaluated in tumors of stage III NSCLC patients. EXPERIMENTAL DESIGN: Before treatment, tumor biopsies from locally advanced NSCLC patients were obtained by bronchoscopy. DR4, DR5, and TRAIL expression were determined immunohistochemically in 87 tumors. Patients were randomized for treatment with 60 Gy radiotherapy with or without carboplatin as radiosensitizer. RESULTS: DR4, DR5, and TRAIL were expressed in 99%, 82%, and 91% of the tumors, respectively. Seventeen percent of the samples expressed only DR4 and no DR5. In NSCLCs with squamous cell differentiation, a typical staining pattern for DR4 and DR5 was observed. Cells from the basal layer were strongly positive, and the more mature cells were less positive or negative. An inverse staining pattern was observed for TRAIL. Poorly differentiated areas showed strong staining intensity for DR4, DR5, and TRAIL. DR5-positive staining was associated with increased risk of death (odds ratio, 5.76; 95% confidence interval, 1.04-31.93; P = 0.045). CONCLUSIONS: The majority of the locally irresectable stage III NSCLCs expressed at least one of the two death receptors for TRAIL. Therefore, these death receptors may provide a target for the use of rhTRAIL as a new adjunct in the treatment of stage III NSCLC.

Therapeutic efficacy of cisplatin-based chemotherapy for advanced-stage urothelial carcinoma (UC) is limited by drug resistance. The nuclear factor (erythroid-derived 2)-like 2 (NRF2) pathway is a major regulator of cytoprotective responses. We investigated its involvement in cisplatin resistance in long-term cisplatin treated UC cell lines (LTTs). Expression of NRF2 pathway components and targets was evaluated by qRT-PCR and western blotting in LTT sublines from four different parental cells. NRF2 transcriptional activity was determined by reporter assays and total glutathione (GSH) was quantified enzymatically. Effects of siRNA-mediated NRF2 knockdown on chemosensitivity were analysed by viability assays, H2AX immunofluorescence, and flow cytometry. Increased expression of NRF2, its positive regulator p62/SQSTM1, and elevated NRF2 activity was observed in 3/4 LTTs, which correlated with KEAP1 expression. Expression of cytoprotective enzymes and GSH concentration were upregulated in some LTTs. NRF2 knockdown resulted in downregulation of cytoprotective enzymes and resensitised 3/4 LTTs towards cisplatin as demonstrated by reduced IC50 values, increased H2AX foci formation, and elevated number of apoptotic cells. In conclusion, while LTT lines displayed diversity in NRF2 activation, NRF2 signalling contributed to cisplatin resistance in LTT lines, albeit in diverse ways. Accordingly, inhibition of NRF2 can be used to resensitise UC cells to cisplatin, but responses in patients may likewise be variable.

1 The intracellular transport of leukotriene Cc (LTC4) in hematopoietic cells such as human monocytes is controlled by an ATP dependent carrier encoded by the multidrug resistance protein1 (MRP1) gene whose function can be blocked by the compound MK-571. Since LTs play a major role in control of cytokine expression in monocytes, we questioned whether blocking of the MRP1 mediated function by MK-571 might affect cytokine production. 2 MK-571 strongly enhanced IL-6 expression at mRNA and protein level in lipopolysaccharide (LPS) and interleukin-l (IL-I) stimulated human monocytes giving rise to 2.0+/-0.4 (x+/-s.d.) and 5.7+/-3.5 fold induction of IL-S protein secretion. The increase in IL-6 secretion was accompanied by an enhanced phosphorylation of p38 but not of c-Jun-N terminal kinase. 3 The involvement of the kinase signalling pathways was further analysed by using SB203580 and PD98059, specific inhibitors of the p38 and ERK1/2 signalling route. MK-571 mediated upregulation of IL-6 in the presence of IL-I was partially attenuated by SB203580 and PD98059. Electrophoretic mobility shift assays demonstrated that MK-571 did not affect the IL-1 induced DNA binding activity of Activator Protein-1 and Nuclear Factor-KB but rather enhanced the transactivational activity of an IL-6 promoter construct. Finally it was shown that the MK-571 mediated effects on IL-6 secretion could not be inhibited by the LT synthesis inhibitor SB203347 or by the anti-oxidant pyrrolidine dithiocarbamate (PDTC). 4 These results indicate that the membrane transporter MRP1 is involved in the regulation of IL-6 expression in activated human peripheral blood monocytes.

1 The intracellular transport of leukotriene Cc (LTC4) in hematopoietic cells such as human monocytes is controlled by an ATP dependent carrier encoded by the multidrug resistance protein1 (MRP1) gene whose function can be blocked by the compound MK-571. Since LTs play a major role in control of cytokine expression in monocytes, we questioned whether blocking of the MRP1 mediated function by MK-571 might affect cytokine production.2 MK-571 strongly enhanced IL-6 expression at mRNA and protein level in lipopolysaccharide (LPS) and interleukin-l (IL-I) stimulated human monocytes giving rise to 2.0+/-0.4 (x+/-s.d.) and 5.7+/-3.5 fold induction of IL-S protein secretion. The increase in IL-6 secretion was accompanied by an enhanced phosphorylation of p38 but not of c-Jun-N terminal kinase.3 The involvement of the kinase signalling pathways was further analysed by using SB203580 and PD98059, specific inhibitors of the p38 and ERK1/2 signalling route. MK-571 mediated upregulation of IL-6 in the presence of IL-I was partially attenuated by SB203580 and PD98059. Electrophoretic mobility shift assays demonstrated that MK-571 did not affect the IL-1 induced DNA binding activity of Activator Protein-1 and Nuclear Factor-KB but rather enhanced the transactivational activity of an IL-6 promoter construct. Finally it was shown that the MK-571 mediated effects on IL-6 secretion could not be inhibited by the LT synthesis inhibitor SB203347 or by the anti-oxidant pyrrolidine dithiocarbamate (PDTC).4 These results indicate that the membrane transporter MRP1 is involved in the regulation of IL-6 expression in activated human peripheral blood monocytes.

Roscovitine and purvalanol are novel cyclin-dependent kinase (CDK) inhibitors that prevent cell proliferation and induce apoptotic cell death in various cancer cell lines. Although a number of studies have demonstrated the potential apoptotic role of roscovitine, there is limited data about the therapeutic efficiency of purvalanol on cancer cells. The natural polyamines (PAs) putrescine, spermidine, and spermine have essential roles in the regulation of cell differentiation, growth, and proliferation, and increased levels of these compounds have been associated with cancer progression. Recently, depletion of intracellular PA levels because of modulation of PA catabolic enzymes was shown to be an indicator of the efficacy of chemotherapeutic agents. In this study, our aim was to investigate the potential role of PA catabolic enzymes in CDK inhibitor-induced apoptosis in HCT 116 colon carcinoma cells. Exposure of cells to roscovitine or purvalanol decreased cell viability in a dose- and time-dependent manner. The selected concentrations of roscovitine and purvalanol inhibited cell viability by 50 % compared with control cells and induced apoptosis by activating the mitochondria-mediated pathway in a caspase-dependent manner. However, the apoptotic effect of purvalanol was stronger than that of roscovitine in HCT 116 cells. In addition, we found that CDK inhibitors decreased PA levels and significantly upregulated expression of key PA catabolic enzymes such as polyamine oxidase (PAO) and spermine oxidase (SMO). MDL-72,527, a specific inhibitor of PAO and SMO, decreased apoptotic potential of CDK inhibitors on HCT 116 cells. Moreover, transient silencing of PAO was also reduced prevented CDK inhibitor-induced apoptosis in HCT 116 cells. We conclude that the PA catabolic pathway, especially PAO, is a critical target for understanding the molecular mechanism of CDK inhibitor-induced apoptosis.

Methotrexate is a widely used cytostatic in chemotherapy cocktails for the treatment of cancer but is associated with cognitive impairment. Previous animal studies indicated that methorexate decreases hippocampal cell proliferation, which might contribute to the observed cognitive impairment. However, clinical studies have shown that cognitive impairment can also be noticed in some cancer patients before any systemic treatment is initiated. We aim in the present study to discern whether hippocampal cell proliferation is negatively affected by tumor growth and if the presence of a tumor amplifies the effects of methotrexate.Buffalo rats were subcutaneously injected with PBS or Morris Hepatoma 7777 cells to induce a tumor. Two weeks after this injection the animals received an intraperitoneal injection of methotrexate or saline. Three weeks later hippocampal cell proliferation was quantified using immunohistochemical staining.Treatment with Morris Hepatoma 7777 cells decreased the number of proliferating cells as compared to control animals. An overall tumor effect was absent mainly because methotrexate treatment significantly decreased cell proliferation with no differences between animals with or without a tumor. Neither methotrexate nor the tumor induced pica behavior.These findings indicate that although the presence of a tumor reduces hippocampal cell proliferation it does not affect the negative effect of methotrexate on this plasticity marker. Since sickness behavior is not induced by methotrexate or tumor presence it does not play a role in the development of cognitive deficits. This study further indicates that the effects of methotrexate on brain and behavior can be studied in healthy animals. (C) 2009 Elsevier Inc. All rights reserved.

Purpose: To evaluate the toxicity, pharmacological, and biological properties of the combination of bortezomib, etoposide, and carboplatin in adults with advanced solid malignancies. Patients and methods: Patients received escalating doses of bortezomib, etoposide, and carboplatin every 21 days. Surrogate markers of angiogenesis were evaluated. Results: Twenty-four patients received 64 courses of therapy. The most common treatment-related adverse events were myelosuppression. Dose-limiting grade 3 and 4 neutropenia and thrombocytopenia were observed when bortezomib was given on days 1, 4, 8, 11. With revised dosing, the maximum tolerated dose (MTD) of bortezomib 0.75 mg/m(2) (days 1, 8), etoposide 75 mg/m(2) (days 1-3), and carboplatin AUC 5 (day 1) was well tolerated, and are the recommended doses for further studies with this combination. No objective responses were observed, however stable disease was noted for greater or equal to four cycles in nine highly refractory patients.

Life expectancy of patients affected by glioblastoma multiforme is extremely low. The therapeutic use of tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) has been proposed to treat this disease based on its ability to kill glioma cell lines in vitro and in vivo. Here, we show that, differently from glioma cell lines, glioblastoma multiforme tumors were resistant to TRAIL stimulation because they expressed low levels of caspase-8 and high levels of the death receptor inhibitor PED/PEA-15. Inhibition of methyltransferases by decitabine resulted in considerable up-regulation of TRAIL receptor-1 and caspase-8, down-regulation of PED/PEA-15, inhibition of cell growth, and sensitization of primary glioblastoma cells to TRAIL-induced apoptosis. Exogenous caspase-8 expression was the main event able to restore TRAIL sensitivity in primary glioblastoma cells. The antitumor activity of decitabine and TRAIL was confirmed in vivo in a mouse model of glioblastoma multiforme. Evaluation of tumor size, apoptosis, and caspase activation in nude mouse glioblastoma multiforme xenografts showed dramatic synergy of decitabine and TRAIL in the treatment of glioblastoma, whereas the single agents were scarcely effective in terms of reduction of tumor mass, apoptosis induction, and caspase activation. Thus, the combination of TRAIL and demethylating agents may provide a key tool to overcome glioblastoma resistance to therapeutic treatments. (Cancer Res 2005; 65(24): 11469-77)

Doxorubicin (DOX) and ifosfamide (IFO) are the most active single agents in soft tissue sarcomas (STS). Tumour necrosis factor-alpha (TNF-alpha) is used for STS in the setting of isolated limb perfusions. Like TNF-alpha, TNF-related apoptosis-inducing ligand (TRAIL) induces apoptosis. In contrast to TNF-alpha preliminary studies suggest that TRAIL lacks systemic side effects. The effects of TRAIL alone and in combination with DOX or 4-hydroxy-IFO were evaluated in the TNF-alpha sensitive rhabdomyosarcoma cell line KYM-1, its 5-fold TNF-alpha sensitive subline KD4 and its150-fold TNF-alpha resistant subline 37B8R. Membrane expression of TRAIL-receptors DR4 (death receptor 4), DR5 (pro-apoptotic), DcR1 (decoy receptor 1), DcR2 (anti-apoptotic) was assessed by flow cytometry. Cytotoxicity was determined by microculture tetrazolium assays. Apoptosis assays were performed with acridine orange. DOX (doxorubicin) and 4-OH-IFO decreased survival in all cell lines; a 2-fold resistance was observed for both drugs in 37B8R. All cell lines expressed DR4 and DR5, but hardly any DcR1 or DcR2. TRAIL was cytotoxic in KYM-1, even more in KD4 and induced massive apoptosis; 37B8R was500-fold resistant to TRAIL and little apoptosis could be observed. TRAIL plus DOX showed synergistic cytotoxicity in KYM-1 and 37B8R. TRAIL plus 4-OH-IFO showed addition in all three cell lines. DOX plus TRAIL-induced more cytotoxicity and apoptosis in all cell lines compared to TRAIL alone. In 37B8R, DOX overcame resistance to TRAIL. In KYM-1, KD4 and 37B8R, sensitivity and resistance to TNF-alpha and TRAIL parallels. TRAIL-resistance was independent from expression of TRAIL-receptors. DOX with TRAIL could overcome TRAIL-resistance in 37B8R cells.

PURPOSE: Several in vitro studies have shown that non-small cell lung cancer (NSCLC) cell lines are sensitive to apoptosis induction by the recombinant human (rh) tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) death ligand, indicating that rhTRAIL might become an attractive molecule for treatment of NSCLCs. To investigate the therapeutic potential of rhTRAIL, the expression of TRAIL and its apoptosis-inducing receptors DR4 and DR5 was evaluated in tumors of stage III NSCLC patients. EXPERIMENTAL DESIGN: Before treatment, tumor biopsies from locally advanced NSCLC patients were obtained by bronchoscopy. DR4, DR5, and TRAIL expression were determined immunohistochemically in 87 tumors. Patients were randomized for treatment with 60 Gy radiotherapy with or without carboplatin as radiosensitizer. RESULTS: DR4, DR5, and TRAIL were expressed in 99%, 82%, and 91% of the tumors, respectively. Seventeen percent of the samples expressed only DR4 and no DR5. In NSCLCs with squamous cell differentiation, a typical staining pattern for DR4 and DR5 was observed. Cells from the basal layer were strongly positive, and the more mature cells were less positive or negative. An inverse staining pattern was observed for TRAIL. Poorly differentiated areas showed strong staining intensity for DR4, DR5, and TRAIL. DR5-positive staining was associated with increased risk of death (odds ratio, 5.76; 95% confidence interval, 1.04-31.93; P = 0.045). CONCLUSIONS: The majority of the locally irresectable stage III NSCLCs expressed at least one of the two death receptors for TRAIL. Therefore, these death receptors may provide a target for the use of rhTRAIL as a new adjunct in the treatment of stage III NSCLC.

1. The intracellular transport of leukotriene C4 (LTC4) in hematopoietic cells such as human monocytes is controlled by an ATP dependent carrier encoded by the multidrug resistance protein1 (MRPI) gene whose function can be blocked by the compound MK-571. Since LTs play a major role in control of cytokine expression in monocytes, we questioned whether blocking of the MRPI mediated function by MK-571 might affect cytokine production. 2. MK-571 strongly enhanced IL-6 expression at mRNA and protein level in lipopolysaccharide (LPS) and interleukin-1 (IL-1) stimulated human monocytes giving rise to 2.0+/-0.4 (x+/-s.d.) and 5.7+/-3.5 fold induction of IL-6 protein secretion. The increase in IL-6 secretion was accompanied by an enhanced phosphorylation of p38 but not of c-Jun-N terminal kinase. 3. The involvement of the kinase signalling pathways was further analysed by using SB203580 and PD98059, specific inhibitors of the p38 and ERK1/2 signalling route. MK-571 mediated upregulation of IL-6 in the presence of IL-1 was partially attenuated by SB203580 and PD98059. Electrophoretic mobility shift assays demonstrated that MK-571 did not affect the IL-1 induced DNA binding activity of Activator Protein-1 and Nuclear Factor-kappaB but rather enhanced the transactivational activity of an IL-6 promoter construct. Finally it was shown that the MK-571 mediated effects on IL-6 secretion could not be inhibited by the LT synthesis inhibitor SB203347 or by the anti-oxidant pyrrolidine dithiocarbamate (PDTC). 4. These results indicate that the membrane transporter MRP1 is involved in the regulation of IL-6 expression in activated human peripheral blood monocytes.