Combined Treatment (Ultraviolet-C/Physapruin A) Enhances Antiproliferation and Oxidative-Stress-Associated Mechanism in Oral Cancer Cells

Physapruin A (PHA), a Physalis peruviana-derived withanolide, exhibits antiproliferation activity against oral and breast cancer cells. However, its potential antitumor effects in combined treatments remain unclear. This investigation focused on evaluating the impact of the combined treatment of ultraviolet-C with PHA (UVC/PHA) on the proliferation of oral cancer cells. The UVC-caused antiproliferation was enhanced by combination with PHA in oral cancer (Ca9-22 and CAL 27) but not normal cells (SG), as evidenced by ATP detection, compared with UVC or PHA alone. UVC/PHA showed a greater extent of subG1 increase, G2/M arrest, annexin-V-assessed apoptosis, caspase 3/7 activation, and reactive oxygen species (ROS) in the UVC or PHA treatment of oral cancer compared to normal cells. Moreover, the mitochondrial functions, such as mitochondrial superoxide bursts and mitochondrial membrane potential destruction, of oral cancer cells were also enhanced by UVC/PHA compared to UVC or PHA alone. These oxidative stresses triggered γH2AX and 8-hydroxyl-2’-deoxyguanosine-assessed DNA damage to a greater extent under UVC/PHA treatment than under UVC or PHA treatment alone. The ROS inhibitor N-acetylcysteine reversed all these UVC/PHA-promoted changes. In conclusion, UVC/PHA is a promising strategy for decreasing the proliferation of oral cancer cells but shows no inhibitory effect on normal cells.


Introduction
Oral cancer ranks among the top 10 cancers worldwide [1]. Oral cancer causes hundreds of thousands of deaths every year around the world [2]. Genetic factors interacting with environmental components can individually and collectively influence a person's susceptibility to cancer, as shown by epidemiological and clinical studies over the past two decades [3]. Surgery, radiation, chemotherapy, and radio-chemotherapy are current oral cancer treatments [4]. Radio-chemotherapy is a treatment that combines anticancer drugs, including radiosensitizers [5], with radiation to improve antiproliferation effects. However, the combined antiproliferation treatment strategy is sometimes ineffective in Cells (Ca9-22, CAL 27, and SG) were stained with an annexin V-FITC (1:1000 dilution) (Strong Biotech Corporation, Taipei, Taiwan)/7AAD (1 µg/mL) solution at 37 • C for 30 min [42]. After PBS resuspension, cells were analyzed by a flow cytometer. Cas 3/7 activation is associated with apoptosis signaling, which was detected using a Glo ® 3/7 commercial system (Promega; Madison, WI, USA) [16]. A Cas 3/7 substrate (DEVD) was added to the medium at 37 • C for 30 min. Consequently, apoptotic cells containing activated Cas 3/7 could cleave DEVD to generate the luminescent signal detected by the luminometer.

Statistics
The significance between multi-comparisons was assessed by one-way ANOVA and Tukey's HSD post hoc evaluation (JMP12, SAS Institute, Cary, NC, USA). Treatments labeled with different lower-case letters showed significantly different results. All assays were performed in three independent experiments.

Proliferation-Modulating Effects of UVC/PHA
The cell viability for all tested combined treatments with different UVC and/or PHA doses is shown in Figure 1A. The synergy determinations (α values) for all of these treatments are provided in Supplementary Table S1. The optimal dose for UVC/PHA of 8 J/m 2 , 0.8 µM was chosen for the following experiments. The α values of UVC/PHA (8 J/m 2 , 0.8 µM) in oral cancer cells for the ATP assay (Ca9-22 vs. CAL 27) were 1.15 ± 0.06 and 1.46 ± 0.20, respectively ( Figure 1A). Accordingly, the UVC/PHA combined treatment showed a synergistic antiproliferation effect on oral cancer cells.
The significance between multi-comparisons was assessed by one-way ANOVA and Tukey's HSD post hoc evaluation (JMP12, SAS Institute, Cary, NC, USA). Treatments labeled with different lower-case letters showed significantly different results. All assays were performed in three independent experiments.

Proliferation-Modulating Effects of UVC/PHA
The cell viability for all tested combined treatments with different UVC and/or PHA doses is shown in Figure 1A. The synergy determinations (α values) for all of these treatments are provided in Supplementary Table S1. The optimal dose for UVC/PHA of 8 J/m 2 , 0.8 μM was chosen for the following experiments. The α values of UVC/PHA (8 J/m 2 , 0.8 μM) in oral cancer cells for the ATP assay (Ca9-22 vs. CAL 27) were 1.15 ± 0.06 and 1.46 ± 0.20, respectively ( Figure 1A). Accordingly, the UVC/PHA combined treatment showed a synergistic antiproliferation effect on oral cancer cells. Oral cancer cells following combined treatment (UVC/PHA) showed a lower cell viability of 45.2% and 28.4% compared to separate treatments (UVC or PHA) in Ca9-22 (82.9% or 59.8%) and CAL 27 (77.5% or 58.4%) cells, respectively, based on the 24 h ATP assay ( Figure 1B). In contrast, neither the UVC, PHA, nor UVC/PHA treatments affected cell viability in normal oral SG cells. NAC reversed the UVC/PHA-enhanced antiproliferation, suggesting that UVC/PHA exerted a stronger oxidative-stress-dependent antiproliferation effect on oral cancer cells compared to the separate treatments. Oral cancer cells following combined treatment (UVC/PHA) showed a lower cell viability of 45.2% and 28.4% compared to separate treatments (UVC or PHA) in Ca9-22 (82.9% or 59.8%) and CAL 27 (77.5% or 58.4%) cells, respectively, based on the 24 h ATP assay ( Figure 1B). In contrast, neither the UVC, PHA, nor UVC/PHA treatments affected cell viability in normal oral SG cells. NAC reversed the UVC/PHA-enhanced antiproliferation, suggesting that UVC/PHA exerted a stronger oxidative-stress-dependent antiproliferation effect on oral cancer cells compared to the separate treatments.
For the trypan blue exclusion assay, UVC/PHA showed lower cell numbers of 37.3% and 33.3% compared to separate treatments (UVC or PHA) in Ca9-22 (70.6% or 76.5%) and CAL 27 (63.6% or 63.6%) cells, respectively ( Figure 1C). UVC/PHA produced a mild change in cell numbers (92.5%) compared to the separate treatments (UVC or PHA) in SG cells (83.2% and 98.1%, respectively). Consequently, the effects of UVC/PHA on cell numbers were similar to the results of the ATP assay ( Figure 1B).

Cell-Cycle-Modulating Effects of UVC/PHA
The cell cycle profile (Figure 2A) showed that UVC/PHA increased subG1 (%) and G2/M (%) but decreased G1 (%) in oral cancer cells (Ca9-22 and CAL 27) compared to the control, UVC, and PHA treatments ( Figure 2B). NAC reversed the UVC/PHAinduced cell cycle changes, suggesting that UVC/PHA exerted a more potent oxidativestress-dependent effect on the cell cycle progression of oral cancer cells compared to the separate treatments.
For the trypan blue exclusion assay, UVC/PHA showed lower cell numbers of 37.3% and 33.3% compared to separate treatments (UVC or PHA) in Ca9-22 (70.6% or 76.5%) and CAL 27 (63.6% or 63.6%) cells, respectively ( Figure 1C). UVC/PHA produced a mild change in cell numbers (92.5%) compared to the separate treatments (UVC or PHA) in SG cells (83.2% and 98.1%, respectively). Consequently, the effects of UVC/PHA on cell numbers were similar to the results of the ATP assay ( Figure 1B).

Cell-Cycle-Modulating Effects of UVC/PHA
The cell cycle profile ( Figure 2A) showed that UVC/PHA increased subG1 (%) and G2/M (%) but decreased G1 (%) in oral cancer cells (Ca9-22 and CAL 27) compared to the control, UVC, and PHA treatments ( Figure 2B). NAC reversed the UVC/PHA-induced cell cycle changes, suggesting that UVC/PHA exerted a more potent oxidative-stress-dependent effect on the cell cycle progression of oral cancer cells compared to the separate treatments.

Apoptosis-Modulating Effects of UVC/PHA
The annexin V/7AAD profile ( Figure 3A) showed that UVC/PHA induced a greater extent of apoptosis in oral cancer cells (%), as displayed by annexin V (+), than the control, UVC, and PHA treatments ( Figure 3B). Moreover, UVC/PHA displayed a greater extent of annexin V (+)-indicated apoptosis (%) in oral cancer cells than in normal cells (SG) (Figure 3B). NAC reversed the UVC/PHA-induced annexin V changes, suggesting that UVC/PHA induced a greater extent of oxidative-stress-dependent apoptosis in oral cancer cells than the separate treatments.

Apoptosis-Modulating Effects of UVC/PHA
The annexin V/7AAD profile ( Figure 3A) showed that UVC/PHA induced a greater extent of apoptosis in oral cancer cells (%), as displayed by annexin V (+), than the control, UVC, and PHA treatments ( Figure 3B). Moreover, UVC/PHA displayed a greater extent of annexin V (+)-indicated apoptosis (%) in oral cancer cells than in normal cells (SG) ( Figure 3B). NAC reversed the UVC/PHA-induced annexin V changes, suggesting that UVC/PHA induced a greater extent of oxidative-stress-dependent apoptosis in oral cancer cells than the separate treatments.
In addition to the annexin V/7AAD examination, the Cas 3/7 activity was also assessed ( Figure 4). The oral cancer cells treated with UVC/PHA displayed a greater extent of Cas 3/7 activity than the control or those treated with UVC or PHA alone. Moreover, UVC/PHA induced higher Cas 3/7 activity in oral cancer cells than in normal cells (SG). NAC and ZVAD reversed the UVC/PHA-induced Cas 3/7 activity changes while showing mild changes in normal cells. Consequently, UVC/PHA induced greater oxidative-stressdependent Cas 3/7 activation in oral cancer cells compared to the separate treatments.  For comparison, treatments labeled with different letters (a to f) produced significantly different results (p < 0.05). Data are presented as mean ± SD (n = 3 independent experiments, with each experiment involving 5000 gated cell counts).
In addition to the annexin V/7AAD examination, the Cas 3/7 activity was also assessed ( Figure 4). The oral cancer cells treated with UVC/PHA displayed a greater extent of Cas 3/7 activity than the control or those treated with UVC or PHA alone. Moreover, UVC/PHA induced higher Cas 3/7 activity in oral cancer cells than in normal cells (SG). NAC and ZVAD reversed the UVC/PHA-induced Cas 3/7 activity changes while showing mild changes in normal cells. Consequently, UVC/PHA induced greater oxidative-stressdependent Cas 3/7 activation in oral cancer cells compared to the separate treatments.

ROS-Modulating Effects of UVC/PHA
The ROS profile ( Figure 5A) showed that the oral cancer cells treated with UVC/PHA displayed a greater extent of ROS (+) (%) than the control or those treated with UVC or PHA ( Figure 5B). Moreover, UVC/PHA induced a greater extent of ROS (+) (%) in oral cancer cells than in normal cells (SG) ( Figure 5B). NAC reversed the UVC/PHA-induced ROS changes, suggesting that UVC/PHA exerted more potent oxidative stress on oral can-

ROS-Modulating Effects of UVC/PHA
The ROS profile ( Figure 5A) showed that the oral cancer cells treated with UVC/PHA displayed a greater extent of ROS (+) (%) than the control or those treated with UVC or PHA ( Figure 5B). Moreover, UVC/PHA induced a greater extent of ROS (+) (%) in oral cancer cells than in normal cells (SG) ( Figure 5B). NAC reversed the UVC/PHA-induced ROS changes, suggesting that UVC/PHA exerted more potent oxidative stress on oral cancer cells than the separate treatments. In the presence or absence of NAC or ZVAD, oral cancer and normal (SG) cells were grouped into four treatments, i.e., control, UVC, PHA, and UVC/PHA, for 24 h. The doses of UVC and PHA treatments were 8 J/m 2 and 0.8 μM, respectively. For comparison, treatments labeled with different letters (a to f) produced significantly different results (p < 0.05). Data are presented as mean ± SD (n = 3 independent experiments, with each experiment performed in three replications).

ROS-Modulating Effects of UVC/PHA
The ROS profile ( Figure 5A) showed that the oral cancer cells treated with UVC/PHA displayed a greater extent of ROS (+) (%) than the control or those treated with UVC or PHA ( Figure 5B). Moreover, UVC/PHA induced a greater extent of ROS (+) (%) in oral cancer cells than in normal cells (SG) ( Figure 5B). NAC reversed the UVC/PHA-induced ROS changes, suggesting that UVC/PHA exerted more potent oxidative stress on oral cancer cells than the separate treatments.

MitoSOX-Modulating Effects of UVC/PHA
The MitoSOX profile ( Figure 6A) showed that the oral cancer cells treated with UVC/PHA displayed a greater extent of MitoSOX (+) (%) than the control and those treated with UVC or PHA ( Figure 6B). Moreover, NAC reversed the UVC/PHA-induced MitoSOX changes, suggesting that UVC/PHA exerted more potent oxidative stress on oral cancer cells than the separate treatments.

MitoSOX-Modulating Effects of UVC/PHA
The MitoSOX profile ( Figure 6A) showed that the oral cancer cells treated with UVC/PHA displayed a greater extent of MitoSOX (+) (%) than the control and those treated with UVC or PHA ( Figure 6B). Moreover, NAC reversed the UVC/PHA-induced MitoSOX changes, suggesting that UVC/PHA exerted more potent oxidative stress on oral cancer cells than the separate treatments.

MitoMP-Modulating Effects of UVC/PHA
The MitoMP profile ( Figure 7A) showed that the oral cancer cells treated with UVC/PHA displayed a greater extent of MitoMP (−) (%) than the control and those treated with UVC or PHA ( Figure 7B). Moreover, NAC reversed the UVC/PHA-induced MitoMP changes, suggesting that UVC/PHA exerted a more potent oxidative-stress-dependent effect on the MitoMP in oral cancer cells than the separate treatments.

MitoMP-Modulating Effects of UVC/PHA
The MitoMP profile ( Figure 7A) showed that the oral cancer cells treated with UVC/PHA displayed a greater extent of MitoMP (−) (%) than the control and those treated with UVC or PHA ( Figure 7B). Moreover, NAC reversed the UVC/PHA-induced MitoMP changes, suggesting that UVC/PHA exerted a more potent oxidative-stress-dependent effect on the MitoMP in oral cancer cells than the separate treatments.

DNA-Damage-Modulating Effects of UVC/PHA
The γH2AX and 8-OHdG profiles ( Figures 8A and 9A) showed that the oral cancer cells treated with UVC/PHA displayed a greater extent of γH2AX and 8-OHdG (+) (%) than the control and those treated with UVC or PHA ( Figures 8B and 9B). Moreover, NAC reversed the UVC/PHA-induced γH2AX and 8-OHdG changes, suggesting that UVC/PHA exerted a more potent oxidative-stress-dependent DNA-damage effect on oral cancer cells than the separate treatments.

DNA-Damage-Modulating Effects of UVC/PHA
The γH2AX and 8-OHdG profiles ( Figures 8A and 9A) showed that the oral cancer cells treated with UVC/PHA displayed a greater extent of γH2AX and 8-OHdG (+) (%) than the control and those treated with UVC or PHA ( Figures 8B and 9B). Moreover, NAC reversed the UVC/PHA-induced γH2AX and 8-OHdG changes, suggesting that UVC/PHA exerted a more potent oxidative-stress-dependent DNA-damage effect on oral cancer cells than the separate treatments.

Discussion
PHA previously showed higher antiproliferation effects in oral cancer cells than in normal cells [27]. Currently, investigations into combined treatments involving PHA are rare. The present study confirmed that UVC/PHA caused a greater extent of antiproliferation in oral cancer than in normal cells. Several UVC/PHA-triggered responses to oral cancer cells were discussed.
Clinical drugs and natural products have been shown to enhance the UVC-induced antiproliferation effects on oral cancer cells. For example, a combined treatment of 10 µM cisplatin/10 J/m 2 UVC inhibited proliferation in colon cancer cells to a greater extent than separate treatments [18]. Coral-derived sinularin [16] and brown-algae-derived fucoidan [20] showed more substantial antiproliferation effects against oral cancer cells when combined with UVC irradiation. Notably, combined treatments of UVC/sinularin [13] or UVC/fucoidan [17] showed higher antiproliferation effects in oral cancer than in normal cells. Similarly, PHA induced greater antiproliferation effects in oral cancer than normal cells [27], providing evidence of the preferential-antiproliferation property of PHA. Due to this preferential-antiproliferation property, PHA combined with UVC showed an enhancement of UVC-and PHA-induced antiproliferation effects in oral cancer cells compared to normal cells (Figure 1). This synergistic antiproliferation effect of UVC/PHA was reversed by NAC, revealing that oxidative stress is crucial in regulating these antiproliferation effects.
Several natural products, such as sinularin [13] and fucoidan [17], have demonstrated the ability to generate oxidative stress. UVC also exhibits the same capacity [30,[45][46][47][48]. Consequently, combined treatments of these natural products and UVC are expected to produce stronger oxidative stress bursts than separate treatments. Similarly, UVC and PHA were confirmed to be oxidative-stress-inducing agents for oral cancer cells, as evidenced by ROS, MitoSOX, and MitoMP ( Figures 5-7). UVC/PHA produced a greater extent of oxidative stresses than the UVC or PHA treatments. This finding raises the possibility that UVC/PHA could provide a higher level of oxidative stress, leading to an increased antiproliferation effect against oral cancer cells. Moreover, UVC/PHA produced higher oxidative stress in oral cancer than in normal cells, confirming the preferentialantiproliferation property of UVC/PHA against oral cancer cells (Figure 1).
Since oxidative stress causes apoptosis [49] and DNA damage [50], these phenomena need to be examined. As expected, the UVC/PHA-triggered oxidative stress bursts may have affected oxidative-stress-associated responses, as shown by the upregulation of γH2AXand 8-OHdG-type DNA damage and caspase 3/7-indicated apoptosis in oral cancer cells compared to the separate treatments (Figures 4, 8 and 9), respectively.
As mentioned above, UVC/PHA induced a greater extent of oxidative-stress-associated responses; therefore, oxidative stress dependence needs further assessment. By applying NAC, all these oxidative-stress-associated responses, such as abnormal cell cycle progression (subG1 increase and G2/M arrest), apoptosis, and DNA damage, were reversed. Notably, NAC had little inhibitory impact on the antiproliferative effect of UVC alone in oral cancer Ca9-22 or CAL 27 cells, depending on the experimental system (annexin V, ROS, or MitoMP). However, the measured impacts of PHA alone or UVC/PHA were suppressed by NAC, which was more effective for UVC/PHA. These results suggest that the combined treatment may have cooperatively enhanced the oxidative stress levels and responses in oral cancer cells compared to the separate treatments. Consequently, UVC/PHA exerted an oxidative-stress-associated mechanism of antiproliferation against oral cancer cells.

Conclusions
Combined treatments benefit cancer therapy by reducing the drug dosages required but improving their effectiveness. This investigation confirmed that UVC/PHA exhibited an improved antiproliferation effect in oral cancer cells compared to UVC or PHA alone. UVC/PHA also reduced the level of proliferation and increased the extent of apoptosis, caspase 3/7 activation, and ROS generation in oral cancer compared to normal cells. In addition to ROS generation, UVC/PHA caused more mitochondrial dysfunction, including MitoSOX generation and MitoMP depletion, in oral cancer cells compared to UVC or PHA alone. NAC reversed these mechanisms triggered by UVC/PHA. Consequently, the role of oxidative stress in regulating these UVC/PHA-induced changes was validated. The synergistic molecular antiproliferation mechanism of UVC/PHA against oral cancer cells is illustrated in Figure 10. The present study investigated the combined treatment of UVC/PHA, which demonstrated an enhanced antiproliferation effect against oral cancer cells and a lack of harmful effects on normal cells, thus providing a potential oral cancer treatment.
Combined treatments benefit cancer therapy by reducing the drug dosages required but improving their effectiveness. This investigation confirmed that UVC/PHA exhibited an improved antiproliferation effect in oral cancer cells compared to UVC or PHA alone. UVC/PHA also reduced the level of proliferation and increased the extent of apoptosis, caspase 3/7 activation, and ROS generation in oral cancer compared to normal cells. In addition to ROS generation, UVC/PHA caused more mitochondrial dysfunction, including MitoSOX generation and MitoMP depletion, in oral cancer cells compared to UVC or PHA alone. NAC reversed these mechanisms triggered by UVC/PHA. Consequently, the role of oxidative stress in regulating these UVC/PHA-induced changes was validated. The synergistic molecular antiproliferation mechanism of UVC/PHA against oral cancer cells is illustrated in Figure 10. The present study investigated the combined treatment of UVC/PHA, which demonstrated an enhanced antiproliferation effect against oral cancer cells and a lack of harmful effects on normal cells, thus providing a potential oral cancer treatment. Figure 10. Synergistic molecular antiproliferation mechanism of UVC/PHA against oral cancer cells. In brief, the UVC/PHA combined treatment cooperatively generated more oxidative stress than separate treatments (UVC or PHA). Subsequently, the UVC/PHA-induced oxidative stress caused higher cell cycle disturbance, apoptosis, and DNA damage than UVC or PHA alone. Finally, these mechanisms of UVC/PHA action led to a synergistic antiproliferation effect against oral cancer cells. Moreover, NAC could suppress the UVC/PHA-associated changes, indicating that UVC/PHA induced an oxidative-stress-mediated synergistic antiproliferation effect against oral cancer cells.

Supplementary Materials:
The following are available online at www.mdpi.com/xxx/s1, Table S1: The synergy determinations (α values) for all tested combined treatments with different UVC and/or PHA doses in oral cancer and normal cells.  In brief, the UVC/PHA combined treatment cooperatively generated more oxidative stress than separate treatments (UVC or PHA). Subsequently, the UVC/PHA-induced oxidative stress caused higher cell cycle disturbance, apoptosis, and DNA damage than UVC or PHA alone. Finally, these mechanisms of UVC/PHA action led to a synergistic antiproliferation effect against oral cancer cells. Moreover, NAC could suppress the UVC/PHA-associated changes, indicating that UVC/PHA induced an oxidative-stress-mediated synergistic antiproliferation effect against oral cancer cells.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/antiox11112227/s1, Table S1: The synergy determinations (α values) for all tested combined treatments with different UVC and/or PHA doses in oral cancer and normal cells.