A Cellular Analysis of the Effects of Garlic Extract on ...
1
A Cellular Analysis of the Effects of Garlic Extract on Cancer Cell Growth Statistical Analysis Unpaired Student t-tests-Spectrophotometry Results: Unpaired t-tests were performed assuming unequal variance between the groups. The absorbance values were expressed as percentages of the control, to normalise data for comparison. ANOVA: Having already decided that Boiled extracts had no effect on cell proliferation, we wanted to establish whether or not there was a difference in effect between the Frozen, Refrigerated and Ambient extracts. ANOVA (Analysis of Variance) can be used to determine the variance between more 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 a significant difference between the groups. P-values of >0.05, mean that Frozen, Refrigerated and Room Temperature-stored extracts had a similar effect on HeLa cell proliferation after 24 hours. Summary • We decided to investigate the claim that the anecdotal health benefits of garlic as a potential anti-cancer agent depend on the method of preparation and storage. We assessed the ability of four preparations of garlic, to inhibit cancer cell growth: Frozen, refrigerated, room temperature-stored and boiled. • We also wanted to know whether aqueous extracts of garlic activate cell death mechanisms such as Apoptosis, Autophagy and Necrosis. • By assessing the viability of the HeLa cells after treatment with garlic, we concluded that boiling garlic kills its anti- cancer abilities completely, whilst most other samples inhibited growth by over 90%. • Through protein biochemistry, we discovered that room- temperature-stored garlic activates apoptosis, (through activation of Caspase 9) whilst refrigerated extracts affect both autophagy and necrosis. (Through activation of LC3B and PARP, respectively). Results Microscopy of Cells: Higher Magnification: After 1 hour, retraction fibres can be observed (see arrows) in the cell sample treated with garlic extract (RT) and after four hours, membrane blebbing can be seen, both of which are signs of apoptosis/cell death. Crystal Violet Assay: The Figure below shows the well plates 24 hours after treatment with garlic extract, having been stained with Crystal Violet. The deeper the colour, the more cells that are present in the sample. Spectrophotometric Quantitation of Crystal Violet Assay: Western Immunoblotting: The cleavage product of Caspase 9 in the RT extracts, indicates that apoptosis has been activated. The cleavage of PARP in the refrigerated extracts at 55 and 37kDa, shows necrotic type cell death. The lower LC3B band increases in signal strength, meaning that refrigerated extract also affects autophagy (the upper band decreases and the lower band increases in intensity, indicating an activation event). 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 extracts were altering expression of cellular proteins, an SDS-PAGE experiment was performed. Once separation of proteins had been achieved, gels were stained with Coomassie dye, de-stained and then scanned using a Bio-Rad Gel Documentation system Western Immunoblotting: Immunoblotting was done to determine the abundance of specific 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, before adding 20ml of distilled water, so that the ratio of garlic to water was 1g/1ml. For further purification, extracts were centrifuged at 2,400 x g, for 10 minutes. The extracts were then passed through sterile syringe filters with a pore size of 0.2 microns to remove large contaminants and any microbial contamination. Spectrophotometry was used in order to find the absorbance of cell samples, which directly corresponds to the amount of cells living in that sample. Absorbance was read at 595nm on a spectrophotometer. To establish whether the garlic extracts were able to cause HeLa cell death, cells were stained after 24 hours with a dye called crystal violet, which has the ability to bind to cellular DNA. Staining with Crystal Violet is a standard method of predicting cell proliferation. Immunoblot Equipment Examining the Results Cell Viability Assay Purified Extracts Spectrophotometry Introduction Garlic: Model System for Study: HeLa cells were used throughout the course of our project. They are a cervical cancer cell line, and the first ever immortal cell line, derived from Henrietta Lacks, after whom they were named. Research Idea: Garlic has been used medicinally for thousands of years- by the ancient Egyptians and as a protective agent against the sun’s heat by African peasantry and Roman soldiers. Due to its versatility and powerful defence mechanisms, the garlic plant has thrived in all environments across the globe. The most important objective of our project, is to decide whether preparing garlic in different ways affects its ability to cause cell death in cancer cells. This factor could potentially have a huge effect on the role of garlic in our diets. Is it being prepared in the most beneficial way possible and more importantly, could garlic be used as a therapeutic agent against cancer? Conclusions • Refrigerated, frozen and room temperature-stored garlic all reduce HeLa cell growth significantly, and there seems to be no difference between the effects of these three preparations after 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 Caspase 9, whilst refrigerated garlic affects both autophagy and necrosis through activation of LC3B and PARP respectively. • Overall, I have proved that the anti-cancer effects of garlic are dependant on preparation and storage methods. Garlic seems to have a significant effect on HeLa cell proliferation, with most samples reducing growth by 90% or more. I strongly recommend that further exploration should be done into this particular topic, so that the unknown anti-cancer property in aqueous garlic extracts could be isolated, and hopefully used as 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
Transcript of A Cellular Analysis of the Effects of Garlic Extract on ...
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
