Cancer/Testis Antigen Expression on Mesenchymal Stem Cells Isolated from Different Tissues

Background/Aims: The expression of cancer/testis antigens (CTAs) on additional normal tissues or stem cells may restrict their use as cancer targets. The objective of the present study was to evaluate the mRNA levels of some CTAs in a variety of tissues. Materials and Methods: mRNA of pericytes, fibroblasts and mesenchymal stem cells (MSCs) derived from adult and fetal tissues, human umbilical vein endothelial cells, MSC-derived adipocytes, selected normal tissues and control cancer cell lines (CLs) were extracted and quantitative polymerase chain reaction was performed for MAGED1, PRAME, CTAG1B, MAGEA3 and MAGEA4. Results: MAGED1 was expressed in all normal tissues and cells evaluated. CTAG1B was expressed at levels comparable to control CLs on MSCs derived from arterial, fetal skin, adipose tissue and saphenous vein, heart, brain and skin tissues. MAGEA4 was detected only in fibroblasts and differentiated adipocytes from MSCs, at levels comparable to the control CLs. Conclusion: The potential use of CTAs in immunotherapy should take into account the potential off-target effects on MSCs.

Low Expression of Human Histocompatibility Soluble Leukocyte Antigen-G (HLA-G5) in Invasive Cervical Cancer With and Without Metastasis, Associated With Papilloma Virus (HPV)

Human leukocyte antigen-G (HLA-G) is a non-classical major histocompatibility complex class lb molecule that acts as a specific immunosuppressor. Some studies have demonstrated that human papillomavirus (HPV) seems to be involved in lower or absent HLA-G expression, particularly in cervical cancer. In this study, we performed a cross-sectional study, systematically comparing the qualitative expression of the HLA-G5 isoform in invasive cervical carcinoma (ICC), stratifying patients according to the presence [ICC with metastasis (ICC(W))) and absence [ICC without metastasis (ICC(WT))] of metastasis, correlating these findings with interference of HPV and demographic and clinical variables. Seventy-nine patients with a diagnosis of ICC were stratified into two groups: ICC(WT) (n=52 patients) and ICC(W) (n=27). Two biopsies were collected from each patient (one from the tumor lesion and one from a lymph node). Immunohistochemistry analyses were performed for the HLA-G5 isoform, for HPV detection, and virus typing. HLA-G5 isoform molecules were detected in 25 cases (31.6%), 17 (32.7%) without metastasis and 8 (29.6%) with metastasis. HPV was detected in the cervical lesions of 74 patients (93.7%), but low expression of the HLA-G5 isoform was observed in all HPV-related cases. These findings are important; however, additional studies are necessary to identify the influence of HPV with HLA-G5 isoform expression on invasive cervical malignancies. (J Histochem Cytochem 58:405-411, 2010)

Granulocyte-Colony Stimulating Factor (G-CSF) induces mechanical hyperalgesia via spinal activation of MAP kinases and PI(3)K in mice

Granulocyte-colony stimulating factor (G-CSF) is a current pharmacological approach to increase peripheral neutrophil counts after anti-tumor therapies. Pain is most relevant side effect of G-CSF in healthy volunteers and cancer patients. Therefore, the mechanisms of G-CSF-induced hyperalgesia were investigated focusing on the role of spinal mitogen-activated protein (MAP) kinases ERK (extracellular signal-regulated kinase). JNK (Jun N-terminal Kinase) and p38, and PI(3)K (phosphatidylinositol 3-kinase). G-CSF induced dose (30-300 ng/paw)-dependent mechanical hyperalgesia, which was inhibited by local post-treatment with morphine. This effect of morphine was reversed by naloxone (opioid receptor antagonist). Furthermore, G-CSF-induced hyperalgesia was inhibited in a dose-dependent manner by intrathecal pre-treatment with ERK (PD98059), JNK (SB600125), p38 (SB202190) or PI(3)K (wortmanin) inhibitors. The co-treatment with MAP kinase and PI(3)K inhibitors, at doses that were ineffective as single treatment, significantly inhibited G-CSF-induced hyperalgesia. Concluding, in addition to systemic opioids, peripheral opioids as well as spinal treatment with MAP kinases and PI(3)K inhibitors also reduce G-CSF-induced pain. (C) 2011 Elsevier Inc. All rights reserved.

Gastroprotective effect of heme-oxygenase 1/biliverdin/CO pathway in ethanol-induced gastric damage in mice

Our objective was to evaluate the role of heme-oxygenase 1 (HO-1)/biliverdin/CO pathway in gastric defense against ethanol-induced gastric damage in mice. Mice were pre-treated with saline, hemin (HO-1 inducer), biliverdin (HO-1 product), dimanganese decacarbonyl (DMDC, CO donor) or zinc protoporphyrin IX (ZnPP IX, HO-1 antagonist). Another group received soluble guanylate cyclase (sGC) inhibitor (ODQ) 30 min before hemin, biliverdin or DMDC. After 30 min, gastric damage was induced by ethanol. After one hour, rats were sacrificed. Gastric lesions were measured using a computer planimetry program, and gastric corpus pieces were assayed for malonylaldehyde (MDA), glutathione (GSH) or bilirubin. HO-1 expression was determined after saline or ethanol administration by polymerase chain reaction (PCR) or immunohistochemistry. Ethanol (25% or 50%) induced gastric damage, increased MDA levels and reduced GSH in the gastric tissue. Ethanol 50% increased HO-1 mRNA transcripts, HO-1 immunoreactivity, and bilirubin concentration in gastric mucosa. Pre-treatment with hemin reduced gastric damage and MDA formation and increased GSH concentration in the gastric mucosa. ZnPP IX amplified the ethanol-induced gastric lesion, increased MDA formation and decreased GSH concentration in gastric mucosa. Biliverdin and DMDC reduced gastric damage and MDA formation and increased GSH concentration in the gastric tissue. ODQ completely abolished the DMDC protective gastric effect However, effects of hemin or biliverdin did not change with ODQ treatment. Our results suggest that HO-1/biliverdin/CO pathway plays a protective role against ethanol-induced gastric damage through mechanisms that can be dependent (CO) or independent (biliverdin) of sGC activation. (C) 2010 Elsevier B.V. All rights reserved.

Differential regulation of TRP channels in a rat model of neuropathic pain

Neuropathic pain is a chronic disease resulting from dysfunction of the nervous system often due to peripheral nerve injury. Hypersensitivity to sensory Stimuli (mechanical, thermal or chemical) is a common source of pain in patients and ion channels involved in detecting these Stimuli are possible candidates for inducing and/or maintaining the pain. Transient receptor potential (TRP) channels expressed on nociceptors respond to different sensory stimuli and a few of them have been studied previously in the models of neuropathic pain. Using real-time PCR for quantification of all known TRP channels we identified several TRP channels, which have not been associated with nociception OF neuropathic pain before, to be expressed in the DRG and to be differentially regulated after spared nerve injury (SNI). Of all TRP channel members, TRPML3 showed the most dramatic change in animals exhibiting neuropathic pain behaviour compared to control animals. fit situ hybridisation showed a widespread increase of expression ill neurons of small, medium and large cell sizes, indicating expression ill multiple subtypes. Co-localisation of TRPML3 with CGRP, NF200 and IB4 staining confirmed a broad Subtype distribution. Expression studies during development showed that TRPML3 is all embryonic channel that is induced upon nerve injury in three different nerve injury models investigated. Thus. the current results link for the first time a re-expression of TRPML3 with the development of neuropathic pain conditions. In addition, decreased mRNA levels after SNI were seen for TRPM6, TRPM8, TRPV1, TRPA1, TRPC3, TRPC4 and TRPC5. (C) 2009 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.

Distinct patterns of somatic alterations in a lymphoblastoid and a tumor genome derived from the same individual

Although patterns of somatic alterations have been reported for tumor genomes, little is known on how they compare with alterations present in non-tumor genomes. A comparison of the two would be crucial to better characterize the genetic alterations driving tumorigenesis. We sequenced the genomes of a lymphoblastoid (HCC1954BL) and a breast tumor (HCC1954) cell line derived from the same patient and compared the somatic alterations present in both. The lymphoblastoid genome presents a comparable number and similar spectrum of nucleotide substitutions to that found in the tumor genome. However, a significant difference in the ratio of non-synonymous to synonymous substitutions was observed between both genomes (P = 0.031). Protein-protein interaction analysis revealed that mutations in the tumor genome preferentially affect hub-genes (P = 0.0017) and are co-selected to present synergistic functions (P < 0.0001). KEGG analysis showed that in the tumor genome most mutated genes were organized into signaling pathways related to tumorigenesis. No such organization or synergy was observed in the lymphoblastoid genome. Our results indicate that endogenous mutagens and replication errors can generate the overall number of mutations required to drive tumorigenesis and that it is the combination rather than the frequency of mutations that is crucial to complete tumorigenic transformation.

DNA mismatch repair gene methylation in gastric cancer in individuals from northern Brazil

Gastric cancer is one of the most common malignancies. DNA methylation is implicated in DNA mismatch repair genes deficiency. In the present study, we evaluated the methylation status of MLH1, MSH2, MSH6 and PMS2 in 20 diffuse- and 26 intestinal-type gastric cancer samples and 20 normal gastric mucosal of gastric cancer patients from Northern Brazil. We found that none of the nonneoplastic samples showed methylation of any gene promoter and 50% of gastric, cancer samples showed at least one methylated gene promoter. Methylation frequencies of MLH1, MSH2, MSH6 and PMS2 promoter were 21.74%, 17.39%, 0% and 28.26% respectively in gastric cancer samples. MLH1 and PMS2 methylation were associated with neoplastic samples compared to nonneoplastic ones. PMS2:? methylation was associated with diffuse- and intestinal-type cancer compared with normal controls. Intestinal-type cancer showed significant association with MLH1 methylation. Diffuse-type cancer was significantly associated with MSH2 methylation. Our findings show differential gene methylation in tumoral tissue, which allows us to conclude that methylation is associated with gastric carcinogenesis. Methylation of mismatch repair genes was associated with gastric carcinogenesis and may be a helpful tool for diagnosis, prognosis and therapies. However, MSH6 does not seem to be regulated by methylation in our samples.

Polymorphisms of Lewis and Secretor genes are related to breast cancer and metastasis in axillary lymph nodes

ABH and Lewis antigen expression has been associated with cancer development and prognosis, tumor differentiation, and metastasis. Considering that invasive ductal breast carcinoma (IDC) presents multiple molecular alterations, the aim of the present study was to determine whether the polymorphism of ABO, Lewis, and Secretor genes, as well as ABO phenotyping, could be associated with tumor differentiation and lymph nodes metastasis. Seventy-six women with IDC and 78 healthy female blood donors were submitted to ABO phenotyping/genotyping and Lewis and Secretor genotyping. Phenotyping was performed by hemagglutination and genotyping by the polymerase chain reaction with sequence-specific primers. ABO, Lewis, and Secretor genes were classified by individual single nucleotide polymorphism at sites 59, 1067, 202, and 314 of the Lewis gene, 428 of the Secretor gene, and 261 (O1 allele), 526 (O2 and B allele), and 703 (B allele). No association was found between breast cancer and ABO antigen expression (P = 0.9323) or genotype (P = 0.9356). Lewis-negative genotype was associated with IDC (P = 0.0126) but not with anatomoclinical parameters. Nonsecretor genotype was associated with axillary lymph node metastasis (P = 0.0149). In conclusion, Lewis and Secretor genotyping could be useful to predict respectively breast cancer susceptibility and axillary lymph nodes metastasis.

Basal levels of DNA damage detected by micronuclei and comet assays in untreated breast cancer patients and healthy women

Breast cancer is the second most frequent type of cancer worldwide and is the most common malignant disease among women. Risk factors for breast cancer include early menarche, late menopause, hormonal therapies, exposure to environmental pollutants, smoking and alcohol use. However, increased or prolonged exposure to estrogen is the most important risk factor. It has been suggested that accumulation of DNA damage may contribute to breast carcinogenesis. Epidemiological studies suggest that cytogenetic biomarkers such as micronuclei in peripheral blood lymphocytes may predict cancer risk because they indicate genomic instability in target tissues. The objective of the present study was to evaluate the frequencies of micronuclei and the extent of DNA damage detected by comet assay in peripheral blood lymphocytes of untreated breast cancer patients and healthy women. The study was conducted using peripheral blood lymphocytes from 45 women diagnosed for Ductal ""in situ"" or invasive breast carcinoma and 85 healthy control women. Micronuclei and comet assays were performed to detect spontaneous DNA damage. The results showed that micronuclei frequencies and tail intensity, detected by comet assay, were significantly higher in the breast cancer group than in controls. The levels of DNA damage were similar in smokers and non-smokers, and aging did not influence the frequencies of micronuclei or tail intensity values observed in either group. In conclusion, the present work demonstrates higher levels of DNA damage in untreated breast cancer patients than in healthy women.

Impact of Surgical Staging in Locally Advanced Cervical Cancer and Subsequent Chemotherapy

Background: Surgical staging (SS) is the gold standard for determination of the true extent of a patient`s disease and is an important prognostic factor in cervical cancer. We investigated whether lymph node dissection (LND) prior to chemotherapy (CT) followed by radical surgery (RS) could modified overall (OS) and disease-free survival (DFS). Methods: We performed a cohort analysis of 98 patients with cervical carcinoma. The experimental group consisted of 36 patients who underwent SS followed by neoadjuvant chemotherapy, and then by RS (objective response) or chemo-radiation therapy (with or without subsequent surgery when not possible). The control group consisted of 62 similarly treated patients without pretreatment SS. The value of this procedure as a diagnostic tool in defining the extent of disease was evaluated. Furthermore, LND/CT-associated treatment complications and the impacts on OS and DFS were also evaluated. Results: Fourteen (38.9%) patients had pelvic LN metastases and three (8.3%) patients had pelvic and para-aortic LN metastases. The 39-month OS and DFS rates for the current study were 90.6% for the staged group and 52% for non-staged treatment (P < 0.001). Conclusion: SS in cervical cancer is a feasible and safe pretreatment procedure, and when associated with CT, it improves OS and DFS. J. Surg. Oncol. 2009;100:505-510. (C) 2009 Wiley-Liss, Inc.

Clinical Criteria as Predictive Factors of Response to Primary Hormone Therapy in Locally Advanced Breast Cancer

This study investigates the efficacy of clinical criteria in selecting patients for primary tamoxifen therapy. A total of 60 breast cancer patients with large primary tumors and unknown hormonal receptor status were subjected to primary hormone therapy. Inclusion criteria were age over 60 years old or menopausal status for at least 10 years and no clinical evidence of inflammatory disease and fast tumor growth. The objective response rate was 55%. There was a positive correlation between the lack of clinical response and axillary lymph node metastasis (p = 0.009). Patients with objective response had significantly improved disease-free (p = 0.045) and overall (p = 0.0002) survival over those who did not have response to hormonal therapy. In multivariate analysis, the clinical response to therapy was the most powerful prognostic factor. This analysis demonstrates that clinical criteria were very effective predictor of response to neo-adjuvant hormone therapy in large breast tumors for postmenopausal women. Response to therapy is the major prognostic factor in primary tamoxifen-treated breast cancer.

Determination of Cyclophosphamide Enantiomers in Plasma by LC-MS/MS: Application to Pharmacokinetics in Breast Cancer and Lupus Nephritis Patients

This article describes the enantioseleclive analysis of cyclophosphamide (CPA) in human plasma using LC-MS/MS. CPA enantiomers were extracted from plasma using a mixture of ethyl acetate and chloroform (75:25, v/v). The enantiomers were separated on a Chiralcel(R) OD-R column, with the mobile phase consisting of a mixture of acetonitrile and water (75:25, v/v) plus 0.2% formic acid. The protonaled ions and their respective product ions were monitored using two functions, 261 > 141 for CPA enantiomers and 189 > 104 for the internal standard (antipyrine). Recovery rates were higher than 95% and the quantification limit was 2.5-ng/ml plasma for both enantiomers. The coefficients of variation and the relative errors obtained for the validation of intra- and interassay precision and accuracy were less than 10%. The method was applied for the investigation of the enantioselective pharmacokinetics of CPA in a lupus nephritis patient treated with 1 g CPA infused over 2 h and in a breast cancer patient treated with 0.9 g infused over 1 h. No stereoselectivity in the pharmacokinetic parameters was observed for either patient. Clearance values of 2.63 and 2.93 l/h and of 3.36 and 3.61 l/h for (-)-(S) and (+)-(R)-CPA were obtained for the breast cancer and lupus nephritis patient., respectively. Chirality 21:383-389, 2009. (C) 2008 Wiley-Liss, Inc.