In vitro studies of the effects of funfal-derived immunosuppressive agents on MCF7 breast cancer cells and Molt4 Leukaemia cells

The therapeutic and aide effects of fungal-derived immunosuppressive drug such as Cyclosporin A (CsA) are still controversial, with the same amount of data published to date that states the beneficial effects of immunosupressants as well as their side effects. The molecular mechanism by which CsA on MCF-7 breast cancer and MOLT-4 human lymphoblastic T-cells were examined and results were compared to the effects of Mycophenolic acid (MPA) and Rapamycin (Rapa) in the search 6for new approaches that may explain controversy of these agents. CsA is a cystostatic drug that has been reported to arrest the cells in the early Gi phase of the cell cycle thus protecting the cells from becoming apoptotic. However, the results obtained from studies in this thesis indicated a proportion of cell death up to 27% after 36 hours of CsA addition to the MOLT-4 cultures. The nature of the cell death was addressed using flow cytometry analysis and it was concluded that up to a 95% of the cell death was due to apoptotic events. The process occurred in a cell cycle dependent manner, where CsA induced apoptosis with the first 48 hours in culture, and cell arrest after this time. As CsA acts through calcium dependent mechanisms, the intercellular free calcium concentration of MOLT-4 cells were measured to establish whether fluctuations in intracellular calcium levels could trigger programmed cell death. A 12-bit cooled CCD camera was used to picture the fura-2 ratiometric intracellular calcium values in MOLT-4 cells, and variations in the intracellular calcium concentrations were associated with cell toxicity when immunosuppressants were added to suspension cultures. CsA induced three-fold increase (428 +12 uM) in the [Ca2+] levels of MOLT-4 cells after 1 hour, compared to control cells (146+5 uM), and then the [Ca2+], levels decreased towards control values after 36 hours. The expression of antigenic CD molecules in the cell surface is a calcium dependent mechanism and differences were found in the pattern of CD antigen expression when CsA was added in culture. CsA (5uM) reduced CD8+ membrane expression in the MOLT-4 cell cultures by 49.1+3.0% and CD4+ membrane expression by 87.4+3.1%. In summary, these studies may bring new insights into the molecular mechanisms of CsA, which involve cell death and prevention of cytolytic T-cell presentation.

Name dc:type.qualificationname

PhD

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

De Montfort University

Year dc:date.issued

2002

Author dc:creator

• Ochando, Jordi Cano

Licence

• https://dora.dmu.ac.uk/bitstreams/672d24e3-cefb-47d6-b4b2-2481aecca4f0/download