Date of Award

2023

Document Type

Thesis

Degree Name

Master of Science in Pharmacology and Toxicology

Department

Pharmacology and Toxicology

First Advisor

Kenza Benzeroual

Committee Chair and Members

Kenza Benzeroual, Chair

Eun-Jung Park

Avinash Kumar

Keywords

Apoptosis, Cancer research, Combination therapy, Glioblastoma, Herbal treatment, In-vitro treatment

Abstract

Background

Glioblastoma Multiform (GBM) is a lethal brain tumor. Despite aggressive treatment, the median survival time is 6 to 18 months. These malignant astrocytic tumors exhibit a high proliferation rate and acquire resistance against many existing therapeutic regimens. Temozolomide is a first line chemotherapeutic treatment prescribed along with surgery and radiation but due to the high resistance to the drug and a low patient survival rate, new drugs and drug combinations must continue to be investigated and developed. GBM cells like any cancer cells avoid differentiation and apoptosis. Thus, the induction of differentiation and apoptosis in glioblastoma cells may be considered as a potential treatment strategy. Evodiamine (Evo), a marketed supplement, and Cucurbitacin B (CuB), a compound obtained from natural plants, have been identified as promising candidates to treat various neoplasms but has not been well studied in GBM, and especially as a combination treatment. Their underlying anti-cancer mechanisms are still under investigation. Targeting the intrinsic PI3K/Akt and extrinsic MAPKs apoptotic pathways can prevent cell proliferation, migration and progression. Finally, the cell cycle is responsible for cancer cell division, and targeting the G2M checkpoint can prevent cancer cells from moving to mitosis. Indeed, recent studies have shown that CuB has an effect on the cell cycle by resting the G2M phase.

Objective

This study investigated the anti-neoplastic effects of Evo and CuB individually and in combination in U-87MG cells. Especially, their effect on the PI3k/Akt intrinsic and the MAPKs extrinsic apoptotic pathways, and the cell cycle targeting the G2M checkpoint.

Methods

The U-87MG cells were grown in a tissue culture plate and cell viability was determined using the MTT assay. The expression and activity of PI3K, Akt, JNK, c-Jun, Erk and MAPK 38, and Bax, Bcl2, and Caspase-3 proteins were measured by western blot analysis. A wound healing assay was performed to study the inhibition of cellular migration by Evo and CuB. G2M cell cycle phase arrest was investigated using the flow cytometry analysis. Evo and CuB, individually and in combinations, were also tested in PC-12 cells (Neuronal cell model) using MTT assay to validate the selectivity of combination towards cancer cells over the normal brain cells.

Results

The results show that Evo and CuB significantly decreased the U-87 cell viability. The effect doubled with the combination treatment. CuB and Evo imparted their effect via the PI3K/Akt intrinsic apoptotic pathway by reduced the expression of p-PI3K, p-Akt, Bcl2 and increasing the expression of cleaved Caspase-3, and via the extrinsic apoptotic pathway by increasing the expression of p-JNK, p-c-Jun, and p-MAPK 38. The combination of Evo and CuB showed significantly higher effect on cell the migration as compared to individual treatments. In the presence of the Evo-CuB combination the percentage of cell population in the G2M phase arrest was significantly higher compared to individual treatments. The cytotoxic effect of the combination was also significantly three times lower in neuronal PC-12 cells compared to U-87MG cells.

Conclusion

This in-vitro study demonstrates that the combination treatment of Evo and CuB reduced cellular proliferation and migration, most likely via the intrinsic and extrinsic apoptotic pathways. Thus, mediating an anti-neoplastic effect that is significantly higher than individual treatment against the Glioblastoma Multiform. Overall, Cucurbitacin and evodiamine in combination proved to be an effective potential anti-neoplastic treatment and may be considered as a potential adjuvant therapy in GBM.

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