The in vivo or in vitro toxicological effects of PG have been previously evaluated [2, 45–47]. However, there have been no reports on the cytotoxicological effects of PG on normal lung cells, such as fibroblast cells. Lung fibroblast cells are essential for maintaining the integrity of the alveoli and restoring injured tissues [35]. The present study focused on evaluating the effects of PG on ROS and GSH levels in normal HPF cells and further on examining the effects of NAC, BSO, and siRNAs against various antioxidants on ROS and GSH levels and cell death following PG treatment. PG induced cell death in HPF cells via caspase-independent apoptosis and/or necrosis (unpublished data). In addition, PG has been shown to induce cell death in various cells via the destruction of MMP (∆Ψm) [6, 12, 27, 48]. As expected, PG induced HPF cell death, which was accompanied by MMP (ΔΨm) loss. The proportion of cells with MMP (∆Ψm) loss among HPF cells treated with 800 µM PG was greater than that of dead cells. These results suggest that PG treatment primarily interrupts mitochondrial membranes, which proceeds to the subsequent stages of cell dath.
PG can be an antioxidant [2, 3, 6, 8, 28] or a pro-oxidant [6, 11, 31]. The present results showed that both ROS levels (as determined by DCF) and O2•− levels (as determined by DHE) were increased in HPF cells treated with 100–800 µM PG at 24 h. Although PG significantly increased the activity of catalase in HPF cells, it did not reduce ROS levels. Interestingly, treatment with 1,600 µM PG led to a high frequency of cell death in HPF cells (data not shown) at 24 h, without increasing the levels of total ROS or O2•−, which was probably due to the leakage of DCF and DHE dyes from the dead cells and the resulting failure to detect ROS. The ROS levels were increased in PG-treated HPF cells at the early time point of 30 min, with 800 and 1,600 µM PG most dramatically increasing the ROS levels at 90 min. By contrast, all doses of PG decreased intracellular O2•− levels at the early time point of 30 min, whereas only 1,600 µM PG increased the O2•− level at 45 min. All doses of PG increased O2•− levels at 90 min. It is possible that PG increased ROS levels in HPF cells by affecting redox enzymes at early time points until 60 min and later increased the levels of total ROS and O2•− by damaging the mitochondria and changing the activity of redox enzymes at 90 min. Since mitochondrial O2•− levels were increased in PG-treated HPF cells at 24 h, the increased O2•− levels at 24 h likely resulted from the generation of O2•− itself from the mitochondria. In addition, PG decreased the activity of SOD in HPF cells at 24 h. It is conceivable that the reduction of SOD activity results in a slower transformation from O2•− to H2O2, consequently leading to the accumulation of O2•− in cells. These results indicate that the doses of PG tested here showed pro-oxidant effects and increased the intracellular ROS levels in HPF cells, whereas higher doses of PG would have both pro-oxidant and pro-apoptotic effects at 24 h. Furthermore, PG caused the loss of MMP (ΔΨm) in HPF cells. It was also reported that PG induces DNA strand breaks in A549 lung carcinoma cells [49]. Taken together, these data suggested that PG treatment induced HPF cell death via oxidative stress by increasing levels of total ROS, especially O2•−.
As a well-known antioxidant, NAC prevents cell death in PG-treated HeLa cervical cancer and endothelial cells, but increases ROS levels [6, 28]. Similarly, PG significantly prevented cell death and MMP (ΔΨm) loss in PG-treated HPF cells, but did not significantly decrease ROS levels and even increased O2•− levels. However, NAC alone significantly decreased basal ROS levels but increased the basal O2•− levels in the control HPF cells. Moreover, NAC has been shown to decrease cell death and MMP (ΔΨm) loss in arsenic trioxide- and MG132-treated HPF cells along with the downregulation of ROS levels [43, 44]. BSO, a GSH synthesis inhibitor, showed a strong enhancement in cell death and MMP (ΔΨm) loss in PG-treated HPF cells without increasing the levels of total ROS and O2•−. By contrast, BSO increased cell death and MMP (ΔΨm) loss in HPF cells treated with arsenic trioxide and MG132 along with the upregulation of ROS levels [43, 44]. BSO alone induced cell death and MMP (ΔΨm) loss in control HPF cells and largely increased the levels of total ROS and O2•−. Thus, an increased ROS level induced by BSO treatment alone is tightly related to HPF cell death. PG is an antioxidant used to downregulate ROS levels in BSO-treated HPF cells. These mixed antioxidant or pro-oxidant effects of PG, NAC, BSO, and their various combinations in different cell types, including cancer and normal cells, are perhaps due to the different basal activities of the mitochondria and antioxidant enzymes present in each cell type.
The present results showed that 800 µM PG slightly increased the number of apoptotic cells and levels of total ROS and O2•− in HPF cells treated with control siRNA. However, the percentage of apoptotic cells in PG-treated HPF cells was lower than expected, compared with that in HPF cells treated with control siRNA. Probably, the presence of LipofectAMINE 2000 agent in the medium and the different cell seeding conditions resulted in the variation in effects of PG on cell death. Antioxidant proteins, especially SOD2 and TXN, have been known to stimulate cell proliferation and promote resistance to anti-growth agents [50–52]. Thus, downregulation of SOD2 or TXN may render cells sensitive to cytotoxic drugs. However, the present results showed that siRNAs against SOD1, SOD2, CAT, or TXN attenuated, instead of enhancing, PG-induced cell death in HPF. In addition, the effects of PG in inducing cell death were lower in HPF cells treated with each siRNA against antioxidants than those treated with control siRNA. Moreover, PG treatment decreased the percentage of apoptotic cells in HPF cells treated with TXN siRNA. The mechanism underlying the effects of SOD1, SOD2, CAT, and TXN on PG-induced HPF cell death remains to be further investigated. Changes in cell death in PG-treated HPF cells following siRNA silencing of antioxidant genes occurred without changes in total ROS levels, but was accompanied by the downregulation of the O2•− levels. Therefore, the alteration of PG-induced HPF cell death by antioxidant-related siRNAs is not tightly related to changes in ROS levels. Moreover, GPX or TXN siRNA increased apoptosis in untreated control HPF cells along with increases in total ROS levels, but not in O2•− levels, whereas SOD1 or SOD2 siRNA decreased apoptosis in the control cells without decreasing the total ROS and O2•− levels. These results suggest that the downregulation of each antioxidant protein by its corresponding siRNA influences ROS levels and cell death differently.
Intracellular GSH level is negatively correlated with progression to cell death [36, 53, 54]. PG induces depletion of GSH in HeLa cervical cancer cells [6] and endothelial cells [28]. In addition, PG depleted intracellular GSH in Calu-6 and A549 lung cancer cells [34]. Similarly, the current result showed that 800 or 1,600 µM PG increased the number of GSH-depleted cells at 24 h. Expectedly, NAC, a known GSH precursor, significantly attenuated GSH depletion in PG-treated HPF cells. By contrast, BSO, an irreversible inhibitor in GSH synthesis [55], did not increase the number of GSH-depleted cells in PG-treated HPF cells. Treatment with 10 µM BSO alone increased cell death in control HPF cells without depleting GSH. In addition, GPX or TXN siRNA induced cell death in control HPF cells without increasing GSH depletion. Moreover, SOD1, SOD2, CAT, or TXN siRNA did not decrease GSH depletion in PG-treated HPF cells, but increased the number of GSH-depleted cells. However, GPX siRNA decreased the proportion of GSH-depleted cells among PG-treated HPF cells. Taken together, these data suggest that the intracellular GSH level plays a role in PG-induced cell death, although changes in its level alone are not sufficient for accurately predicting cell death. Notably, the GSH level in PG-treated HPF cells, except those CMF-negative cells, was generally decreased at early time points of 30–180 min and 24 h. The decreased GSH level probably resulted from its rapid consumption when reducing ROS levels at each corresponding time point.
In conclusion, PG induced cell death and MMP (ΔΨm) loss in HPF cells, which were accompanied by an increase in ROS levels and depletion of GSH. NAC treatment attenuated PG-induced cell death with decreased GSH depletion, and BSO increased cell death without altering ROS generation or GSH depletion. In addition, siRNA silencing of SOD1, SOD2, or CAT attenuated cell death in PG-treated HPF cells, whereas silencing of GPX enhanced cell death, which were not tightly related to ROS or GSH levels. The data from the current study provide useful information for understanding the cytotoxic and molecular effects of PG on normal lung cells, specifically HPF cells, by regulating ROS and GSH levels.