Over three decades have passed since the first report on the expression of CA125 by ovarian tumors. Since that time our understanding of ovarian cancer biology has changed significantly to the point that these tumors are now classified based on molecular phenotype and not purely on histological attributes. However, CA125 continues to be, with the recent exception of HE4, the only clinically reliable diagnostic marker for ovarian cancer. Many large-scale clinical trials have been conducted or are underway to determine potential use of serum CA125 levels as a screening modality or to distinguish between benign and malignant pelvic masses. CA125 is a peptide epitope of a 3–5 million Da mucin, MUC16. Here we provide an in-depth review of the literature to highlight the importance of CA125 as a prognostic and diagnostic marker for ovarian cancer. We focus on the increasing body of literature describing the biological role of MUC16 in the progression and metastasis of ovarian tumors. Finally, we consider previous and on-going efforts to develop therapeutic approaches to eradicate ovarian tumors by targeting MUC16. Even though CA125 is a crucial marker for ovarian cancer, the exact structural definition of this antigen continues to be elusive. The importance of MUC16/CA125 in the diagnosis, progression and therapy of ovarian cancer warrants the need for in-depth research on the biochemistry and biology of this mucin. A renewed focus on MUC16 is likely to culminate in novel and more efficient strategies for the detection and treatment of ovarian cancer.

The monoterpenoid, citral, when delivered through PEG-b-PCL nanoparticles inhibits in vivo growth of 4T1 breast tumors. Here, we show that citral inhibits proliferation of multiple human cancer cell lines. In p53 expressing ECC-1 and OVCAR-3 but not in p53-deficient SKOV-3 cells, citral induces G1/S cell cycle arrest and apoptosis as determined by Annexin V staining and increased cleaved caspase3 and Bax and decreased Bcl-2. In SKOV-3 cells, citral induces the ER stress markers CHOP, GADD45, EDEM, ATF4, Hsp90, ATG5, and phospho-eIF2α. The molecular chaperone 4-phenylbutyric acid attenuates citral activity in SKOV-3 but not in ECC-1 and OVCAR-3 cells. In p53-expressing cells, citral increases phosphorylation of serine-15 of p53. Activation of p53 increases Bax, PUMA, and NOXA expression. Inhibition of p53 by pifithrin-α, attenuates citral-mediated apoptosis. Citral increases intracellular oxygen radicals and this leads to activation of p53. Inhibition of glutathione synthesis by L-buthionine sulfoxamine increases potency of citral. Pretreatment with N-acetylcysteine decreases phosphorylation of p53 in citral-treated ECC-1 and OVCAR-3. These results define a p53-dependent, and in the absence of p53, ER stress-dependent mode of action of citral. This study indicates that citral in PEG-b-PCL nanoparticle formulation should be considered for treatment of breast and other tumors.

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

The terpene-enriched steam distilled extract of ginger rhizomes has potent anti-tumor activity. Screening of the components of ginger extract demonstrated citral, a commonly used food flavoring agent, as the major monoterpene that inhibits proliferation of human and murine cancer cell lines A2780, ECC-1, OVCAR-3, SKOV-3, ID8 and MOVCAR. Citral initiates a rapid decrease in intracellular levels of glutathione and a corresponding increase in oxygen radicals. In wild-type p53-expressing ECC-1 cells, citral treatment increases phosphorylation of the Ser-15 residue of p53 and causes apoptosis. Inhibition of intracellular oxygen radicals by N-acetylcysteine and p53 activity by pifithrin-α inhibits citral-induced apoptosis in ECC-1 cells. Apoptosis in mutant p53 (Arg248Glu) expressing OVCAR-3 cells was also inhibited by N-acetylcysteine and was associated with a decrease in phosphorylation of Ser-15 of p53. Finally, in cells not expressing p53, citral inhibited proliferation, but not through apoptosis. Instead in the p53-negative SKOV-3 cells, citral treatment caused endoplasmic reticulum stress indicated by increase in phosphorylation of eIF2α and the expression of CHOP, GADD45, EDEM, ATF4 and Hsp90 and increased ATG5 and LC3-II suggesting the cells were undergoing autophagy. The study identifies the mechanism that allows citral to produce its cytotoxic effects. Combined with our recent results that nanoparticle-encapsulated citral decreases in vivo growth of 4T1 murine breast tumors, the results from the current study suggest that citral and its analogs be considered as chemotherapeutic agents. Since citral mediates its cytotoxic effects primarily by inducing oxidative stress, it is likely that this monoterpenes and its derivatives can be used for the treatment of a variety of solid tumors including ovarian cancer. Citation Format: Arvinder Kapur, Mildred Felder, Lucas Fass, Justanjyot kaur, Austin Czarnecki, Kavya Rathi, Manish Patankar. The monoterpene, citral, increases intracellular oxygen radicals and inhibits cancer cell proliferation by inducing apoptosis and endoplasmic reticulum stress . [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr B73.

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