Synergistic Antiproliferation of Cisplatin and Nitrated [6,6,6]Tricycle Derivative (SK2) for a Combined Treatment of Oral Cancer Cells

SK2, a nitrated [6,6,6]tricycle derivative with an n-butyloxy group, showed selective antiproliferation effects on oral cancer but not on normal oral cells. This investigation assessed for the first time the synergistic antiproliferation potential of cisplatin/SK2 in oral cancer cells. Cell viability assay at 24 h showed that a low dose of combined cisplatin/SK2 (10 μM/10 μg/mL) provided more antiproliferation than cisplatin or SK2 alone. Cisplatin/SK2 triggered also more apoptosis inductions in terms of subG1 accumulation, annexin V, pancaspase, and caspase 3/8/9 measurements. Moreover, cisplatin/SK2 provided more oxidative stress and DNA damage in oral cancer cells than independent treatments. Oxidative stress inhibitors rescued the cisplatin/SK2-induced antiproliferation and oxidative stress generation. Moreover, cisplatin/SK2 induced more antiproliferation, apoptosis, oxidative stress, and DNA damage in oral cancer cells than in normal oral cells (S-G). In conclusion, low-dose cisplatin/SK2 combined treatment promoted selective and synergistic antiproliferation in oral cancer cells depending on oxidative-stress-associated responses.


Introduction
Oral cancer is the top three cancer in Taiwan [1], and it also has a global impact [2]. It shows a low five-year survival rate when diagnosed late [3]. Typical oral cancer therapy includes surgery, chemotherapy, and radiation, which all have adverse side effects [4]. Moreover, chemotherapy and radiotherapy may provide resistance problems that limit the effectiveness of cancer therapy [5]. Recently, a low dose strategy was reported to overcome chemoresistance [6][7][8][9][10]. In an animal study, the low-dose anticancer drug doxorubicin overcomes the chemoresistance of patient-derived leukemia stem cells for prolonging survival [11]. Accordingly, low-dose drug treatments may overcome chemoresistance in cancer therapy.
In addition to dosage concerns [11], a combined treatment also provides an effective strategy to reduce resistance problems in cancer treatment [12][13][14][15][16][17][18]. For the complex causation and etiology of cancer, multiple therapies, including combinations of natural products, radiation, and anticancer agents, have been applied to oral cancer treatment [12,13]. Combination treatment may sensitize cancer cells to clinical drugs and reduce their potential adverse effects. For example, cisplatin is one of the effective anticancer drugs against oral, bladder, ovarian, leukemia, prostate, lung, and other cancers [19]. Cisplatin is commonly used for combined treatment applications with natural products [20], which may decrease the side effects of cisplatin. However, some studies for cisplatin combined treatments did not concern the responses of normal cells [21][22][23][24], and their potential side effects were not investigated.
The dioxabicyclo [3.3.1]nonane core is a central and bioactive structure in many natural products. We previously reported a novel benzo-fused dioxabicyclo [3.3.1]nonane-namely, SK2, exhibiting selective antiproliferation effects against oral cancer cells with a mild adverse effect on normal cells [25]. SK2 also triggers oxidative stress, apoptosis, and DNA damage in oral cancer cells but these changes were not examined in normal oral cells [25]. An antioral cancer application of combined treatment with SK2 has not been reported thus far.
The aim of the present study is to investigate the synergistic effects of antiproliferation of a combined SK2/cisplatin treatment. This includes the pursuit of detailed mechanisms for possible synergistic antiproliferation.

Statistics
In multi-comparison analysis, the Tukey post hoc test was performed by JMP 12 software (SAS Institute, Cary, NC, USA). The significance was indicated by not overlapping lowercased letters. In contrast, data with overlapping lower-cased letters showed a nonsignificant difference. Triplicate experiments were performed and designated as means ± SDs.
To assess the effects of oxidative stress, the ROS inhibitor NAC was applied. The antiproliferation of cisplatin/SK2 single and combined treatments was suppressed by NAC, suggesting that synergistic antiproliferation of cisplatin/SK2 was mediated by oxidative stress.

Cisplatin/SK2 Causes Synergistic subG1 Accumulation in Oral Cancer Cells
To assess the involvement of apoptosis in the antiproliferation of SK2, the subG1 status in cell cycle analysis was observed. The oral cell cycle patterns of four treatments-the control, cisplatin, SK2, and cisplatin/SK2-were demonstrated for oral cancer cells (Ca9-22 and HSC-3 cells) and normal cells (S-G) ( Figure 2). A combined cisplatin/SK2 showed Cell viability for cisplatin (CDDP) and/or SK2. Following NAC preprocessing (10 mM for 1 h) or not, oral cancer (Ca9-22 and HSC-3) and normal oral (S-G) cells were processed with four treatments-control (0.1% DMSO), CDDP (10 μM), SK2 (10 μg/mL), and CDDP/SK2 (10 μM/10 μg/mL)-for 24 h and assessed by an MTS assay. For multi-comparisons, data without overlapping letters differ significantly (p < 0.05). The highest value is marked with a, and others are shown in descending order. The data are shown as means ± SD (n = 3). In the example of Ca9-22 cells, the cell viabilities between the control (black) and NAC (gray) for CDDP/SK2 (e vs. d), showing nonoverlapping characters, differed significantly. Similarly, the control (black) and CDDP/SK2 (black) (a vs. e) were significantly different. In contrast, the controls (black) for CDDP and SK2 (bc vs. c) did not differ significantly.

Cisplatin/SK2 Causes Synergistic subG1 Accumulation in Oral Cancer Cells
To assess the involvement of apoptosis in the antiproliferation of SK2, the subG1 status in cell cycle analysis was observed. The oral cell cycle patterns of four treatments-the control, cisplatin, SK2, and cisplatin/SK2-were demonstrated for oral cancer cells (Ca9-22 and HSC-3 cells) and normal cells (S-G) ( Figure 2). A combined cisplatin/SK2 showed higher sub-G1 (%) than independent treatments (cisplatin or SK2) and control. Moreover, a combined cisplatin/SK2 increased more subG1 (%) in oral cancer cells than in normal cells (S-G), indicating that cisplatin/SK2 induces selectively subG1 accumulation in oral cancer cells indicating apoptosis.  For multi-comparisons, data without overlapping letters differ significantly (p < 0.05). The highest value is marked with a, and others are shown in descending order. The data are shown as means ± SD (n = 3). In the example of Ca9-22 cells, the cell viabilities between the control (black) and NAC (gray) for CDDP/SK2 (e vs. d), showing nonoverlapping characters, differed significantly. Similarly, the control (black) and CDDP/SK2 (black) (a vs. e) were significantly different. In contrast, the controls (black) for CDDP and SK2 (bc vs. c) did not differ significantly.
NAC, suggesting that synergistic antiproliferation of cisplatin/SK2 was mediated by dative stress. Cell viability for cisplatin (CDDP) and/or SK2. Following NAC preprocessing (10 m 1 h) or not, oral cancer (Ca9-22 and HSC-3) and normal oral (S-G) cells were processed with treatments-control (0.1% DMSO), CDDP (10 μM), SK2 (10 μg/mL), and CDDP/SK2 (10 μ μg/mL)-for 24 h and assessed by an MTS assay. For multi-comparisons, data without overlap letters differ significantly (p < 0.05). The highest value is marked with a, and others are show descending order. The data are shown as means ± SD (n = 3). In the example of Ca9-22 cells, th viabilities between the control (black) and NAC (gray) for CDDP/SK2 (e vs. d), showing non lapping characters, differed significantly. Similarly, the control (black) and CDDP/SK2 (black) e) were significantly different. In contrast, the controls (black) for CDDP and SK2 (bc vs. c) di differ significantly.

Cisplatin/SK2 Causes Synergistic subG1 Accumulation in Oral Cancer Cells
To assess the involvement of apoptosis in the antiproliferation of SK2, the subG1 tus in cell cycle analysis was observed. The oral cell cycle patterns of four treatmentscontrol, cisplatin, SK2, and cisplatin/SK2-were demonstrated for oral cancer cells ( 22 and HSC-3 cells) and normal cells (S-G) ( Figure 2). A combined cisplatin/SK2 sho higher sub-G1 (%) than independent treatments (cisplatin or SK2) and control. More a combined cisplatin/SK2 increased more subG1 (%) in oral cancer cells than in no cells (S-G), indicating that cisplatin/SK2 induces selectively subG1 accumulation in cancer cells indicating apoptosis.  and normal (S-G) cells were processed with four treatments-control (0.1% DMSO), CDDP (10 µM), SK2 (10 µg/mL), and CDDP/SK2 (10 µM/10 µg/mL)-for 24 h and were assessed by a cell cycle assay. For multi-comparisons, data without overlapping characters differ significantly (p < 0.05). The highest value is marked with a, and others are shown in descending order. The data are provided as means ± SD (n = 3). In the example of Ca9-22 and HSC-3 cells, the subG1 (%) for control, SK2, and CDDP/SK2 (c, b, a) differed significantly. In contrast, the controls for CDDP showing the overlapping character "c" did not differ significantly.

Cisplatin/SK2 Causes Synergistic Annexin-V-Detected Apoptosis in Oral Cancer Cells
The apoptosis potential of subG1 accumulation in Figure 2 was further examined by an annexin-V assay, which detected the phosphatidylserine in the outer plasma membrane of apoptotic cells. Cisplatin/SK2 combined treatment displayed higher apoptosis (%) in oral cancer cells (Ca9-22 and HSC-3) than the independent treatments (cisplatin or SK2) and control ( Figure 3). Moreover, a combined cisplatin/SK2 decreased apoptosis (annexin V) in oral cancer cells more than in normal cells (S-G), indicating that cisplatin/SK2 induces selective apoptosis in oral cancer cells.
For multi-comparisons, data without overlapping characters differ significantly (p < 0.05). The highest value is marked with a, and others are shown in descending order. The data are provided as means ± SD (n = 3). In the example of Ca9-22 and HSC-3 cells, the subG1 (%) for control, SK2, and CDDP/SK2 (c, b, a) differed significantly. In contrast, the controls for CDDP showing the overlapping character "c" did not differ significantly.

Cisplatin/SK2 Causes Synergistic Annexin-V-Detected Apoptosis in Oral Cancer Cells
The apoptosis potential of subG1 accumulation in Figure 2 was further examined by an annexin-V assay, which detected the phosphatidylserine in the outer plasma membrane of apoptotic cells. Cisplatin/SK2 combined treatment displayed higher apoptosis (%) in oral cancer cells (Ca9-22 and HSC-3) than the independent treatments (cisplatin or SK2) and control ( Figure 3). Moreover, a combined cisplatin/SK2 decreased apoptosis (annexin V) in oral cancer cells more than in normal cells (S-G), indicating that cisplatin/SK2 induces selective apoptosis in oral cancer cells. and normal (S-G) cells were processed with four treatments-control (0.1% DMSO), CDDP (10 μM), SK2 (10 μg/mL), and CDDP/SK2 (10 μM/10 μg/mL)-for 24 h and assessed by an annexin-V assay. The annexin-V-positive (%) is defined as apoptosis (%). For multi-comparisons, data without overlapping characters differ significantly (p < 0.05). The highest value is marked with a, and others are shown in descending order. Data are shown as means ± SD (n = 3). In the example of Ca9-22 cells, the apoptosis (%) for control, CDDP, SK2, and CDDP/SK2 (d, c, b, a), showing nonoverlapping characters, differed significantly. For S-G cells, the controls and CDDP showing overlapping character "c" did not differ significantly.

Cisplatin/SK2 Shows Synergistic Apoptosis (Caspase Activation) in Oral Cancer Cells
Apoptosis is triggered by the signaling of several caspases. The caspase assay was further applied to examine the apoptosis potential of subG1 accumulation in Figure 2 in terms of flow cytometry (pancaspase and Cas 3) and luminescence detection. A cisplatin/SK2 combined treatment displayed higher pancaspase (+) (%) in oral cancer cells (Ca9-22 and HSC-3) than the independent treatments (cisplatin or SK2) and control ( Figure 4A). Moreover, a combined cisplatin/SK2 increased more pancaspase (+) (%) in oral cancer cells and normal (S-G) cells were processed with four treatments-control (0.1% DMSO), CDDP (10 µM), SK2 (10 µg/mL), and CDDP/SK2 (10 µM/10 µg/mL)-for 24 h and assessed by an annexin-V assay. The annexin-V-positive (%) is defined as apoptosis (%). For multi-comparisons, data without overlapping characters differ significantly (p < 0.05). The highest value is marked with a, and others are shown in descending order. Data are shown as means ± SD (n = 3). In the example of Ca9-22 cells, the apoptosis (%) for control, CDDP, SK2, and CDDP/SK2 (d, c, b, a), showing nonoverlapping characters, differed significantly. For S-G cells, the controls and CDDP showing overlapping character "c" did not differ significantly.

Cisplatin/SK2 Shows Synergistic Apoptosis (Caspase Activation) in Oral Cancer Cells
Apoptosis is triggered by the signaling of several caspases. The caspase assay was further applied to examine the apoptosis potential of subG1 accumulation in Figure 2 in terms of flow cytometry (pancaspase and Cas 3) and luminescence detection. A cisplatin/SK2 combined treatment displayed higher pancaspase (+) (%) in oral cancer cells (Ca9-22 and HSC-3) than the independent treatments (cisplatin or SK2) and control ( Figure 4A). Moreover, a combined cisplatin/SK2 increased more pancaspase (+) (%) in oral cancer cells than in normal cells (S-G), indicating that cisplatin/SK2 induces selective pancaspase activation in oral cancer cells. than in independent treatments ( Figure 4B). Moreover, a combined cisplatin/SK2 increased more Cas 3 (+) (%) in oral cancer cells than in normal cells (S-G).
Similarly, luminescence detection of Cas 3/7 ( Figure 4C) showed the same tendency as flow cytometry ( Figure 4B). The Cas 3 induction of cisplatin/SK2 combined treatment was suppressed by ZVAD, suggesting that caspase signaling was triggered by apoptosis. Moreover, cisplatin/SK2 combined treatment induced more Cas 3/7 activity in oral cancer cells than in normal cells (S-G), suggesting that cisplatin/SK2 induces selective caspase activation in oral cancer cells ( Figure 4C).

Cisplatin/SK2 Shows Synergistic Induction of Extrinsic and Intrinsic Apoptosis in Oral Cancer Cells
The extrinsic and intrinsic apoptosis signaling was mainly triggered by Cas 8 and Cas 9, and they were further examined by Cas 8 and Cas 9 flow cytometry, respectively. A combined cisplatin/SK2 showed higher Cas 8 and Cas 9 (+) (%) in oral cancer cells (Ca9-22 and HSC-3) than the independent treatments (cisplatin or SK2) and control ( Figure 5A and 5B). Moreover, a combined cisplatin/SK2 increased more Cas 8 and Cas 9 (+) (%) in oral cancer cells than in normal cells (S-G), indicating cisplatin/SK2 induces selective extrinsic and intrinsic caspase activations in oral cancer cells. Since the pancaspase assay provides a broad-spectrum detection for several caspases (Cas 1 and Cas 3 to 9) [25], a specific Cas 3 assay was performed. In terms of flow cytometry, a combined cisplatin/SK2 treatment displayed higher Cas 3 (+) in oral cancer cells than in independent treatments ( Figure 4B). Moreover, a combined cisplatin/SK2 increased more Cas 3 (+) (%) in oral cancer cells than in normal cells (S-G).
Similarly, luminescence detection of Cas 3/7 ( Figure 4C) showed the same tendency as flow cytometry ( Figure 4B). The Cas 3 induction of cisplatin/SK2 combined treatment was suppressed by ZVAD, suggesting that caspase signaling was triggered by apoptosis. Moreover, cisplatin/SK2 combined treatment induced more Cas 3/7 activity in oral cancer cells than in normal cells (S-G), suggesting that cisplatin/SK2 induces selective caspase activation in oral cancer cells ( Figure 4C).

Cisplatin/SK2 Shows Synergistic Induction of Extrinsic and Intrinsic Apoptosis in Oral Cancer Cells
The extrinsic and intrinsic apoptosis signaling was mainly triggered by Cas 8 and Cas 9, and they were further examined by Cas 8 and Cas 9 flow cytometry, respectively. A combined cisplatin/SK2 showed higher Cas 8 and Cas 9 (+) (%) in oral cancer cells (Ca9-22 and HSC-3) than the independent treatments (cisplatin or SK2) and control ( Figure 5A and 5B). Moreover, a combined cisplatin/SK2 increased more Cas 8 and Cas 9 (+) (%) in oral cancer cells than in normal cells (S-G), indicating cisplatin/SK2 induces selective extrinsic and intrinsic caspase activations in oral cancer cells.

Cisplatin/SK2 Combined Treatment Shows Synergistic Induction of ROS in Oral Cancer Cells
Since NAC rescued the synergistic antiproliferation of cisplatin/SK2 in oral cancer cells (Figure 1

Cisplatin/SK2 Shows Synergistic Induction of Extrinsic and Intrinsic Apoptosis in Oral Cancer Cells
The extrinsic and intrinsic apoptosis signaling was mainly triggered by Cas 8 and Cas 9, and they were further examined by Cas 8 and Cas 9 flow cytometry, respectively. A combined cisplatin/SK2 showed higher Cas 8 and Cas 9 (+) (%) in oral cancer cells (Ca9-22 and HSC-3) than the independent treatments (cisplatin or SK2) and control ( Figure 5A and 5B). Moreover, a combined cisplatin/SK2 increased more Cas 8 and Cas 9 (+) (%) in oral cancer cells than in normal cells (S-G), indicating cisplatin/SK2 induces selective extrinsic and intrinsic caspase activations in oral cancer cells.

Cisplatin/SK2 Combined Treatment Shows Synergistic Induction of ROS in Oral Cancer Cells
Since NAC rescued the synergistic antiproliferation of cisplatin/SK2 in oral cancer cells (Figure 1), oxidative stress was further examined by ROS flow cytometry. Cisplatin/SK2 combined treatment displayed higher ROS (+) (%) in oral cancer cells (Ca9-22 and HSC-3) than the independent treatments (cisplatin or SK2) and control ( Figure 6). Moreover, cisplatin/SK2 combined treatment induced more ROS (+) (%) in oral cancer cells than in normal cells (S-G), suggesting that cisplatin/SK2 induces selective ROS generation in oral cancer cells. Moreover, a combined cisplatin/SK2 treatment increased more ROS (+) (%) in oral cancer cells than in normal cells (S-G), indicating that cisplatin/SK2 induced the generation of more ROS in oral cancer cells.
To assess the involvement of oxidative stress, the ROS inhibitor NAC was applied. The ROS (+) (%) of cisplatin/SK2 separate and combined treatments was suppressed by NAC, suggesting that synergistic ROS generation of cisplatin/SK2 is mediated by oxidative stress. To assess the involvement of oxidative stress, the ROS inhibitor NAC was applied. The ROS (+) (%) of cisplatin/SK2 separate and combined treatments was suppressed by NAC, suggesting that synergistic ROS generation of cisplatin/SK2 is mediated by oxidative stress.

Cisplatin/SK2 Shows Synergistic Induction of MitoSOX in Oral Cancer Cells
As described (Figure 1), NAC rescued the synergistic antiproliferation of cisplatin/SK2 in oral cancer cells. In addition to cellular oxidative stress, NAC can also suppress the generation of mitochondrial reactive species such as mitochondrial superoxide (MitoSOX) [36]. Accordingly, the oxidative stress of mitochondria was further examined by MitoSOX flow cytometry. Cisplatin/SK2 combined treatment displayed a higher MitoSOX (+) (%) in oral cancer cells (Ca9-22 and HSC-3) than independent treatments (cisplatin or SK2) and control ( Figure 7). Moreover, a combined cisplatin/SK2 increased more MitoSOX (+) (%) in oral cancer cells than in normal cells (S-G), indicating cisplatin/SK2 induces selective MitoSOX generation in oral cancer cells.
To assess the involvement of oxidative stress of mitochondria, the MitoSOX inhibitor MitoTEMPO was applied. The MitoSOX (+) (%) of cisplatin/SK2 single and combined treatments was suppressed by MitoTEMPO, suggesting that synergistic MitoSOX generation of cisplatin/SK2 is mediated by oxidative stress.

Cisplatin/SK2 Shows Synergistic MMP Destruction of Oral Cancer Cells
MMP destruction is associated with oxidative stress [37]. Since NAC rescued the synergistic antiproliferation of cisplatin/SK2 in oral cancer cells (Figure 1

Cisplatin/SK2 Shows Synergistic Induction of MitoSOX in Oral Cancer Cells
As described (Figure 1), NAC rescued the synergistic antiproliferation of cisplatin/SK2 in oral cancer cells. In addition to cellular oxidative stress, NAC can also suppress the generation of mitochondrial reactive species such as mitochondrial superoxide (MitoSOX) [36]. Accordingly, the oxidative stress of mitochondria was further examined by MitoSOX flow cytometry. Cisplatin/SK2 combined treatment displayed a higher MitoSOX (+) (%) in oral cancer cells (Ca9-22 and HSC-3) than independent treatments (cisplatin or SK2) and control (Figure 7). Moreover, a combined cisplatin/SK2 increased more

Cisplatin/SK2 Shows Synergistic Induction of γH2AX and 8-OHdG in Oral Cancer Cells
The DNA damage effects of cisplatin and/or SK2-induced oxidative stress were examined. Cisplatin/SK2 combined treatment displayed greater DNA damages such as γH2AX and 8-OHdG (+) (%) in oral cancer cells (Ca9-22 and HSC-3) than in independent treatments (cisplatin or SK2) and control (Figures 9 and 10).  To assess the involvement of oxidative stress of mitochondria, the MitoSOX inhibitor MitoTEMPO was applied. The MitoSOX (+) (%) of cisplatin/SK2 single and combined treatments was suppressed by MitoTEMPO, suggesting that synergistic MitoSOX generation of cisplatin/SK2 is mediated by oxidative stress.

Cisplatin/SK2 Shows Synergistic MMP Destruction of Oral Cancer Cells
MMP destruction is associated with oxidative stress [37]. Since NAC rescued the synergistic antiproliferation of cisplatin/SK2 in oral cancer cells (Figure 1), oxidative stress was further examined by MMP flow cytometry. Cisplatin/SK2 combined treatment displayed higher MMP ( ) (%) in oral cancer cells (Ca9-22 and HSC-3) than in independent treatments (cisplatin or SK2) and control (Figure 8). Moreover, a combined cisplatin/SK2 increased more MMP ( ) (%) in oral cancer cells than in normal cells (S-G), indicating that

Cisplatin/SK2 Shows Synergistic Induction of γH2AX and 8-OHdG in Oral Cancer Cells
The DNA damage effects of cisplatin and/or SK2-induced oxidative stress were examined. Cisplatin/SK2 combined treatment displayed greater DNA damages such as γH2AX and 8-OHdG (+) (%) in oral cancer cells (Ca9-22 and HSC-3) than in independent treatments (cisplatin or SK2) and control (Figures 9 and 10).

Discussion
The present study evaluated the modulating proliferation ability of SK2-cisplatin combined treatment in oral cancer cells. The drug cytotoxicity of this combined treatment was examined in normal oral cells. In addition to cell viability, the responses of oxidative stress, apoptosis, and DNA damage were investigated and compared between oral cancer and normal oral cells following SK2 and/or cisplatin treatments.
Natural products and synthetic chemicals can sensitize oral cancer cells for chemotherapy and radiotherapy [35,38,39]. For example, the combined treatment of ethyl acetate extract of Nepenthes ventricosa x maxima (EANV)/cisplatin synergistically inhibited the proliferation of oral cancer cells [38]. Sulfonyl chromen-4-ones/UVC [35] and sulfonyl chromen-4-ones/X-ray [40] can synergistically inhibit proliferation against oral cancer cells. A combined treatment of cordycepin/cisplatin synergistically enhanced apoptosis in oral cancer OC3 cells [39]. Combined treatments may, therefore, effectively improve oral cancer therapies.
A dioxabicyclo [3.3.1]nonane core commonly appears in several natural products, such as in the marine algal toxins azaspiracids [41], epicoconigrone A [42], and epicoccolide A [43]. However, the modulating antiproliferation ability of dioxabicyclo [3.3.1]nonane derivatives has rarely been investigated. Our previous study first developed a novel

Discussion
The present study evaluated the modulating proliferation ability of SK2-cisplatin combined treatment in oral cancer cells. The drug cytotoxicity of this combined treatment was examined in normal oral cells. In addition to cell viability, the responses of oxidative stress, apoptosis, and DNA damage were investigated and compared between oral cancer and normal oral cells following SK2 and/or cisplatin treatments.
Natural products and synthetic chemicals can sensitize oral cancer cells for chemotherapy and radiotherapy [35,38,39]. For example, the combined treatment of ethyl acetate extract of Nepenthes ventricosa x maxima (EANV)/cisplatin synergistically inhibited the proliferation of oral cancer cells [38]. Sulfonyl chromen-4-ones/UVC [35] and sulfonyl chromen-4-ones/X-ray [40] can synergistically inhibit proliferation against oral cancer cells. A combined treatment of cordycepin/cisplatin synergistically enhanced apoptosis in oral cancer OC3 cells [39]. Combined treatments may, therefore, effectively improve oral cancer therapies.
A dioxabicyclo [3.3.1]nonane core commonly appears in several natural products, such as in the marine algal toxins azaspiracids [41], epicoconigrone A [42], and epicoccolide A [43]. However, the modulating antiproliferation ability of dioxabicyclo [3.3.1]nonane derivatives has rarely been investigated. Our previous study first developed a novel chemical with a dioxabicyclo [3.3.1]nonane core (SK2) and showed the antiproliferation effect on oral cancer cells [25]. Nevertheless, SK2-induced oxidative stress, DNA damage, and apoptosis have not been assessed in normal oral cells thus far. Moreover, to date, the combined treatment for most chemicals with a dioxabicyclo [3.3.1]nonane core has rarely been reported.
Several selective killing agents have been reported, which show higher antiproliferation ability in cancer cells than in normal cells [44][45][46][47]. Combining the selective killing agents with clinical drugs may potentially avoid the side effects of anticancer drugs. For example, cisplatin and FK228 (romidepsin, a histone deacetylase inhibitor) [48] show the selective killing of breast cancer cells with low cytotoxicity to normal breast cells and cause synergistic antiproliferation in breast cancer cells [49]. Accordingly, these combined treatments may exhibit synergistic and selective antiproliferation against cancer cells. SK2 is also a selective killing agent [25], inhibiting the proliferation of oral cancer cells more pronouncedly than those of normal cells. As expected, the present study first demonstrated that a structure with dioxabicyclo [3.3.1]nonane core such as SK2 could sensitize the cisplatin response to oral cancer cells (Figure 1). It warrants a detailed assessment of the synergistic antiproliferation for combined treatment of SK2 and other clinical drugs such as curcumin, doxorubicin, and 5-fluorouracil in the future.
Several studies have reported synergistic enhancement of oxidative stress in combined treatments could increase antiproliferation. For example, cisplatin and FK228 combined treatment cooperatively induces ROS levels and causes synergistic antiproliferation in breast cancer cells [49]. Salinomycin is a monocarboxylic polyether ionophore that shows synergistic ROS induction combined with resveratrol, causing synergistic antiproliferation in breast cancer cells [50]. A combined treatment of dihydroartemisinin and cisplatin provides a synergistic effect of ROS generation and antiproliferation in pancreatic cancer cells [17]. Additionally, sulfonyl chromen-4-ones/X-ray induces higher ROS generation and antiproliferation in oral cancer cells [40].
SK2 contains an N-O bond and NO 2 group, showing free radical potential [51,52]. This radical character also demonstrates oxidative stress in oral cancer cells [25], such as the generation of ROS and MitoSOX and MMP destruction. However, the role of oxidative stress for SK2 was not examined [25]. In contrast, in the present study, all changes in oxidative stress, DNA damage, and apoptosis of SK2 were examined in normal oral cells in detail. The combined treatment of SK2/cisplatin showed synergistic ROS and MitoSOX generation in oral cancer cells but not in normal oral cells (Figures 6 and 7). NAC pretreatment suppresses synergistic antiproliferation ( Figure 1) and the ROS induction ( Figure 6) of SK2/cisplatin in oral cancer cells. Similarly, MitoTEMPO pretreatment suppresses synergistic MitoSOX induction (Figure 7). These results confirm the role of oxidative stress in synergistic antiproliferation and oxidative stress of SK2/cisplatin. Therefore, synergistic oxidative stress contributes to synergistic antiproliferation in SK2/cisplatin combined treatment of oral cancer cells.
Moreover, several anticancer agents generate oxidative stress, improving apoptosis [53][54][55] and DNA damage [27,54]. Except for synergistic oxidative stress, the combined treatments show synergistic apoptosis and DNA damage, such as cisplatin/FK228 in breast cancer [49], salinomycin/resveratrol in breast cancer [50], dihydroartemisinin/cisplatin in pancreatic cancer [17], and sulfonyl chromen-4-ones/X-ray in oral cancer cells. Similarly, SK2/cisplatin combined treatment show synergistic intrinsic and extrinsic apoptosis ( Figure 5) and DNA damage (γH2AX and 8-OHdG) (Figures 9 and 10) in oral cancer cells but not in normal oral cells. Additionally, a combined SK2/cisplatin treatment shows synergistic inductions of annexin V, pancaspase, Cas 3/8/9, and Cas 3/7 (Figures 3 and 4) in oral cancer cells but not in normal oral cells. Therefore, it is likely oxidative stress that contributes to synergistic apoptosis and DNA damage in SK2/cisplatin combined treatment of oral cancer cells.

Conclusions
Our previous research showed that the nitrated [6,6,6]tricycle-derived compound (SK2) exhibited a selective antiproliferation ability in oral cancer cells [25]. However, its potential combined treatment with clinical drugs was not reported. Taking cisplatin as an example, the combined treatment of SK2 was examined in the present study. Combined treatment with cisplatin/SK2 shows synergistic and selective antiproliferation in oral cancer cells but not in normal oral cells. Moreover, cisplatin/SK2 shows a synergistic induction of oxidative stress, caspase-dependent apoptosis, and DNA damage in oral cancer cells at low concentrations of cisplatin and SK2. This finding suggests that SK2 has the potential to be applied in combined treatment with clinical drugs.