1. Effects of Traditional Chinese MedicineEffects of Traditional Chinese Medicine
on breast cancer cells (MCF-7)on breast cancer cells (MCF-7)
Done by Loh Kai Li and Cheong Yu Shan
With assistance from Dr. Tan Hong Kiat
AbstractAbstract
The extract of five herbs, namely, Forsythiae Fructus, Taraxaci Herba, Persicae
Semen, Cryperi Rhizoma and Codonopsis Radix, were tested for possible
anticancer effects on breast cancer cell line, MCF-7. Forsythiae Fructus,
Codonopsis Radix and Persicae Semen showed the best growth inhibition
effect on MCF-7, with Vero used as control. Forsythiae Fructus had shown
significant the lowest percentage cell viability on MCF-7 at 100µg/ml after
72 hours of incubation. Cell viability was reduced by 27% with minimum
inhibition on Vero.
IntroductionIntroduction
According to the World Health Organization, cancer is a major public
health problem in developed countries like United States and Singapore
[1]. Breast cancer has the highest occurrence rate, accounting for 32% (211,
240) of all new cancer cases [1].
Currently, oncologists are researching to find anticancer drugs in Chinese
herbal medicine. Experiments had shown that Chinese herbal medicine
played an anticancer role by inducing apoptosis and differentiation in
cells, enhancing immune system, inhibiting angiogenesis and reversing
multi-drug resistance (MDR) [2].
By identifying the potential drugs responsible for the anticancer effects in
herbs will assist researchers to develop and optimize chemotherapy
treatments. This will greatly increase the use of herbs as anticancer drugs.
It was hypothesized that the five chosen herbs should produce similar
results. It was also hypothesized that a combination of herbs could
produce an even better result in inhibition or apoptosis of breast cancer
cells.
Results and DiscussionsResults and Discussions
Inhibition of MCF-7 Proliferation and Cell Toxicity on Vero cells
Percentage cell viability based on each herb were calculated and compared.
Graphs below represented the effects of different herbs on percentage cell
viability. This was compared against control which was untreated MCF-7
and Vero cells respectively.
Fig. 1A & B (Left to Right): Effect of Inhibition on MCF-7 and Vero with Treatment of Five Herb Extracts at Three
Concentrations, incubated over 72 hours.
Legend: LQ: Lian Qiao; Forsythiae Fructus, TR: Tao Ren; Persicae Semen, PGY: Pu Gong Ying; Taraxaci Herba,
DS: Dang Shen; Codonopsis Radix, XF: Xiang Fu, Cryperi Rhizoma.
At 72 hours of incubation, Tao Ren, at 25µg/ml, gave the highest growth
inhibition of 31.77%, while Lian Qiao, at 100µg/ml, showed 27.09% growth
inhibition (Fig. 1A). There was absence of linear relationship between
concentration of herb and the length of incubation hours due to herb
properties. A wider range of concentration of herb could be carried out to
study the trend in this occurrence.
The graph depicted that all concentrations of Tao Ren showed decrease in
percentage cell viability by about 10% (Fig. 1B). Lian Qiao, Pu Gong Ying
and Xiang Fu reduced Vero’s cell viability by about 5% to 15%. Only Dang
Shen did not show any inhibitory effects across the three incubation time.
Effect of Combination of Herb Extracts
It was hypothesized that by combining herbs together could result in even
better cell inhibition. Therefore, three combinations of herbs were tested
and statistically analyzed.
Fig. 2A & B (Left to Right): Effect of Growth Inhibition on MCF-7 and Vero respectively with Treatment of
Individual & Combination of Herb Extracts at 100µg/ml, incubated over 72 hours.
All three different combinations of herb extracts could only reduce viability
by at most 10%, which was half that of Lian Qiao and Tao Ren (Fig. 2A). An
explanation for this could be when two herbs were added together, they
might inhibit each others anticancer properties, causing lesser inhibitory
effects. While the herb combinations did not produce better growth
inhibition on MCF-7, they did not show much decrease in cell viability in
Vero (Fig. 2B). However, ongoing of research is still being carried out to
prove these results.
Materials and MethodsMaterials and Methods
Cell Proliferation Assay
A seeding density of ten thousand MCF-7 cells and Vero cells were plated
into 96-well plates in triplicate. The plates were incubated at 37ºC in 5%
CO2 incubator for three different durations (24, 48 and 72 hours) at three
different concentrations of 25, 50 and 100µg/ml. 100µl of MTT reagent to
each well. After incubating for four hours, MTT formazan crystals formed
were completely dissolved in 200µl DMSO. Plates were read with ELISA
reader (Anthos 2001) at 570nm with a reference wavelength of 620nm in a
plate reader.
DAPI Staining
A drop of MCF-7 cells were cultured and treated with the best herb. This
method was used for Vero too. DAPI stain was then used to stain the
nucleus of both treated and untreated cells. The wells were completely
dried in drying oven and were viewed with inverted microscope
(Olympus CK40) under ultra violet (UV) light to detect signs of apoptosis
in cells.
Growth Inhibition with Combination of Herbs
Herbs were combined in the format of DS+LQ, LQ+TR, and DS+TR. 50µL
of each herb, at concentration of 100µg/ml, were added together. The
method used was the same in Cell Proliferation Assay. Ten thousand of
cells were plated into 96-well plate and the plate was incubated for 72
hours straight.
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ConclusionConclusion
Lian Qiao was proved to be the most effective herb for this project. Results
achieved had satisfied the hypothesis that all the five herbs chosen based
on previous project had at least some inhibitory effects on cancer cells.
However, the hypothesis that combination of herbs would produce better
growth inhibition on MCF-7 was not yet satisfied.
This project could be further extended by detecting presence of cytochrome
C in treated cells for confirmation in cell apoptosis. Bioactive compounds
from Lian Qiao, Dang Shen and Tao Ren should also be isolated in order
for deeper chemistry analysis for each herb’s anticancer properties.
ReferencesReferences
[1]: Jemal A., Murray T., Ward E., Samuels A., Tiwari R.C., Ghafoor A., Feuer E.J., and Thun M.J.. 2005. Cancer
Statistics, 2005. A Cancer Journal for Clinicians, 55(1), 10-30.
[2]: Ruan W.J., Lai M.D., and Zhou J.G.. 2006. Anticancer effects of Chinese herbal medicine, science or myth?.
Science B 2006, 7(12), 1006-1014.