The Membrane as a barrier or target in cancer chemotherapy
The overall aim of the project was to investigate the role of the cell membrane as a barrier and/or target for drug action and relate this to the development of strategies for overcoming multiple drug resistance (MDR). The effects of doxorubicin on various bacterial strains expressing different levels of anionic phospholipid were compared. Giowth of wild-type Echerichia coli (E. coli) strain MRE600 was severely affected up to 9 hours following doxorubicin treatment (15uM), but resistance occurred after 9 hours. E. coli strain FIDL1 1 was resistant to doxorubicin (1 O0piM) over 9 hours, however, increasing the anionic lipid content showed little difference in sensitivity. The mouse mammary tumour cell line (EMT6-S) and MDR sub-line (EMT6-R) were characterised with regard to growth kinetics, susceptibility to doxorubicin and membrane lipid composition. The log phase doubling times (h) were found to be 21.8 (EMT6-S)and 25.0 (EMT6-R) and the IC 50 values for doxorubicin to be 2.2 x 10-8 M and 1.8 x 10-6 M for EMT6-S and EMT6-R cells, respectively. No difference was observed between the phospholipid profiles of the two cell lines and total fatty acid composition was similar, however, the level of linoleic acid appeared to be higher in the resistant cells. The photocytotoxicity of the cationic dyes methylene blue (MB), toluidine blue (TBO) and Victoria blue BO (VBBO) against the EMT6 cell lines was compared to the cyotoxic effect of doxorubicin and cis-platinurn. The cytotoxic effect of VBBO was enhanced 10-fold by illumination (7.2 J cm2) in both EMT6-S and EMT6-R cells. In order to overcome resistance, however, the EMT6-R cells required a 10-fold greater level of the dye than the parental cells to reach an IC50 value. By contrast, doxorubicin required almost a 100-fold increase in concentration to overcome this resistance. Pre-treatment of EMT6-S and EMT6-R cells with low concentrations of VBBO resulted in a 2-fold increase in doxorubicin toxicity in both cell lines. Pre-treatment with MB and TBO resulted in a 1.4-fold and 2-fold increase in doxorubicin toxicity, respectively, in the sensitive cells, increasing to 2-fold and 3-fold, respectively in the resistant cells. Glutathione (GSH) depletion of EMT6-S and EMT6-R cells did not enhance the photocytotoxicity of VBBO, suggesting that the primary site of action of VBBO is at an intracellular site not protected by GSH or that the mechanism of action is not via the in situ generation of singlet oxygen. Addition of the chemosensitizer, verapamil (7gM), increased the efficacy of doxorubicin by 2-fold in EMT6-S cells and by 18-fold in EMT6-R cells. By contrast, the presence of verapamil did not increase the cytotoxicity of YBBO in either cell line. A series of compounds, PVB, MVB and MOVB, based on the skeleton of VBBO was examined. VBBO was found to be the most effective photosensitizer. The rate of uptake for VBBO, MVB and PVB appeared to be very similar, whereas that of MOVB was slower. The uptake/dose trend was also similar four all four drugs tested and conelated to the levels of lipophilicity of the agents. Confocal microscopy studies showed all the photosensitizers to be distributed widely throughout the cytoplasm, with considerable accumulation of VBBO and PVB in the perinuclear region. Time course studies showed the intracellular distribution of VBBO in both cell lines to be similar, although uptake of the drug appeared slower in the resistant cell line. VBBO was clearly localised throughout the cytoplasm, in a punctate pattern, which may be consistent with the widespread distribution of mitochondria. No interaction with the plasma membrane was evident. By contrast, doxorubicin was found to localise mainly in the nucleus of the sensitive cell line, whereas no nuclear involvement was seen in the resistant cells. The drug was also effluxed more rapidly from EMT6-R cells than EMT6-S cells. Time course studies with EMT6-S cells showed that the drug clearly interacts with both the plasma membrane and the nucleus. These results indicate that the main modes of action for the two drugs differ markedly, suggesting interaction with both the membrane and the nucleus in the case of doxorubicin, but possibly mitochondrial involvement for VBBO.
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