Objective: Antimitotic drugs, like paclitaxel and vinblastine, share a common mechanism of action, suppressing microtubule dynamics in mitotic spindles and arresting cells in metaphase. These processes frequently lead to programmed cell death (apoptosis). Although it is well known that antimitotics bind to their target tubulin, the major protein in microtubules, the mechanisms by which drug activity leads to apoptosis are not well understood. Our major objective was to identify alternate intracellular antimitotic targets that might be directly linked to apoptotic events.
Design: The study design was experimental basic laboratory research.
Sample and Setting: Epithelial cell lines (human breast cancer and chinese hamster ovarian) were examined using antibodies that recognize tubulin and possible alternate targets such as: centrosomes. Other alternate targets were visualized using dyes that bind to mitochondria or lysosomes. Experiments were carried out in a basic science laboratory with cell culture, flow cytometry and fluorescence microscopy facilities.
Variables: The variables were vinblastine, fluorescent vinblastine (vinblastine bodipy), bodipy, monoclonal antibodies against centrosomal proteins and the cytoskeletal protein tubulin, rhodamine 123 (R123) that binds to mitochondrial membranes and lysotracker dye that interacts with lysosomes.
Methods: The potential apoptotic effects of vinblastine bodipy or bodipy alone were studied using flow cytometry. Cells were grown overnight in the presence or absence of vinblastine bodipy or bodipy alone. The fluorescent bodipy label permits quantification of drug levels throughout the cell cycle. Propidium iodide labeling of DNA in these experiments allows us to quantifiy drug effects on phases of the cell cycle. For example, we can determine whether the drug increases the percentage of cells in mitosis.
Alternate drug targets were examined using fluorescence microscopy techniques with appropriate controls. Cells in log phase were grown on coverslips overnight and then exposed to vinblastine bodipy (plus/minus unlabeled vinblastine) for 24 hours. Control cells were also grown in the presence of the bodipy moiety alone or in the complete absence of drug. For single label experiments cells in the absence of drug were briefly exposed to R123 or lysotracker (5 min) and then prepared for visualization by fluorescence microscopy. Using single antibodies or dyes, we were able to study common patterns of organelle staining. In addition, single and double labeling experiments were carried out with vinblastine bodipy plus minus fluorescent antibodies that recognize tubulin or centrosomes. From these experiments cells could be examined for co-localization of drug and organelle-labeled antibodies.
Findings: Flow cytometry experiments indicate that vinblastine bodipy induces apoptosis in cells. This result agrees with experiments using unlabelled vinblastine. Bodipy alone does not induce apoptosis. Fluorescence microscopy studies show that vinblastine bodipy directly interacts with mitochondria, often appearing as mitochondrial clustering that increases in competition experiments with unlabelled vinblastine. The bodipy moiety alone shows only very weak mitochondrial staining, without mitochondrial clustering.
Conclusions: In addition to targeting the cytoskeletal protein tubulin, vinblastine appears to directly interact with mitochondria. Disruption of mitochondrial membranes and release of cytochrome c are steps that lead to apoptosis. It is possible that this direct interaction between drug and mitochondria could weaken mitochondrial membranes leading to cytochrome c release and apoptosis.
Implications: Nurses are responsible for administering and monitoring drug therapy. Antimitotics are highly toxic to normal tissues especially in the bone marrow and the nervous system. Identification of novel drug targets may help to explain drug action and toxic effects. This information could contribute to developing more selective antimitotics or chemopreventive agents to reduce toxicity. (Supported by NIH Grant, NINR, NR04780)
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