siRNA inhibition of telomerase enhances the anti-cancer effect of doxorubicin in breast cancer cells

<p>Abstract</p> <p>Background</p> <p>Doxorubicin is an effective breast cancer drug but is hampered by a severe, dose-dependent toxicity. Concomitant administration of doxorubicin and another cancer drug may be able to sensitize tumor cells to the cytotoxicity of doxorubicin and lowers the therapeutic dosage. In this study, we examined the combined effect of low-dose doxorubicin and siRNA inhibition of telomerase on breast cancer cells. We found that when used individually, both treatments were rapid and potent apoptosis inducers; and when the two treatments were combined, we observed an enhanced and sustained apoptosis induction in breast cancer cells.</p> <p>Methods</p> <p>siRNA targeting the mRNA of the protein component of telomerase, the telomerase reverse transcriptase (hTERT), was transfected into two breast cancer cell lines. The siRNA inhibition was confirmed by RT-PCR and western blot on hTERT mRNA and protein levels, respectively, and by measuring the activity level of telomerase using the TRAP assay. The effect of the hTERT siRNA on the tumorigenicity of the breast cancer cells was also studied <it>in vivo </it>by injection of the siRNA-transfected breast cancer cells into nude mice.</p> <p>The effects on cell viability, apoptosis and senescence of cells treated with hTERT siRNA, doxorubicin, and the combined treatment of doxorubicin and hTERT siRNA, were examined <it>in vitro </it>by MTT assay, FACS and SA-β-galactosidase staining.</p> <p>Results</p> <p>The hTERT siRNA effectively knocked down the mRNA and protein levels of hTERT, and reduced the telomerase activity to 30% of the untreated control. <it>In vivo</it>, the tumors induced by the hTERT siRNA-transfected cells were of reduced sizes, indicating that the hTERT siRNA also reduced the tumorigenic potential of the breast cancer cells. The siRNA treatment reduced cell viability by 50% in breast cancer cells within two days after transfection, while 0.5 μM doxorubicin treatment had a comparable effect but with a slower kinetics. The combination of hTERT siRNA and 0.5 μM doxorubicin killed twice as many cancer cells, showing a cumulative effect of the two treatments.</p> <p>Conclusion</p> <p>The study demonstrated the potential of telomerase inhibition as an effective treatment for breast cancer. When used in conjunction to doxorubicin, it could potentiate the cytotoxic effect of the drug to breast cancer cells.</p

Up-Regulation of Mcl-1 and Bak by Coronavirus Infection of Human, Avian and Animal Cells Modulates Apoptosis and Viral Replication

Virus-induced apoptosis and viral mechanisms that regulate this cell death program are key issues in understanding virus-host interactions and viral pathogenesis. Like many other human and animal viruses, coronavirus infection of mammalian cells induces apoptosis. In this study, the global gene expression profiles are first determined in IBV-infected Vero cells at 24 hours post-infection by Affymetrix array, using avian coronavirus infectious bronchitis virus (IBV) as a model system. It reveals an up-regulation at the transcriptional level of both pro-apoptotic Bak and pro-survival myeloid cell leukemia-1 (Mcl-1). These results were further confirmed both in vivo and in vitro, in IBV-infected embryonated chicken eggs, chicken fibroblast cells and mammalian cells at transcriptional and translational levels, respectively. Interestingly, the onset of apoptosis occurred earlier in IBV-infected mammalian cells silenced with short interfering RNA targeting Mcl-1 (siMcl-1), and was delayed in cells silenced with siBak. IBV progeny production and release were increased in infected Mcl-1 knockdown cells compared to similarly infected control cells, while the contrary was observed in infected Bak knockdown cells. Furthermore, IBV infection-induced up-regulation of GADD153 regulated the expression of Mcl-1. Inhibition of the mitogen-activated protein/extracellular signal-regulated kinase (MEK/ERK) and phosphoinositide 3-kinase (PI3K/Akt) signaling pathways by chemical inhibitors and knockdown of GADD153 by siRNA demonstrated the involvement of ER-stress response in regulation of IBV-induced Mcl-1 expression. These results illustrate the sophisticated regulatory strategies evolved by a coronavirus to modulate both virus-induced apoptosis and viral replication during its replication cycle

Time to definitive diagnosis of breast cancer in Latina and non-Hispanic white women: the six cities study

Time delay after an abnormal screening mammogram may have a critical impact on tumor size, stage at diagnosis, treatment, prognosis, and survival of subsequent breast cancer. This study was undertaken to evaluate disparities between Latina and non-Hispanic white (NHW) women in time to definitive diagnosis of breast cancer after an abnormal screening mammogram, as well as factors contributing to such disparities. As part of the activities of the National Cancer Institute (NCI)-funded Redes En Acción research network, clinical records of 186 Latinas and 74 NHWs who received abnormal screening mammogram results were reviewed to determine the time to obtain a definitive diagnosis. Data was obtained from participating clinics in six U.S. cities and included demographics, clinical history, and mammogram characteristics. Kaplan-Meier estimates and Cox proportional hazards models were used to test differences in median time to definitive diagnosis by ethnicity after adjusting for clinic site, demographics, and clinical characteristics. Time-to-event analysis showed that Latinas took 2.2 times longer to reach 50% definitively diagnosed with breast cancer relative to NHWs, and three times longer to reach 80% diagnosed (p=0.001). Latinas’ median time to definitive diagnosis was 60 days compared to 27 for NHWs, a 59% gap in diagnosis rates (adjusted Hazard Ratio [aHR] = 1.59, 95% CI = 1.09, 2.31; p=0.015). BI-RADS-4/5 women’s diagnosis rate was more than twice that of BI-RADS-3 (aHR = 2.11, 95% CI = 1.18, 3.78; p=0.011). Disparities in time between receipt of abnormal screening result and definitive diagnosis adversely affect Latinas compared to NHWs, and remain significant after adjusting for demographic and clinical variables. With cancer now the leading cause of mortality among Latinos, a greater need exists for ethnically and culturally appropriate interventions like patient navigation to facilitate Latinas’ successful entry into, and progression through, the cancer care system

Uso de software como ferramenta pedagógica no processo de ensino-aprendizagem da mamografia digital Educational software as a tool for teaching & learning of digital mammography

OBJETIVO: Avaliar o impacto sobre o treinamento de residentes utilizando uma ferramenta computacional dedicada à avaliação do desempenho da leitura de imagens radiológicas convencionais e digitais. MATERIAIS E MÉTODOS: O treinamento foi realizado no Laboratório de Qualificação de Imagens Médicas (QualIM). Os residentes de radiologia efetuaram cerca de 1.000 leituras de um total de 60 imagens obtidas de um simulador estatístico (Alvim®) que apresenta fibras e microcalcificações de dimensões variadas. O desempenho dos residentes na detecção dessas estruturas foi avaliado por meio de parâmetros estatísticos. RESULTADOS: Os resultados da probabilidade de detectabilidade foram de 0,789 e 0,818 para os sistemas convencional e digital, respectivamente. As taxas de falso-positivos foram de 8% e 6% e os valores de verdadeiro-positivos, de 66% e 70%, respectivamente. O valor de kappa total foi 0,553 para as leituras em negatoscópio e 0,615 em monitor. A área sob a curva ROC foi de 0,716 para leitura em filme e 0,810 para monitor. CONCLUSÃO: O treinamento proposto mostrou ser efetivo e apresentou impacto positivo sobre o desempenho dos residentes, constituindo-se em interessante ferramenta pedagógica. Os resultados sugerem que o método de treinamento baseado na leitura de simuladores pode produzir um melhor desempenho dos profissionais na interpretação das imagens mamográficas.<br>OBJECTIVE: The present study was aimed at evaluating the performance of residents trained in the reading of conventional and digital mammography images with a specific computational tool. MATERIALS AND METHODS: The training was accomplished in the Laboratory of Medical Images Qualification (QualIM - Laboratório de Qualificação de Imagens Médicas). Residents in radiology performed approximately 1,000 readings of a set of 60 images acquired from a statistical phantom (Alvim®) presenting microcalcifications and fibers with different sizes. The analysis of the residents' performance in the detection of these structures was based on statistical parameters. RESULTS: Values for detection probability were respectively 0.789 and 0.818 for conventional and digital systems. False-positive rates were 8% and 6%, and true-positive rates, 66% and 70% respectively. The total kappa value was 0.553 for readings on the negatoscope (hard-copy readings), and 0.615 on the monitor (soft-copy readings). The area under the ROC curve was 0.716 for hard-copy readings and 0.810 for soft-copy readings. CONCLUSION: The training has showed to be effective, with a positive impact on the residents' performance, representing an interesting educational tool. The results of the present study suggest that this method of training based on the reading of images from phantoms can improve the practitioners' performance in the interpretation of mammographic images