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Background: Great interest has been raised recently by the design of new adoptive immunotherapeutic strategies based on the in vivo infusion of ex vivo-expanded and activated natural killer (NK) cells. The development of good manufacturing practice (GMP) methods for the efficient production of fully functional NK cells is mandatory for clinical application., Materials and Methods: Peripheral blood mononuclear cells were obtained by leukapheresis and processed in the GMP facility. For NK-cell enrichment, a two-step immunomagnetic procedure consisting of CD3(+) T-cell depletion followed by CD56(+) cell positive selection was used. Isolated NK cells were suspended in serum-free medium containing autologous plasma, interleukin (IL)-2 and IL-15 in the presence of irradiated autologous feeder cells and cultured for 14 days at 37 °C. IL-2 and IL-15 were also added during the last 24 hours of culture. Expanded cells underwent full quality control testing for cytogenetic characteristics, viability, sterility, phenotype and endotoxin status; functional tests, such as degranulation assays and cytotoxicity, were performed on expanded NK cells before cryopreservation and after thawing., Results: NK-cell populations expanded on average 15.7±4.7 fold by day 14, with a viability of 96% ±0.5. At the end of the incubation period, 97% ±1.1 of the expanded population was CD56(+) NK cells; these effector cells showed significant up-regulation of the activating receptors NKG2D and DNAM-1. Functional tests demonstrated that expanded NK cells are fully functional with no difference whether tested before cryopreservation or after thawing., Discussion: These data provide the basis for developing new NK-cell-based immunotherapeutic strategies for the treatment of patients with cancer.

Objective: To identify a phenotype that could be informative and prognostic in patients with renal cell carcinoma (RCC) peripheral blood was evaluated for TH1, TH2, regulatory T cells (Tregs), natural killer (NK) and NKT cells and for cytokines/chemokines., Patients and Methods: Peripheral blood from 77 patients with RCC and 40 healthy controls was evaluated by flow cytometry using monoclonal antibodies against CD4, CD25, FoxP3, CD45RA, CD45RO, CD152, CD184, CD279, CD3, CD16, CD56, CD161, CD158a, CD4, CD26, CD30, CD183 and CD184. A concomitant evaluation of 38 molecules was conducted in patients' serum using a multiplex biometric ELISA-based immunoassay., Results: The number of NK cells CD3⁻/CD16⁺, CD3⁻/CD16⁺/CD161⁺ (NK) and CD3⁻/CD16⁺/CD161⁺/CD158a⁺ (NK- Kir 2+) was greater in the patients with RCC (P < 0.05); and the number of Treg cells CD4⁺/CD25(high+)/FOXP3⁺ and the subset CD4⁺/CD25(high+)/FOXP3⁺/CD45RA⁺ (naïve) and CD45R0⁺(memory) cells, were greater in the patients with RCC (P < 0.001). An increase in the following was observed in the serum of patients with RCC compared with healthy controls: interleukin (IL)-4, IL-6, IL-8, IL-10, G-CSF, CXCL10, CXCL11, hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF). According to Ingenuity Pathway Analysis (IPA), CXCL10, IL-6, IL-8, epidermal growth factor (EGF), HGF and VEGF were associated with a network that controls cellular movement, tissue development and cellular growth. Kaplan-Meier analysis for disease-free survival showed that high numbers of CD4⁺/CD25(high+)/FOXP3⁺/CD45RA⁺ (Treg naïve) and low numbers of CD3⁻/CD16⁺/CD161⁺/CD158a⁺ (NK-Kir+) cells predict short disease-free survival in patients with RCC., Conclusion: Concomitant evaluation of Treg (CD4⁺/CD25(high+)/FOXP3⁺ and CD4⁺/CD25(high+)/FOXP3⁺/CD45RA⁺) and of six soluble factors (IL-6, IL-8 ,VEGF, CXCL10, CXCL11, EGF, HGF) might be a surrogate marker of host immunity in patients with RCC., (© 2013 BJU International.)

Malignant melanoma is a highly aggressive tumor that frequently resists chemotherapy, so the search for new agents for its treatment is of great importance. In the present study, the antiproliferative propensity against human melanoma cell lines of lauroside B (1), a megastigmane glycoside isolated from Laurus nobilis (bay laurel) leaves, was investigated. This compound suppressed the proliferation of three human melanoma cell lines, namely, A375, WM115, and SK-Mel-28. The 1-induced inhibition of human melanoma cell proliferation was due to the induction of apoptosis, as demonstrated by FACS analysis with annexin V/PI staining and confirmed by activation of caspase-3 and by the cleavage of poly(ADP-ribose) polymerase (PARP). Growing evidence implicates NF-κB as an important contributor to metastasis and increased chemoresistance of melanoma. Thus, it was hypothesized that 1-induced apoptosis could be associated with suppression of NF-κB activation. The results showed that exposure of human melanoma cells to 1 inhibited IκB-α degradation and constitutive NF-κB DNA-binding activity as well as the expression, regulated by NF-κB, of two antiapoptotic genes, XIAP and c-FLIP. Induction of apoptosis by 1 in human aggressive melanoma cell lines has a potential high biological value.

Treatment of breast cancer cells with a combination of the EGFR-tyrosine kinase inhibitor (EGFR-TKI) gefitinib and the anti-ErbB-2 monoclonal antibody trastuzumab results in a synergistic antitumor effect. In this study, we addressed the mechanisms involved in this phenomenon. The activation of signaling pathways and the expression of cell cycle regulatory proteins were studied in SK-Br-3 and BT-474 breast cancer cells, following treatment with EGFR and/or ErbB-2 inhibitors. Treatment with the gefitinib/trastuzumab combination produced, as compared with a single agent, a more prolonged blockade of AKT and MAPK activation, a more pronounced accumulation of cells in the G0/G1 phase of the cell cycle, a more significant increase in the levels of p27(kip1) and of hypophosphorylated pRb2, and a decrease in the levels of Cyclin D1 and survivin. Similar findings were observed with the EGFR/ErbB-2 inhibitor lapatinib. Gefitinib, trastuzumab, and their combination increased the stability of p27(kip1), with the combination showing the highest effects. Blockade of both receptors with gefitinib/trastuzumab or lapatinib induced a significant increase in the levels of p27(kip1) mRNA and in the nuclear levels of the p27(kip1) transcription factor FKHRL-1. Inhibition of PI3K signaling also produced a significant raise in p27(kip1) mRNA. Finally, down-modulation of FKHRL-1 with siRNAs prevented the lapatinib-induced increase of p27(kip1) mRNA. The synergism deriving from EGFR and ErbB-2 blockade is mediated by several different alterations in the activation of signaling proteins and in the expression of cell cycle regulatory proteins, including transcriptional and posttranscriptional regulation of p27(kip1) expression.

Background: Despite theoretical and empirical support for an association between maternal control and child anxiety, few studies have examined the origins of this association. Furthermore, none use observer-ratings of maternal control within a genetically informative design. This study addressed three questions: 1) do children who experience maternal control report higher anxiety levels than those who do not?; 2) to what extent do genetic and environmental factors influence maternal control and child anxiety?; 3) to what extent do genetic and environmental factors influence the associations between child anxiety and maternal control?, Method: Five hundred and thirty 8-year-old children (from 265 twin pairs) and their mothers were observed participating in an 'etch-a-sketch' task from which maternal control was rated. Children rated their anxiety using the Screen for Child Anxiety Related Emotional Disorders., Results: Children who experienced maternal behaviour rated as 'extreme control' reported higher anxiety levels than those who did not. Maternal control was highly heritable (A = .63), high self-rated anxiety less so (h(2)(g) = .36). The overlap between high child anxiety and maternal control was primarily due to shared genetic factors., Conclusions: These results suggest that maternal control is likely to have been elicited by children with high levels of anxiety.

The role played by the microenvironment in cancer induction, promotion and progression is crucial. Emerging evidence suggests that cytokines, chemokines and growth factors are major players in carcinogenesis. Therefore, a detailed understanding of factors and mechanisms associated with the processes leading from inflammation to cancer could improve the therapeutic strategies against this disease. We have used hepatocarcinoma as our model in this study. We evaluated the serum levels of 50 different cytokines, chemokines and growth factors in patients affected by HCC with chronic HCV-related hepatitis and liver cirrhosis using multiplex biometric ELISA-based immunoassay. Our data showed that some pro-inflammatory molecules were significantly up-regulated in these patients, and highlighted the complexity of the cytokine network in this disease. This work suggests the need to monitor these proteins in order to define a profile that could characterize patients with HCC or to help identify useful markers. This could lead to better definition of the disease state, and to an increased understanding of the relationships between chronic inflammation and cancer.

The chemokine receptor CXCR4 was described as an independent predictor of poor prognosis in primary human melanoma. To investigate on a possible role of CXCR4 expression on peripheral blood lymphocytes (PBL) subsets, 195 patients with melanoma were evaluated for correlations between PBL subsets CXCR4 expressing and clinicopathological and prognostic features. One hundred ninety-five patients with stages I-III melanoma were enrolled in this study. Lymphocytes subsets were assayed by the direct fluorescence method for whole blood and staining with fluorochrome-conjugated monoclonal antibodies. Correlations between PBL subsets, baseline patient, and tumor features were studied by contingency tables and the chi(2) test. The Kaplan-Meier product limit method was applied to plot disease-free- and overall-survival curves. Univariate analysis was performed with the log-rank test. Cox proportional-hazards regression was used to analyze the effect of multiple risk factors on disease-free survival (DFS). Melanoma patients characterized by CD4(+)CD45RA(+)CXCR4(+) higher than 25% of PBL showed a longer DFS. Conversely, CD4(+)CD45RA(+)CXCR4(+) <25% increased the risk of relapse. The 5-year DFS rate was 76% for patients with CD4(+)CD45RA(+)CXCR4(+) lymphocytes <25% of PBL, and 94% for patients with CD4(+)CD45RA(+)CXCR4(+) >25% (p = 0.030 at log-rank test). Univariate and multivariate analysis for DFS confirmed the prognostic value of the CD4(+)CD45RA(+)CXCR4(+) lymphocytes. Although further studies are needed to better define the involved subpopulation, the detection of cellular subset CD4(+)CD45RA(+)CXCR4(+) is an easy and feasible evaluation of melanoma patients in concomitance with the established melanoma prognostic markers.

AKT and MAPK signaling are involved in the resistance of breast cancer cells to the EGFR tyrosine kinase inhibitor gefitinib. RAS proteins are upstream mediators that transfer messages from surface receptors to intracellular signal transducers including MAPK and AKT pathways. AZD3409 is a novel prenyl inhibitor that has shown activity against both farnesyl transferase and geranylgeranyl transferase in isolated enzyme studies. We explored the activity of AZD3409 on breast cancer cell lines with high (SK-Br-3), intermediate (MDA-MB-361) or low (MDA-MB-468) sensitivity to gefitinib. We found that AZD3409 inhibits the growth of breast cancer cells in a dose-dependent manner, with the MDA-MB-468 and MDA-MB-361 cell lines showing higher sensitivity as compared with SK-Br-3 cells. Treatment with AZD3409 produced a significant reduction in the levels of activation of AKT in the three cell lines. AZD3409 also induced an increase in the expression of p27kip-1 and of hypophosphorylated forms of pRb2 in MDA-MB-468 cells that was associated with accumulation of cells in G0/G1 and the appearance of a sub-G1 peak suggestive of apoptosis. In contrast, AZD3409 produced a G2 arrest associated with reduced expression of pRb2 in MDA-MB-361 cells. A synergistic anti-tumor effect was observed when MDA-MB-468 or MDA-MB-361 cells were treated with both AZD3409 and gefitinib, whereas this combination was only additive in SK-Br-3 cells. However, treatment of breast cancer cells with AZD3409 and gefitinib did not produce a more significant blockade of AKT signaling as compared with gefitinib alone. These data suggest that AZD3409 might be active in gefitinib-resistant breast carcinoma.

Cyclic nucleotide phosphodiesterase 3B (PDE3B) has been suggested to be critical for mediating insulin/IGF-1 inhibition of cAMP signaling in adipocytes, liver, and pancreatic beta cells. In Pde3b-KO adipocytes we found decreased adipocyte size, unchanged insulin-stimulated phosphorylation of protein kinase B and activation of glucose uptake, enhanced catecholamine-stimulated lipolysis and insulin-stimulated lipogenesis, and blocked insulin inhibition of catecholamine-stimulated lipolysis. Glucose, alone or in combination with glucagon-like peptide-1, increased insulin secretion more in isolated pancreatic KO islets, although islet size and morphology and immunoreactive insulin and glucagon levels were unchanged. The beta(3)-adrenergic agonist CL 316,243 (CL) increased lipolysis and serum insulin more in KO mice, but blood glucose reduction was less in CL-treated KO mice. Insulin resistance was observed in KO mice, with liver an important site of alterations in insulin-sensitive glucose production. In KO mice, liver triglyceride and cAMP contents were increased, and the liver content and phosphorylation states of several insulin signaling, gluconeogenic, and inflammation- and stress-related components were altered. Thus, PDE3B may be important in regulating certain cAMP signaling pathways, including lipolysis, insulin-induced antilipolysis, and cAMP-mediated insulin secretion. Altered expression and/or regulation of PDE3B may contribute to metabolic dysregulation, including systemic insulin resistance.

In the present study, we have characterized several human thyroid cancer cell lines of different histotypes for their responsiveness to contact inhibition. We found that cells derived from differentiated carcinoma (TPC-1, WRO) arrest in G(1) phase at confluence, whereas cells derived from anaplastic carcinoma (ARO, FRO and FB1) continue to grow after reaching confluence. Furthermore, we provide experimental evidence that the axis, E-cadherin/beta-catenin/p27(Kip1), represents an integral part of the regulatory mechanism that controls proliferation at a high cell density, whose disruption may play a key role in determining the clinical behaviour of thyroid cancer. This conclusion derives from the finding that: (i) the expression of p27(Kip1) is enhanced at high cell density only in cells responsive to contact inhibition (TPC-1, WRO), but not in contact-inhibition resistant cells (ARO, FRO or FB1 cells); (ii) the increase in p27(Kip1) also resulted in increased levels of p27(Kip1) bound to cyclin E-Cdk2 complex, a reduction in cyclin E-Cdk2 activity and dephosphorylation of the retinoblastoma protein; (iii) antisense inhibition of p27(Kip1) upregulation at high cell density in confluent-sensitive cells completely prevents the confluence-induced growth arrest; (iv) proper expression and/or membrane localization of E-cadherin is observed only in cells responsive to contact inhibition (TPC-1, NPA, WRO) but not in unresponsive cells (ARO, FRO or FB1); (v) disruption of E-cadherin-mediated cell-cell contacts at high cell density induced by an anti-E-cadherin neutralizing antibody, inhibits the induction of p27(kip1) and restores proliferation in contact-inhibited cells; (vi) re-expression of E-cadherin into cells unresponsive to contact inhibition (ARO, FB1) induces a p27(kip1) expression and growth arrest. In summary, our data indicate that the altered response to contact inhibition exhibited by thyroid anaplastic cancer cells is due to the failure to upregulate p27(Kip1) in response to cell-cell interactions.

Background: Vascular endothelial growth factor (VEGF) is involved in angiogenesis. We investigated the association of VEGF serum levels (pre-treatment and follow-up) with outcome in patients with melanoma., Patients and Methods: Serum levels of VEGF in melanoma patients at diagnosis and during follow-up were analysed with enzyme-linked immunoassays. Patients were followed up with physical examination and ultrasound scans of the liver every three months and thorax X-ray annually. The VEGF serum level was evaluated six-monthly., Results: From February 1996 to February 2000, 33 patients were enrolled. Ninety-two serum blood samples were collected. Patients had a median age of 60 years (range 32-82). Twenty patients were males, 13 females. One patient presented with stage IA disease, 2 with stage IB, 11 with stage IIA, 4 with stage IIB, 8 with stage III and 5 with stage IV. Two patients were affected by uveal melanoma. The melanomas were predominantly located at the extremities or trunk (26/33). The median serum level of VEGF at diagnosis was 249 ng/ml (minimum: 9 ng/ml, maximum: 1215 ng/ml). The median survival of all 33 patients was 45.1 months. The median time-to-progression was 36.7 months. Patients with lower or higher serum VEGF values showed no statistically significant differences in survival. In contrast, high serum VEGF values were associated with shorter disease-free survival as compared with lower values (median DFS: 25 vs 60 months, p = 0.048 at log-rank test)., Conclusion: Our results suggest that serum VEGF could be of prognostic value in melanoma.

Purpose: Factors that are predictive of prognosis in patients who are diagnosed with malignant melanoma (MM) are widely awaited. Detection of circulating melanoma cells (CMCs) by reverse transcriptase-polymerase chain reaction (RT-PCR) has recently been postulated as a possible negative prognostic factor. Two main questions were addressed: first, whether the presence of CMCs, defined as the patient being positive for any of the three markers, had a prognostic role; and second, what the predictive value of each individual marker was., Patients and Methods: A consecutive series of 200 melanoma patients observed between January 1997 and December 1997, with stage of disease ranging from I to IV, was analyzed by semiquantitative RT-PCR. Tyrosinase, p97, and MelanA/MART1 were used as markers to CMCs on baseline peripheral blood samples. Progression-free survival (PFS) was used as a unique end point and was described by the product limit method. Multivariable analysis was applied to verify whether the auspicated prognostic value of these markers was independent of the stage of disease, and a subgroup analysis was performed that excluded patients with stage IV disease., Results: Overall, 32% (64 of 200) of patients progressed, and a median PFS of 52 months in the whole series was observed. The presence of CMCs and the markers individually or combined was predictive of prognosis in the univariate analysis but did not provide additional prognostic information to the stage of disease in multivariable models. In the subgroup analysis of stage (ie, I-III subgroup), similar results were observed., Conclusion: Detection of CMCs in peripheral blood samples at the time of MM diagnosis by semiquantitative RT-PCR does not add any significant predictive value to the stage of disease. Thus, this approach should not be used in clinical practice, and further studies are required to determine its usefulness.

Growth factors of the glial cell line-derived neurotrophic factor (GDNF) family control the differentiation of neuronal cells of the central and peripheral nervous systems. Intracellular signalling of these growth factors is, at least in part, mediated by activation of the RET receptor tyrosine kinase. Here, we demonstrate that GDNF triggering inhibits the proliferation of the embryonal carcinoma cell line NT2/D1. This anti-proliferative effect is accompanied by down-regulation of the SSEA-3 antigen, a marker typical of undifferentiated NT2/D1 cells. We show that these effects are mediated by activation of RET signalling. The block of RET by a kinase-deficient dominant negative mutant impairs GDNF-dependent growth inhibition, whereas the adoptive expression of a constitutively active RET, the RET-MEN2A oncogene, promotes effects similar to those exerted by GDNF. We show that RET signalling increases the expression of the cyclin-dependent kinase inhibitor p27(kip1) in NT2/D1 cells. Both DNA synthesis inhibition and SSEA-3 down-regulation are prevented if p27(kip1) expression is blocked by an antisense construct, which demonstrates that RET-triggered effects are mediated by p27(kip1).

Oncogenic activation of the RET receptor tyrosine kinase is common in different human cancers. We found that the pyrazolo-pyrimidine PP1 inhibited RET-derived oncoproteins with a half maximal inhibitor concentration of 80 nM. Furthermore, RET/PTC3-transformed cells treated with 5 microM of PP1 lost proliferative autonomy and showed morphological reversion. PP1 prevented the growth of two human papillary thyroid carcinoma cell lines that carry spontaneous RET/PTC1 rearrangements and blocked anchorage-independent growth and tumorigenicity in nude mice of NIH3T3 fibroblasts expressing the RET/PTC3 oncogene. These findings suggest targeting RET oncogenes with PP1 or related compounds as a novel treatment strategy for RET-associated neoplasms.