An Electron paramagnetic resonance (EPR) spin labeling study in HT-29 Colon adenocarcinoma cells after Hypericin-mediated photodynamic therapy

Abstract

Background: Colon cancer affects 1.23 million people worldwide and is the third most common malignant disease in men and the second in women. The only curative treatment is surgical resection, but a significant number of patients develop local recurrence or distant metastases. One of the alternative treatment methods for colon cancer is photodynamic therapy (PDT). In recent years, hypericin (HYP) derived from Hypericum perforatum has been suggested as a strong candidate photosensitizer for PDT. Our interest is focused on the biophysical changes in colon cancer cells in relation to HYP-mediated PDT.

Results: In this study, HYP-mediated PDT at 0.04, 0.08 or 0.15 μM HYP concentrations was performed in HT-29 colon adenocarcinoma cells and the Electron Paramagnetic Resonance (EPR) spectra of the spin labeled cells were obtained. Plasma membranes are already heterogeneous structures; the presence of cancer cells increased the heterogeneity and also fluidity of the plasma membranes. Therefore, the obtained spectra were evaluated by EPRSIMC program, which provides the calculation of heterogeneous structures up to four spectral components with different fluidity characteristics. Generally, two motional patterns were obtained from calculations and the number of them increased at the highest concentration. As the order parameters of the most populated components compared, an increase was observed depending on the HYP concentration. However, because of the heterogeneous structure of membrane, the order parameters of the less populated components did not exhibit a regular distribution.

Conclusion: After HYP-mediated PDT, concentration dependent changes were observed in the domain parameters indicating an increase in the HYP accumulation.

Keywords: EPR spin labeling; Hypericin; Membrane fluidity; Photodynamic therapy.

Fig. 1

Chemical structure of hypericin

Fig. 2

Experimental (black lines) and calculated spectra (colored lines) of 16-DS spin labeled control and HYP mediated HT-29 cells at a) 298 K, b) 310 K

Fig. 3

Concentration dependent change in the order parameter of HT-29 colon cancer cells at 298 and 310 K

Fig. 4

Examples of the GHOST diagrams, defined by the RGB specification where the intensity of each color component (red, green, and blue, respectively) represents the relative value of the spectral parameters (τ_c, W, pa), for control and HYP treated HT-29 cells at 298 K. The red points show the weighted mean and peripheral colored points show the distribution of solutions

Fig. 5

Concentration dependent change in the rotational correlation time (τ_c) of spin label in HT-29 colon cancer cells at 298 and 310 K: a) from computer simulation, b) calculated from direct evaluation of spectra. The degree of significance was denoted as follows: *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 with respect to control sample

Fig. 6

Experimental, calculated, and domain spectra of 16-DS spin labeled 0.4 μM HYP mediated HT-29 cells at a) 298 K and b) 310 K