Chinitz

EffectsofMagnesiumSulfateonBreastCancerCells
KaylaChinitz
NilesNorthHighSchool

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TableofContents
Acknowledgements.3
PurposeandHypothesis..4
ReviewofLiterature5
Materials...12
Procedure.15
Variables...16
Results..17
DataAnalysis....19
Discussion.20
ExperimentalErrors...22
Conclusion....24
Resources.25

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Acknowledgements

IwouldliketothankMs.Camelformentoringmeextensivelythroughouttheproject,especially
whiletesting.IwouldliketothankProfessorJamesLodolceforhelpingmemakeimportant
decisionsforstartingofftheprojectandforeditingmyprocedure.IwouldalsoliketothankMr.
Thielsenforgivinginputontheresultsofmyexperiment.

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Purpose

Thepurposeofthisprojectistodeterminewhethermagnesiumsulfateaffectsthegrowthand
developmentofbreastcancercells,celllineMCF7,andifitcausesapoptosistoleadtoother
studiesinvolvingtheeffectsofothervegetablenutrientsoncancercellgrowth.

Hypothesis

Ifbreastcancercells,celllineMCF7,aretreatedwithvaryingamountsofmagnesium,thenthe
amountofsurvivingcancercellswilldecreaseasthemagnesiumlevelsincrease.Priorresearch
hasshownthatexcessamountsofmagnesiumdecreaseriskofcolorectalcancercellsinvivo,
andmagnesiumdeficienciesincreaseriskofcolorectalcancer.Additionally,itisavegetable
nutrientwhichhelpsmaintainnormalbodyfunctionsandisgenerallybeneficialtothebody.

Chinitz

ReviewofLiterature
Anappleadaykeepsthedoctorawayisawellknownphrasemeaningeatyour
vegetables,orelse.Itseemscontradictorythen,thatseverelyillcancerpatientsgoingthrough
chemotherapymaybeexperiencingnutrientdeficienciesduetolackofappetite.Thecombined
effectsofchemotherapyandthecanceritselfonthepatientsappetiteanddigestivesystemalong
withthedietaryrestrictionsinvolvedinchemotherapyoftenleadtothepatientsdiminished
nutrientintake.Vegetablenutrients,suchasmagnesium,helpthebodysdefensesystemfightoff
disease.Sowhydontchemotherapypatientsreceiveincreaseddosesofthesecriticalvitamins
andminerals?Therehasnotbeenenoughresearchcarriedoutonthesubjectofvitamins
cancerfightingabilities.Currentresearchonmagnesiumslinktocancerandcarcinogenesis
suggests,notonlythatitcouldhelpstrengthenthebodysimmunesystem,butthatitcould
combatthecancerdirectly.Ifmagnesiumdoeshavetheabilitytofightagainstcancer,itcould
helpprotecttheover290,000adultsdiagnosedwithoneoftheworldsmostmercilessdiseases:
breastcancer.
In2013,approximately232,340womenwerediagnosedwithinvasivebreastcancerand
64,640werediagnosedwithinsitubreastcancerintheUnitedStates.Therewereapproximately
39,620deaths,mostofwhichoccurredinwomenoverage65.Prognosesaregivenbasedon
severalfactors:tumorsize,spreadtolymphnodes,andmetastases(theTNMclassification),as
wellasthestages(Breastcancerfactsandfigures,n.d.).ForStage0(insitu)andStageIbreast
cancer,thefiveyearsurvivalrateis100%.ForStageII,thefiveyearsurvivalrateis93%.For
StageIII,itis72%.ForStageIV,itis22%(Breastcancersurvivalrates,2014).

Chinitz

Therearethreemajortypesofbreastcancer:ductalcarcinomainsitu(DCIS),invasive
ductalcarcinoma,andinvasivelobularcarcinoma.DCISisanoninvasiveformofbreastcancer,
meaningthattheabnormalcellgrowthisonlyfoundintheductsandhasnotspreadtothebreast
tissue.Themostcommonisinvasiveductalcarcinoma,whichstartsinthemilkductandspreads
tootherbreasttissue.Invasivelobularcarcinomastartsinthelobules,orthemilkproducing
glands.Invasiveformsofbreastcancerhaveachanceofmetastasizingtootherpartsofthebody.
Thereareseveralfactorsthatincreasetheriskofcontractingordevelopingbreastcancer,oneof
themostprominentbeinggender.Estrogenandprogesteroneinducebreastcancercellgrowth,so
womenaremorelikelythanmentodevelopbreastcancer.Riskalsoincreasesasageincreases.
Hereditarymutationsarethoughttocausebetween5%10%ofbreastcancercases,mainlyinthe
BRCA1andBRCA2genes.Inhealthycells,thesegenesmakeproteinswhichpreventabnormal
cellgrowth.Thesemutationsdonothavetobeinheritedandaretheleadingcauseofbreast
cancer.(Breastcancer:Detailedguide,2014).Breastcancerisdiagnosedprimarilythrough
medicalexams,mammograms,breastultrasounds,andbreastMRIs.Treatmentsincludesurgery,
radiationtherapy,chemotherapy,andhormonetherapy.Surgeryinvolvesapartialmastectomyor
fullmastectomy.Inapartialmastectomy,onlythetumorandasmallringofnormalbreasttissue
areremoved,whilethefullbreastisremovedinafullmastectomy.Mostwomenwillneed
radiationorchemotherapytofollowapartialmastectomy.Forcancersthatarehormonereceptor
positive,hormonetherapymaybeusedtolowerestrogenlevelstoslowcancercellgrowth.
Targetedtherapyandbonedirectedtherapymayalsobeusedastreatments.(Breastcancer:
Detailedguide,2014).

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Chemotherapyhasbeenshowntolowerlevelsofantioxidantsandmicronutrientsin
leukemiapatients.Skolinmeasuredoxidativestressinthebloodandbonemarrowandthe
antioxidantsvitaminA,vitaminE,andcarotenoidsatdifferentstagesofchemotherapy
treatment.Astheoxidativestressincreased,theantioxidantlevelsdecreased.Oxidativestress
referstoanimbalanceoffreeradicalsandantioxidantsinthebody(Skolin,2005).Freeradicals
areatomsthathaveunpairedelectronsandstealelectronsfromtheneareststablemolecule.This
createsachainreactionoffreeradicals.Excessiveamountsoffreeradicalscanleadtocelland
tissuedamage.InSkolinsstudy,hespecificallytestedformononuclearbloodandbonemarrow
celldamage.Antioxidantsregulatethembygivingupanelectron(UnderstandingFreeRadicals,
1997).
Patientsmayexperiencenutrientdeficiencyduetoagenerallackofappetite.Depending
onthetypeofcancer,thelackofappetitemayresultfromcancerinducedalterationsin
metabolism.Inaddition,medicationsforalltypesofcancer,usuallychemotherapy,cancause
mouthsores,severenausea,difficultyswallowing,changesintasteandsmell,aswellasother
factorswhichdecreaseappetite.Whenpatients
are
abletoeat,theyareinstructedtoeatfoods
highinproteinandcalories.Whenapersonsonlynutritionforseveraldayshasbeenthroughan
IV,thecommonbeliefisthatthemainconcernisforthemtoeatfoodswithsubstance,andadd
inextracalorieswhereverpossible.Whilevegetablesmaybehighlybeneficial,itismore
importanttokeepthepatientfrombeingmalnourished(AppetiteLoss2012).Accordingto
DavidDugdaleandYiBinChen,consumingrawvegetableswhileonchemotherapycanbe
dangerous(2012).Duringchemo,theimmunesystembecomesvulnerabletothingsnotusually
consideredharmfultothebody.Rawvegetablesareconsiderednotsterileenoughforthebodyto

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digest,andallfruitandvegetablejuiceshavetobepasteurized.Duetothisextremelimitationon
vegetableconsumption,manychemotherapypatientsaresusceptibletonutrientdeficiencies.
Magnesiumhasseveralqualitieswhichhighlightitasanimportantnutrientthatmaybe
lackinginpatientsundergoingtreatment.Itisfoundprimarilyindark,leafygreenssuchas
spinach,kale,andswisschard.Itisalsopresentinmuchsmalleramountsinothervegetables,
fruits,legumes,nuts,andgrains.Over300biochemicalreactionsinthebodyrequiremagnesium
inordertooccur.Furthermore,ithelpsmaintainahealthyimmunesystem,healthybones,and
normalnerve,muscle,andheartfunctions.Morerecentresearchofmagnesiumsuggeststhatit
playsaroleinhighbloodpressure,heartdisease,anddiabetesprevention(Evert,2013).
Inadditiontoresearchwhichshowshowmagnesiumdeficiencymayhavenegative
outcomesinrelationtocancer,Larsson,Bergkvist,andWolkconductedalongtermstudyto
observehowmagnesiumimprovescancerrisk.Theystudiedthedietsofalargegroupofhealthy
women,andspecificallyobservedthenumberofwomenwhowerediagnosedwithcolorectal
canceroveramedianfollowupperiodof14.8years.Theyfoundthatabout1.3%ofthe
participantswerediagnosedwithcolorectalcancer.Theygroupedthesediagnosesbythe
participantsmagnesiumintake(milligrams/day).Theresultsshowaconstantinverse
relationshipbetweenmagnesiumintakeandcancerdiagnoses.Thelowerthemagnesiumintake,
thehigherthenumberofpeoplediagnosedwithcolorectalcancer(2005).Mercola(2014)also
conductedastudyoncolorectalcancerriskassociatedwithmagnesiumintake.Magnesiumwas
testedonbothcolorectalcancerandcolorectalcancertumorsinincrementswith100mg
increases.Forevery100mgincrease,theriskofthetumorsdecreasedby13%,andtheriskof

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cancerdecreased12%.Thisrelationshipshowsanegativecorrelationbetweenmagnesiumintake
andcolorectalcancerandtumorrisk.
Magnesiumalsohasanindirectrelationshiptocancerthroughinsulinresistance.Several
studieshaveanalyzedtheeffectsofbothmagnesiumsupplementsaswellasmagnesium
deficiencyoninsulinresistance.Thesestudieshavelookedatlongtermeffectsdealingwith
insulinsyndromeriskfactors,haveworkeddirectlywithpeoplewithinsulinsyndromes,and
havealsoanalyzedmagnesiumseffectoninsulinrelatedhormones.Insulinresistanceincludes
hypertension,atherosclerosis,hyperinsulinemia,aswellasothersyndromes.Thefirst
relationshipslinksmagnesiumwithinsulinresistance.Oneexperimentconcludedhigher
magnesiumconcentrationsinpeoplewithhypertensionandtypetwodiabetesregulatesinsulin
resistance(Paolisso&Barbagallo,1996).Moreindepthstudieshavebeenconductedshowing
relationshipswhichfocusonlowmagnesiumconcentrations.Anotherstudyanalyzedresearch
involvingtherelationshipofinsulinlikegrowthfactor1(IGF1),ahormonewhosestructureis
similartothatofinsulin,withbreastcancerinwomen.Ithasbeenshowntoincreaseriskof
adenocarcinomaandcarcinogenesisbecauseitcanalsosuppressapoptosis.Thesuppressionof
apoptosisisanecessarysurvivalfactorinnormalepithelialcells,butcanbeunfavorablewhenin
thepresenceofcancertumorsorcancercellsbecauseitpreventsthedeathofharmfultumorsand
cells.(Zeng&Yee,2011).Anotherstudythatconnectedinsulinresistancetocancertestedwith
colorectalcancer,whichissignificantbecausethestudieswhichlinkmagnesiumdirectlyto
canceralsousecolorectalcancerintheexperiment(Colangelo,2002).

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Theconclusionsofthesestudiesthatworkwithcancerpatientsaredependablebecause,
sincethestudiesareinvivo,theresearchersknowhowmagnesiumaffectsthecancercellsinthe
body.However,thereareseveraldiscrepanciesbetweenthewaycellsbehaveinamicroplate
versusinthehumanbody.Invitrostudiescannotreplicatetheexactconditionsofthehuman
body.Inaddition,cellandothersubstanceconcentrationsmaydiffer.Theproportionofcancer
cellstomagnesiumwhichwillbeusedinthisexperimentmaynotaccuratelyrepresentthe
proportionsthatwouldnaturallyoccurinthebody.Invitro,theonlyfactorsplayingintothe
resultsarethesubstancesonthematerialslist.Whereas,invivo,thepatientspersonalmedical
history,stageofcancer,andalltheprocessesthebodyisalwaysgoingthrougharefactorswhich
couldaffecthowthemagnesiumaffectsthecancer.Althoughinvitrostudiescannotmake
definiteconfirmationsaboutcellbehavior,theyareanimportantstepintheprocessoftesting
usingcells,andtheyprovidefurtherinformationforfutureinvivostudies(DifferencesBetween
InVitroandInVivocells,2012).
Inthisexperiment,cellconcentrationwillbecomparedbytheuseofahemocytometer.
Hemocytometersarecellcountingchamberswithgridswhichallowsmallcellsamplestobe
testedforviability.Trypanbluedyeisaddedtothecellstodistinguishbetweenviableand
nonviable,orapoptotic,cells(Caprette,2006).Apoptosisistheprogrammeddeathofcells,the
rateofwhichisusuallydeterminedbytherateofcelldivision.(Alberts,Johnson,Lewis,Raff,
Roberts,Walter,2002).Cancerprogressionislargelyduetotheirabilitytoresistapoptosis
(Resistingapoptosis,n.d.).

10

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Researchonvarioustypesofcancerpatientshaveconcludedsimilarlyandsupportthe
statementthatmagnesium,bothdirectlyandindirectly,decreasestheriskofcancerand
carcinogenesis.In201339,620peoplediedofbreastcancerintheUnitedStates.Prognosesvary
bystage.Therearethreeprimarytypesofbreastcancer,ductalcarcinomainsitu(DCIS),
invasiveductalcarcinoma,andinvasivelobularcarcinoma.Themaincauseofbreastcancerare
mutationsintheBRCA1andBRCA2genes.Treatmentincludesurgery,radiationtherapy,
chemotherapy,hormonetherapy,targetedtherapy,andbonedirectedtherapy.Thewaysinwhich
chemotherapyhasthepotentialtocausenutrientdeficienciesarethatchemolowersantioxidant
levels,decreasesappetite,andlimitstheabilitytoconsumefreshfruitsandvegetables.
Magnesiumisfoundprimarilyindark,leafygreens.Itisrequiredforseveralbiochemical
reactionsinthebody,andithelpsmaintainnormalnerve,bone,andmusclefunctioning.Some
studieshavelinkedmagnesiumdirectlytoadecreaseincancerrisk.Ifmagnesiumcould
potentiallyhaveanimpactonthegrowthrateofcancer,cancerpatientsshouldtrytoincorporate
magnesiumrichfoodsintotheirdietsortakemagnesiumsupplements.Uptothispoint,
magnesiumhasnotbeenextensivelyresearchedinitsrelationtocanceroritssideeffects.Ifit
doesmakeadifference,itwouldbeaveryimportantfind.

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Materials

20uL200uLmicropipetteandtips
2uL20uLmicropipetteandtips
gloves
BreastcancercellscelllineMCF7
hemocytometer
microscope
96wellmicroplate
1920uLmagnesiumsulfatesolution
Trypanbluedye
biologicalsafetycabinet
microcentrifugetubes
centrifugetubes
o
CO
incubatorat37
C
2
wastecontainerformedia
trypsin
RPMI1640Medium(10%FetalCalfSerum1%Pen/Strep)
waterbath
70%ethanolspray
cultureflask

12

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Procedure

CareandCultureofCells
1. Cleanthebiohoodwith70%ethanol.
2. Beginbyputtingthecompleteandplainmediaintoawaterbathat37degreesC.
3. ThawthetrypsinbyputtingitintotheCO2incubator.
4. Removemediafromflaskandpourintowastecontainer.
5. Carefullyadd6mlofplainmedia(dontpipettedirectlyontothecells),andgentlywash
thecells.Removethistothewaste.
6. Pour(ifalreadyaliquoted)orpipette3mLoftrypsinintoflaskovercells.
7. Incubatetheflaskat37degreesCintheincubatorforabout10minutes.
8. Examinethecellsundertheinvertedmicroscopetodetermineifthecellshavereleased
fromtheplastic.
9. Iftheyhaventreleased,gentlysmacktheflaskontheworkingsurfaceofthehoodto
helpreleasethecells.
10. Pipettetheliquid,nowcontainingthecells,intoalabeledtesttube.
11. Washtheflaskbypipetting3mLofcompletemediatothecellsbeforeremovingthem.
Pipetteupanddownafewtimesagainstthebottomoftheflask(wherethecellswere
stuckdown).Washwithanadditional3mlofcompletemedia.
12. Centrifugethetesttubeat1000RPMsfor7minutes.Remember,thecentrifugeneedsto
bebalanced.
13. Pouroffthemediaintothewastecontainer.
14. Resuspendthecellsbyaddingapproximately7mLofcompletemediatothetesttube
withthepelletofcells.
15. Ifsplittingcells,put50mLofcompletemediaineachof(2)cultureflasks.
16. Addhalf(approximately5mL)oftheresuspendedcellstoeachcultureflask.Besureto
mixwellbeforepipettingintothecultures.
17. Putintotheincubator.
18. Cleanthebiohoodwith70%ethanolanddisposegloves.
19. Disposeofwastemediabyautoclavingat121degreesC.

DataCollection
20. Sprayallmaterialswith70%ethanol
21. Useamicropipettetoinsert80uLofmagnesiumsulfatesolutionand80uLofmediainto
rowA,wells18ofa96wellmicroplate.
22. Useamicropipettetoinsert40uLofmagnesiumsulfatesolutionand120uLofmedia
intorowB,wells18ofa96wellmicroplate.
23. Useamicropipettetoinsert160uLofculturedcellsintorowAandB,wells18ofthe
microplate.
13

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24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.

Putthewellplateintotheincubatorandincubatefor24hours.Returntheremainingcells
totheincubatoraswell.
Cleanthebiohoodwith70%ethanol.
After24hours,insertacoupledropsoftrypsinintoeachwellinrowAandletthem
trypsinizefor45minutes.
UseamicropipettetoobtainthesamplesfromtherowAandinserteachsampleintoa
microcentrifugetube.
Add320uLofTrypanbluedyetoeachmicrocentrifugetube.
LoadonechamberofthehemocytometerwiththesamplefromrowA,well1andplaceit
underthemicroscope.
Countthecellsinthecornersquaresandthecentersquare.Withinthesquares,countthe
cellnumberofviableandnonviablecells.
Recordresultsforlateranalysis.
Cleanthehemocytometer.Disposegloves.
Repeatsteps2932witheachofthesamplesinrowsAandB.
Sprayallmaterialswith70%ethanol
Useamicropipettetoinsert120uLofmagnesiumsulfatesolutionand40uLofmedia
intorowC,wells18ofa96wellmicroplate.
Useamicropipettetoinsert160uLofmediaintorowD,wells18ofa96well
microplate.
Useamicropipettetoinsert160uLofculturedcellsintorowCandD,wells18ofthe
microplate.
Putthewellplateintotheincubatorandincubatefor24hours.Returntheremainingcells
totheincubatoraswell.
Cleanthebiohoodwith70%ethanol.
After24hours,useamicropipettetoobtainthesamplesfromtherowCandinserteach
sampleintoamicrocentrifugetube.
Add320uLofTrypanbluedyetoeachmicrocentrifugetube.
LoadonechamberofthehemocytometerwiththesamplefromrowC,well1andplaceit
underthemicroscope.
Countthecellsinthecornersquaresandthecentersquare.Withinthesquares,countthe
cellnumberofviableandnonviablecells.
Recordresultsforlateranalysis.
Cleanthehemocytometer.Disposegloves.
Repeatsteps2932witheachofthesamplesinrowsCandD.

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Organizationofmagnesiumsulfatesolutionandmediainthewellplate
ALLWELLSHAVE160uLofcells

10

11

12

80uL
MgSO

4
80uL
media

80uL
MgSO

4
80uL
media

80uL
MgSO

4
80uL
media

80uL
MgSO

4
80uL
media

80uL
MgSO

4
80uL
media

80uL
MgSO

4
80uL
media

80uL
MgSO

4
80uL
media

80uL
MgSO

4
80uL
media

40uL
MgSO

4
120uL
media

40uL
MgSO

4
120uL
media

40uL
MgSO

4
120uL
media

40uL
MgSO

4
120uL
media

40uL
MgSO

4
120uL
media

40uL
MgSO

4
120uL
media

40uL
MgSO

4
120uL
media

40uL
MgSO

4
120uL
media

120uL
MgSO

4
40uL
media

120uL
MgSO

4
40uL
media

120uL
MgSO

4
40uL
media

120uL
MgSO

4
40uL
media

120uL
MgSO

4
40uL
media

120uL
MgSO

4
40uL
media

120uL
MgSO

4
40uL
media

120uL
MgSO

4
40uL
media

160uL
media

160uL
media

160uL
media

160uL
media

160uL
media

160uL
media

160uL
media

160uL
media

15

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Variables
Independent:Ratioofmagnesiumsulfatesolutiontomediaaddedtocells
Dependent:Cellconcentrationafterincubationwithmagnesiumsulfate
Control:Rowofcellsincubatedwithoutmagnesium
Constants:Timeinincubator,temperatureofincubator,allmaterialsunderthebiohoodsprayed
with70%ethanolspray.

16

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Results
Theresultsofthisexperimentwereinconclusivebecausethecellcountingswiththemagnesium
wereunreadable.Cellviabilitywasunabletobedeterminedduetoagglutinationand
precipitationmostlikelycausedbyareactioninvolvingthemagnesiumsulfatesolution.

160uLofbreastcancercellsincubatedwith120uLMgSO
and40uLmediafor24hours.
4

Topchamber:160uLofbreastcancercellsincubatedwith120uLMgSO
and40uLmediafor24
4
hours.
Bottomchamber:160uLofbreastcancercellsincubatedwith160uLofculturemedia(
RPMI1640
Medium10%FetalCalfSerum1%Pen/Strep)
for24hours.

ReactionsofTestedWellsandCellViability
Well

Viability

Well

Viability

A1

Unreadable

C1

Nottested

A2

Unreadable

C2

Nottested

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A3

Unreadable

C3

Nottested

A4

Nottested

C4

Nottested

A5

Nottested

C5

Nottested

A6

Nottested

C6

Nottested

A7

Nottested

C7

Unreadable

A8

Nottested

C8

Unreadable

B1

Unreadable

D1

Nottested

B2

Unreadable

D2

Nottested

B3

Nottested

D3

Nottested

B4

Nottested

D4

Nottested

B5

Nottested

D5

Nottested

B6

Nottested

D6

Nottested

B7

Nottested

D7

Viable:5
Nonviable:0

B8

Nottested

D8

Nottested

18

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DataAnalysis

AllthreeofthecellreadingsfromrowAagglutinatedandcouldnotberead.Boththe
cellsreadingsfromrowBagglutinatedandcouldnotberead.BoththecellreadingsfromrowC
agglutinatedandcouldnotberead.ThecellreadingfromrowD,thecontrol,didnotagglutinate
andcouldberead.Ithad5viablecellsandnononviablecells.Thisreadingsuggeststhatthe
magnesiumsulfatecausedthesomeparticlestoclumptogetherandotherstoprecipitate.Evenin
thereadablecount,thecelldensitywasextremelylow.Thisimpliesthatthecellsdidnothave
enoughtimetoculture,thecellsdidnothaveenoughtimetoincubatewiththemagnesium
sulfatesolution,oranotherfactorinhibitedthecellgrowth.

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Discussion
Thereasonfortheabnormalcellactivityandunreadablecellcountsisstillunknown.
However,thereareseveralfactorswhichcanberuledout.Itisnotduetocontaminationofthe
cellsbecausethecontrolgroupdidnotappeartohaveanyabnormalactivity.Inaddition,the
cellsusedfortestingwerefromtwodifferentsamples,bothofwhichactedthesameway.
Therefore,italsocouldnothavebeenanabnormalityinthespecificcellsample.Sincethe
sampleswiththemagnesiumsulfateappearedtoformaprecipitate,thissuggestsareaction
occurredbetweenthemagnesiumsulfateandtheTrypanbluedye.However,otherstudieshave
beenconductedinvolvingthemixtureofthesetwosubstancesandnocoagulationoccurred.
Althoughthecellswerenotcontaminated,theymaynothaveactednormallybecausetheyhad
beenfrozenforoneyear.
Ifthisexperimentweretoberunagain,severalstepsintheprocedurewouldneedtobe
modified.First,beforethetesting,Trypanbluedyewouldbeaddedtosmallsamplesofseveral
formsofmagnesiumtoruleoutthepossibilitythatareactionwhichproducesaprecipitate
occurswhenthesetwosubstancesarecombined.Theseothertypesofmagnesiuminclude(but
arenotlimitedto)magnesiumsulfate.Magnesiumglycinate,magnesiumoxide,magnesium
carbonate,andmagnesiumcitrateareallcommonmagnesiumsupplements.
Oneofthemajorproblemswiththecellsthemselveswastheirextremelylowconfluency
anddensity.Throughoutthetestingprocess,itwasdifficulttoobtainasufficientsampleofcells,
evenaftertrypsinizingandcentrifuging.Ifthisexperimentwerererun,thecellswouldbenewer,
meaningtheywouldnothavebeenunfrozenfrompastusage.Theywouldalsobegivenmore
incubationtime,bothintheculturingandtestingportionsoftheprocedure.Inthetestingportion
20

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withthemagnesiumsolution,thecellswouldbeincubatedforaround72hoursinsteadof24
hours.Inaddition,tostrengthenthecredibilityoftheresults,allthewellswouldbetestedinstead
ofjustacouplefromeachrow.Itcannotbeassumedthattwosamplesfromaroware
representativeoftheentirerow.Thecellswouldalsobeexaminedmorethoroughlywiththe
hemocytometer.
Allinall,asecondtrialofthisstudywouldnotonlybebeneficialinclearingupsome
unclearpatches,butitwouldbenecessaryinordertoassumethatthistypeofreactionwould
occureverytimemagnesiumsulfatesolution,breastcancercells,trypsin,andTrypanbluedye
weremixed.Withonlyonetrialitisstillpossiblethatacompletelydifferentunknownfactor
causedthereaction,anditisunknownwhetheritwouldcauseitineverytrial.

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ExperimentalError

CultureandCareofCells:
Thecellsusedwereunfrozenfromoneyearago,sotheymayhavebehaveddifferently
thancellswhichwereneverfrozen.Thematerialsinthebiohoodcouldhavebeencontaminated
dueto1)insufficientsanitizationofmaterials,and2)otherexperimentswerealsoconducted
underthebiohood,sotherecouldhavebeensomecrosscontaminationifnotproperly
disinfected.
Whencentrifuged,mosttimesthecelldensitywassolowthatapelletcouldnotbeseenatthe
bottomofthecentrifugetube.Therefore,ifthecellsamplewasmajoritymedia,itwouldnotbe
likelytoobtainanaccuratecellcount.Ifthecellswerenottrypsinizedforlongenough,not
mixedproperlywiththetrypsin,ornotcentrifugedlongenough,theirgrowthratemayhave
slowed.
Plating:
Themagnesiumsulfatesolutionmaynothavecausedthesamereactionifasmaller
amountofithadbeenused.Conversely,theconcentrationofthesolutionmayhavebeentoo
high.
Themagnesiumandmediawereplatedonedayinadvancetothecells,sothismayhavechanged
thewaythesubstancesreactedtooneanother.Ifthemicropipettetipcameincontactwitha
sampleandwasreusedtoplatethenextsample,thenitcouldhavecontaminatedthenextsample.
24hoursmaynothavegiventhecellsenoughtimetoincubatewiththemagnesiumsulfate
solution.
DataCollection:

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Duetotimeconstraints,notallofthewellsweretested.Theresultisalackofdata,
whichweakensthecertaintyandaccuracyoftheresults.Thecellsmaynothavebeentrypsinized
longenoughormixedwellenough,whichwouldhaveledtolowcelldensitybecauseonlya
smallsampleofcellsweretakenfromeachwell.Ifthecellsclumpedinanareawhichwasnot
tested,thereadingswouldbeinaccurate.

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Conclusion
Thehypothesisandrationalestated,Ifbreastcancercells,celllineMCF7,aretreated
withvaryingamountsofmagnesium,thantheamountofsurvivingcancercellswilldecreaseas
themagnesiumlevelsincrease.Magnesiumhasbeenshowntodecreaseriskoflivercancercells
invivoanditisavegetablenutrientwhichhelpsmaintainnormalbodyfunctionsandis
generallybeneficialtothebody.Thishypothesiscouldnotbesupportedorrefutedbecausethe
resultswereinconclusive.Thereisnodefiniteconclusionforthisexperimentbecausethe
viabilityofthecellscouldnotbedetermined.Inconclusiveresultsstillhavethepotentialto
motivatefurtherresearch,especiallyonatopicsuchastherelationshipbetweenvegetable
nutrientsandcancerdevelopmentwhichhasnotbeenextensivelyresearched.

24

Chinitz

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