3.2. Determination of Cell Viability and Clonogenic Survival in Irradiated, PpIX-Treated HeLa Cells
To determine the effects of PpIX + X-ray treatments on HeLa cell viability, we performed a WST-1 assay (Figure 2). HeLa cells were incubated with increasing concentrations of PpIX for 6 h prior to irradiation. PpIX-treated cells were subjected to increasing levels of X-ray exposure, and then, cell viability was analyzed at 1 h, 24 h and 72 h post-irradiation. The percent of survival was expressed with reference to non-irradiated control cells. No change in cell viability was observed with increasing X-ray doses in the absence of PpIX. Furthermore, no change in cell viability was observed by increasing PpIX concentration at any of the X-ray doses at 1 h and 24 h post-irradiation (Figure 4a,b). However, at 72 h post-irradiation, cell viability decreased as the PpIX concentration and X-ray irradiation dose increased (Figure 4c).
Figure 2  Viability of HeLa cells treated with increasing PpIX concentration and X-ray exposure. The data correspond to the mean ± SD at 1 h post-irradiation (a) (n = 6), at 24 h post-irradiation (b) (n = 4) and 72 h post-irradiation (c) (n = 4). The cell proliferation reagent 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1) was used to quantify cell viability. The percent survival is shown relative to untreated control cells. No change was observed in cell viability upon increasing the X-ray dose and PpIX concentration in control cells. Statistical significance (p < 0.01) relative to the experiment performed without PpIX treatment at the same irradiation dose is indicated by (TT). Statistical significance (p < 0.01) relative to the experiment performed without irradiation at the same PpIX concentration is indicated by (+).   Figure 3 shows the effects of PpIX on clonogenic survival at different X-ray doses. HeLa cells were incubated with PpIX for 6 h prior to irradiation. Clonogenic survival is expressed with reference to non-irradiated control cells at the same PpIX concentration. Clonogenic survival decreased with increasing X-ray doses and PpIX concentration. ANOVA revealed a significant difference (p < 0.01) in clonogenic survival between the non-irradiated and the 1, 3 and 5 Gy-irradiated samples at equivalent PpIX concentrations. A significant difference (p < 0.01) was also found between control cells (no PpIX) and cells treated with 1 μg/mL PpIX in the 1 and 3 Gy-irradiated samples, as well as between the control (no PpIX) and cells treated with 3 μg/mL PpIX in the 1 Gy-irradiated sample.
Figure 3  HeLa cell colony forming was measured by the clonogenic assay. Cells were grown in six-well plates and exposed to various concentrations of PpIX and doses of X-ray irradiation. After irradiation, the cell culture medium was removed, the cells were washed with PBS and fresh medium was added. Cells were then cultured for seven days, and the cell colony formation was quantified. Clonogenic survival was standardized to a non-irradiated control (100%) treated with the same PpIX concentration. The data correspond to the mean ± SD (n = 4). Statistical significance (p < 0.01) relative to the experiment performed without PpIX at the same irradiation dose is indicated by (TT). Statistical significance (p < 0.01) relative to the experiment performed without irradiation at the same PpIX concentration is indicated by (+).   Taken together, these results indicate that PpIX treatment does not affect cellular metabolic activity at 1 h and 24 h post-irradiation, but it does affects clonogenic cell survival. Thus, for the microarray analysis, we selected the condition corresponding to 3 Gy-irradiated cells. This radiation dose affected clonogenic survival, but did not affect cell viability after 24 h, when X-ray-irradiation was the only treatment involved. Moreover, to study the effect of PpIX at the same X-ray dose, we chose to use 1 μg/mL, as this dose affected clonogenic survival.
The effect of radiosensitization becomes noticeable when the effect of PpIX becomes relatively greater than the X-ray radiation damage alone. In our study, we consider that the contribution of PpIX to the cell viability and the clonogenic survival was dominant at 1 and 3 Gy, but the X-ray radiation damage was exceedingly greater than the radiosensitization at 5 Gy.