1. Introduction
Oral squamous cell carcinoma (OSCC) is the 11th most common malignancy globally, with over 300,000 new cases and 140,000 deaths reported every year [1]. Tobacco use, alcohol, betel nut, and sexually acquired human papilloma virus are risk factors for OSCC [2]. The standard treatment for OSCC relies on surgery, radiotherapy, chemotherapy, and molecular-targeted therapy. However, the five-year survival rate is only 48%, and recurrence occurs in 20% of patients within two to three years [3]. Therefore, to provide better treatment for OSCC, new drugs or strategies are urgently needed.
Phytochemicals have served as a source of chemopreventive and chemotherapeutic agents for centuries [4]. For example, in 1992, paclitaxel from Taxus brevifolia was approved by the U.S. Food and Drug Administration (FDA) for the treatment of patients with recurrent ovarian cancer and breast cancer [5]. Smith et al. reported that vinorelbine tartrate, a vinca alkaloid, is effective as a first-line treatment for advanced non-small-cell lung cancer [6]. Interestingly, in the past decade, increasing attention has focused on cardiac glycosides (CGs) in cancer treatment [7], as many CGs, including bufalin, ouabain, and digoxin, have been reported to suppress tumor cell growth by inducing apoptosis [8,9,10]. Originally, CGs were used for the treatment of congestive heart failure for their ability to block the activity of Na+/K+-ATPase, which has been linked to the selective antiproliferative activity of CGs in tumor cells, without affecting normal cell growth [11,12]. Moreover, a number of antitumor targets have been reported for different CGs (oleandrin: nuclear factor-κB (NF-κB) [13] and signal transducer and activator of transcription (STAT)3 [14]; ouabain, digoxin, and proscillaridin: DNA topoisomerase II [15]; divaricoside: myeloid cell leukemia 1 (Mcl-1) [16], suggesting that individual CG might mediate their antitumor effect through distinct mechanisms. From a clinical perspective, a couple of CGs, including Anvirzel and PBI-05204, have been evaluated for their safety and pharmacokinetic profiles in patients with refractory solid tumors in a phase I clinical trial [17,18].
Strophanthus divaricatus (Apocynaceae) is an indigenous plant found in Taiwan, from which we have isolated and characterized different CGs with potent antitumor activities [16]. As part of our natural product-based drug development effort, we previously demonstrated the unique ability of one of the CGs isolated from this indigenous plant, divaricoside, to induce apoptosis in OSCC cells [16]. In this study, we further investigated the efficacy and antitumor mechanism of another CG, α
2. Results
2.1. α
To investigate the antiproliferative activity of α
2.2. α
Previous studies reported that cardiac glycosides cause cell-cycle arrest in tumor cells [19,20]. We examined the impact of α
2.3. α
To further confirm whether apoptosis was involved in α
2.4. α
Dysregulation of mitogen-activated protein kinases (MAPKs), and signal transducer and activator of transcription (STAT) is correlated with tumorigenesis of multiple types of cancer, including OSCC [21,22,23]. Western blotting showed that α
2.5. Mcl-1 Is Involved in α
Several studies have shown that the antiapoptotic protein Mcl-1 is involved in bufalin- or ouabain-induced apoptosis in cancer cells [10,24]. To evaluate the role of Mcl-1, we examined the effects of α
3. Discussion
Natural products have served as a rich resource, providing medicinal agents with structural complexity for centuries. Recent studies showed that some CGs, including oleandrin, digoxin, and ouabain, possess antitumor activity [25]. Epidemiological studies have shown lower mortality rates in breast cancer patients receiving CG therapy [26]. Frankel et al. reported that digoxin plus trametinib induces a 20% greater response than trametinib alone in patients with metastatic melanomas [27]. In this study, α
Takai et al. found that bufalin inhibits cell-cycle arrest at G1 phase, with downregulation of the expression of cyclin A and cyclin D3 in ovarian cancer cells [28]. Ouabain and cinobufagin have been reported to cause S phase arrest and apoptosis in hepatoma cells [29]. It is well known that the cyclin E/cyclin-dependent kinase 2 (CDK2) complex drives DNA replication through the S phase of the cell cycle [30]. In this study, accumulation of α
In addition to cell cycle arrest, apoptosis is one of the main causes of cell-growth inhibition [34]. Yong et al. reported that oleandrin induces apoptosis through caspase activation in osteosarcoma cells [35]. Evidence shows that CGs induce apoptosis by modulating mitogen-activated protein kinase (MAPK) signaling pathways in cancer cells [36]. For example, strophanthidin downregulated the expression of MAPK kinase (MEK) in hepatoma cells [37]. Yong et al. reported that oleandrin inhibits cell growth by activating p38 in osteosarcoma cells [35]. Our results showed that α
The effect of α
Our data demonstrated that α
4. Materials and Methods
4.1. Reagents, Antibodies, and Plasmids
α
4.2. Cell Culture
Both SCC2095 and SCC4 cells (American Type Cell Culture, human tongue primary tumor) were kindly provided by Professor Susan R. Mallery (The Ohio State University). Cells were cultured in DMEM/F12 (Invitrogen, Carlsbad, CA, USA) with 10% heat-inactivated fetal bovine serum (FBS; Gibco, Grand Island, NY, USA), 100 μg/mL streptomycin, and 100 IU/mL penicillin in a humidified incubator with 5% CO2 and 95% relative humidity at 37 °C.
4.3. Cell Viability Analysis
To assess cell proliferation, cells (5 × 103/200 μL) were seeded in 96-well plates and incubated with 10% FBS medium overnight, and then cells were exposed to DMSO or α
4.4. Western Blot
Cell lysates were prepared with a lysis buffer (50 mM Tris, 150 mM NaCl, 1.0 mM EDTA, 1% sodium deoxycholate, 0.1% Triton X-100, 1 mM PMSF, protease inhibitor cocktail) [48]. Proteins were separated using 8–10% SDS-PAGE gels and transferred to PVDF (Bio-Rad, Herfordshire, UK) membranes. After blocking with skim milk, the transblotted membranes were probed with primary antibodies at 4 °C overnight, followed by secondary antibodies conjugated to horseradish peroxidase at room temperature for 1 h. Protein bands were detected using enhanced chemiluminescence detection kit (Little Chalfont, Buckinghamshire, UK).
4.5. Flow Cytometry
Cells (2 × 105/3 mL) were seeded in six-well plates and treated with α
4.6. Transient Transfection for Overexpression
For overexpression of Mcl-1, cells (2 × 105/3 mL) were seeded in six-well plates and transiently transfected with Myc-DDK-tagged plasmids using Fugene HP (Roche, Basel, Switzerland) according to the manufacture’s protocol [48]. Transfected cells were maintained in culture medium for 24 h, and were treated with DMSO or α
4.7. Reverse Transcriptase-PCR (RT-PCR)
Total RNA was extracted from cells treated with Trizol reagent (Invitrogen) and cDNA was prepared by using the RevertAid First strand cDNA Synthesis kit (Ferments, Thermo Scientific) according to the manufacture’s instruction [50]. The primers used were as follows: Mcl-1 mRNA: (forward) 5′-TGCTTCGGAAACTGGACATC-3′, (reverse) 5′-TAGCCACAAAGGCACCAAAAG-3′ and GADPH mRNA: (forward) 5′-AGGTCATCCCTGAGCTGAACGG-3′, (reverse) 5′-CGCCTGCTTCACCACCTTCTTG-3′.
4.8. Statistical Analysis
All data are presented as mean ± S.E.M. from three independent experiments. The statistical analyses were determined using Student’s t test. Differences were considered significant at * p < 0.05 or ** p < 0.01.
Supplementary Materials
The following are available online at
Author Contributions
J.-R.W. conducted the extraction, isolated α
Funding
This work was supported by grants from the Ministry of Science and Technology (MOST 106-2320-B-110-003-MY3, MOST 107-2313-B-110-003-MY3), the Ministry of Health and Welfare, China Medical University Hospital Cancer Research Center of Excellence (MOHW109-TDU-B-212-010001, MOHW109-TDU-B-212-134026), NSYSU-KMU Joint Research Project (NSYSUKMU 109-P004), China Medical University Hospital (DMR-102-010, DMR-105-016, DMR-109-014), the National Health Research Institutes, Taiwan (NHRI-109A1-CACO-13202002), and the Ministry of Health and Welfare (MOHW 10858, MOHW 10968).
Conflicts of Interest
The authors declare that there are no conflict of interest.
Abbreviations
FBS | Fetal bovine serum |
PBS | Phosphate-buffered saline |
MAPKs | Mitogen-activated protein kinases |
STAT | Signal transducer and activator of transcription |
OSCC | Oral squamous cell carcinoma |
CGs | Cardiac glycosides |
JAK | Janus kinase |
PI | Propidium iodide |
PARP | Poly ADP-ribose polymerase |
ERK | Extracellular signal-regulated kinases |
MEK | MAPK kinase |
MTT | 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide |
PVDF | Polyvinylidene difluoride |
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Figures
Figure 1. Effect of αldiginoside on the viability of oral cancer cells. (A) Chemical structure of αldiginoside. (B) SCC2095 and (C) SCC4 cells. Cells were treated with αldiginoside in 96-well plates for 24 or 48 h, and cell viability was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Points represent means; bars represent standard deviation (S.D.) (n = 3–6). * p < 0.05, ** p < 0.01 relative to the control group.
Figure 2. Effect of αldiginoside on cell cycle and cell cycle-regulating proteins. (A) Cell cycle analysis showed an increase in the sub G1 and S phase cell populations after treatment of SCC2095 cells with αldiginoside, followed by propidium iodide (PI) staining. Three independent experiments were performed; data are presented in (B) as means ± S.D. * p < 0.05, ** p < 0.01 when compared with the control group. (C) Western blotting of lysates of αldiginoside-treated SCC2095 cells showing the phosphorylation and expression of cyclin E and CDC25C. Cells were treated with αldiginoside for 48 h.
Figure 3. Effect of αldiginoside treatment on apoptosis. (A) SCC2095 cells were treated with dimethyl sulfoxide (DMSO) or αldiginoside for 48 h and stained with propidium iodide (PI)/annexin V. (B) The percentage of apoptotic cells (Q2 + Q4) is shown. Cells were analyzed using flow cytometry after staining with fluorescein-conjugated annexin V and PI. Columns represent means; bars represent standard deviations (S.D.) (n = 3). * p < 0.05, ** p < 0.01 when compared with the control group. (C) Levels of caspase-3, caspase-8 activation and poly (ADP-ribose) polymerase (PARP) cleavage of αldiginoside-treated SCC2095 cells.
Figure 4. Phosphorylation/expression of p38, extracellular signal-related kinase (ERK), Janus kinase (JAK)2, and signal transducers and activators of transcription (STAT)3 after αldiginoside treatment of SCC2095 cells.
Figure 5. Effect of αldiginoside treatment on expression of the B-cell lymphoma 2 (Bcl-2) family of proteins. (A) Upper panel, the expression of myeloid cell leukemia 1 (Mcl-1), Bcl-2, Bcl-2 associated X-protein (Bax), and Bcl-2 homologous antagonist/killer (Bak) after 48 h αldiginoside treatment in SCC2095 cells. Lower panel, the Bax/Bcl-2 ratio. Densitometric quantification of the autoradiograms for Bax and Bcl-2 was performed and calculated. (n = 2). (B) Time-dependent effect of αldiginoside treatment on the expression of Mcl-1. (C) Expression of Mcl-1 with 250 nM αldiginoside alone or in combination with 200 nM MG132. (D) Mcl-1 RNA expression measured by RT-PCR in αldiginoside-treated cells. (E) Effect of ectopic Mcl-1 expression after αldiginoside treatment. SCC2095 cells were transfected with control vector or Mcl-1 plasmid for 24 h and treated with 100 nM αldiginoside for 48 h. Whole-cell extracts were subjected to Western blotting. (F) Effect of Mcl-1 overexpression on the viability of SCC2095 cells treated with 100 nM αldiginoside for 48 h. After incubation, cells were analyzed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Columns represent means; bars represent standard deviations (S.D.) * p < 0.05.
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Abstract
We recently isolated a cardiac glycoside (CG), α
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1 Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung 80424, Taiwan; Department of Biotechnology, College of Pharmacy, Kaohsiung Medical University, Kaohsiung 80708, Taiwan; Graduate Institute of Pharmacognosy, College of Pharmacy, Taipei Medical University, Taipei 11042, Taiwan
2 Department of Pharmacy, Kinmen Hospital, Kinmen 89142, Taiwan;
3 Division of Hematology and Oncology, Department of Internal Medicine, China Medical University Hospital, Taichung 40447, Taiwan;
4 Division of Hematology and Oncology, Department of Internal Medicine, China Medical University Hospital, Taichung 40447, Taiwan;
5 Department of Fragrance and Cosmetic Science, College of Pharmacy, Kaohsiung Medical University, Kaohsiung 80708, Taiwan;