Ginkgolide B Regulates CDDP Chemoresistance in Oral Cancer via the Platelet-Activating Factor Receptor Pathway

Simple Summary The platelet-activating factor receptor (PAFR) is a key molecule that participates in intracellular signaling pathways. It is involved in cancer progression, but the detailed mechanism of its chemosensitivity is unknown. The purpose of the current study was to elucidate the mechanism regulating cisplatin (CDDP) sensitivity through PAFR functions in oral squamous cell carcinoma (OSCC). These results suggest that PAFR is a therapeutic target for modulating CDDP sensitivity in OSCC cells. In addition, we found that ginkgolide B (GB), a specific inhibitor of PAFR, enhanced both CDDP chemosusceptibility and apoptosis. Thus, GB may be a novel drug that could enhance combination chemotherapy with CDDP for OSCC patients. Abstract The platelet-activating factor receptor (PAFR) is a key molecule that participates in intracellular signaling pathways, including regulating the activation of kinases. It is involved in cancer progression, but the detailed mechanism of its chemosensitivity is unknown. The purpose of the current study was to elucidate the mechanism regulating cisplatin (CDDP) sensitivity through PAFR functions in oral squamous cell carcinoma (OSCC). We first analyzed the correlation between PAFR expression and CDDP sensitivity in seven OSCC-derived cell lines based upon cell viability assays. Among them, we isolated 2 CDDP-resistant cell lines (Ca9-22 and Ho-1-N-1). In addition to conducting PAFR-knockdown (siPAFR) experiments, we found that ginkgolide B (GB), a specific inhibitor of PAFR, enhanced both CDDP chemosusceptibility and apoptosis. We next evaluated the downstream signaling pathway of PAFR in siPAFR-treated cells and GB-treated cells after CDDP treatment. In both cases, we observed decreased phosphorylation of ERK and Akt and increased expression of cleaved caspase-3. These results suggest that PAFR is a therapeutic target for modulating CDDP sensitivity in OSCC cells. Thus, GB may be a novel drug that could enhance combination chemotherapy with CDDP for OSCC patients.


Introduction
Oral squamous cell carcinoma (OSCC) is one of the most common cancers in the head and neck regions, constituting approximately 3% of all cancers [1]. Although progress has been made in recent years, the overall 5-year survival rate of OSCC patients remains unsatisfactory, standing at less than 50% [2].
In some cases, chemotherapy is an efficient adjuvant treatment for OSCC patients. However, the emergence of resistance to anti-cancer drugs hampers the curative effect to a large extent [3]. Cisplatin (CDDP) is a platinum-based anti-cancer drug used for a broad range of cancers. However, the severe side effects and frequent chemoresistance often limit its clinical application [4]. Therefore, understanding the molecular mechanisms of CDDP chemoresistance acquisition is critical and essential for improving the therapeutic outcome of OSCC patients.
Platelet activating factor (PAF), synthesized by various types of cells, is implicated in inflammation, carcinogenesis, and tumor metastasis [5,6]. PAF binds and induces biological activities through a unique 7-transmembrane G-protein-coupled receptor, the PAF-receptor (PAFR), which possesses exceptionally high affinity for its ligand [7][8][9]. The PAFR is expressed on the surface of various mammalian cells, including leukocytes, tissue macrophages and cancer cells [10,11]. PAFR expression directly regulates tumor growth via induction of systemic immunosuppressive effects and by positive feed-forward mechanisms. Upregulation of PAFR is also detected in primary tumors as well as tumors metastasizing to lymph nodes [12,13]. PAFR expression is positively correlated with advancing tumor stage, invasiveness, and poor prognosis in several types of cancer [12,13]. PAFR function is also closely related to cancer chemotherapy in epithelial carcinoma [14]. Therefore, we hypothesized here that PAFR regulates the effect of CDDP chemotherapy in OSCC patients.
In the current study, we found that PAFR expression status was involved in CDDP sensitivity in OSCC cells based upon PAFR knockdown experiments. In addition, we found that ginkgolide B (GB), an inhibitor of PAFR, increased the CDDP chemosusceptibility, suggesting that GB may be a novel drug that could enhance CDDP chemotherapy.

Ethics Statement
This study protocol has been approved by the Ethics Committee of the Graduate School of Medicine, Chiba University (approval number, 680).

Cellular Proliferation Assay
The cells were treated with the indicated concentrations of CDDP (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) and/or GB, a PAFR inhibitor (Selleck Chemicals, Houston, TX, USA) for the indicated time periods. Cell viability was determined as previously described [17]. Half-maximal inhibitory concentrations (IC 50 ) values were calculated from semi-logarithmic dose-response curves by linear interpolation.

Apoptosis Assays
The FITC Annexin V Apoptosis Detection Kit I (Becton-Dickinson, Franklin Lakes, NJ, USA) was used to measure apoptosis. Briefly, cells were treated with trypsin and collected. The collected cells were washed, and the cell suspension was adjusted to 1 × 10 5 /100 µL. Annexin V-FITC and PI were added to 100 µL of cell suspension and assayed according to the manufacturer's instructions.

Statistical Analysis
The data were analyzed using an unpaired t-test or one-way analysis of variance (ANOVA), followed by a post hoc Tukey's test (ANOVA with Tukey's multiple comparison test). All statistical analyses were performed with Microsoft Excel (Microsoft, Redmond, WA, USA). The data are expressed as the mean ± standard error of the mean.

PAFR Expression in OSCC Cells
To analyze the expression status of PAFR, the seven OSCC cell lines and HNOKs were subjected to qRT-PCR and immunoblot analyses. PAFR mRNA expression was upregulated significantly (ANOVA with Tukey's multiple comparison test, p < 0.05.) in the four OSCC cells (except HSC-3, Sa3, and Ho-1-u-1) compared with the HNOKs ( Figure 1A). PAFR protein expression was also significantly upregulated in six OSCC cell lines compared with the HNOKs ( Figure 1B).

CDDP Sensitivity of OSCC Cells
To investigate the susceptibility of OSCC cells (Ca9-22, Ho-1-N-1, HSC-2, HSC-3-M3, Sa3, HSC-3, and Ho-1-u-1) to CDDP, we assessed cell viability after treatment with the drug (Figure 2A). Figure 2B shows the IC 50 of OSCC cells for CDDP. These results indicated that Ca9-22 was the most resistant and Ho-1-u-1 was the most sensitive to CDDP. From the results of Figures 1 and 2, the resistance to CDDP in OSCC cells had positive correlations to PAFR expression status. Considering these results, Ho-1-N1 and Ca9-22 cells were selected for further experiments. Representative immunoblot analyses of PAFR protein expression. PAFR protein expression was upregulated in six OSCC cell lines compared to HNOKs. Densitometry data were normalized to αtubulin protein levels. Values are expressed as percentages of HNOKs. Detailed information about the Western blotting can be found at Figure S1 and Table S1. OSCC, oral squamous cell carcinoma; HNOKs, human normal oral keratinocytes.

CDDP Sensitivity of OSCC Cells
To investigate the susceptibility of OSCC cells (Ca9-22, Ho-1-N-1, HSC-2, HSC-3-M3, Sa3, HSC-3, and Ho-1-u-1) to CDDP, we assessed cell viability after treatment with the drug (Figure 2A). Figure 2B shows the IC50 of OSCC cells for CDDP. These results indicated that Ca9-22 was the most resistant and Ho-1-u-1 was the most sensitive to CDDP. From the results of Figures 1 and 2, the resistance to CDDP in OSCC cells had positive correlations to PAFR expression status. Considering these results, Ho-1-N1 and Ca9-22 cells were selected for further experiments.  Values are expressed as percentages of HNOKs. Detailed information about the Western blotting can be found at Figure S1 and Table S1. OSCC, oral squamous cell carcinoma; HNOKs, human normal oral keratinocytes. Representative immunoblot analyses of PAFR protein expression. PAFR protein expression was upregulated in six OSCC cell lines compared to HNOKs. Densitometry data were normalized to αtubulin protein levels. Values are expressed as percentages of HNOKs. Detailed information about the Western blotting can be found at Figure S1 and Table S1. OSCC, oral squamous cell carcinoma; HNOKs, human normal oral keratinocytes.

CDDP Sensitivity of OSCC Cells
To investigate the susceptibility of OSCC cells (Ca9-22, Ho-1-N-1, HSC-2, HSC-3-M3, Sa3, HSC-3, and Ho-1-u-1) to CDDP, we assessed cell viability after treatment with the drug (Figure 2A). Figure 2B shows the IC50 of OSCC cells for CDDP. These results indicated that Ca9-22 was the most resistant and Ho-1-u-1 was the most sensitive to CDDP. From the results of Figures 1 and 2, the resistance to CDDP in OSCC cells had positive correlations to PAFR expression status. Considering these results, Ho-1-N1 and Ca9-22 cells were selected for further experiments.

Effect of PAFR Knockdown on Cell Proliferation
The expression levels of PAFR mRNA and PAFR protein in siPAFR-transfected cells decreased significantly (Unpaired t-test, p < 0.001; n.s., no significant difference) compared with the control cells ( Figure 3A-D). Based upon cellular proliferation assays, there was no significant effect of siPAFR transfection on cell proliferation ( Figure 3E,F).

Effect of PAFR Knockdown on Cell Proliferation
The expression levels of PAFR mRNA and PAFR protein in siPAFR-transfected cells decreased significantly (Unpaired t-test, p < 0.001; n.s., no significant difference) compared with the control cells ( Figure 3A-D). Based upon cellular proliferation assays, there was no significant effect of siPAFR transfection on cell proliferation ( Figure 3E,F). Unpaired t-test; *, p < 0.001; n.s., no significant difference). (C,D) Expression of PAFR protein was markedly reduced by siPAFR transfection compared with control (Ca9-22 (C) and Ho-1-N-1 (D); Unpaired t-test; *, p < 0.001; n.s., no significant difference). Densitometry data were normalized to α-tubulin protein levels. Values are expressed as percentages of control. (E,F) To determine the effect of siPAFR on cellular proliferation, siPAFR-transfected cells were seeded in 96-well plates at a density of 1 × 10 4 cells/well, followed by an assessment of cellular viability. The cells were counted at the indicated times. Cell proliferation after siPAFR transfection was not significantly different  N-1 (B); Unpaired t-test; *, p < 0.001; n.s., no significant difference). (C,D) Expression of PAFR protein was markedly reduced by siPAFR transfection compared with control (Ca9-22 (C) and Ho-1-N-1 (D); Unpaired t-test; *, p < 0.001; n.s., no significant difference). Densitometry data were normalized to α-tubulin protein levels. Values are expressed as percentages of control. (E,F) To determine the effect of siPAFR on cellular proliferation, siPAFR-transfected cells were seeded in 96-well plates at a density of 1 × 10 4 cells/well, followed by an assessment of cellular viability. The cells were counted at the indicated times. Cell proliferation after siPAFR transfection was not significantly different from controls (Ca9-22 (E) and Ho-1-N-1 (F)). Detailed information about the Western blotting can be found at Figure S2 and Table S2.

Effect of PAFR Knockdown on CDDP Sensitivity
We investigated CDDP susceptibility in siPAFR-transfected cells ( Figure 4A,B). After siPAFR transfection, cells were treated with CDDP (0-1000 µM) for 48 h. siPAFR-transfected cells had higher susceptibility to CDDP than control cells ( Figure 4C,D; Unpaired t-test; *, p < 0.01; **, p < 0.001). These data suggested that PAFR expression was involved in the regulation of CDDP susceptibility in OSCC cells. To further investigate apoptosis of OSCC cells after CDDP treatment, we performed flow cytometric analysis. The apoptosis frequency of siPAFR-transfected OSCC cells was higher than that of the control group ( Figure 5A,B; Unpaired t-test; *, p < 0.05; **, p < 0.01). Since the activation of ERK and Akt signaling pathways are common attributes of apoptosis or survival [19], we investigated ERK and Akt activation in siPAFR-transfected OSCC cells after treatment with CDDP. We also investigated cleaved caspase-3 to confirm apoptosis. Decreased ERK and Akt phosphorylation was observed in siPAFR-transfected Ca9-22 and Ho-1-N-1 cells after treatment with CDDP. In addition, highly cleaved caspase-3, an apoptosis marker, was observed in both Ca9-22 and Ho-1-N-1 cells after treatment with CDDP ( Figure 5C).

Effect of GB on Cell Proliferation
We examined the effect of GB on cell growth in OSCC cells by cellular proliferation assay. After treatment with 200 µM GB for the indicated duration, no effect on cell proliferation of OSCC cells was found ( Figure 6A,B).

Effect of PAFR Knockdown on CDDP Sensitivity
We investigated CDDP susceptibility in siPAFR-transfected cells ( Figure 4A,B). A siPAFR transfection, cells were treated with CDDP (0-1000 µM) for 48 h. siPAFR-tra fected cells had higher susceptibility to CDDP than control cells ( Figure 4C,D; Unpai t-test; *, p < 0.01; **, p < 0.001). These data suggested that PAFR expression was invol in the regulation of CDDP susceptibility in OSCC cells. To further investigate apopto of OSCC cells after CDDP treatment, we performed flow cytometric analysis. The ap tosis frequency of siPAFR-transfected OSCC cells was higher than that of the con group ( Figure 5A,B; Unpaired t-test; *, p < 0.05; **, p < 0.01). Since the activation of E and Akt signaling pathways are common attributes of apoptosis or survival [19], we vestigated ERK and Akt activation in siPAFR-transfected OSCC cells after treatment w CDDP. We also investigated cleaved caspase-3 to confirm apoptosis. Decreased ERK a Akt phosphorylation was observed in siPAFR-transfected Ca9-22 and Ho-1-N-1 cells a treatment with CDDP. In addition, highly cleaved caspase-3, an apoptosis marker, w observed in both Ca9-22 and Ho-1-N-1 cells after treatment with CDDP ( Figure 5C).   (C) Immunoblot analysis of the phosphorylation of ERK, phosphorylation of Akt and cleaved caspase-3. Decreased ERK and Akt phosphorylation was observed in siPAFR-transfected cells in both Ho-1-N-1 and Ca9-22 after treatment with CDDP. In addition, highly cleaved caspase-3, an apoptosis marker, was observed in both Ca9-22 and Ho-1-N-1 after treatment with CDDP. α-tubulin protein levels were used as loading control. Detailed information about the Western blotting can be found at Figure S3 and Table S3. CDDP, cisplatin.

Effect of GB on Cell Proliferation
We examined the effect of GB on cell growth in OSCC cells by cellular proliferation assay. After treatment with 200 µM GB for the indicated duration, no effect on cell proliferation of OSCC cells was found ( Figure 6A,B). (C) Immunoblot analysis of the phosphorylation of ERK, phosphorylation of Akt and cleaved caspase-3. Decreased ERK and Akt phosphorylation was observed in siPAFR-transfected cells in both Ho-1-N-1 and Ca9-22 after treatment with CDDP. In addition, highly cleaved caspase-3, an apoptosis marker, was observed in both Ca9-22 and Ho-1-N-1 after treatment with CDDP. α-tubulin protein levels were used as loading control. Detailed information about the Western blotting can be found at Figure S3 and Table S3. CDDP, cisplatin.  N-1 (B)). GB, ginkgolide B; OSCC, oral squamous cell carcinoma.

Effect of GB on CDDP Sensitivity
First, we assessed the capability of GB as a specific inhibitor of PAFR and found that PAF-induced IL-1β expression significantly decreased after treatment with GB (Unpaired t-test, p < 0.001, Figure S5). Cellular viability assay, flow cytometry and immunoblot anal-  N-1 (B)). GB, ginkgolide B; OSCC, oral squamous cell carcinoma.

Effect of GB on CDDP Sensitivity
First, we assessed the capability of GB as a specific inhibitor of PAFR and found that PAF-induced IL-1β expression significantly decreased after treatment with GB (Unpaired t-test, p < 0.001, Figure S5). Cellular viability assay, flow cytometry and immunoblot analyses were performed to investigate the effect of CDDP plus GB combination therapy on OSCC cells ( Figure 7A,B). The CDDP sensitivity of OSCC cells increased in a GB dosedependent manner ( Figure 7C,D, Unpaired t-test, p < 0.05). The apoptosis frequency of the cells treated with CDDP plus GB treatment was higher than that of treatment with CDDP alone (Figure 8A,B; Unpaired t-test; *, p < 0.05; **, p < 0.01; n.s., no significant difference). We next investigated downstream signaling of PAFR after CDDP and GB treatments. In both Ca9-22 and Ho-1-N1 cells, reduced phosphorylation of ERK and Akt and highly cleaved caspase-3 were observed in cells treated with CDDP plus GB combination therapy ( Figure 8C).   ; Unpaired t-test; *, p < 0.05; **, p < 0.01; n.s., no significant difference). (C) Immunoblot analysis of the phosphorylation of ERK, phosphorylation of Akt, and cleaved caspase-3. In both Ca9-22 and Ho-1-N1, reduced phosphorylation of ERK and Akt and highly cleaved caspase-3 were observed in cells treated with CDDP plus GB combination therapy. α-tubulin protein levels were used as loading control. Detailed information about the Western blotting can be found at Figure S4 and Table S4. CDDP, cisplatin; GB, ginkgolide B; OSCC, oral squamous cell carcinoma.

Discussion
CDDP is a widely used chemotherapeutic drug used for cancer, including OSCC [20] however, the acquisition of CDDP resistance markedly restricts its application. Since the severe side effects and chemoresistance are important factors for CDDP chemotherapy for OSCC patients [21], an agent that reduces such complications is required. In the present study, we found that the suppression of PAFR by siRNA (Figures 4 and 5) and GB ( Figures  7 and 8) enhanced the CDDP sensitivity of OSCC cells. Furthermore, our data indicated that ERK and Akt signaling, downstream of PAFR, may be key pathways in CDDP treatment ( Figures 5 and 8).

Discussion
CDDP is a widely used chemotherapeutic drug used for cancer, including OSCC [20] however, the acquisition of CDDP resistance markedly restricts its application. Since the severe side effects and chemoresistance are important factors for CDDP chemotherapy for OSCC patients [21], an agent that reduces such complications is required. In the present study, we found that the suppression of PAFR by siRNA (Figures 4 and 5) and GB (Figures 7 and 8) enhanced the CDDP sensitivity of OSCC cells. Furthermore, our data indicated that ERK and Akt signaling, downstream of PAFR, may be key pathways in CDDP treatment (Figures 5 and 8).
In various cancers, PAFR overexpression accelerates cell proliferation, migration, and invasion relative to control cells [12,13,[22][23][24][25][26][27][28]. In addition, high-PAFR tumors showed significantly decreased overall survival compared to low-PAFR tumors [20,21]. These studies suggested that tumors' PAFR expression levels are closely related to not only tumor progression but also cancer prognosis. The PAFR signaling pathway has been shown to activate ERK and Akt, both of which mediate important signals for cell proliferation, survival, and differentiation in several types of cancer cells [29,30]. Similarly to the previous data [31,32], our study demonstrated that the inducible activation of the ERK and Akt pathways is associated with chemotherapy resistance in OSCC cells (Figures 5C and 8C).
Ginkgolides have been isolated from Ginkgo biloba, a Chinese herb, and used in traditional Chinese medicine for thousands of years [33]. Currently, ginkgolides are used for analgesia, suppression of wheezing, and treatment of cerebrovascular disease, coronary artery disease, and hypertension [34]. Ginkgolides, including ginkgolide A, B, C, J, K, L, and M were found to be specific and selective antagonists of PAFR. Of them, GB has the most potent inhibitory effect on PAFR [35,36]. GB has many beneficial characteristics, such as anti-inflammatory properties, as well as anti-allergic, antioxidant, and neuroprotective effects. Thus, it offers significant therapeutic actions in many diseases [37]. To date, no serious side effects directly attributable to GB have been reported [38,39].
We focused on GB as a selective inhibitor of PAFR, and we investigated the effect of GB on CDDP sensitivity in OSCC. To clarify whether GB enhances CDDP sensitivity to Ca9-22 and Ho-1-N-1 cell lines, cell viability assays, flow cytometric analyses, and immunoblot analyses were performed. The results showed that CDDP combined with GB reduced cell viability and increased cell apoptosis, while GB alone had no such impact (Figures 7 and 8). Side effects of anticancer drugs, especially CDDP, have become a serious problem. Our data indicated that GB not only enhanced the sensitivity to CDDP but also achieved the same efficacy with lower doses of CDDP for the patients of several types of cancer.

Conclusions
These results suggest that PAFR is involved in the regulation of CDDP sensitivity in OSCC. In addition, GB increased CDDP sensitivity through its effects on the PAFR signaling pathways. Our results suggest that GB may have therapeutic efficacy when used in combination with CDDP in OSCC.

Supplementary Materials:
The following are available online at https://www.mdpi.com/article/ 10.3390/cancers13246299/s1, Figure S1: Full-length blots of Figure 1B, Figure S2: Full-length blots of Figure 3C,D, Figure S3: Full-length blots of Figure 5C, Figure S4: Full-length blots of Figure 8C, Figure S5: Confirmation of GB inhibition, Table S1: Densitometry readings/intensity ratio of each band for blots of Figure 1B, Table S2: Densitometry readings/intensity ratio of each band for blots of Figure 3C,D, Table S3: Densitometry readings/intensity ratio of each band for blots of Figure 5C, Table S4: Densitometry readings/intensity ratio of each band for blots of Figure 8C.
Author Contributions: Project administration, K.U. and A.K.; conducted experiments, K.K. and T.A.; biochemical analyses, K.K., T.N. and R.N.; data analysis, K.U., K.K., A.K., M.I., T.S. and T.A.; wrote the manuscript, K.U., A.K. and K.K. All authors have read and agreed to the published version of the manuscript. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.
Data Availability Statement: All relevant data are within the paper and its Supplementary Materials.

Conflicts of Interest:
The authors declare no conflict of interest.