EXPRESSION OF HEAT SHOCK PROTEIN 27 IN PROSTATE CANCER CELL LINES ACCORDING TO THE EXTENT OF...
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Transcript of EXPRESSION OF HEAT SHOCK PROTEIN 27 IN PROSTATE CANCER CELL LINES ACCORDING TO THE EXTENT OF...
EXPRESSION OF HEAT SHOCK PROTEIN 27 IN
PROSTATE CANCER CELL LINES ACCORDING TO THE EXTENT OF MALIGNANCY
AND DOXAZOSIN TREATMENT
Seung Wook LeeJeong Woo LeeJae Hoon ChungJung Ki Jo María Isabel Pérez Palacio
Camilo Ruiz Mejía
INTRODUCTION
HEAT SHOCK PROTEIN 27
It is important in the control of
apoptosis (anti-apoptic agent) in
tumor and cell protection by
interacting with both mitochondrial
dependent and independent
pathways of apoptosis
HSP 27 interacts with caspase 9 pathways , interrupting caspase 3 activation and therefore inhibiting the apoptosis process. `
FUNCTIONS
HSP is related to the folding, activation, trafficking, and
transcriptional activity of most steroid receptors including
androgen receptor
Prostate cancer is becoming an increasingly common disease in men.
PROSTATE CANCER
During the normal aging process, the prostate gland grows , however in prostate cancer, malignant cells proliferate and cause adenocarcinoma
PROSTATE CANCER
Prostate cancer may cause Pain Difficulty
in urinating, Problems during
sexual intercourse Erectile dysfunction Death
PROSTATE CANCER
Many people have been treated successfully however it is as dangerous as any other cancer because of the risk that it may metastasize
PROSTATE CANCER
POSSIBLE TREATMENTSSurgeryRadiation therapyHormone therapyChemotherapy
PROSTATE CANCER
DOXAZOSIN (CARDURA)
Antitumor activity
It has a potent antigrowth effect against androgen
independent human prostate
cells
It is an R1 B adrenoreceptor
antagonist which confers it an
antiproliferative property
RELATIONSHIP
Prostate cancer is a common disease in men today
Possible treatment : Doxazosin
which is used as an anti proliferative in
malignant prostate cells
It can be hindered
by the expression of HSP 27
Who's purpose is to play a role in the regulation of apoptosis acting as an anti apoptotic agent.
GENERAL OBJECTIVE
The objective of this study was to analyze the
expression of HSP 27 in prostate cancer cells both
treated and not treated with Doxazosin.
MÉTODOS Y MATERIALES
SUJETOS
RWPE-1
LNCaP
Pc-3
TSU-Pr1
COLECCIÓN DE CULTIVOS
DE TIPO AMERICANO
SUJETOS
RWPE-1 LNCaP Pc-3 TSU-Pr1
Grupo control
Grupo tratado con 10 o 25 microM de Doxazosina
Grupo de control de vectores
tratado con DMSO
CULTIVO DE CÉLULAS
TSU-Pr1PC-3LNCaPRWPE-
1
Medio nutritivo F12(10% FBS)(Penicilina)
(Estreptomicina) • 37 C• 5% Co2
ANTES DE TRATAMIENTO
FRAGMENTACIÓN DNA
Se realizó esta prueba para analizar la apoptosis en la PC-3 tratada con doxazosina
FRAGMENTACIÓN DNA
Esta es una prueba o método cualitativo para la evaluación de la muerte celular mediante la detección de fragmentos de ADN.
Se basa en la utilización de electroforesis en gel de agarosa.
260 nm
FRAGMENTACIÓN DNA
Una de las características clásicas de la apoptosis es la escisión del ADN genómico en fragmentos oligonucleosomales
La visualización de estos fragmentos puede ayudar a la caracterización de un evento apoptótico.
RT- PCR
Esta prueba se utilizó con el fin de aislar el RNA total.
Se usa RT-PCR cuantitativa para cuantificar mRNA específicos, este análisis permite para una prueba más rápida, con una mayor sensibilidad, es simple y sencilla.
RT- PCR
TUNEL En el articulo se utilizó para analizar el efecto de la doxazosina en la apoptosis de PC-3
TUNEL es un método común para la detección de la fragmentación del ADN que resulta
de cascadas de señalización de apoptosis.
TRATAMIENTO DOXASOZINA
Todas las células fueron tratadas con una dosis letal
media de doxasozina y 0.25 % DMSO como el control.
INMUNOTINCIÓN Se pretendía observar la tinción de HSP27 en cada cultivo celular.
Se utilizó una inmunotinción directa.
Se comparo la intensidad de la tinción en los cuatro cultivos celulares.
INMUNOTINCIÓN
Absorben luz= 200-400 nm (luz
UV).
Emiten= 400-700 nm (luz
visible).
La luz es proporcional a la
cantidad de Ag.
Ag= HSP27.
RESULTADOS
NIVEL DE EXPRESIÓN DE HSP27
NIVEL DE EXPRESIÓN DE HSP27
•INMUNOFLUORECENSIA.
•La tinción para TSU-Pr1 fue la más intensa.
•El orden fue el siguiente:
•RWPE-1 <LNCaP <PC-3 <TSU-Pr1
EXPRESIÓN DE HSP27 EN TRATAMIENTO CON DOXAZOSINA
RT-PCR:
Control 50 %.
CV (DMSO) 60%.
10 µM 110 %.
25 µM 128%.
DOXAZOSIN INDUCE EXPRESIÓN DE HSP27
• INMUNOFLUORESCENCIA DE HSP27:
• Células HSP27 positivas Más intenso 10 µM y 20 µM.
DOXAZOSINA INDUCE APOPTOSIS.
• TUNEL:• La HSP se expresa en células con núcleo condensado
y escaso citoplasma (apoptosis).• Células que expresan HSP27 TUNEL
POSITIVO.
DISCUSSION
36
AUTHOR THEORY RESULTS
Rochi et al. The increase in HSP27 protected the cells in the process of CRPC. YES
Miyake et al. The expression of HSP27 is useful to predict
biochemical recurrence in prostate cancer
YES
37
AUTHOR THEORY RESULTS
Rochi et al. They found that HSP27 played a role as the apoptosis controller. YES
Teimourian et al. After irradiation, there was less viability in cells
that reduced the expression of HSP27,
that in those who didn´t.
YES
CONLUSIONS
CONCLUSIONS
It is important to study the expression of HSP27, because it
gives us a chance to look for new therapeutic options.
Prostate cancer is really common disease, and we need to
investigate more about it.
39
The study of heat shock proteins might give us a
chance to understand the development of tumoral
processes.
If we find new ways of treatment, we might reduce the
mortality level due to prostate cancer.
CAMILO RUIZ MEJÍA
María Isabel Pérez Palacio
BIBLIOGRAFIA
1. Xanthoudakis S., & Nicholson D.W. Heat-shock proteins as death determinants. nature (internet). 2000 (citado marzo 7 de 2014);2:1-9. Disponible en:http://www.nature.com/ncb/journal/v2/n9/fig_tab/ncb0900_E163_F1.html
2. Martínez L.M., Vargas N., Toro A.E., Pamplona A.P., Quevedo E. Biologia Molecular, 7ma edición. Medellín, Colombia: UPB. 2012;31(3): 247-243.
3. Seung W.L.,Jeong W.L., Jae H.C., Jung K.J. Expression of heat shock protein 27 in prostate cancer cell lines according to the extent of malignancy and doxazosin treatment. World J Mens Health; 2014