RESUMO

Head and neck squamous cell carcinoma (HNSCC) is a remarkably heterogeneous disease with around 50% mortality, a fact that has prompted researchers to try new approaches to improve patient survival. Hemoxygenase-1 (HO-1) is the rate-limiting step for heme degradation into carbon monoxide, free iron and biliverdin. We have previously reported that HO-1 protein is upregulated in human HNSCC samples and that it is localized in the cytoplasmic and nuclear compartments; additionally, we have demonstrated that HO-1 nuclear localization is associated with malignant progression. In this work, by using pharmacological and genetic experimental approaches, we begin to elucidate the mechanisms through which HO-1 plays a role in HNSCC. We found that high HO-1 mRNA was associated with decreased patient survival in early stages of HNSCC. In vitro experiments have shown that full-length HO-1 localizes in the cytoplasm, and that, depending on its enzymatic activity, it increases cell viability and promotes cell cycle progression. Instead, HO-1 does not alter migration capacity. Furthermore, we show that C-terminal truncated HO-1 localizes into the nucleus, increases cell viability and promotes cell cycle progression. In conclusion, we herein demonstrate that HO-1 displays protumor activities in HNSCC that depend, at least in part, on the nuclear localization of HO-1.

RESUMO

NF-κB regulates the expression of Cyclin D1 (CD1), while RAC3 is an NF-κB coactivator that enhances its transcriptional activity. In this work, we investigated the regulatory role of CD1 on NF-κB activity. We found that CD1 inhibits NF-κB transcriptional activity through a corepressor function that can be reverted by over-expressing RAC3. In both, tumoral and non-tumoral cells, the expression pattern of RAC3 and CD1 is regulated by the cell cycle, showing a gap between the maximal expression levels of each protein. The individual increase, by transfection, of either CD1 or RAC3 enhances cell proliferation. However the simultaneous and constitutive over-expression of both proteins has an inhibitory effect. Our results suggest that the relative amounts of CD1 and RAC3, and the timing of expression of these oncogenes could tilt the balance of tumor cell proliferation in response to external signals.

Assuntos

RESUMO

RAC3 belongs to the family of p160 nuclear receptors coactivators and it is over-expressed in several tumors. We have previously shown that RAC3 is a NF-kappaB coactivator. In this paper, we investigated the role of RAC3 in cell-sensitivity to apoptosis, using H2O2 in the human embryonic kidney cell line (HEK293), and tumor necrosis factor-related apoptosis inducing ligand (TRAIL) in a human chronic myeloid leukemia cell line (K562) naturally resistant to TRAIL. We observed that the tumoral K562 cells have high levels of RAC3 if compared with the non-tumoral HEK293 cells. The normal or transfected coactivator over-expression inhibits apoptosis through a diminished caspase activity and AIF nuclear translocation, increased NF-kappaB, AKT and p38, and decreased ERK activities. In contrast, inhibition of RAC3 by siRNA induced sensitivity of K562 to TRAIL-induced apoptosis. Such results suggest that over-expression of RAC3 contributes to tumor development through molecular mechanisms that do not depend strictly on acetylation and/or steroid hormones, which control cell death. This could be a possible target for future tumor therapies.

Assuntos

RESUMO

The nuclear receptor coactivator RAC3 plays important roles in many biological processes and tumorigenesis. We found that RAC3 is over-expressed in human chronic myeloid leukemia cells K562, which are normally resistant to TRAIL-induced apoptosis. RAC3 down-regulation by siRNA rendered these cells sensitive to TRAIL-induced cell death. In addition to the up-regulation of TRAIL receptors, the process involves Bid, caspases and PARP activation, loss of mitochondrial membrane potential, and release of AIF, cytochrome c and Smac/DIABLO to the cytoplasm. We conclude that RAC3 is required for TRAIL resistance and that this anti-apoptotic function is independent of its role in hormone receptor signaling.

Assuntos

RESUMO

RAC3 pertenece a la familia de coactivadores de receptores nucleares p160, y se encuentra sobreexpresado en varios tumores. Demostramos previamente que RAC3 es coactivador del factor de transcripción anti-apoptótico NF-kB. En este trabajo investigamos su rol en la apoptosis inducida por H2O2 en una línea celular no tumoral derivada de riñón embrionario humano (HEK293), y por el ligando inductor de apoptosis relacionado a TNF (TRAIL) en una línea de leucemia mieloide crónica humana (K562), naturalmente resistente a la muerte por este estímulo. Observamos que las células tumorales K562 poseen niveles altos de RAC3 comparados con las células no tumorales HEK293. La sobreexpresión normal de coactivador o por transfección, inhibe la apoptosis mediante una disminución de la activación de caspasas, translocación del factor inductor de apoptosis (AIF) al núcleo, aumento de la actividad de NF-kB y las quinasas AKT y p38 y disminución de la quinasa ERK. Lo opuesto fue observado por disminución de RAC3 mediante la técnica de ARN interferente (RNAi) en K562, aumentando así la apoptosis inducida por TRAIL. Estas evidencias sugieren que una sobreexpresión de RAC3 contribuye al desarrollo de tumores, participando en las cascadas que controlan la muerte celular por mecanismos no estrictamente dependientes de hormonas esteroideas y/o de acetilación, constituyendo esto un posible blanco de ataque para el tratamiento de tumores.

RAC3 belongs to the family of p160 nuclear receptors coactivators and it is over-expressed in several tumors. We have previously shown that RAC3 is a NF-kB coactivator. In this paper, we investigated the role of RAC3 in cell-sensitivity to apoptosis, using H2O2 in the human embryonic kidney cell line (HEK293), and tumor necrosis factor-related apoptosis inducing ligand (TRAIL) in a human chronic myeloid leukemia cell line (K562) naturally resistant to TRAIL. We observed that the tumoral K562 cells have high levels of RAC3 if compared with the non-tumoral HEK293 cells. The normal or transfected coactivator over-expression inhibits apoptosis through a diminished caspase activity and AIF nuclear translocation, increased NF-kB, AKT and p38, and decreased ERK activities. In contrast, inhibition of RAC3 by siRNA induced sensitivity of K562 to TRAIL-induced apoptosis. Such results suggest that over-expression of RAC3 contributes to tumor development through molecular mechanisms that do not depend strictly on acetylation and/or steroid hormones, which control cell death. This could be a possible target for future tumor therapies.

Assuntos

RESUMO

We have previously shown that nuclear receptor coactivator overexpression significantly enhanced NF-kappaB activity in a dose response manner. We studied the mechanism by which TIF2 regulates NF-kappaB activity. We determined that: 1) the p38 specific inhibitor reduces 50% NF-kappaB transcriptional activity, even in cells that overexpress distinct TIF2 deletions; 2) there is a physical interaction between TIF2 and p38 and RelA determined through in vitro translated protein binding assays; 3) TIF2 is a p38 substrate; 4) there is a physical interaction between TIF2 and IKK in TNF-alpha 20 ng/ml stimulated or not HEK 293 cell protein extract, and IkappaB only in basal conditions, determined by binding pull down assays. This NF-kappaB complex regulates its activity and targets gene expression in a determined physiologic context depending on the coactivator complex content.

Assuntos

RESUMO

We have previously shown that nuclear receptor coactivator overexpression significantly enhanced NF-kappaB activity in a dose response manner. We studied the mechanism by which TIF2 regulates NF-kappaB activity. We determined that: 1) the p38 specific inhibitor reduces 50

NF-kappaB transcriptional activity, even in cells that overexpress distinct TIF2 deletions; 2) there is a physical interaction between TIF2 and p38 and RelA determined through in vitro translated protein binding assays; 3) TIF2 is a p38 substrate; 4) there is a physical interaction between TIF2 and IKK in TNF-alpha 20 ng/ml stimulated or not HEK 293 cell protein extract, and IkappaB only in basal conditions, determined by binding pull down assays. This NF-kappaB complex regulates its activity and targets gene expression in a determined physiologic context depending on the coactivator complex content.