Membrane transport of camptothecin: facilitation by human P-glycoprotein (ABCB1) and multidrug resistance protein 2 (ABCC2)

BACKGROUND: The purpose of the present study was to continue the investigation of the membrane transport mechanisms of 20-(S)-camptothecin (CPT) in order to understand the possible role of membrane transporters on its oral bioavailability and disposition. METHODS: The intestinal transport kinetics of CPT were characterized using Caco-2 cells, MDCKII wild-type cells and MDCKII cells transfected with human P-glycoprotein (PGP) (ABCB1) or human multidrug resistance protein 2 (MRP2) (ABCC2). The effects of drug concentration, inhibitors and temperature on CPT directional permeability were determined. RESULTS: The absorptive (apical to basolateral) and secretory (basolateral to apical) permeabilities of CPT were found to be saturable. Reduced secretory CPT permeabilities with decreasing temperatures suggests the involvement of an active, transporter-mediated secretory pathway. In the presence of etoposide, the CPT secretory permeability decreased 25.6%. However, inhibition was greater in the presence of PGP and of the breast cancer resistant protein inhibitor, GF120918 (52.5%). The involvement of additional secretory transporters was suggested since the basolateral to apical permeability of CPT was not further reduced in the presence of increasing concentrations of GF120918. To investigate the involvement of specific apically-located secretory membrane transporters, CPT transport studies were conducted using MDCKII/PGP cells and MDCKII/MRP2 cells. CPT carrier-mediated permeability was approximately twofold greater in MDCKII/PGP cells and MDCKII/MRP2 cells than in MDCKII/wild-type cells, while the apparent K(m )values were comparable in all three cell lines. The efflux ratio of CPT in MDCKII/PGP in the presence of 0.2 μM GF120918 was not completely reversed (3.36 to 1.49). However, the decrease in the efflux ratio of CPT in MDCKII/MRP2 cells (2.31 to 1.03) suggests that CPT efflux was completely inhibited by MK571, a potent inhibitor of the Multidrug Resistance Protein transporter family. CONCLUSIONS: The current results provide evidence that PGP and MRP2 mediate the secretory transport of CPT in vitro. However, the involvement of other transporters cannot be ruled out based on these studies. Since these transporters are expressed in the intestine, liver and kidney variations in their expression levels and/or regulation may be responsible for the erratic oral absorption and biliary excretion of CPT observed in human subjects

DNA methylation regulates expression of VEGF-R2 (KDR) and VEGF-R3 (FLT4)

Abstract Background Vascular Endothelial Growth Factors (VEGFs) and their receptors (VEGF-Rs) are important regulators for angiogenesis and lymphangiogenesis. VEGFs and VEGF-Rs are not only expressed on endothelial cells but also on various subtypes of solid tumors and leukemias contributing to the growth of the malignant cells. This study was performed to examine whether VEGF-R2 (KDR) and VEGF-R3 (FLT4) are regulated by DNA methylation. Methods Real-time (RT) PCR analysis was performed to quantify KDR and FLT4 expression in some ninety leukemia/lymphoma cell lines, human umbilical vein endothelial cells (HUVECs) and dermal microvascular endothelial cells (HDMECs). Western blot analyses and flow cytometric analyses confirmed results at the protein level. After bisulfite conversion of DNA we determined the methylation status of KDR and FLT4 by DNA sequencing and by methylation specific PCR (MSP). Western blot analyses were performed to examine the effect of VEGF-C on p42/44 MAPK activation. Results Expression of KDR and FLT4 was observed in cell lines from various leukemic entities, but not in lymphoma cell lines: 16% (10/62) of the leukemia cell lines expressed KDR, 42% (27/65) were FLT4 positive. None of thirty cell lines representing six lymphoma subtypes showed more than marginal expression of KDR or FLT4. Western blot analyses confirmed KDR and FLT4 protein expression in HDMECs, HUVECs and in cell lines with high VEGF-R mRNA levels. Mature VEGF-C induced p42/44 MAPK activation in the KDR- /FLT4+ cell line OCI-AML1 verifying the model character of this cell line for VEGF-C signal transduction studies. Bisulfite sequencing and MSP revealed that GpG islands in the promoter regions of KDR and FLT4 were unmethylated in HUVECs, HDMECs and KDR + and FLT4 + cell lines, whereas methylated cell lines did not express these genes. In hypermethylated cell lines, KDR and FLT4 were re-inducible by treatment with the DNA demethylating agent 5-Aza-2'deoxycytidine, confirming epigenetic regulation of both genes. Conclusions Our data show that VEGF-Rs KDR and FLT4 are silenced by DNA methylation. However, if the promoters are unmethylated, other factors (e.g. transactivation factors) determine the extent of KDR and FLT4 expression

Murine 5T multiple myeloma cells induce angiogenesis in vitro and in vivo

Multiple myeloma is a B cell malignancy. Recently, it has been demonstrated that bone marrow samples of patients with multiple myeloma display an enhanced angiogenesis. The mechanisms involved seem to be multiple and complex. We here demonstrate that the murine 5T multiple myeloma models are able to induce angiogenesis in vitro by using a rat aortic ring assay and in vivo by determining the microvessel density. The rat aortic rings cultured in 5T multiple myeloma conditioned medium exhibit a higher number of longer and more branched microvessels than the rings cultured in control medium. In bone marrow samples from 5T multiple myeloma diseased mice, a statistically significant increase of the microvessel density was observed when compared to bone marrow samples from age-matched controls. The angiogenic phenotype of both 5T multiple myeloma cells could be related, at least in part, to their capacity to produce vascular endothelial growth factor. These data clearly demonstrate that the 5T multiple myeloma models are good models to study angiogenesis in multiple myeloma and will allow to unravel the mechanisms of neovascularisation, as well as to test new putative inhibitors of angiogenesis

Increased accumulation of doxorubicin and doxorubicinol in cardiac tissue of mice lacking mdr1a P-glycoprotein

To gain more insight into the pharmacological role of endogenous P-glycoprotein in the metabolism of the widely used substrate drug doxorubicin, we have studied the plasma pharmacokinetics, tissue distribution and excretion of this compound in mdr1a(–/– and wild-type mice. Doxorubicin was administered as an i.v. bolus injection at a dose level of 5 mg kg−1. Drug and metabolite concentrations were determined in plasma, tissues, urine and faeces by high-performance liquid chromatography. In comparison with wild-type mice, the terminal half-life and the area under the plasma concentration–time curve of doxorubicin in it>mdr1a(–/–) mice were 1.6- and 1.2-fold higher respectively.The retention of both doxorubicin and its metabolite doxorubicinol in the hearts of mdr1a(–/–) mice was substantially prolonged. In addition, a significantly increased drug accumulation was observed in the brain and the liver of mdr1a(–/–) mice. The relative accumulation in most other tissues was not or only slightly increased. The differences in cumulative faecal and urinary excretion of doxorubicin and metabolites between both types of mice were small. These experiments demonstrate that the absence of mdr1a P-glycoprotein only slightly alters the plasma pharmacokinetics of oxorubicin. Furthermore, the substantially prolonged presence of both doxorubicin and doxorubicinol in cardiac tissue of mdr1a(–/–) mice suggests that a blockade of endogenous P-glycoprotein in patients, for example by a reversal agent, may enhance the risk of cardiotoxicity upon administration of doxorubicin. © 1999 Cancer Research Campaig

Risk factor screening to identify women requiring oral glucose tolerance testing to diagnose gestational diabetes : a systematic review and meta-analysis and analysis of two pregnancy cohorts

BACKGROUND: Easily identifiable risk factors including: obesity and ethnicity at high risk of diabetes are commonly used to indicate which women should be offered the oral glucose tolerance test (OGTT) to diagnose gestational diabetes (GDM). Evidence regarding these risk factors is limited however. We conducted a systematic review (SR) and meta-analysis and individual participant data (IPD) analysis to evaluate the performance of risk factors in identifying women with GDM. METHODS: We searched MEDLINE, Medline in Process, Embase, Maternity and Infant Care and the Cochrane Central Register of Controlled Trials (CENTRAL) up to August 2016 and conducted additional reference checking. We included observational, cohort, case-control and cross-sectional studies reporting the performance characteristics of risk factors used to identify women at high risk of GDM. We had access to IPD from the Born in Bradford and Atlantic Diabetes in Pregnancy cohorts, all pregnant women in the two cohorts with data on risk factors and OGTT results were included. RESULTS: Twenty nine published studies with 211,698 women for the SR and a further 14,103 women from two birth cohorts (Born in Bradford and the Atlantic Diabetes in Pregnancy study) for the IPD analysis were included. Six studies assessed the screening performance of guidelines; six examined combinations of risk factors; eight evaluated the number of risk factors and nine examined prediction models or scores. Meta-analysis using data from published studies suggests that irrespective of the method used, risk factors do not identify women with GDM well. Using IPD and combining risk factors to produce the highest sensitivities, results in low specificities (and so higher false positives). Strategies that use the risk factors of age (>25 or >30) and BMI (>25 or 30) perform as well as other strategies with additional risk factors included. CONCLUSIONS: Risk factor screening methods are poor predictors of which pregnant women will be diagnosed with GDM. A simple approach of offering an OGTT to women 25 years or older and/or with a BMI of 25kg/m2 or more is as good as more complex risk prediction models. Research to identify more accurate (bio)markers is needed. Systematic Review Registration: PROSPERO CRD42013004608

Reduction of serum IGF-I levels in patients affected with Monoclonal Gammopathies of undetermined significance or Multiple Myeloma. Comparison with bFGF, VEGF and K-ras gene mutation

<p>Abstract</p> <p>Background</p> <p>Serum levels of IGF-I in patients affected with multiple myeloma (MM) have been scarcely studied. The present study is aimed to explore this point comparing 55 healthy subjects, 71 monoclonal gammopaties of uncertain significance (MGUS) and 77 overt MM patients. In the same subjects, basic FGF and VEGF, have been detected. All three mediators were analyzed in function of K-<it>ras </it>mutation and melphalan response. Concerning IGF-I, two representative monitoring examples have also been added.</p> <p>Methods</p> <p>Cytokine determinations were performed by commercially available ELISA kits, while K12-<it>ras </it>mutation was investigated on genomic DNA isolated from bone marrow cell specimens by RFLP-PCR assay.</p> <p>Results</p> <p>Significant reductions of IGF-I levels were observed in MGUS and MM as compared with healthy controls. In addition, MM subjects showed significantly decreased serum IGF-I levels than MGUS. Conversely, increasing levels were observed for bFGF and VEGF, molecules significantly correlated. A multivariate analysis corrected for age and gender confirmed the significant difference only for IGF-I values (P = 0.01). K12-<it>ras </it>mutation was significantly associated with malignancy, response to therapy and with significantly increased serum bFGF levels.</p> <p>Conclusion</p> <p>IGF-I reduction in the transition: Controls→MGUS→MM and changes observed over time suggest that IGF-I should be furtherly studied in future clinical trials as a possible monitoring marker for MM.</p

Vascular endothelial growth factor receptor-1 mediates migration of human colorectal carcinoma cells by activation of Src family kinases

Vascular endothelial growth factor (VEGF) is the predominant pro-angiogenic cytokine in human malignancy, and its expression correlates with disease recurrence and poor outcomes in patients with colorectal cancer. Recently, expression of vascular endothelial growth factor receptors (VEGFRs) has been observed on tumours of epithelial origin, including those arising in the colon, but the molecular mechanisms governing potential VEGF-driven biologic functioning in these tumours are not well characterised. In this report, we investigated the role of Src family kinases (SFKs) in VEGF-mediated signalling in human colorectal carcinoma (CRC) cell lines. Vascular endothelial growth factor specifically activated SFKs in HT29 and KM12L4 CRC cell lines. Further, VEGF stimulation resulted in enhanced cellular migration, which was effectively blocked by pharmacologic inhibition of VEGFR-1 or Src kinase. Correspondingly, migration studies using siRNA clones with reduced Src expression confirmed the requirement for Src in VEGF-induced migration in these cells. Furthermore, VEGF treatment enhanced VEGFR-1/SFK complex formation and increased tyrosine phosphorylation of focal adhesion kinase, p130 cas and paxillin. Finally, we demonstrate that VEGF-induced migration is not due, at least in part, to VEGF acting as a mitogen. These results suggest that VEGFR-1 promotes migration of tumour cells through a Src-dependent pathway linked to activation of focal adhesion components that regulate this process

Galectin-3C Inhibits Tumor Growth and Increases the Anticancer Activity of Bortezomib in a Murine Model of Human Multiple Myeloma

Galectin-3 is a human lectin involved in many cellular processes including differentiation, apoptosis, angiogenesis, neoplastic transformation, and metastasis. We evaluated galectin-3C, an N-terminally truncated form of galectin-3 that is thought to act as a dominant negative inhibitor, as a potential treatment for multiple myeloma (MM). Galectin-3 was expressed at varying levels by all 9 human MM cell lines tested. In vitro galectin-3C exhibited modest anti-proliferative effects on MM cells and inhibited chemotaxis and invasion of U266 MM cells induced by stromal cell-derived factor (SDF)-1α. Galectin-3C facilitated the anticancer activity of bortezomib, a proteasome inhibitor approved by the FDA for MM treatment. Galectin-3C and bortezomib also synergistically inhibited MM-induced angiogenesis activity in vitro. Delivery of galectin-3C intravenously via an osmotic pump in a subcutaneous U266 cell NOD/SCID mouse model of MM significantly inhibited tumor growth. The average tumor volume of bortezomib-treated animals was 19.6% and of galectin-3C treated animals was 13.5% of the average volume of the untreated controls at day 35. The maximal effect was obtained with the combination of galectin-3C with bortezomib that afforded a reduction of 94% in the mean tumor volume compared to the untreated controls at day 35. In conclusion, this is the first study to show that inhibition of galectin-3 is efficacious in a murine model of human MM. Our results demonstrated that galectin-3C alone was efficacious in a xenograft mouse model of human MM, and that it enhanced the anti-tumor activity of bortezomib in vitro and in vivo. These data provide the rationale for continued testing of galectin-3C towards initiation of clinical trials for treatment of MM