br Introduction Surface Plasmon Resonance imaging

Introduction Surface Plasmon Resonance imaging (SPRi) can be used to measure the expression of cantharidin on the cell surface and antigens or proteins secreted by cells [12,13]. In recent years a larger number of potential therapeutic agents were identified of which only few entered into the clinic and even fewer showed therapeutic efficacy with a tolerable toxicity profile. All drugs have in common that they ultimately have to kill the cancer cells. These cells will undergo a process baptized apoptosis and during this process specific alterations in the cells take place. Measurement of apoptosis in cancer cells can therefore be used to screen the effectiveness of potential therapeutic agents on cancer cell lines with properties that can be related to the cancers to be treated [4] or to measure the response to therapy in patients [16]. A number of different compounds are excreted during apoptosis and one such compound is cytochrome C. Cytochrome C is a so called hemoprotein that is associated with the inner membrane of the mitochondrion where it is part of the electron transport chain [11]. Under normal conditions cytochrome C is bound to cardiolipin in the inner mitochondrial membrane. This prevents the initiation of apoptosis. Cytochrome C can become detached from cardiolipin in early apoptosis by its oxidization due to the production of mitochondrial reactive oxygen species. Cytochrome C plays an intermediary role in early apoptosis and upon release into the cytoplasm it binds apoptotic protease activating factor-1 (Apaf-1) and activates caspase 9 [6], which in term starts a cascade of events with apoptosis as the end result. As cytochrome C is highly water soluble it can easily dissolve into the cytoplasm and disperse throughout the cell. As apoptosis progresses cytochrome C will inevitably also leak from the cell. This makes the detection by SPR possible. A variety of methods are described and are commercially available. Characteristics of apoptosis such as cell loss, nuclear morphology, DNA content, cell membrane permeability, mitochondrial membrane potential changes and cytochrome C localization and release are measured. However in order to detect all of these characteristics a number of components are mixed together in a complex and laborious protocol. Measurements of apoptosis are usually end point measurements and cells cannot be followed in real time. The most commonly used techniques to measure apoptosis are microscopy and flow cytometry [1–3,7,9,14,15,17,18]. Though robust and specific, these methods have their own unique challenges and downsides. SPR can be a potential alternative technique to study apoptosis. Previously it was shown that SPR can be used to detect apoptosis-associated genes [5], but this was not done using a cell sample. In a different study cells were brought into apoptosis and their morphological changes were monitored using SPR [8].
Materials and methods
Results
Discussion We explored whether SPRi could be used to follow apoptosis of cells by measuring the cytochrome C release as a progression marker of the apoptotic process. Before conducting real time cell measurements we harvested the culture medium of MCF7 cells exposed for two days to different concentrations of paclitaxel and showed an SPRi response on anti-cytochrome C spots. Cytochrome C was detected in cells not exposed to paclitaxel indicating the natural apoptosis of cells in culture and with increasing doses of paclitaxel Cytochrome C release increased (see Fig. 1). Unexpectedly SPRi responses were also observed on anti-EpCAM spots that were intended as negative controls. As MCF7 expresses EpCAM on their cell surface a possible explanation for the interactions could be that during the apoptosis process the MCF7 cells excreted vesicles in the supernatant. These vesicles have the potential to express similar markers as its cell of origin, but also have the capability to be distinct from their origin [19] and therefore can be the reason for the interactions seen on the EpCAM spots. To exclude nonspecific binding a separate experiment was conducted in which no cells were added to the RPMI 1640 complete culture medium with paclitaxel (data not shown). Except for a large bulk refractive index shift in the raw data, no interaction was detected, indicating that the presence of the MCF7 cells during culture was responsible for the SPRi response obtained from the supernatant. This further strengthens our conclusion that indeed we are detecting the apoptosis specific cytochrome C high response values up to 14,000 RU as well as EpCAM but the SPRi responses of the latter did not increase with increasing concentrations of paclitaxel.