A Cellular Analysis of the Effects of Garlic Extract on Cancer Cell Growth

Statistical AnalysisUnpaired Student t-tests-Spectrophotometry Results:Unpaired t-tests were performed assuming unequal variancebetween the groups. The absorbance values were expressedas percentages of the control, to normalise data forcomparison.

ANOVA:Having already decided that Boiled extracts had no effect oncell proliferation, we wanted to establish whether or notthere was a difference in effect between the Frozen,Refrigerated and Ambient extracts. ANOVA (Analysis ofVariance) can be used to determine the variance betweenmore than two sets of data. ANOVA also generates a p-value.Similar to a t-test, when the p-value is <0.05, then there is asignificant difference between the groups.

P-values of >0.05, mean that Frozen, Refrigerated andRoom Temperature-stored extracts had a similar effect onHeLa cell proliferation after 24 hours.

Summary• We decided to investigate the claim that the anecdotal

health benefits of garlic as a potential anti-cancer agentdepend on the method of preparation and storage. Weassessed the ability of four preparations of garlic, toinhibit cancer cell growth: Frozen, refrigerated, roomtemperature-stored and boiled.

• We also wanted to know whether aqueous extracts ofgarlic activate cell death mechanisms such as Apoptosis,Autophagy and Necrosis.

• By assessing the viability of the HeLa cells after treatmentwith garlic, we concluded that boiling garlic kills its anti-cancer abilities completely, whilst most other samplesinhibited growth by over 90%.

• Through protein biochemistry, we discovered that room-temperature-stored garlic activates apoptosis, (throughactivation of Caspase 9) whilst refrigerated extracts affectboth autophagy and necrosis. (Through activation of LC3Band PARP, respectively).

ResultsMicroscopy of Cells:Higher Magnification: After 1 hour, retraction fibres can beobserved (see arrows) in the cell sample treated with garlicextract (RT) and after four hours, membrane blebbing can beseen, both of which are signs of apoptosis/cell death.

Crystal Violet Assay:The Figure below shows the well plates 24 hours aftertreatment with garlic extract, having been stained withCrystal Violet. The deeper the colour, the more cells that arepresent in the sample.

Spectrophotometric Quantitation of Crystal Violet Assay:

Western Immunoblotting:

The cleavage product of Caspase 9 inthe RT extracts, indicates thatapoptosis has been activated. Thecleavage of PARP in the refrigeratedextracts at 55 and 37kDa, showsnecrotic type cell death. The lowerLC3B band increases in signalstrength, meaning that refrigeratedextract also affects autophagy (theupper band decreases and the lowerband increases in intensity, indicatingan activation event).

Spectrophotometry

Experimental Methods

Generation of Aqueous Extracts:

Cell Viability Assay:

Spectrophotometric Quantitation of Crystal Violet Assay:

SDS-PAGE:In order to decide whether or not the aqueous extractswere altering expression of cellular proteins, an SDS-PAGEexperiment was performed. Once separation of proteinshad been achieved, gels were stained with Coomassie dye,de-stained and then scanned using a Bio-Rad GelDocumentation system

Western Immunoblotting:Immunoblotting was done to determine the abundance ofspecific proteins known to be associated with Apoptosis,Autophagy and Necrosis. We blotted for PARP, Caspase 9,SQSTM1 and LC3B. Pre-cast 4-12% gels were used.

We crushed 20g of garlic, beforeadding 20ml of distilled water, so thatthe ratio of garlic to water was1g/1ml. For further purification,extracts were centrifuged at 2,400 xg, for 10 minutes. The extracts werethen passed through sterile syringefilters with a pore size of 0.2 micronsto remove large contaminants and anymicrobial contamination.

Spectrophotometry was used in orderto find the absorbance of cell samples,which directly corresponds to theamount of cells living in that sample.Absorbance was read at 595nm on aspectrophotometer.

To establish whether the garlicextracts were able to cause HeLa celldeath, cells were stained after 24hours with a dye called crystal violet,which has the ability to bind tocellular DNA. Staining with CrystalViolet is a standard method ofpredicting cell proliferation.

Immunoblot Equipment Examining the Results

Cell Viability Assay

Purified Extracts

Spectrophotometry

IntroductionGarlic:

Model System for Study:HeLa cells were used throughout the course of our project.They are a cervical cancer cell line, and the first everimmortal cell line, derived from Henrietta Lacks, afterwhom they were named.

Research Idea:

Garlic has been used medicinally forthousands of years- by the ancientEgyptians and as a protective agentagainst the sun’s heat by Africanpeasantry and Roman soldiers. Dueto its versatility and powerful defencemechanisms, the garlic plant hasthrived in all environments across theglobe.

The most important objective of ourproject, is to decide whether preparinggarlic in different ways affects its ability tocause cell death in cancer cells. This factorcould potentially have a huge effect on therole of garlic in our diets. Is it beingprepared in the most beneficial waypossible and more importantly, could garlicbe used as a therapeutic agent againstcancer?

Conclusions• Refrigerated, frozen and room temperature-stored garlic all

reduce HeLa cell growth significantly, and there seems to beno difference between the effects of these three preparationsafter 24 hours. (p-value >0.05). Boiling seems to kill this anti-cancer property in garlic altogether.

• The Western Immunoblot established that room-temperature-stored garlic triggers apoptosis through activation of Caspase9, whilst refrigerated garlic affects both autophagy andnecrosis through activation of LC3B and PARP respectively.

• Overall, I have proved that the anti-cancer effects of garlic aredependant on preparation and storage methods. Garlic seemsto have a significant effect on HeLa cell proliferation, withmost samples reducing growth by 90% or more. I stronglyrecommend that further exploration should be done into thisparticular topic, so that the unknown anti-cancer property inaqueous garlic extracts could be isolated, and hopefully usedas a therapeutic agent against cancer in the future.

R100 VS CON R CON

Mean 8.025878 101.4988

Variance 4.708976 20.65874

Observations 6 6

Pooled Variance 12.68386

Hypothesized Mean

Difference

0

df 10

t Stat -45.4591

P(T<=t) one-tail 3.19E-13

t Critical one-tail 1.812461

P(T<=t) two-tail 6.39E-13

t Critical two-tail 2.228139

R50 VS CON R CON

Mean 15.68665 101.4988

Variance 265.211 20.65874

Observations 6 6

Pooled Variance 142.9349

Hypothesized Mean

Difference

0

df 10

t Stat -12.432

P(T<=t) one-tail 1.05E-07

t Critical one-tail 1.812461

P(T<=t) two-tail 2.09E-07

t Critical two-tail 2.228139

ANOVA RT100 VS F100 VS R100

Source of Variation SS df MS F P-value F crit

Between Groups 5.29412 4 1.32353 0.146715 0.961223 3.179117

Within Groups 117.2746 13 9.021126

Total 122.5688 17

ANOVA RT50 VS R50 VS F50

Source of Variation SS df MS F P-value F crit

Between Groups 308.9704 2 154.4852 1.716763 0.213119 3.68232

Within Groups 1349.795 15 89.98633

Total 1658.765 17

T-test p-value

Refrigerated 50µl vs Control <0.0001

Refrigerated 100µl vs Control <0.0001

Room Temperature 50µl vs Control <0.0001

Room Temperature 100µl vs Control <0.0001

Frozen 50µl vs Control <0.0001

Frozen 100µl vs Control <0.0001

Boiled 50µl vs Control 0.3244

Boiled 100µl vs Control 0.2488