Single-cell gel electrophoresis (comet assay) is one of the most common methods used to measure oxidatively damaged DNA in peripheral blood mononuclear cells (PBMC), as a biomarker of oxidative stress in vivo. However, storage, extraction, and assay workup of blood samples are associated with a risk of artifactual formation of damage. Previous reports using this approach to study DNA damage in PBMC have, for the most part, required the isolation of PBMC before immediate analysis or freezing in cryopreservative. This is very time-consuming and a significant drain on human resources. Here, we report the successful storage of whole blood in ~250 μl volumes, at -80°C, without cryopreservative, for up to 1 month without artifactual formation of DNA damage. Furthermore, this blood is amenable for direct use in both the alkaline and the enzyme-modified comet assay, without the need for prior isolation of PBMC. In contrast, storage of larger volumes (e.g., 5 ml) of whole blood leads to an increase in damage with longer term storage even at -80°C, unless a cryopreservative is present. Our "small volume" approach may be suitable for archived blood samples, facilitating analysis of biobanks when prior isolation of PBMC has not been performed.

Ovarian cancer causes significant mortality, the five year survival rate is very low compared to other cancers, and most of the cases are diagnosed late, at stage III-IV. Most of the patients relapse and develop resistance to first line chemotherapy, therefore new strategies are urgently needed to improve survival and overcome resistance. Extracts from Boswellia sp. (Frankincense) used for centuries as a herbal medicine in Asia, are known to have anti-inflammatory effects and have more recently gained interest as potential anti-cancer agents. The active ingredients of Boswellia sp., boswellic acids, have a many effects on various cancer cells including induction of apoptosis, highlighting a putative role of boswellic acids as novel anti-cancer drugs, alone or in combination with other chemotherapies. This study examines the cytotoxic properties of the principal biologically active component of Frankincense, 3-O-acetyl-11-keto-β-boswellic acid (AKBA), in epithelial ovarian cancer cells, ultimately to evaluate its potential to subvert resistance to cisplatin. This study represents the first report of the potential utility of AKBA in the treatment of ovarian cancer. Using cisplatin-sensitive and resistant cell lines we have conducted a preliminary investigation into the cytotoxic mechanism of AKBA on ovarian cancer cells, focusing on DNA damage, the role of reactive oxygen species (ROS) and cellular responses, including induction of apoptosis and cell cycle perturbations. Methods: Ovarian cancer cell lines [UWB1.289 (BRCA1 mutant), A2780, and the cisplatin-resistant counterpart, A2780cis] were exposed to different concentrations of AKBA (5, 10, 15, 25 50 μM added as single dose) for different times (0, 6, 16, 24, 48 h). Flow cytometry was used to assess cell death and cell cycle status (propidium iodide staining) and apoptosis (propidium iodide/Annexin V staining). Analysis of DNA damage was examined using the alkaline comet assay. Further examinations of cell cycle perturbations using immunocytochemical analysis of α- and γ-tubulin; and ROS production using 2,7′-dichlorofluorescein diacetate and spectrofluorimetry are ongoing. Results: The data shows that 50 μM AKBA induced significant (P<0.001) DNA damage (strand breaks and alkali-labile sites) immediately in all cell lines compared to control exposure [0.5% v/v DMSO (AKBA solvent) or cell culture medium only], whereas 15 μM and 25 μM AKBA also induced significant DNA damage after longer than 16 h exposure. Significant cell death (PI staining; subG1/G0) was observed between 25 and 50 μM AKBA dependent on the exposure time in all cell lines, with UWB1.289 and A2780 showing greater cell death overall compared to A2780cis. These observations were confirmed by Annexin V staining, except that apoptosis was observed at the lower concentrations of AKBA (after 48 h exposure) compared to simple PI staining. Cell cycle analysis showed evidence of cell cycle arrest at G2/M in all cell lines following AKBA treatment, dependent on concentration and time of exposure. Conclusions: AKBA is cytotoxic to ovarian cancer cells, at pharmacologically realistic concentrations. AKBA also induces DNA damage and G2/M arrest, which may be related to its cytotoxic effects. AKBA may form the basis of a novel anticancer treatment for ovarian cancer perhaps alongside conventional chemotherapy. Citation Format: Kamla KSA AlSalmani, Marcus S. Cooke, Ikram A. Burney, Mark D. Evans. Evaluation of the cytotoxic effects of 3-O-acetyl-11-keto-β-boswellic acid in ovarian cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr A43.