OROncology Reports1021-335X1791-2431D.A. Spandidos10.3892/or.2014.3023or-31-04-1738ArticlesRC-6 ribonuclease induces caspase activation, cellular senescence and neuron-like morphology in NT2 embryonal carcinoma cellsYIANGGIOU-TENG12*TSAIHSIU-FENG1*CHENJER-RONG3*CHOUPEI-LUN45WUTSAI-KUN67LIUHSIAO-CHUN8CHANGWEI-JUNG9LIULIANG-CHIH310TSENGHSU-HUNG11YUYUNG-LUEN7912
Department of Emergency Medicine, Taipei Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, New Taipei 231, Taiwan, R.O.C.
Department of Emergency Medicine, School of Medicine, Tzu Chi University, Hualien 970, Taiwan, R.O.C.
Department of Surgery, China Medical University Hospital, Taichung 404, Taiwan, R.O.C.
Division of Allergy-Immunology-Rheumatology, Department of Internal Medicine, Saint Mary’s Hospital Luodong, Yilan 265, Taiwan, R.O.C.
Department of Internal Medicine, School of Medicine, College of Medicine, Taipei Medical University, Taipei 110, Taiwan, R.O.C.
Division of Renal Medicine, Tungs’ Taichung Metroharbor Hospital, Taichung 435, Taiwan, R.O.C.
The Ph.D. program for Cancer Biology and Drug Discovery, China Medical University, Taichung 404, Taiwan, R.O.C.
Department of Nursing, Taipei Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, New Taipei 231, Taiwan, R.O.C.
Graduate Institute of Cancer Biology and Center for Molecular Medicine, China Medical University, Taichung 404, Taiwan, R.O.C.
School of Medicine, China Medical University, Taichung 404, Taiwan, R.O.C.
Division of General Surgery, Taichung Hospital, Ministry of Health and Welfare, Taichung 403, Taiwan, R.O.C.
Department of Biotechnology, Asia University, Taichung 413, Taiwan, R.O.C.Correspondence to: Dr Yung-Luen Yu, Graduate Institute of Cancer Biology and Center for Molecular Medicine, China Medical University, Taichung 404, Taiwan, R.O.C., E-mail: ylyu@mail.cmu.edu.tw. Dr Hsu-Hung Tseng, Division of General Surgery, Taichung Hospital, Ministry of Health and Welfare, Taichung 403, Taiwan, R.O.C., E-mail: qn3020@yahoo.com.tw
Frog ribonucleases have been demonstrated to have anticancer activities. However, whether RC-6 ribonuclease exerts anticancer activity on human embryonal carcinoma cells remains unclear. In the present study, RC-6 induced cytotoxicity in NT2 cells (a human embryonal carcinoma cell line) and our studies showed that RC-6 can exert anticancer effects and induce caspase-9 and -3 activities. Moreover, to date, there is no evidence that frog ribonuclease-induced cytotoxicity effects are related to cellular senescence. Therefore, our studies showed that RC-6 can increase p16 and p21 protein levels and induce cellular senescence in NT2 cells. Notably, similar to retinoic acid-differentiated NT2 cells, neuron-like morphology was found on some remaining live cells after RC-6 treatment. In conclusion, our study is the first to demonstrate that RC-6 can induce cytotoxic effects, caspase-9/-3 activities, cellular senescence and neuron-like morphology in NT2 cells.
Human NTERA-2 D1 cells (NT2 cells) belong to embryonal carcinoma cells that have cancer and stem cell characteristics (1–4). Therefore, NT2 cells are often used as cell models for cancer therapy and neuron differentiation studies (5–7). Currently, cisplatin, fisetin and nucleoside drugs are applied to NT2 cell treatment. These drugs can activate the MAPK and caspase-dependent pathway resulting in NT2 cell death (8,9). On the other hand, NT2 cells can be induced to differentiate into neuron cells by treatment with differentiation agents including retinoic acid, AraC, DAC, valproic acid and berberine (7,8,10,11). Among these differentiation agents, retinoic acid is generally used for neuron differentiation studies (3,6,12). Previous studies have demonstrated that retinoic acid can induce NT2 cells to give rise to cell aggregation, neuron-like morphology and to express neuronal markers (13–15). Although the mechanisms of retinoic acid-induced neuron differentiation in NT2 cells remain to be elucidated, it has been reported that retinoic acid induced NT2 cells to differentiate into neural cells via Wnt, Nitric oxide and cGMP signal pathways (16,17).
Many ribonucleases have been demonstrated to have anticancer activities (18–21). RC-RNase and onconase are frog ribonucleases purified from Rana catesbeiana and Rana pipiens, respectively. Both belong to the RNase A superfamily (22–25). RC-RNase and onconase exert cytotoxic effects on various cancer cells such as hepatoma, cervical cancer, breast cancer, leukemia, mesothelioma, lung cancer, lymphoma, myeloma and prostate carcinoma (21,26–31). Although the mechanisms of frog ribonuclease-exerted cytotoxicity remain to be elucidated, it is noteworthy that onconase has been used as an anticancer drug in clinical trails (32–35). Previous studies indicated that RC-RNase and onconase exert different cytotoxic effects in different cancer cell types (28,30,31,36–41). In addition, several studies suggested that RC-RNase- and onconase-induced cell cytotoxicity may be related to the caspase cascade and MAPK signal pathway (36,38,42–44). Similar to RC-RNase, RC-6 (Rana catesbeiana ribonuclease-6) is also a frog ribonuclease derived from Rana catesbeiana (29,45). Previous studies demonstrated that onconase and RC-RNase can induce cell death in various cancer cells (21,26–31). However, only few studies showed that RC-6 inhibited cell growth in cervical cancer and hepatoma cells (29,45). Therefore, whether RC-6 can exert anticancer activities in various cancer cell types and the mechanisms of RC-6-induced cytotoxic effects on cancer cells remain unclear. In addition, to date, there are no studies to demonstrate whether frog ribonucleases (RC-RNase, onconase, RC-6) can inhibit cell growth in embryonal carcinoma cells.
Here, RC-6-induced anticancer effects on NT2 cells were investigated. The present study demonstrated that RC-6 can inhibit cell growth and induce caspase-9/-3 cascade activation in NT2 cells. On the other hand, some NT2 cells remained alive after RC-6 treatment. Of note, these remaining live cells displayed cell aggregation and neuron-like morphology similar to retinoic acid-differentiated NT2 cells. However, when we compared RC-6-treated NT2 cells with retinoic acid-treated NT2 cells, neuron marker was found in retinoic acid-treated NT2 cells but not in RC-6-treated NT2 cells. In addition, senescence characteristics were observed in a small fraction of NT2 cells after RC-6 treatment. Taken together, our study is the first to indicate that RC-6 can induce caspase-9/-3 activities and senescence characteristics in NT2 cells and it can also induce cell aggregation, neuron-like morphology in the remaining live cells.
Materials and methodsMaterials
RC-6 was kindly provided by Dr Jaang-Jiun Wang (Division of Pediatric Infectious Diseases, Emory University School of Medicine, Atlanta, GA, USA). p16 and Tau antibodies were purchased from BD Biosciences (San Jose, CA, USA). p21 and p27 antibodies were bought from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Horseradish peroxidase-conjugated secondary antibodies were from Sigma-Aldrich (St. Louis, MO, USA). FITC-conjugated secondary antibody was obtained from Jackson ImmunoResearch (West Grove, PA, USA). Ac-LEHD-pNA (acetyl-Leu-Glu-His-Asp-p-nitroanilide; caspase-9 substrate), Ac-DEVD-pNA (Acetyl-Asp-Glu-Val-Asp-p-nitroanilide; caspase-3-like substrate), and Ac-IETD-pNA (acetyl-Ile-Glu-Thr-Asp-p-nitroanilide; caspase-8 substrate) were purchased from AnaSpec (San Jose, CA, USA). Fetal bovine serum, DMEM, non-essential amino acid, L-glutamine and penicillin/streptomycin were purchased from Gibco-BRL.
Cell lines and cell cultures
The NT2 cell line was obtained from Bioresources Collection and Research Center (BCRC, Hsinchu, Taiwan). NT2 cells were cultured in DMEM supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 IU/ml penicillin/streptomycin, and 0.1 mM non-essential amino acids and maintained at 37°C in a humidified atmosphere containing 5% CO2.
Cell viability assay
Cell viability assay was performed as previously described (31,44). In brief, cells were grown on 6-well cell culture plates overnight. After 24 h, cells were treated with 50 μg/ml RC-6 (experimental group) or without RC-6 (control group). Every 24 h, cells were collected and stained with trypan blue and counted on a hemocytometer.
Caspase substrate cleavage assay
Caspase activities were determined using caspase substrate cleavage assay (31,44). Briefly, cells were lysed with lysis buffer (50 mM Tris-HCl, 120 mM NaCl, 1 mM EDTA, 1% NP-40, pH 7.5) and were then treated with protease inhibitors. Subsequently, cell pellets were cleaned through centrifugation (15,000 × g, at 4°C, for 30 min). Caspase-3, −8 and −9 activities were determined. The working solutions were prepared involving experimental sample (80 μg total protein), 158 μl reaction buffer (20% glycerol, 0.5 mM EDTA, 5 mM dithiothreitol, 100 mM HEPES, pH 7.5), and 2 μl fluorogenic substrate (Ac-LEHD-pNA, Ac-DEVD-pNA or Ac-IETD-pNA). Then, the working solutions were incubated for 8 h at 37°C. The fluorogenic substrate was determined at 405 nm in an ultra-microplate reader (Bio-Tek Instruments). Fold increase in caspase activity was calculated using the following formula: (A405sample− A405control)/A405control.
SA-β-gal activity was assessed as described in a previous study (46). In brief, cells were fixed with 0.5% glutaraldehyde solution for 15 min. Next, cells were treated with 0.02% NP-40 and 0.1% sodium deoxycholate for 15 min. Then, cells were incubated with 1 mg/ml X-gal substrate solution (5-bromo-4-chloro-3-indolyl-bd-galactopyranoside) containing 5 mM potassium ferricyanide and 2 mM magnesium chloride at an acidic pH 6.0 for 16 h under CO2-free and dark conditions.
Western blot assay
Cells were treated with lysis buffer (50 mM Tris-HCl, 120 mM NaCl, 1 mM EDTA, 1% NP-40 and pH 7.5). After centrifugation, (16,000 × g) at 4°C for 10 min, the suspension fraction containing protein was collected. The protein concentration of the cell lysates was measured with a Bio-Rad protein assay kit (Bio-Rad Laboratories) according to the manufacturer’s instructions. Next, proteins were separated by 13.3% SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5% skim milk for 4 h at room temperature then probed with primary antibodies overnight at 4°C. Membranes were washed three times with 0.1% Tween-20 (15 min/every time), then incubated with HRP-conjugated secondary antibody (1:1,000 dilution) for 2 h at room temperature. All proteins were observed using Western Lightning Chemiluminescence reagent plus (Perkin-Elmer, Waltham, MA, USA).
Immunofluorescent assay
Cells were fixed with 4% paraformaldehyde for 15 min then treated with 0.03% Triton X-100 and blocked with 3% BSA. The cells were incubated with Tau antibody overnight at 4°C. After washing three times with PBS (15 min/time), cells were incubated with a secondary goat anti-rabbit FITC antibody at room temperature for 1 h. After washing three times with PBS (15 min/time), the cells were observed under fluorescent microscopy.
Statistical analysis
Data were obtained from four independent triplicate experiments and are presented as the mean values of the chosen triplicate groups. The data are shown as means ± standard deviations (SD).
ResultsCell growth inhibition and caspase-9/-3 cascade are induced in NT2 cells with RC-6 treatment
In the present study, whether RC-6 had antitumor effects on NT2 cells was determined by cell number observation. Following RC-6 treatment, viable cells were counted by using cell viability assay with trypan blue stain under a hemocytometer (31,44). When comparing RC-6-treated cells with control cells, the cell number continuously increased in control cells while cell growth was clearly inhibited in RC-6-treated cells (Fig. 1). In addition, as shown in Fig. 1, there were few remaining live cells after RC-6 treatment on day 7. Next, caspase activation was determined by using substrate cleavage assay (30,36). Our results showed that caspase-9, −8 and −3 were not activated in NT2 cells with RC-6 treatment at day 2, whereas caspase-9 and −3 activities were observed on day 4 (Fig. 2B and C). However, caspase-8 activity was not clearly found in RC-6-treated cells on day 2 and day 4 (Fig. 2A). These results suggested that RC-6 exerts an antitumor effect in NT2 cells and induces cell cytotoxicity related to the caspase-9/-3 cascade pathway.
Senescence characteristics in NT2 cells after RC-6 treatment
Our observations of cell morphology and cell growth of RC-6-treated cells under a microscope showed that a small fraction of NT2 cells had flat phenotype and these cells were not able to proliferate similar to cellular senescence as in previous studies (46–49). In order to investigate whether RC-6 induced cellular senescence, SA-β-Gal assay was performed. Our results showed that some flat, enlarged RC-6-treated cells had SA-β-Gal activity which appeared as blue color inside the cells (Fig. 3B). However, blue color was not found in control cells (Fig. 3A). Thus, RC-6 induced some cells to give rise to cellular senescence. Previous studies indicated that p16, p21 and p27 are related to cellular senescence in senescence cells (49–53). Therefore, these proteins were determined in the present study. Western blotting assay (Fig. 4) demonstrated that p16 and p21 levels were increased in RC-6-treated cells whereas p27 level was not increased in RC-6-treated cells. Hence, RC-6 was able to induce p16 and p21 protein increase and cause cellular senescence in a small fraction of NT2 cells.
Neuron-like morphology in the remaining live cells following RC-6 treatment
After RC-6 treatment for 7 days, most cells died, while few cells survived. These remaining live cells were collected and re-cultured with fresh media under RC-6-free conditions. Notably, the remaining live cells aggregated and presented a neural sphere-like phenotype similar to retinoic acid-differentiated NT2 cells (Fig. 5). Subsequently, the remaining live cells presented a neuron-like morphology (Fig. 6A) similar to retinoic acid-differentiated NT2 cells (Fig. 6B). When comparing the remaining live cells with retinoic acid-differentiated cells, neuron marker Tau only appeared in retinoic acid-differentiated cells (Fig. 6C); however, Tau was not found in the remaining live cells after RC-6 treatment (data not shown). The present study suggests that RC-6 induced NT2 cells to form neuron-like cells whereas these cells did not have neuron function.
Discussion
RC-6 has been reported to exert anticancer effects on human cervical cancer (HeLa) and hepatoma (HepG2) cells (29,45). In the present study, we further demonstrated that RC-6 also exerted anticancer effects on human embryonal carcinoma cells (NT2 cells). The results suggested that RC-6 may exert anticancer effects on various types of cancer, such as the RC-RNase- and onconase-exerted anticancer effects (21,26–31). Although the mechanisms of RC-6-induced cell cytotoxicity on cervical cancer cells has not been studied (29), RC-6-induced cell cytotoxicity in HepG2 cells was related to caspase-9/-3 cascade (45). Here, caspase-9 and −3 activities were found in RC-6-treated NT2 cells similar to RC-6-treated-HepG2 cells. Thus, we considered that the caspase-9/-3 cascade is an important signal pathway in RC-6-induced cell death. RC-6, RC-RNase and onconase are all frog ribonucleases (22–25). RC-RNase and onconase can induce caspase-9 and −3 activities in cancer cells; however, they cannot induce caspase-8 activity in cancer cells (31,36,38,54). In the present study, RC-6 also induced caspase-9 and −3 activities but did not induce caspase-8 activity. Based on these findings, we suggested that frog ribonuclease-induced cell death was related to the caspase-9/-3 cascade but not the caspase-8/-3 cascade.
Retinoic acid induces NT2 cells to differentiate into neuron-like cells, as has previously been reported (2,6,55–57). Numerous studies have demonstrated that cell aggregation and neuron-like morphology appear in retinoic acid-differentiated NT2 cells (13–15). Moreover, caspase activities were observed in NT2 cells during retinoic acid treatment (8,56,58). Here, cell aggregation, neuron-like morphology was also observed in the remaining live NT2 cells after RC-6 treatment. In addition, caspase activities were activated in NT2 cells during RC-6 treatment. Our results indicated that RC-6-treated NT2 cells were similar to retinoic acid-treated NT2 cells. Therefore, we consider that RC-6 and retinoic acid may induce certain similar signal pathways in NT2 cells, whereas these signal pathways are still unknown and remain to be studied in the future. On the other hand, retinoic acid induced neuron marker expression in NT2 cells in the present study as well as in previous studies (4,13). However, neuron markers were not clearly found in RC-6-treated NT2 cells in the present study. A previous study showed that RC-6 having ribonuclease activity could cleave RNAs (29). There is no evidence to show that retinoic acid can induce RNA degradation similar to ribonucleases. Based on these findings, we speculated that neuron marker RNAs may be degraded in NT2 cells during RC-6 treatment; therefore, neuron markers cannot be found in RC-6-treated NT2 cells unlike retinoic acid-differentiated NT2 cells.
Recent studies demonstrated that senescence can be induced in many cell types under various conditions and treatments such as hypoxia, uremia, UVB, radiation, H2O2 and oxidized low-density lipoprotein (59–64). Previous studies indicated that β-galactosidase activity is a major characteristic found in senescence cells and is generally applied to senescence detection (65–67). In addition, many studies have reported that p16 and p21 protein levels are increased in senescence cells (50–53). At present, a small fraction of RC-6-treated NT2 cells can express β-galactosidase activity. Moreover, p16 and p21 protein levels are increased in RC-6-treated NT2 cells. Thus, our results indicated that RC-6 can induce senescence characteristics in NT2 cells. Previous studies demonstrated that p27 protein increase was also found in senescence cells (49,68,69). However, p27 protein levels were not increased significantly in RC-6-treated NT2 cells. Our results indicated that p16 and p21 play more important roles than p27 in RC-6-induced senescence.
In summary, the present study is the first to show that RC-6 can induce embryonal carcinoma cells to give rise to caspase-9/-3 activation, senescence characteristics and neuron-like morphology.
Acknowledgements
The present study was supported by the following grants: NSC99-2320-B-039-030-MY3, NSC99-2632-B-039-001-MY3, NSC101-2321-B-039-004 and NHRI-EX102-10245BI.
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RC-6 inhibits cell growth in NT2 cells. Data were obtained from four triplicate groups and are displayed as means ± SD.
Caspase-9 and −3 activities are induced with RC-6 treatment. (A) Caspase-8, (B) caspase-9 and (C) caspase-3 activities were measured in RC-6-treated cells. Data were determined from four triplicate groups and are displayed as means ± SD.
RC-6 induces senescence-associated β-galactosidase activity. β-galactosidase activity was determined in (A) control and (B) RC-6-treated cells. Note that blue color was found in RC-6-treated cells.
RC-6 induces senescence-associated protein expression. NT2 cells were treated with RC-6 for 0 days (lane 1), 2 days (lane 2), 4 days (lane 3) or 6 days (lane 4). Detection of actin served as internal control. Note that p16 and p27 protein levels were increased, but p27 protein level was not increased.
RC-6-induced neuron-like morphology observed under a phase-contrast microscope. (A) RC-6-treated cells and (B) retinoic acid-differentiated cells displayed similar neuron sphere phenotype. Retinoic acid-differentiated cells served as positive control.
Neuron marker only appeared in retinoic acid-differentiated cells but not in RC-6 induced cells. Neuron marker (Tau) was detected by immunoblotting assay and observed under a fluorescent microscope. (A) RC-6-treated cells and (B) retinoic acid-differentiated cells had similar neuron-like morphology. (C) Tau protein was only found in retinoic acid-differentiated cells. Retinoic acid-differentiated cells served as positive control.