Introduction
Oral cancers which affect the oral cavity and oropharynx represent a significant health concern. Particularly, in certain developing countries, they rank among the top three for cancer incidence [1]. Among oral malignancies, oral squamous cell carcinoma (OSCC) predominates, constituting roughly 90% of cases. Unfortunately, most OSCC diagnoses occur at advanced stages, resulting in the five-year disease-specific survival rate of only 50–60% [2]. The location of OSCC significantly impacts patients’ quality of life, often impairing critical functions such as speech and taste [3]. Despite the advance in conventional treatments, including chemotherapy and radiotherapy, approximately one-third of patients experience recurrence and develop resistance to therapy [4,5]. Therefore, there is a pressing need for novel and optimized treatment approaches.
Ophiopogon japonicus, a traditional Chinese medicine, has drawn attention for its therapeutic properties in managing cardiovascular and inflammatory conditions [6-9]. Its beneficial effects on cardiovascular health include anti-ischemic and anti-arrhythmic activities, platelet aggregation inhibition, and microcirculation enhancement [10,11]. Ophiopogonin D (OPD), a steroidal glycoside derived from O. japonicus, also demonstrated diverse biological activities, including anti-thrombotic and anti-inflammatory effects, as well as antitussive properties [12-15]. However, its potential anticancer activity against OSCC remains unexplored. Here, we present the cellular effects of OPD on cell viability, proliferation, apoptosis, and metastasis in YD38 OSCC cells. OPD strongly inhibited the cell proliferation and metastasis, and induced apoptosis in YD38 cells, suppressing vital proteins in the integrin/matrix metalloproteinases (MMPs)/FAK and AKT/PI3K/mTor pathways.
Materials and Methods
1. Chemicals and reagents
OPD (≥98% of purity) was purchased from Chengdu Biopurify Phytochemicals Co., Ltd.. 3-(4,5-Dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide (MTT), bovine serum albumin were obtained from Sigma-Aldrich. BCA protein assay kit was purchased from Thermo Fisher Scientific. RPMI-1640 media, 10% fetal bovine serum (FBS), trypsin ethylene diamine tetraacetic acid and antibiotic-antimycotic solution were obtained from Welgene. Primary antibodies of cyclins, cdk6, poly ADP ribose polymerase (PARP), MMP-2, MMP-9, fibronectin, p-AKT, p-mTor, and beta actin were provided by Santa Cruz Biotechnology. Integrin α5, p-FAK, cleaved caspase-3, PI3Kβ, PI3K p85, p-PI3K, p-ERK, p-JNK, p-p38 were purchased from Cell Signaling Technology Inc.. Secondary antibodies were from Novus Biologicals.
2. Cell lines and cell culture
YD38 gingival cancer cells (including FaDu hypopharyngeal adenocarcinoma and YD15 tongue cancer) were purchased from Korean cell line bank. Cells were maintained in RPMI- 1640 supplemented with 10% FBS, 100 U/mL penicillin and 100 mg/mL streptomycin and cultured in a humidified incubator with 5% CO2 at 37℃.
3. MTT assay
Cell viability was measured by an MTT assay. Briefly, cells were seeded in 96-well plates (1 × 103 cells/well) and incubated overnight. OPD was treated at various concentrations and incubated for 24 hours or 48 hours at 37℃. After treatment, 10 μL of MTT (5 mg/mL) solution was added into each well and cells were further incubated at 37℃ for 4 hours. Finally, 100 μL of dimethyl sulfoxide was added to each well. The absorbance at 540 nm was measured in each well with a plate reader (Multiskan; Thermo Fisher Scientific).
4. Western blot analysis
The cells were seeded at the density of 5 × 105 cells/mL of medium in 100 mm culture dishes and incubated for 24 hours. Then, cells were treated with OPD (0, 50, 100, 150 and 200 μmol/L) for 48 hours. After that, whole cells were homogenized and the protein concentrations were determined using a BCA kit. The total proteins were separated by running on SDS-PAGE and transferred onto PVDF membrane (Merck Millipore). The PVDF membranes were blocked with 5% skim milk in PBS with Tween-20 for 1 hours and then incubated overnight at 4°C with primary antibodies of target proteins. The primary antibodies and β-actin were diluted in 1:1,000 and 1:2,000, respectively. After washing with PBS-T (phosphate buffered saline containing 0.1% Tween-20) for 15 minutes, the membranes were incubated with corresponding secondary antibodies for 2 hours at room temperature. The signals were detected and analyzed by a Kodak Digital Science Image Station (Bruker BioSpin).
Results
1. OPD selectively exerted cytotoxic effects against YD38 cells
The effects of OPD on cell viability were determined by an MTT assay. YD38 cells treated with various concentrations of OPD (Fig. 1) for 48 hours significantly decreased the percent cell viability at the concentration above 75 μM. On contrary, cells of FaDu and YD15 were not affected by OPD, indicating that OPD selectively exerted cytotoxic effects against YD38 cells (Fig. 2).
2. OPD downregulated the cell cycle regulators
The hyperactivation of cell cycle check points plays an essential role in regulating uncontrolled proliferation in various types of OSCC. In Western blotting results, the overexpressed cell cycle regulators of cyclin A, E, B1, D and cdk6 were dosedependently suppressed by the treatment of OPD (Fig. 3).
3. OPD induced apoptosis on YD38 cells
The effect of OPD on the programed cell death was investigated by detecting the expressions of apoptotic markers, cleaved caspase-3 and PARP (116 and 89 kDa). At the last phase of caspase-dependent apoptotic process, executioner cleaved caspase-3 propagates an apoptotic signal by the enzymatic action on downstream targets, including PARP and other substrates. As show in Fig. 4, OPD strongly activated caspase-3 and PARP dose-dependently with the increased expressions of cleaved caspase-3 and PARP. In addition, OPD dramatically increased the expression of light chain-3B (LC3BII), a microtubule associated protein in autophagy induction, in a dose-dependent manner (Fig. 4).
4. OPD suppressed metastasis through integrin/ MMPs/FAK pathway
The treatment with OPD significantly suppressed the expression of MMP enzymes (MMP-2 and MMP-9), which play significant roles in extracellular matrix remodeling and angiogenesis of tumor generation (Fig. 5). OPD also suppressed the upstream factors of fibronectin and integrin α5, and a downstream regulator of p-FAK, leading to the inhibition of cell adhesion, mobility, and metastasis.
5. OPD inhibited the PI3K/AKT/mTor and JNK pathways
We further investigated whether OPD could affect the signaling pathway of PI3K/AKT/mTor, a major oncogenic signaling cascade implicated in cell proliferation and survival. Our results showed that OPD suppressed the constitutive activation of PI3Kβ and PI3K p85 as well as the PI3K/AKT/mTor phosphorylation in a dose-dependent manner. In addition, in a MAPK signaling pathway, OPD significantly downregulated the phosphorylation of c-Jun N-terminal kinase (JNK) while less affecting the expressions of p-p38 and p-ERK (Fig. 6).
Discussion
In the present study, we investigated the cellular effects and mechanism of OPD on YD38 OSCC. OPD exhibited significant inhibitory effects on cell proliferation and potential cytotoxicity in YD38 cells by interrupting cell cycle and multiple cellular signaling pathways. Among three OSCC cell lines tested, YD38 gingival cancer cells showed the highest sensitivity to OPD in cell viability, suggesting that OPD has selective cytotoxicity towards YD38 cells. This could be beneficial for the treatment of gingival cancer, minimizing undesirable effects on other tissues. Cell cycle progression is regulated by a number of proteins in the cdk/cyclin family. The onset and progression of various types of cancers, including OSCC, are attributed to the overexpression and activation of these regulators [16-18]. Interestingly, in our results, the treatment of YD38 cells with OPD markedly reduced the expression of cyclin A, E, B1, and D, as well as cdk6, thereby inhibiting cell cycle progression. Cellular apoptosis is abnormally dysregulated at various stages in OSCC, particularly at the last phase of apoptosis when executioner caspases are activated to commit programmed cell death [3,19,20]. In this work, we confirmed OPD-induced apoptosis by detecting increased expression of both cleaved caspase-3 and cleaved PARP via Western blotting. This is consistent with an earlier report showing that OPD-induced apoptosis involved intrinsic and extrinsic apoptotic pathways in human breast cancer cells [21]. OPD also inhibited the expression of MMPs in YD38 cells, which play vital roles in the metastasis of cancer [22]. This inhibitory effect on metastasis was further confirmed by the inhibition of fibronectin, integrin α5, and FAK phosphorylation. Interestingly, the inhibition of MMP- 2 and MMP-9 was associated with the strong suppression of p-JNK, not p-p38 or p-ERK, in YD38 cells. This suggests that the inhibitory effects of OPD on metastasis, as well as cytotoxic effect, might be mediated through the JNK pathway. In addition, OPD suppressed the PI3K/AKT/mTor pathway, an intracellular signaling pathway related to cellular quiescence, proliferation, and cancer. OPD’s suppression of this pathway might have caused apoptosis induction as well as cell proliferation inhibition [23]. Most notably, the activation of PI3Kβ and PI3K p85 was markedly suppressed by OPD. Considering that PI3K has been strongly implicated in oral tumorigenesis, as 7% of oral pre-cancer patient samples bear mutations in the PI3K gene [24], the anti-tumor effects of OPD on YD38 gingival cancer cells may have worked through the PI3K/AKT/mTor pathway.
In conclusion, OPD strongly inhibited cell proliferation and metastasis and induced apoptosis in YD38 cells. The cellular effects of OPD were associated with the inhibition of cell cycle regulators, caspase-dependent apoptosis, and cell mobility inhibition by downregulating the integrin/MMPs/FAK pathway and PI3K/AKT/mTor pathways. Thus, OPD could be an effective adjuvant for treating gingival cancer.
