1. Antiproliferative effects of trans-resveratrol on HepG2 cells and an evaluation of cell viability method sensitivities.
Niousha Ghamami, Mark Gichuru, Kayla Merim & Ireena Soleas
Honours Biology & Pharmacology Program, McMaster University

2. Hepatocellular Carcinoma
Types of liver cancer
Primary liver cancer
6th most common cancer and 3rd leading cause of cancer in the world
increasing incidence in Asia Pacific, sub-saharan Africa, southern Europe and North America
Bile duct cancer
Cancer in the liver blood vessels
Hepatoblastomas
Hepatocellular carcinoma (HCC)

3. Hepatocellular Carcinoma
Infectious agents
Toxic compounds
Inflammation
Cytokines, Chemokines, Reactive oxygen and nitrogen species
Hepatic Neoplasia

4. Trans-resveratrol (3, 5, 4’-trihydroxystilbene)
Prevents or delays the onset of cancer
Cardioprotective agent
Down regulates COX 1
Potent Antioxidant

5. Objectives 1° Objective 2° Objective
Determine the antiproliferative effects of trans-resveratrol (resveratrol) and other hydroxystilbene derivatives on the HepG2 cell line.
2° Objective
Investigate the sensitivity of various methods of cell viability quantification.

6. Experimental approach
Three colorimetric based methods of determining cell viability
MTS Assay
BCA Assay
Bradford Assay
Tetrazolium based salt (MTS) bioreduced to purple formazan product
Conversion uses dehydrogenase enzymes found only in metabolically active cells
Quantity of formazan product measured via absorbance reading, and is directly proportional to live cell number
Two step reaction:
Protein in solution reduces Cu2+ to Cu1+ ion in alkaline environment, amount of Cu2+ reduced is proportional to protein in solution
Two molecules of bicinchoninic acid (BCA) chelate with each Cu1+ ion and form a purple coloured product which can be measured via absorbance
In acidic environment of Bradford reagent, proteins bind to Coomassie dye
Dye is originally reddish/brown, binding of proteins causes spectral shift to blue form
Absorbance measured and proportional to protein content

7. HepG2 cells seeded at a density of 20 000
Methods
Cell Quantification
HepG2 cells seeded at a density of
cells/well
Adhered for 24 hrs
Treatment
Control (1% DMSO), mM Resveratrol 24 hr incubation
Trypsonized
Resuspended in PBS and Trypan blue (1:1)
Haematocytometer
Used for counting to determine cell viability

8. HepG2 cells seeded at a density of 5000
Methods
MTS Assay
HepG2 cells seeded at a density of
cells/well
Adhered for 24 hrs
Treatment
High control (1% DMSO), low control (cell media and FBS) mM resveratrol 1 hr incubation
MTS reagent
Incubate for 2 hrs
Absorbance reading
At 482 nm on ELISA spectrophotometer

9. Bradford Total Protein Assay BCA Total Protein Assay
Standards:0-2 mg/mL
Aliquots of lysed samples and standards plated in a 96-well plate
Bradford Coomassie Blue reagent added
5 min incubation
Absorbance read at 540 nm
Standards: g/mL
Aliquots of lysed samples and standards plated in a 96-well plate
BCA reagent added
Half hour incubation
Absorbance read at 540 nm
Sample protein concentration for both BCA and Bradford protein assays was determined from standard curves

10. Results
MTS Assay
Figure 1. Effects of resveratrol (1-100µM) on MTS reduction in HepG2 cells cultured with trans-resveratrol over a 1 hour incubation period. Increase in cell viability is observed.

11. BCA Assay
y = x
r² =
Figure 2. Effect of trans-resveratrol (1-100µM) on protein content in HepG2 cells over a 1 hour incubation period as determined by BCA assay. Decrease in cell viability is observed.

12. Manual Cell Count
Figure 4. Effect of trans-resveratrol ( µM) on cell viability in HepG2 cells over a 24 hour incubation period as determined by manual cell counts. A decrease in viability from control is observed.

13. Comparison of BCA & Bradford Assays
y = x
r² =
y = x
r² = A B
Figure 3. Comparison of sensitivity of protein quantitation as determined by BCA assay (A) and Bradford assay (B). Both A and B display cell number dependent increases in protein concentration. B displayed a larger r² value. A represents average of 2 trials, B represents average of 1 trial.

14. Discussion
MTS Assay
Cell viability determination via the MTS assay did not demonstrate growth inhibiting property of resveratrol on HepG2 cell line
Discrepancy between this observation and studies confirming resveratrol’s antiproliferative effects exist (Colin et al., 2008; Notas et al., 2006); Strevbo et al., 2006)
BCA assay confirmed that cell viability was not increasing
Increasing formazan production occurring through mechanism other than increased HepG2 cell growth

15. Thus, determined MTS not suitable for this investigation
Possible Mechanisms
Holian & Walter (2000) state that tetrazolium compound reduction to formazan product depends on:
1. Cellular NAD(P)H levels
2. Requires metabolism of glucose
3. Coupled with respiratory chain
Due to resveratrol’s inhibition of proliferation through cell cycle arrest, it is suggested that an increase in cell volume and mitochondrial content is induced.
All resulting in an increased production of formazan, without an increase in cell number.
Result:
Altered glucose utilization
Increased intracellular NAD(P)H levels
Also suggests resveratrol interacts with enzymes in respiratory chain which can further potentiate reduction of MTS.
Thus, determined MTS not suitable for this investigation

16. Comparison of BCA & Bradford Assays
Bradford assay displayed a higher r2 value of 0.42, therefore concluded to be a more sensitive cell viability measure as compared to BCA assay
Low r2 indicates variability, thus more trials must be performed to confirm validity of Bradford assay as a cell viability measure in future studies.

17. Manual Cell Count
Dose-dependent antiproliferative response was observed
Best demonstrates resveratrol’s antiproliferative properties
Continue to use manual count in future studies but perform more trials to increase confidence and decrease variability.

18. Future Directions
Study hydroxystilbene derivatives and compare potency of their potential antiproliferative effects to resveratrol
Continue to measure cell viability via manual cell counts
Explore other methods of cell viability, ie. [3H]-thymidine incorporation

19. Thank you!