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An Hendrix,
Raija Sormunen,
Wendy Westbroek,
Kathleen Lambein, Hannelore Denys,
Gwen Sys,
Geert Braems,
Rudy Van den Broecke,
Veronique Cocquyt,
Christian Gespach,
Marc Bracke,
Olivier De Wever
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ABSTRACT: The secretory Rab27B small GTPase promotes invasive growth and metastasis in estrogen receptor (ER)α-positive breast cancer cells by orchestrating the peripheral targeting of vesicles secreting pro-invasive growth regulators. Increased Rab27B expression is associated with poor prognosis in breast cancer patients. The molecular mechanisms of peripheral Rab27B secretory vesicle distribution are poorly understood. Mass spectometry analysis on secretory green fluorescent protein (GFP)-Rab27B vesicles prepared from GFP-Rab27B transfected MCF-7 human breast cancer cells detected eight subunits of the vacuolar H(+)-ATPase (V-ATPase) and presence of V(0) a1 and V(0) d1 subunits was confirmed by Western blot analysis. Reversible inhibition of V-ATPase activity by bafilomycin A1 or transient silencing of V(0) a1 or V(0) d1 subunits demonstrated that V-ATPase controls peripheral localization and size of Rab27B vesicles. V-ATPase expression and activity further controls Rab27B-induced collagen type I invasion, cell cycle progression and invasive growth in the chorioallantoic membrane (CAM) assay. In agreement, Rab27B-dependent extracellular heat-shock protein (HSP)90α release and matrix metalloprotease (MMP)-2 activation is markedly reduced by bafilomycin A1 and transient silencing of V(0) a1 and V(0) d1 subunits. Poor prognosis ERα-positive primary breast tumors expressing high levels of Rab27B also expressed multiple V-ATPase subunits and showed a strong cytoplasmic and peripheral V-ATPase V(1) E expression. In conclusion, inhibiting V-ATPase activity by interfering agents and drugs might be an effective strategy for blocking Rab27B-dependent pro-invasive secretory vesicle trafficking in ERα-positive breast cancer patients. © 2013 Wiley Periodicals, Inc.
International Journal of Cancer 02/2013; · 5.44 Impact Factor
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Astrid De Boeck,
Patrick Pauwels,
Karen Hensen,
Jean-Luc Rummens,
Wendy Westbroek,
An Hendrix,
Dawn Maynard, Hannelore Denys,
Kathleen Lambein,
Geert Braems,
Christian Gespach,
Marc Bracke,
Olivier De Wever
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ABSTRACT: OBJECTIVE: Bone marrow-derived mesenchymal stem cells (BM-MSC) migrate to primary tumours and drive tumour progression. This study aimed to identify the molecular mechanisms associated with these heterotypic cellular interactions and analyse their relevance in colorectal cancer (CRC). DESIGN: Paracrine interactions of BM-MSC with CRC cells were studied using collagen invasion assays, cell counts, flow cytometric cell-cycle analysis and tumour xenograft models. The role of neuregulin 1 (NRG1) and the human epidermal growth factor receptor (HER) family pathways were investigated using tyrosine kinase assays, mass spectrometry, pharmacological inhibition, antibody-mediated neutralisation and RNA interference. Transmembrane neuregulin 1 (tNRG1), HER2 and HER3 expression was analysed in primary CRC (n=54), adjacent normal colorectal tissues (n=4), liver metastases (n=3) and adjacent normal liver tissues (n=3) by immunohistochemistry. RESULTS: BM-MSC stimulate invasion, survival and tumorigenesis of CRC through the release of soluble NRG1, activating the HER2/HER3-dependent PI3K/AKT signalling cascade in CRC cells. Similarly, tumour-associated mesenchymal cells (T-MC) in CRC demonstrate high tNRG1 expression, which is significantly associated with advanced Union for International Cancer Control stage (p=0.005) and invasion depth (p=0.04) and decreased 5-year progression-free survival (p=0.01). HER2 and HER3 show membrane localisation in cancer cells of CRC tissue. CONCLUSION: Paracrine NRG1/HER3 signals initiated by BM-MSC and T-MC promote CRC cell progression, and high tNRG1 expression is associated with poor prognosis in CRC.
Gut 04/2012; · 10.11 Impact Factor
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Katrien Vandecasteele,
Philippe Tummers,
Amin Makar,
Marc van Eijkeren,
Louke Delrue, Hannelore Denys,
Bieke Lambert,
Anne-Sophie Beerens,
Rudy Van den Broecke,
Kathleen Lambein,
Valérie Fonteyne,
Gert De Meerleer
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ABSTRACT: To report on toxicity after postoperative intensity-modulated arc therapy (IMAT) for cervical (CC) and endometrial cancer (EC).
Twenty-four CC and 41 EC patients were treated with postoperative IMAT. If indicated, para-aortic lymph node irradiation (preventive or when affected, PALN) and/or concomitant cisplatin (40 mg/m(2), weekly) was administered. The prescribed dose for IMAT was 45 Gy (CC, 25 fractions) and 46 Gy (EC, 23 fractions), followed by a brachytherapeutic boost if possible. Radiation-related toxicity was assessed prospectively. The effect of concomitant cisplatin and PALN irradiation was evaluated.
Regarding acute toxicity (n = 65), Grade 3 and 2 acute gastrointestinal toxicity was observed in zero and 63% of patients (79% CC, 54% EC), respectively. Grade 3 and 2 acute genitourinary toxicity was observed in 1% and 18% of patients, respectively. Grade 2 (21%) and 3 (12%) hematologic toxicity (n = 41) occurred only in CC patients. Seventeen percent of CC patients and 2% of EC patients experienced Grade 2 fatigue and skin toxicity, respectively. Adding cisplatin led to an increase in Grade >2 nausea (57% vs. 9%; p = 0.01), Grade 2 nocturia (24% vs. 4%; p = 0.03), Grade ≥ 2 hematologic toxicity (38% vs. nil, p = 0.003), Grade ≥ 2 leukopenia (33% vs. nil, p = 0.009), and a strong trend toward more fatigue (14% vs. 2%; p = 0.05). Para-aortic lymph node irradiation led to an increase of Grade 2 nocturia (31% vs. 4%, p = 0.008) and a strong trend toward more Grade >2 nausea (44% vs. 18%; p = 0.052). Regarding late toxicity (n = 45), no Grade 3 or 4 late toxicity occurred. Grade 2 gastrointestinal toxicity, genitourinary toxicity, and fatigue occurred in 4%, 9%, and 1% of patients. Neither concomitant cisplatin nor PALN irradiation increased late toxicity rates.
Postoperative IMAT for EC or CC is associated with low acute and late toxicity. Concomitant chemotherapy and PALN irradiation influences acute but not late toxicity.
International journal of radiation oncology, biology, physics 02/2012; 84(2):408-14. · 4.59 Impact Factor
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ABSTRACT: To report on the value of magnetic resonance imaging (MRI) and 2-deoxy-2-[18] fluoro-D-glucose positron emission tomography computed tomography (¹⁸FDG PET-CT) in predicting resectability and pathological response of primary locally advanced cervical cancer after neoadjuvant intensity-modulated arc therapy (IMAT) with or without cisplatin (C).
Twenty-seven patients with International Federation of Gynecology and Obstetrics stages IB2 to IVA cervical cancer were treated with IMAT-C followed by extrafascial hysterectomy (EH). All patients received MRI and ¹⁸FDG PET-CT after IMAT-C. The end points of this study were to: 1. Assess the ability of MRI to predict negative surgical margins (R0). 2. Assess the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of MRI in predicting the following situation at the EH specimen: "no residual disease or minimal microscopically visible residual tumor." 3. Assess the sensitivity, specificity, PPV, and NPV value of ¹⁸FDG PET-CT in predicting "no residual viable tumor cells" at the EH specimen.
An R0 resection was obtained in all patients. None of the EH specimens contained macroscopically visible tumor. In 13 patients, no viable tumor cells were found and only 14 had residual microscopic disease. Twenty-four of 27 MRIs were able to correctly predict R0 resection. A negative MRI was 100% predictive for the end point "R0 resection." The specificity and NPV of MRI (end point 2) were 74% and 100%, respectively. No sensitivity or PPV could be calculated. The sensitivity, specificity, PPV, and NPV of ¹⁸FDG PET-CT were 29%, 62%, 44%, and 44%, respectively (end point 3).
A negative MRI after IMAT-C predicts 100% correctly for R0 resection. The role of FDG PET-CT in predicting viable tumor cells at EH specimen is at least debatable.
International Journal of Gynecological Cancer 01/2012; 22(4):630-7. · 1.65 Impact Factor
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Gert De Meerleer,
Katrien Vandecasteele,
Piet Ost,
Louke Delrue, Hannelore Denys,
Amin Makar,
Bruno Speleers,
Simon Van Belle,
Rudy Van den Broecke,
Valérie Fonteyne,
Wilfried De Neve
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ABSTRACT: To retrospectively review our experience with whole abdominopelvic radiotherapy (WAPRT) using intensity-modulated arc therapy in the palliative treatment of chemotherapy-resistant ovarian cancer with bulky peritoneal disease.
Between April 2002 and April 2008, 13 patients were treated with WAPRT using intensity-modulated arc therapy. We prescribed a dose of 33 Gy to be delivered in 22 fractions of 1.5 Gy to the abdomen and pelvis. All patients had International Federation of Gynecology and Obstetrics Stage III or IV ovarian cancer at the initial diagnosis. At referral, the median age was 61 years, and the patients had been heavily pretreated with surgery and chemotherapy. All patients had symptoms from their disease, including gastrointestinal obstruction or subobstruction in 6, minor gastrointestinal symptoms in 2, pain in 4, ascites in 1, and vaginal bleeding in 2. A complete symptom or biochemical response required complete resolution of the patient's symptoms or cancer antigen-125 level. A partial response required ≥50% resolution of these parameters. The actuarial survival was calculated from the start of radiotherapy.
The median overall survival was 21 weeks, with a 6-month overall survival rate of 45%. The 9 patients who completed treatment obtained a complete symptom response, except for ascites (partial response). The median and mean response duration (all symptoms grouped) was 24 and 37 weeks, respectively. Of the 6 patients presenting with obstruction or subobstruction, 4 obtained a complete symptom response (median duration, 16 weeks).
WAPRT delivered using intensity-modulated arc therapy offers important palliation in the case of peritoneal metastatic ovarian cancer. WAPRT resolved intestinal obstruction for a substantial period.
International journal of radiation oncology, biology, physics 03/2011; 79(3):775-81. · 4.59 Impact Factor
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ABSTRACT: A few reports have assessed HER2 status in breast cancer by both dual-probe fluorescence in situ hybridisation (FISH) and immunohistochemistry (IHC) in an unselected and consecutive fashion, but CEP17 and HER2 copy number were not evaluated separately in these studies. Therefore, the aim of this study was to perform FISH testing for HER2 in a large number of breast tumours, irrespective of the IHC scores, which were also determined in all cases.
Both FISH and IHC were applied to 200 tumours from 196 consecutive patients who underwent resection of primary breast cancer with the sentinel procedure and/or axillary dissection. Not only the ratio, but also mean HER2 and CEP17 copy number were determined and used in statistical analyses to evaluate relationships between FISH, IHC and clinicopathological features.
The amplification status based solely on HER2 signals was 98% concordant with results of dual-probe FISH. In non-amplified tumours, the mean CEP17 and HER2 copy number correlated, possibly because of cell cycling. Amplified tumours were histopathologically more aggressive than non-amplified tumours, and features of aggressiveness increased with the mean HER2 copy number. In both amplified and non-amplified tumours, a gene dosage effect was observed: an increase in the mean HER2 copy number was associated with a higher IHC score.
This working method and analysis enabled new insights to be obtained into the pathobiology of HER2 in breast cancer. The findings may be helpful in optimising the methodology of HER2 testing.
Journal of clinical pathology 03/2011; 64(3):200-7. · 2.43 Impact Factor
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ABSTRACT: For premenopausal patients with receptor-positive early breast cancer, administration of tamoxifen for 5 years constitutes the main adjuvant endocrine therapy. During pregnancy, tamoxifen and its metabolites interact with rapidly growing and developing embryonic or fetal tissues. Information about tamoxifen and pregnancy was gathered by searching PubMed. In addition, we had access to the records of the pharmaceutical company AstraZeneca. Because these observations are retrospective and other therapies and diagnostic measures are possible confounders, a causal relationship was not established between tamoxifen treatment and pregnancy outcome. The records from AstraZeneca documented three live births with congenital anomalies and four live births without congenital anomalies related to tamoxifen treatment before pregnancy. Tamoxifen therapy during pregnancy resulted in 16 live births with congenital malformations and a total of 122 live births without malformations. The 122 live births without malformations included 85 patients from a prevention trial that did not record a single anomaly, whereas the AstraZeneca Safety Database alone reported 11 babies with congenital malformations of 44 live births. Additionally, there were: 12 spontaneous abortions, 17 terminations of pregnancy without known fetal defects, six terminations of pregnancy with fetal defects, one stillbirth without fetal defects, two stillbirths with fetal defects, and 57 unknown outcomes. The relatively high frequency of severe congenital abnormalities indicates that reliable birth control during tamoxifen treatment is mandatory. After tamoxifen use, a washout period of 2 months is advisable based on the known half-life of tamoxifen. In case of an inadvertent pregnancy, risks and options should be discussed.
The Oncologist 01/2011; 16(11):1547-51. · 3.91 Impact Factor
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An Hendrix,
Dawn Maynard,
Patrick Pauwels,
Geert Braems, Hannelore Denys,
Rudy Van den Broecke,
Jo Lambert,
Simon Van Belle,
Veronique Cocquyt,
Christian Gespach,
Marc Bracke,
Miguel C Seabra,
William A Gahl,
Olivier De Wever,
Wendy Westbroek
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ABSTRACT: BACKGROUND Secretory GTPases like Rab27B control vesicle exocytosis and deliver critical proinvasive growth regulators into the tumor microenvironment. The expression and role of Rab27B in breast cancer were unknown. METHODS Expression of green fluorescent protein (GFP) fused with wild-type Rab3D, Rab27A, or Rab27B, or Rab27B point mutants defective in GTP/GDP binding or geranylgeranylation, or transient silencing RNA to the same proteins was used to study Rab27B in estrogen receptor (ER)-positive human breast cancer cell lines (MCF-7, T47D, and ZR75.1). Cell cycle progression was evaluated by flow cytometry, western blotting, and measurement of cell proliferation rates, and invasion was assessed using Matrigel and native type I collagen substrates. Orthotopic tumor growth, local invasion, and metastasis were analyzed in mouse xenograft models. Mass spectrometry identified proinvasive growth regulators that were secreted in the presence of Rab27B. Rab27B protein levels were evaluated by immunohistochemistry in 59 clinical breast cancer specimens, and Rab3D, Rab27A, and Rab27B mRNA levels were analyzed by quantitative real-time polymerase chain reaction in 20 specimens. Statistical tests were two-sided. RESULTS Increased expression of Rab27B promoted G(1) to S phase cell cycle transition, proliferation and invasiveness of cells in culture, and invasive tumor growth and hemorrhagic ascites production in a xenograft mouse model (n = 10; at 10 weeks, survival of MCF-7 GFP- vs GFP-Rab27B-injected mice was 100% vs 62.5%, hazard ratio = 0.26, 95% confidence interval = 0.08 to 0.88, P = .03). Mass spectrometric analysis of purified Rab27B-secretory vesicles identified heat-shock protein 90alpha as key proinvasive growth regulator. Heat-shock protein 90alpha secretion was Rab27B-dependent and was required for matrix metalloproteinase-2 activation. All Rab27B-mediated functional responses were GTP- and geranylgeranyl-dependent. Presence of endogenous Rab27B mRNA and protein, but not of Rab3D or Rab27A mRNA, was associated with lymph node metastasis (P < .001) and differentiation grade (P = .001) in ER-positive human breast tumors. CONCLUSIONS Rab27B regulates invasive growth and metastasis in ER-positive breast cancer cell lines, and increased expression is associated with poor prognosis in humans.
CancerSpectrum Knowledge Environment 06/2010; 102(12):866-80. · 14.07 Impact Factor
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ABSTRACT: Limited data are available on the use of cytoreductive surgery with hyperthermic intraperitoneal chemoperfusion (HIPEC) in patients with recurrent stage III ovarian cancer.
Patients with recurrent, heavily pretreated ovarian cancer were enrolled onto a phase II multimodal protocol consisting of extensive cytoreduction followed by HIPEC.
Forty-two women were treated from October 2002 until January 2009. Chemoperfusion was performed with cisplatin in 59% and oxaliplatin in 41% of patients. A macroscopically complete resection was achieved in 50% of patients. No mortality occurred, and the major morbidity rate was 21%. After a mean follow-up of 21 months, median overall survival (OS) was 37 months (95% confidence interval 12.2-61.8) and median progression-free survival was 13 months (95% confidence interval 6.9-19.1). In univariate analysis, OS was influenced by completeness of cytoreduction, type of chemoperfusion drug, nodal status, and tumor grade. In a Cox regression model, only completeness of cytoreduction (hazard ratio 0.06-0.8, P=.022) and tumor grade (hazard ratio 1.23-12.6, P=.021) were independent predictors of OS.
In selected patients with heavily pretreated recurrent ovarian cancer, cytoreduction combined with HIPEC may provide a meaningful OS with acceptable morbidity. Optimal results are achieved in patients with a macroscopically complete resection and biologically favorable disease.
Annals of Surgical Oncology 12/2009; 19(7):2352-9. · 4.17 Impact Factor
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ABSTRACT: A quantitative HPLC method with fluorescence detection has been developed for the simultaneous determination of four anthracyclines (doxorubicin, epirubicin, daunorubicin and idarubicin) and their respective 13-S-dihydro metabolites (doxorubicinol, epirubicinol, daunorubicinol and idarubicinol) in plasma and saliva, using epidaunorubicin as internal standard. A progressive optimization matrix led to a two-step extraction based on a protein precipitation with ethanol followed by a liquid-liquid extraction with dichloromethane after pH adjustment to 8.5. The chromatographic separation was performed in 14min on a C18 column, applying gradient elution with a mixture of 0.1% formic acid in water and 0.1% formic acid in acetonitrile. The analytes were detected and quantified at an excitation and emission wavelength of 480 and 555nm, respectively. Limit of detection (LOD) and lower limit of quantification (LLOQ) were 0.3 or 0.75, and 1 or 2.5ng/mL, respectively. Linearity by means of weighted (1/x) regression was obtained from the LLOQ up to 1000 or 2500ng/mL for the parent drugs and up to 400 or 1000ng/mL for the metabolites. Intra-assay and inter-assay relative standard deviation values were all less than 14% at low, medium and high levels, and below 17% at the LLOQ. Accuracy ranged between 91 and 113% at low, medium and high concentrations and between 83 and 118% at the LLOQ. Absolute recoveries were between 78 and 88% in plasma, and between 70 and 79% in saliva, respectively. Autosampler, benchtop, freeze-thaw and long-term stability samples fulfilled acceptance criteria. This selective method was applied successfully to the analysis of plasma and saliva samples from patients administered epirubicin intravenously.
Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 11/2009; 877(30):3907-15. · 2.78 Impact Factor
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ABSTRACT: Emerging evidence points towards a key role of the extracellular matrix (ECM) during tumor progression and therapy resistance. Paradoxically, in today's routine of cancer management the ECM is not taken into account. It is the aim of the present review to broaden our understanding of the mechanisms of therapy resistance, taking the ECM as a presumptive central regulator. The stromal ecosystem drives the accumulation of ECM at the invasion front. Therefore, we address the question whether the detection of ECM signatures in histopathology and biofluids may help predicting therapy resistance and determining the prognosis of cancer. Since the ECM is an attractive target for tumor therapy, current therapeutic strategies in preclinical or clinical development will be discussed.
Current Pharmaceutical Design 03/2009; 15(12):1373-1384. · 3.87 Impact Factor
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ABSTRACT: Emerging evidence points towards a key role of the extracellular matrix (ECM) during tumor progression and therapy resistance. Paradoxically, in today's routine of cancer management the ECM is not taken into account. It is the aim of the present review to broaden our understanding of the mechanisms of therapy resistance, taking the ECM as a presumptive central regulator. The stromal ecosystem drives the accumulation of ECM at the invasion front. Therefore, we address the question whether the detection of ECM signatures in histopathology and biofluids may help predicting therapy resistance and determining the prognosis of cancer. Since the ECM is an attractive target for tumor therapy, current therapeutic strategies in preclinical or clinical development will be discussed.
Current pharmaceutical design 02/2009; 15(12):1373-84. · 4.41 Impact Factor
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ABSTRACT: Cardiac toxicity remains an important side effect of anthracyclines. New drug formulations (eg, pegylated liposomal doxorubicin [PL-DOX]) seem to be a successful strategy for reducing it. Changes in cardiac function induced by early chemotherapy, however, are subtle and difficult to quantitate by conventional imaging methods. Doppler myocardial imaging-based velocity, strain, and strain rate measurements have been shown to sensitively quantify abnormalities in cardiac function in other settings.
We evaluated the feasibility and sensitivity of strain rate imaging compared with conventional echocardiography in detecting cardiac effects of PL-DOX therapy in elderly patients with cancer. In a pilot study, we examined 16 elderly women (age 69.8 +/- 3.1 years) with breast cancer receiving 6 cycles of PL-DOX. Conventional and Doppler myocardial imaging echocardiography were obtained at baseline and after 3 and 6 cycles of treatment. Segmental peak systolic longitudinal and radial velocity, strain, and strain rate were measured.
Left ventricular dimensions, ejection fraction, and systolic myocardial velocity did not change throughout the follow-up. In contrast, a significant reduction in longitudinal and radial strain and strain rate was found after 6 cycles (longitudinal strain -18.8% +/- 2.8% vs -22.7% +/- 2.8%, P < .001 vs baseline and P = .001 vs after 3 cycles; radial strain 32.3% +/- 8.1% vs 50.1% +/- 11.6%, P < .001 vs baseline). Changes in radial function appeared earlier and were more pronounced than in longitudinal direction.
In contrast with conventional echocardiography and myocardial velocity measurements, myocardial deformation parameters allowed detecting subtle changes in longitudinal and radial left ventricular function after 6 cycles of PL-DOX. We suggest that Doppler-based myocardial deformation imaging should be used for cardiac function monitoring during chemotherapy.
Journal of the American Society of Echocardiography: official publication of the American Society of Echocardiography 01/2009; 21(12):1283-9. · 2.98 Impact Factor
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ABSTRACT: Several studies indicate that cancer-associated fibroblasts play a critical role in cancer cell invasion and metastasis, the hallmarks of malignancy. To better understand the mechanisms underlying such effects, we established a heterotypic model of human fibroblasts (primary colon fibroblasts and immortalized human dermal fibroblasts) in co-culture with human colon cancer cells (HCT-8/E11), using three-dimensional collagen type-I and Matrigel matrices. We report that TGF-beta is the unique and dominant factor to provide pro-invasive signals to HCT-8/E11 colon cancer cells from TGF-beta-treated human fibroblasts in three-dimensional collagen type I and Matrigel matrices. These effects are not mimicked or reversed by EGF or bFGF, and are associated with the TGF-beta-mediated induction of myofibroblast differentiation and functional markers, such as alpha-SMA, the haptotactic matrix molecule TNC, collagen type 1 maturation enzyme P4H, serine protease FAP, and myofibroblast contractility. Accordingly, TGF-beta induced a strong activation of RhoA and stress fiber formation in fibroblasts, with no impact on Rac1-GTP levels. In contrast, EGF down-regulated Rho-GTP levels in fibroblasts, giving permissive signals for Rac1 activation, fibroblast polarization, and invasion. Taken together, our data imply that TGF-beta and EGF exert invasive growth-promoting actions in human colon tumors through a differential and cumulative impact on the stromal and cancer cell compartments. Our data predict that inhibitors directed at this reciprocal molecular and cellular crosstalk will have therapeutic applications for targeting the invasive growth of human primary tumors and their metastatic spread.
Cancer Letters 09/2008; 266(2):263-74. · 4.24 Impact Factor
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ABSTRACT: Desmoid tumors (aggressive fibromatosis) are locally invasive soft tissue tumors in which beta-catenin-mediated TCF-3-dependent transcription is activated. To provide more insight into the pathophysiology of these tumors, expression profiles were generated using oligonucleotide arrays (Affymetrix). In total, 69 differentially expressed genes were identified in desmoids compared to normal fibroblasts (fascia) from the same patients. The differential expression of a selection of genes was confirmed using RT-PCR and Northern blotting. We further evaluated the insulin-like growth factor-binding protein 6 (IGFBP-6), a gene that was consistently downregulated in all desmoids tested. Promotor studies and electromobility shift assays revealed two functional beta-catenin/TCF-responsive elements in the human IGFBP-6 promoter. These findings suggest that IGFBP-6 is directly downregulated by the beta-catenin/TCF complex in desmoid tumors, and imply a role for the IGF axis in the proliferation of desmoid tumors.
Oncogene 02/2004; 23(3):654-64. · 6.37 Impact Factor
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ABSTRACT: A quantitative HPLC method with fluorescence detection has been developed for the simultaneous determination of four anthracyclines (doxorubicin, epirubicin, daunorubicin and idarubicin) and their respective 13-S-dihydro metabolites (doxorubicinol, epirubicinol, daunorubicinol and idarubicinol) in plasma and saliva, using epidaunorubicin as internal standard. A progressive optimization matrix led to a two-step extraction based on a protein precipitation with ethanol followed by a liquid–liquid extraction with dichloromethane after pH adjustment to 8.5. The chromatographic separation was performed in 14 min on a C18 column, applying gradient elution with a mixture of 0.1% formic acid in water and 0.1% formic acid in acetonitrile. The analytes were detected and quantified at an excitation and emission wavelength of 480 and 555 nm, respectively. Limit of detection (LOD) and lower limit of quantification (LLOQ) were 0.3 or 0.75, and 1 or 2.5 ng/mL, respectively. Linearity by means of weighted (1/x) regression was obtained from the LLOQ up to 1000 or 2500 ng/mL for the parent drugs and up to 400 or 1000 ng/mL for the metabolites. Intra-assay and inter-assay relative standard deviation values were all less than 14% at low, medium and high levels, and below 17% at the LLOQ. Accuracy ranged between 91 and 113% at low, medium and high concentrations and between 83 and 118% at the LLOQ. Absolute recoveries were between 78 and 88% in plasma, and between 70 and 79% in saliva, respectively. Autosampler, benchtop, freeze–thaw and long-term stability samples fulfilled acceptance criteria. This selective method was applied successfully to the analysis of plasma and saliva samples from patients administered epirubicin intravenously.
Journal of Chromatography B.
