Seminario de Biologia Molecular UPB 2015 (p53)

Presentado por: Juana Hoyos Palacio Jennifer Posada Caro

INTRODUCTION

CSN1-CSN8COP9 CSN5

Catalytic center of COP9

Regulation of tumor suppressors ad oncogen

productosstability of intracellular regulators of cell process

Overexpressed in a variety of human cancers

Ubiquitin proteasome pathway

Removal of NEDD8 from CRL

CANCER

Cancer is a potentially fatal disease caused by environmental factors that mutate genes encoding critical cell-regulatory proteins. The resultant aberrant cell behavior leads to an abnormal cell proliferation that destroy surrounding normal tissue and can spread to vital organs resulting in a disseminated disease.

GASTRIC CANCER Gastric cancer (GC ) is an uncontrolled growth of cells in the inner surface of the stomach. Early gastric cancer has no associated symptoms; so most symptoms of gastric cancer reflect advanced disease and by the time they develop, the disease is almost invariably too far advanced for curative procedures.

• Indigestion• Nausea or vomiting• Dysphagia• Postprandial fullness• Loss of appetite• Melena or pallor from anemia• Hematemesis• Weight loss

GASTRIC CANCER Gastrointestinal stromal tumor Carcinoid tumor

Adenocarcinoma

p53P53 is a nuclear phosphoprotein of 53 kDa, encoded by a 2 gene located on the small arm of chromosome 17. It contains five functional domains: N‐terminal  domain, Proline  rich  domain, the  large,  central  core  domain, the  oligomerization  domain and the  basic  C‐terminal domain.

CSN5

CSN5

Proteasome

APOPTOSIS

OBJECTIVE

Investigate the molecular mechanisms by which CSN5 is involved in gastric cancer.

Materiales y Métodos

Características de los pacientes

Cáncer gástrico +

diagnostico preoperatorio

+ ausencia de tratamiento

preoperatorio con radio o quimioterapia.

Materiales y Métodos Cultivo

Celulas GES + AGS + MKN-45

Cultivo

Anexos 10% suero bobino

37º 5% CO2

Todas las celulas tuvieron un 50% de

crecimiento antes de la transfección

Cultivo

Materiales y Métodos Transfección

CSN5 siRNA siLentFect Lipid Reagent

Non-specific siRNA = control

-se encubaron muestras en un nuevo medio

- después se lisaron

Materiales y Métodos Homogenización

ANGT + GCT Lavado 2 veces con PBS Homogenizacion Centrifugar

Centrifugar sobrenadantes

Remover sobrenadante

Medicion proteinas simples Leupeptina

AprotininaPepsatina

Materiales y Métodos SDS-PAGE Incubación con ac

Western Blot

Materiales y Métodos Variabilidad celular

- 3000 cèlulas de AGS- 2000 cèlulas de MKN45- Ya con su tranfecciòn- Labilidad medida con Cell

count kit

Materiales y Métodos FITC

- Se cultivaron las células del cáncer gástrico con V-FITC

- Se analizò la actividad apoptótica con FACScan Cytometer / microscopio fluorescencte

RESULTADOS

Figura 1

Figura 2

RESULTADOS

Figura 4

RESULTADOS

Figura 4

RESULTADOS

Figura 5

Discusión Author Exposure Opinion

Brady, Gil-Gomez “Bax can be directly regulated by p53 and showed increased expression in certain tissues after apoptotic stimule “

true

Zhang, Chen, Su, Yang, Lee “CSN5 has been reported as a negative regulator of p53”

true

Discusión Author Exposure Opinion

Reed “Bax is a pro-apoptotic member of the Bcl2 famimly proteins, which serve as critical regulators of pathways involved in apoptosis, acting to either inhibit or promote cell death”

True

Roder “Gastric cancer remains one of the leading causes of cancer death worldwide”

True

Conclusion -We think this study is important because now researchers can go more over and think about a key to prevent or fight against gastric cancer

-There are many people that could be beneficiated with this investigation because is another step foreward to know more about gastric cancer, one of the most deathly cancer ever.

-Investigators should apply this investigation to other type of cancer, so they can know more out it and how to handle with it

-Science should invest in new treatments for patient so that they can get more succesfull results in treatment.

Jennifer

Juana