Table 1. Oleic acid content determined from the HAMLET preparations using LC-MS method. Protein aD-hLA mg-hLA mg-hLA-ext mg-hLA-ext mg-hLA-ext-keto Protein to OA ratio Used to make HAMLET 1:10 1:10 1:10 1:25 1:10 Determined by LC-MS method 1 :10.4 1:8.2 1:9.2 1:34.5 1:6.1 1:7.1 mg-hLA-ext-keto-Alexa Fluor 488 1:10 doi:10.1371/journal.pone.0026093.t001 derived from the mg-hLA-ext, indicating that the C-terminal portion of the hLA does not interfere with its tumoricidal activity (Figure 7A). Incubation with OA alone had a mild influence on the viability of the SK-BR-3 cells. Similar results were obtained with the MDA-MB-468 cells (data not shown). The tumoricidal activity was studied using Annexin V-FITC staining of the cell lines SK- BR-3 (Figure 7B) and MDA-MB-468 (data not shown) after exposure to hLA in a molten globule state complexed with OA. Cells were incubated for 1 h with 30 mM protein and then stained according to the manufacturer’s protocol. Five-thousand events were analyzed by flow cytometry. Phosphatidylserine exposure (detected cell surface by Annexin V binding) was higher in cells incubated with OA complexes derived from mg-hLA and mg- hLA-ext than that in cells incubated with protein without complex with OA or media alone, as evidenced by the shift in fluorescence emission (Anexin V-FITC). In conclusion, the OA complexes of both mg-hLA and mg-hLA-ext-showed comparable tumoricidal activities. The addition of a 17–amino acid C-terminal tag to hLA doesn’t affect its biological activity when complexed with OA, making it a suitable substrate for subsequent labeling. In addition, we have tested other human cell lines, Jurkat (T-cell lymphoma), A549 (lung adenocarcinoma), MCF-7 (breast adeno- carcinoma), U-87 (glioblastoma), and HeLa (cervical adenocarci- noma) (data not shown), and studied the specificity of the complex toward tumor cell lines and the MCF-10A immortalized, non- transformed mammary epithelial cells. We found the complex is not cytotoxic when MCF-10A cells were cultured to confluence Table 2. Cell lines tested for the tumoricidal activity with HAMLET prepared with hLA-ext in 1:10 oleic acid ratio. Cell lines SK-BR-3 (breast adenocarcinoma) MDA-MB-468 (breast adenocarcinoma) Jurkat (T-cell lymphoma) A549 (lung adenocarcinoma) MCF-7 (breast adenocarcinoma) U-87 (glioblastoma) HeLa (cervical adenocarcinoma) MCF-10A (immortalized, non-transformed mammary - epithelial cells) doi:10.1371/journal.pone.0026093.t002 Tumoricidal activity HAMLET + + + + + + + Studies with Site-Specific Labeled HAMLET (data not shown), as previously described for other non-tumor cells [14]. Confocal microscopy studies For confocal microscopy studies, we labeled the C-terminal extension of hLA-ext first with C2-keto-Gal and then conjugated it with Alexa Fluor 488 (Figure 4). The labeled complex tested on A549 cells showed tumoricidal activity (Figure 8A).Using the labeled complex or molten globule protein alone; we studied internalization of the mg-hLA-ext by fluorescence confocal microscopy on an MCF-7 cell line (Figure 8). We found that the Alexa Fluor 488–labeled mg-hLA-ext when complexed with OA (Figure 8C) internalizes to cytoplasm and cell nuclei, while the Alexa Fluor 488–labeled molten globule protein of hLA-ext alone was not internalized (Figure 8B). The internalization process could be followed by live confocal microscopy on SK-BR-3 cells (Figure 8D). SK-BR-3 cells were treated with mg-hLA-ext-Alexa Fluor 488/OA (green). Nuclei were stained in blue with Hoechst 33342. With time, the complex bound to the cell membrane and was internalized. Accumulation of a green fluorescent signal could be detected in the cytoplasm and cell nuclei. The DNA was fragmented when this process occurred, and chromatin was seen on the nuclear margins. Inside the cell nucleus, an accumulation of green fluorescent signal at defined structures was seen. In a different study, co-localization of the green fluorescent signal corresponding to the complex and fibrillarin protein was found at the cell nuclei in SK-BR-3 cells (Figure 9) and MDA-MB- 468 cells (data not shown) treated with mg-hLA/OA complex. a-Domain hLA (aD-hLA) Alpha domain of hLA (aD-hLA) with and without OA complex were tested on SK-BR-3 cells and MDA-MD-468 cells (data not shown) and biological activities were observed using Trypan Blue and Annexin V (Figure 10 A and B). The aD-hLA and aD-hLA with extension (aD-hLA-ext) complexed with OA showed comparable tumoricidal activities, indicating that the beta domain of hLA is not required for the complex to acquire cytotoxic properties. The aD-hLA-ext was glycosylated, labeled with Alexa Fluor 488, and complexed with OA. To visualize the internali- zation of this alpha domain–derived complex, cells were incubated with labeled proteins, with (Figure 10D) and without (Figure 10C) complexation with OA and fixed. Nuclei were stained with Hoechst 33342. Only complexed protein was internalized after 3 h of incubation. These results indicate that we have been successful in expressing, labeling, and imaging, by confocal microscopy, a reduced size HAMLET-like molecule. Discussion Since the discovery 16 years ago of HAMLET [14], a tumoricidal complex originally isolated from human milk, investigators have been challenged to deepen the knowledge of its nature. Extensive work has already been achieved, such as defining the need for both components, the molten globule state of a-LA and OA as a lipid cofactor, and defining the cellular targets, which include mitochondria [17], proteasomes [20], nuclei [33,34], lysosomes [35], and the cell membrane [21]. Still, many aspects remain unexplained, such as HAMLET’s specificity for tumor cells, the possible presence of a HAMLET receptor, possible mechanism of action, and the nature of the complex’s structure. Studies have shown that a-LA without any disulfide bond, where all the cysteine residues are mutated to alanine residues, or PLoS ONE | www.plosone.org 6 October 2011 | Volume 6 | Issue 10 | e26093