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