Epigallocatechin-3-gallate and chemoprevention of melanoma

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.

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.

As humans, we possess no natural protection against solar ultraviolet radiation.

When unprotected skin is bombarded with UVB, DNA repair is incomplete→mutations occur.

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.

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.

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.

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.

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.

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.

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.3. Treated with only acetone and exposed to 180mJ/cm2

UVB.4. Treated with EGCG/acetone and exposed to 180 mJ/cm2

UVB.

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).

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.