Antiproliferation- and Apoptosis-Inducible Effects of a Novel Nitrated [6,6,6]Tricycle Derivative (SK2) on Oral Cancer Cells

The benzo-fused dioxabicyclo[3.3.1]nonane core is the central framework in several natural products. Using this core, we had developed a novel nitrated [6,6,6]tricycle-derived compound containing an n-butyloxy group, namely, SK2. The anticancer potential of SK2 was not assessed. This study aimed to determine the antiproliferative function and investigated possible mechanisms of SK2 acting on oral cancer cells. SK2 preferentially killed oral cancer cells but caused no harmful effect on non-malignant oral cells. After the SK2 exposure of oral cancer cells, cells in the sub-G1 phase accumulated. This apoptosis-like outcome of SK2 treatment was validated to be apoptosis via observing an increasing annexin V population. Mechanistically, apoptosis signalers such as pancaspase, caspases 8, caspase 9, and caspase 3 were activated by SK2 in oral cancer cells. SK2 induced oxidative-stress-associated changes. Furthermore, SK2 caused DNA damage (γH2AX and 8-hydroxy-2′-deoxyguanosine). In conclusion, a novel nitrated [6,6,6]tricycle-derived compound, SK2, exhibits a preferential antiproliferative effect on oral cancer cells, accompanied by apoptosis, oxidative stress, and DNA damage.


Introduction
Oral cancer is a malignancy that develops in the lips, mouth, or throat tissues. In Taiwan, oral cancer represents the third most common malignancy and is the fourth leading cause of death due to cancer in males [1]. Oral cancer also occurs globally [2]. Due to diagnosis at advanced stages, the five-year survival rate for oral cancer is low [3], giving prominence to the benefit of refining our understanding of the pathogenesis of human oral carcinogenesis [4]. In addition to surgery, chemo-and radiotherapy are alternative

SK2 Preferentially Kills Oral Cancer Cells
The structure of SK2 is provided ( Figure 1A). The cell viability was dose-dependently inhibited by SK2 treatments of oral cancer cells (CAL 27 and OECM-1). Moreover, the cytotoxicity for non-malignant oral cells was examined. SK2 showed a minor cytotoxic effect (>90% viability) on non-malignant oral cells (HGF-1) ( Figure 1B). These results reveal that SK2 exerts preferential antiproliferative effects on oral cancer cells but does not affect non-malignant oral cells. ways for curing oral cancer, but they commonly generate severe side effects [5]. Therefore, anticancer drugs with low side effects can improve the effectiveness of oral cancer therapy. The dioxabicyclo[3.3.1]nonane core exists in several natural products and chemical drugs. For example, 1,3-disubstituted 2,9-dioxabicyclo [3.3.1]nonane is the core of rings F/G in the marine algal toxins azaspiracids [6]. The 2,nonane core can be used for anticoagulants [7]. Natural products, such as epicoccolide A [8] and epicoconigrone A [9], exhibit the benzo-fused dioxabicyclo [3.3.1]nonane core as the central framework. Epicoccolide A shows antifungal activity [10]. However, the anticancer effects of dioxabicyclo[3.3.1]nonane derivatives remain unclear.
Using this benzo-fused dioxabicyclo[3.3.1]nonane core, we had developed a novel nitrated [6,6,6]tricycle-derived compound containing an n-butyloxy group, namely, SK2 [11]. SK2 possesses an N-O bond similar to the O-O bond, which is prone to chemical cleavage [12] and forms a free-radical structure. Moreover, SK2 possesses a NO2 group that is a radical initiator or promoter [13]. Accordingly, the N-O bond and NO2 group in SK2 may generate free radicals. Therefore, we expect that it may potentially modulate oxidative or nitrative stresses. The nitration-mediated inhibition of antioxidant enzymes, such as manganese superoxide dismutase, enhances superoxide levels and oxidative/nitrative stress [14], causing DNA damage [15], autophagy, and apoptosis [16,17]. This warrants the detailed investigation of the potential anticancer effect of SK2.
The purpose of the present study was to assess the antiproliferative functions and mechanisms of SK2 acting on oral cancer cells by measuring proliferation, the cell cycle, cellular and mitochondrial oxidative stress, apoptosis, and the DNA-damage status.

SK2 Disturbs Cell Cycle Progression
After the SK2 treatment of oral cancer cells, the cell cycle distribution was assessed ( Figure 2). The SK2 treatments dose-dependently induced the accumulation of the sub-G1 population of oral cancer cells.

SK2 Disturbs Cell Cycle Progression
After the SK2 treatment of oral cancer cells, the cell cycle distribution was assessed ( Figure 2). The SK2 treatments dose-dependently induced the accumulation of the sub-G1 population of oral cancer cells. Figure 2. SK2 causes sub-G1 accumulation in oral cancer cells. DNA-content detections and statistics are provided. Cells were exposed to SK2 (0 (0.1% DMSO), 7.5, and 10 µg/mL) for 24 h. Data (means ± SDs; n = 3) without overlapping characters differed significantly (p < 0.05). For an example of CAL 27 and OECM-1 cells, different concentrations with different characters produced significantly different results. For CAL 27 and OECM-1 cells, for SK2 at 0, 7.5, and 10 µg/mL, "c, b, and a" indicate significant differences because the results did not overlap.

SK2 Increases Annexin V/7AAD-Assessed Apoptosis
After the SK2 treatment of oral cancer cells, the distribution of annexin V/7AAD was assessed ( Figure 3). The SK2 treatments dose-dependently induced the annexin V-positive (+) population of oral cancer cells. . SK2 triggers apoptosis in oral cancer cells. Annexin V/7AAD detections and statistics are provided. Cells were exposed to SK2 (0 (0.1% DMSO), 7.5, and 10 µg/mL) for 24 h. Annexin V(+)/7AAD (+ or −) was regarded as the apoptosis %. Data (means ± SDs; n = 3) without overlapping characters differed significantly (p < 0.05). For an example of CAL 27 and OECM-1 cells, different concentrations with different characters produced significantly different results. For CAL 27 and OECM-1 cells, for SK2 at 0, 7.5, and 10 µg/mL, "c, b, and a" indicate significant differences because the data did not overlap. . SK2 causes sub-G1 accumulation in oral cancer cells. DNA-content detections and statistics are provided. Cells were exposed to SK2 (0 (0.1% DMSO), 7.5, and 10 µg/mL) for 24 h. Data (means ± SDs; n = 3) without overlapping characters differed significantly (p < 0.05). For an example of CAL 27 and OECM-1 cells, different concentrations with different characters produced significantly different results. For CAL 27 and OECM-1 cells, for SK2 at 0, 7.5, and 10 µg/mL, "c, b, and a" indicate significant differences because the results did not overlap.

SK2 Increases Annexin V/7AAD-Assessed Apoptosis
After the SK2 treatment of oral cancer cells, the distribution of annexin V/7AAD was assessed ( Figure 3). The SK2 treatments dose-dependently induced the annexin V-positive (+) population of oral cancer cells.

SK2 Increases Caspase-Signaling Activation
Apoptosis is initiated and promoted by apoptotic signaling such as caspases [18]. After the SK2 treatment of oral cancer cells, the pancaspase expression was assessed ( Figure 4A). Based on flow cytometry analysis, the pancaspase-positive (+) population, i.e., pancaspase activation, of oral cancer cells was dose-dependently induced by SK2 treatments.

SK2 Increases ROS Induction
The oxidative-stress status of the oral cancer cells was assessed by the ROS level. After the SK2 treatment of oral cancer cells, the ROS levels were assessed ( Figure 5). Based on flow cytometry analysis, the ROS-positive (+) population of oral cancer cells was dosedependently induced by SK2 treatments.

SK2 Increases Mitochondrial Superoxide (MitoSOX) Induction
The oxidative-stress status of oral cancer cells was also assessed by the MitoSOX level. After the SK2 treatment of oral cancer cells, the MitoSOX levels were assessed ( Figure 6). The MitoSOX-positive (+) population of oral cancer cells was dose-dependently induced by SK2 treatments based on flow cytometry analysis. ferent results. For CAL 27 and OECM-1 cells, for SK2 at 0, 7.5, and 10 µg/mL, "c, b, and a" indicate significant differences because the results did not overlap.

SK2 Increases Caspase-Signaling Activation
Apoptosis is initiated and promoted by apoptotic signaling such as caspases [18]. After the SK2 treatment of oral cancer cells, the pancaspase expression was assessed (Figure 4A). Based on flow cytometry analysis, the pancaspase-positive (+) population, i.e., pancaspase activation, of oral cancer cells was dose-dependently induced by SK2 treatments.
Since pancaspase only enables detection for general caspases such as caspase-1 and 3 to 9 [19], specific caspases such as the extrinsic, intrinsic, and executor caspases (Cas 8, 9, and 3) were further examined ( Figure 4B-D). Based on flow cytometry analysis, the Cas 8, 9, and 3 (+) populations were dose-dependently induced by SK2 treatments.  , for SK2 at 0, 7.5, and 10 µg/mL, "c, b, and a" indicate significant differences because the data did not overlap. For an example of CAL 27 cells (B,C), for SK2 at 7.5 and 10 µg/mL, the same letter "a" indicates non-significant differences because the data overlapped.

SK2 Increases ROS Induction
The oxidative-stress status of the oral cancer cells was assessed by the ROS level. After the SK2 treatment of oral cancer cells, the ROS levels were assessed ( Figure 5). Based on flow cytometry analysis, the ROS-positive (+) population of oral cancer cells was dosedependently induced by SK2 treatments. detections, and statistics are provided. Cells were exposed to SK2 (0 (0.1% DMSO), 7.5, and 10 µg/mL) for 24 h. (+) shown in each panel represents pancaspase and Cas 3, 8, and 9 (+). Data (means ± SDs; n = 3) without overlapping characters differed significantly (p < 0.05). For CAL 27 and OECM-1 cells (D), for SK2 at 0, 7.5, and 10 µg/mL, "c, b, and a" indicate significant differences because the data did not overlap. For an example of CAL 27 cells (B,C), for SK2 at 7.5 and 10 µg/mL, the same letter "a" indicates non-significant differences because the data overlapped.

SK2 Increases Mitochondrial Membrane Potential (MMP) Destruction
The oxidative-stress status of oral cancer cells was further measured by the MMP level. After the SK2 treatment of oral cancer cells, the MMP levels were assessed (Figure 7). Based on flow cytometry analysis, the MMP-negative (−) population of oral cancer cells was induced by SK2 treatments.

SK2 Increases γH2AX Induction
The DNA damage of oral cancer cells was assessed by the γH2AX level, a DNAdouble-strand-break biomarker [20]. After the SK2 treatment of oral cancer cells, the γH2AX levels were assessed ( Figure 8). The γH2AX-positive (+) population of oral cancer cells was dose-dependently induced by SK2 treatments based on flow cytometry analysis.

SK2 Increases 8-Hydroxy-2 -deoxyguanosine (8-OHdG) Induction
The DNA-damage status of oral cancer cells was assessed by the 8-OHdG level [21]. After the SK2 treatment of oral cancer cells, the 8-OHdG levels were assessed ( Figure 9). The 8-OHdG-positive (+) population of oral cancer cells was dose-dependently induced by SK2 treatments based on flow cytometry analysis. For an example of CAL 27 and OECM-1 cells, different concentrations with different characters produced significantly different results. For CAL 27 and OECM-1 cells, for SK2 at 0, 7.5, and 10 µg/mL, "c, b, and a" indicate significant differences because the data did not overlap.

SK2 Increases Mitochondrial Superoxide (MitoSOX) Induction
The oxidative-stress status of oral cancer cells was also assessed by the MitoSOX level. After the SK2 treatment of oral cancer cells, the MitoSOX levels were assessed (Figure 6). The MitoSOX-positive (+) population of oral cancer cells was dose-dependently induced by SK2 treatments based on flow cytometry analysis.   For an example of CAL 27 and OECM-1 cells, different concentrations with different characters produced significantly different results. For CAL 27 and OECM-1 cells, for SK2 at 0, 7.5, and 10 µg/mL, "c, b, and a" indicate significant differences because the data did not overlap.

SK2 Increases Mitochondrial Superoxide (MitoSOX) Induction
The oxidative-stress status of oral cancer cells was also assessed by the MitoSOX level. After the SK2 treatment of oral cancer cells, the MitoSOX levels were assessed (Figure 6). The MitoSOX-positive (+) population of oral cancer cells was dose-dependently induced by SK2 treatments based on flow cytometry analysis. Figure 6. SK2 prompts MitoSOX induction in oral cancer cells. MitoSOX detections and statistics are provided. Cells were exposed to SK2 (0 (0.1% DMSO), 7.5, and 10 µg/mL) for 24 h. (+) in each panel represents MitoSOX (+). Data (means ± SDs; n = 3) without overlapping characters differed significantly (p < 0.05). For an example of CAL 27 and OECM-1 cells, different concentrations with different characters produced significantly different results. For CAL 27 and OECM-1 cells, for SK2 at 0, 7.5, and 10 µg/mL, "c, b, and a" indicate significant differences because the data did not overlap. Figure 6. SK2 prompts MitoSOX induction in oral cancer cells. MitoSOX detections and statistics are provided. Cells were exposed to SK2 (0 (0.1% DMSO), 7.5, and 10 µg/mL) for 24 h. (+) in each panel represents MitoSOX (+). Data (means ± SDs; n = 3) without overlapping characters differed significantly (p < 0.05). For an example of CAL 27 and OECM-1 cells, different concentrations with different characters produced significantly different results. For CAL 27 and OECM-1 cells, for SK2 at 0, 7.5, and 10 µg/mL, "c, b, and a" indicate significant differences because the data did not overlap.

SK2 Increases Mitochondrial Membrane Potential (MMP) Destruction
The oxidative-stress status of oral cancer cells was further measured by the MMP level. After the SK2 treatment of oral cancer cells, the MMP levels were assessed ( Figure  7). Based on flow cytometry analysis, the MMP-negative (−) population of oral cancer cells was induced by SK2 treatments. For an example of OECM-1 cells, for SK2 at 7.5 and 10 µg/mL, the same letter "a" indicates non-significant differences because the results overlapped. For CAL 27 cells, for SK2 at 0, 7.5, and 10 µg/mL, "c, b, and a" indicate significant differences because the results did not overlap. For OECM-1 cells, for SK2 at 7.5 and 10 µg/mL, "a and a" indicate the results are not significantly different because they overlapped.

SK2 Increases γH2AX Induction
The DNA damage of oral cancer cells was assessed by the γH2AX level, a DNA-double-strand-break biomarker [20]. After the SK2 treatment of oral cancer cells, the γH2AX levels were assessed (Figure 8). The γH2AX-positive (+) population of oral cancer cells was dose-dependently induced by SK2 treatments based on flow cytometry analysis. Data (means ± SDs; n = 3) without overlapping characters differed significantly (p < 0.05). For an example of CAL 27 cells, different concentrations with different characters indicate significant differences. For an example of OECM-1 cells, for SK2 at 7.5 and 10 µg/mL, the same letter "a" indicates non-significant differences because the results overlapped. For CAL 27 cells, for SK2 at 0, 7.5, and 10 µg/mL, "c, b, and a" indicate significant differences because the results did not overlap. For OECM-1 cells, for SK2 at 7.5 and 10 µg/mL, "a and a" indicate the results are not significantly different because they overlapped. 7). Based on flow cytometry analysis, the MMP-negative (−) population of oral cancer cells was induced by SK2 treatments. Data (means ± SDs; n = 3) without overlapping characters differed significantly (p < 0.05). For an example of CAL 27 cells, different concentrations with different characters indicate significant differences. For an example of OECM-1 cells, for SK2 at 7.5 and 10 µg/mL, the same letter "a" indicates non-significant differences because the results overlapped. For CAL 27 cells, for SK2 at 0, 7.5, and 10 µg/mL, "c, b, and a" indicate significant differences because the results did not overlap. For OECM-1 cells, for SK2 at 7.5 and 10 µg/mL, "a and a" indicate the results are not significantly different because they overlapped.

SK2 Increases γH2AX Induction
The DNA damage of oral cancer cells was assessed by the γH2AX level, a DNA-double-strand-break biomarker [20]. After the SK2 treatment of oral cancer cells, the γH2AX levels were assessed (Figure 8). The γH2AX-positive (+) population of oral cancer cells was dose-dependently induced by SK2 treatments based on flow cytometry analysis.

Discussion
The present study examined the proliferation-modulating effects and investigated the anticancer mechanisms of SK2 in oral cancer cells. The oxidative-stress generation, apoptosis signaling, and DNA-damage induction were assessed in oral cancer cells following SK2 treatment.

Discussion
The present study examined the proliferation-modulating effects and investigated the anticancer mechanisms of SK2 in oral cancer cells. The oxidative-stress generation, apoptosis signaling, and DNA-damage induction were assessed in oral cancer cells following SK2 treatment.
The IC 50 values for several clinical drugs for oral cancer cells are reported for comparison. For cisplatin, the IC 50 values were 5.3 and 5 µM for oral cancer cells (CAL 27) based on the 24 h MTT [22] and MTS [23] methods. For curcumin, the IC 50 was 12 µM for CAL 27 cells based on the 24 h sulforhodamine B method [24]. For 5-fluorouracil, the IC 50 was 95.6 µM for CAL 27 cells based on the 24 h Alamar blue method [25]. Moreover, cisplatin [26], curcumin [27], and 5-fluorouracil [28] showed side effects.
Although dioxabicyclo[3.3.1]nonane derivatives have frequently been synthesized, their anticancer effects have rarely been investigated. Recently, peniciketal A, a Penicillium raistrickii-derived spiroketal compound containing a benzo-fused 2,8-dioxabicyclo[3.3.1] nonane core, was discussed [29,30]. Peniciketal A had IC 50 values of 33.50, 56.85, and 60.64 µM for the 24 h treatment and 18.99, 45.14, and 39.94 µM for the 48 h treatment of leukemia cells (THP-1, K562, and HL60). Peniciketal A induced apoptosis in and preferential antiproliferative effects on leukemia cells but not for primary mouse embryonic fibroblasts (MEFs) [29]. A sanctis B-containing dibenzo-2,8-dioxabicyclo[3.3.1]nonane scaffold exhibited antiproliferative effects on breast cancer cells, but no detailed anticancer mechanism was reported [31]. Similarly, the IC 50 value of SK2 at 24 h of exposure for oral cancer cells (CAL 27 and OECM-1) was 7.5 µg/mL (25.68 µM), but it showed noncytotoxicity to non-malignant oral cells (HGF-1) based on the MTS assay. These results suggest that dioxabicyclo[3.3.1]nonane derivatives have a preferential antiproliferative ability for cancer cells rather than normal cells. This low cytotoxic property for normal cells may potentially contribute to its low side effects. This warrants a detailed examination of the in vivo antitumor effects of SK2 by using an animal model.
The cellular ROS-induction effects of dioxabicyclo[3.3.1]nonane derivatives remain unclear. SK2 contains a N-O bond and NO 2 group that are competent in forming a freeradical structure [12,13]. Accordingly, oxidative-stress effects of SK2 in oral cancer cells were examined. The present study first reported that SK2 induced cellular and mitochondrial oxidative stress and MMP destruction in oral cancer cells. Several natural products and chemical agents can preferentially kill cancer cells by oxidative-stress induction in cancer cells rather than normal cells [32,33]. The basal ROS level in most cancer cells is generally higher than that in normal cells, making cancer cells unable to tolerate the exogenous ROS generated by ROS-modulating drugs, causing the preferential antiproliferative effects on cancer cells [33]. Accordingly, this rationale may partially explain the preferential antiproliferative effect of SK2 on oral cancer cells rather than non-malignant oral cells.
A combined treatment approach has benefits for targeting different molecular mechanisms [41,42] for anticancer therapy. For example, the natural product cordycepin combined with the clinical drug cisplatin showed synergistic apoptotic effects on oral cancer cells [43]. A synthesized chemical, sulfonyl chromen-4-ones (CHW09), had synergistic effects of inhibiting oral cancer cell proliferation when combined with either UVC [44] or X-ray [45] irradiation. Since SK2 is noncytotoxic to non-malignant oral cells (HGF-1), the potential synergistic effects of combined treatment with SK2 and other anticancer drugs or radiation warrant a detailed evaluation of oral cancer therapy in the future.
The MetaCore/MetaDrug platform [46,47] uses QSAR models to predict the input molecule's molecular pathway and pharmacokinetic activity. The potential targets were unavailable after data retrieval on 27 November 2021. Moreover, a similar structure was unavailable for data mining using MetaCore/MetaDrug. Therefore, the potential targets cannot be predicted using this platform. It is noted that ROS are also generated when the antioxidant machinery is overloaded [48,49]. When antioxidant signaling is suppressed, ROS are generally developed. For example, pomegranate extract downregulated several antioxidant signaling pathways, enhancing oxidative stress, in oral cancer cells [50]. Accordingly, SK2 may downregulate the antioxidant signaling pathway, inducing ROS generation. The possibility that antioxidant signaling proteins were the potential targets needs to be further assessed in the future.

Cell Cultures and Cell Viability
Oral cancer cell lines (CAL 27) and non-malignant oral cell lines (HGF-1) were obtained from ATCC (Manassas, VA, USA). Another oral cancer cell line, OECM-1 [51], was provided by Dr. Wan-Chi Tsai at Kaohsiung Medical University, Kaohsiung, Taiwan. They were cultured in a standard medium mixed with 10% fetal bovine serum (FBS) and standard antibiotics at 37 • C in a 5% CO 2 chamber [52]. The cell viability was evaluated using the tetrazolium dye of the MTS reagent (Promega, Madison, WI, USA) [34].

Cell Cycle Assay
The cell cycle phases were determined by their different cellular DNA contents as revealed by probing with 7-aminoactinomycin D (7AAD) (Biotium Inc., Hayward, CA, USA) [53]. The cells were fixed using 75% ethanol overnight. Subsequently, the cells were maintained with 1 µg/mL of 7AAD for 30 min at 37 • C. Finally, flow cytometry analysis was performed using an Accuri C6 flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) using the FL1 channel.

MitoSOX Assay
The MitoSOX was probed using MitoSOX™ Red (Molecular Probes, Invitrogen, Eugene, OR, USA), producing fluorescence for flow cytometry [55]. In brief, cells were treated with 5 µM MitoSOX at 37 • C for 30 min. Finally, flow cytometry analysis was performed using an Accuri C6 using the FL3 channel.

Statistics
The data were multi-compared, and the significance was determined using the JMP 12 software (SAS Institute, Cary, NC, USA) with ANOVA and the Tukey post hoc test. Data without overlapping small characters differed significantly. The data are shown as the means ± SDs for triplicate experiments.

Conclusions
We previously used the benzo-fused dioxabicyclo[3.3.1]nonane core to synthesize a novel nitrated [6,6,6]tricycle-derived compound containing an n-butyloxy group (SK2). However, the biological function of SK2 was not investigated. The present study validated that SK2 preferentially killed oral cancer cells but showed no harmful effect on non-malignant oral cells. Mechanistically, SK2 induced oxidative stress, triggered apoptosis, and caused DNA damage to oral cancer cells ( Figure 10). Elucidating these mechanisms may improve the anticancer application of SK2 to oral cancer therapy.

Statistics
The data were multi-compared, and the significance was determined using the JMP 12 software (SAS Institute, Cary, NC, USA) with ANOVA and the Tukey post hoc test. Data without overlapping small characters differed significantly. The data are shown as the means ± SDs for triplicate experiments.

Conclusions
We previously used the benzo-fused dioxabicyclo[3.3.1]nonane core to synthesize a novel nitrated [6,6,6]tricycle-derived compound containing an n-butyloxy group (SK2). However, the biological function of SK2 was not investigated. The present study validated that SK2 preferentially killed oral cancer cells but showed no harmful effect on non-malignant oral cells. Mechanistically, SK2 induced oxidative stress, triggered apoptosis, and caused DNA damage to oral cancer cells ( Figure 10). Elucidating these mechanisms may improve the anticancer application of SK2 to oral cancer therapy.

Conflicts of Interest:
The authors confirm that this article's content has no conflict of interest.
Sample Availability: Samples of the compounds are not available from the authors.