Abstract
Background/Aim: Hepatocellular carcinoma (HCC) is a highly prevalent disease and treatment is limited. Therefore, development of new therapeutic agents is urgent. The aim of this study was to investigate the in vitro and in vivo anti-cancer effects of Nardostachys jatamansi root extract (NJRE) against HCC and underlying mechanisms involved in such effects. Materials and Methods: Effects of NJRE on viability of HCC cell lines were determined by MTT analysis and annexin/PI apoptosis assays. Expression levels of proteins in MAPK and STAT3 pathways and caspase-3 and PARP after treatment with NJRE in HCC cell lines were determined by western blotting. In a syngeneic model using mouse HCC cells Hepa1-6, inhibition of tumor formation after oral administration of NJRE was determined and expression levels of phospho-ERK and phospho-STAT3 in liver tissues were analyzed by immunohistochemical staining. Results: NJRE reduced the activation of STAT3 by inhibiting the expression of ERK and finally attenuated the proliferation of HCC. Conclusion: NJRE has anti-cancer effects against HCC. It has potential to be used in the treatment of human HCC.
Hepatocellular carcinoma (HCC) is the third- highest cause of cancer-related death and the sixth most common cancer in the world. It has a very poor survival rate (1). HCC is highly prevalent in Southeast Asia and Africa. Its occurrence is also increasing in Western countries (2). The most of HCC progresses as a result of chronic liver damage caused by hepatitis B and hepatitis C, alcohol misuse, exposure to liver toxins, and non-alcoholic steatohepatitis. Currently possible treatment options for HCC include chemotherapy, surgical intervention, radiation therapy, local ablation, and immunooncology drugs. They can be used in the treatment depending on the clinical stage of HCC. However, the efficacy of treatment regimens for HCC remains limited despite significant developments made in chemotherapeutic agents used for treating HCC.
Natural compound-derived drugs have recently been recognized as new sources of anti-cancer drugs and neoadjuvant chemotherapy, enhancing chemotherapeutic efficacy and alleviate its side-effects (3). Among various natural compounds, curcumin, a type of polyphenol, has shown anti-cancer effects by interacting with various cell signaling proteins, cytokine signaling receptors, and growth factors (4). A recent study showed that curcumin can suppress cell proliferation and induce apoptosis in human liver cancer cells when administered alone or in combination with doxorubicin and cisplatin (5). Quercetin, a polyphenolic flavonoid rich in fruits and vegetables, is also known to have anti-oxidant, anti-inflammatory, and anti-cancer effects (6). Quercetin has inhibitory effect against HCC through apoptosis and cell-cycle arrest. Its combination with cisplatin exhibits a synergistic inhibitory effect on cell growth and induces apoptosis (7). Anti-cancer effects of natural compound-derived drugs have been continuously studied and considered an alternative strategy for treating HCC. Nardostachys jatamansi, a Himalayan herb belonging to the valerian family, has since long been used as a medicine. Nardostachys jatamansi root extract (NJRE) has been reported to have anti-cancer effects by inhibiting the growth of cancer cells through MYCN-mediated regulation of mouse double minute 2 homolog (MDM2) and p53 in neuroblastoma (8). Previous studies have shown that it can also inhibit angiogenesis, proliferation, and invasion of cancer cells in neuroblastoma and glioblastoma (9). In addition, recent studies have shown that Nardostachys jatamansi can prevent doxorubicin-induced cytotoxicity (10). It is expected to have a great potential not only due to its anti-cancer effect, but also due to its ability to prevent toxicity of combination drugs. The objective of the present study was to explore the anti-cancer activity of NJRE by studying its biological effects and underlying mechanisms both in vitro and in vivo.
Materials and Methods
Preparation of NJRE. Nardostachys jatamansi roots were purchased from a standard commercial source (Omni Herb, Seoul, Republic of Korea). The identity of the herb was confirmed by the Korean drug test laboratory. Voucher specimens (NO; Oh/wh/nj-43) were deposited at the College of Oriental Medicine Herbarium of Wonkwang University. These roots of Nardostachys jatamansi were then used to prepare NJRE by decocting dried herbs (10 kg) through twice extraction using 80 L of distilled 30% ethanol and refluxing at 86±2°C for 3 h. The extract was filtered, and the liquid in the filtrate was removed using a rotary evaporator and a spray dryer (KEMIMEDI Company, Seoul, Republic of Korea).
Cell culture. Human HCC cell lines (PLC/PRF-5, Huh7, Hep3B, SK-Hep1), mouse HCC cell line (Hepa1-6) and mouse normal hepatocyte (FL83B) were purchased from American Type Culture Collection (ATCC, VA, USA). FL83B and PLC/PRF-5 were cultured in Ham’s F-12K medium (Gibco, MA, USA). Huh7, Hep3B and Hepa1-6 were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Hyclone, UT, USA). SK-Hep1 was cultured in Minimum Essential Medium (MEM, Gibco). All media were supplemented with 10% Fetal Bovine Serum (Invitrogen, MA, USA), 100 μg/ml penicillin, and 0.25 μg/ml streptomycin.
Cell viability assay. Effects of NJRE on cell viability were determined using MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) assay. Briefly, cells (1×106/well) were seeded into a 96-well plate and incubated at 37°C for 12h. After treatment with NJRE (0, 50, 100 and 200 μg/ml) and negative control for 48 h, the viability of cells was determined by MTT assay. After adding 100 μl of MTT assay solution (0.5 mg/ml in a medium) to each well, the plate was incubated at 37°C for 1 h. After incubation, MTT solution in each well was removed and 100 μl of DMSO was added to each well. Absorbance was measured at a wavelength of 570 nm using a microplate spectrofluorometer (Spectramax PLUS 384, Molecular Devices Corp., CA, USA).
Annexin V/propidium iodide (PI) apoptosis assay. Cellular apoptosis assay was performed using an annexin V/PI apoptosis detection kit (BD Biosciences) following the manufacturer’s instruction. Briefly, after treatment with NJRE for 48 h, cells were collected, washed with cold PBS (Corning, MA, USA), and resuspended in 500 μl binding buffer containing annexin V-FITC and PI before analysis with a flow cytometry (BD Biosciences, NJ, USA) and a FlowJo software (TreeStar, Ashland, OR, USA).
Western blot analysis. Proteins were extracted using RIPA lysis buffer (20 mM Tris-HCl at pH 7.5, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, and 0.1% SDS) containing protease inhibitor (Roche, Basel, Swiss) and phosphatase inhibitor cocktail (Sigma, MO, USA). Equal amounts of proteins were loaded onto 8 and 12% SDS-PAGE gel, and transferred onto nitrocellulose membranes (GE Healthcare, WI, USA). Membranes were blocked with 5% skim milk for 1 h at room temperature and incubated with primary antibodies cleaved-PARP, PARP, cleaved-caspase-3, casepase-3, phospho-STAT3 (Serine 727 and Tyrosine 705), STAT3, phospho-p38, p38, phospho-JNK (Cell signaling, MA, USA), JNK, phospho-ERK, ERK, cyclin D1 (Santa cruz, TX, USA), and β-actin (Sigma) overnight at 4°C, followed by incubation with relevant secondary antibodies at room temperature for 2 h. Protein bands were visualized using a chemiluminescent ECL solution (GE Healthcare). Band intensities were analyzed using a Multi Gauge version V3.0 software (Fuji Film, Tokyo, Japan).
Animal studies. All animal care and experimental protocols were performed in accordance with the guidelines for the Care and Use of Laboratory Animals provided by the Research Supporting Center for Medical Science of the Catholic University of Korea (2015-0179-04). To generate a mouse model with HCC, 6-week-old male C57BL/6 mice were injected with 2×106 Hepa1-6 cells into the liver portal vein using an insulin syringe. At 3days after injection of cell, mice were randomly divided into two groups for treatment: 1) mice injected with Hepa1-6 cells were treated with PBS, and 2) mice injected with Hepa1-6 cells received daily oral administration of NJRE (100 mg/kg). Normal mice were also divided into two groups. One group was treated with PBS and the other group was orally administered with NJRE at 100 mg/kg. After 3 weeks, mice were sacrificed by anesthesia with zoletile (50 mg/ml) and xylazine (23.32 mg/ml).
Histological analysis and immunohistochemistry (IHC). Paraffin-embedded blocks were sectioned to a thickness of 5 μm and transferred to silanized glass slides. These slides were then deparaffinized in xylene and rehydrated in a series of concentrations of alcohol. Antigen retrieval was performed by heating samples at 121°C for 15 min in 0.01 M citrate buffer (pH 6.0) using a microwave vacuum histo processor (RHS-1; Milestone, Bergamo, Italy). These slides were incubated with 3% hydrogen peroxide in methanol for 10 min to block endogenous peroxidase activity. They were then incubated with Alfa-fetoprotein (AFP) (Abcam, Cambridge, UK), phospho-STAT3 (Cell signaling), and phospho-ERK (Santa cruz) antibodies diluted at 1:100 in antibody diluent (Dako, Carpinteria, CA, USA) with background-reducing components at 4°C overnight. After washing, the Dako envision Plus system (Dako) was adjusted to room temperature for 30 min, and the immune reaction was performed with diaminobenzidine at room temperature for 1 min 30 sec, followed by hematoxylin staining. The staining area was determined using ImageJ (National Institutes of Health, MD, USA).
Statistical analysis. All results were obtained from three independent experiments. All data are expressed as mean±standard error of the mean (SEM). Comparisons between two groups were performed using two-tailed Student’s t-test. All statistical analyses were performed using GraphPad Prism 6.0 (GraphPad Software, CA, USA). Statistical significance was indicated by *p<0.05, **p<0.01 and ***p<0.001.
Results
Effects of NJRE on cell viability. In a preliminary screening, we initially evaluated four novel natural compounds as potential anti-cancer agents against four HCC cell lines (Hep3B, Huh7, PLC/PRF-5 and SK-Hep1) using cell viability assays and annexin V/PI apoptosis assays. Among these compounds, compound A, an ethanol extract of Nardostachys jatamansi roots, showed the most significant cytotoxicity and apoptotic activity for all four HCC cells (Figure 1). Therefore, we further studied the anti-cancer effect of NJRE on HCC. First, we exposed human HCC cells (Hep3B, Hu7, PLC/PRF-5), mouse HCC cells (Hepa1-6), and mouse normal hepatocytes (FL83B) to NJRE at various concentrations (0, 50, 100, 200 μg/ml) for 48 h and determined cell viability using MTT assay. Median effective doses of NJRE for inhibiting proliferation of Huh7, Hep3B, PLC/PRF-5, and Hepa1-6 cells were 64.16, 91.8, 133.6, and 68.46 μg/ml, respectively. Therefore, NJRE concentrations in the range of 0-200 μg/ml were used in this experiment. As shown in Figure 2, NJRE significantly reduced the viability of HCC cells in a dose-dependent manner. However, no cytotoxicity was observed for FL83B cells at these same concentrations (0-100 μg/ml). These results suggest that NJRE shows potent cytotoxic effect and that it can inhibit the proliferation of HCC cells.
NJRE inhibit HCC proliferation through ERK/STAT3 pathway. Previous studies have shown that expression of MAPK pathway related proteins (ERK, JNK, p38) can regulate the proliferation and apoptosis of HCC cells (11-13). To study the mechanism by which NJRE could inhibit the growth of HCC cells, NJRE at different concentrations (0, 50, 100 μg/ml) was used to treat Huh7 cells for 12 h and expression levels of ERK, JNK, and p38 in MAPK pathways were determined by western blot. Results showed that the expression of phospho-ERK was gradually decreased by half, while expression levels of phospho-p38 and JNK were gradually increased by treatment with NJRE in a dose-dependent manner (Figure 3A). These results suggest that the inhibition of proliferation and the induction of apoptosis of cancer cells by NJRE are mediated through the regulation of MAPK signal pathways. Several reports have shown that ERK can enhance the proliferation of HCC by activating STAT3 (14, 15). Activated STAT3 enters the nucleus and transcribes several genes, including genes such as cyclin D1 involved in the proliferation of HCC (16, 17). To determine whether the activation of STAT3 was affected by NJRE treatment, expression levels of STAT3, phospho-STAT3 (Ser 727 and Tyr 705), and cyclin D1 in Huh7 cells treated with NJRE at different concentration (0, 50, 100 μg/ml) for 12 h were determined by western blot. As shown in Figure 2B, NJRE reduced the expression of phospho-STAT3 (Ser 727) and phospho-STAT3 (Tyr 705) in a dose-dependent manner. We also found that the expression of cyclin D1 was significantly reduced after treatment with NJRE (Figure 3B). Thus, NJRE can inhibit the proliferation of HCC by reducing the ERK/STAT3 signaling pathways.
NJRE promotes HCC cell death via apoptosis. To determine the effect of NJRE on apoptosis of HCC cells, annexin V/PI apoptosis analysis was performed using flow cytometry after Huh7 and Hep3B cells were treated with NJRE at different concentrations (0, 50, and 100 μg/ml) for 48 h. We found that the percentage of apoptotic cells increased after treatment with NJRE in a dose-dependent manner in huh7 cells and Hep3B cells (Figure 4A). We also measured expression levels of caspase-3 and PARP in Huh7 and Hep3B cells by western blot after treatment with NJRE for 48 h. Results demonstrated that NJRE increased the activity of caspase-3 and cleavage of PARP in both Huh7 cells and Hep3B cells (Figure 4B) in a dose-dependent manner. These results indicate that NJRE can induce apoptosis of HCC cells by JNK and p38 MAPK pathway.
Effects of NJRE on HCC in vivo. To investigate whether NJRE could suppress HCC proliferation in vivo, a syngeneic HCC mouse model was established using Hepa1-6 cells. We orally administered NJRE to mice for 3 weeks daily at a concentration of 100 mg/kg. The concentration was determined based on the fact that NJRE at 100 μg/ml, which was the most effective concentration for HCC cells, was not toxic to mouse normal liver cells in vitro (Figure 5A). As a result, we found that tumors in the NJRE-treated group almost disappeared by hematoxylin and eosin (H&E) staining of mouse liver tissue (Figure 5B). In addition, NJRE treatment reduced the liver weight to body weight ratio to a value similar to that of the control group (Figure 5C). It also increased mouse survival rate compared to the control without NJRE treatment (Hepa1-6 group) (Figure 5D). These results demonstrate that NJRE can inhibit the proliferation of HCC in vivo.
NJRE suppresses tumor proliferation through ERK and STAT3 pathways in vivo. We investigated the mechanisms attributed to tumor growth inhibition by NJRE in vivo. We determined the expression of AFP in liver tissues through IHC analysis. We found that NJRE reduced AFP expression by IHC analysis and quantitative analysis (Figure 6A and B). We also evaluated expression levels of p-ERK and p-STAT3 (Ser 727) in liver tissues via IHC analysis to check whether tumor growth inhibition by NJRE was due to reduction of expression of proteins in ERK/STAT3 pathways. We found that NJRE decreased the phosphorylation of ERK and STAT3 (Ser 727) compared to the control (Hepa1-6 group) (Figure 6A and B). Additionally, among proteins involved in HCC proliferation through the STAT3 pathway, the expression of PCNA in liver tissues was evaluated. As shown in Figure 6A and B, the expression of PCNA was down-regulated in NJRE treated group compared to that in the control (Hepa1-6 group), indicating that NJRE suppressed HCC proliferation via ERK and STAT3 pathways. Taken together, these results suggest that NJRE can suppress the expression of MAPK/ERK, thereby inhibiting the activation of STAT3 and consequently reducing HCC proliferation. Therefore, NJRE has the potential to be used in anti-tumor therapy for HCC.
Discussion
In this study, we investigated whether NJRE could attenuate tumor progression in HCC via inhibition of ERK and STAT3 signaling pathways. NJRE has been reported to have anti-cancer effects on breast cancer and neuroblastoma (8, 18). Therefore, we examined whether NJRE might have anti-cancer effects in HCC. HCC is a primary malignant tumor in the liver. It is the second major cause of cancer-related deaths and the fifth most malignant tumor in the world (19). Many studies have been conducted to characterize the molecular mechanisms of HCCs to develop new treatments and develop and design drugs that can affect multiple targets or pathways (20, 21). Natural products such as curcumin, quercetin and berberine have been used in various cancer diseases due to their chemical and structural diversities (4). Therefore, the discovery of new drugs through natural product screening is considered one of the effective strategies for development of new anti-cancer drugs (22). In addition, several reports have shown that natural products-derived chemotherapy drugs possess cancer chemopreventive activity, whereas common chemotherapy drugs such as doxorubicin, cisplatin, and 5-FU (5-fluorouracil) have toxic side-effects (23-25). Therefore, discovering new drug candidates through natural compounds is considered an effectual strategy for the development of new anti-cancer drugs.
Anti-cancer agents are often limited in use due to their toxicities to normal tissues and organs (26). Since almost all anti-cancer agents have side-effects due to their toxicity, new anti-cancer drugs must overcome this limitation. In this study, NJRE showed IC50 values of 50-150 μg/ml for various HCC cells (Huh7, PLC/PRF-5, Hep3B, and Hepa1-6) without showing any toxicity to normal mouse hepatocyte (FL83B) in this concentration range (Figure 2). In addition, no toxicity was observed even after oral administration of NJRE to mice, suggesting that NJRE has potential anti-cancer effects against HCC without toxicity.
HCC proliferation is regulated by complex molecular mechanisms. Many studies have shown that MAPKs such as ERK, p38 and JNK are involved in HCC cell proliferation (27-29). In this study, NJRE treatment on HCC cells led to a decrease of the phospho-STAT3 (Ser 727) levels, along with a decrease in ERK phosphorylation (Figure 3). STAT3 is known to regulate proliferation and apoptosis as a transcription factor. Phosphorylation of STAT3 at Tyr 705 and Ser 727 is crucial for transcriptional activation of STAT3 and may involve several pathways, including the MAPK pathway (30, 31). Our results showed that NJRE treatment causes a decrease in the phosphorylation of both Tyr 705 and Ser 727 (Figure 3B). Inhibition of STAT3 was associated with decreased levels of cyclin D1 and PCNA expression, which are associated with proliferation of HCC cell (32, 33). Collectively, NJRE inhibited the phosphorylation of ERK and STAT3 both at Tyr 705/Ser 727 and thus reduced the expression of cyclin D1 and PCNA (Figure 3 and 6). Our results also demonstrated that NJRE increases the phosphorylation of p38 and JNK, which induces apoptosis in HCC cell (Figure 3A). Phosphorylation of p38 and JNK promotes caspase-3 activation, which leads to cleavage of PARP, ultimately leading to apoptosis (34). Taken together, NJRE increased the activity of caspase-3 and cleavage of PARP via the activation of p38 and JNK (Figure 4). Although further molecular studies are required, these studies show that inhibition of these pathways is a crucial strategy for anti-cancer therapies.
In conclusion, results of this study suggest that the anti-proliferative effect of NJRE is mediated by suppression of ERK and STAT3 pathways. Although additional chemical and structural studies are required to confirm the possible active ingredients of NJRE responsible for its anti-cancer effects, our results demonstrate that NJRE has potential as an effective treatment for HCC.
Acknowledgements
This research was supported by the Brain Korea (BK21) PLUS program, the Basic Science Research Program, and Creative Materials Discovery Program through grants [2019R1I1A1A01056842, 2019R1I1A1A01059642] of the National Research Foundation (NRF) funded by the Ministry of Science and ICT, Republic of Korea.
Footnotes
Authors’ Contributions
SKY conceived and directed the study and wrote the manuscript. WHH designed the study and wrote the manuscript. JHK and GWL designed the experiments, performed the experiments, analyzed data, wrote the manuscript. DJP, PSS, SMK, BYK contributed to data acquisition and interpretation.
↵* This Author is currently affiliated to AM Science, Seoul, Republic of Korea.
Conflicts of Interest
The Authors declare that they have no conflicts of interest.
- Received January 25, 2021.
- Revision received February 26, 2021.
- Accepted March 1, 2021.
- Copyright © 2021 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.