These results suggest denaturation and degradation of proteins and DNA fragmentation (all cell death-related processes). Therefore, Raman spectroscopy, with PCA, provides a noninvasive, label-free tool for assessment of cellular changes on the anti-cancer effect of BV. 1. Introduction Bee venom (BV) extracted from honey bees is commonly used in Korean medicine to treat diseases, including pain, arthritis, tumor, and skin diseases [1C3]. It is composed of a complex mixture of biologically active peptides, including melittin (a major component of BV), apamin, adolapin, mast-cell-degranulating (MCD) peptide; enzymes (phospholipase A2, and hyaluronidase), and non-peptide components (histamine, dopamine, and norepinephrine), which have a variety of pharmaceutical properties [2C5]. Recent studies have shown that BV has anti-cancer effects, including induction of apoptosis and inhibition of proliferation in various cancer cells, such as prostate, breast, lung, liver, ovarian, and bladder [3,6C9]. The efficacy of BV appears to be due to the synergistic effect and selective cytotoxicity of melittin, and this anti-cancer peptide might be a better choice than the native form of BV [10,11]. The interaction of anti-cancer agents with the affected cells is considered to be Piceatannol very important for the selection and optimization of a drug to attain the most effective cancer treatment. It is well known that the function of an anti-cancer agent is to induce apoptosis of the target cancer cells. Apoptosis is characterized Piceatannol by cellular morphological changes, such as shrinkage, membrane blebbing, DNA cleavage, caspase activation, and mitochondrial dysfunction [12C14]. Assays for apoptosis, such as MTT and western blot that measure enzymatic activity and protein synthesis as endpoints and are associated with cell viability, have been investigated for chemosensitivity testing [15]. These assays are invasive, destructive, time-consuming, labor-intensive, and involve complicated procedures. Furthermore, it requires large amounts of material, while the product yield is low. The dynamics of the interactions cannot be examined directly with these assays as the introduction of fluorescent labels during measurement can change the biological conditions. Therefore, a non-invasive, label-free analytical technique is needed for the real-time monitoring of live cells. Raman spectroscopy is a non-invasive and rapid detection technique that requires no sample labeling prior to analysis [16C19]. Thus, this technique is being explored extensively for the analysis of biological systems. Raman spectroscopy provides quantitative information about the molecular structure, chemical composition, and molecular interactions within the cells, with high sensitivity and selectivity. The intracellular information about nucleic acids, proteins, lipids, and other components can be explored using variations in spectral shape or intensity [20C23]. Raman spectroscopy has been applied for the analysis of the effect of external agents on the cells, causing specific time-dependent biochemical changes associated with the process of cell death [24C29]. Notingher tests were performed for comparing 2 groups, and differences among groups were analyzed by one-way ANOVA. The statistical analyses were performed using the Prism 5.0 program. Differences were considered statistically significant at 0.05. 3. Results and discussion 3.1 Cytotoxic effect of BV on MDA-MB-231 cells and PBMLs cells To evaluate the cytotoxic effect of BV on the human MDA-MB-231 breast cancer cells and normal cells, cell viability was determined using the CCK-8 assay. Bee venom significantly inhibited the proliferation of MDA-MB-231 cells; IC50 values of 5.2, 4.5, 3.0, and 2.8 g/mL were obtained for 12, 24, 48, and 72 h incubation post-treatment, respectively, as shown in Fig. 1(A). To differentiate between the susceptibilities of cancer cells and normal cells, we explored the effects of BV in PBMLs. As shown in Fig. 1(B), BV did not show any significant cytotoxic effect on PBMLs till doses of 12.5 g/mL and 72 Piceatannol h incubation. Open in a separate window Fig. 1 Effects of bee venom on the viability of human MDA-MB-231 breast cancer cells and peripheral blood mononuclear lymphocytes (PBMLs). Cells were seeded in 96-well plates at a density of 2 105 cells/well and allowed to attach for 12 h in growth medium. MDA-MB-231 cells (A) PDK1 and PBMLs (B) were then treated with various doses of BV (0.7, 1.5, 3.0, 6.0 and 12.5 g/mL) for 12, 24, 48, and 72 h. Cell viability was assessed using Cell Counting Kit-8. Values are expressed as means S.E.M. of 3 independent experiments. *** 0.001 compared with control; n.s., nonsignificant difference compared with control. 3.2 BV-induced apoptotic cell death in human MDA-MB-231 cells To identify the apoptotic effect of BV in MDA-MB-231 cells, we performed the TUNEL assay. We observed many TUNEL-positive cells with small, dense, and fragmented morphology emitting yellow fluorescence, whereas the PI stained nucleus in the control cells exhibited round morphology and emitted red fluorescence as shown in Fig. 2(A). To confirm the mode of BV-induced cell death, we stained the MDA-MB-231 cells with annexin V/PI for 12 h, 24 h and 48 h.