A Raman Spectroscopic Study of Thymoquinone Antitumor Action

Fluorescence assay and Raman spectroscopy were used to study the mechanisms of action of 2-isopropyl-5-methyl- 1,4-benzoquinone (thymoquinone) on HEp-2 human larynx carcinoma cells. Thymoquinone was found to have a more pronounced toxic effect than 1,4-benzoquinone and 2,3,5-trimethyl-1,4-benzoquinone. The action of thymoquinone leads to a decrease in the mitochondrial membrane potential and release of cytochrome c from the mitochondria, indicating activation of apoptosis of tumor cells through the mitochondrial-mediated pathway. These results indicate the possibility of using Raman spectroscopy in the study of programmed cell death.

Cytochrome c absorbs light with λ = 530 nm and a strong Raman spectrum can be observed due to the resonance absorption of exciting light with λ = 532 nm [15]. The Raman spectral band with maximum at 750 cm -1 is used to study the intracellular distribution of protein. This band is related to the vibrational mode of the pyrrole ring in the cytochrome c molecule [16,17].
Materials and Methods. HEp-2 Human larynx epidermal carcinoma cells were used. The cells were cultured in DMEM medium from SigmaAldrich (USA) with added 8-10% embryonic bovine serum and 0.08 mg/mL gentamycin at 37 o C in an atmosphere of 5% CO 2 . The culture was carried out in sterile plastic fl asks (25 cm 2 bottom) equipped with air fi lters. The cells were cultured in sterile plastic Petri dishes with diameter 35 mm to study the effect of para-benzoquinones (1,4-benzoquinone, 2-isopropyl-5-methyl-1,4-benzoquinone, and 2,3,5-trimethyl-1,4-benzoquinone) on proliferational activity. Upon reseeding, the cell concentration was 1·10 -5 cells/ml. The compounds were added to the Petri dish 24 h after reseeding of the cells. Cell counting was carried out 96 h after culturing.
For the studies using fl uorescence analysis and Raman spectroscopy, the cell culture was carried out on silica plates in sterile Petri dishes. The cell concentration upon reseeding was 0.3·10 5 cells/mL. On the third day of the culture, the cell monolayer on the silica plate was washed twice with BBSS.
The Raman spectroscopic study of the intracellular distribution of cytochrome c was carried out using a spectroanalytical unit consisting of a LOTISTII Nanofi nder HighEnd scanning confocal microscope (Belarus-Japan) and a 3.2-W laser with λ = 532 nm. The signal accumulation time was 2 s and the scan step was 1 mm.
The ethyl ester of tetramethylrhodamine (TMRE) has been used to measure the mitochondrial membrane potential. Cells in BBSS were incubated with 0.l-μM probe for 30 min at 37 o C. The change in the mitochondrial membrane potential (ΔΨ) vs. elapsed time was measured using a Solar CM2203 spectrofl uorimeter (manufactured in Belarus). The intensity of the TMRE fl uorescence was measured at λ exc = 545 nm and λ rec = 590 nm. The protonophore carbonyl cyanide mchlorophenylhydrazone (CCCP) (5 μM) was used to determine the direction of change of the mitochondrial membrane potential. A study of the distribution of mitochondria in cancer cells and a qualitative evaluation of the change in the mitochondrial membrane potential were carried out using a spectro-analytical unit consisting of a LOTISTII Nanofi nder HighEnd scanning confocal microscope (Belarus-Japan) and a 3.2 MW laser with λ = 532 nm (signal accumulation time 1 s and scan step 0.4 μm). The TMRE fl uorescence was recorded at λ = 590 nm.
The kinetic curves presented are typical for a series of from three-to-fi ve independent experiments. The results are given as mean values of the plus-minus standard deviation from the mean for from three-to-fi ve independent experiments. The reliability was determined using Student's t-criterion at signifi cance level p < 0.05 ( * p < 0.05 in comparison with the control).
The change in the mitochondrial membrane potential was studied to establish the mechanisms of the toxic action of these para-benzoquinones. The addition of the para-benzoquinones to the indicated suspension of cancer cells led to a decrease in the TMRE fl uorescence intensity, indicating a decrease in the mitochondrial membrane potential. Figure 2a gives curves for the dependence of the mitochondrial membrane potential of these cells on the concentration of thymoquinone and 2,3,5-trimethyl-1,4-benzoquinone. The decrease in the mitochondrial membrane potential becomes more pronounced with increasing concentration of the para-benzoquinones, especially for thymoquinone. The difference in the dependence of the decrease in the mitochondrial membrane potential on the concentration of the indicated compounds suggests a difference in the mechanisms of the their toxic action on cancer cells. Cyclosporin A, which is an inhibitor of the opening of mitochondrial permeability transition pores, was used to study the role of such pores in the quinone-induced decrease in the mitochondrial membrane potential. Cyclosporin A previously introduced into the cell suspension inhibited the decrease in the mitochondrial membrane potential by the action of thymoquinone (Fig. 2b). In contrast, the decrease in the mitochondrial membrane potential by the action of 1,4-benzoquinone and 2,3,5-trimethyl-1,4-benzoquinone was not blocked by cyclosporin A.
In order to elucidate the details of the mechanism of programmed cell death by the action of thymoquinone, a study was carried out on the intracellular distribution of cytochrome c before and after addition of this reagent. Figure 3a,b gives im-ages of cancer cells converted relative to the intensity of the Raman spectral peak at 750 cm -1 characteristic for cytochrome c before and after the addition of thymoquinone. Figure 3c,d gives the corresponding curves for the intensity of this peak for cross section y = 15 μm. The intensity curves of the Raman spectral peak at 750 cm -1 show that prior to thymoquinone stimulation, intensity peaks are seen for regions in the vicinity of the cell membrane, indicating protein compartmentalization. Figure 3b shows that the addition of thymoquinone leads to intracellular redistribution of cytochrome c. In this case, the spectra of the thymoquinone-stimulated cells show a relatively homogeneous distribution of the intensity of the Raman spectral peak at 750 cm -1 , indicating the release of cytochrome c from the mitochondrial matrix into the cell cytosol (Fig. 3b,d).
A study of the role of mitochondria in the compartmentalization of cytochrome c was carried out using a laser confocal microscope and TMRE as a fl uorescence probe. TMRE is a lipophilic cation with a delocalized charge, which is selectively accumulated in the most electronegative region of the cells, namely, the mitochondrial matrix. The amount of TMRE probe in the mitochondria depends on the potential on the inner mitochondrial membrane. Figure 4 gives fl uorescent images of cancer cells colored by TMRE in the control and after the addition of thymoquinone. The predominant accumulation of the probe indicates localization of the mitochondria in the membrane regions of the control cells. The addition of thymoquinone leads to a decrease in the mitochondrial membrane potential accompanied by partial removal of the fl uorescent probe from the mitochondria and a corresponding change in the intracellular distribution of the probe fl uorescence intensity.  The data obtained on the distribution of cytochrome c and the intracellular localization of the mitochondria indicate that thymoquinone induces programmed cell death of cancer cells through a mitochondrial-mediated pathway.
Conclusions. The data obtained using fl uorescence analysis and Raman spectroscopy on the release of cytochrome c from the mitochondrial matrix upon the action of thymoquinone indicate activation of mitochondrial-mediated apoptosis in cancer cells. A Raman spectral study of the intracellular distribution of cytochrome c permitted us to follow the early stages of apoptosis, which is an important advantage of this method in comparison with biochemical and morphological methods for the study of programmed cell death.