Abstract 3211: Citral is the major component of ginger-derived terpenes to mediate p53-dependent apoptosis in cancer cells

Steam distillation of ginger yields a mixture of twenty two terpenes. This mixture has anti-proliferative effect on over fifteen different human and mouse cancer cell lines tested in our previous studies. Since the mixture is heterogeneous, it cannot be developed into a therapeutic drug. Hence, we tested individual terpenes from the ginger extract to identify its bioactive component(s). Camphene and alpha-pinene together constitute 10% of the ginger terpene extract and have no effect on cancer cell proliferation. On the other hand, citral a mixture of the two isomers, neral and geranial which constitute 30-50% of the ginger terpene extract, inhibits proliferation of ECC-1, OVCAR-3, OVCAR5, OVCAR 433 with IC50 between 20-50 μM. The decrease in proliferation of the cells is due to induction of apoptosis by citral as measured by monitoring Annexin V and propidium iodide staining and expression of cleaved caspase3. Treatment of cancer cells with citral results in a rapid increase in intracellular Reactive Oxygen Species (ROS). The cellular stress resulting from ROS generation leads to phosphorylation of the Ser-15 residue of p53. Activation of p53 via this phosphorylation event leads to apoptosis in cancer cells. When cells are pre-treated with ROS inhibitor, N-acetyl cysteine (NAC), citral induced apoptosis is inhibited. Similarly inhibition of p53 by pifithrin-α or knockdown of the tumor suppressor by siRNA attenuates the pro-apoptotic response of citral. There is a significant increase in p53 protein expression in cancer cells when treated with Citral This increase in p53 is likely due to an increase in p53 gene expression as determined in our qPCR assays, even though increase due to inhibition of proteasomal degradation of the protein cannot be completely ruled out at this point. An increase in p53 expression in response to citral is associated with an increase in expression of the p53 responsive genes BAX, PUMA and NOXA. All of these experiments demonstrate a novel p53-dependent mechanism for citral to induce apoptosis in cancer cells. Anti-proliferative effects of citral were observed in cancer cells expressing wild-type p53 (ECC-1) as well as in cell lines with R248Q (OVCAR3), ins224 (OVCAR5) p53 mutations. Based on these experimental results we propose that citral should be considered as a novel p53 activating agent that can be used as an adjunct to conventional chemo- and biologic therapies against cancer. Our on-going efforts will identify sensitivity of commonly found p53 mutations to activation by citral and also the specific p53 kinases triggered subsequent to the ROS surge mediated by this terpene. Citation Format: Lucas Fass, Mildred Felder, Manish S. Patankar, Arvinder K. Kapur. Citral is the major component of ginger-derived terpenes to mediate p53-dependent apoptosis in cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3211. doi:10.1158/1538-7445.AM2014-3211