Research ArticleExperimental Studies

Pipoxolan Exhibits Antitumor Activity Toward Oral Squamous Cell Carcinoma Through Reactive Oxygen Species-mediated Apoptosis

PEI-YU CHOU, MIN-MIN LEE, SUNG-YUAN LIN and MING-JYH SHEU
Anticancer Research November 2017, 37 (11) 6391-6400;

Abstract

Pipoxolan is frequently prescribed as a smooth muscle relaxant. Pipoxolan has also been shown to have anticancer activity. Our study investigated whether pipoxolan induced apoptosis in oral squamous cell carcinoma (OSCC). Cell cytotoxicity was evaluated by the MTT assay. Cell apoptosis and cell-cycle distribution were measured by annexin V/propidium iodide (PI) double staining and flow cytometry, respectively. Apoptotic-related proteins were assessed by western blotting. Reactive oxygen species (ROS) generation and mitochondrial membrane potential (MMP) were measured with fluorescent probes. Following exposure of TW206 OSCC cells to pipoxolan, a time-dependently decrease in MMP and an increase in ROS were observed. However, these effects were significantly abrogated by the free radical scavenger N-acetyl-L-cysteine. Since high levels of ROS were produced early in the treatment, intracellular ROS seemed to play a key role in pipoxolan-induced apoptosis. In HSC-3 OSCC cells, our results demonstrated that pipoxolan treatment caused a time-dependent increase of protein expression of active caspase-3 and -9, cytosolic cytochrome c, cleavage of poly (ADP-ribose) polymerase, and B-cell lymphoma 2 (BCL2)-like protein 4 (BAX). However, expression of BCL2 itself was reduced. Clearly, such an increase in BAX/BCL2 ratio would be associated with apoptosis. In addition, pipoxolan markedly suppressed the protein expression of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) and phosphorylation of protein kinase B (AKT). These data suggest that pipoxolan acts against HSC-3 in vitro via intrinsic apoptotic signaling pathways, and inhibition of PI3K/AKT signaling.

Oral squamous cell carcinoma (OSCC) is a major health issue and mostly predominant in South East Asia. More than 200,000 new cases are reported annually worldwide. each year. Although much research on treatment strategies have been carried out, the 5-year survival rate of patients with OSCC is still less than 50% (1). Indeed, combined surgical resection with chemotherapy significantly improves the effectiveness of treatment and reduces the recurrence rate in patients with OSCC (2), however, cancer cells become resistant to chemotherapeutic agents. Accordingly, it is necessary to search for new agents for the treatment of OSCC.

Oxidative stress occurs when there is an imbalance between reactive oxygen species (ROS) and the body's antioxidant-scavenging mechanisms (3). Studies have shown that elevated oxidative stress can induce carcinogenesis in various types of cancer, and potential treatment strategies that target ROS have been investigated (4). Additionally, studies have confirmed that oxidative stress is correlated with the development of OSCC (5). However, anticancer agents have been demonstrated to induce the death of OSCC through apoptosis (6), that can be activated by DNA damage and an increase in the level of ROS (7). Upon such stimulation, cytochrome c is released from mitochondria into the cytoplasm. This leads to the activation of caspase-9, caspase-3, and poly (ADP-ribose) polymerase (PARP) and causes apoptosis (8). The cleavage at Asp residues that separate the large and small subunits (i.e. caspase-9) leads to the activation of an effector caspase (i.e. caspase-3) (9).The caspases are mainly produced as zymogens, and become activated by two pathways during apoptosis (10). Regulated by the B-cell lymphoma 2 (BCL2) family, the signal of the intrinsic pathway stimulates cytochrome c translocation from the mitochondria to the cytoplasm, and stimulates caspase-9 activation. The extrinsic pathway involves the activation of ligands of the tumor necrosis factor (TNF) family or FAS (11). FAS-associated death domain protein (FADD) activates caspase-8. Thereafter, both caspase-8 and -9 have been shown to cleave and activate the proenzyme of caspase-3, which initiates cellular destruction by cleaving PARP (12).

Study suggests that the epidermal growth factor receptor (EGFR)/phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin pathway is strongly linked with the progression and development of head and neck squamous cell carcinoma (HNSCC) (13). Targeting EGFR-PI3K-AKT-mTOR pathway activation increased radiosensitivity in HNSCC, showing promising outcome (14). The anticancer agent esculetin alone (15) or in combination with chemotherapeutic agents (16) was shown to induce apoptosis of OSCC cells.

5,5-Diphenyl-2-(β-N-piperidinoethyl)-1,3-dioxolan-4-one-hydrochloride (pipoxolan hydrochloride, Rowapraxin®; MW=387.5; Figure 1) is commonly prescribed as a smooth muscle relaxant, and has many different activities including anti-spasmodic, digestive analgesic and anti-migraine (17,18). Pipoxolan has also been shown to have anticancer activity, including against human leukemia HL-60 (19, 20) and non-small lung cancer cells (21). Pipoxolan has also been shown to protect from ischemia/reperfusion-induced cerebral infarction, carotid artery ligation-induced intimal hyperplasia and affects vascular smooth muscle cell migration both in vivo and in vitro (22). In this study, we aimed to investigate the impact of pipoxolan on cytotoxicity toward OSCC cells and explored possible molecular mechanisms of action.

Materials and Methods

Materials. Antibodies to caspase-3, -8 and -9 were purchased from Cell Signaling (Boston, MA, USA); those to cleaved PARP, PI3K, AKT, pAKT, B-cell lymphoma 2 (BCL2)-like protein 4 (BAX) and BCL2 were obtained from Santa Cruz (Santa Cruz, CA, USA); antibody to cytochrome c was purchased from BioLegend (San Diego, CA, USA). Secondary antibodies were acquired from Amersham Biosciences (Freiburg, Germany). All other chemicals were obtained from Sigma (Saint Louis, MO, USA). Pipoxolan was provided by Chin Teng Pharmaceutical Ind. Co., Ltd. (Dachia, Taiwan).

Cell culture. The cancer cell lines were obtained from the Food Industry Research and Development Institute (Hsinchu, Taiwan). Cal-27, HSC-3 and TW206 cells were cultured in RPMI-1640 medium containing 2 mM L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin (Sigma–Aldrich, St. Louis, MO, USA), and 10% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA) in a humidified atmosphere containing 5% CO2 at 37°C. All experiments were conducted using exponentially growing cells cultured in complete medium with 1% FBS. To prevent cell overgrowth, the initial cell density was 1×105 cells/mL for 48 h treatment. Pipoxolan was prepared freshly in dimethyl sulfoxide (DMSO; Sigma–Aldrich, St. Louis, MO, USA) before each experiment. The final DMSO concentration in all experiments was 0.1% and had negligible effect on the measured parameters, including mitochondrial membrane potential (MMP). Negative controls were treated with 0.1% DMSO only. For treatments with inhibitor, 2.5 × 105 cells/mL were seeded into 24-well plates for 24 h, then pretreated with 10 mM N-acetylcysteine (NAC, antioxidant; Sigma–Aldrich) for 1 h, followed by treatment with or without pipoxolan (20 μg/mL).

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. This assay was conducted according to our previous report (20) with slight modification. Exponentially growing human OSCC cells were seeded at a density of 2×105 cells/mL for 24 h, and then cells were treated with different doses of pipoxolan (1.6, 3.2, 6.25, 12.5, 25, 50 and 100 μg/mL) for 24 h. Then, 20 μL of MTT solution (5 mg/mL) was added, and the cells were incubated for an additional 3 h. The resulting formazan precipitate was dissolved in 200 μL of DMSO, and the solution was vigorously mixed to dissolve the reacted dye. The absorbance of each well was read on a multi-plate reader at 570 nm by a spectrophotometer (BioTek, Winooski, VT, USA).

Quantification of apoptosis using flow cytometry. This assay was conducted according to our previous report (20) with slight modification. Cells were seeded at a density of 2×105 cells/well in a 24-well plate (Falcon, Franklin Lakes, NJ, USA) and treatment with different doses of pipoxolan for 24 h. In the time course study, TW206 and HSC-3 cells were treated with 20 μg/mL in and 50 μg/mL, respectively, for different time periods (0, 8, 12, 24 and 48 h). The cells were washed twice with ice-cold 1× phosphate-buffered saline (PBS) before being fixed in 70% ethanol overnight. Cells were centrifuged (3000 × g for 5 min at room temperature) and resuspended in 0.3 ml of DNA staining solution [100 μg/ml propidium iodide (PI), 0.2% NP-40, and 1 mg/mL RNase A (DNase-free) in PBS lacking Ca++ and Mg++; at a 1:1:1 ratio by volume] for a minimum of 30 min in a dark room and analyzed within 2 h. Cells were analyzed using flow cytometry (FACS) Calibur™ system (Becton Dickinson, San Jose, CA, USA). Ten thousand events were acquired and DNA content was determined using DNA analysis software ModFitLT, version 3.0 (Verity Software, Topsham, ME, USA).

Annexin-V/PI double staining. This assay was conducted according to our previous report (20) with slight modification. TW206 cells were seeded at a density of 2×105 cells/well in a 24-well plate (Falcon) and treated with 20 μg/ml pipoxolan for different time periods (2, 4, and 6 h). For annexin-V staining, a commercially available kit for the FACS Calibur™ system was used (BioSource, Camarillo, CA, USA). In brief, the cells were washed twice in PBS buffer and resuspended at 100 μl/tube 1x annexin-V binding buffer. The cells were then stained with 5 μl of annexin-V fluorescein isothiocyanate and 10 μl of PI buffer to each tube. After incubation for 15 min in the dark at room temperature, cells were diluted with 400 μl 1x annexin-V binding buffer and then measured without gating within 1 h with a FACS Calibur™ system (Becton Dickinson).

Figure 1.
Figure 1.

Chemical structure of pipoxolan.

Flow cytometric analysis of ROS and MMP. This assay was conducted according to our previous report with modification (20). Cells were cultured at a density of 2.5×105/ml in 24-well plates, then pipoxolan (20 μg/ml) was added and plates incubated for a further 1, 3 and 6 h respectively. After pipoxolan treatment, ROS were assessed using a fluorescent dye, 2’,7’-dichlorodihydrofluorescein diacetate (H2-DCF-DA) (Molecular Probes, Eugene, OR, USA), which is converted to 2’,7’-dichlorofluorescein (DCF) by esterases when taken up. ROS were then analyzed with the FACS Calibur™ system (Becton Dickinson). A fluorometric probe, 3,3’-dihexyloxacarbocyanine iodide (DiOC6), was applied to determine the MMP (23). Briefly, OSCC cells were plated in 6-well culture dishes. When reaching confluence, the cells were treated with vehicle or pipoxolan for 24 h. OSCC cells were subsequently stained with DiOC6 (40 nM) for 15 min at 37°C. Cells were then collected, washed twice in PBS, and analyzed with FACS Calibur™ system (Becton Dickinson).

Western blot analysis. This assay was conducted according to our previous report with slight modification (20). TW206 and HSC-3 cells were plated in 10-cm dishes in RPMI containing 1% FBS at a density of 5×106 cells and incubated with 20 μg/ml or 50 μg/ml of pipoxolan, respectively for 2, 4, 8, 12, 24 and 48 h. The cells were collected, then lysed in a lysis solution (6.25 ml Tris-HCl, pH 6.8. 10 ml 10% sodium dodecyl sulfate 8.3 ml dithiothreitol) followed by incubation at 95°C for 5 min. Samples were separated in a 12% polyacrylamide gel and then transferred onto polyvinylidene difluoride membrane. The membrane was blocked in 5% non-fat milk in PBS-Tween20 buffer for 1 h. and probed with antibodies specific for p53, BAX, BCL2, cytochrome c, caspase-9, caspase-8, caspase-3 and cleaved PARP overnight at 4°C. The blots were then incubated with horseradish peroxidase-linked secondary antibody for 1 h. followed by development with the electrochemi-luminsence reagent (Invitrogen, Carlsbad, CA, USA) and exposure to Hyperfilm (Amersham, Arlington Height, IL, USA). The data were analyzed by Fujifilm Las-4000 system (Fujifilm Co. Tokyo, Japan).

Statistical analysis. Values are presented as the mean±SEM. Statistical analysis of differences between two groups was performed using Student's t-test. Statistical comparison of more than two groups was performed using one-way analysis of variance (ANOVA) with Bonferroni's post hoc test. In all cases, p<0.05 was considered significant.

Results

Effects of pipoxolan on cell viability of human TW206, HSC-3 and Cal-27 cells. In the MTT assay, the relative cell viability at different concentrations of pipoxolan after 24 h was measured. Pipoxolan displayed cytotoxicity toward OSCC (TW206, HSC-3) and HNSCC (Cal-27) cells. The IC50 values for TW206, HSC-3 and Cal-27 were 13.13 μg/mL, 42.28 μg/mL and 52.69 μg/mL, respectively (Figure 2A and B). TW206 and HSC-3 cell lines were especially sensitive to pipoxolan, further studies were performed on these cell lines.

Effects of pipoxolan on phosphatidylserine externalization of TW206 cells. Treatment of TW206 and HSC-3 cells with 20 μg/mL of pipoxolan induced the translocation of phosphatidylserine from the cell membrane (inner layer) to the outer layer as shown by annexin V/PI double staining assay. Our results demonstrate that apoptosis was detected at 2 h of pipoxolan treatment, however, the highest level was not reached until 6 h of pipoxolan treatment (Figure 2C).

Effects of pipoxolan on the cell cycle in TW206 and HSC-3 cells. To determine whether pipoxolan-induced cytotoxicity involves alterations in cell-cycle progression of TW206 and HSC-3 cells, flow cytometric analyses were conducted. Our results show that pipoxolan (15 μg/mL) caused significant apoptosis of TW206 cells (Figure 3A). Pipoxolan at 20 and 25 μg/mL for 24 h led to an even higher percentage of hypodiploid cells (sub-G1 fraction) than in control cells. The maximal degree of apoptosis in TW206 cells was seen after 24 h of pipoxolan treatment at 25 μg/ml (Figure 3B). Moreover, pipoxolan (20 and 25 μg/ml) treatment for 24 h caused a significant decrease of TW206 cells at G0/G1 phase (Figure 3B). Moreover, pipoxolan induced HSC-3 cell apoptosis in dose- and time-dependent manners (Figure 3D and E). The results show that pipoxolan (25 μg/ml) caused significant apoptosis of HSC-3 cells. Pipoxolan at 50 and 100 μg/ml for 24 h led to a higher percentage of hypodiploid cells than in control cells (Figure 3E). Furthermore, these treatments caused a significant decrease of HSC-3 cells at G0/G1 phase (Figure 3D).

Pipoxolan-induced apoptosis in TW206 cells was accompanied by an increase in ROS production and the loss of MMP. We examined the effects of pipoxolan on ROS production and loss of MMP by using the ROS-specific dye DCFH-DA and mitochondria-specific dye DiOC6, respectively. Although the highest levels were not reached until 6 h, an increase in DCFH fluorescence in TW206 cells was detected of early as 2 h after pipoxolan adminstration (Figure 4A). In order to explore whether oxidative stress is essential for pipoxolan-mediated apoptosis, cells were pre-treated with the free radical scavenger NAC (10 mM). As shown, NAC significantly reduced pipoxolan-induced TW-206 cytotoxicity (Figure 4B). These findings suggest that ROS generation maybe crucial for pipoxolan-induced cell death. A remarkable decrease in MMP was observed in TW206 cells after 1-6 h of exposure to pipoxolan (20 μg/ml) (Figure 4C). Our data showed that pipoxolan triggered apoptosis of TW206 cells through the increase of ROS production and the disruption of MMP.

Figure 2.
Figure 2.

A: Pipoxolan induces apoptosis of human oral squamous cell carcinoma cells. TW206, HSC-3 and Cal-27 cells were treated with pipoxolan at the indicated concentrations for 24 h. Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. Optical density was determined at 570 nm and cell survival data are expressed as a percentage of that relative to the control. B: Cytotoxic effects of pipoxolan on TW206 cells. TW206 cells were was treated with different concentrations of pipoxolan for 24 and 48 h. C: Effect of pipoxolan on phosphatidylserine externalization (annexin V binding) in TW206 cells. TW206 cells were treated with 20 μg/ml pipoxolan for 2, 4 and 6 h. Cells were incubated with annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) followed by flow cytometry.

Pipoxolan-induced apoptosis is mediated through mitochondrial apoptotic pathways in TW206 and HSC-3 cells. We determined whether pipoxolan induces apoptosis through the intrinsic signaling pathway. Pipoxolan treatment at 50 μg/ml caused a time-dependent increase of cytochrome c in HSC-3 cells. Pipoxolan induced the release of cytochrome c from mitochondria and stimulated the cleavage of pro-caspase-9 into 35 kDa active fragments of caspase-9 (Figure 5) and of pro-caspase-3 into 17 kDa fragments of caspase-3 (Figure 5). Finally, PARP was effectively hydrolyzed to the 85-kDa fragment of cleaved PARP in HSC-3 cells (Figure 5). Our data show that pipoxolan treatment caused OSCC apoptosis through the activation of caspases-9, and -3, however, caspases-8 was not involved in pipoxolan-induced apoptosis (data not shown).

Figure 3.
Figure 3.

Determination of the proportion of sub-G1 cells following pipoxolan treatment of TW206 and HSC-3 cells, as determined by flow cytometry. A: TW206 cells were treated with 15, 20 and 25 μg/ml pipoxolan for 24h. B: TW206 cells were treated with 20 μg/mL pipoxolan for 4, 8, 12, 24 and 48 h. C: HSC-3 cells were treated with 25, 50 and 100 μg/ml pipoxolan for 24 h. D: HSC-3 cells were treated with 50 μg/mL pipoxolan for 4, 8, 12, 24 and 48 h. Data are means±SEM of three experiments. *p<0.05, **p<0.01, ***p<0.001, significantly increased versus control. PE-A: Phycoerythrin-A.

We examined whether pipoxolan-induced apoptosis is associated with BCL2 family proteins, which are the important regulator proteins for the activation of the caspase cascade. The expression of the pro-apoptotic factor BAX significantly increased in TW206 and HSC-3 cells after 24 h incubation with 20 and 50 μg/ml pipoxolan, respectively. That of BCL2 significantly decreased after 24 h incubation with 20 and 50 μg/ml pipoxolan treatment in the TW206 and HSC-3 cancer cell lines, respectively (Figure 5).

Figure 4.
Figure 4.

Pipoxolan-induced reactive oxygen species (ROS) generation and apoptosis are blocked by N-acetyl-L-cysteine (NAC). A. TW206 cells were incubated with 20 μg/ml of pipoxolan for 1, 3 and 6 h. The fluorescence of oxidized 2’,7’-dichlorofluorescin was determined by flow cytometry. B: Cells were pre-treated with NAC (10 mM) for 1 h and then incubated with 20 μg/ml pipoxolan for 24 h. C: TW206 cells were incubated with 20 μg/ml of pipoxolan for 1, 3 and 6 h. Mitochondrial membrane permeability potential was measured by using a fluorescent dye, DiOC6, and was determined by flow cytometry. Values represent means±SEM of three-independent experiments each performed in triplicate. *p<0.01, significantly different from the corresponding pipoxolan treatment alone. FITC-A: Fluorescein isothiocyanate-A.

Effect of pipoxolan on PI3K, AKT, and phosphorylated AKT expression in TW206 and HSC-3 cells. To further investigate the involvement of PI3K/AKT, an experiment was conducted to measure the expression of PI3K/AKT signaling upon pipoxolan treatment. Our results demonstrate that incubation of TW206 and HSC-3 cells with pipoxolan (20 and 50 μg/ml) led to a time-dependent decrease of PI3K and pAKT levels. Notably, after incubation for 24 h, pipoxolan had significantly suppressed PI3K and pAKT (Figure 5).

Discussion

Initial treatment for OSCC is surgery, then, radiation with/without chemoradiation is added postoperatively for high-risk patients. Currently, docetaxel, cisplatin-based and 5-fluorouracil are the standard therapeutical regimen for OSCC, however, resistance develops as the treatment continues, causing tumor relapse and treatment failure (24). Hence it is critical to explore other effective agents used in combination with present chemotherapeutic agents to sensitize their therapeutic effect. Pipoxolan could have the potential to be a novel anti-OSCC drug as it contains a dioxolan moiety which has been reported to induce apoptosis of various cancer cells (20, 21). In this study, we explored the role of pipoxolan in cytotoxicity toward OSCC in vitro, and elucidated its underlying mechanisms. Compared to Cal-27 (HNSCC) cells, TW206 and HSC-3 (OSCC) were found to be more sensitive to pipoxolan (Figure 2). Encouragingly, pipoxolan exerted its cytotoxicity by apoptosis of both TW206 (Figure 2C, 3A and B) and HSC-3 cell (Figure 3C and D). Pipoxolan induces the generation of ROS, in turn, triggering mitochondrial dysfunction and activating apoptotic components, leading to apoptosis (Figure 4). The molecular signals suggest that pipoxolan might initiate apoptosis through the intrinsic apoptotic pathway and PI3K/AKT signaling (Figure 5).

Figure 5.
Figure 5.

Pipoxolan affected the levels of the associated proteins of both the intrinsic and apoptotic pathways in TW206 and HSC-3 cells. TW-206 (20 μg/ml) and HSC-3 (50 μg/ml) were treated with pipoxolan for 0, 2, 4, 8, 12, 24 and 48 h. Then cytosolic proteins or whole-cell lysates were prepared and subjected to western blotting. A: Protein expression of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K), phosphorylated protein kinase B (pAKT), B-cell lymphoma 2 (BCL2), BCL2-like protein 4 (BAX), cytochrome c, caspase-9 and caspase-3 were analyzed in TW206 and HSC-3 cells by bestern blotting. β-Actin served as the loading control. The number above each blot indicates the intensity of the protein expression.

Under physiological conditions, the maintenance of a stable intracellular ROS level is essential keeping cell proliferation and redox balance (25). If the redox equilibrium becomes imbalanced, persistent oxidative stress favoring carcinogenesis will develop (26). Moreover, patients suffering from OSCC have been reported to carry higher rates of lipid peroxidation and low antioxidant levels (27). The cell-damaging property of ROS and the increased ROS generation by cancer cells may provide an opportunity to develop the cell killing potential of ROS by using exogenous ROS-stressing agents to increase the intracellular ROS to a toxic level, or the threshold that triggers cell death. Because it has been demonstrated that there is a causal relationship between ROS production and a loss of MMP (28). Evidence supports that ROS-inducing anticancer agents may affect various signaling pathways and genes that modulate carcinogenesis (29). ROS is reported to be involved in the early stages of apoptosis (30), causing a loss of MMP (31). Upon ROS stimulation, cytochrome c is released from mitochondria into the cytoplasm. This leads to the activation of caspase-9, caspase-3, and PARP, resulting in apoptosis (32). Our observations suggest that pipoxolan treatment causes a redox imbalance, leading to overproduction of ROS. Based on the flow cytometric results, we demonstrated that pipoxolan induced ROS level in the OSCC in a time-dependent manner (Figure 4A). Accordingly, ROS seems to be involved in pipoxolan-induced cytotoxicity in oral cancer cells. Meanwhile, our result demonstrate that pipoxolan significantly reduced the MMP in OSCC in a time-dependent manner (Figure 4C). These results suggest that pipoxolan is capable of induction of mitochondrial depolarization, and that MMP loss could be affected directly or indirectly by ROS generation in oral cancer cells. The mechanism accounting for ROS overproduction in OSCC remains uncertain. There are several factors contributing to the increased production of ROS in cancer cells such as oncogenic signals, mitochondrial dysfunction, and active metabolism (33).

Figure 6.
Figure 6.

Hypothetic schematic of signaling transduction for the effects of pipoxolan on oral squamous cell carcinoma cells.

The effects of pipoxolan on TW206 and HSC-3 OSCC cells are through both intrinsic but not extrinsic apoptotic pathways. Many investigators have suggested that FAS/CD95 is involved in the induction of apoptosis in mammalian cells (34). Binding of FAS to the FADD stimulates the recruitment of caspase-8. Caspase-8 then activates caspase-3 and other downstream caspases (35). Our results demonstrated that pipoxolan induced a time-dependent increase of FAS/CD95 proteins (data not shown). However, alteration of caspase-8 protein expression in these cells was not found under pipoxolan treatment (data not shown). This information implies the involvement the intrinsic but not extrinsic apoptotic signaling in both TW206 and HSC-3 cells pathway in the effects of pipoxolan.

BCL2 family, including BCL2 and BAX, play important roles in regulating proteins to activate the caspase cascade (36). The expression of BAX (pro-apoptotic protein) was significantly increased in TW206 and HSC-3 cells after 24 h incubation with 20 and 50 μg/ml pipoxolan, respectively (Figure 5). In contrast, BCL2 (antiapoptotic protein) considerably decreased. Cytochrome c was also increased in both TW206 and HSC-3 cells after 12 h incubation with pipoxolan, respectively (Figure 5). If mitochondria recognize apoptotic signaling, BCL2 family members located on the mitochondrial membrane alter the MMP and trigger the release of cytochrome c (37). Once released into the cytoplasm during the early stages of apoptosis, cytochrome c activates post-mitochondrial caspase cascade, causes the DNA fragmentation and leads to apoptotic cell death (38). As shown, pipoxolan (20, and 50 μg/ml) treatment caused a time-dependent increase in the cytosolic cytochrome c in both TW206 and HSC-3 cells (Figure 5). Following treatment with pipoxolan, the cleavage of pro-caspase-9 and pro-caspase-3 into catalytically active forms were also clearly detected in a time-dependent manner (Figure 5). Our data suggest that pipoxolan activates the intrinsic apoptotic pathway of caspase-9/caspase-3 cascade in both TW206 and HSC-3 cells.

The activation of factors of the PI3K/AKT signaling pathway are noted in many cancer types, and 35-92% of OSCC tumors stain positively for phosphorylation of AKT (39). PI3K, a dimeric enzyme, composes of a regulatory (p85) and a catalytic (p110) subunit. The p85 subunit is anchored to ERBB receptor docking sites, and the p110 subunit is responsible for the phosphorylation and activation of AKT (39). The pAKT is an important factor for the prediction of poor clinical outcome in OSCC, because it is correlated with lymph node metastasis, clinical stage and E-cadherin expression (40). The activation of AKT signaling in OSCC is probably associated with the activation of epidermal growth factors or other factors (41). Inhibition of EGFR signaling appears to be a novel therapeutic strategy for the treatment of OSCC. Currently, the EGFR-directed inhibitor cetuximab is the solely approved targeted therapy for the treatment of OSCC. EGFR status may affect patient response to cetuximab treatment (41). Evidence also suggests that anthracycline therapies can be more effective for treatment of OSCC when combined with inhibitors of the PI3K/AKT pathway (7). Knockdown of fibroblast activation protein inactivated PTEN/PI3K/AKT and its downstream signaling, regulating proliferation, migration, and invasion of OSCC cells (42).

In conclusion, pipoxolan is cytotoxic against human OSCC cells, and this involves caspase-dependent apoptosis by a ROS-mediated mechanism and PI3K/AKT signal transduction. Fuurther study on the synthesis of compounds with dioxolane might develop novel antitumor agents against OSCC cells.

Acknowledgements

This work was supported by Grants from the Ministry of Science and Technology (MOST 103-2320-B-039-026; MOST104-2320-B-039-007) and China Medical University (CMU100-ASIA-18). The Authors also appreciate English assistance from Stephen Hsu, and technical assistance from Hsiao Jun-Han and Sung Jie-Ru.

Footnotes

  • Conflicts of Interest

    The Authors declare no conflicts of interest interest in regard to this study.

  • Received April 10, 2017.
  • Revision received May 1, 2017.
  • Accepted May 2, 2017.

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Pipoxolan Exhibits Antitumor Activity Toward Oral Squamous Cell Carcinoma Through Reactive Oxygen Species-mediated Apoptosis
PEI-YU CHOU, MIN-MIN LEE, SUNG-YUAN LIN, MING-JYH SHEU
Anticancer Research Nov 2017, 37 (11) 6391-6400;
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