microRNA-3129 promotes cell proliferation in gastric cancer cell line SGC7901 via positive regulation of pRb

Several microRNAs (miRNAs) have been reported as oncogenes or tumor suppressors in many cancers, including gastric cancer (GC). However, the role and molecular mechanism of miR-3129 in GC is largely unknown. We aimed to explore the function and the underlying molecular mechanism of miR-3129 in GC. Cancer tissues and corresponding adjacent tissues were collected from 50 patients with GC, and the expression of miR-3129 was detected by RT-qPCR. The expression of miR-3129 and pRb in human GC cell line SCG7091 was altered by transient transfection. Thereafter, MTT and flow cytometry assays were used to analyze cell viability and cell cycle. The expression of cyclin E, CDK2, CDK2 inhibitors (p16 and 21), and pRb were detected by RT-qPCR and western blot. A significant up-regulation of miR-3129 was observed in GC tissues compared to adjacent tissues. Overexpression of miR-3129 significantly improved cell viability after 4 days of post-transfection. Flow cytometry assay results showed that the miR-3129 overexpression arrested more SGC7901 cells at S phase. Moreover, overexpression of miR-3129 down-regulated the expression of CDK2 inhibitors while it up-regulated the expression levels of cyclin E, CDK2, and pRb. Interestingly, we found that pRb inhibition reversed the effect of miR-3129 inhibitor on cell proliferation in SGC7901 cells, increased cell viability, reduced cells at G0/1 phase, and modulated the expression of proliferation-related factors. Our results revealed that miR-3129 functioned as an oncogene through positive regulation of pRb and may prove to be a promising option for molecular therapy of GC.

Maltrato institucional hacia el adulto mayor: percepciones del prestador de servicios de salud y de los ancianos / Institutional abuse toward the elderly: Perceptions of health care providers and older adults

Objetivo. Analizar la percepción que el prestador de servicios de salud y el adulto mayor (AM) tienen sobre el maltrato al AM en los servicios públicos de salud, en ciudades seleccionadas de México. Material y métodos. De 2009 a 2012 se realizó un estudio con diseño cualitativo y estrategia de triangulación de fuentes de datos; se efectuaron entrevistas semiestructuradas a 13 prestadores y a 12 ancianos para recuperar su experiencia en el tema. El análisis utilizó procedimientos de la Teoría Fundamentada. Resultados. El maltrato contra el AM es una práctica naturalizada por el personal y por el anciano, la cual se manifiesta de formas diversas. Conclusiones. La institucionalización, profesionalización histórica y falta de conciencia sobre las necesidades de los AM demandan cambios de planeación, organización y supervisión del Sistema de Salud. El personal requiere intervenciones de formación, capacitación y cambio de actitudes/comportamiento, para otorgar atención integral, digna, humana y de respeto a los Derechos Humanos de los AM.

Objective. To analyze the health care providers (HCP) and elderly patients' perceptions about abuse of the elderly by health personnel of public health services, in selected cities in Mexico. Materials and methods. A qualitative study and a strategy of data triangulation were performed during 2009 and 2012; 13 HCPs and 12 elders were interviewed, in order to obtain their experience regarding elder abuse. Grounded Theory proceedings were used for the analysis. Results. Elder abuse is a naturalized practice, from HCP and elderly people's point of view; these perceptions are showed in different ways. Conclusion. Institutionalization, historical professionalization and lack of consciousness about needs of the elderly (sociocultural and economic), require changes in planning, organization and monitoring process in the Health System; training and educational interventions on staff and exchange attitudes and behavior are necessary in order to offer a health care that is comprehensive, decent, human and with respect for the human rights.

The role of micro RNAs let7c, 100 and 218 expression and their target RAS, C-MYC, BUB1, RB, SMARCA5, LAMB3 and Ki-67 in prostate cancer

OBJECTIVE: The aim of this study is to verify the expression of proteins that are controlled by miR-let7c, 100 and 218 using immunohistochemistry in tissue microarray representative of localized and metastasized the lymph nodes and bone prostate cancer. METHODS: To verify the expression of proteins that are controlled by miR-let7c (C-MYC, BUB1, RAS) 100 (SMARCA5, RB) and 218 (LAMB3) and cell proliferation (Ki-67) we used immunohistochemistry and computerized image system ImageJ MacBiophotonics in three tissue microarrays representative of localized prostate cancer and lymph node and bone metastases. miRNA expression was evaluated by qRT-PCR using 60 paraffin blocks to construct the tissue microarray representative of localized disease. RESULTS: RAS expression was increased in localized prostate cancer and bone metastases compared to the lymph nodes (p=0.017). RB showed an increase in expression from localized prostate cancer to lymph node and bone metastasis (p=0.036). LAMB3 was highly expressed in localized and lymph node metastases (p<0.001). Cell proliferation evaluated by Ki-67 showed an increase from localized prostate cancer to metastases (p<0.001). We did not found any relationship between C-MYC (p=0.253), BUB1 (p=0.649) and SMARCA5 (p=0.315) protein expression with prognosis or tumor behavior. CONCLUSION: We found that the expression of RAS, RB, LAMB3 and Ki-67 changed in the different stages of prostate cancer. Furthermore, we confirmed the overexpression of the miRNAs let7c, 100 and 218 in localized prostate cancer but failed to show the control of protein expression by the putative controller miRNAs using immunohistochemistry. .

Gamma-tocotrienol modulation of senescence-associated gene expression prevents cellular aging in human diploid fibroblasts

OBJECTIVE: Human diploid fibroblasts undergo a limited number of cellular divisions in culture and progressively reach a state of irreversible growth arrest, a process termed cellular aging. The beneficial effects of vitamin E in aging have been established, but studies to determine the mechanisms of these effects are ongoing. This study determined the molecular mechanism of γ-tocotrienol, a vitamin E homolog, in the prevention of cellular aging in human diploid fibroblasts using the expression of senescence-associated genes. METHODS: Primary cultures of young, pre-senescent, and senescent fibroblast cells were incubated with γ-tocotrienol for 24 h. The expression levels of ELN, COL1A1, MMP1, CCND1, RB1, and IL6 genes were determined using the quantitative real-time polymerase chain reaction. Cell cycle profiles were determined using a FACSCalibur Flow Cytometer. RESULTS: The cell cycle was arrested in the G0/G1 phase, and the percentage of cells in S phase decreased with senescence. CCND1, RB1, MMP1, and IL6 were upregulated in senescent fibroblasts. A similar upregulation was not observed in young cells. Incubation with γ-tocotrienol decreased CCND1 and RB1 expression in senescent fibroblasts, decreased cell populations in the G0/G1 phase and increased cell populations in the G2/M phase. γ-Tocotrienol treatment also upregulated ELN and COL1A1 and downregulated MMP1 and IL6 expression in young and senescent fibroblasts. CONCLUSION: γ-Tocotrienol prevented cellular aging in human diploid fibroblasts, which was indicated by the modulation of the cell cycle profile and senescence-associated gene expression.

Testosterone therapy delays cardiomyocyte aging via an androgen receptor-independent pathway

The testicular feminized (Tfm) mouse carries a nonfunctional androgen receptor (AR) and reduced circulating testosterone levels. We used Tfm and castrated mice to determine whether testosterone modulates markers of aging in cardiomyocytes via its classic AR-dependent pathway or conversion to estradiol. Male littermates and Tfm mice were divided into 6 experimental groups. Castrated littermates (group 1) and sham-operated Tfm mice (group 2, N = 8 each) received testosterone. Sham-operated Tfm mice received testosterone in combination with the aromatase inhibitor anastrazole (group 3, N = 7). Castrated littermates (group 4) and sham-operated untreated Tfm mice (group 5) were used as controls (N = 8 and 7, respectively). An additional control group (group 6) consisted of age-matched non-castrated littermates (N = 8). Cardiomyocytes were isolated from the left ventricle, telomere length was measured by quantitative PCR and expression of p16INK4α, retinoblastoma (Rb) and p53 proteins was detected by Western blot 3 months after treatment. Compared with group 6, telomere length was short (P < 0.01) and expression of p16INK4α, Rb and p53 proteins was significantly (P < 0.05) up-regulated in groups 4 and 5. These changes were improved to nearly normal levels in groups 1 and 2 (telomere length = 0.78 ± 0.05 and 0.80 ± 0.08; p16INK4α = 0.13 ± 0.03 and 0.15 ± 0.04; Rb = 0.45 ± 0.05 and 0.39 ± 0.06; p53 = 0.16 ± 0.04 and 0.13 ± 0.03), but did not differ between these two groups. These improvements were partly inhibited in group 3 compared with group 2 (telomere length = 0.65 ± 0.08 vs 0.80 ± 0.08, P = 0.021; p16INK4α = 0.28 ± 0.05 vs 0.15 ± 0.04, P = 0.047; Rb = 0.60 ± 0.06 vs 0.39 ± 0.06, P < 0.01; p53 = 0.34 ± 0.06 vs 0.13 ± 0.03, P = 0.004). In conclusion, testosterone deficiency contributes to cardiomyocyte aging. Physiological testosterone can delay cardiomyocyte aging via an AR-independent pathway and in part by conversion to estradiol.

Expression of pRb, p53, p16 and Cyclin D1 and Their Clinical Implications in Urothelial Carcinoma

The aim of this study was to assess immunohistochemical expression of p53, pRb, p16, and cyclin D1, alone or in combination, as prognostic indicators and to investigate their correlation with clinocopathologic features of urothelial carcinoma. Immunohistochemical staining for p53, pRb, p16, and cyclin D1 was performed on a tissue microarray from 103 patients with urothelial carcinoma who underwent radical cystectomy. Of the patient samples analyzed, 36 (35%), 61 (59%), 47 (46%) and 30 (29%) had altered expression of p53, pRb, p16, and cyclin D1, respectively. Abnormal expression of p53 and pRb correlated with depth of invasion (P=0.040 and P=0.044, respectively). Cyclin D1 expression was associated with tumor stage and recurrence (P=0.017 and P=0.036, respectively). Altered pRb was significantly correlated with overall survival (P=0.040). According to the expression pattern of pRb and p53, p53/pRb (altered/normal) had worse survival than p53/pRb (normal/altered) (P=0.022). Alteration of all markers had worse survival than all normal (P=0.029). As determined by multivariate analysis, tumor stage, lymph node metastasis and the combined expression of p53 and pRb are independent prognostic factors. In conclusion, immunohistochemical evaluation of cell cycle regulators, especially the p53/pRb combination, might be useful in planning appropriate treatment strategies.

Prognostic Significance of Immunohistochemical Expression of p53 and Retinoblastoma Gene Protein (pRB) in Curatively Resected Gastric Cancer

BACKGROUND: The aim of this study was to determine the prognostic significance of the expression of p53 and retinoblastoma (Rb) gene products in cases of curatively resected gastric adenocarcinoma, by immunohistochemical analysis. METHODS: Between January 1996 and December 2001, 736 curatively resected gastric cancer patients underwent immunohistochemical staining for p53 or Rb proteins (pRb), and we retrospectively analyzed the correlation of our results with the clinical outcomes of these cases. RESULTS: High levels of expression of p53 (> 25% p53-positive cells) and Rb (> 50% Rb-positive cells) proteins were detected in 40.1% and 43.7% of cases, respectively. Tubular type was found to frequently exhibit higher levels of p53 expression (high expression in 44.2%) than signet ring cell type (high expression in 26.0%) (p=0.042). The incidence of vascular invasion was lower in the high pRb expressors (43.2%) than in the pRb low expressors (56.8%), but this was not a statistically significant discrepancy (p=0.063). Preoperative CEA levels were found to be low in high pRb expressors: initial CEA level in the high pRb expressors was 2.31 +/- 3.30 ng/mL, and was 5.18 +/- 24.80 ng/mL in the low pRb expressors (p=0.033). Tumor depth and node metastasis were both independent of the levels of expression of p53 and Rb proteins. The seven-year overall survival rate and relapse-free survival rates of patients were 87.2% and 75.7%, respectively. Multivariate Cox regression analysis indicated that tumor stage, tumor size, patient age and pRb expression were the significant prognostic factors with regard to overall survival, and tumor stage and age were both significant factors with regard to relapse-free survival. CONCLUSION: Immunohistochemical staining of retinoblastoma gene products was an independent prognostic factor for the prediction of overall survival in curatively resected gastric cancer patients.

The Levels of MDM2 Protein Are Decreased by a Proteasome-Mediated Proteolysis Prior to Caspase-3-Dependent pRb and PARP Cleavages

MDM2 is a substrate of caspase-3 in p53-mediated apoptosis. In addition, MDM2 mediates its own ubiquitination in a RING finger-dependent manner. Thus, we investigated whether MDM2 is degraded through a ubiquitin-dependent proteasome pathway in the absence of p53. When HL-60 cells, p53 null, were treated with etoposide, MDM2 was markedly decreased prior to caspase-3-dependent retinoblastoma tumor suppressor protein (pRb) and poly (ADP- ribose) polymerase (PARP) cleavages. Moreover, down-regulation of MDM2 level was not coupled with its mRNA down-regulation. However, the level of MDM2 was partially restored by proteasome inhibitors such as LLnL and lactacystin, even in the presence of etoposide. Our results suggest that, in the p53 null status, MDM2 protein level is decreased by proteasome-mediated proteolysis prior to caspase-3-dependent PARP and pRb cleavages.

Changes in the expression of c-myc, RB and tyrosine-phosphorylated proteins during proliferation of NIH 3T3 cells induced by hyaluronic acid

We have shown that hyaluronic acid stimulates the proliferation of quiescent NIH 3T3 cells. We have shown that treatment of 1 mg/ml hyaluronic acid results in increase of tyrosine phosphorylation of two proteins, MW 124 kDa and 60 kDa as detected by anti-tyrosine antibodies by Western blot analysis. Maximum phosphorylation occurred within 2 h after addition of 1 mg/ml hyaluronic acid. Stimulation of proliferation was also accompanied by increase in c-Myc protein, which was inhibited by amlloride, an inhibitor of Na+/H+ antiporter and EGTA and increase in the steady state level of pRb, the RB gene product. These results suggest that the intracellular signal transduction pathways that mediate the stimulatory effects of hyaluronic acid on cellular proliferation are similar to those of growth factors.