2. The increase in melanoma incidence in U.S.
Caucasians is among the greatest for any cancer.
• Melanoma incidence rates
increased 120.5% from
1973 to 1994, while
mortality rates increased
38.9%.
• Once melanoma has
metastasized, it is almost
always fatal.
3. Current treatments for advanced melanoma are
still inadequate:
Chemotherapy with cytotoxic agents has a cure
rate of only 1%.
Interleukin-2 therapy has a similar cure rate.
4. As humans, we possess no natural protection against
solar ultraviolet radiation.
When unprotected skin is bombarded with UVB, DNA
repair is incomplete→mutations occur.
5. The Ras/Raf/Mek/Erk pathway
• Regulates cell
proliferation, gene
expression, and
apoptosis.
• Sustained ERK activation
is required to pass the
G1-restriction
point, which commits the
cell to DNA replication.
6. Melanoma cell lines commonly have high constitutive
ERK activity.
Activation of this pathway may be involved in melanoma metastasis,
through:
matrix degradation
regulation of cell adhesion
drug resistance
This suggests that the MAPK pathway would be an excellent therapeutic
target for melanoma.
7. Hypothesis: When human melanoma M14 WT cell lines
are treated with the green tea polyphenol
epigallocatechin-3-gallate (EGCG), phosphorylation of the
mitogen-activated protein kinases ERK 1/2 is significantly
inhibited compared with untreated cells.
Numerous studies have demonstrated that EGCG
blocks activation of several proteins in the MAPK
signaling pathway.
This suggests that inactivation of ERK 1/2 by EGCG
may hinder proliferation and spread of melanoma
cells.
8. Logic
• An extract of EGCG was applied to mouse skin prior to UVB
exposure, resulting in significant inhibition of ERK 1/2
phosphorylation.
• Specific, direct inhibition of ERK 1/2 and AKT activity was
discovered upon administration of EGCG to human cervical
cancer cell lines.
• EGCG was found to inhibit matrix metalloproteinase activity
in human prostate cancer cell lines through inactivation of
ERK 1/2.
9. Supporting paper #1 hypothesis: Topical application of EGCG
to SKH-1 hairless mice in vivo inhibits UVB-mediated
phosphorylation of MAPKs.
It was demonstrated that UVB
exposure increases ERK
phosphorylation while
EGCG attenuates it.
This connects with my hypothesis
because I will show an EGCG-
mediated block of ERK
phosphorylation in a cell line in
which ERK activation is higher
than normal.
10. The authors wanted to investigate the molecular
basis of the chemopreventive effects of EGCG in
vivo.
The SKH-1 hairless mouse model was used
because it resembles humans who are
chronically exposed to sunlight. Female mice
and younger mice are more sensitive to sunlight,
so 6-week-old female mice were used.
11. Western blot analysis of MAPK phosphorylated protein
demonstrated a significant in vivo inhibition of ERK 1/2
activation in EGCG-pretreated UVB-exposed mouse skin
when compared with unpretreated controls.
48 mice were divided into 4 groups (n=12):
1. Treated topically with 200 µl acetone.
2. Treated topically with 5 mg EGCG/200 µl acetone.
Treated with only acetone and exposed to 180mJ/cm2
3.
UVB.
Treated with EGCG/acetone and exposed to 180 mJ/cm2
4.
UVB.
12. Results/Implications:
• Repeated UVB exposure resulted
in an increased phosphorylation
of ERK 1/2, while preapplication
of EGCG prior to repeated UVB
exposure significantly inhibited
ERK 1/2 phosphorylation.
• Relative density of the bands was
measured from four individual
blots, confirming a significant
reduction of ERK 1/2 activation with
EGCG treatment (p<0.01).
13. Critique of Experiment
• The effects of EGCG on UVB irradiation are tested in vivo,
confirming that the molecular mechanisms of ERK
inactivation are relevant in an intact mammal.
• Scanning densitometry was used to compare band intensity,
but there is still a level of objectivity in comparing Western
blot bands. The blots shown in the paper may not be
representative of all blots obtained.
14. Summary
• This is the first paper to examine the cell signaling effects of
repeated UVB exposure and topical EGCG pretreatment in
vivo.
• Comparison of clinical and molecular changes in skin treated
with EGCG and exposed to UVB light enhance the credibility
of this paper.
• The authors suggest that topical EGCG treatment prior to UV
exposure results in significant decreases in clinical and
molecular markers of UVB damage.
15. Supporting paper #2 hypothesis:
• EGCG inhibits the proliferation of human cervical tumor cells
through the inactivation of EGFR and several other
downstream MAPKs.
• How this connects to my hypothesis: this paper points to
very specific proteins in the MAPK cascade that are inhibited
by EGCG.
16. EGFR, ERK 1/2, and AKT all play key roles in the
ability of cervical tumor cells to resist apoptosis
and proliferate.
EGCG, by blocking these cell signaling proteins,
may halt uncontrolled cell proliferation.
17. The most supportive evidence-kinase
immunoprecipitation
• Shows a direct effect on ERK 1/2
phosphorylation by EGCG.
• Precipitated kinases were
incubated in in vitro kinase
reactions in the presence of 0-50
µM EGCG.
• Low concentrations of EGCG (5
µM) caused a substantial
reduction in protein kinase B and
ERK 1/2 activity.
18. Critique
• Immunoprecipitation is an excellent means for an
investigator to analyze changes in individual signaling
pathways brought about by chemopreventive and
chemotherapeutic agents.
• However, this is an in vitro method, and the results could
prove to be quite different in an in vivo system.
19. Summary
• These investigators show significantly diminished EGFR, ERK
1/2, and AKT activation with treatment of cervical cancer
cells with EGCG.
• They also demonstrate that attenuated ERK activation is
associated with G1 arrest and apoptosis.
20. Supporting paper #3 hypothesis:
• EGCG inhibits tumor invasion and angiogenesis of prostate
cancer by inhibiting MMPs through the obstruction of MAPK
phosphorylation and subsequent blocking of transcription
factors AP-1 and NF-κB.
• Logical connection to my hypothesis: The authors suggest
from the results of this paper that EGCG may prevent tumor
metastasis. Melanoma is the most metastatic of all human
skin tumors, and I hypothesize that EGCG will halt metastasis
by putting the brakes on ERK 1/2 phosphorylation.
21. The most supportive evidence-Western blotting after
treatment of DU-145 cells with EGCG.
• DU-145 cells were cultured in
FCM and treated with 0, 5, 10,
20, or 40 µg/ml EGCG for 24
hours.
• Western blot showed that
treatment of EGCG to cells dose-
dependently inhibited FCM-
induced phosphorylation of ERK
1/2 and p38, but not JNK.
22. Critique
• This shows a specific inhibition of the ERK 1/2 and p38
signaling cascades by EGCG, contrasting with a lack of
inhibition of the JNK pathway.
• Only a representative blot from three independent
experiments is shown. The authors may have shown three
blots that best demonstrate what they were hoping to
confirm in this study.
23. Summary
• This paper shows a clear connection between EGCG
treatment and reduction in ERK 1/2 phosphorylation with
subsequent growth arrest in human prostate cancer cell
lines.
• The authors suggest that EGCG has a clear inhibitory effect
on MMP activity, and that more studies should be conducted
in an in vivo prostate cancer system to further elucidate
EGCG’s anti-metastatic, anti-angiogenic effects.
24. When human melanoma M14 cell lines are treated with the
green tea polyphenol EGCG, phosphorylation of ERK 1/2 is
significantly inhibited compared with untreated cells.
• To analyze interactions between ERK 1/2 and its associated
proteins, a kinase immunoprecipitation assay will be
performed.
• Cells will be treated with 1, 5, 10, and 20 µM EGCG.
• Controls: Cells untreated with EGCG.
25. Limitations
• Data will be shown as relative density of bands. This is
somewhat subjective; an experiment in which statistics can
be used would be more objective.
• This experiment is performed in vitro; we will not know
whether the molecular changes elicited by EGCG would be
the same in an in vivo system.
26. Literature Cited
Afaq F., Ahmad N., Mukhtar H. (2003). Suppression of UVB-induced
mitogen-activated protein kinases and nuclear factor kappa B by green
tea polyphenol in SKH-1 hairless mice. Oncogene 22: 9254-9264.
Sah J., Balasubramanian S., Eckert R., Rorke E. (2004). Epigallocatechin-3-
gallate inhibits epidermal growth factor receptor signaling pathway.
Evidence for direct inhibition of ERK 1/2 and AKT kinases. The Journal of
Biological Chemistry 279 (13): 12755-12762.
Vayalil P.K., Katiyar S.K. (2004). Treatment of epigallocatechin-3-gallate
inhibits matrix metalloproteinases-2 and -9 via inhibition of activation of
mitogen-activated protein kinases, c-jun and NF-κB in human prostate
carcinoma DU-145 cells. The Prostate 59: 33-42.
