c-Myc protein expression is not an independent prognostic predictor in cervical squamous cell carcinoma

The c-myc protein is known to regulate the cell cycle, and its down-regulation can lead to cell death by apoptosis. The role of c-myc protein as an independent prognostic determinant in cervical cancer is controversial. In the present study, a cohort of 220 Brazilian women (mean age 53.4 years) with FIGO stage I, II and III (21, 28 and 51%, respectively) cervical squamous cell carcinomas was analyzed for c-myc protein expression using immunohistochemistry. The disease-free survival and relapse-rate were analyzed using univariate (Kaplan-Meier) survival analysis for 116 women who completed the standard FIGO treatment and were followed up for 5 years. Positive c-myc staining was detected in 40% of carcinomas, 29% being grade 1, 9% grade 2, and 2% grade 3. The distribution of positive c-myc according to FIGO stage was 19% (17 women) in stage I, 33% (29) in stage II, and 48% (43) in stage III of disease. During the 60-month follow-up, disease-free survival in univariate (Kaplan-Meier) survival analysis (116 women) was lower for women with c-myc-positive tumors, i.e., 60.5, 47.5 and 36.6% at 12, 36, and 60 months, respectively (not significant). The present data suggest that immunohistochemical demonstration of c-myc does not possess any prognostic value independent of FIGO stage, and as such is unlikely to be a useful prognostic marker in cervical squamous cell carcinoma.

Changes in red blood cell osmotic fragility induced by total plasma and plasma fractions obtained from rats bearing progressive and regressive variants of the Walker 256 tumor

Two variants (A and B) of the widely employed Walker 256 rat tumor cells are known. When inoculated sc, the A variant produces solid, invasive, highly metastasizing tumors that cause severe systemic effects and death. We have obtained a regressive variant (AR) whose sc growth is slower, resulting in 70-80% regression followed by development of immunity against A and AR variants. Simultaneously with the beginning of tumor regression, a temporary anemia developed (~8 days duration), accompanied by marked splenomegaly (~300%) and changes in red blood cell osmotic fragility, with mean corpuscular fragility increasing from 4.1 to 6.5 g/l NaCl. The possibility was raised that plasma factors associated with the immune response induced these changes. In the present study, we identify and compare the osmotic fragility increasing activity of plasma fractions obtained from A and AR tumor bearers at different stages of tumor development. The results showed that by day 4 compounds precipitating in 60% (NH4)2SO4 and able to increase red blood cell osmotic fragility appeared in the plasma of A and AR tumor bearers. Later, these compounds disappeared from the plasma of A tumor bearers but slightly increased in the plasma of AR tumor bearers. Furthermore, by day 10, compounds precipitating between 60 and 80% (NH4)2SO4 and with similar effects appeared only in plasma of AR tumor bearers. The salt solubility, production kinetics and hemolytic activity of these compounds resemble those of the immunoglobulins. This, together with their preferential increase in rats bearing the AR variant, suggest their association with an immune response against this tumor.

Complement system in cutaneous squamous cell carcinoma

Cutaneous squamous cell carcinoma (cSCC) consists 20% of keratinocytederived non-melanoma skin cancers (NMSC), the incidence of which is increasing globally. cSCC is the most common metastatic skin cancer and it causes approximately 20% of skin cancer-related deaths. At present, there are no molecular markers for predicting which cSCC lesions are aggressive or metastasize rapidly. UV radiation is the most important risk factor for cSCC. During the development of cSCC, normal epidermal keratinocytes are transformed and form actinic keratosis (AK), which progresses to cSCC in situ (cSCCIS, Bowen’s disease) and finally to invasive and metastatic cSCC. Inflammatory factors and cells are a part of cancer microenvironment and cSCC can develop in the chronically irritated skin or in the context of chronic inflammation. The complement system is a central part of innate immunity and it regulates normal immunological and inflammatory processes. In this study, the role of complement system components and inhibitors were studied in the progression of cSCC in culture and in vivo. Elevated expression of complement factor H (CFH), complement factor I (CFI), complement component C3 and complement factor B (CFB) was noted in cSCC cells in culture. The analysis with immunohistochemistry (IHC) revealed that the expression of CFH, CFI, C3 and CFB was specifically noted in tumor cells in vivo. The staining intensity of CFH, CFI, C3 and CFB was also stronger in invasive cSCC than in AK or cSCCIS samples. The knockdown of CFH, CFI and CFB with specific siRNAs decreased cSCC cell viability and migration, whereas the knockdown of C3 reduced only cSCC cell migration. Moreover, the knockdown of CFI, C3 and CFB inhibited growth of cSCC xenograft tumors established in SCID mice in vivo. In these tumors, CFI, C3 and CFB knockdown decreased the number of proliferating cells. Moreover, the knockdown of CFI increased local inflammation and complement activation. This study provides evidence for the roles of CFH, CFI, C3 and CFB in the tumor progression indicating these as molecular biomarkers and putative therapeutic targets of cSCC.

Antagonist G-mediated targeting and cytotoxicity of liposomal doxorubicin in NCI-H82 variant small cell lung cancer

The aim of the present study was to characterize the interactions of antagonist G (H-Arg-D-Trp-NmePhe-D-Trp-Leu-Met-NH 2)-targeted sterically stabilized liposomes with the human variant small cell lung cancer (SCLC) H82 cell line and to evaluate the antiproliferative activity of encapsulated doxorubicin against this cell line. Variant SCLC tumors are known to be more resistant to chemotherapy than classic SCLC tumors. The cellular association of antagonist G-targeted (radiolabeled) liposomes was 20-30-fold higher than that of non-targeted liposomes. Our data suggest that a maximum of 12,000 antagonist G-targeted liposomes were internalized/cell during 1-h incubation at 37ºC. Confocal microscopy experiments using pyranine-containing liposomes further confirmed that receptor-mediated endocytosis occurred, specifically in the case of targeted liposomes. In any of the previously mentioned experiments, the binding and endocytosis of non-targeted liposomes have revealed to be negligible. The improved cellular association of antagonist G-targeted liposomes, relative to non-targeted liposomes, resulted in an enhanced nuclear delivery (evaluated by fluorimetry) and cytotoxicity of encapsulated doxorubicin for incubation periods as short as 2 h. For an incubation of 2 h, we report IC50 values for targeted and non-targeted liposomes containing doxorubicin of 5.7 ± 3.7 and higher than 200 µM doxorubicin, respectively. Based on the present data, we may infer that receptors for antagonist G were present in H82 tumor cells and could mediate the internalization of antagonist G-targeted liposomes and the intracellular delivery of their content. Antagonist G covalently coupled to liposomal drugs may be promising for the treatment of this aggressive and highly heterogeneous disease.

c-erbB-2 expression and nuclear pleomorphism in canine mammary tumors

The objective of the present investigation was to study the expression of c-erbB-2 and MIB-1 and try to associate them with morphological features of the cell such as nuclear pleomorphism, mitotic count and histological grade in a series of 70 canine mammary gland tumors, 22 of them benign and 48 malignant. Tumors were collected at the Veterinary Hospital of UFMG (Brazil) and the Veterinary Faculty of Porto University (Portugal). c-erbB-2 expression was determined according to the guidelines provided by the manufacturer of the HercepTest system and nuclear pleomorphism, mitotic count and histological grade according the Elston and Ellis grading system. The HercepTest is the FDA-approved in vitro diagnostic test marketed by Dako. It is a semi-quantitative immunohistochemical assay used to determine overexpression of HER2 protein (human epidermal growth factor receptor) in breast cancer tissue. MIB-1 expression was also evaluated in 28 malignant tumors. Seventeen (35.4%) of the malignant tumors were positive for c-erbB-2 expression, which was positively associated with nuclear pleomorphism (P < 0.0001), histological grade (P = 0.0017) and mitotic count (P < 0.05). Nuclear pleomorphism also showed a positive association with MIB-1 index (P < 0.0001). These results suggest that some of the biological and morphological characteristics of the tumor are associated in canine mammary gland tumors, as also reported for human breast cancer. It was also possible to show that the immunoexpression of c-erbB-2 can be a factor in mammary carcinogenesis. This fact opens the possibility of using anti-c-erbB-2 antibodies in the treatment of canine mammary tumors.

The role of osteoblasts in regulating hematopoietic stem cell activity and tumor metastasis

Bone marrow stromal cells are critical regulators of hematopoiesis. Osteoblasts are part of the stromal cell support system in bone marrow and may be derived from a common precursor. Several studies suggested that osteoblasts regulate hematopoiesis, yet the entire mechanism is not understood. It is clear, however, that both hematopoietic precursors and osteoblasts interact for the production of osteoclasts and the activation of resorption. We observed that hematopoietic stem cells (HSCs) regulate osteoblastic secretion of various growth factors, and that osteoblasts express some soluble factors exclusively in the presence of HSCs. Osteoblasts and hematopoietic cells are closely associated with each other in the bone marrow, suggesting a reciprocal relationship between them to develop the HSC niche. One critical component regulating the niche is stromal-derived factor-1 (SDF-1) and its receptor CXCR4 which regulates stem cell homing and, as we have recently demonstrated, plays a crucial role in facilitating those tumors which metastasize to bone. Osteoblasts produce abundant amounts of SDF-1 and therefore osteoblasts play an important role in metastasis. These findings are discussed in the context of the role of osteoblasts in marrow function in health and disease.

Dexamethasone blocks the migration of the human neuroblastoma cell line SK-N-SH

Glucocorticoids (Gc) influence the differentiation of neural crest-derived cells such as those composing sympathoadrenal tumors like pheochromocytomas, as well as neuroblastomas and gangliomas. In order to obtain further information on the effects of Gc on cells evolving from the neural crest, we have used the human neuroblastoma cell line SK-N-SH to analyze: 1) the presence and the binding characteristics of Gc receptors in these cells, 2) the effect of dexamethasone (Dex) on the migration of SK-N-SH cells, and 3) the effect of Dex on the organization of the cytoskeleton of SK-N-SH cells. We show that: 1) receptors that bind [³H]-Dex with high affinity and high capacity (Kd of 9.6 nM, Bmax of 47 fmol/mg cytosolic protein, corresponding to 28,303 sites/cell) are present in cytosolic preparations of SK-N-SH cells, and 2) treatment with Dex (in the range of 10 nM to 1 µM) has an inhibitory effect (from 100% to 74 and 43%, respectively) on the chemotaxis of SK-N-SH cells elicited by fetal bovine serum. This inhibition is completely reversed by the Gc receptor antagonist RU486 (1 µM), and 3) as demonstrated by fluorescent phalloidin-actin detection, the effect of Dex (100 nM) on SK-N-SH cell migration is accompanied by modifications of the cytoskeleton organization that appear with stress fibers. These modifications did not take place in the presence of 1 µM RU486. The present data demonstrate for the first time that Dex affects the migration of neuroblastoma cells as well as their cytoskeleton organization by interacting with specific receptors. These findings provide new insights on the mechanism(s) of action of Gc on cells originating in the neural crest.

Multiple factor analysis of metachronous upper urinary tract transitional cell carcinoma after radical cystectomy

Transitional cell carcinoma (TCC) of the urothelium is often multifocal and subsequent tumors may occur anywhere in the urinary tract after the treatment of a primary carcinoma. Patients initially presenting a bladder cancer are at significant risk of developing metachronous tumors in the upper urinary tract (UUT). We evaluated the prognostic factors of primary invasive bladder cancer that may predict a metachronous UUT TCC after radical cystectomy. The records of 476 patients who underwent radical cystectomy for primary invasive bladder TCC from 1989 to 2001 were reviewed retrospectively. The prognostic factors of UUT TCC were determined by multivariate analysis using the COX proportional hazards regression model. Kaplan-Meier analysis was also used to assess the variable incidence of UUT TCC according to different risk factors. Twenty-two patients (4.6%). developed metachronous UUT TCC. Multiplicity, prostatic urethral involvement by the bladder cancer and the associated carcinoma in situ (CIS) were significant and independent factors affecting the occurrence of metachronous UUT TCC (P = 0.0425, 0.0082, and 0.0006, respectively). These results were supported, to some extent, by analysis of the UUT TCC disease-free rate by the Kaplan-Meier method, whereby patients with prostatic urethral involvement or with associated CIS demonstrated a significantly lower metachronous UUT TCC disease-free rate than patients without prostatic urethral involvement or without associated CIS (log-rank test, P = 0.0116 and 0.0075, respectively). Multiple tumors, prostatic urethral involvement and associated CIS were risk factors for metachronous UUT TCC, a conclusion that may be useful for designing follow-up strategies for primary invasive bladder cancer after radical cystectomy.

p53 immunostaining is correlated with reduced survival and is not correlated with gene mutations in resected pulmonary large cell carcinomas

Malignancy of pulmonary large cell carcinomas (LCC) increases from classic LCC through LCC with neuroendocrine morphology (LCCNM) to large cell neuroendocrine carcinomas (LCNEC). However, the histological classification has sometimes proved to be difficult. Because the malignancy of LCC is highly dependent on proteins with functions in the cell cycle, DNA repair, and apoptosis, p53 has been targeted as a potentially useful biological marker. p53 mutations in lung cancers have been shown to result in expression and protein expression also occurs in the absence of mutations. To validate the importance of both p53 protein expression (by immunostaining) and p53 gene mutations in lung LCC (by PCR-single strand conformational polymorphism analysis of exons 5, 6, 7, and 8) and to study their relationships with clinical factors and sub-classification we investigated the correlation of p53 abnormalities in 15 patients with LCC (5 classic LCC, 5 LCNEC, and 5 LCCNM) who had undergone resection with curative intent. Of these patients, 5/15 expressed p53 and none had mutant p53 sequences. There was a negative survival correlation with positive p53 immunostaining (P = 0.05). After adjustment for stage, age, gender, chemotherapy, radiotherapy, and histological subtypes by multivariate analysis, p53 expression had an independent impact on survival. The present study indicates that p53 assessment may provide an objective marker for the prognosis of LCC irrespective of morphological variants and suggests that p53 expression is important for outcome prediction in patients with the early stages of LCC. The results reported here should be considered to be initial results because tumors from only 15 patients were studied: 5 each from LCC, LCNEC and LCCNM. This was due to the rarity of these specific diseases.

Primary mechanism of apoptosis induction in a leukemia cell line by fraction FA-2-b-ß prepared from the mushroom Agaricus blazei Murill

Agaricus blazei Murill is a native Brazilian mushroom which functions primarily as an anticancer substance in transplanted mouse tumors. However, the mechanism underlying this function of A. blazei Murill remains obscure. The present study was carried out to investigate the effect of fraction FA-2-b-ß, an RNA-protein complex isolated from A. blazei Murill, on human leukemia HL-60 cells in vitro. Typical apoptotic characteristics were determined by morphological methods using DNA agarose gel electrophoresis and flow cytometry. The growth suppressive effect of fraction FA-2-b-ß on HL-60 cells in vitro occurred in a dose- (5-80 µg/mL) and time-dependent (24-96 h) manner. The proliferation of HL-60 cells (1 x 10(5) cells/mL) treated with 40 µg/mL of fraction FA-2-b-ß for 24-96 h and with 5-80 µg/mL for 96 h resulted in inhibitory rates ranging from 8 to 54.5%, and from 4.9 to 86.3%, respectively. Both telomerase activity determined by TRAP-ELISA and mRNA expression of the caspase-3 gene detected by RT-PCR were increased in HL-60 cells during fraction FA-2-b-ß treatment. The rate of apoptosis correlated negatively with the decrease of telomerase activity (r = 0.926, P < 0.05), but correlated positively with caspase-3 mRNA expression (r = 0.926, P < 0.05). These data show that fraction FA-2-b-ß can induce HL-60 cell apoptosis and that the combined effect of down-regulation of telomerase activity and up-regulation of mRNA expression of the caspase-3 gene could be the primary mechanism of induction of apoptosis. These findings provide strong evidence that fraction FA-2-b-ß could be of interest for the clinical treatment of acute leukemia.

The role of the SLAM-SAP signaling pathway in the modulation of CD4+ T cell responses

The signaling lymphocytic activation molecule (SLAM), present on the surface of hematopoietic cells, can regulate some events of the immune responses. This modulatory action is associated with the capacity of SLAM to interact with an intracytoplasmic adapter, such as SLAM-associated protein (SAP). SLAM is constitutively expressed in most of these cells, is rapidly induced after antigenic or inflammatory stimuli, and participates in the immunological synapse. Defects in the function of the SLAM-SAP pathway contribute to immunological abnormalities, resulting in autoimmune diseases, tumors of the lymphoid tissues and inadequate responses to infectious agents. Initially, the role of SLAM was investigated using an anti-SLAM monoclonal antibody (α-SLAM mAb) identified as an agonist of the SLAM-SAP pathway, which could induce the production of interferon-γ and could redirect the immune response to a T helper 1 (Th1) cell profile. However, in this review we postulate that the SLAM-SAP pathway primarily induces a Th2 response and secondarily suppresses the Th1 response.

MMP-1/PAR-1 signal transduction axis and its prognostic impact in esophageal squamous cell carcinoma

The matrix metalloprotease-1 (MMP-1)/protease-activated receptor-1 (PAR-1) signal transduction axis plays an important role in tumorigenesis. To explore the expression and prognostic value of MMP-1 and PAR-1 in esophageal squamous cell carcinoma (ESCC), we evaluated the expression of two proteins in resected specimens from 85 patients with ESCC by immunohistochemistry. Sixty-two (72.9%) and 58 (68.2%) tumors were MMP-1- and PAR-1-positive, respectively, while no significant staining was observed in normal esophageal squamous epithelium. MMP-1 and PAR-1 overexpression was significantly associated with tumor node metastasis (TNM) stage and regional lymph node involvement. Patients with MMP-1- and PAR-1-positive tumors, respectively, had poorer disease-free survival (DFS) than those with negative ESCC (P = 0.002 and 0.003, respectively). Univariate analysis showed a significant relationship between TNM stage [hazard ratio (HR) = 2.836, 95% confidence interval (CI) = 1.866-4.308], regional lymph node involvement (HR = 2.955, 95%CI = 1.713-5.068), MMP-1 expression (HR = 2.669, 95%CI = 1.229-6.127), and PAR-1 expression (HR = 1.762, 95%CI = 1.156-2.883) and DFS. Multivariate analysis including the above four parameters identified TNM stage (HR = 2.035, 95%CI = 1.167-3.681), MMP-1 expression (HR = 2.109, 95%CI = 1.293-3.279), and PAR-1 expression (HR = 1.967, 95%CI = 1.256-2.881) as independent and significant prognostic factors for DFS. Our data suggest for the first time that MMP-1 and PAR-1 were both overexpressed in ESCC and are novel predictors of poor patient prognosis after curative resection. The MMP-1/PAR-1 signal transduction axis might be a new therapeutic target for future therapies tailored against ESCC.

p16 (INK4a) has clinicopathological and prognostic impact on oropharynx and larynx squamous cell carcinoma

CDKN2A encodes proteins such as p16 (INK4a), which negatively regulate the cell-cycle. Molecular genetic studies have revealed that deletions in CDKN2A occur frequently in cancer. Although p16 (INK4a) may be involved in tumor progression, the clinical impact and prognostic implications in head and neck squamous cell carcinoma (HNSCC) are controversial. The objective of this study was to evaluate the frequency of the immunohistochemical expression of p16 (INK4a) in 40 oropharynx and 35 larynx from HNSCC patients treated in a single institution and followed-up at least for 10 years in order to explore potential associations with clinicopathological outcomes and prognostic implications. Forty cases (53.3%) were positive for p16 (INK4a) and this expression was more intense in non-smoking patients (P = 0.050), whose tumors showed negative vascular embolization (P = 0.018), negative lymphatic permeation (P = 0.002), and clear surgical margins (P = 0.050). Importantly, on the basis of negative p16 (INK4a) expression, it was possible to predict a probability of lower survival (P = 0.055) as well as tumors presenting lymph node metastasis (P = 0.050) and capsular rupture (P = 0.0010). Furthermore, increased risk of recurrence was observed in tumors presenting capsular rupture (P = 0.0083). Taken together, the alteration in p16 (INK4a) appears to be a common event in patients with oropharynx and larynx squamous cell carcinoma and the negative expression of this protein correlated with poor prognosis.

Predictive significance of standardized uptake value parameters of FDG-PET in patients with non-small cell lung carcinoma

18F-fluoro-2-deoxyglucose (FDG) positron emission tomography (PET)/computed tomography (CT) is widely used to diagnose and stage non-small cell lung cancer (NSCLC). The aim of this retrospective study was to evaluate the predictive ability of different FDG standardized uptake values (SUVs) in 74 patients with newly diagnosed NSCLC. 18F-FDG PET/CT scans were performed and different SUV parameters (SUVmax, SUVavg, SUVT/L, and SUVT/A) obtained, and their relationship with clinical characteristics were investigated. Meanwhile, correlation and multiple stepwise regression analyses were performed to determine the primary predictor of SUVs for NSCLC. Age, gender, and tumor size significantly affected SUV parameters. The mean SUVs of squamous cell carcinoma were higher than those of adenocarcinoma. Poorly differentiated tumors exhibited higher SUVs than well-differentiated ones. Further analyses based on the pathologic type revealed that the SUVmax, SUVavg, and SUVT/L of poorly differentiated adenocarcinoma tumors were higher than those of moderately or well-differentiated tumors. Among these four SUV parameters, SUVT/Lwas the primary predictor for tumor differentiation. However, in adenocarcinoma, SUVmax was the determining factor for tumor differentiation. Our results showed that these four SUV parameters had predictive significance related to NSCLC tumor differentiation; SUVT/L appeared to be most useful overall, but SUVmax was the best index for adenocarcinoma tumor differentiation.

Roles of novel biomarkers in progression of cutaneous squamous cell carcinoma

Roles of novel biomarkers was studied in progression of cutaneous squamous cell carcinoma (cSCC) as the most common metastatic skin cancer. The incidence of cSCC is increasing worldwide due to lifestyle changes such as recreational exposure to sunlight and the aging of the population. Because of an emerging need for molecular markers for the progression of cSCC, we set our goal to characterize three distinct novel markers overexpressed in cSCC cells. Our results identified overexpression of serpin peptidase inhibitor clade A member 1 (SerpinA1), EphB2 and absent in melanoma 2 (AIM2) in cSCC cell lines compared with normal human epidermal keratinocytes (NHEKs). Immunohistochemical analysis of SerpinA1, EphB2 and AIM2 revealed abundant tumor cell-specific expression of cytoplasmic SerpinA1 and AIM2 and cytoplasmic and membranous EphB2 in cSCC tumors in vivo. The staining intensity of SerpinA1, EphB2 and AIM2 was significantly stronger in cSCC as compared with carcinoma in situ (cSCCIS) and actinic keratosis (AK). Tumor cell-associated SerpinA1 and EphB2 was noted in chemically induced mouse skin SCC, and the staining intensity was stronger in mouse cSCCs than in untreated skin. AIM2 staining intensity was significantly more abundant in cSCC of organ transplant recipients (OTR) than in sporadic cSCC in vivo. EphB2 knockdown resulted in inhibition of migration in cSCC cells. In addition, knockdown of EphB2 and AIM2 was found to inhibit the proliferation and invasion of cSCC cells and to delay the growth and vascularization of cSCC xenografts in vivo. Altogether, these findings identify SerpinA1 as a novel biomarker for cSCC. In addition, characterization of the roles of EphB2 and AIM2 in the progression of cSCC was implicated them as possible therapeutic targets for the treatment of cSCC particularly in unresectable and metastatic tumors.