● 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 BlotWestern blot analyses were

performed to observe the effect of the ERβ agonists on the receptors. More signal indicates a higher level of protein.

AcknowledgmentsWe 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.

ResultsRationale: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 Pathology5. 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, 1174–11826. 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.