dna fragmentation and cell cycle arrest: a hallmark of apoptosis induced by crocin from kashmiri...

DNA fragmentation and cell cycle arrest: a hallmark of apoptosis

induced by crocin from Kashmiri Saffron in a human pancreatic cancer

cell line

Hamid Bakshi et ál.

Alejandro Cardona PalacioMallory Fernández Zurita

Medical School Molecular Biology

III SemesterUPB

INTRODUCTION

CELL CYCLE

In eukaryotes it is divided in two phases:

• The cell grow.

• The cell replicates the DNA.

Intherphase

• The cell split.

• The cell create two daughter cells.

Mitosis

CELL CYCLE

• Organized process

• Events occur one after the other

• It’s regulated

• p53

CANCER

• Abnormal cells tend to multiply.

• Surrounding tissue.

• Metastasis: cancer emigrates from one organ to the other.

PANCREATIC CANCER

• The cases has increased in recent

years.

• Has not yet found a truly effective

treatment.

• The symptoms appear when the

disease has progressed.

PANCREATIC CANCER

Exocrine pancreas:

It segregates pancreatic juice that contain enzymes that are involved in the process of the absortion of the nutrients by making some parts of the GI tract more alcaline.

Endocrine pancreas:

It segregates insuline and glucagon, hormones that regulate the levels of glucose in the blood.

PANCREATIC CANCER

Exocrine pancreas:

It segregates pancreatic juice that contain enzymes that are involved in the process of the absortion of the nutrients by making some parts of the GI tract more alcaline.

Endocrine pancreas:

It segregates insuline and glucagon, hormones that regulate the levels of glucose in the blood.

APOPTOSIS

• Programmed death of the cell.

• In cancer this death doesn’t happen and the cell keeps growing.

DNA FRAGMENTATION

• Considered the underlying cause of the whole process of apoptosis.

• In most cases the DNA appears fragmented and, in fact, this fragmentation is used as a marker of cell death by apoptosis.

• The first fragmentation in DNA conformation leads to the last enzyme to act between the joints of the nucleosomes.

As we all can see if the elements that regulate the cell cycle are affected in some way, the DNA fragmentation and apoptosis may

not occur.

So those abnormal cells proliferate and Cancer can appear.

GENERAL OBJECTIVE

Find out if a natural element extracted by a plant that grows in India can induce apoptosis in a pancreatic cancer cell line and if also can stop the proliferation.

MATERIALES Y MÉTODOS

PLANTAS

• Uso Crocus sativus.

• Plantas recolectadas en India.

• Preservaron en el laboratorio bajo la referencia BUBT-1705.

AISLAMIENTOS

• Se separo la crocina del Crocus sativus.

• Es el elemento que realmente tiene una acción que induce la apoptosis en la células cancerígenas pancreáticas.

CULTIVOS CELULARES

o Se realizó un cultivo de células cancerígenas pancreáticos humanas de una línea donada.

o En un medio RPMI-1640.

o Obtención de la cantidad de células necesarias para el estudio.

o Pertenecientes a una misma línea.

VIABILIDAD CELULAR

Se realizo una evaluación para determinar si las células cumplen los estándares específicos basados en una absorción de 630 nm, para ser utilizadas en este estudio y tener resultados fiables.

DETECCIÓN DE LA APOPTOSIS

Este estudio esta basado en las alteraciones que puede causar la crocina en la apoptosis de una célula.

Es necesario realizar una adecuada detección de la apoptosis normal de estas células.

Después de aplicar el compuesto natural podremos saber si hubo algún cambio en el proceso.

ANÁLISIS DEL CICLO CELULAR

Lavadas PBS.

Incubadas con PI.

Citometria de Flujo.

Programa M Cycle software.

ANÁLISIS DE FRAGMENTACIÓN

Las células fueron tratadas con 10 µg/mL de crocina.

Se añadieron 10 µg/mL de Actinomicina D como análisis positivo.

Las células fueron intervenidos en un momento específico después de la aplicación de crocina.

Su DNA fue analizado a través de electroforesis por gel.

RESULTADOS

VIABILIDAD CELULAR

La Crocina indujo apoptosis de la línea BxPC-3, produciendo una disminución de la proliferación.

Pico de Incremento surge después 72 horas de aplicado.

Contribuye al potencial antiproliferativo.

ANÁLISIS DEL CICLO CELULAR

Incremento de la fase G1 al aplicar la Crocina por 2 días.

La detención del ciclo 48 horas después fue menor y el mayor incremento se dio a las 72 horas.

DETECCIÓN MORFOLÓGICA DE LA APOPTOSIS

La crocina indujo un patrón de apoptosis tras 72 horas de incubación.

La acumulación de células en G1 parece llevar a la apoptosis en una etapa posterior.

brillantes núcleos condensados azulfluorescente y reducción de volumen.

ANÁLISIS DE LA FRAGMENTACIÓN DE DNA

Se examinó la muerte celular por fragmentación del DNA de 0 a 36 horas.

10 µg/mL de Crocina fueron más que suficientes para inducir la fragmentación.

ELECTROFORESIS EN GEL AGAROSA DEMOSTRANDO LA FRAGMENTACIÓN

DEL DNA

INDUCCIÓN DE LA APOPTOSIS Y FRENO DEL CICLO CELULAR

DISCUSSION

Author Agree or

disagree

Guzman M. Agents capable of inducing apoptosis, inhibiting cell proliferation, or modulating signal transduction are currently used for the treatment of cancer. A combination of multiple chemo-preventive agents or agents with multiple targets is considered to be more effective than single aggent.

Hamid Bakshi et ál. Crocus sativus has been shown to induce apoptosis in human cancer cell lines.

Author Agree or disagree

Nair et ál.Rios et ál.Winterhalter et ál.

Crocus sativus has been used to treat several medical condition, such as gastrointestinal disorders, urological infections, as well as in treating malignancies.

Nair et ál.Winterhalter et ál. Crocus sativus contains components like

safranal, alpha crocin or crocin is the major component. Adiccionaly Crocus sativus contains amino acids, flavoniods and other chemical compounds.

Winterhalter et ál. Among these, crocin is the most important since it is the major component in Crocus sativus and has shown significant biological activities.

PERSONAL OPINION

This kind of investigation have an inmense impact in the live and treatments of people with pancreatic cancer, that’s why they should be done more often and should be known, so the patients and doctors can used them when they have no other choice.

The conclusions of this study shows us that natural elements can help us cure or treat some of the worst diseases that had increased on the recent years.

The medical advantage in the 21st century, predicts that diseases, that were considered incurable as the cancer, could have a successful treatment. A great example is the use of the plant Crocus sativus for the treatment of one of most aggressive cancer.

It is necessary to start experimenting on human beings , to observe the advantages that this new study presents. And investigate the adverse effects, pharmacological interactions and dosings.

MAPAS CONCEPTUALES

Alejandro Cardona Palacio

Mallory FernándezZurita