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Characterizing the Mechanism of Action of a Potential Targeted Therapy, Triptolide, in Small Cell Carcinoma of the Ovary

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Small cell carcinoma of the ovary (SCCOHT) is a rare ovarian cancer affecting young women and characterized by mutation in SMARCA4 and silencing of SMARCA2, two tumor suppressors that function as ATPases in the SWItch/Sucrose Non-Fermentable (SWI/SNF) chromatin remodeling complex.

Small cell carcinoma of the ovary (SCCOHT) is a rare ovarian cancer affecting young women and characterized by mutation in SMARCA4 and silencing of SMARCA2, two tumor suppressors that function as ATPases in the SWItch/Sucrose Non-Fermentable (SWI/SNF) chromatin remodeling complex. SCCOHT patients face a 5-year survival rate of only 26%, but recently we have identified sensitivity of SCCOHT models to a natural product, triptolide. This study aims to ascertain the mechanism of action of triptolide. Previous SCCOHT epigenetic drug research has shown that some drugs reverse SMARCA2 epigenetic silencing to inhibit tumor growth, therefore it is hypothesized that triptolide acts the same and restores SWI/SNF function. Cells treated with triptolide have no change in SMARCA2 expression, suggesting that re-expression of epigenetically silenced tumor suppressor gene does not underlie its mechanism of action. Growth rates following triptolide treatment were observed in the presence and absence of SMARCA4, but no difference in sensitivity was observed. Thus, it is not likely that triptolide acts by restoring SWI/SNF. Others have observed that triptolide acts on xeroderma pigmentosa type B protein (XPB), a component of super-enhancers, which are DNA regions with high levels of transcription that regulate genes responsible for cell identity and oncogenes driving tumorigenesis. Both SCCOHT-1 and BIN67 cell lines treated with triptolide displayed lower expression of the super-enhancer associated MYC oncogene compared to untreated cells, supporting the theory that triptolide could be inhibiting super-enhancers regulating oncogenes.. A western blot confirmed reduced protein levels of RNA polymerase II and bromodomain 4 (BRD4), two essential components found at high levels at super-enhancers, in BIN67 cells treated with triptolide. ChIP-sequencing of Histone H3 Lysine-27 Acetylation (H3K27ac) marks in BIN67 and SCCOHT-1 cell lines identified super-enhancers in SCCOHT using tools CREAM and ROSE, which were mapped to neighboring genes associated genes and compared with the COSMIC database to identify oncogenes, of which the top 11 were examined by qRT-PCR to ascertain whether triptolide reduces their expression. It has been found that 6 out of 11 of the oncogenes examined (SALL4, MYC, SGK1, HIST1H3B, HMGA2, and CALR) decreased in expression when treated with triptolide. Thus, there is reason to believe that triptolide’s mechanism of action is via inhibition of super-enhancers that regulate oncogene expression.

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Date Created
2020-05

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DIRECTED ENZYME PRODRUG THERAPY: THE SYNTHESIS OF A Β-GLUCURONIDE LINKER AND ITS COUPLING WITH Z-IODOCOMBSTATIN

Description

The purpose of this project is to explore the benefit of using prodrugs in chemotherapy, as well as to explain the concept of angiogenesis and the importance of this process to tumor development. Angiogenesis is the formation of new

The purpose of this project is to explore the benefit of using prodrugs in chemotherapy, as well as to explain the concept of angiogenesis and the importance of this process to tumor development. Angiogenesis is the formation of new blood capillaries that are necessary for the survival of a tumor, as a tumor cannot grow larger than 1-2 mm3 without developing its own blood supply. Vascular disrupting agents, such as iodocombstatin, a derivative of combretastatin, can be used to effectively cut off the blood supply to a growing neoplasm, effectively inhibiting the supply of oxygen and nutrients needed for cell division Thus, VDAs have a very important implication in terms of the future of chemotherapy. A prodrug, defined as an agent that is inactive in the body until metabolized to yield the drug itself, was synthesized by combining iodocombstatin with a β-glucuronide linker. The prodrug is theoretically hydrolyzed in the body to afford the active drug by β-glucuronidase, an enzyme that is produced five times as much by cancer cells as by normal cells. This effectively creates a “magic-bullet” form of chemotherapy, known as Direct Enzyme Prodrug Therapy (DEPT).

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2015-05