1. ● Glioblastoma Multiforme (GBM) is the most common
and malignant primary tumor in adults. Five year
survival rate is less than 5% in adults.4
● Incidence rates of GBM suggest a “sex protection”
for women over men. It has been proposed that
estrogen receptor agonists play a role in the etiology
of GBM.1, 5
● In this study, we investigated the synergistic effects of
beta-estradiol and estrogen receptor β agonists, such
as liquiritigenin and isoliquiritigenin, GBM
proliferation.
Beta-estradiol Isoliquiritigenin Liquiritigenin
Glioblastoma Cell lines
pcGBM2: Patient derived cell line
CellTiter-Blue® Cell Viability Assay
The CellTiter-Blue® reagent detects
mitochondrial activity in living cells
via fluorescence. Elevated fluorescent
signals indicate more living cells and
less effective drugs.
Drug Extraction
To compare the effects of purified
liquiritigenin versus a crude extract,
licorice bark was ground and boiled.
Western Blot
Western blot analyses were
performed to observe the effect
of the ERβ agonists on the
receptors. More signal indicates
a higher level of protein.
Acknowledgments
We want to thank Dr. Sanjiv S. Gambhir, Google and Ben
& Catherine Ivy Foundation for supporting the internship
program. We also want to thank Drs. Edwin Chang,
Arutselvan Natarajan, and Paulmurugan Ramasamy for
their guidance and support.
Results
Rationale:
Beta-estradiol ((β-E2): The primary female sex hormone. We investigated estradiol because estradiol levels fall
after menopause, the same time that GBM incidence rates for women begin to rise 3
.
Liquiritigenin (LQ): The most active estrogenic compound from the root of Glycyrrhizae uralensis Fisher
(licorice). It is a plant-derived selective estrogen receptor beta agonist 2
.
Isoliquiritigenin (ILQ): A flavonoid that is also derived from licorice bark 6
.
GC-5b: A crude extract of the licorice bark, containing liquiritigenin, isoliquiritigenin, and other compounds
Figure 1: Comparative effect of crude extract vs purified single drug
Figure 2: Synergistic effects of β-E2+LQ, β-E2+ILQ, LQ+ILQ
Beta-estradiol +
Liquiritigenin
Beta-estradiol +
Isoliquiritigenin
Liquiritigenin +
Isoliquiritigenin
Conclusions
● Beta-estradiol, liquiritigenin, and
isoliquiritigenin have a relatively weak dose
response.
● However, we found that in combination,
these agonists are very potent.
● These findings indicate that combinations of
beta-estradiol, liquiritigenin, and
isoliquiritigenin could be useful in clinical
applications.
1. Kabat, Etgen, and Rohan (2010). Do Steroid Hormones Play a Role in the Etiology of Glioma? Cancer Epidemiology Biomarkers & Prevention 19, 24212427.
2. Mersereau, J., Levy, N., Staub, R., Baggett, S., Zogric, T., Chow, S., . . . Bjeldanes, L. (2008). Liquiritigenin is a plant-derived highly selective estrogen receptor β agonist. Molecular and Cellular Endocrinology, 49-57.
3. Normal Testosterone and Estrogen Levels in Women. (n.d.). Retrieved August 14, 2015.
4. Olar, A, and Aldape, KD (2014). Using the molecular classification of glioblastoma to inform personalized treatment. The Journal of Pathology
5. Sareddy, G., Nair, B., Gonugunta, V., Zhang, Q., Brenner, A., Brann, D., Tekmal, R., and Vadlamudi, R. (2012). Therapeutic significance of estrogen receptor β agonists in gliomas. Mol. Cancer Ther.11, 1174–1182
6. Takahashi, T., Takasuka, N., Iigo, M., Baba, M., Nishino, H., Tsuda, H., & Okuyama, T. (2004). Isoliquiritigenin, a flavonoid from licorice, reduces prostaglandin E2 and nitric oxide, causes apoptosis, and suppresses aberrant crypt foci development. Cancer Science, 448-453.
7. 1-Click Docking. Mcule. <https://mcule.com/apps/1-click-docking/>.
Future Directions
● Preliminary Western analysis suggests no
induction of ERβ expression by LQ or ILQ.
(See figure 3).
Next Steps:
● Analyze the effect of LQ and ILQ on ERα
expression level
● Analyze the interaction between ERα and
ERβ.
● Assess effect of LQ, ILQ, estradiol on
ERβ activity and downstream gene
expression.
● Test agonist combinations on other
glioma cell lines and in an animal model.
Therapeutic Effects of Estrogen Receptor Beta
Agonists on Glioblastoma Proliferation
Introduction
Materials and Methods
Results Conclusions
Literature cited
Future Directions
Acknowledgments
Figure 3: Western Blot for antibodies to ERβ
in untreated and drug treated pcGBM2.