Background
Hepatocellular carcinoma (HCC) is a common malignant tumor with characteristics of poor prognosis and high morbidity and mortality worldwide [
1,
2]. Various factors have been identified to contribute to the occurrence and progression of HCC, including the infection by hepatitis B or C virus, the inactivation of tumor suppressor genes such as p53, the abnormal activation of oncogenes such as K-ras and some signaling molecules such as PI3K, ERK/MAPK and Wnt/β-catenin as well as the evasion of the host immune system [
3,
4]. For the management of HCC, resection, liver transplantation and radiofrequency ablation are options for early-stage HCC patients, yet with high rates of recurrence and metastasis [
3,
5,
6]. In addition, only a few systemic treatment options are available for advanced HCC patients, and include sorafenib and the recently described atezolizumab plus bevacizumab regimen as possible first-line treatments [
7]. In this context, it is of importance and interest to either develop new agents targeting multiple molecular targets or set up a new strategy such as application of combined therapy in the treatment of advanced HCC.
Acteoside is a phenylethanoid glycoside widely distributed in many medicinal plants, including
Ligustrum purpurascens [
8],
Rehmannia glutinosa [
9] and
Ligstrum purpurascens [
10]. Mounting evidence has shown that acteoside can exert various biological activities, including its antitumor activity. For example, acteoside showed strong antiproliferative effects on prostatic cancer cells [
11]. Intraperitoneal delivery of acteoside suppressed tumor growth in a melanoma mouse model possibly through inhibiting protein kinase C [
12]. In addition, acteoside potentiated the sensitization of colorectal cancer cells to 5-Fluorouracil via targeting PI3K/Akt signaling [
13]. Our previous data have also shown an inhibition of melanogenesis by acteoside in B16 melanoma cells [
14]. Despite that, little information is available regarding the possible efficacy of acteoside in the treatment of HCC and the potential underlying mechanisms.
In the present study, we have shown that acteoside inhibits cell proliferation, colony formation and migration in all the three human HCC cell lines BEL7404, HLF and JHH7. The angiogenesis is also inhibited following treatment with acteoside, as shown by the inhibition of tube formation and cell migration of HUVECs. The in vivo antitumor efficacy of acteoside is further demonstrated in BEL7404 or JHH-7 xenograft nude mice model, with an enhancement when combined with sorafenib in inhibiting the growth of JHH-7 xenograft. The antitumor effects of acteoside could be ascribed to its up-regulation of p53 as well as suppression of KLKs and angiogenesis.
Methods
Cell culture
The human hepatocellular carcinoma (HCC) cell lines BEL7404, HLF and JHH7 were obtained from the American Type Culture Collection and were routinely cultured in DMEM medium containing 10% fetal bovine serum supplemented with 100 units/ml penicillin G and 100 μg/ml streptomycin in a humidified incubator at 37 °C with 5% carbon dioxide. Human umbilical vein endothelial cells (HUVECs) were isolated from the human umbilical cord veins (provided by ScienCell Research Laboratories, San Diego, USA) and maintained in an endothelial cell medium as described previously [
15].
Reagents
Acteoside (A01) was purchased from Jiangsu Yongjian Medicine Technology Co., Ltd. (Taizhou, China; Lot No. 100581; Purity≥99% by HPLC). Sorafenib was obtained from Selleck Chemicals (Houston, USA). We dissolved acteoside in 0.9% saline. The stock solution of sorafenib was prepared in dimethyl sulfoxide (DMSO; 10 mM), aliquotted and stored at − 20 °C.
Animals
Male BALB/c (Nu/Nu) mice (18-20 g, 6–8 weeks at the time of receipt) were used in the study (Shanghai Slac Laboratory Animal Co. Ltd., Shanghai, China). Animals were housed in individually ventilated polysulfone cages in a temperature- and light-controlled room (12 h light-12 h dark cycle, lights on at 7:00 a.m.) with free access to food and water. All experimental protocols were approved by the Institutional Animal Care and Use Committee and performed in accordance with the guidelines of the International Association for the Study of Pain concerning the use of laboratory animals.
Cell proliferation assay
The anti-proliferative activity was performed using the MTT assay. Briefly, cells were plated at a density of 4–5 × 103 cells per well in 96-well plates. After attached overnight, cells were treated with a series of concentrations of acteoside. Following the drug treatments at the indicated time point, cells were incubated with 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Roche Diagnostic Corporation, Indianapolis, IN, USA. Final concentration:5 μg/ml) for 3–4 h at 37 °C. The formazan product was dissolved in DMSO for quantitation at a wavelength of 570 nm.
Cells were seeded in 24-well plates at a density of 200 cells per well and allowed to attach to the bottom of the well overnight. Cells were then treated with indicated drugs at the given concentration. The culture medium was replaced every 3 days until colonies were visible at day 12 post-culturing. For colonies counting, the cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. Colonies> 10 cells were counted under a light microscope (× 100 magnification; Olympus, Tokyo, Japan).
Wound healing assay
The cells were seeded in 6-well plates (1 × 105 cells per well) and cultured to 90% confluency. The cell layer was scratched with a sterile 200 μl pipette tip to produce a wound gap. Cell migration into the wounded area was visualized at indicated time points (0, 10 and 24 h) under an inverted light microscope at a magnification of × 100. Each experiment was performed in triplicate.
The HUVECs were cultured as previously described [
15]. In brief, the 96-well plates were coated with Matrigel (BD Biosciences, Franklin Lakes, USA) according to the manufacturer’s instructions and then returned to the incubator to polymerize for 30–40 min. The HUVECs were then seeded at a density of 2 × 10
5 cells/mL and treated with various given drugs for 8 h. Tube formation was photographed with an inverted microscope at the indicated time points and analyzed using an ImageJ software.
Western blotting analysis
Total cell lysates were prepared with radio-immunoprecipitation assay buffer (RIPA) (50 mM Tris-HCl at pH 8.0, 150 mM NaCl, 1% NP40, 1% sodium deoxycholate, 0.1% SDS, 10 μg/ml leupeptin, 10 μg/ml aprotinin and 2 mM PMSF). Protein samples with equal amounts (20 μg/lane) were separated on SDS-PAGE gel and transferred to PVDF membranes (Bio-Rad, Hercules, CA). The membranes were incubated overnight with p53 antibody (Cell Signaling Technology, Boston, MA). For loading controls, the membranes were rinsed with a stripping buffer and reprobed with the β-actin antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Proteins were detected using an ECL detection system (Perkin Elmer Life Sciences, Boston, USA).
qRT-PCR
Total RNA was extracted from cell lines using the Trizol reagent (Invitrogen, USA) and underwent a reverse transcription-polymerase chain reaction (RT-PCR) to synthesize cDNA using a commercially available kit. The real-time qRT-PCR using 2xSYBR green qPCR Supermix was conducted on the Stratagene Mx3000P PRC machine (Agilent Technologies, USA). The primer sequences were shown below: sense 5′-CACCATGTGGTTCCTGGTTC-3′ and anti-sense 5′- CAAACAAGTTGTGGCGACCC-3′ for KLK1; sense 5′-GTGTACAGTCATGGATGGGC-3′ and anti-sense 5′-CCCAGAATCACCCCCACAAG-3′ for KLK2; sense 5′- CCACACCCGCTCTACGATATGA-3′ and anti-sense 5′-CCCAGAATCACCCGAGCAG-3′ for KLK3; sense 5′-CGCACACTGTTTCCAGAACTC-3′ and anti-sense 5′- GTTGCAGGAGTCCTTCTGGT-3′ for KLK4; sense 5′-CCTGCACCCACATCTTTCTCT-3′ and anti-sense 5′-GGTAAGCATCCTCGCACCTT-3′ for KLK5; sense 5′- CTCTCTCCTGGGGACACAGA-3′ and anti-sense 5′-TCCGCCATGCACCAACTTAT-3′ for KLK6; sense 5′-TTTTGGAGCCCAGCTGTGTG-3′ and anti-sense 5′-GTCACCATTGCAGGCGTTTT-3′ for KLK7; sense 5′-CTGGGCAGGACACTCCAG-3′ and anti-sense 5′-ACACCGCCACAGAGTAGTTG-3′ for KLK8; sense 5′-GTAGGGGGTTCTCGTAGGGT-3′ and anti-sense 5′-CGGTGACGTCATAGAGACGG-3′ for KLK9; sense 5′-CAGGAGTGCCAGCCTCAC-3′ and anti-sense 5′- CTGGGGAGGAAGAGGATGGA-3′ for KLK10; sense 5′-CCCACCCCTTGGATTCTGTCT-3′ and anti-sense 5′-GTGAACTATGTAGCGGGGCT-3′ for KLK11; sense 5′-TGTTCTTGGTGAGTTCTCCCG-3′ and anti-sense 5′-GGATCCAGTCCACATACTTGC-3′ for KLK12; sense 5′-CCTGAACCACGACCATGACA-3′ and anti-sense 5′-GCAGGGTTTGGATGTAGCCT-3′ for KLK13; sense 5′-CCCAACTACAACTCCCGGAC-3′ and anti-sense 5′-CCTGAAGCAACTGCTCGTGA-3′ for KLK14; sense 5′-AGTTGCTGGAAGGTGACGAG-3′ and anti-sense 5′-TGGCTAACATCTGGGCCTTG-3′ for KLK15; sense 5′-GACAGTCAGCCGCATCTTCT-3′ and anti-sense 5′- GCGCCCAATACGACCAAATC-3′ for GAPDH. The cycle threshold (Ct) values of each KLK gene were normalized to that of GAPDH and analyzed with the MxPro software.
In vivo antitumor activity of A01 alone or in combination with sorafenib
The BEL7404 cell suspensions were prepared (3 × 10
6 cells) and injected subcutaneously into the right flank of athymic nude mice. Ten days after the inoculation, tumor-bearing mice (body weight(g):22–26; mean ± SEM:23.8 ± 0.14) were screened and selected for randomization into 7 treatment groups in accordance with tumor volume (70-120 mm
3). A total of 38 mice were assigned to different treatment groups (
n = 5 per group except for the model group (
n = 8)). Animals were then treated via oral gavage with acteoside (A01; 12.5, 25 and 50 mg/kg), sorafenib alone (50 mg/kg) or in combination with A01 (50 mg/kg) once daily for 14 days. Moreover, a dose of 20 mg/kg A01 was also delivered intravenously. Doses were selected based on previous studies and our preliminary data [
15]. The tumor growth was monitored every 3 or 4 days using an electronic caliper, with the volume calculated as 0.5xlengthxwidth
2 (mm
3). In addition, a parallel experiment using JHH-7 inoculation was included. Seven days after the inoculation, tumor-bearing mice (body weight(g):20–26; mean ± SEM:24.0 ± 0.20) were screened and selected for randomization into treatment groups in accordance with tumor volume (80-130 mm
3). A total of 23 mice were assigned to 4 treatment groups (
n = 5 per group except for the model group (
n = 8)). Animals then received A01 (20 mg/kg, iv), sorafenib alone (50 mg/kg, ig) or combined with A01 (20 mg/kg, iv) once daily for 14 consecutive days. All mice were anesthetized with chloral hydrate (350 mg/kg) and sacrificed by neck dislocation at the end of the observation period. And tumor mass was removed and pictured with a digital camera.
Statistical analysis
All data are expressed as mean ± standard deviation (SD). Unless otherwise stated, statistical analyses were performed using analysis of variance (ANOVA) followed by a post-hoc Tukey test. Tumor volume values are shown as mean ± standard error of mean (SEM). Two-way ANOVA followed by Tukey tests were used to analyze tumor volume data. The GraphPad Prism software was used for statistical analyses. A value of p < 0.05 was considered statistically significant.
Discussion
The management of advanced HCC has long been intractable due to the inability of diagnosis at earlier stages and the chemoresistance of the tumor [
5,
6]. Sorafenib, the multikinase inhibitor reported to inhibit cell proliferation and angiogenesis, together with another oral multikinase inhibitor lenvatinib has become the standard treatment of advanced HCC [
3,
5,
20,
21]. However, the overall survival rates are modest in light of the low response rates and unsuitability in the clinical settings [
3,
22]. A recent study showed that atezolizumab plus bevacizumab produced better clinical outcomes than sorafenib in patients with unresectable HCC, making this combination a possible new first-line treatment option for advanced HCC [
7]. In this context, we have identified that the phenylethanoid glycoside, acteoside inhibited cell proliferation, colony formation and migration in the three human HCC cell lines BEL7404, HLF and JHH-7 as well as prohibited angiogenesis of HUVECs. The combination of acteoside and sorafenib produced stronger inhibition of cell colony formation and migration of the HCC cells as well as of angiogenesis of HUVECs. Acteoside presented the antitumor efficacy in BEL7404 or JHH-7 xenograft nude mice model, with an enhancement when combined with sorafenib in inhibiting the growth of JHH-7 xenograft. Further analyses revealed an increase in p53 as well as a decrease of some KLK genes. Accordingly, the possible use of acteoside as adjuncts in the treatment of advanced HCC in the clinic should be considered.
Mounting evidence has shown the antitumor effects of acteoside in various tumor cells such as B16 melanoma cells, glioblastoma cells, colorectal cancer cells, oral squamous cell carcinoma cells, prostate cancer cells and human breast adenocarcinoma cells [
11‐
13,
23‐
25] or in animals bearing tumors [
12]. Despite that, few studies have focused on the effects of acteoside on HCC in vitro or in vivo except one study using diethylnitrosamine as the carcinogen to induce hepatocarcinogenesis in rats and revealing the chemoprevention by acteoside [
26]. In the current study, we showed the inhibition by acteoside of cell proliferation, colony formation and migration in HCC cell lines as well as tumor growth in mice bearing HCC cell line xenografts, thereby directly demonstrating the antitumor effects of acteoside in HCC. Furthermore, the combination of acteoside and sorafenib produced even stronger antitumor effects. Combined with the evidence that acteoside is protective against liver injuries [
27‐
29] with no toxic effects in animals and no mutagenicity [
30], it seems to be feasible for the future application of the combination therapy of acteoside and sorafenib in patients with advanced HCC in the clinic.
The human kallikrein family comprises 15 homologous, single-chain, secreted trypsin- or chymotrypsin-like serine proteases (KLK1–15) and has been implicated in regulation of tumor growth, neoplastic progression, angiogenesis and metastasis [
18,
31]. In particular, the KLKs have been suggested as a cancer biomarker in the diagnosis and prognosis of various cancers, including hepatocellular carcinoma due to its dysregulated expression [
17‐
19]. For example, KLK1 is proposed as the biomarker of gastric cancer due to the elevation of KLK1 levels in gastric neoplasm and the prevention of tumor growth in gastric cancer by inhibition of KLK1 activity with kallikrein-binding protein (KBP) [
32]. In this scenario, we observed a significant inhibition of KLK mRNA levels by acteoside (KLK1, 2, 4, 9 and 10). Application of KBP, which specifically binds to tissue kallikrein and inhibits kallikrein activity, was reported to suppress growth of HCC in mice possibly via its anti-angiogenic activity [
33], suggesting the possible involvement of KLKs in the progression of HCC. In addition, the tumor suppressor protein p53 is able to induce senescence, apoptosis as well as cell cycle arrest. Increased p53 expression confers enhanced chemosensitivity in cancer cells [
34]. In this regard, our western results showed an increase in p53 levels following treatment with acteoside. Taken together, it is reasonable to infer that acteoside exerts the antitumor effects on HCC possibly through its up-regulation of p53 as well as inhibition of KLK and angiogenesis.
It should be noted that the direct molecular target to which acteoside could directly bind is still unclear. Our preliminary data using molecular docking revealed a direct binding of acteoside with kallikrein (data not shown). Further experiments would be necessary to clarify this presumption. In addition, as for the doses of acteoside used for cell assay in the study 10–50 times higher than that of sorafenib (10 μM), firstly we chose those doses of acteoside in accordance with the dose-response relationship in the MTT assay. Second, the doses we used (100, 500 μM) were comparable to previous studies [
35], in which acteoside (100 μM) inhibited cell proliferation of colorectal cancer cell. Last, despite the higher dose in the cell assay, acteoside produced a pronounced inhibition of tumor growth in the HCC xenograft nude mice model in our study (25 and 50 mg/kg), effects being similar to sorafenib (50 mg/kg). For sorafenib, it was used in our study at the dose of 10 μM at the cellular level. This dose is comparable to previous studies in which sorafenib was found to inhibit the cell viability with an IC
50 of 4.0–6.5 μM in the HCC cell lines of PLC/PRF/5 and HepG2 cells. In contrast, the dose of sorafenib used to suppress the growth of HCC xenografts in SCID female mice was 30 mg/kg (p.o. by gavage) [
36]. In the clinic, the recommended dosage of sorafenib for patient is 400 mg per time (2*0.2 g), twice a day (800 mg per day in total). This dosage for sorafenib used in the clinic is in contrast to that used in cell assay (IC50 value in μM level), possibly due to its poor absorbability under in vivo conditions. For acteoside, based on its dosage in mice (50 mg/kg, i.g.), its equivalent dose for human (60 kg bw) is about 250 mg. This dosage is comparable to that of sorafenib used in the clinic and should be acceptable for the patients.
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