Research ArticleExperimental Studies

Over-expression of PDE5 in Oral Squamous Cell Carcinoma – Effect of Sildenafil Citrate

TAKAHIRO IWASAKI, TAKESHI ONDA, HIRONA HONDA, KAMICHIKA HAYASHI, TAKAHIKO SHIBAHARA, TAKESHI NOMURA and MASAYUKI TAKANO
Anticancer Research May 2021, 41 (5) 2297-2306; DOI: https://doi.org/10.21873/anticanres.15005

Abstract

Background/Aim: Phosphodiesterase 5 (PDE5) holds clinical relevance in several pathological states, including lung, breast, and prostate cancer. In this study, we examined PDE5 expression in oral squamous cell carcinoma (OSCC)-derived cell lines and tissues, and the anti-tumour effect of PDE5 inhibitor, sildenafil citrate (SC). Materials and Methods: Cell proliferation, cell invasion, and gap closure assays were performed in six OSCC-derived cell lines upon treatment with varying concentrations of SC. PDE5 expression was determined in primary OSCC tissues by western blotting and immunohistochemistry. Results: Elevated PDE5 expression was observed in all cell lines. A concentration-dependent decrease in cell viability, invasion rate, and migration was observed after SC treatment. A significant correlation (p=0.05) was observed between elevated PDE5 expression and lymphatic infiltration in OSCC tissues. Conclusion: PDE5 plays an important role in carcinogenesis of OSCC, and the specific inhibition of PDE5 may be an effective chemotherapeutic strategy.

Key Words:

Oral cancer includes cancers of the tongue, gingiva, floor of the mouth, buccal mucosa, lips, and other parts of the oral cavity, and ranks sixth among the most commonly occurring types of malignant tumours worldwide (1). As the oral cavity, excluding teeth, is covered with squamous epithelium, approximately 90% of oral cancers are oral squamous cell carcinomas (OSCC), with others including glandular tumours, malignant melanomas and lymphomas, sarcomas, and metastatic cancers (2). Although oral cancer is detected by visual examination of the oral cavity, OSCC is often discovered at an advanced stage (3). The stage at which cancer is diagnosed influences treatment options and has a great impact on patient survival (4). At early stages of cancer, the five-year survival rate averages to more than 80% (5). The survival rate for advanced cancers is less than 50%, whereas for metastatic cases, it decreases to an even lower rate of 39% (1). Therefore, early detection and treatment of OSCC is vital for improving patient survival (6).

Although the carcinogenesis and progression of OSCC are closely linked to the abnormal activation of oncogenes, inactivation of tumour suppressor genes, and epigenetic anomalies, the molecular mechanisms operating therein remain unexplained (7). This has impeded the development of highly sensitive biomarkers for the early detection of OSCC and strategies for early treatment (7). Therefore, it is imperative to elucidate the underlying molecular mechanisms of OSCC and identify novel therapeutic targets to improve the prognosis of OSCC (8).

Recently, malignant tumours with altered expression of phosphodiesterase 5 (PDE5) have been reported. PDE5 cleaves the 3’, 5’-cyclic phosphate moiety of cyclic guanosine monophosphate (cGMP), a intracellular second messenger, to its 5’ nucleotide form, and thereby influences signal transduction in various biological systems (9). Aberrant expression of PDE5 has been reported in lung (10), prostate (11), breast (12), colorectal (13), brain (14), and thyroid cancer (15), and malignant melanoma (16). Furthermore, the antitumor effect of PDE5 inhibitors on several types of malignant tumours and cancer cell lines has been established, indicating that PDE5 may be a useful biomarker for diagnosis and a valuable therapeutic target (9). However, the expression level and role of PDE5 in OSCC remain unclear. In this study, we analysed the expression level of PDE5 in OSCC-derived cell lines and primary OSCC tissues. In addition, the in vitro anti-tumour effect of the PDE5 inhibitor sildenafil citrate was analysed on OSCC-derived cell lines.

Materials and Methods

Cell lines. The six OSCC-derived cell lines used in this study were SAS, KON, OSC-20, HSC-3, HSC-3-M3, and Ca9-22 (Japanese Collection of Research Bioresources Cell Bank, Osaka, Japan). The human epidermal keratinocyte line HaCaT (CLS Cell Lines Service GmbH, Eppelheim, Germany) was used as a control. All cell lines were cultured in 150×20 mm culture dishes (Nunc, Roskilde, Denmark) at 37°C in a 5% CO2 and 95% air environment. Foetal bovine serum (FBS) was purchased from Sigma-Aldrich (St. Louis, MO, USA). SAS, OSC-20, KON, and HaCaT cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM)/F-12 (Sigma-Aldrich) with 10% FBS. HSC-3, HSC-3-M3, and Ca9-22 cells were cultured in minimum essential medium (MEM) (Sigma-Aldrich) containing 10% FBS. Penicillin-streptomycin (Thermo Fisher Scientific, Tokyo, Japan) was added to the culture media at a concentration of 50 IU/ml.

Western blot analysis. Protein expression of PDE5 in the six OSCC-derived cell lines was analysed by western blotting (WB) as described previously (17). HaCaT cells were used as the control. Briefly, cells from each cell line were dissolved in Mammalian Protein Extraction Reagent (Thermo Fisher Scientific) and protease inhibitor cocktail (Wako Pure Chemical Industries, Ltd., Osaka, Japan) for extraction. Total protein was quantified using a Qubit Protein Assay kit (Thermo Fisher Scientific). After electrophoresing 15 μg of protein in a 12% SDS-polyacrylamide gel, the proteins were transferred to a polyvinylidene difluoride membrane. Anti-PDE5 antibody (diluted, 1:500, ab64179; Abcam plc, Cambridge, UK) was used as the primary antibody. Anti- Rabbit IgG, HRP-linked whole Ab (diluted, 1:1000, NA934-1ML; GE Healthcare, Buckinghamshire, UK) was used as the secondary antibody. Anti-β-actin antibody (diluted, 1:1000, No. #4970, Cell Signaling Technology, CA, USA) was used as the internal standard. Bands were detected by enhanced chemiluminescence using West Dura Extended Duration Substrate (Thermo Fisher Scientific). The bands obtained in the western blots were quantified using ImageJ software (18, 19).

Reagents. Sildenafil citrate (SC, Viagra® OD Film, Pfizer, Tokyo, Japan) was used as a PDE5 inhibitor. SC was dissolved in the culture medium in concentrations of 0 μM, 10 μM, and 50 μM (20).

Cell proliferation assay. The cell proliferation assay was performed as previously described (21, 22). Briefly, cells from each OSCC-derived cell line were treated with SC (0 μM, 10 μM, or 50 μM), and the viable cell count was measured 12 and 24 h after treatment using the RealTime-Glo™ MT Cell Viability Assay (Promega Corporation, Madison, WI, USA). Results are presented as percentages relative to the control (0 μM). Five technical replicates and three biological replicates were assayed.

Matrigel cell invasion assay. Cell invasion assays were performed as described previously (21). Briefly, the invasive capacity of each OSCC-derived cell line was measured using a CultreCoat 96-well Basement Membrane Extract-Coated Invasion Assay Kit (Trevigen, Gaithersburg, MD, USA). SC was dissolved in serum-free medium at 0 μM, 10 μM, and 50 μM SC, and 1×105 cells per well were seeded on the surface of the insert chambers. The chambers were filled with culture medium containing 10% FBS, and the kits were incubated at 37°C in an atmosphere containing 5% CO2. After 48 h, cells that migrated to the other side of the membrane were detected using a cell dissociation solution (Calcein-AM). Fluorescence was measured at 480-520 nm. Five technical replicates and three biological replicates were assayed.

Gap closure assay. The gap closure assay was performed as described previously (22). Briefly, each OSCC-derived cell line was seeded into a culture insert (3.0×105 cells/insert; cat. no. 80206; Ibidi GmbH). When cells reached confluency, the inserts were removed to create gaps. The cell sheets were removed by washing with PBS, and cells were treated with SC (0 μM, 10 μM, and 50 μM) and incubated at 37°C in an atmosphere containing 5% CO2. The area of the gaps at each time point was measured using ImageJ software (ver. 1.50; National Institutes of Health) and MRI Wound healing tool (18, 23).

Selection of OSCC tissue samples. The cases were selected in accordance with the selection criteria listed below: 1) examined at the Tokyo Dental College Oral Cancer Centre between April 2010 and March 2016, and histopathologically diagnosed as OSCC, 2) primary cases initially originating in the tongue (ICD-0 codes are C02.0, C02.1, C02.2, C02.3, and C02.9), 3) surgical resection was conducted, but preoperative radiation therapy or chemotherapy was not performed, 4) existing usable formalin-fixed paraffin-embedded tissue samples are available, 5) important clinical records corresponding to the tumour exist. Exclusion criteria were as follows: 1) lack of essential data, 2) lack of sufficient tumour tissue in the paraffin block, and 3) microinvasive carcinoma or carcinoma in situ. Fifty cases were selected that met the above standards, and PDE5 protein expression levels and clinical indices were evaluated. Informed consent was obtained from each patient. This study was conducted with the approval of the Tokyo Dental College Ethics Committee (Approval number I 19-22).

Immunohistochemistry (IHC). IHC was performed as described previously (17). The anti-PDE5 antibody used for IHC was the same as that used in WB (1:100 dilution), and was conducted as previously described at 4°C. The IHC score was calculated using a previously described method (24). The IHC scores for each cancer tissue were compared with that of normal tissue samples proximal to the resection stump. The cut-off value was set as a maximum value of 127.33 for a normal tissue IHC score, with high PDE5 expression indicated by a higher IHC score. Scores were determined by two independent experts who were not informed of the patients’ clinical information.

Statistical analysis. In vitro analysis results were evaluated using ANOVA with student’s t-test and Bonferroni correction (21). The values are represented as mean±SD. All assays were performed in triplicate. Significant differences in PDE5-IHC score and pathological characteristics were evaluated using the Mann–Whitney U-test and Fisher’s exact test (24). Overall survival rates were calculated using Kaplan–Meier analysis (21). All the p-values were two-tailed, and p≤0.05 was deemed statistically significant.

Results

Increased expression of PDE5 in OSCC-derived cell lines WB analysis was performed using six OSCC-derived cell lines and the human epidermal keratinocyte line HaCaT to evaluate the expression status of PDE5. The molecular weight of PDE5 was determined to be 95 kDa. PDE5 protein expression was confirmed to be significantly higher in OSCC-derived cell lines (100%) than in HaCaT cells (Figure 1A and B, p≤0.05).

Figure 1.
Figure 1.

PDE5 protein expression in control epidermal keratinocytes (HaCaT) and oral squamous cell carcinoma (OSCC)-derived cell lines. A) Western blot analysis showed that OSCC-derived cell lines display higher PDE5 expression relative to the control. B) Quantification of PDE5 protein in OSCC-derived cell lines and HaCaT. Elevated PDE5 protein expression was observed in all OSCC-derived cell lines. Assays were performed in triplicate; values are represented as mean±SD, *p≤0.05.

SC decreased cell viability in OSCC-derived cell lines. The in vitro effect of SC, a PDE5 inhibitor, on OSCC-derived cell lines was examined. OSCC-derived cell lines were treated with varying concentrations of SC (0 μM, 10 μM, and 50 μM), and cell viability was evaluated with an MTT Cell Viability Assay kit at 12 h and 24 h after treatment. As shown in Figure 2, a concentration-dependent decrease in cell viability was confirmed 12 and 24 h after SC treatment in all six OSCC-derived cell lines (p≤0.05).

Figure 2.
Figure 2.

Effect of sildenafil citrate (SC) on cell viability. Cell lines A) SAS, B) KON, C) OSC-20, D) HSC-3, E) HSC-3-M3, and F) Ca9-22 were incubated with varying concentrations of SC (0 μM, 10 μM, or 50 μM), and cell viability was measured 12 and 24 h following treatment. Five technical replicates and three biological replicates were assayed, *p≤0.05.

SC decreased invasive capacity of OSCC-derived cell lines. To assess the effect of SC on the invasive capacity of OSCC cells, OSCC-derived cell lines were treated with SC (0 μM, 10 μM, and 50 μM), and invasive capacity was measured 48 h later through an in vitro assay. Five out of six cell lines (83.3%) displayed a concentration-dependent statistically significant decrease in the invasion rate upon SC treatment compared to the control (0 μM) (Figure 3, p<0.05). In the gap closure assay, the six OSCC-derived cell lines were treated with SC (0 μM, 10 μM, and 50 μM), and the closure of gaps was observed over time. The assay was completed when the gap in the corresponding control group (0 μM) was completely closed. In the SAS, OSC-20, HSC-3, HSC-3-M3, and Ca9-22 control groups, the gaps closed completely after 10 h, while the gap closed completely after 30 h in the KON control group. All cell lines treated with SC (10 μM and 50 μM) showed a statistically significant delay in gap closure (Figure 4A-G, p≤0.05).

Figure 3.
Figure 3.

Effect of sildenafil citrate (SC) on invasive capacity. SAS, KON, OSC-20, HSC-3, HSC-3-M3, and Ca9-22 were incubated with SC concentrations of 0 μM, 10 μM, or 50 μM. Fluorescence was measured, 48 h after treatment, to quantify the number of invasive cells. Five technical replicates and three biological replicates were assayed, *p≤0.05.

Figure 4.
Figure 4.

Effect of sildenafil citrate (SC) on invasive capacity and mobility. OSCC-derived cell lines were incubated with SC at varying concentrations (0 μM, 10 μM, or 50 μM), and cell migration was evaluated with a gap closure assay. A) Representative image of gap closure assay (KON). B-G) Width of the gaps at time t = 0 h was set as 100%. Five technical replicates and three biological replicates were assayed, *p≤0.05.

PDE5 protein expression in normal oral and primary OSCC tissue. Compared with normal oral tissue, a high frequency and volume of PDE5 expression was observed in OSCC tissues, similar to that of OSCC-derived cell lines. Among the 50 OSCC tissue samples subjected to IHC staining, 38 samples (76%) showed high expression of PDE5. In contrast, normal tissues showed weak staining for PDE5. Figure 5 (A-C) shows representative images of immunohistochemical staining in normal and primary OSCC tissues. PDE5 protein levels in normal oral and primary OSCC tissues are shown in Figure 5D. PDE5-IHC scores for normal tissues fell within the range 0.33-127.3 (average 46.54), whereas scores for OSCC tissues fell in the range 91.38−267.4 (average 152.68). PDE5 expression status in OSCC tissues and its association with clinical pathological indices is shown in Table I. A statistically significant correlation was observed between PDE5 expression and the presence or absence of lymphatic infiltration. In cases with high PDE5 expression, more lymphatic infiltration was observed (p=0.05). However, when a Kaplan–Meier survival analysis was employed to determine the 5-year overall and disease-free survival rates, and a log-rank test was performed between each of the groups, no statistically significant differences were observed in the overall survival rate (p=0.69) or the disease-free survival rate (p=0.64) (data not shown).

Figure 5.
Figure 5.

Immunohistochemistry (IHC) scores for normal oral tissue and primary OSCC tissue. A) Normal oral tissue (representative image). B) OSCC tissue with low PDE5 expression (representative images). C) OSCC tissue with high PDE5 expression (representative image). Normal magnification, 400×. D) PDE5 IHC scores for normal oral and OSCC tissues. A statistically significant difference was seen between the two groups, *p≤0.05.

View this table:
Table I.

Association of PDE5 and clinicopathological characteristics of oral squamous cell carcinoma patients.

Discussion

The superfamily of mammalian PDE enzymes are divided into 11 families. PDE cleaves cyclic adenosine monophosphate (cAMP) and cGMP into 5’-AMP and 5’-GMP, respectively, and regulates cAMP- and cGMP-mediated signalling (25). Among all the isoforms of PDE, PDE5 is expressed in numerous tissues and can be identified in nearly all cultured cells (26). PDE5 is a major isoform involved in the hydrolysis of cGMP, and its activity is strictly regulated by cGMP (27). cGMP regulates many biological processes, such as cell growth and adhesion, energy homeostasis, neurotransmission, and muscle relaxation (28). In addition, dysregulation of cGMP homeostasis has been observed in various pathological conditions, including cancer (29). cGMP signal transmission plays an important role in the induction of apoptosis and inhibition of cell growth (28). Over-expression of PDE5 has been reported in a number of human cancers, including lung (10), prostate (11), breast (12), colorectal (13), brain (14), and thyroid cancer (15). These findings suggest that PDE5 may play an important role in carcinogenesis, and that increased expression of PDE5 may be an effective indicator of cancer diagnosis and may be therapeutically targeted (30). In fact, a number of in vitro studies have shown that PDE5 inhibitors exhibit an anti-proliferative effect on cancer cell lines, and inhibit their invasive capacity (31). It has also been reported that PDE5 inhibitors have a synergistic effect when used in combination with chemotherapy drugs (32).

SC (Viagra®) is a powerful and selective PDE5 inhibitor routinely used to treat erectile dysfunction and pulmonary arterial hypertension (33). Because SC has a chemical structure similar to cGMP, it competes with cGMP to bind to PDE5, raising cGMP levels and activating protein kinase G, thereby relaxing vascular smooth muscle and increasing blood flow (34). The antitumor effects of SC have been studied by a number of research groups (9). SC directly induces caspase-dependent apoptosis in B chronic lymphocytic leukaemia cells in vitro (35), and may augment endogenous antitumor immunity by inhibiting the function of bone marrow-derived suppressor cells (36). In a rat brain tumour model, SC increased the permeability of the blood-brain tumour barrier, boosting the efficacy of doxorubicin (14). Furthermore, SC enhances the chemotherapeutic effect of doxorubicin in prostate and breast cancer cells without increasing the toxicity of the drug (37, 38). SC may increase the efficacy of chemotherapy drugs such as mitomycin C, doxorubicin, cisplatin, and gemcitabine in bladder and pancreatic cancer cells (38). In addition, SC may augment the cytotoxicity of celecoxib in colorectal cancer, hepatocytoma, glioblastoma, and medulloblastoma (39). However, the inhibition of PDE5 expression by SC in OSCC-derived cell lines and OSCC tissues has not been studied in detail.

This study demonstrated for the first time that PDE5 protein expression is increased at a high frequency in OSCC, and that SC has an antitumor effect on OSCC in vitro. Moreover, a relationship was observed between PDE5 expression status and clinical pathological characteristics. Most of the tissue samples of primary OSCC displayed high PDE5 expression accompanied by lymphatic infiltration, however, in tumours with no lymphatic infiltration, PDE5 expression levels were low (p=0.05). In breast cancer tissues, a significant correlation between enhanced PDE5 expression, tumour grade, disease stage, and lymph node metastasis was observed (12). A significant correlation between age, lymph node metastasis, and distant metastasis has also been reported for thyroid cancer (40). In both reports, tumours displaying high PDE5 expression also tended to be positive for lymph node metastasis. The results of the present study did not confirm a correlation between PDE5 expression and the presence or absence of lymph node metastasis, however, it did confirm a statistically significant correlation with the presence or absence of lymphatic infiltration. The circulatory and lymphatic systems are the main routes by which tumour metastasis occurs (41). Cancer cells derived from the primary tumour infiltrate into the surrounding lymphatic system, and are carried by lymphatic vessels to the lymph nodes. Thus, lymphatic infiltration and metastasis are intricately associated with the formation of metastatic tumours (42). One limitation of this study was the small sample size, which may have affected the interpretation of results. Further studies with a larger sample size may elucidate the significance of PDE5 in OSCC diagnosis, prevention, and development of treatment strategies. However, in vitro analysis showed that compared to epidermal keratinocytes, PDE5 protein expression was significantly higher in all six OSCC-derived cell lines used in this study. Treatment of the six cell lines with PDE5-inhibiting SC resulted in decreased cell viability, invasive capacity, and mobility in all cell lines. This suggests that, as in other malignant tumours, PDE5 may also serve as a molecular target for the diagnosis and treatment of OSCC. The anti-tumour effect of SC must be confirmed by in vivo experiments, and clinical trials in OSCC patients are required. Currently, the use of PDE5 as a therapeutic target for SC is in preclinical stages, and few clinical trials have been completed or are in progress (9). As SC is widely used in the treatment of erectile dysfunction and pulmonary arterial hypertension, the safety and pharmacokinetics of this drug has already been confirmed in humans. Thus, SC holds promise as a candidate for drug repositioning for cancer treatment.

Acknowledgements

The Authors would like to thank Editage (www.editage.com) for English language editing.

Footnotes

  • Conflicts of Interest

    The Authors report no conflicts of interest in relation to this study.

  • Authors’ Contributions

    TI and TO contributed equally to this work. TI, TO and MT were involved in the conception and design of the present study. TI and TO performed experiments, analyzed data and drafted the manuscript. HH, KH, TS and TN interpreted the data and assisted in manuscript preparation. All Authors read and approved the final manuscript.

  • Funding

    The present study was supported by JSPS KAKENHI (grant no. 16K11701 and 19K10366).

  • Received March 10, 2021.
  • Revision received March 30, 2021.
  • Accepted March 31, 2021.

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Over-expression of PDE5 in Oral Squamous Cell Carcinoma – Effect of Sildenafil Citrate
TAKAHIRO IWASAKI, TAKESHI ONDA, HIRONA HONDA, KAMICHIKA HAYASHI, TAKAHIKO SHIBAHARA, TAKESHI NOMURA, MASAYUKI TAKANO
Anticancer Research May 2021, 41 (5) 2297-2306; DOI: 10.21873/anticanres.15005
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