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

DNA fragmentation and cell cyclearrest: a hallmark of apoptosis

induced by crocin from KashmiriSaffron in a human pancreatic cancer

cell line

Hamid Bakshi et ál.

Alejandro Cardona PalacioMallory Fernández Zurita

Medical SchoolMolecular Biology

III SemesterUPB

INTRODUCTION

CELL CYCLE

In eukaryotes it is divided in two phases:

• The cellgrow.

• The cellreplicatesthe DNA.

Intherphase

• The cellsplit.

• The cellcreate twodaughtercells.

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 juicethat contain enzymes thatare involved in the process ofthe absortion of thenutrients by making someparts of the GI tract morealcaline.

Endocrine pancreas:

It segregates insuline andglucagon, hormones that regulatethe levels of glucose in the blood.

PANCREATIC CANCER

Exocrine pancreas:

It segregates pancreatic juicethat contain enzymes thatare involved in the process ofthe absortion of thenutrients by making someparts of the GI tract morealcaline.

Endocrine pancreas:

It segregates insuline andglucagon, hormones that regulatethe 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 wholeprocess of apoptosis.

• In most cases the DNA appears fragmented and, infact, this fragmentation is used asa marker of cell death by apoptosis.

• The first fragmentation in DNA conformation leads tothe 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 elementextracted by a plant that growsin India can induce apoptosis ina pancreatic cancer cell line andif also can stop the proliferation.

MATERIALES Y MÉTODOS

PLANTAS

• Uso Crocus sativus.

• Plantas recolectadas enIndia.

• Preservaron en ellaboratorio bajo lareferencia BUBT-1705.

AISLAMIENTOS

• Se separo la crocina del Crocus sativus.

• Es el elemento que realmente tiene una acción queinduce la apoptosis en la células cancerígenaspancreáticas.

CULTIVOS CELULARES

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

o En un medio RPMI-1640.

o Obtención de la cantidad de células necesarias para elestudio.

o Pertenecientes a una misma línea.

VIABILIDAD CELULAR

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

DETECCIÓN DE LA

APOPTOSIS

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

Es necesario realizar una adecuada detección de laapoptosis normal de estas células.

Después de aplicar el compuesto natural podremos sabersi 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 despuésde 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 deaplicado.

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 horasde incubación.

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

brillantes núcleos condensados azulfluorescente yreducción de volumen.

ANÁLISIS DE LA

FRAGMENTACIÓN DE DNA

Se examinó la muertecelular por fragmentacióndel DNA de 0 a 36 horas.

10 µg/mL de Crocina fueronmás que suficientes parainducir la fragmentación.

ELECTROFORESIS EN GEL AGAROSA

DEMOSTRANDO LA FRAGMENTACIÓN DEL DNA

INDUCCIÓN DE LA APOPTOSIS Y FRENO

DEL CICLO CELULAR

DISCUSSION

Author Agreeor

disagree

Guzman M. Agents capable of inducing apoptosis,inhibiting cell proliferation, or modulatingsignal transduction are currently used for thetreatment of cancer. A combination of multiplechemo-preventive agents or agents withmultiple targets is considered to be moreeffective than single aggent.

Hamid Bakshi et ál. Crocus sativus has been shown to induceapoptosis 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 severalmedical condition, such as gastrointestinaldisorders, urological infections, as well as intreating malignancies.

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

safranal, alpha crocin or crocin is the majorcomponent. Adiccionaly Crocus sativus containsamino acids, flavoniods and other chemicalcompounds.

Winterhalter et ál. Among these, crocin is the most important sinceit is the major component in Crocus sativus andhas shown significant biological activities.

PERSONAL OPINION

This kind of investigation have an inmense impact inthe live and treatments of people with pancreaticcancer, that’s why they should be done more oftenand should be known, so the patients and doctors canused them when they have no other choice.

The conclusions of this study shows us that naturalelements can help us cure or treat some of the worstdiseases that had increased on the recent years.

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

It is necessary to start experimenting on human beings , toobserve the advantages that this new study presents. Andinvestigate the adverse effects, pharmacologicalinteractions and dosings.

MAPAS CONCEPTUALES

Alejandro Cardona Palacio

Mallory FernándezZurita