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Cells and cancerDr Lisa Chopin

School of Life Sciences, QUT

Leader, Ghrelin Research Group, IHBI,

QUT

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Cells and cancer

• The building blocks of cells

• The basic cell• Cancer• Cancer processes• Hormone dependent

cancer

http://www.newscentre.bham.ac.uk/press/2007/10/Cancer_Cold_Virus_03_10_07.shtml4

Cells• The building blocks of cells

– Proteins, carbohydrates and fats (lipids)

• The basic cell– The plasma membrane– Organelles– The cytoskeleton

• Cells form tissues– The nucleus

• DNA and RNA

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Building blocks of life

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Levels of structural organisation• many levels of organisation

• smallest = chemical level

• atoms

• molecules

•Cells

•Tissues

•organs

• largest = organism

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Building blocks – we are what we eat

• The body– Carbohydrates– Proteins– Lipids (fat)– Nucleic acids– Water – Vitamins and

minerals

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• sugar and starches

• Large part of our diet

• make up 1-2% of a cell•C = carbon

•H = Hydrogen

•O = oxygen

• form a ring structure

•Monosaccharide•Simple sugar

•Building blocks of sugars

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•Disaccharides •= 2 sugars joined•eg lactose (milk)

•Polysaccharides•Poly = many•Long chains of simple sugars

•Starch = plant polymer of glucose

•potatoes 10

Protein

• We eat 125g/day• adults need 40-50g/day• replace proteins• 20-60g/day lost

– secretions– shedding of gut cells into

lumen

• In diet– Fish, meat, eggs, nuts

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•Building blocks = amino acids

•20 amino acids in animals

•Joined to make proteins

•Can form very large polymers = proteins•Can form complex structures •or function as amino acids•Short = peptides

http://www.bio.davidson.edu/Courses/Molbio/MolStudents/spring2005/Heiner/hemoglobin.html 12

•Basic material making up the body structure

•Hair •Fingernails•Skin•Muscle

•Also important for function•Hormones•Enzymes•Neurotransmitters•Oxygen transport•antibodies

http://srs.dl.ac.uk/Annual_Reports/AnRep95_96/Protein%20crystallography.html

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Lipids (fats)•25-160g/day

•Plant and animal fat

•Not soluble in water

•C, H, O (P)

•Lipids

•Triglycerides

•Phospholipids

•steroids14

Lipids (fats)

•Triglycerides•Most common fats•Building blocks

•Fatty acids•3 Long chains

•+ 1 Glycerol

http://www.chemistryland.com/ElementarySchool/BuildingBlocks/BuildingOrganic.htm

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Phospholipids

• Modified triglycerides• 2 fatty acid chains• Phosphate group (P)• Special properties• Very important in biology

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Cells• all living things made of cells

– (if they can replicate)

•Trillions in the body•Hundreds of different types

http://www.icr.ac.uk/ieu/projects/CancerScienceWebsite/AnimGallery/AnimGallery.htm

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The basic cellPlasma membrane

(cell membrane)

Cytoplasm

Semi-fluid

Nucleus

Control centre18

The plasma membrane• made up of phospholipids

•Head = likes water

•Tail = hates water

• In water (in the body)

•tails repel water

•Clump together

http://www.aber.ac.uk/gwydd-cym/cellbiol/cellmembrane/index.htm

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Plasma membrane

• contains other lipids

• cholesterol

•Proteins

•Sugars

Cytoplasm = watery

Extracellular fluid = watery environment

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• Cytoplasm– Intracellular fluid

• Outside the cell– Extracellular fluid– Different to

intracellular fluid– Ions– Nutrients– Waste

• Plasma membrane– Barrier between

intracellular fluid + extracellular fluid

Extracellular fluid

Intracellular fluid

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• Some substance must pass between 2– Nutrients into cell– Wastes out of cell

• Not freely as they would become equal

• Plasma membrane = selective barrier

• = selectively permeable

Extracellular fluid

Intracellular fluid

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Passive transport –simple diffusion

• Important in all cells

• Molecules always moving– Thermal motion

• Collide with each other

• Scatter evenly throughout

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Brownian motion

http://www.microscopy-uk.org.uk/dww/home/hombrown.htm24

Passive transport - Diffusion• Molecules move from higher

concentration lower concentration– collisions

• Until equally distributed– Reach equilibrium

• concentration inside and outside cell and inside cell are equal

• Molecules diffuse along their concentration gradient– Chemical driving force

http://bio.winona.edu/berg/ANIMTNS/Directry.htm

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Osmosis

• http://www.biologycorner.com/resources/osmosis.gif

• Water diffuses from high water concentration to low water concentration– Water diffuses by

osmosis towards higher solute concentration

• Membrane is semi-permeable– Not freely

permeable to solutes

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Tonicity• the effect that a solution will have on a cell

• hypertonic (Hyper = above)•Higher solute concentration than in the cell

•Hyperosmotic

•Water moves out by osmosis

•Iso-tonic (iso = the same)•Iso-osmotic

•Same solute concentration

•Hypotonic (hypo = below)•Less solute in the solution than in the cell

•Hypo-osmotic

•Water moves in by osmosis

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The cytoplasm • Cytoplasm

– Cellular material – Between nucleus and

plasma membrane• cytosol

– Fluid• Cytoplasmic organelles• cytoskeleton

http://www.aber.ac.uk/gwydd-cym/cellbiol/transport/index.htm

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Cytoplasmic organelles• little organs• Cell machinery• Include

– Mitochondria– ribosomes– Lysosomes– Endoplasmic

reticulum– Golgi apparatus

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Cytoplasmic organelles -Mitochondria

• power plant of cell•energy source for cell

•Makes ATP

•Ancient Bacterial origin•Has its own DNA

•Inherit from mother

http://www.mitochondrial.net/ 30

Energy

• ATP is made in mitochondria – adenosine triphosphate– Energy stored in the cell as ATP

• Made from energy from food– Carbohydrates– Fats products• Amino acids

• Products – energy (ATP) + O2+ water

adenosine P P P

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Energy

adenosine P P P

adenosine P P P+ + EnergyADP

•Break High energy bonds

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Cytoplasmic organelles •Organelles work

together • Produce• Store• export substances• Degrade substances

http://cmweb.pvschools.net/~bbecke/newell/Cells.html

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Endoplasmic reticulum (ER)• “within cell

network”• Network of tubes

and membranes• Fluid filled sacs • Continuous with

nuclear membrane

• 2 types– Smooth – Rough

http://www.unipv.it/webbio/anatcomp/freitas/2006-2007/smooth%20&%20rough.jpg

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• studded on outside with ribosomes

•Proteins synthesised

•On ribosomes

•Outside of RER

•Enter RER

•Processed and modified

Rough Endoplasmic reticulum (RER)

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Cytoplasmic organelles -ribosomes

• small

•Dark staining granules

•Contain RNA

•Ribosomal RNA

•Site of protein synthesis

• bound or free

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• extension of RER

• No ribosomes

• No protein synthesis

•Many roles •Lipid metabolism and synthesis

•Steroid synthesis

•Drug detoxification

Smooth endoplasmic reticulum (SER)

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Golgi apparatus• membranous sacs

• Stacks of dinner plates

•Tiny vesicles•Fluid filled spheres

•Function:•Modify

•Concentrate

•Package

ER products

• Proteins

•lipids

http://www.nih.gov/sigs/cellbio/38

Golgi• protein/lipid vesicles reach Golgi from RER

•Fuse with receiving side of Golgi

•Contents emptied into Golgi

•Move through Golgi

•Enzymes modify proteins

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3 Golgi pathways

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The cytoskeleton• Cell skeleton• Support

structures of the cell

• Attach cells to each other

• Allows cells to move

• To move things in the cell

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The cytoskeleton

• 3 types of cytoskeletal elements– 1. microtubules– 2. microfilaments– 3. intermediate

filaments• Support rods• Network in the

cell42

The cytoskeleton-microfilaments•actin

•Functions:

•Mechanical strength for cell

•Attach cytoplasm to the cytoplasmic membrane

•Needed for cell movement (muscle)

http://www.cerebrospinalfluidresearch.com/content/3/1/13/figure/F1

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The cytoskeleton-microtubules

• Function– Shape of cell– Strength– Motor- move organelles

along microtubules– Form structures

• Cilia• flagella

– Cell divisionMicrotubules in green, actin in red

http://migration.wordpress.com/2007/07/11/basics-the-cytoskeleton/

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The cytoskeleton-intermediate filaments

• intermediate between the 2

• rope-like

•Fibrous

•Very strong

•Resist forces applied to cell

•Cell shapehttp://www.olympusfluoview.com/applications/protocols/cellsintermediatefilaments.html

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Cytoskeleton – joining cells together

• Epithelium = a tissue– 90% of human

cancers are epithelial• Sheets of cells

– Skin– Mucous membranes

• linings• Nests of cells

– glands

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Epithelium• Made from epithelial cells• Forms glands• Covers all surfaces of the

body• Boundary between

environments– Skin and outside body– Bladder wall and urine in

the bladder

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Epithelium - characteristics

• Many cells, tightly packed

• Make special contacts with each other– Tight junctions– Desmosomes

• Connect with cytoskeleton

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Tight junctions and

desmosomes–attach epithelial

cells together

• Tight junctions - like a zipper, Proteins in the plasma membrane, Joins plasma membranes• Desmosomes – Anchors, Like buttons and thread,

Attach to cytoskeleton

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Extracellular matrix (ECM)• Epithelium sits

on ECM• Lots of space • Extracellular

matrix secreted by cells– Water– Proteins– Carbohydrates

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Basement membrane

• Specialised extracellular matrix

• Connects cells to connective tissue underneath– Glycoprotein

• Contains no cells• Protects epithelial

layer• Helps resist forces• Anchors cells• Affects cell

behaviour

Basement membrane

Epithelial cells

http://fig.cox.miami.edu/~cmallery/150/memb/epithelial_cell.jpg

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Collagen

• Major component– Basement

membrane– ECM

• Protein– Long polymer

http://www.accessexcellence.org/RC/VL/GG/ecb/collagen_elastin.html

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Glands• Cell or group of

epithelial cells that make a product

• Product is exported – Secreted

• Product = a secretion• 2 types of glands

– Endocrine• Produce hormones

– Exocrine• Many types• Sweat, digestive enzymes,

saliva, mucus

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Mucus• Slimy coating protects

epithelial surfaces• Protective

– Eg stomach protected from acid

• Lubricating– Saliva– Swallow food

• Mucins– Protein with many sugar

groups coating it

http://712designs.com/animations.htm 54

The nucleusPlasma membrane

(cell membrane)

Cytoplasm

Semi-fluid

Nucleus

Control centre

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The nucleus

• Cell’s computer• Stores genetic

information• Instructions for

making proteins• Usually round or

oval• Centre of the cell

http://www.york.ac.uk/res/sots/activities/diydna.htm56

Nucleic acids

• DNA• RNA• Largest

molecules in the body

• building blocks = nucleotides

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DNA and RNA• nucleotides

•phosphate

•Sugar•Deoxyribose

•ribose

•Base = code•AGCT – DNA

•AGCU - RNA 58

• Double stranded• Double helix• carries genetic

material• Genes

http://www.scar.utoronto.ca/~olaveson/A01F-Fall2007.html

http://www.biologycorner.com/bio1/DNA.html

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RNA• No double helix• Single stranded

– Can base-pair with itself

• Different roles– Protein synthesis– Gene regulation

• May be very important– Differences between

species

http://www.biologycorner.com/bio1/DNA.html60

Nucleus -chromatin

•Chromatin•DNA stored in the nucleus as chromatin•Packaged DNA•Very long molecules

http://openlearn.open.ac.uk/file.php/2645/S377_1_039i.jpg

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Chromosomes

• DNA stored as chromatin• Even more packaged into chromosomes

for cell division• Chromosomes 50 000 times shorter than

DNA unpackaged

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Cell division = mitosis• Cell proliferation

– Happens about 25 million times per second

• Depends on cell type– Embryo

• Very rapid– Gut cells and skin cells

• Rapidly turnover• Prone to damage

– Brain cells• Up to 100 years old

– Liver cells• In response to signal

http://www.cancerhelp.org.uk/help/default.asp?page=95http://www.biophysik.uni-bremen.de/radmacher/animations.html 64

Cell division

• blue = DNA

• green = Microtubules

• red = intermediate filaments http://www.biophysik.uni-bremen.de/radmacher/animations.html

• cells divide into 2 daughter cells

•Double DNA content

• DNA condenses into chromosomes

•Change in cytoskeleton

•Forms mitotic spindle

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Cell division

• chromosomes moved to opposite poles

• nucleus reforms

• cells form 2 daughter cells

•Equal number of chromosomes

•Cells growhttp://www.biologycorner.com/bio1/cellcycle.html http://www.learner.org/channel/courses/essential/life/session3/closer1.html#animation

66http://www.linkpublishing.com/video-mitosis.htm

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The cell cycle – control of cell division

• Very tight control

• Growth factors• If DNA

damaged cycle is halted

http://www.emc.maricopa.edu/faculty/farabee/BIOBK/BioBookmito.html

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Protein synthesis

• DNA (genes) code for all proteins

• Must be decoded• DNA RNA protein

• Controlled by nucleus

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DNA RNA• Transcription• DNA copied into

RNA• mRNA

– Messenger RNA• RNA is further

processed• Exported from the

nucleus

http://www.biologycorner.com/bio1/DNA.html

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RNA protein• Translation• RNA (nucleic acids)

translated proteins (amino acids)

• Made by ribosomes• Into rough

endoplasmic reticulum

• To Golgi for processing

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DNA damage causes cancer• DNA damage

– Sunlight– radiation– Chemicals– Smoke– Car exhaust– Normal life processes

• Using oxygen!

• A few cancers inherited– Mutation in gene– Further damage required

• Often no clear causehttp://www.dmacdigest.com/cancer.html

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DNA damage• Repeated damage to DNA

– Constantly occurring• DNA in one cell damaged 10 000

times a day– More damage with age

• Protection against cancer– DNA repair mechanisms– Cells commit suicide

• apoptosis– Immune system destroys cells

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Apoptosis• Active process• RNA and proteins

made• Break down of

cytokeleton• Breakdown of nucleus

and DNA laddering• Cytoplasm thrown off• Mitochondria involved• Engulfed by white

blood cellshttp://www.dadamo.com/wiki/wiki.pl/Apoptosis http://student.ccbcmd.edu/courses/bio141/lecguide/unit5/cellular/cmidefense/ctls/ctlapop.html

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• Proliferation

• Invasion

• Migration

• Anti-apoptosis

• Angiogenesis

• Metastasis

Changes in cancer cell behaviour – the hallmarks of cancer

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Proliferation• Cell division usually

tightly controlled• Cancer = uncontrolled

cell division– Divide rapidly– Don’t mature properly

• Tumour = mass of cells– Benign– malignant

http://www.bme.gatech.edu/vcl/Tissue_Engineering/Background/5_cell_seeding.htm

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Cell death

• Cancer cells become immortal

• Evade apoptosis– Don’t undergo cell

death when DNA is damaged

• = survival

http://www.cat.cc.md.us/courses/bio141/lecguide/unit3/intro/nk/nkapop.html 78

Angiogenesis

• Synthesis of new blood vessels– Angio– genesis

• Normal – Growth as children

• Abnormal– Cancer

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Angiogenesis • Cells need a blood supply for oxygen to generate energy

• Cancer cells outgrow their blood supply– Start to starve– Make energy without

oxygen– Need a lot of energy

due to proliferation• Cancer cells send

signals• Encourage more

blood vessels to form and grow = angiogenesis

http://www.gene.com/gene/research/focusareas/oncology/angiogenesis.html

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Cell Migration• Some cells can move• Cytoskeleton

changes• Forms new anchors

– adhesions• to crawl along

extracellular matrix– Action of actin– Part of cytoskeleton

http://www.biolsci.org/v03p0303.htm

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Some normal human cells can crawl

• Cells in the embryo• Brain development• Cells in wound healing

and blood clotting– Platelets– Skin cells

• White blood cells

http://www.rockefeller.edu/labheads/hatten/mechanism.htmlhttp://www.youtube.com/watch?v=eTWc4PFSpzQ

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Cancer cell migration• Cancer cells migrate

– Most normal cells dont• Must break anchors

– Neighbouring cells– Extracellular matrix

• Invasion– through

• the basement membrane• Extracellular matrix

– Enzymes break down structures

• Pathway for cell migration

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Metastasis• Migrating cells

reach blood vessel or lymphatic vessel– Travel to distant

sites– = metastasis

• Few survive• Exit vessel• Colonise distant

sites– Form metastases

http://www.nature.com/embor/journal/v7/n11/full/7400839.html

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Metastasis

• Major cause of cancer death

• Spread of cancer cells to distant sites

• No cure

http://www.nature.com/nrc/journal/v3/n1/fig_tab/nrc967_F1.html

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Hormone dependent cancers

• Prostate and breast cancer

• Prostate cancer – most commonly diagnosed

cancer in men– Affects 1 in 11 Australian

men by the age of 80• Androgens stimulate

growth– testosterone

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Hormone dependent cancers

• Breast cancer – most commonly

diagnosed cancer in women

– Affects about 1 in 11 Australian women

• Oestrogens stimulate growth

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ANDROGENS in ANDROGENS in males males (testosterone)(testosterone)

EPITHELIUM

Extracellular matrix

GROWTH FACTORSGROWTH FACTORS

Basement membrane OESTROGEN OESTROGEN

in femalesin females

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Ghrelin

• Peptide hormone • Discovered in 1999• Causes growth hormone release• Role in increasing appetite

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• http://www.gghjournal.com/volume20/1/featureArticle.cfm

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Ghrelin action• Acts by binding

a receptor on the cell surface– Protein

embedded in the plasma membrane

– Cannot pass through the lipid bilayer

• Signal passed on through the receptor– Signaling

molecules in the cell

• Study role of receptor in cancer

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Ghrelin is made in prostate and breast cancer cells

• Stain cells in cancer specimens – ghrelin peptide present in tissues– Epithelial cells

negative

positive92

ANDROGENS in ANDROGENS in males males (testosterone)(testosterone)

EPITHELIUM

Extracellular matrix

GhrelinGhrelin

Basement membrane OESTROGEN OESTROGEN

in femalesin females

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Proliferation assays

• Breast and prostate cancer cells produce ghrelin– Cancer growth factor?

• Grow breast and prostate cancer cells – 37C– Media with nutrients– Immortal– Sterile conditions

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Grow in normal culture conditions for 4, 24, 48, 72 and 96 hrs

2000cells

Media blank

Cells

Add WST-1

2 hrs

Read plate at 650-405 nm

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Proliferation assays

• Breast and prostate cancer cells grow faster in the test tube when treated with ghrelin

05

101520253035404550

0 0.1 1 10 100 1000

Ghrelin concentration (nM)

% A

bove

con

trol

*

**

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Migration - ghrelin

• Migration assays• Double well

– Cells migrate through a membrane

– 8μm pores – Cells rearrange

cytoskeleton to pass through

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Tissue cultureinsert

Well

Cells in medium

20% FCS

Remove unmigrated cellsFix and stain migrated cells

Readplate at595 nm

24hr Incubation

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Migration assays

• Cancer cells migrate faster in response to ghrelin treatment– Breast cancer– Ovarian cancer

Mean SKOV3 Migration Over 24hr

00.10.20.30.40.50.60.70.80.9

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media 10nMGhrelin

100nMGhrelin

10nM Ghr10nM Obe

10nM Ghr100nM Obe

10nMObestatin

100nMObestatin

Treatment

Abs

orba

nce * * * * *

B. Mean SKOV3 Migration Over 24hr

00.10.20.30.40.50.60.70.80.9

1

media 10nMGhrelin

100nMGhrelin

10nM Ghr10nM Obe

10nM Ghr100nM Obe

10nMObestatin

100nMObestatin

Treatment

Abs

orba

nce * * * * *

B.

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Invasion assays

Matrigel

PBSTissue cultureinsert

Well

Cells in medium

20% FCS

Remove uninvaded cells and MatrigelFix and stain invaded cells

Readplate at595 nm

24hr Incubation

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Ghrelin

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Cancer biomarkers• Compare proteins

produced by normal breast and breast cancer

• Breast cancer very varied disease

• At least 5 different diseases– Need to be able to diagnose

better– Find new tests– New treatments

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References and source of figures

• Marieb (2004) Human anatomy and physiology 6th edition

• Martini (2004) Fundamentals of human anatomy and physiology

• http://www.cancerquest.org/index.cfm?page=3102#

• http://cellbase.ascb.org/education.html• Thanks to: Carina Walpole, Rachael Murray,

Penny Jeffery,