Gyeong Bok Jung, Jeong-Eun Huh, Hyo-Jung Lee, Dohyun Kim, Gi-Ja Lee, Hun-Kuk Park, and Jae-Dong Lee
We demonstrated the apoptotic effect of bee venom (BV) on human MDA-MB-231 breast cancer cells using Raman spectroscopy and principal component analysis (PCA). Biochemical changes in cancer cells were monitored following BV treatment; the results for different concentrations and treatment durations differed markedly. Significantly decreased Raman vibrations for DNA and proteins were observed for cells treated with 3.0 µg/mL BV for 48 h compared with those of control cells. These results suggest denaturation and degradation of proteins and DNA fragmentation (all cell death-related processes). The Raman spectroscopy results agreed with those of atomic force microscopy and conventional biological tests such as viability, TUNEL, and western blot assays. Therefore, Raman spectroscopy, with PCA, provides a noninvasive, label-free tool for assessment of cellular changes on the anti-cancer effect of BV.
Bee venom (BV) extracted from honey bees is commonly used in Korean medicine to treat diseases, including pain, arthritis, tumor, and skin diseases [1–3]. 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 [2–5]. 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 [3,6–9]. 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 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 by cellular morphological changes, such as shrinkage, membrane blebbing, DNA cleavage, caspase activation, and mitochondrial dysfunction [12–14].
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 . 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 [16–19]. 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 [20–23]. 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 [24–29]. Notingher et al. used Raman spectroscopy to measure the time-dependent molecular changes in cells during apoptosis . Byrne et al. evaluated the effect of the chemotherapeutic drug Actinomycin D in A549 lung cancer cell line using Raman spectroscopy . However, the anti-cancer effect of BV on breast cancer cells has not been evaluated by Raman spectroscopy.
In this study, we investigated the biochemical changes at the molecular level in human MDA-MB-231 breast cancer cells using Raman spectroscopy following exposure to BV. In addition, we examined the correlation between the Raman data and the results from conventional cytotoxicity assays and apoptotic DNA damage. We also investigated the morphological characteristics of MDA-MB-231 cells following BV treatment using atomic force microscopy (AFM).
In this study, we demonstrated the effects of concentration- and time-dependent BV treatment using Raman spectroscopy along with multivariate analysis. The Raman spectrum for the cells treated with 3.0 µg/mL of BV for 48 h corresponded to the Raman bands assigned to DNA and protein, and demonstrated a decrease in signal intensity, which was attributed to nuclear fragmentation and protein degradation. Differences in Raman spectra between the control and BV-treated cells correlated with the cellular events during apoptosis. The results of Raman spectroscopy showed good agreement with AFM and the conventional biological assays, such as viability, TUNEL, and western blot assays performed on the same types of cells. This study provides a new method to monitor the concentration- and time-dependent multi-molecular events via measurement of the vibrations of various biomolecules in living cells. Therefore, Raman spectroscopy along with multivariate analysis might be a useful tool for a label-free and noninvasive investigation of the anti-cancer effect of BV on human breast cancer cells.