3. Results and Discussion

3.1. PpIX Uptake by HeLa Cells
To study the behavior of PpIX in HeLa cells, we examined intracellular porphyrin levels. During porphyrin-mediated PDT, PpIX is absorbed into tumor cells, where it them accumulates [14]. While other porphyrins are detected using this method, PpIX is the major porphyrin found in cells and tissues [15]. HeLa cells were incubated with increasing concentrations of PpIX for 6 h, and the levels of porphyrin uptake were measured (Figure 1). We found that PpIX was rapidly taken up by the cells, and intracellular porphyrin accumulation increased with increasing PpIX concentrations.
Figure 1  Total porphyrin levels in HeLa cells. Cells were incubated with different concentrations of PpIX for 6 h. The data shown correspond to the mean ± SD (n = 4).

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.

3.3. Generation of ROS by PpIX and X-Ray Irradiation
Intracellular ROS production was assessed by pre-incubation of HeLa cells with cell-permeable fluorescent probes (APF or DHE) that show different sensitivities to specific ROS species (∙OH, O2− and 1O2). HeLa cells were incubated with PpIX for 6 h prior to X-ray irradiation. Cells without PpIX treatment were irradiated under the same conditions as those used for controls. ROS levels are expressed in terms of fluorescence intensity. Figure 4a shows the effects of PpIX on intracellular ROS levels at different X-ray doses in the presence of APF. X-ray irradiation in the absence of PpIX increased ROS levels in HeLa cells. After pre-incubation with PpIX, ROS levels increased with the X-ray dose and PpIX concentration. Figure 4b shows the effects of PpIX on the intracellular ROS levels for various X-ray doses in the presence of DHE. No change was observed in the intracellular ROS by increasing X-ray irradiation in the absence of PpIX. After pre-incubation with PpIX, the ROS levels increased with PpIX concentration and X-ray dose.
Figure 4  Detection of intracellular ROS levels in HeLa cells exposed to increasing PpIX concentrations and X-ray doses, using aminophenyl fluorescein (APF) (a) or dihydroethidium (DHE) (b). PpIX was added 6 h prior to X-ray irradiation. Before X-ray irradiation, APF or DHE was added to HeLa cells at a final concentration of 10 μM and incubated in the dark for 30 min at 37 °C. After X-ray irradiation, the medium was removed, and the cells were washed with PBS. The resulting fluorescence was measured using a microplate reader. 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 (+).   In our previous study, using standard reactions, we showed that APF had a higher sensitivity for detecting ∙OH, unlike DHE, which showed higher sensitivity for detecting O2− and s1O2 [3]. Therefore, these results suggest that X-rays increase ∙OH generation and that PpIX enhances the production of ∙OH, O2− and 1O2.

3.4. Microarray Gene Expression Analysis
For the microarray analyses, RNA was extracted from HeLa cells with no treatment (NT), cells treated with 1 μg/mL PpIX without X-ray irradiation (PpIXT), cells that had been irradiated with 3 Gy without PpIX treatment (XT) and cells that had been treated with both 1 μg/mL PpIX and 3 Gy-irradiation (PpIX-XT). In this experiment, the cells were collected 24 h after irradiation. These specific conditions were chosen, because X-ray exposure at a dose of 3 Gy did not affect cell survival 24 h post-irradiation, regardless of the concentration of PpIX treatment (Figure 2b). Furthermore, 1 μg/mL PpIX-treated cells were used because PpIX enhanced ROS generation induced by X-ray irradiation at this concentration (Figure 4b). Moreover, clonogenic survival was affected by 1 μg/mL PpIX treatment and X-ray irradiation (Figure 3). Transcriptional changes occurred as early as 0.5 h to 2 h after X-ray exposure and peaked shortly thereafter [16,17]. After that time, transcriptional changes were observed 12 to 24 h after irradiation [16,18,19]. In each group, the RNA sample was analyzed using a human gene expression microarray consisting of 43,376 oligonucleotide probes.

3.4.1. Analysis of Microarray Gene Expression Profiles via the Classification of Upregulated and Downregulated Genes
We characterized the genes that were upregulated or downregulated by PpIX treatment prior to X-ray irradiation. Accordingly, we first selected genes with altered expression in each treatment group with respect to the NT group based on their p-values (p < 0.01). Then, these genes were classified as upregulated or downregulated after each treatment. In the PpIX-XT group, 290 genes met the selection criteria for upregulation, and 203 genes met the selection criteria for downregulation. In the XT group, 196 genes were upregulated and 146 genes were downregulated. In the PpIXT group, 28 genes were upregulated and 39 genes were downregulated (Table 1). The number of differentially-expressed genes was highest in the PpIX-XT group, followed by the XT and PpIXT groups. We selected genes for the subsequent functional analysis based on their p-value. This selection method was mathematically proven to consider both the fold, as well as the differences.
microarrays-04-00025-t001_Table 1 Table 1  Summary of the microarray data with p-values < 0.01 in the PpIX treatment alone (PpIXT), 3 Gy X-ray irradiation alone (XT) and 3 Gy X-ray irradiation (PpIX-XT) groups, versus the non-treatment (NT) group.

3.4.2. Functional Analysis
The selected genes were analyzed using the functional annotation chart in the Database for Annotation, Visualization and Integrated Discovery (DAVID, version 6.7). Using this analysis, we extracted the GO terms based on the number of genes in each GO category. Table 2 shows the biological processes (as identified by the GO terms) that were differentially-represented with respect to the NT group (FDR < 0.01). Eighteen GO terms were identified among the upregulated genes in the PpIX-XT group, and six GO terms were identified among the downregulated genes. Twenty GO terms were identified among the upregulated genes in the XT group, and four GO terms were identified among the downregulated genes. Moreover, no GO terms were identified among the upregulated or downregulated genes in the PpIXT group. These GO terms corresponded to four functions: cell cycle regulation (15 GO terms), DNA metabolic processes (five GO terms), chromosome organization (10 GO terms) and cellular response to stress (two GO terms). The changes in gene expression after PpIX administration without X-ray irradiation (PpIXT) were less extensive than those in other treatment groups and were not systematic.
For each GO term corresponding to cell cycle regulation, the number of genes was higher and the p-value of the FDR was lower in the PpIX-XT group than in the XT group. These results indicated that cell cycle disturbances were induced in both the PpIX-XT and XT groups; however, the extent of these disturbances was higher in the PpIX-XT group than in the XT group. For a more detailed analysis of the molecular processes disrupted in XT and PpIX-XT groups, we next evaluated the genes that corresponded to the GO terms.

3.5. Functional Validation Using Marker Genes
Based on the results of the functional analysis, we examined the expression levels of genes involved in cell cycle regulation, DNA metabolic processes and chromosome organization. We selected genes with well-described functions in order to best characterize the corresponding biological responses.
microarrays-04-00025-t002_Table 2 Table 2  Biological processes based on GO terms that are differentially represented in the PpIXT, XT and PpIX-XT groups, with reference to the NT group. The data were generated using the functional annotation chart provided in Database for Annotation, Visualization and Integrated Discovery (DAVID).   We first examined marker genes involved in cell cycle regulation, because the functional analysis identified 15 GO terms related to this process. Figure 5a shows the heat map of marker gene expressions for cell cycle regulation. Each row represents a gene, and each column represents the ratio of the average fold change in gene expression relative to the NT group, as well as associated p-values. The color intensity depicts the degree of expression; the blue color indicates highly negative expression, while red indicates highly positive expression. Cyclins, cyclin-dependent kinases and cyclin-dependent kinase inhibitors, among other genes, were selected as markers of the cell cycle. CCNE1, CCNE2 and CDK4 were downregulated, whereas Cdkn1a was upregulated in the XT and PpIX-XT groups. Furthermore, upregulation of ATM, CHK2 and CDKN1A indicated inhibition of DNA replication. Figure 5b shows the heat map of the selected genes for the DNA metabolic process (GO:0006259). Key genes involved in DNA replication, such as PCNA, the MCM complex (MCM5, MCM8 and MCM10), LIG1 and DNA polymerase (POLA1, POLD3), were downregulated relative to the NT group. Therefore, XT and PpIX-XT treatment arrested the tumor cells at the G1 transition of the cell cycle and inhibited DNA replication, whereas PpIX treatment showed no effect on the cell cycle or on the DNA replication compared to the NO group.
Figure 5  Heat map of marker genes representing (a) cell cycle regulation and (b) DNA metabolic processes. Each row represents a gene, and each column represents the average fold change in gene expression relative to the NT group (* p < 0.01). The colors indicate the intensity based on the log expression ratio from blue (highly negative) to red (highly positive).   Ionizing radiation typically induces direct and/or indirect damage to DNA, triggering various cellular responses, including cell-cycle arrest, transformation and cell death [20,21,22]. The results of our study suggest a similar pattern in response to DNA damage. The PpIX-XT group differed from the XT group in terms of quantitative gene expression, but qualitative gene expression was the same in both groups.
In the case of genes selected for chromosome organization, we found that several core histones (HIST1H2BB, HIST1H2BD, HIST1H2BE, HIST1H2BH, HIST1H2BJ, HIST1H2BL, HIST1H2BO, HIST2H2BE and HIST3H2BB) were upregulated in both the XT and PpIX-XT groups. A significant amount of the histone synthesis occurs during the S phase. The synthesis of the histone proteins is tightly coupled to DNA synthesis [23,24]. Histone synthesis in cycling tissue culture cells can be separated into basal synthesis and S phase synthesis. The upregulation of histone gene expressions in this study was caused by the basal synthesis or by transitional response. The genes responsible for cellular responses to stress were identical to the genes involved in DNA metabolic processes.
In our previous in vivo study, treatment with 3-Gy irradiation at 10 intervals significantly suppressed tumor growth. However, in this study, we irradiated the cells with a single dose (3 Gy) following administration of PpIX. This dose appeared to be weak and ineffective at suppressing tumor growth. Therefore, we evaluated microarray data, which revealed systematic changes in cellular biological responses, as well as differences between the PpIX-XT and XT treatments.