Ten recommendations for sarcoma surgery: consensus of the surgical societies based on the German S3 guideline "Adult Soft Tissue Sarcomas".

PURPOSE: The evidence-based (S3) guideline "Adult Soft Tissue Sarcomas" (AWMF Registry No. 032/044OL) published by the German Guideline Program in Oncology (GGPO) covers all aspects of sarcoma treatment with 229 recommendations. Representatives of all medical specialties involved in sarcoma treatment contributed to the guideline. This paper compiles the most important recommendations for surgeons selected by delegates from the surgical societies. METHODS: A Delphi process was used. Delegates from the surgical societies involved in guideline process selected the 15 recommendations that were most important to them. Votes for similar recommendations were tallied. From the resulting ranked list, the 10 most frequently voted recommendations were selected and confirmed by consensus in the next step. RESULTS: The statement "Resection of primary soft tissue sarcomas of the extremities should be performed as a wide resection. The goal is an R0 resection" was selected as the most important term. The next highest ranked recommendations were the need for a preoperative biopsy, performing preoperative MRI imaging with contrast, and discussing all cases before surgery in a multidisciplinary sarcoma committee. CONCLUSION: The evidence-based guideline "Adult Soft Tissue Sarcomas" is a milestone to improve the care of sarcoma patients in Germany. The selection of the top ten recommendations by surgeons for surgeons has the potential to improve the dissemination and acceptance of the guideline and thus improve the overall outcome of sarcoma patients.

QSEA-modelling of genome-wide DNA methylation from sequencing enrichment experiments.

Genome-wide enrichment of methylated DNA followed by sequencing (MeDIP-seq) offers a reasonable compromise between experimental costs and genomic coverage. However, the computational analysis of these experiments is complex, and quantification of the enrichment signals in terms of absolute levels of methylation requires specific transformation. In this work, we present QSEA, Quantitative Sequence Enrichment Analysis, a comprehensive workflow for the modelling and subsequent quantification of MeDIP-seq data. As the central part of the workflow we have developed a Bayesian statistical model that transforms the enrichment read counts to absolute levels of methylation and, thus, enhances interpretability and facilitates comparison with other methylation assays. We suggest several calibration strategies for the critical parameters of the model, either using additional data or fairly general assumptions. By comparing the results with bisulfite sequencing (BS) validation data, we show the improvement of QSEA over existing methods. Additionally, we generated a clinically relevant benchmark data set consisting of methylation enrichment experiments (MeDIP-seq), BS-based validation experiments (Methyl-seq) as well as gene expression experiments (RNA-seq) derived from non-small cell lung cancer patients, and show that the workflow retrieves well-known lung tumour methylation markers that are causative for gene expression changes, demonstrating the applicability of QSEA for clinical studies. QSEA is implemented in R and available from the Bioconductor repository 3.4 (www.bioconductor.org/packages/qsea).

Influence of natural killer cells and perforin­mediated cytolysis on the development of chemically induced lung cancer in A/J mice.

One alternative approach for the treatment of lung cancer might be the activation of the immune system using vaccination strategies. However, most of clinical vaccination trials for lung cancer did not reach their primary end points, suggesting that lung cancer is of low immunogenicity. To provide additional experimental information about this important issue, we investigated which type of immune cells contributes to the protection from lung cancer development. Therefore, A/J mice induced for lung adenomas/ adenocarcinomas by the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) were depleted of CD4+ or CD8+ T cells, CD11b+ macrophages, Gr-1+ neutrophils and asialo GM1+ natural killer (NK) cells. Subsequent analysis of tumour growth showed an increase in tumour number only in mice depleted of NK cells. Further asking by which mechanism NK cells suppressed tumour development, we neutralized several death ligands of the tumour necrosis factor (TNF) family known to be involved in NK cell-mediated cytotoxicity. However neither depletion of TNF-α, TNF-related apoptosis-inducing ligand, TNF-like weak inducer of apoptosis or FasL alone nor in combination induced an augmentation of tumour burden. To show whether an alternative cell death pathway is involved, we next generated A/J mice deficient for perforin. After challenging with NNK, mice deficient for perforin showed an increase in tumour number and volume compared to wild-type A/J mice. In summary, our data suggest that NK cells and perforin-mediated cytolysis are critically involved in the protection from lung cancer giving promise for further immunotherapeutic strategies for this disease.

Increased binding of anti-dsDNA antibodies to short oligonucleotides modified with topoisomerase I reveals a potential new enzyme function independent from DNA relaxation.

OBJECTIVE: Topoisomerase I (topo I) is a highly conserved enzyme which is known to reduce torsional stress at double-stranded (ds) DNA. Torsional stress induced by supercoiling of dsDNA requires either very long dsDNA existing in genomic DNA or circulation as presented in plasmid DNA. To enable DNA relaxation, topo I induce a transient single-strand break followed by stress-relieving rotation of the released DNA strand. Our group found by serendipity that the topo I inhibitor irinotecan is able to suppress murine systemic lupus erythematosus (SLE), an autoimmune disease which is characterized by the existence of pathogenic anti-dsDNA antibodies (abs). As a possible mechanism we demonstrated in the absence of immunosuppression an increased binding of anti-dsDNA abs to long genomic or circulated plasmid dsDNA modified with topo I. RESULTS: Here we show that this effect requires active site tyrosine of topo I which is known to facilitate DNA relaxation activity. Moreover, topo I enhanced anti-dsDNA abs binding to short linear oligonucleotides down to a size of 42 bp. Since oligonucleotides of such length are devoid of torsional stress and relaxation respectively, our results suggest a new and unknown function for the enzyme topo I.

Prospective Evaluation of Quantitative F-18-FDG-PET/CT for Pre-Operative Thoracic Lymph Node Staging in Patients with Lung Cancer as a Target for Computer-Aided Diagnosis.

PURPOSE: Pre-operative assessment of thoracic lymphonodal (LN) involvement in patients with lung cancer (LC) is crucial when choosing the treatment modality. Visual assessment of F-18-FDG-PET/CT (PET/CT) is well established, however, there is still a need for prospective quantitative data to differentiate benign from malignant lesions which would simplify staging and guide the further implementation of computer-aided diagnosis (CAD). METHODS: In this prospective study, 37 patients with confirmed lung cancer (m/f = 24/13; age: 70 [52-83] years) were analyzed. All patients underwent PET/CT and quantitative data (standardized uptake values) were obtained. Histological results were available for 101 thoracic lymph nodes. Quantitative data were matched to determine cut-off values for delineation between benign vs. malignant lymph nodes. Furthermore, a scoring system derived from these cut-off values was established. Statistical analyses were performed through ROC analysis. RESULTS: Quantitative analysis revealed the optimal cut-off values (p < 0.01) for the differentiation between benign and malignant thoracic lymph nodes in patients suffering from lung cancer. The respective areas under the curve (AUC) ranged from 0.86 to 0.94. The highest AUC for a ratio of lymph node to healthy lung tissue was 0.94. The resulting accuracy ranged from 78.2% to 89.1%. A dedicated scoring system led to an AUC of 0.93 with a negative predictive value of 95.4%. CONCLUSION: Quantitative analysis of F-18-FDG-PET/CT data provides reliable results for delineation between benign and malignant thoracic lymph nodes. Thus, quantitative parameters can improve diagnostic accuracy and reliability and can also facilitate the handling of the steadily increasing number of clinical examinations.

Reversal of established lupus nephritis and prolonged survival of New Zealand black x New Zealand white mice treated with the topoisomerase I inhibitor irinotecan.

Systemic lupus erythematosus is a chronic autoimmune disorder that predominantly affects women of childbearing age. Lupus-associated glomerulonephritis is a major cause of mortality in these patients. Current treatment protocols for systemic lupus erythematosus include cyclophosphamide, prednisolone, azathioprine, and mycophenolate mofetil. However, in mice none of these agents alone or in combination were shown to reverse established proteinuria. Using New Zealand Black x New Zealand White F1 mice, we report that administration of the topoisomerase I inhibitor irinotecan from week 13 completely prevented the onset of proteinuria and prolonged survival up to at least 90 wk without detectable side effects. Furthermore, application of irinotecan to mice with established lupus nephritis, as indicated by grade 3+ (> or =300 mg/dl) and grade 4+ (> or =2000 mg/dl) proteinuria and, according to a median age of 35 wk, resulted in remission rates of 75% and 55%, respectively. Survival was significantly prolonged with 73 wk (grade 3+ and 4+ combined) versus 40 wk for control animals. Although total IgG and anti-dsDNA Abs in the serum and mesangial IgG deposits in the kidneys were not reduced in irinotecan-treated mice, subendothelial immune deposits were considerably diminished, suggesting a prevention of glomerular basement membrane disruption. This effect was accompanied by increased rates of ssDNA breaks and inhibition of renal cell apoptosis being different to what is known about irinotecan in anticancer therapy. In conclusion, our data provide evidence that irinotecan might represent an entirely new strategy for the treatment of systemic lupus erythematosus.

Superior vena cava syndrome in thoracic malignancies.

The superior vena cava syndrome (SVCS) comprises various symptoms due to occlusion of the SVC, which can be easily obstructed by pathological conditions (eg, lung cancer, due to the low internal venous pressure within rigid structures of the thorax [trachea, right bronchus, aorta]). The resulting increased venous pressure in the upper body may cause edema of the head, neck, and upper extremities, often associated with cyanosis, plethora, and distended subcutaneous vessels. Despite the often striking clinical presentation, SVCS itself is usually not a life-threatening condition. Currently, randomized controlled trials on many clinically important aspects of SVCS are lacking. This review gives an interdisciplinary overview of the pathophysiology, etiology, clinical manifestations, diagnosis, and treatment of malignant SVCS.

Death receptor 5 mediated-apoptosis contributes to cholestatic liver disease.

Chronic cholestasis often results in premature death from liver failure with fibrosis; however, the molecular mechanisms contributing to biliary cirrhosis are not demonstrated. In this article, we show that the death signal mediated by TNF-related apoptosis-inducing ligand (TRAIL) receptor 2/death receptor 5 (DR5) may be a key regulator of cholestatic liver injury. Agonistic anti-DR5 monoclonal antibody treatment triggered cholangiocyte apoptosis, and subsequently induced cholangitis and cholestatic liver injury in a mouse strain-specific manner. TRAIL- or DR5-deficient mice were relatively resistant to common bile duct ligation-induced cholestasis, and common bile duct ligation augmented DR5 expression on cholangiocytes, sensitizing mice to DR5-mediated cholangitis. Notably, anti-DR5 monoclonal antibody-induced cholangitis exhibited the typical histological appearance, reminiscent of human primary sclerosing cholangitis. Human cholangiocytes constitutively expressed DR5, and TRAIL expression and apoptosis were significantly elevated in cholangiocytes of human primary sclerosing cholangitis and primary biliary cirrhosis patients. Thus, TRAIL/DR5-mediated apoptosis may substantially contribute to chronic cholestatic disease, particularly primary sclerosing cholangitis.

TRAIL receptor-mediated JNK activation and Bim phosphorylation critically regulate Fas-mediated liver damage and lethality.

TNF-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family with potent apoptosis-inducing properties in tumor cells. In particular, TRAIL strongly synergizes with conventional chemotherapeutic drugs to induce tumor cell death. Thus, TRAIL has been proposed as a promising future cancer therapy. Little, however, is known regarding what the role of TRAIL is in normal untransformed cells and whether therapeutic administration of TRAIL, alone or in combination with other apoptotic triggers, may cause tissue damage. In this study, we investigated the role of TRAIL in Fas-induced (CD95/Apo-1-induced) hepatocyte apoptosis and liver damage. While TRAIL alone failed to induce apoptosis in isolated murine hepatocytes, it strongly amplified Fas-induced cell death. Importantly, endogenous TRAIL was found to critically regulate anti-Fas antibody-induced hepatocyte apoptosis, liver damage, and associated lethality in vivo. TRAIL enhanced anti-Fas-induced hepatocyte apoptosis through the activation of JNK and its downstream substrate, the proapoptotic Bcl-2 homolog Bim. Consistently, TRAIL- and Bim-deficient mice and wild-type mice treated with a JNK inhibitor were protected against anti-Fas-induced liver damage. We conclude that TRAIL and Bim are important response modifiers of hepatocyte apoptosis and identify liver damage and lethality as a possible risk of TRAIL-based tumor therapy.

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.

Epigenomic profiling of non-small cell lung cancer xenografts uncover LRP12 DNA methylation as predictive biomarker for carboplatin resistance.

BACKGROUND: Non-small cell lung cancer (NSCLC) is the most common cause of cancer-related deaths worldwide and is primarily treated with radiation, surgery, and platinum-based drugs like cisplatin and carboplatin. The major challenge in the treatment of NSCLC patients is intrinsic or acquired resistance to chemotherapy. Molecular markers predicting the outcome of the patients are urgently needed. METHODS: Here, we employed patient-derived xenografts (PDXs) to detect predictive methylation biomarkers for platin-based therapies. We used MeDIP-Seq to generate genome-wide DNA methylation profiles of 22 PDXs, their parental primary NSCLC, and their corresponding normal tissues and complemented the data with gene expression analyses of the same tissues. Candidate biomarkers were validated with quantitative methylation-specific PCRs (qMSP) in an independent cohort. RESULTS: Comprehensive analyses revealed that differential methylation patterns are highly similar, enriched in PDXs and lung tumor-specific when comparing differences in methylation between PDXs versus primary NSCLC. We identified a set of 40 candidate regions with methylation correlated to carboplatin response and corresponding inverse gene expression pattern even before therapy. This analysis led to the identification of a promoter CpG island methylation of LDL receptor-related protein 12 (LRP12) associated with increased resistance to carboplatin. Validation in an independent patient cohort (n = 35) confirmed that LRP12 methylation status is predictive for therapeutic response of NSCLC patients to platin therapy with a sensitivity of 80% and a specificity of 84% (p < 0.01). Similarly, we find a shorter survival time for patients with LRP12 hypermethylation in the TCGA data set for NSCLC (lung adenocarcinoma). CONCLUSIONS: Using an epigenome-wide sequencing approach, we find differential methylation patterns from primary lung cancer and PDX-derived cancers to be very similar, albeit with a lower degree of differential methylation in primary tumors. We identify LRP12 DNA methylation as a powerful predictive marker for carboplatin resistance. These findings outline a platform for the identification of epigenetic therapy resistance biomarkers based on PDX NSCLC models.

PU.1 supports TRAIL-induced cell death by inhibiting NF-κB-mediated cell survival and inducing DR5 expression.

The hematopoietic Ets-domain transcription factor PU.1/SPI1 orchestrates myeloid, B- and T-cell development, and also supports hematopoietic stem cell maintenance. Although PU.1 is a renowned tumor suppressor in acute myeloid leukemia (AML), a disease characterized by an accumulation of immature blast cells, comprehensive studies analyzing the role of PU.1 during cell death responses in AML treatment are missing. Modulating PU.1 expression in AML cells, we found that PU.1 supports tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis via two mechanisms: (a) by repressing NF-κB activity via a novel direct PU.1-RelA/p65 protein-protein interaction, and (b) by directly inducing TRAIL receptor DR5 expression. Thus, expression of NF-κB-regulated antiapoptotic genes was sustained in PU.1-depleted AML cells upon TRAIL treatment and DR5 levels were decreased. Last, PU.1 deficiency significantly increased AML cell resistance to anthracycline treatment. Altogether, these results reveal a new facet of PU.1's tumor suppressor function during antileukemic 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.

Transoral endoscopic mediastinal surgery (TOEMS)-results of a first clinical study for scarless mediastinal lymph node biopsies.

BACKGROUND: Video-assisted mediastinoscopy (VAM) represents the standard procedure for mediastinal lymph node biopsies. This operation results in a scar at prominent position at the anterior neck. Since there is a trend to less invasive procedures, natural orifice transluminal endoscopic surgery (NOTES) was introduced to different fields of surgery. Based on NOTES we developed a new approach for mediastinoscopy: transoral endoscopic mediastinal surgery (TOEMS). In previous studies using human cadavers and living pigs the feasibility of TOEMS was shown. It was unclear whether TOEMS could be safely applied in patients requiring mediastinal lymph node biopsies. METHODS: We conducted a clinical phase I study recruiting ten patients with unclear mediastinal lymphadenopathy not resolved by prior bronchoscopy. All patients underwent TOEMS for mediastinal lymph node biopsy. The duration of the procedure and complications were monitored. In addition, all patients were examined for pain, swallowing dysfunction and sensation disturbance. RESULTS: TOEMS was accomplished in eight patients. In two patients operation was converted to VAM due to technical problems. Mediastinal lymph nodes were dissected in all patients who finished with TOEMS. On average, two separate lymph stations were reached by TOEMS. Duration of the procedure was 159±22 min. Permanent palsy of the right recurrent laryngeal nerve was noticed in one patient postoperatively. CONCLUSIONS: This is the first report for a human application of NOTES in thoracic surgery. In fact, transoral endoscopic surgery seems to be a feasible approach for mediastinal lymph node biopsies. Further studies are needed to show whether this procedure has an advantage over VAM in terms of pain, complications and accessibility of mediastinal lymph node stations.

Suppression of lupus nephritis and skin lesions in MRL/lpr mice by administration of the topoisomerase I inhibitor irinotecan.

BACKGROUND: Since the precise mechanism for the pathogenesis of systemic lupus erythematosus (SLE) is unknown, no targeted therapies in addition to immunosuppression are available so far. We recently demonstrated that administration of the topoisomerase I (topo I) inhibitor irinotecan at extremely low concentrations reversed established lupus nephritis in NZB/NZW mice. While profound immunosuppression was absent, we proposed changes in DNA relaxation and anti-double-stranded (ds)DNA antibody binding as the underlying mechanism. To exclude that these effects were restricted to NZB/NZW mice, irinotecan was used in a genetically different strain of lupus-prone mice. METHODS: MRL/lpr mice were treated with high- and low-dose irinotecan beginning at 8 weeks of age. Treatment was repeated every fourth week. In vitro, DNA was relaxed by recombinant topo I, and altered anti-dsDNA antibody binding was measured by enzyme-linked immunosorbent assay. RESULTS: Administration of both high- and low-dose irinotecan prevented proteinuria and prolonged survival in MRL/lpr mice. Moreover, both concentrations of irinotecan significantly improved histopathology of the skin at 18 weeks of age. While only high-dose irinotecan diminished the numbers of plasmablasts and double-negative T cells, no changes in IgG-secreting cells or anti-dsDNA IgG were observed. In vitro, relaxation of DNA by topo I increased the binding of anti-dsDNA IgG but not the binding of anti-dsDNA IgM derived from the plasma of MRL/lpr mice. CONCLUSION: The beneficial effects of topo I inhibition in a second, genetically different strain of lupus-prone mice strongly implicate irinotecan as a new therapeutic option for human SLE.

Comparison of volatile organic compounds from lung cancer patients and healthy controls-challenges and limitations of an observational study.

This paper outlines the design and performance of an observational study on the profiles of volatile organic compounds (VOCs) in the breath of 37 lung cancer patients and 23 healthy controls of similar age. The need to quantify each VOC considered as a potential disease marker on the basis of individual calibration is elaborated, and the quality control measures required to maintain reproducibility in breath sampling and subsequent instrumental trace VOC analysis using solid phase microextraction-gas chromatography-mass spectrometry over a study period of 14 months are described. Twenty-four VOCs were quantified on the basis of their previously suggested potential as cancer markers. The concentration of aromatic compounds in the breath was increased, as expected, in smokers, while lung cancer patients displayed significantly increased levels of oxygenated VOCs such as aldehydes, 2-butanone and 1-butanol. Although sets of selected oxygenated VOCs displayed sensitivities and specificities between 80% and 90% using linear discriminant analysis (LDA) with leave-one-out cross validation, the effective selectivity of the breath VOC approach with regard to cancer detection is clearly limited. Results are discussed against the background of the literature on volatile cancer marker investigations and the prospects of linking increased VOC levels in patients' breath with approaches that employ sniffer dogs. Experience from this study and the literature suggests that the currently available methodology is not able to use breath VOCs to reliably discriminate between cancer patients and healthy controls. Observational studies often tend to note significant differences in levels of certain oxygenated VOCs, but without the resolution required for practical application. Any step towards the exploitation of differences in VOC profiles for illness detection would have to solve current restrictions set by the low and variable VOC concentrations. Further challenges are the technical complexity of studies involving breath sampling and possibly the limited capability of current analytical procedures to detect unstable marker candidates.

PG490-mediated sensitization of lung cancer cells to Apo2L/TRAIL-induced apoptosis requires activation of ERK2.

Tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) belongs to the family of programmed cell death-inducing cytokines. Apo2L/TRAIL induces apoptosis in a wide variety of tumor cells. Tumor cells that are resistant to Apo2L/TRAIL-induced apoptosis can be sensitized by chemotherapeutic drugs and other agents via an unknown mechanism. Here we report that PG490 (triptolide), a diterpene triepoxide extracted from the Chinese herb Tripterygium wilfordii and used in traditional Chinese medicine, sensitizes lung cancer but not normal human bronchial epithelial cells to Apo2L/TRAIL-induced apoptosis. Sensitization was accompanied by caspase-3 and caspase-8 activation, whereas no cleavage of caspase-9 was observed. Determination of cell surface receptors by flow cytometry demonstrated no difference in Apo2L/TRAIL-R1 and -R2 expression, the two receptors with functional death domains, between resistant and sensitized cells. In cells treated with the combination of Apo2L/TRAIL and PG490, we observed activation of ERK2, a member of the mitogen-activated protein kinase family. Furthermore, sensitization could be blocked by the ERK inhibitor U0126 but not the p38 inhibitor SB203580, suggesting that activation of ERK2 is required for this effect. In addition, sensitization of lung cancer cells was also seen in ex vivo culture of lung cancer tissue from four patients who underwent surgery. Immunohistochemical staining showed a clear reduction in proliferation cell nuclear antigen (PCNA) in tissue treated with Apo2L/TRAIL and PG490. In conclusion, apoptosis induced by the combination of Apo2L/TRAIL and PG490 warrants further evaluation as a potential new strategy for the treatment of lung cancer.

The Topoisomerase I Inhibitor Irinotecan and the Tyrosyl-DNA Phosphodiesterase 1 Inhibitor Furamidine Synergistically Suppress Murine Lupus Nephritis.

OBJECTIVE: The treatment of lupus nephritis is still an unmet medical need requiring new therapeutic approaches. Our group found recently that irinotecan, an inhibitor of topoisomerase I (topo I), reversed proteinuria and prolonged survival in mice with advanced lupus nephritis. While irinotecan is known to stabilize the complex of topo I and DNA, the enzyme tyrosyl-DNA phosphodiesterase 1 (TDP-1) functions in an opposing manner by releasing topo I from DNA. Therefore, we undertook this study to test whether the TDP-1 inhibitor furamidine has an additional effect on lupus nephritis when used in combination with irinotecan. METHODS: NZB/NZW mice were treated with low-dose irinotecan and furamidine either alone or in combination beginning at age 26 weeks. DNA relaxation was visualized using gel electrophoresis. Binding of anti-double-stranded DNA (anti-dsDNA) antibodies to DNA modified by topo I, TDP-1, and the topo I inhibitor camptothecin was determined by enzyme-linked immunosorbent assay. RESULTS: Compared to treatment with either agent alone, simultaneous treatment with low-dose irinotecan and furamidine significantly improved survival of NZB/NZW mice. Similar to what has been previously shown for irinotecan alone, the combination treatment did not change the levels of anti-dsDNA antibodies. In vitro, recombinant TDP-1 increased topo I-mediated DNA relaxation, resulting in enhanced binding of anti-dsDNA antibodies. In combination with topo I and camptothecin, TDP-1 reversed the inhibitory effects of camptothecin on DNA relaxation and anti-dsDNA binding. CONCLUSION: Affecting DNA relaxation by the enzymes topo I and TDP-1 and their inhibitors may be a promising approach for the development of new targeted therapies for systemic lupus erythematosus.

Enhancement of Apo2L/TRAIL (tumor necrosis factor-related apoptosis-inducing ligand)-induced apoptosis in non-small cell lung cancer cell lines by chemotherapeutic agents without correlation to the expression level of cellular protease caspase-8 inhibitory protein.

OBJECTIVE: Apo2L/tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potential anticancer drug that promotes apoptosis specifically in tumor cells. Because not all cancer cells are susceptible to Apo2L/TRAIL, the aim of our study was to determine whether non-small cell lung cancer cells can be sensitized by chemotherapeutic agents for Apo2L/TRAIL-induced apoptosis. In addition, endogenous expression levels of the caspase-inhibiting cellular protease caspase-8 inhibitory protein (C-FLIP) were measured to investigate partial resistance to Apo2L/TRAIL. METHODS: Six human lung cancer cell lines (A549, NCI-H358, Calu1, Calu6, SkMes1, and SkLu1) were incubated with soluble Apo2L/TRAIL and two different concentrations each of cisplatin, paclitaxel, doxorubicin, 5-fluorouracil, and camptothecin. After 24 hours the rate of apoptosis was measured by annexin V/propidium iodide staining followed by FACScan analysis. Expression levels of C-FLIP in cell lines and lung cancer biopsy specimens were determined by Western blotting. RESULTS: Treatment of lung cancer cells with Apo2L/TRAIL alone resulted in apoptotic cell death in four cell lines (P <.001). Combining Apo2L/TRAIL and chemotherapeutic agents enhanced the rate of apoptosis significantly. Statistical analysis revealed a synergistic effect of Apo2L/TRAIL in combination with 1.8 mmol/L camptothecin and 100 micromol/L cisplatin, each in four of the six cell lines (P <.002). Western blot analysis showed that sensitization to Apo2L/TRAIL did not correlate with the expression of cellular protease caspase-8 inhibitory protein. Furthermore, no increased cellular protease caspase-8 inhibitory protein levels relative to those in normal lung tissue could be found in non-small cell lung cancer specimens from 12 patients. CONCLUSION: Apo2L/TRAIL-induced apoptosis in non-small cell lung cancer cell lines is significantly enhanced by chemotherapeutic agents. Resistance and sensitization to Apo2L/TRAIL are not correlated with the endogenous expression level of cellular protease caspase-8 inhibitory protein, implying that in non-small cell lung cancer other mechanisms are responsible for inhibition of the Apo2L/TRAIL pathway. Even though the molecular mechanism remains unclear, the combination of Apo2L/TRAIL with chemotherapy may be a promising treatment modality for non-small cell lung cancer.

Cell death induced by down-regulation of heat shock protein 70 in lung cancer cell lines is p53-independent and does not require DNA cleavage.

OBJECTIVE: The inducible form of heat shock protein 70 is known to be overexpressed in tumors and seems to be necessary for the survival of tumor cells via an unknown mechanism. We therefore evaluated whether selective depletion of heat shock protein 70 induces cell death in lung cancer cells. METHODS: An adenovirus expressing antisense heat shock protein 70 and an adenovirus with beta-galactosidase were used for transduction of the lung cancer cell lines A549, NCI-H358, LXF-289, LOU-NH91, normal human bronchial epithelial cells, and normal lung fibroblasts IMR90. Cell death was determined by morphology, propidium iodide uptake, and trypan blue staining; DNA cleavage was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling. Expression of heat shock protein 70, heat shock cognate 70, and phosphorylated p53 was determined by Western blot analysis. RESULTS: Transduction of lung cancer cells with adenovirus expressing antisense heat shock protein 70 but not with adenovirus with beta-galactosidase resulted in extensive cell death after 96 hours (A549: 53.2 +/- 9.44% versus 12.9 +/- 6.6%; NCI-H358: 48.4 +/- 7.2% versus 25.2 +/- 1.4%; LXF-289: 58.8 +/- 6.5% versus 24.7 +/- 5.4%; LOU-NH91: 82.5 +/- 1.8% versus 38.5 +/- 2.6%). In contrast, adenovirus expressing antisense heat shock protein 70 showed much less cytotoxicity in normal human bronchial epithelial cells (16.0 +/- 0.5% versus 17.1 +/- 7.3%) and in normal lung fibroblasts IMR90 (17.2 +/- 3.6% versus 8.2 +/- 1.6%). After treatment with adenovirus expressing antisense heat shock protein 70, transactivation of p53 in A549 but not in NCI-H358, a cell line deleted for p53 has been observed. Furthermore, 22.0 +/- 3.0% of A549 cells treated for adenovirus expressing antisense heat shock protein 70 stained positive with terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling versus 10.2 +/- 4.0% treated with control virus. This effect but not cell death itself was blocked by treatment with 10 micromol/L zVAD-fmk, a broad caspase inhibitor. CONCLUSIONS: Selective down-regulation of heat shock protein 70 induces cell death in lung cancer but not in normal lung cells. The demonstrated effect is p53-independent and does not require DNA cleavage. The data suggest that gene transfer of antisense heat shock protein 70 might be useful in developing new strategies for the treatment of lung cancer.