Multifunctional analysis of cultivated primary adenocarcinoma cells after treatment with different chemotherapeutic drugs

A47

Background: The discovery of new anticancer drugs is currently performed by using common cancer cell lines. However, it has been shown that long term cultured cell lines are limited in forecasting in vivo drug sensitivities. Therefore, we developed a standardized procedure to isolate pure epithelial cells directly from gastrointestinal tumor tissues and used them to examine chemotherapeutic effects of various cancer agents.
 Methods: Primary epithelial cells were freshly derived from adenocarcinoma patients, cultivated for 0, 24, 48 and 72 hours and either treated with various single tumor therapeutics such as Oxaliplatin, Gemcitabine, Iressa and 5-FU or by using drug combinations (Leucovorin/5-FU; FOLFOX; IFOX; and GOLF). Drug responses were analysed by immunofluorescence, ATP measurement and electrochemiluminescence and compared to either similarly treated HT29 cells or short and long term cultivated primary cells.
 Results: Application of the GOLF regimen showed in all cell cultures the strongest effect on cell proliferation and metabolism. We observed that compared to the single drug treatments, the different drugs composing GOLF were highly synergistic in inducing growth inhibition. The GOLF combination induced apoptosis in all colorectal cancer cells as measured by caspase-3 activation, PARP cleavage, as well as increased levels of total and phosphorylated p53. Since apoptosis onset in eukaryotic cells is substantially regulated by the balance between apoptotic and survival pathways the effects of the different drug treatments on expression and activity of the components of the Erk and Akt dependent pathways were also evaluated. In all cell cultures the induction of apoptosis due to GOLF treatment was paralleled by increased levels of total as well as phosphorylated ERK, whereas the expression and activation of EGFR, MEK, P70s6K, GSK3β and especially AKT were decreased. All these effects were also seen in cells treated with the single drugs or combinations thereof although less pronounced.
 Conclusions: Primary cells from gastrointestinal patients could provide a cellular platform beyond the common cancer cell lines for in vitro drug screening and might allow individualization of chemotherapies for patient treatments.