Abstract
Background/Aim: Previous studies have indicated that hyperforin inhibits tumor growth of hepatocellular carcinoma. However, the anticancer effects of hyperforin in non-small cell lung cancer (NSCLC) are ambiguous. The aim of the present study was to investigate the anticancer effect of hyperforin in NSCLC. NSCLC CL1-5-F4 cells were treated with different concentrations of hyperforin or NF-κB inhibitor (QNZ) for different time periods. Materials and Methods: Change of cell viability, NF-κB activation, apoptotic signaling pathways, expression of anti-apoptotic proteins, and cell invasion were detected using the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, NF-κB reporter gene assay, flow cytometry, western blotting, and cell invasion assay. Results: The results demonstrated that hyperforin significantly promotes extrinsic and intrinsic apoptotic pathways, and inhibits cell viability and NF-κB activation. In addition, results also indicated that blockage of NF-κB activation reduces the levels of anti-apoptotic proteins and cell invasion in CL1-5-F4 cells. Conclusion: These results suggested hyperforin induces apoptosis and inhibits NF-κB-modulated anti-apoptotic and invasive potential in NSCLC.
Globally, lung cancer is the leading cause of cancer-related death, and non-small cell lung cancer (NSCLC) is the most common type of lung cancer which occupies 85% (1, 2). Approximately 70% of lung cancer patients have locally advanced or metastatic disease at the time of diagnosis (3). Even after common treatment strategy such as curative resection, chemotherapy and radiotherapy, 5-year overall survival remains relative poor (4). Therefore, development of potential anticancer agents is critical for patients with NSCLC.
Tumor progression was activated by multiple intracellular signaling pathways. Currently, treatment target of NSCLC is focused on specific molecules which modulate tumor progression (5). Nuclear factor-kappaB (NF-κB), the transcriptional factor that regulates expression of a number of oncogenes, plays an essential role in NSCLC progression (6). Active NF-κB induces expression of anti-apoptotic proteins, such as X-linked inhibitor of apoptosis protein (XIAP), myeloid leukemia cell differentiation protein 1 (MCL-1) and Cellular FLICE (FADD-like IL-1β-converting enzyme)-inhibitory protein (c-FLIP) (7). Ferreira et al. suggested that XIAP expression is a prognostic marker in NSCLC (8). MCL-1 is also identified as a critical factor for NSCLC survival (9). Moreover, c-FLIP expression is an indicator of NSCLC with poor prognosis. In addition, sustained activation of NF-κB has been associated with increasing NSCLC invasive ability (10). Among these, the alteration of NF-κB-mediated anti-apoptotic mechanism and invasion capacity are the sticking point of NSCLC therapy design.
Hyperforin, a major lipophilic constituent extracted from plant Hyperforin perforatum L., has been used for centuries as a treatment of depression (11). Besides the anti-depression, anti-inflammation, and antimicrobial effects, hyperforin was recently found to have anti-tumoral potential (12). Hyerforin may inhibit tumor growth by prompted expression of apoptotic proteins in breast cancer and hepatocellular carcinoma (13, 14). Hyperforin triggers pro-apoptotic activities and inhibits expression of anti-apoptotic proteins in leukemic cells from patients with B-cell chronic lymphocytic leukemia (15). In addition to apoptosis induction, hyperforin was also found to have an inhibitory effect on cancer invasive potential (16). However, whether hyperforin showed anti-tumor effect on NSCLC remains vague and the putative underlying mechanism may also need to be elucidated.
Materials and Methods
Chemical reagents and antibodies. Hyperforin and NF-κB inhibitor 4-N-[2-(4-phenoxyphenyl) ethyl] quinazoline-4, 6-diamine (QNZ) were purchased from Sigma-Aldrich (St. Louis, MO, USA) and Selleckchem (Houston, TX, USA), respectively, both of them are prepared as 1 mM stock in Dimethyl sulfoxide (DMSO). Primary antibodies to MCL-1 and cellular FLICE (FADD-like IL-1β-converting enzyme)-inhibitory protein (c-FLIP) were bought from BioVision (Milpitas, CA, USA) and Cell Signaling Technology (Beverly, MA, USA), respectively. Primary antibody to Caspase-8 and X-linked inhibitor of apoptosis protein (XIAP) were obtained from Thermo Fisher Scientific (Fremont, CA, USA). Primary antibodies to β-actin and the antibiotic hygromycin B were bought from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Secondary antibodies were purchased from Jackson ImmunoResearch (West Grove, PA, USA). D-luciferin was bought from Caliper (Hopkinton, MA, USA).
Cell culture. The highly metastatic human lung adenocarcinoma cell line, CL1-5-F4, was provided by Dr. Chia-Lin Hsieh (Taipei Medical University, Taiwan). Culture medium of CL1-5-F4 was Dulbecco's Modified Eagle's Medium (DMEM) and Ham's F-12 Nutrient Mixture and supplemented with 10% fetal bovine serum (FBS), 1% L-glutamine, penicillin, and streptomycin. (FBS), L-glutamine (2 mM), and penicillin (100 U/ml)-streptomycin (100 mg/ml) were obtained from Gibco/Life Technologies (Carlsbad, CA, USA). Cells were incubated at 37°Cin an atmosphere of 5% CO2, 95% (17).
MTT assay. 3-(4, 5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma-Aldrich and prepared as 5 mg/ml stock in phosphate-buffered saline buffer. Cells were plated in 96-well (2×104/well) for 24 hr and treated with various dose of hyperforin or QNZ for 48 hr. After treatment, medium was replaced with MTT solution (1:9=MTT stock: medium) and maintained in the incubator for another four hours. Before ELISA (Tecan Group Ltd., Männedorf, Switzerland) reader analysis, MTT solution was replaced by 100 μl DMSO. Absorbance wavelength of MTT is 570 nm and reference wavelength is 650 nm, blank value was defined as zero (+/− 0.1).
Establishment of CL1-5-F4 NF-κB reporter gene stable clone. 1×106 CL1-5-F4 cells were plated in 6 cm dish to growth for 24 hr before transfection. JetPEI™ transfection agent and NF-κB-luciferrase2 vector (pNF-κB/luc2) were obtained from Polyplus Transfection (Illkirch, Bas-Rhin, France) and Promega (Madison, WI, USA), respectively. The transfection protocol of pNF-κB/luc2 was described in detail in a previous study (18). CL1-5-F4 NF-κB reporter gene stable clones were selected by IVIS 200 Imaging System (Xenogen, Alameda, CA, USA) and named CL1-5-F4/NF-κB-luc2 cells.
NF-κB reporter gene assay. CL1-5-F4/NF-κB-luc2 cells were plated into 96-well (2×104/well) for 24 h and treated with various concentrations of hyperforin or 0.5 μM QNZ for 48 h. After treatment, 100 μl D-luciferin solution (500 μM D-luciferin in 100 μl PBS) was added into each well before image acquisition, photon signal was collected for 1 min by IVIS 200 Imaging System. Cell viability was evaluated by using MTT assay and used to correct relative NF-κB activity (19, 20).
Cell cycle analysis. CL1-5-F4 cells were seeded into 12-well (2×105/well) for 24 h and treated with various concentrations of hyperforin for 48 h. After hyperforin treatment, cells were harvested by centrifugation and fixed by 75% ethanol gently and stored at -20°C overnight. Then, cells were centrifuged and labeled by 40 μg/ml Propidium iodide (PI) dye (contained with 100 μg/ml RNase and 1% Triton X-100) in phosphate-buffered saline buffer (PBS) for thirty minutes in the dark at 37°C. Ultimately, the signal intensity of PI dye in CL1-5-F4 cells were validated by flow cytometry (FACS101, Becton Dickinson FACScan, Franklin Lakes, NJ, USA) (21).
Detection of mitochondria membrane potential (MMP). CL1-5-F4 cells were plated into 12-well plates (2×105/well) for 24 h and treated with various concentrations of hyperforin for 48 h. After hyperforin treatment, cells were harvested by centrifugation and labeled with 4 μM DiOC6 in 500 μl PBS for 30 min at 37°C. Detection of MMP was performed by using flow cytometry as described by Hsu et al. (18).
Caspase-3 and caspase-8 activation analysis. CL1-5-F4 cells were plated into 12-well plates (2×105/well) for 24 h and treated with various concentrations of hyperforin for 48 h. Cells were harvested by centrifugation and labeled with caspase-3 activation probe (1 μl FITC-DEVD-FMK in 300 μl PBS) or caspase-8 activation probe (1 μl Red-IETD-FMK in 300 μl PBS) for 30 min in 37°C incubator, respectively. Labelled cells were harvested, washed and then sent to flow cytometry system. The effect of hyperforin on caspase-3 and caspase-8 activation were finally validated by flow cytometry on FL-1 or FL-2 channel (21).
Western blotting assay. 2×106 CL1-5-F4 cells were plated into 10 cm dishes for 24 h and treated with various concentrations of hyperforin or 0.5 μM QNZ for 48 h. After treatment, protein was extracted from each group by lysis buffer (50 mM Tris-HCl pH 8.0, 120 mM NaCl, 0.5% NP-40, and 1 mM phenylmethanesulfonyl fluoride). Proteins were separated by 10-15% SDS-PAGE, and transferred to 0.2 μm polyvinylidene difluoride membrane. Then PVDF membrane stained by primary and secondary antibodies. Lastly, proteins were identified by ECL reagents (Pierce, Rockford, IL, USA), and the protein was visualized using the ChemiDoc MP Imaging System (Bio-Rad Laboratories, Inc., CA, USA). The level of protein bands was measured by Bio-Rad Image Lab software (19).
Invasion assay. Matrigel and 8 mm pore transwells were purchased from Corning (Tewksbury, MA, USA). Matrigel was coated at upper channel of transwell one day before invasion experiment. CL1-5-F4 cells were treated with different concertations of hyperforin or 0.5 μM QNZ for 48 h and then collected into upper channel of transwell at number of 1×106. Allow 24 h invasion effect of CL1-5-F4 cells, followed with fixation (methanol and acetic acid 3:1) and staining (0.5% crystal violet) step. The light microscope Nikon ECLIPSE Ti-U was used to photograph the invaded cells at ×100. The number of invaded cells was calculated using ImageJ software version 1.50 (National Institutes of Health, Bethesda, MD, USA) (21).
Statistical analysis. Student's t-test was performed in this study to compare the difference between control and hyperforin treatment groups. p-Value smaller than 0.05 was defined as significant difference. Each value in this study was displayed as mean±standard error. Details of each statistical analysis is recorded in the figure legends.
Results
Hyperforin effectively induces cytotoxicity via suppression of NF-κB activation in CL1-5-F4 cells. MTT assay was used to examine the cytotoxicity of hyperforin in CL1-5-F4 cells. As showed in Figure 1A, hyperforin dose-dependently enhanced cytotoxicity of CL1-5-F4 cells. Through NF-κB reporter gene assay, we found that hyperforin also significantly inhibited NF-κB activation in CL1-5-F4 cells (Figure 1B). NF-κB inhibitor, QNZ, was used to examine whether blockage of NF-κB activation inhibits cell growth in CL1-5-F4 cells. Figure 1 C-D shows that QNZ significantly inhibits of tumor growth and NF-κB activation in CL1-5-F4 cells.
Hyperforin triggers apoptosis through induction of extrinsic and intrinsic pathways in CL1-5-F4 cells. After confirming the effect of hyperforin on cell growth and NF-κB activation, we evaluated mechanism of hyperforin-induced apoptosis in CL1-5-F4 cells by using flow cytometry. Caspase-8 activation and loss of MMP are characteristic markers of extrinsic and intrinsic apoptotic pathways, respectively. We found that hyperforin significantly augments caspase-8 activation and loss of MMP as compared to control group (Figure 2A-B). Accumulation of subG1 population and caspase-3 activation are related to apoptosis. Figure 2C-D indicats that hyperforin significantly promotes accumulation of subG1 population and caspase-3 activation by 25-50% and 30-60% as compared to control group, respectively.
Hyperforin suppresses expression of NF-κB-modulated anti-apoptotic proteins in CL1-5-F4 cells. Next, Western blotting assay was used to investigate whether hyperforin alters expression of NF-κB-modulated anti-apoptotic proteins in CL1-5-F4 cells. Protein levels of XIAP, MCL-1, and C-FLIP were diminished after hyperforin treatment (Figure 3A). QNZ also suppressed the expression of XIAP, MCL-1, and C-FLIP (Figure 3B). In sum, both hyperforin and QNZ reduced expression of anti-apoptotic proteins (MCL-1, C-FLIP, and XIAP) by 30-90% compared to control group.
Hyperforin markedly reduces NF-κB-modulated invasive ability of CL1-5-F4 cells. CL1-5-F4 cells are belonged to NSCLC with high metastatic potential (17). We used invasion assay to investigate the effect of hyperforin and QNZ on the invasive ability of CL1-5-F4 cells. Here, our results indicated that hyperforin inhibited CL1-5-F4 cells invasion (Figure 4A). QNZ treatment also showed similar inhibition effect on CL1-5-F4 cells invasion (Figure 4B).
Discussion
Hyperforin has been suggested to have the potential to block the growth of liquid and solid tumor, including leukemia, hepatocellular carcinoma, and breast cancer (11, 13, 14). In these cancer types, multiple apoptotic mechanisms were found to be markedly activated by hyperforin. In addition to the induction of apoptosis, hyperforin has also been suggested to exert anti-invasive and anti-metastatic properties during cancer treatment (16). Regarding NSCLC, the challenge for both clinicians and scientists is how to tailor treatment to achieve the best response and avoid unnecessary toxicities remains. There is no report to discuss the anti-tumor function of hyperforin on any type of lung cancer. In this study, we examined whether hyperforin may also serve as a novel agent for NSCLC therapy.
Although some studies suggested that hyperforin may be functioning on apoptosis pathways, the molecular mechanism of hyperforin on NSCLC has not been defined yet. At the molecular level, in lung cancer, NF-κB, in not only a leading mediator for lung carcinogenesis but also a target for prevention and therapy (6). Gehan H. et al. reported that expression of NF-κB/p65 has a prognostic value and impact on survival of NSCLC patients, and that it is also a suitable target for development of new lung cancer therapies (22). The NF-κB reporter gene assay applied in this study indicated that hyperforin significantly reduces NF-κB activation in CL1-5-F4 cells (Figure 1B). Furthermore, the NF-κB inhibitor (QNZ) confirmed the effect of hyperforin indicating that blockage of NF-κB activation is critical anti-growth factor in NSCLC (Figure 1C-D). In extrinsic apoptotic mechanisms, death signal is delivered from Fas-associated death domain to caspase-8, which activates downstream effector caspases (23). Increased caspase-8 activation was found in hyperforin treated groups by flow cytometry (Figure 2A). NF-κB mediated anti-apoptotic proteinsc-FLIP which negatively alters caspase-8 signaling, was also down-regulated by hyperforin (Figure 3A). In intrinsic apoptotic mechanisms, the potential loss of MMP mediates apoptosis (23). Our flow cytometry results indicated that hyperforin triggers the loss of MMP (Figure 2B). Furthermore, MCL-1-mediated impairment of the intrinsic apoptosis pathways was also diminished by hyperforin (Figure 3A). In Figure 2C and 2D, apoptosis activation by hyperforin was also confirmed by increasing subG1 population and caspase-3 activation. We provide evidence that hyperforin eliminated the activation of NF-κB and NF-κB-mediated anti-apoptotic proteins expression, as well as facilitated extrinsic and intrinsic apoptotic signaling transduction.
While one of the important characteristic of tumor progression is invasion capacity, previous study also suggested that NF-κB is an important modulator on this property (6). High capacity of invasion correlates with more aggressive behavior as well as much shorter overall survival in lung cancer patient (24). Therefore, reducing invasion ability may offer benefits in NSCLC therapeutic efficacy. Our (Figure 4B) results indicate that NF-κB inhibition may effectively decrease the number of invaded cells. Most importantly, hyperforin may also act as an invasion inhibitor of NSCLC through inhibition of NF-κB activation (Figure 4A). In conclusion, hyperforin is a novel agent for NSCLC treatment. Hyperforin can not only induces apoptosis in NSCLC, but also successfully decreases the invasive potential of NSCLC.
Acknowledgements
The study was supported by Taipei Medical University and Taipei Medical University Hospital (grant no. grant no. TMU105-AE1-B49 and 105TMU-TMUH-23). This project was also supported by the National Health Research Institutes (MG-105-SP-07, MG-106-SP-07). This study was also funded by Zuoying Branch of Kaohsiung Armed Forces General Hospital, Kaohsiung, Taiwan, R.O.C (grant no. ZBH106-17).
Footnotes
↵* These Authors contributed equally to this study.
Conflicts of Interest
The Authors disclose no potential conflicts of interest.
- Received January 30, 2018.
- Revision received February 20, 2018.
- Accepted February 21, 2018.
- Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved