| Abstract: | A major limitation to the clinical usefulness of cancer chemotherapy is the onset of acquired drug resistance, in which the effectiveness of a drug such as Doxorubicin (DOX), used in a wide variety of neoplasms, diminishes following repeated administration. Resistance is associated with drug exclusion from tumour cell nuclei. New analogues of DOX have been synthesised to reduce patient cardiotoxicity and to increase anti-tumour activity. More recently, a 2-fluoroglycoside of DOX (ME2303) has been shown to be more resistant to glycolysis and has marked anti-proliferative effects on DOX-resistant tumours. The aim of the current study was therefore to determine the intracellular localisation of ME2303 in drug sensitive and resistant human ovarian cancer cell aggregates by mapping fluorine, as a means of understanding the complex mechanisms of drug resistance. Cell aggregates of the human ovarian cell line A2780 and its DOX-resistant subline 2780AD were cultured for 1hr in 5μM of the drug ME2303, then chemically fixed and embedded in Lowicryl K4M using alcohol dehydration at ?20°C. Ultrathin sections (40-50nm) were examined on a Zeiss TEM 902 energy filtering electron microscope using 80Kv at a magnification of 12,000x. Fluorine maps were generated using the two window method by collecting two integrated images above and two integrated images below the K ionisation edge for fluorine (685eV) with a 15eV window. Image sequences were collected within 20sec to minimise the effects of mass loss from the specimen via a Dage SIT 66 video camera connected to a DT 2861 video interface board (512x512 pixels with 256 grey levels) within a 486 PC. In the A2780 cell line, fluorine was found to be distributed diffusely within the cytoplasm and at discrete sites within the nucleus. In contrast, fluorine in the 2780AD subline (co-cultured with 1.4μM DOX to maintain resistance) was found to be largely associated with the peri-nuclear Golgi region and with mitochondria, but was also found within cell nuclei, along the inner nuclear envelope and in the nucleolus. The intra-nuclear localisation of fluorine suggests that even in the presence of DOX, ME2303 can mediate anti-proliferative activity in DOX resistant human ovarian cancer cells by effective nuclear translocation. |
