Date of Award

8-2017

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Medical Education

First Advisor

Thomas E. Adrian

Second Advisor

Basil Ramadi

Third Advisor

Milos Lijubisavijevic

Abstract

Among hematological malignancies, acute leukemia is the major cause of mortality. Despite improvement of survival with current chemotherapies, some patients still diefrom the disease or the treatment side effects. Thus, new therapeutic agents are needed. Anti-cancer drugs derived from natural products are of interest. Frondoside A is a triterpenoid glycoside form the sea cucumber, Cucumaria frondosa that has shown potent antitumor effects in various cancers. Previous studies in acute leukemia are limited. The current study investigated the effects of frondoside a in acute leukemia cell lines alone and in combination with drugs currently used for this malignancy. This study is the first to attempt comparing the efficacy of this compound to available conventional drugs.

The acute leukemia cell lines used were CCRF-CEM, HL-60 and THP-1. Cells were cultured and treated with different concentrations of frondoside A, vincristine sulphate, asparaginase and prednisolone each compound alone and in combination with frondoside A. Experiments were conducted with treatment incubation periods of 24, 48 and 72h. The inhibitory concentration 50 (IC50) for each compound at each time point was determined for the three cell lines using the CellTiter-Glo luminescence assay. Induction of apoptosis was examined using Annexin V test and expression of apoptosis-related genes (low-density expression array) was investigated in two acute leukemia cell lines. The expression of protein products of selected genes was also investigated. The effect of frondoside A combined with NFκB pathway inhibitor, andrographolide in acute leukemia cell lines was also examined.

CCRF-CEM cells were very sensitive to frondoside a treatment while HL-60 and THP-1 were less sensitive. Frondoside A markedly enhanced the anticancer effects of all of the conventional drugs in all cell lines. Synergistic effects were seen in some of the combination concentrations. Induction of apoptosis was confirmed morphologically and by Annexin V in CCRF-CEM and THP-1 treated cells. Analysis of the effect of frondoside a on expression of apoptosis-related genes showed marked changes in multiple pro- and anti-apoptotic genes. Expression of some genes coding for both pro-apoptosis and anti-apoptosis proteins were increased, suggesting that a survival pathway was also activated in the frondoside A-treated cells. Frondoside A treatment increased the gene expression of multiple members of both the intrinsic and extrinsic as well as the executioner pathways.

The compound also up regulated the genes encoding multiple death receptors and death effect or domains. In THP-1 cells, frondoside a treatment resulted in the increased expression of the tumor suppressor protein p21 and it decreased the expression of the mutated p53 protein in CCRF-CEM cells. Frondoside A treatment also markedly up regulated multiple genes in the NFκB pathway with changes being more marked in the THP-1 cell line, which is more resistant to the effects of frondoside A. Combining andrographolide IC50 concentration with frondoside A in the treatment of acute leukemia cell line resulted in marked enhancement of frondoside A anti-leukemia effect.

Frondoside A has marked anti-leukemia effects. It decreased the viability of acute leukemia blasts and induces apoptosis. The apoptosis appeared to be due to the activation of both extrinsic and intrinsic pathways. Resistance to frondoside A can be due to the activation of the NFκB survival pathway in the treated cells and combining the treatment with NFκB pathway inhibitors results in dramatic enhancement of the anti-leukemic effect of frondoside A. Frondoside A affected different genes and pathways in leukemia blast cells and inhibiting malignant cells by targeting multiple pathways might be more beneficial in the treatment strategy. It potentiates the anticancer effects of all three drugs currently used to treat acute leukemias and it may be a valuable addition to the therapeutic options in these deadly diseases especially in high-risk patients by sparing the side effects of high dose therapy and bone marrow transplantation.

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