A protein kinase Cβ inhibitor attenuates multidrug resistance of neuroblastoma cells

BACKGROUND: The acquisition of drug resistance is a major reason for poor outcome of neuroblastoma. Protein kinase C (PKC) has been suggested to influence drug resistance in cancer cells. The aim of this study was to elucidate whether inhibition of PKCβ isoforms influences drug-resistance of neuroblastoma cells. METHODS: The effect of the PKCβ inhibitor LY379196 on the growth-suppressing effects of different chemotherapeutics on neuroblastoma cells was analyzed with MTT assays. The effect of LY379196 on the accumulation of [(3)H]vincristine was also investigated RESULTS: The PKCβ inhibitor LY379196 suppressed the growth of three neuroblastoma cell lines. LY379196 also augmented the growth-suppressive effect of doxorubicin, etoposide, paclitaxel, and vincristine, but not of carboplatin. The effect was most marked for vincristine and for the cell-line (SK-N-BE(2)) that was least sensitive to vincristine. No effect was observed on the non-resistant IMR-32 cells. Two other PKC inhibitors, Gö6976 and GF109203X, also enhanced the vincristine effect. The PKC inhibitors caused an increased accumulation of [(3)H]vincristine in SK-N-BE(2) cells. CONCLUSIONS: This indicates that inhibition of PKCβ could attenuate multidrug resistance in neuroblastoma cells by augmenting the levels of natural product anticancer drugs in resistant cells

The use of cystatin C to inhibit epithelial–mesenchymal transition and morphological transformation stimulated by transforming growth factor-β

INTRODUCTION: Transforming growth factor-β (TGF-β) is a potent suppressor of mammary epithelial cell (MEC) proliferation and is thus an inhibitor of mammary tumor formation. Malignant MECs typically evolve resistance to TGF-β-mediated growth arrest, enhancing their proliferation, invasion, and metastasis when stimulated by TGF-β. Recent findings suggest that therapeutics designed to antagonize TGF-β signaling may alleviate breast cancer progression, thereby improving the prognosis and treatment of breast cancer patients. We identified the cysteine protease inhibitor cystatin C (CystC) as a novel TGF-β type II receptor antagonist that inhibits TGF-β binding and signaling in normal and cancer cells. We hypothesized that the oncogenic activities of TGF-β, particularly its stimulation of mammary epithelial–mesenchymal transition (EMT), can be prevented by CystC. METHOD: Retroviral infection was used to constitutively express CystC or a CystC mutant impaired in its ability to inhibit cathepsin protease activity (namely Δ14CystC) in murine NMuMG MECs and in normal rat kidney (NRK) fibroblasts. The effect of recombinant CystC administration or CystC expression on TGF-β stimulation of NMuMG cell EMT in vitro was determined with immunofluorescence to monitor rearrangements of actin cytoskeletal architecture and E-cadherin expression. Soft-agar growth assays were performed to determine the effectiveness of CystC in preventing TGF-β stimulation of morphological transformation and anchorage-independent growth in NRK fibroblasts. Matrigel invasion assays were performed to determine the ability of CystC to inhibit NMuMG and NRK motility stimulated by TGF-β. RESULTS: CystC and Δ14CystC both inhibited NMuMG cell EMT and invasion stimulated by TGF-β by preventing actin cytoskeletal rearrangements and E-cadherin downregulation. Moreover, both CystC molecules completely antagonized TGF-β-mediated morphological transformation and anchorage-independent growth of NRK cells, and inhibited their invasion through synthetic basement membranes. Both CystC and Δ14CystC also inhibited TGF-β signaling in two tumorigenic human breast cancer cell lines. CONCLUSION: Our findings show that TGF-β stimulation of initiating metastatic events, including decreased cell polarization, reduced cell–cell contact, and elevated cell invasion and migration, are prevented by CystC treatment. Our findings also suggest that the future development of CystC or its peptide mimetics hold the potential to improve the therapeutic response of human breast cancers regulated by TGF-β

Integrin α5β1 Function Is Regulated by XGIPC/kermit2 Mediated Endocytosis during Xenopus laevis Gastrulation

During Xenopus gastrulation α5β1 integrin function is modulated in a temporally and spatially restricted manner, however, the regulatory mechanisms behind this regulation remain uncharacterized. Here we report that XGIPC/kermit2 binds to the cytoplasmic domain of the α5 subunit and regulates the activity of α5β1 integrin. The interaction of kermit2 with α5β1 is essential for fibronectin (FN) matrix assembly during the early stages of gastrulation. We further demonstrate that kermit2 regulates α5β1 integrin endocytosis downstream of activin signaling. Inhibition of kermit2 function impairs cell migration but not adhesion to FN substrates indicating that integrin recycling is essential for mesoderm cell migration. Furthermore, we find that the α5β1 integrin is colocalized with kermit2 and Rab 21 in embryonic and XTC cells. These data support a model where region specific mesoderm induction acts through kermit2 to regulate the temporally and spatially restricted changes in adhesive properties of the α5β1 integrin through receptor endocytosis

Therapeutic Implications of GIPC1 Silencing in Cancer

GIPC1 is a cytoplasmic scaffold protein that interacts with numerous receptor signaling complexes, and emerging evidence suggests that it plays a role in tumorigenesis. GIPC1 is highly expressed in a number of human malignancies, including breast, ovarian, gastric, and pancreatic cancers. Suppression of GIPC1 in human pancreatic cancer cells inhibits in vivo tumor growth in immunodeficient mice. To better understand GIPC1 function, we suppressed its expression in human breast and colorectal cancer cell lines and human mammary epithelial cells (HMECs) and assayed both gene expression and cellular phenotype. Suppression of GIPC1 promotes apoptosis in MCF-7, MDA-MD231, SKBR-3, SW480, and SW620 cells and impairs anchorage-independent colony formation of HMECs. These observations indicate GIPC1 plays an essential role in oncogenic transformation, and its expression is necessary for the survival of human breast and colorectal cancer cells. Additionally, a GIPC1 knock-down gene signature was used to interrogate publically available breast and ovarian cancer microarray datasets. This GIPC1 signature statistically correlates with a number of breast and ovarian cancer phenotypes and clinical outcomes, including patient survival. Taken together, these data indicate that GIPC1 inhibition may represent a new target for therapeutic development for the treatment of human cancers

Impact of TGF-ß1 -509C/T and 869T/C polymorphisms on glioma risk and patient prognosis

Transforming growth factor beta (TGF-ß) plays an important role in carcinogenesis. Two polymorphisms in the TGF-ß1 gene (-509C/T and 869T/C) were described to influence susceptibility to gastric and breast cancers. The 869T/C polymorphism was also associated with overall survival in breast cancer patients. In the present study, we investigated the relevance of these TGF-ß1 polymorphism in glioma risk and prognosis. A case-control study that included 114 glioma patients and 138 cancer-free controls was performed. Single nucleotide polymorphisms (SNPs) were evaluated by polymerase chain reaction followed by restriction fragment length polymorphism (PCR-RFLP). Univariate and multivariate logistic regression analyses were used to calculate odds ratio (OR) and 95 % confidence intervals (95 % CI). The influence of TGF-ß1 -509C/T and 869T/C polymorphisms on glioma patient survival was evaluated by a Cox regression model adjusted for patients' age and sex and represented in Kaplan-Meier curves. Our results demonstrated that TGF-ß1 gene polymorphisms -509C/T and 869T/C are not significantly associated with glioma risk. Survival analyses showed that the homozygous -509TT genotype associates with longer overall survival of glioblastoma (GBM) patients when compared with patients carrying CC + CT genotypes (OR, 2.41; 95 % CI, 1.06-5.50; p = 0.036). In addition, the homozygous 869CC genotype is associated with increased overall survival of GBM patients when compared with 869TT + TC genotypes (OR, 2.62; 95 % CI, 1.11-6.17; p = 0.027). In conclusion, this study suggests that TGF-ß1 -509C/T and 869T/C polymorphisms are not significantly associated with risk for developing gliomas but may be relevant prognostic biomarkers in GBM patients.This work was supported by Fundação para a Ciência e Tecnologia, Portugal (PTDC/SAU-GMG/113795/2009 and SFRH/BPD/33612/2009 to B.M.C.; SFRH/BD/88121/2012 to J.V.C.; SFRH/BD/92786/2013 to C.S.G.; PTDC/SAU-ONC/115513/2009 to R.R.)

Induction of transforming growth factor beta receptors following focal ischemia in the rat brain

Transforming growth factor-βs (TGF-βs) regulate cellular proliferation, differentiation, and survival. TGF-βs bind to type I (TGF-βRI) and II receptors (TGF-βRII), which are transmembrane kinase receptors, and an accessory type III receptor (TGF-βRIII). TGF-β may utilize another type I receptor, activin-like kinase receptor (Alk1). TGF-β is neuroprotective in the middle cerebral artery occlusion (MCAO) model of stroke. Recently, we reported the expression pattern of TGF-β1-3 after MCAO. To establish how TGF-βs exert their actions following MCAO, the present study describes the induction of TGF-βRI, RII, RIII and Alk1 at 24 h, 72 h and 1 mo after transient 1 h MCAO as well as following 24 h permanent MCAO using in situ hybridization histochemistry. In intact brain, only TGF-βRI had significant expression: neurons in cortical layer IV contained TGF-βRI. At 24 h after the occlusion, no TGF-β receptors showed induction. At 72 h following MCAO, all four types of TGF-β receptors were induced in the infarct area, while TGF-βRI and RII also appeared in the penumbra. Most cells with elevated TGF-βRI mRNA levels were microglia. TGF-βRII co-localized with both microglial and endothelial markers while TGF-βRIII and Alk1 were present predominantly in endothels. All four TGF-β receptors were induced within the lesion 1 mo after the occlusion. In particular, TGF-βRIII was further induced as compared to 72 h after MCAO. At this time point, TGF-βRIII signal was predominantly not associated with blood vessels suggesting its microglial location. These data suggest that TGF-β receptors are induced after MCAO in a timely and spatially regulated fashion. TGF-β receptor expression is preceded by increased TGF-β expression. TGF-βRI and RII are likely to be co-expressed in microglial cells while Alk1, TGF-βRII, and RIII in endothels within the infarct where TGF-β1 may be their ligand. At later time points, TGF-βRIII may also appear in glial cells to potentially affect signal transduction via TGF-βRI and RII

Cytokine Plasma Levels: Reliable Predictors for Radiation Pneumonitis?

BACKGROUND: Radiotherapy (RT) is the primary treatment modality for inoperable, locally advanced non-small-cell lung cancer (NSCLC), but even with highly conformal treatment planning, radiation pneumonitis (RP) remains the most serious, dose-limiting complication. Previous clinical reports proposed that cytokine plasma levels measured during RT allow to estimate the individual risk of patients to develop RP. The identification of such cytokine risk profiles would facilitate tailoring radiotherapy to maximize treatment efficacy and to minimize radiation toxicity. However, cytokines are produced not only in normal lung tissue after irradiation, but are also over-expressed in tumour cells of NSCLC specimens. This tumour-derived cytokine production may influence circulating plasma levels in NSCLC patients. The aim of the present study was to investigate the prognostic value of TNF-alpha, IL-1beta, IL-6 and TGF-beta1 plasma levels to predict radiation pneumonitis and to evaluate the impact of tumour-derived cytokine production on circulating plasma levels in patients irradiated for NSCLC. METHODOLOGY/PRINCIPAL FINDINGS: In 52 NSCLC patients (stage I-III) cytokine plasma levels were investigated by ELISA before and weekly during RT, during follow-up (1/3/6/9 months after RT), and at the onset of RP. Tumour biopsies were immunohistochemically stained for IL-6 and TGF-beta1, and immunoreactivity was quantified (grade 1-4). RP was evaluated according to LENT-SOMA scale. Tumour response was assessed according to RECIST criteria by chest-CT during follow-up. In our clinical study 21 out of 52 patients developed RP (grade I/II/III/IV: 11/3/6/1 patients). Unexpectedly, cytokine plasma levels measured before and during RT did not correlate with RP incidence. In most patients IL-6 and TGF-beta1 plasma levels were already elevated before RT and correlated significantly with the IL-6 and TGF-beta1 production in corresponding tumour biopsies. Moreover, IL-6 and TGF-beta1 plasma levels measured during follow-up were significantly associated with the individual tumour responses of these patients. CONCLUSIONS/SIGNIFICANCE: The results of this study did not confirm that cytokine plasma levels, neither their absolute nor any relative values, may identify patients at risk for RP. In contrast, the clear correlations of IL-6 and TGF-beta1 plasma levels with the cytokine production in corresponding tumour biopsies and with the individual tumour responses suggest that the tumour is the major source of circulating cytokines in patients receiving RT for advanced NSCLC

Pseudorapidity dependence of long-range two-particle correlations in pPb collisions at root sNN=5.02 TeV

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