Abstract 1390: Honokiol, a bioactive component from Magnolia plant, promotes DNA demethylation and reactivates silenced tumor suppressors in pancreatic cancer cells through TET-dependent mechanism
Authors: Prasad Ram , Katiyar Santosh 
Published: August 2018

Pancreatic cancer is the fourth leading cause of cancer-related deaths in the United States. American Cancer Society estimated that in 2017, approximately 53,670 new cases of pancreatic cancer will be diagnosed and 43,090 deaths will occur. In addition to genetic changes, epigenetic alterations are commonly associated with cancers including pancreatic cancer. Studies have shown that DNA hypermethylation contributes to tumor growth by inactivating tumor suppressors. DNA methyltransferases (DNMTs) regulate DNA methylation, but mechanisms underlying DNA demethylation have not fully elucidated and have enormous translational potential. Ten-eleven translocations (TETs) have been shown to convert 5-methylcytosine (5-mC) into 5-hydroxymethylcytosine (5-hmC) and eventually reactivates tumor suppressors. Bioactive components from plants have shown their efficacy as potential chemopreventive and therapeutic agents. Here, we investigated the effect of honokiol on DNA methylation using in vitro cell culture model. We first checked the expression levels of global DNA methylation in pancreatic cancer cell lines (PANC-1, AsPC-1) and compared with normal human pancreas duct epithelial cells. Our data revealed that the levels of global DNA methylation were higher in pancreatic cancer cell lines. Treatment of AsPC-1 and PANC-1 cells with honokiol for five days resulted in significant decrease in global DNA methylation levels (60-80%, p&lt;0.001) compared to non-honokiol treated cells. To detect the comparative levels of 5-mC/5-hmC in AsPC-1 and PANC-1 cells immunofluorescence staining was used. Honokiol treatment increases 5-hmC levels in pancreatic cancer cells compared to non-honokiol-treated cells. The number of 5-hmC positive cells were significantly higher in honokiol-treated AsPC-1 (12-61%; p&lt;0.01-0.001) and PANC-1 (42-77%; p&lt;0.001) cells in a dose-dependent manner compared to non-honokiol treated control group as verified by flow cytometry. Honokiol treatment also significantly increases the levels of TET activity and protein expression in cancer cell lines. We also found that concomitant treatment of cancer cells with cycloheximide (proteins synthesis blocking agent) prevented honokiol-induced TET expression and formation of 5-hmC and therefore suggest that honokiol promotes 5-hmC formation (i.e., demethylation) through TET activation. Ascorbate, an inducer of TETs, also promotes 5-hmC formation in pancreatic cancer cells. Additionally, honokiol treatment reactivates tumor suppressor genes/proteins levels, such as p16INK4a and RASSF1A in cancer cells. Together, this study provides evidence that honokiol acts as a DNA demethylating agent and is able to reactivate an epigenetically silenced tumor suppressors and thus inhibits the growth of pancreatic cancer cells.

Citation Format: Ram Prasad, Santosh Katiyar. Honokiol, a bioactive component from Magnolia plant, promotes DNA demethylation and reactivates silenced tumor suppressors in pancreatic cancer cells through TET-dependent mechanism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1390.