Endostatin gene therapy stimulates upregulation of ICAM-1 and VCAM-1 in a metastatic renal cell carcinoma model

One of the greatest challenges in urological oncology is renal cell carcinoma (RCC), which is the third leading cause of death in genitourinary cancers. RCCs are highly vascularized and respond positively to antiangiogenic therapy. Endostatin (ES) is a fragment of collagen XVIII that possesses antiangiogenic activity. In this study, we examined the potential of ES-based antiangiogenic therapy to activate tumor-associated endothelial cells in metastatic RCC (mRCC). Balb/c-bearing Renca cells were treated with NIH/3T3-LendSN or, as a control, with NIH/3T3-LXSN cells. The T-cell subsets and lymphocyte populations of tumors, mediastinal lymph nodes and the spleen were assessed by flow cytometry. The expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) was assessed by real-time PCR, flow cytometry and immunohistochemistry analysis. ES gene therapy led to an increase in the percentage of infiltrating CD4-interferon (IFN)-gamma cells (P<0.05), CD8-IFN-gamma cells (P<0.01) and CD49b-tumor necrosis factor-alpha cells (P<0.01). In addition, ES therapy caused an increase at the mRNA level of ICAM-1 (1.4-fold; P<0.01) and VCAM-1 (1.5-fold) (control vs treated group; P<0.001). Through flow cytometry, we found a significant increase in the CD34/ICAM-1 cells (8.1-fold; P<0.001) and CD34/VCAM-1 cells (1.6-fold; P<0.05). ES gene therapy induced a significant increase in both T CD4 and CD8 cells in the lymph nodes and the spleen, suggesting that ES therapy may facilitate cell survival or clonal expansion. CD49b cells were also present in increased quantities in all of these organs. In this study, we demonstrate an antitumor inflammatory effect of ES in an mRCC model, and this effect is mediated by an increase in ICAM-1 and VCAM-1 expression in tumor-associated endothelial cells.

Fibronectin expression is decreased in metastatic renal cell carcinoma following endostatin gene therapy

Tumor cells induce the disruption of homeostasis between cellular and extracellular compartments to favor tumor progression. The expression of fibronectin (FN), a matrix glycoprotein, is increased in several carcinoma cell types, including renal cell carcinoma (RCC). RCC are highly vascularized tumors and are often amenable to antiangiogenic therapy. Endostatin (ES) is a fragment of collagen XVIII that possesses antiangiogenic activity. In this study, we examined the modulation of FN gene expression by ES gene therapy in a murine metastatic renal cell carcinoma (mRCC) model. Balb/C mice bearing Renca cells were treated with NIH/3T3-LXSN cells or NIH/3T3-LendSN cells. At the end of the experiment, the ES serum levels were measured, and the FN gene expression was assessed using real-time PCR. The tissue FN was evaluated by western blotting and by immunofluorescence analysis. The ES serum levels in treated mice were higher than those in the control group (P < 0.05). ES treatment led to significant decreases at the FN mRNA (P < 0.001) and protein levels (P < 0.01). Here, we demonstrate the ES antitumor effect that is mediated by down-regulation of FN expression in mRCC. (C) 2012 Elsevier Masson SAS. All rights reserved.

Expression analysis and in silico characterization of intronic long noncoding RNAs in renal cell carcinoma: emerging functional associations

Abstract Background Intronic and intergenic long noncoding RNAs (lncRNAs) are emerging gene expression regulators. The molecular pathogenesis of renal cell carcinoma (RCC) is still poorly understood, and in particular, limited studies are available for intronic lncRNAs expressed in RCC Methods Microarray experiments were performed with custom-designed arrays enriched with probes for lncRNAs mapping to intronic genomic regions. Samples from 18 primary RCC tumors and 11 nontumor adjacent matched tissues were analyzed. Meta-analyses were performed with microarray expression data from three additional human tissues (normal liver, prostate tumor and kidney nontumor samples), and with large-scale public data for epigenetic regulatory marks and for evolutionarily conserved sequences. Results A signature of 29 intronic lncRNAs differentially expressed between RCC and nontumor samples was obtained (false discovery rate (FDR) <5%). A signature of 26 intronic lncRNAs significantly correlated with the RCC five-year patient survival outcome was identified (FDR <5%, p-value ≤0.01). We identified 4303 intronic antisense lncRNAs expressed in RCC, of which 22% were significantly (p <0.05) cis correlated with the expression of the mRNA in the same locus across RCC and three other human tissues. Gene Ontology (GO) analysis of those loci pointed to 'regulation of biological processes’ as the main enriched category. A module map analysis of the protein-coding genes significantly (p <0.05) trans correlated with the 20% most abundant lncRNAs, identified 51 enriched GO terms (p <0.05). We determined that 60% of the expressed lncRNAs are evolutionarily conserved. At the genomic loci containing the intronic RCC-expressed lncRNAs, a strong association (p <0.001) was found between their transcription start sites and genomic marks such as CpG islands, RNA Pol II binding and histones methylation and acetylation. Conclusion Intronic antisense lncRNAs are widely expressed in RCC tumors. Some of them are significantly altered in RCC in comparison with nontumor samples. The majority of these lncRNAs is evolutionarily conserved and possibly modulated by epigenetic modifications. Our data suggest that these RCC lncRNAs may contribute to the complex network of regulatory RNAs playing a role in renal cell malignant transformation.

High-throughput sequencing of small RNA transcriptomes reveals critical biological features targeted by microRNAs in cell models used for squamous cell cancer research

Abstract Background The implication of post-transcriptional regulation by microRNAs in molecular mechanisms underlying cancer disease is well documented. However, their interference at the cellular level is not fully explored. Functional in vitro studies are fundamental for the comprehension of their role; nevertheless results are highly dependable on the adopted cellular model. Next generation small RNA transcriptomic sequencing data of a tumor cell line and keratinocytes derived from primary culture was generated in order to characterize the microRNA content of these systems, thus helping in their understanding. Both constitute cell models for functional studies of microRNAs in head and neck squamous cell carcinoma (HNSCC), a smoking-related cancer. Known microRNAs were quantified and analyzed in the context of gene regulation. New microRNAs were investigated using similarity and structural search, ab initio classification, and prediction of the location of mature microRNAs within would-be precursor sequences. Results were compared with small RNA transcriptomic sequences from HNSCC samples in order to access the applicability of these cell models for cancer phenotype comprehension and for novel molecule discovery. Results Ten miRNAs represented over 70% of the mature molecules present in each of the cell types. The most expressed molecules were miR-21, miR-24 and miR-205, Accordingly; miR-21 and miR-205 have been previously shown to play a role in epithelial cell biology. Although miR-21 has been implicated in cancer development, and evaluated as a biomarker in HNSCC progression, no significant expression differences were seen between cell types. We demonstrate that differentially expressed mature miRNAs target cell differentiation and apoptosis related biological processes, indicating that they might represent, with acceptable accuracy, the genetic context from which they derive. Most miRNAs identified in the cancer cell line and in keratinocytes were present in tumor samples and cancer-free samples, respectively, with miR-21, miR-24 and miR-205 still among the most prevalent molecules at all instances. Thirteen miRNA-like structures, containing reads identified by the deep sequencing, were predicted from putative miRNA precursor sequences. Strong evidences suggest that one of them could be a new miRNA. This molecule was mostly expressed in the tumor cell line and HNSCC samples indicating a possible biological function in cancer. Conclusions Critical biological features of cells must be fully understood before they can be chosen as models for functional studies. Expression levels of miRNAs relate to cell type and tissue context. This study provides insights on miRNA content of two cell models used for cancer research. Pathways commonly deregulated in HNSCC might be targeted by most expressed and also by differentially expressed miRNAs. Results indicate that the use of cell models for cancer research demands careful assessment of underlying molecular characteristics for proper data interpretation. Additionally, one new miRNA-like molecule with a potential role in cancer was identified in the cell lines and clinical samples.

Carvedilol protects against apoptotic cell death induced by cisplatin in renal tubular epithelial cells

Cisplatin is a highly effective chemotherapeutic drug; however, its use is limited by nephrotoxicity. Studies showed that the renal injury produced by cisplatin involves oxidative stress and cell death mediated by apoptosis and necrosis in proximal tubular cells. The use of antioxidants to decrease cisplatin-induced renal cell death was suggested as a potential therapeutic measure. In this study the possible protective effects of carvedilol, a beta blocker with antioxidant activity, was examined against cisplatin-induced apoptosis in HK-2 human kidney proximal tubular cells. The mitochondrial events involved in this protection were also investigated. Four groups were used: controls (C), cisplatin alone at 25 mu M (CIS), cisplatin 25 mu M plus carvedilol 50 mu M (CV + CIS), and carvedilol alone 50 mu M (CV). Cell viability, apoptosis, caspase-9, and caspase-3 were determined. Data demonstrated that carvedilol effectively increased cell viability and minimized caspase activation and apoptosis in HK-2 cells, indicating this may be a promising drug to reduce nephrotoxicity induced by cisplatin.

Cediranib: a VEGF receptor tyrosine kinase inhibitor

Cediranib is a potent inhibitor of the VEGF family receptor tyrosine kinases, and a new agent in cancer treatment. The drug has shown promising activity in a variety of solid malignancies, in preclinical models and in clinical trials. Its pharmacokinetics allow for a convenient once-daily administration, with a toxicity profile that is very similar to other VEGF inhibitors. Its main side effects include hypertension, nausea, dysphonia, fatigue and diarrhea. Adverse events seem to be manageable, especially when used in doses lower than 45 mg/day. Studies have shown some activity as a single agent or in combination in advanced tumors, but not enough to secure its approval for routine use up to now. Clinical trials are still evaluating the role of cediranib in combination chemotherapy with cytotoxic agents.

Moxonidine into the lateral parabrachial nucleus reduces renal and hormonal responses to cell dehydration

The deactivation of the inhibitory mechanisms with injections of moxonidine (alpha(2)-adrenoceptor/imidazoline receptor agonist) into the lateral parabrachial nucleus (LPBN) increases hypertonic NaCl intake by intra- or extracellular dehydrated rats. In the present study, we investigated the changes in the urinary sodium and volume, sodium balance, and plasma vasopressin and oxytocin in rats treated with intragastric (i.g.) 2 M NaCl load (2 ml/rat) combined with injections of moxonidine into the LPBN. Male Holtzman rats (n=5-12/group) with stainless steel cannulas implanted bilaterally into LPBN were used. Bilateral injections of moxonidine (0.5 nmol/0.2 mu l) into the LPBN decreased i.g. 2 M NaCIinduced diuresis (4.6 +/- 0.7 vs. vehicle: 7.4 +/- 0.6 ml/120 min) and natriuresis (1.65 +/- 0.29 vs. vehicle: 2.53 +/- 0.17 mEq/120 min), whereas the previous injection of the alpha(2)-adrenoceptor antagonist RX 821002 (10 nmol/0.2 mu l) into the LPBN abolished the effects of moxonidline. Moxonidine injected into the LPBN reduced i.g. 2 M NaCl-induced increase in plasma oxytocin and vasopressin (14.6 +/- 2.8 and 2.2 +/- 0.3 vs. vehicle: 25.7 +/- 7 and 4.3 +/- 0.7 pg/ml, respectively). Moxonidine injected into the LPBN combined with i.g. 2 M NaCl also increased 0.3 M NaCl intake (7.5 +/- 1.7 vs. vehicle: 0.5 +/- 0.2 mEq/2 h) and produced positive sodium balance (2.3 +/- 1.4 vs. vehicle: -1.2 +/- 0.4 mEq/2 h) in rats that had access to water and NaCl. The present results show that LPBN alpha(2)-adrenoceptor activation reduces renal and hormonal responses to intracellular dehydration and increases sodium and water intake, which facilitates sodium retention and body fluid volume expansion. (C) 2012 IBRO. Published by Elsevier Ltd. All rights reserved.

Antifibrotic Effect of Tamoxifen in a Model of Progressive Renal Disease

Tamoxifen, a selective estrogen receptor modulator, has antifibrotic properties; however, whether it can attenuate renal fibrosis is unknown. In this study, we tested the effects of tamoxifen in a model of hypertensive nephrosclerosis (chronic inhibition of nitric oxide synthesis with L-NAME). After 30 days, treated rats had significantly lower levels of albuminuria as well as lower histologic scores for glomerulosclerosis and interstitial fibrosis than untreated controls. Tamoxifen was renoprotective despite having no effect on the sustained, severe hypertension induced by L-NAME. Tamoxifen prevented the accumulation of extracellular matrix by decreasing the expression of collagen I, collagen III, and fibronectin mRNA and protein. These renoprotective effects associated with inhibition of TGF-beta 1 and plasminogen activator inhibitor-1, and with a significant reduction in a-smooth muscle actin-positive cells in the renal interstitium. Furthermore, tamoxifen abrogated IL-1 beta- and angiotensin-II-induced proliferation of fibroblasts from both kidney explants and from the NRK-49F cell line. Tamoxifen also inhibited the expression of extracellular matrix components and the production and release of TGF-beta 1 into the supernatant of these cells. In summary, tamoxifen exhibits antifibrotic effects in the L-NAME model of hypertensive nephrosclerosis, likely through the inhibition of TGF-beta 1, suggesting that it may have therapeutic use in CKD treatment.

Differential expression of CD90 and CD14 stem cell markers in malignant breast cancer cell lines

The recently emerged concept of cancer stem cell (CSC) has led to a new hypothesis on the basis for tumor progression. Basically, the CSC theory hypothesizes the presence of a hierarchically organized and relatively rare cell population, which is responsible for tumor initiation, self-renewal, and maintenance, in addition to accumulation of mutation and resistance to chemotherapy. CSCs have recently been described in breast cancer. Different genetic markers have been used to isolate breast CSCs, none of which have been correlated with the tumorigenicity or metastatic potential of the cells, limiting their precise characterization and clinical application in the development of therapeutic protocols. Here, we sought for subpopulations of CSCs by analyzing 10 judiciously chosen stem cell markers in a normal breast cell line (MCF10-A) and in four human breast cancer cell lines (MCF-7, MDA-MB-231, MDA-MB-435, and Hs578-T) displaying different degrees of metastatic and invasiveness potential. We were able to identify two markers, which are differentially expressed in nontumorigenic versus tumor cells. The CD90 marker was highly expressed in the malignant cell lines. Interestingly, the CD14 molecule displayed higher expression levels in the nontumorigenic cell line. Therefore, we demonstrated that these two markers, which are more commonly used to isolate and characterize stem cells, are differentially expressed in breast tumor cells, when compared with nontumorigenic breast cells. (C) 2012 International Society for Advancement of Cytometry

Angiotensin II Facilitates Breast Cancer Cell Migration and Metastasis

Breast cancer metastasis is a leading cause of death by malignancy in women worldwide. Efforts are being made to further characterize the rate-limiting steps of cancer metastasis, i.e. extravasation of circulating tumor cells and colonization of secondary organs. In this study, we investigated whether angiotensin II, a major vasoactive peptide both produced locally and released in the bloodstream, may trigger activating signals that contribute to cancer cell extravasation and metastasis. We used an experimental in vivo model of cancer metastasis in which bioluminescent breast tumor cells (D3H2LN) were injected intra-cardiacally into nude mice in order to recapitulate the late and essential steps of metastatic dissemination. Real-time intravital imaging studies revealed that angiotensin II accelerates the formation of metastatic foci at secondary sites. Pre-treatment of cancer cells with the peptide increases the number of mice with metastases, as well as the number and size of metastases per mouse. In vitro, angiotensin II contributes to each sequential step of cancer metastasis by promoting cancer cell adhesion to endothelial cells, trans-endothelial migration and tumor cell migration across extracellular matrix. At the molecular level, a total of 102 genes differentially expressed following angiotensin II pretreatment were identified by comparative DNA microarray. Angiotensin II regulates two groups of connected genes related to its precursor angiotensinogen. Among those, up-regulated MMP2/MMP9 and ICAM1 stand at the crossroad of a network of genes involved in cell adhesion, migration and invasion. Our data suggest that targeting angiotensin II production or action may represent a valuable therapeutic option to prevent metastatic progression of invasive breast tumors.

Topoisomerase expression in oral squamous cell carcinoma: relationship with cancer stem cells profiles and lymph node metastasis

BACKGROUND: The relationship between predictive proteins and tumors presenting cancer stem cells (CSCs) profiles in oral tumors is still poorly understood. This study aims to identify the relationship between topoisomerases I, II alpha, and III alpha and putative CSCs immunophenotype in oral squamous cell carcinoma (OSCC) and determine its influence on prognosis. METHODS: The following data were retrieved from 127 patients: age, gender, primary anatomic site, smoking and alcohol intake, recurrence, metastases, histologic classification, treatment, and survival. An immunohistochemical study for topoisomerases I, II alpha, and III alpha was performed in a tissue microarray containing 127 paraffin blocks of OSCCs. RESULTS: In univariate analysis, topoisomerases expression showed significant differences according to CSCs profiles and p53 immunoexpression, but not with survival. Topoisomerases II alpha and III alpha also showed significant relationship with lymph node metastasis. The multivariate test confirmed these associations. CONCLUSIONS: The results that all topoisomerases correlates with OSCC CSCs may indicate a role for topoisomerases in head and neck carcinogenesis. Notwithstanding, it is plausible that other members of topoisomerases family could represent novel therapeutical targets in oral squamous cell carcinoma. J Oral Pathol Med (2012) 41: 762-768

Expression of cancer-testis antigens MAGE-A4 and MAGE-C1 in oral squamous cell carcinoma

Background Tumor markers are genes or their products expressed exclusively or preferentially in tumor cells and cancer-testis antigens (CTAs) form a group of genes with a typical expression pattern expressed in a variety of malignant neoplasms. CTAs are considered potential targets for cancer vaccines. It is possible that the CTA MAGE-A4 (melanoma antigen) and MAGE-C1 are expressed in carcinoma of the oral cavity and are related with survival. Methods This study involved immunohistochemical analysis of 23 patients with oral squamous cell carcinoma (SCC) and was carried out using antibodies for MAGE-A4 and MAGE-C1. Fisher's exact test and log-rank test were used to evaluate the results. Results The expression of the MAGE-A4 and MAGE-C1 were 56.5% and 47.8% without statistical difference in studied variables and survival. Conclusion The expression of at least 1 CTA was present in 78.3% of the patients, however, without correlation with clinicopathologic variables and survival. (c) 2011 Wiley Periodicals, Inc. Head Neck, 2012

Preserving the B-Cell Compartment Favors Operational Tolerance in Human Renal Transplantation

Transplanted individuals in operational tolerance (OT) maintain long-term stable graft function after completely stopping immunosuppression. Understanding the mechanisms involved in OT can provide valuable information about pathways to human transplantation tolerance. Here we report that operationally tolerant individuals display quantitative and functional preservation of the B-c ell compartment in renal transplantation. OT exhibited normal numbers of circulating total B cells, naive, memory and regulatory B cells (Bregs) as well as preserved B-cell receptor repertoire, similar to healthy individuals. In addition, OT also displayed conserved capacity to activate the cluster of differentiation 40 (CD40)/signal transducer and activator of transcription 3 (STAT3) signaling pathway in Bregs, in contrast, with chronic rejection. Rather than expansion or higher activation, we show that the preservation of the B-cell compartment favors OT. Online address: http://www.molmed.org doi: 10.2119/molmed.2011.00281

Adipose Tissue-Derived Stem Cell Treatment Prevents Renal Disease Progression

Adipose tissue-derived stem cells (ASCs) are an attractive source of stem cells with regenerative properties that are similar to those of bone marrow stem cells. Here, we analyze the role of ASCs in reducing the progression of kidney fibrosis. Progressive renal fibrosis was achieved by unilateral clamping of the renal pedicle in mice for 1 h; after that, the kidney was reperfused immediately. Four hours after the surgery, 2 x 10(5) ASCs were intraperitoneally administered, and mice were followed for 24 h posttreatment and then at some other time interval for the next 6 weeks. Also, animals were treated with 2 x 10(5) ASCs at 6 weeks after reperfusion and sacrificed 4 weeks later to study their effect when interstitial fibrosis is already present. At 24 h after reperfusion, ASC-treated animals showed reduced renal dysfunction and enhanced regenerative tubular processes. Renal mRNA expression of IL-6 and TNF was decreased in ASC-treated animals, whereas IL-4. IL-10, and HO-1 expression increased despite a lack of ASCs in the kidneys as determined by SRY analysis. As expected, untreated kidneys shrank at 6 weeks, whereas the kidneys of ASC-treated animals remained normal in size, showed less collagen deposition, and decreased staining for FSP-1, type I collagen, and Hypoxyprobe. The renal protection seen in ASC-treated animals was followed by reduced serum levels of TNF-alpha, KC, RANTES, and IL-1 alpha. Surprisingly, treatment with ASCs at 6 weeks, when animals already showed installed fibrosis, demonstrated amelioration of functional parameters, with less tissue fibrosis observed and reduced mRNA expression of type I collagen and vimentin. ASC therapy can improve functional parameters and reduce progression of renal fibrosis at early and later times after injury, mostly due to early modulation of the inflammatory response and to less hypoxia, thereby reducing the epithelial-mesenchymal transition.

Cancer cell proliferation is inhibited by specific modulation frequencies

BACKGROUND: There is clinical evidence that very low and safe levels of amplitude-modulated electromagnetic fields administered via an intrabuccal spoon-shaped probe may elicit therapeutic responses in patients with cancer. However, there is no known mechanism explaining the anti-proliferative effect of very low intensity electromagnetic fields. METHODS: To understand the mechanism of this novel approach, hepatocellular carcinoma (HCC) cells were exposed to 27.12 MHz radiofrequency electromagnetic fields using in vitro exposure systems designed to replicate in vivo conditions. Cancer cells were exposed to tumour-specific modulation frequencies, previously identified by biofeedback methods in patients with a diagnosis of cancer. Control modulation frequencies consisted of randomly chosen modulation frequencies within the same 100 Hz-21 kHz range as cancer-specific frequencies. RESULTS: The growth of HCC and breast cancer cells was significantly decreased by HCC-specific and breast cancer-specific modulation frequencies, respectively. However, the same frequencies did not affect proliferation of nonmalignant hepatocytes or breast epithelial cells. Inhibition of HCC cell proliferation was associated with downregulation of XCL2 and PLP2. Furthermore, HCC-specific modulation frequencies disrupted the mitotic spindle. CONCLUSION: These findings uncover a novel mechanism controlling the growth of cancer cells at specific modulation frequencies without affecting normal tissues, which may have broad implications in oncology. British Journal of Cancer (2012) 106, 307-313. doi:10.1038/bjc.2011.523 www.bjcancer.com Published online 1 December 2011 (C) 2012 Cancer Research UK