Alterations in membrane fluidity and dynamics in experimental colon cancer and its chemoprevention by diclofenac

Abstract

We examined the role of membrane fluidity and dynamics as important early events in the carcinogenic transformation of colonic epithelial cells. 1,2-Dimethylhydrazine dihydrochloride (DMH) was used to induce initial stages of colon cancer and diclofenac was used for chemoprevention. To determine alterations of membrane fluidity of rat colonic epithelial cells, fluidity (inverse of fluorescence polarization) and order parameter were studied with 1,6-diphenylhexatriene (DPH) polarization. Order parameter as well as fluorescence polarization was found to be significantly decreased, thus demonstrating an increase in the fluidity of the membrane. To further confirm the fluidity changes, microviscosity of the cell membrane was studied using pyrene excimer formation, which showed a significant decrease in microviscosity and hence elevated membrane fluidity (translational diffusion). The colonocytes were stained with merocyanine 540 (MC540) to further elaborate the changes in membrane fluidity and lipid packing. The increased number of colonocytes showing high MC540 fluorescence pointed towards the wide spaces and hence, high fluidity in the membrane after DMH treatment. Membrane dynamics studies, i.e., lipid phase separation and membrane phase state were carried out using N-NBD-PE and Laurdan, respectively. We saw a transition from the gel to a more liquid crystalline state of the membrane in the Laurdan experiment. Further more percentage quenching (%Q) value of N-NBD-PE showed less phase separation (or domain formation). Diclofenac co-administration with DMH was successful in reverting the changes observed, confirming the role of these anti-inflammatory drugs in considerable lipid affinity and consequently in the chemoprevention of early stages of colon cancer.