The Effects of Thymoquinone on CT26 Cells Observed Through Cell Culture

Final Report

Yvone Shametaj

Kandy T. Velazquez, Ph.D., FACSM

SCHC 497

USC-SOM

December 7, 2020
Introduction

Cancer is one of the most complex and elusive diseases. Although scientific research has made tremendous gains

in pharmacological treatments and therapies, there is yet to be a universally effective treatment or cure due to genetic

variations in tumorigenic cells and variations between individuals’ immune response. Many more researchers are turning

to homeopathic medicine for possible solutions.

Thymoquinone, a compound derived from black seed oil of the nigella sativa plant, has been historically known

as an herbal remedy in India and Arabian countries, and has been shown to have promising therapeutic effects in research

studies published over the past few decades1. This experiment focuses on the purported antiproliferative and proapoptotic

properties of thymoquinone (TQ). Scientific literature suggests that thymoquinone interferes with carcinogenic processes

and can make tumor cells more susceptible to cancer treatments such as radiotherapy, chemotherapy, and

immunotherapy1. Using CT26 colon carcinoma cells cultured in media on a 96-well plate, maximum cytolysis, maximum

cell barrier index, and electrical resistance were measured using impedance-based cell analysis software.

Materials and Methods

The experiment consisted of 5 experimental groups with varying concentrations of thymoquinone (10uM, 20uM,

40uM, 60uM, and 80uM) and 3 control groups (TQ lysis using alcohol, TQ 0uM or media only, and wells with CT26

cells). An electrode-equipped 96-well plate was mapped out with the Maestro Zedge by Axion Biosystems and data was

measured over 46.67 hours under controlled CO2 and temperature. CT26 cells were grown in media composed of RPMI,

10% FBS, and 1% pen/strep in an incubator for multiple days until approximately 10 million cells were counted prior to

distribution in the wells with a micropipette. Water was pipetted into humidity wells in the plate, media in all the wells,

and then the cells and thymoquinone as shown in the Figure 1 plate map. The experiment was ended earlier than

anticipated due to premature cell death.

Figure 1. 96-well plate map of experimental thymoquinone concentrations and controls.

Plate
Map
1 2 3 4 5 6 7 8 9 10 11 12
A TQ Lysis TQ Lysis TQ Lysis CT26 cells
(0) (0) (0)
B TQ Lysis TQ Lysis TQ Lysis CT26 cells CT26 cells
 (0) (0) (0)
C TQ (80) TQ (60) TQ (40) TQ (20) TQ (10) TQ (0)

D TQ (80) TQ (60) TQ (40) TQ (20) TQ (10) TQ (0)

E TQ TQ (80) TQ (60) TQ (40) TQ (20) TQ (10) TQ (0)
(0)
F TQ TQ (80) TQ (60) TQ (40) TQ (20) TQ (10) TQ (0)
(0)
G TQ TQ (80) TQ (60) TQ (40) TQ (20) TQ (10) TQ (0)
(0)
H TQ TQ (80) TQ (60) TQ (40) TQ (20) TQ (10) TQ (0)
(0)
Note: Concentrations were spread out across the map after cells were counted in the Maestro Zedge in order to maintain

similar average counts across the groups.

Results

Figure 2. Cytolysis at t = 46.67 hours in Maestro Zedge. Note: TQ (0 uM) left and right indicates which side of the map is

being referred to.

Figure 3. Cytolysis at t = 24.46 hours in Maestro Zedge.

Figure 4. Length of time (hours) until 50% of cell death occurred.

Note: Kill time 50 may be skewed due to high cell death prior to 48 hours.

Figure 5. Maximum barrier index until t = 44.62 hours

Note: Resistance and cytolysis graphs for thymoquinone unavailable.

Discussion

The resistance and cytolysis line graphs over the 46-hour period for thymoquinone were unavailable to include in

this report, but in summary, there was a general downward trend in resistance over time and cytolysis remained low at 24

hours and rose to a maximum of almost 100% at the end of the 46 hours. A downward trend in electrical resistance

suggests decreased cell proliferation. This is further elaborated through the maximum barrier index in Figure 5. As cell

proliferation was lowest in the CT26 cells, 0 uM TQ group on the right side of the plate, and the 10 uM TQ group, the

maximum barrier index values were negative. Resistance was lowered in all experimental groups with small differences.

More research is certainly needed to better analyze the effects of different concentrations of thymoquinone.

Unusually, the kill time 50 in Figure 4 was high for TQ 0 uM on the right side of the plate and almost zero for

TQ 0 uM on the left side of the plate. This drastic difference may have been due to human error in placing the cells in the

well or due to counting errors by the Zedge. It appears there were multiple counting errors with the control groups in

general. Cytolysis at t = 24.46 hours in Figure 3 was 100% in the left side TQ 0 uM group, but approximately 20% in the

TQ lysis group, a group that should have experienced 100% cytolysis.

The data suggests that a concentration of 10uM may be effective in killing CT26 cancer cells due to a negative

MBI, lowest kill time 50 of the other experimental groups, and highest percentage cytolysis at 24.46 hours and 46.67

hours among the experimental groups. Again, more extensive research is necessary, preferably with other types of cancer

cells and non-cancerous cells used as a basis for comparison.

Conclusion

Thymoquinone seems to have presented some efficacy in killing CT26 cancer cells through increased cytolysis

and lowered cell proliferation, however the mechanism by which it does this is still unclear. It is also uncertain whether it

can specifically target cancer cells and not just any cells. Experimentation in mice may be a better predictor of the efficacy

of thymoquinone in cancer patients after assessing its effects on healthy cells in more cell culture experiments.

References

1. Mostofa, A., Hossain, M. K., Basak, D., & Sayeed, M. S. (2017). Thymoquinone as a Potential Adjuvant Therapy

for Cancer Treatment: Evidence from Preclinical Studies. Frontiers in Pharmacology, 8.

doi:10.3389/fphar.2017.00295