Abstract POSTER-THER-1417: Citral is the major component of ginger-derived terpenes to mediate p53-dependent apoptosis in cancer cells
Authors: Kapur Arvinder , Felder Mildred , Faas Lucas , Patankar Manish S. 
Published: August 2015

Abstract
Ginger extracts and ginger powder have been previously studied for their anti-cancer effects. It is generally believed that phenolic compounds present in ginger- especially shogaol and gingerol- are the active components that are cytotoxic to cancer cells. In our previous work we demonstrated that steam distilled extracts of ginger that are primarily composed of terpenes and do not contain any phenolic compounds, can induce cancer cell death with a potency that is significantly higher than gingerol and shogaol. In the current study we conduct a thorough analysis of the steam distilled extract of ginger to identify the bioactive terpene that induces cell death in ovarian cancer cells and also describe the molecular mechanisms leading to apoptosis. First, we screened the major components present in the extract for their ability to induce cancer cell death and determined that terpenes such as α-pinene and camphene are not active in the cell death assays. Instead, the major killing of ovarian and other cancer cells is mediated by the two monoterpene isomers, neral and geranial. Citral is a mixture of neral and geranial and is commercially available. Using citral in our assays we now demonstrate that these isomers can increase the expression of cleaved caspase 3 and apoptosis is also indicated in annexin V labeling assays. Treatment of cancer cells with citral results in a rapid rise in intracellular reactive oxygen species (ROS). In fact, inhibition of ROS by N-acetylcysteine blocks the ability of citral to produce apoptotic cell death. Another major observation is that citral induces rapid phosphorylation of the serine-15 residue of the tumor suppressor p53. Inhibition of ROS attenuates citral-induced phosphorylation of p53. Inhibition of p53 by pifithrin-α, a specific inhibitor, inhibits citral-mediated apoptosis. These experiments indicate that citral triggers ROS in cancer cells causing double stranded DNA breaks. DNA damage activates p53-mediated apoptotic signaling. Indeed, in cancer cells lacking p53 (SKOV3), while the ginger-derived terpene decreases proliferation, apoptosis is not observed. Instead after treatment with citral, SKOV3 cells undergo autophagy as determined by an increase in autophagolysosomes and increased expression of LC3B. We have also studied the mechanisms behind ROS increase in citral-treated cancer cells. Our studies show that immediately after exposure of cancer cells to citral, there is a rapid decrease in the intracellular levels of glutathione. This decrease is likely caused due to the enzymatic as well as non-enzymatic covalent coupling of glutathione and citral. As a result, citral works as a glutathione sink whereby the cancer cells are unable to control ROS. Concluding from these studies, we are using established concepts from medicinal chemistry to develop drug conjugates of citral that have higher bioavailability and can be targeted specifically to ovarian tumors. Even though p53 is mutated in ovarian tumors, not all mutations result in complete loss of function. We are screening for p53 mutations that result in only partial loss of tumor suppressor function. Patients stratified for such mutations will be good candidates for citral-based therapy. Citral has been approved by FDA as a safe food additive and hence can likely serve as an agent for maintenance therapy in ovarian cancer patients.
Citation Format: Arvinder Kapur, Mildred Felder, Lucas Faas, Manish S. Patankar. Citral is the major component of ginger-derived terpenes to mediate p53-dependent apoptosis in cancer cells [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-THER-1417.