Antimetastatic effects of cordycepin mediated by the inhibition of mitochondrial activity and estrogen-related receptor α in human ovarian carcinoma cells

Cordycepin (3′-deoxyadenosine) is a compound for antitumor, which has been found to exert antiangiogenic, antimetastatic, and antiproliferative effects, as well as inducing apoptosis. However, the association between cancer metastasis and mitochondrial activity in cordycepin-treated ovarian carcinoma cells remains unclear. The 50 and 100 μM of cordycepin inhibits mitochondrial fusion and induces mitochondrial fission, respectively. These suggested that cordycepin showed the down-regulation of mitochondrial function and limitation of energy production. Because of activation of mitochondria and generation of energy are needed in cancer cell migration/invasion. After 24 h treatment, cordycepin suppresses epithelial–mesenchymal transition and migration in ovarian carcinoma cells through inhibiting estrogen-related receptor (ERR)-α. The ERRα is a co-transcription factor for gene expressions associated with mitochondrial fusion. Our results indicate that cordycepin suppresses metastasis and migration of ovarian carcinoma cells via inhibiting mitochondrial activity in non-toxic concentrations, and cordycepin has potential benefits in ovarian cancer therapy.


INTRODUCTION
Ovarian cancer is a common gynecological cancer and is always found in woman worldwide. A high mortality rate is found in ovarian cancer patients when tumor invasion and metastasis. Clinically, onsurgical therapies such as chemotherapy or radiotherapy is always used to treat patients with ovarian cancer [1]. Ovarian cancer could be categorized into three subtypes, including (I) epithelial carcinomas, (II) stromal carcinomas, and (III) germ cell tumors [2], and the epithelial ovarian carcinomas is most found in patients in ovarian cancer cases [3,4]. In addition, this ovarian epithelial tumor cells would result in migration/ invasion through epithelial-mesenchymal transition (EMT) thereby entering into blood steam [5][6][7][8]. Several epithelial markers such as (I) epithelial keratins included E-cadherin, occludins, claudins, and desmoplakin are down-regulated and (II) acquire mesenchymal traits included vimentin, N-cadherin, fibronectin, and α-smooth muscle actin are upregluated while development of EMT in cancer cells, these results will increase metastatic ability [9].
Cordycepin (3′-deoxyadenosine) is an antitumor compound isolated from Cordyceps. Recently, many studies have been reported that cordycepin shows antiangiogenic, antimetastatic, antiproliferative effects and apoptosis induction [10][11][12][13][14]. The association between migration and invasion and mitochondrial activity in cordycepin-treated ovarian carcinoma cells remains unclear, hence, cordycepin was tested for suppressing the migration and invasion of ovarian carcinoma cells and determined the inhibitory effects of cordycepin on the mitochondrial activity and EMT. Moreover, we Research Paper www.impactjournals.com/oncotarget have demonstrated that EMT and mitochondrial fusion induction were involved in metastasis in this study.
In the MTT assay, both mitochondrial morphology and membrane potential are indices for mitochondrial function. Fission and fusion are need to have balance for regulations of cell growth, mitochondrial redistribution, and energy production. These circumstances plays important roles in apoptosis and mitophagy [16]. Data showed that treating with 50 and 100 μM cordycepin changed the mitochondrial distribution and induced mitochondrial fission, respectively ( Figure 2A). Mitochondrial membrane potential is a crucial parameter of mitochondrial function that is used as an indicator of cell health. JC-1 is a lipophilic, cationic dye that can selectively enter mitochondria and reversibly change its color from green to red with increasing membrane potential. In healthy cells with high levels of mitochondria, JC-1 spontaneously forms complexes known as J-aggregates, with intense red fluorescence. By contrast, in apoptotic or unhealthy cells with low mitochondrial membrane potential, JC-1 remains in the monomeric form, which shows only green fluorescence. Figure 2B indicated that 50 and 100 μM cordycepin treatment markedly decreased the mitochondrial membrane potential. (C) The statistical significance was evaluated and showed in mitochondrial membrane potential. Data were shown as mean ± SD (n = 3). www.impactjournals.com/oncotarget

Effects of cordycepin on EMT and mitochondria fusion of OVCAR-3 cells
EMT is a major mechanism involved in cancer metastasis [17], it also triggers cell proliferation and drug resistance [18,19]. Therefore, inhibition of EMT mediated by a novel medicine is a complementary therapeutic method for suppressions of metastasis and chemotherapy resistance [19]. 50 and 100 μM cordycepin treatment for 24 h markedly increased E-cadherin level and decreased vimentin level in OVCAR-3 cells (Figure 3).

Effects of cordycepin on the migration in OVCAR-3 cells
Transcription factor ERRα regulates mitochondrial function and cancer cell viability [22]. ERRα promotes mitochondrial fusion via elevating Mfn-1 and Mfn-2 proteins [21]. In addition, ERRα suppression results in EMT inhibition in breast cancer cells [23]. Metastasis, proliferation, and migration of A549 lung cancer cells were increased when ERRα overexpression [24]. We speculate that cordycepin inhibits mitochondrial activity and EMT in OVCAR-3 cells may due to ERRα regulation. Indeed, ERRα expression was decreased after cordycepin treatment at 100 μM for 24 h ( Figure 5A). We used specific small interfering (si) RNA for ERRα in OVCAR-3 cells to confirm our results ( Figure 5B).
We subsequently investigated the migration of OVCAR-3 cells after ERRα knockdown and cordycepin treatment. As illustrated in Figure 6, cordycepin treatment for 24 h significantly suppressed the migration of OVCAR-3 cells; this result was similar to that obtained after ERRα knockdown. Collectively, the data revealed that cordycepin inhibited EMT and migration in OVCAR-3 cell by inducing mitochondrial fission and suppressing the mitochondrial membrane potential, and then decreasing mitochondrial activity.

DISCUSSION
Because of chemotherapy resistance and metastasis, many complementary and alternative medicine are developed in applications of cancer prevention and therapy. Traditional Chinese medicine is one of treatment for complementary and alternative therapy. Cordycepin is an active compound and has been used in cancer treatment in past clinical study.
Loss of intercellular junctions and raise of cell motility are casue by EMT in cancer cell, thereby resuting in metastasis [25]. The E-cadherin is an adhesion molecules on cell membrane, which expression is going to affect cell junction. A study has reported that reduction of E-cadherin is an target index for first stage of cancer cell metastasis [26,27]. Subsequently, other biomarkers are also change involved in EMT, including N-cadherin, fibronectin, and vimentin [27,28], leading to cancer cell migration and invasion [28][29][30]. We recently reported that EMT upregulation was induced by mitochondrial fusion in ovarian carcinoma cells [21].
Mitochondria undergo fission and fusion change called mitochondrial dynamics, which regulates cell metabolism, survival, and proliferation [31]. Fusion unifies the mitochondrial compartments, whereas fission generates morphologically and functionally distinct mitochondria. Mitochondrial fission often occurs early in an apoptotic event [32] and the autophagic process [33]. Mitochondrial fusion is associated with increased cell survival [34].
Our results suggest that ERRα elevated Mfn-1 and Mfn-2 to induce EMT, resulting in cancer cell migration. and vimentin elevation) in OVCAR-3 cells were inhibited by noncytotoxic concentration of cordycepin (100 μM) treatment. Cordycepin suppressed mitochondrial activity, thereby avoiding EMT and ovarian carcinoma cells migration (Figure 7). This is the first study to confirm the inhibition of metastasis and migration in cordycepin-treated ovarian carcinoma cells. Moreover, our results suggested that cordycepin inhibited mitochondrial activity at noncytotoxic concentrations and suppressed ovarian carcinoma cells migration.

Cell viability
The cytotoxic effect of cordycepin on ovarian carcinoma cells was measured using crystal violet staining assay. The cells were seeded on 24-well plates (3 × 10 4 cells/well) and treated with various concentrations of cordycepin for 24 h. The cells were subsequently washed and stained with 2 g/L of crystal violet in phosphatebuffered formaldehyde for 20 min. Crystal violet bound to the cells was dissolved in 20 g/L of SDS solution, and the corresponding absorbance was measured at 600 nm [16].

MTT assay
The mitochondrial activity of cordycepin in ovarian carcinoma cells was measured using the MTT staining assay. The cells were seeded on 24-well plates (3 × 10 4 cells/well) and treated with various concentrations of cordycepin for 24 h. The cells were reacted with MTT (0.5 mg/mL) for 2 h and then washed, after which the tetrazolium was dissolved with dimethyl sulfoxide, the absorbance was measured at 570 nm.

Western blot
Cells were rinsed and lysed using RIPA buffer with protease and phosphatase inhibitors for 20 min on ice. The cells were then centrifuged at 12,000 ×g for 10 min at 4°C. Protein extracts were resolved through SDSpolyacrylamide gel electrophoresis. The protein bands were electrotransferred to nitrocellulose membranes and treated with enhanced chemiluminescence (ECL) blocking agent (GE Healthcare Bio-Sciences) in saline buffer (T-TBS) for 1 h and then incubated with a primary antibody overnight at 4°C. Subsequently, the membranes were washed three times in T-TBS, and the bound antibodies were detected using appropriate horseradish peroxidase-conjugated secondary antibodies, followed by analysis in an ECL plus Western blotting detection system (GE Healthcare Bio-Sciences).

Mitochondrial morphology and mitochondrial membrane potential
Cells were treated with 250 nM Mitotracker Deep-Red FM (Invitrogen) for 30 min in a serum-free culture medium to determine the mitochondrial morphology. After washed the cells, the nuclei were stained with Hochest 33342 [21]. The mitochondrial morphology was observed using a confocal microscope. Mitochondrial membrane potential was measured by JC-1 staining, and the flourscent intensity was determined through microscopy.

ERRα knockdown
ERRα interference in OVCAR-3 cells was determined using a lipofectamine RNAiMAX transfection reagent, according to the manufacturer's protocols (Invitrogen). The sequence of specific siRNAs for ERRα was purchased from Santa Cruz (Santa Cruz). Cell lysates were subjected to Western blotting with an ERRα antibody to confirm the inhibition of ERRα expression.

Real-time PCR
Total RNA was obtained using the Trizol reagent (Gibco BRL Life Technologies, Inc., Gaithersburg, MD, USA), according to the manufacturer's instructions. Primers were synthesized by MD-Bio Inc. (Taipei, Taiwan). The gene expression level was determined through relative quantitative real-time polymerase chain reaction (CFX Cycler System, Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Statistical analysis
The statistical significance was determined by one-way analysis of variance (ANOVA) using the general linear model procedure of SPSS software (SPSS Institute, Inc., Chicago, IL, USA), followed by ANOVA with Duncan's test. The results were considered to be statistically significant if the p value was < 0.05.

CONCLUSIONS
Cordycepin is an active compound in various Cordyceps spp. and widely used in traditional Chinese medicine. It can considerably inhibit human ovarian carcinoma cells by suppressing EMT and mitochondrial activity through ERRα inhibition. Our results indicate that cordycepin has potential benefits for anti-metastasis and anti-migration in ovarian cancer therapy.