Artemisia Capillaris leaves inhibit cell proliferation and induce apoptosis in hepatocellular carcinoma

The dried leaves of Artemisia capillaris were purchased from Jung Do Herbal Drug Co. (Gyeonggi Province, Korea) and the voucher specimen (DBH16011101) was deposited in the Herb Resource Bank of Traditional Korean Medicine (http://​herb-bank.​com), Kyung Hee University (Seoul, Korea). The dried material (5 g) was extracted with 50 mL of 70% ethanol for 24 h at room temperature. Next, the extract was filtered, concentrated on a rotary vacuum evaporator, and completely freeze-dried (yield: 7.12%). The powder was stored at 4 °C.

An Agilent 1100 series HPLC system (Agilent Corp., Santa Clara, CA) was used to acquire chromatograms. All the chromatographic analysis was performed on a Phenomenex Kinetex C18 column (100 mm × 4.6 mm i.d. 2.6 μm). The mobile phase was composed of 0.1% formic acid in distilled water and 0.1% formic acid in methanol. The conditions of solvent gradient elution were 30% in 0–2 min, 30–90% in 2–12 min, 90% in 12–22 min, 90–30% in 22–22.1 min, 30% in 22.1–30 min, at a flow rate of 0.5 mL/min. The column temperature was maintained at 40 °C, and all the injection volumes of sample solutions were fixed at 2 μL. The eluent was directed to an ESI-LTQ-XL-Linear Ion Trap (Thermo Scientific) mass spectrometer and the data was acquired in full-scan and positive mode with mass range from 100 to 800 m/z.

HCC cells (Huh7 and HepG2) were purchased from JCRB (Shinjuku, Japan) and American Type Culture Collection (Manassas, VA), respectively. Huh7 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM), and HepG2 cells were cultured in Minimum Essential Media Eagle (MEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS, cat.n. 26,140–079) and 1% penicillin/streptomycin. FBS and all other reagents used for cell culture were purchased from Invitrogen (Carlsbad, CA). The cultures were maintained at 37 °C in an incubator with a controlled humidified atmosphere composed of 95% air and 5% CO₂.

Cell viability was determined using an MTT assay. In brief, cells were seeded at a density of 4 × 10³ cells/well in 96-well plates, followed by overnight incubation. On the following day, the media were removed, and the cells were treated with either vehicle as a negative control or various concentrations of LAC117 (1–100 μg/mL) and incubated for 72 h. After incubation of respective time, 10% of an MTT solution (2 mg/mL, Sigma, cat.n.M2128) was added to each well, and the cells were incubated for another 4 h at 37 °C. The formazan crystals were dissolved in DMSO (100 μL/well, Sigma, cat.n.D2160) with constant shaking for 5 min. The absorbance of the plate was then read with a microplate reader at 540 nm. Three replicate wells were evaluated for each analysis.

To measure the cell proliferation activity of LAC117 in Huh7 and HepG2 cells, 8 × 10³ cells were plated per well onto 96-well plates. Following overnight culture, LAC117 was added at specified concentrations. After 24 h of incubation, cell proliferation was measured with a BrdU assay kit (Cell Signaling, cat.n.6813) per the manufacturer’s instructions. Plates were read at 450 nm by using a spectrometer.

The cells were washed with DPBS before being lysed in a lysis buffer containing protease and phosphatase inhibitors. Equal amounts of proteins were separated using 8, 10 or 12% sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride (PVDF) membranes. Protein transfer was confirmed using a Ponceau S staining solution (Sigma, cat.n.P7170). The blots were then immunostained with the appropriate primary antibodies (1:1000) followed by appropriate secondary antibodies (1:5000) conjugated to horseradish peroxidase. The primary antibodies specific to the interested proteins were used and detected manually using an X-Ray film by enhanced chemiluminescence (Amersham Biosciences, Piscataway, NJ).

Huh-7 and HepG2 cells were plated onto chamber slides at a density of 5 × 10⁴ cells per chamber. At 24 h post-incubation, cells were treated with LAC117 (100 μg/mL) at 37 °C for 24 h. Coverslips with adherent cells were fixed in 4% paraformaldehyde (PFA) for 15 min at room temperature, and then rinsed in distilled PBS and incubated with equilibration for 1 min. TUNEL assay was subsequently performed by using a TUNEL kit ApopTag® Peroxidase In Situ Apoptosis Detection Kit (Merck Millipore, Temecula, CA. cat.n.S7100) in accordance with the manufacturer’s instructions. Huh7 and HepG2 cells were plated on 18-mm cover glasses for 24 h and treated with LAC117 (100 μg/mL). Apoptotic cells were visually identified in 10 randomly selected fields and photographed at a magnification of × 200. Apoptotic cells were counted to calculate the percentage of TUNEL-positive cells.

All animal experiments were performed in accordance with the guidelines of the INHA Institutional Animal Care and Use Committee (INHA IACUC) of the Medical School of Inha University (approval ID: INHA 150915–379-1). The cells (HepG2) were harvested and mixed in PBS. Six-week-old male BALB/c nu/nu mice (Orient Bio, Seoul, Korea) were inoculated with 1 × 10⁷ cells in the flank. When the tumor size reached approximately 50–100 mm³, mice were randomly divided into 2 groups (4 mice per group). Next, LAC117 (100 mg/kg) or vehicle (1% Tween 80) was administrated intraperitoneally once daily for 15 days. At the end of the 15 days treatment period, the animals were anaesthetized with mixture of ketamine (100 mg/kg) and xylazine (2%, 20 mg/kg). Tumor size was measured every 2 days, and it was calculated using the following formula: 0.5 × length × width².

Male BALB/c nu/nu mice (4 weeks old, weighing 18–20 g) were obtained from Orient Bio. Animal Inc. (Seoul, Republic of Korea). The animals were fed standard rat chow and tap water ad libitum, and were maintained under a 12 h dark/light cycle at 21 °C. After one week of adaptation, Huh7 (8 × 10⁶ cells/mice) was inoculated into the right flanks of mice. When the tumor size reached approximately 300–500 mm³, they were surgically removed (n = 5). A 2-mm diameter section was excised and explanted on 2% agarose-coated 24 well plates with culture medium at 37 °C. After overnight incubation, the tumor spheroids were subjected to treatment with or without LAC117 (100 μg/mL) for 7 days. For IHC analysis, tissues were immediately fixed in 4% PFA overnight and paraffin-embedded slides were prepared for further analysis.

Immunohistochemical staining of fixed paraffin-embedded specimens was performed using 8-μm-thick sections. Heat-induced epitope retrieval (HIER) was performed in a citrate buffer (pH 6.0) for 5 min before peroxidase quenching with 3% hydrogen peroxide (H₂O₂) in PBS for 10 min. The tissue sections were washed with PBS and blocked with normal goat or horse serum for 1 h and incubated at 4 °C overnight in 1:50 dilutions of primary antibodies against cleaved caspase-3, PCNA, p-AKT, and p-mTOR. The sections were then incubated with biotinylated secondary antibodies (1:100) for 1 h. The sections were visualized with an avidin-biotin peroxidase complex solution using an ABC kit (Vector Laboratories, cat.n.PK-6101), washed in PBS and developed with a diaminobenzidine tetrahydrochloride (DAB) substrate for 5 min, and then counterstained with hematoxylin. At least 3 randomly selected fields for each section were examined at x 200 magnification and analyzed.

The protective effects of the AC against cancer have previously been reported (14). AC contains potent compounds as anti-inflammatory or anti-cancer components such as chlorogenic acid, esculetin, scopoletin, isochlorogenic acid, scoparone, hyperin, isorhamnetin-1,6-diglucoside, artepillin, and capillin, which are marker components of AC. Therefore, we identified whether LAC117 isolated from leaves of AC contains these components using HPLC-MS analysis. The chromatogram revealed the strong presence of the scoparone (RT = 9.29 min, 207 m/z; peak 4), chlorogenic acid (RT = 3.41 min, 355 m/z; peak 1), isorhamnetin-1,6-diglucoside (RT = 10.42 min, 625 m/z; peak 6), and hyperin (RT = 9.38 min, 465 m/z; peak 5, Fig. 1). The molecules responsible for these signals were identified by comparing them to the spectra and retention time of known standard compounds.

To examine the effects of LAC117 on cell growth and viability, we performed the MTT assay using two HCC cell lines (HepG2 and Huh7). The cells were exposed to the indicated concentrations of LAC117 (1 to 100 μg/mL) for 72 h. LAC117 reduced cell viability of both HepG2 and Huh7 cells in a dose-dependent manner (Fig. 2a). In particular, LAC117 treatment inhibited cell growth by 60~ 90% at dose of 50 μg/mL depending on the cell type. To further evaluate this result, we determined cell proliferation by using the BrdU cell proliferation assay (Fig. 2b). In agreement with the MTT assay, LAC117 dose-dependently inhibited the proliferation of HCC cells. In the both studies, we found that Huh7 cells were more sensitive to LAC117 than HepG2 cells.

During apoptosis, mitochondrial membrane potential regulates matrix configuration and the rapid release of cytochrome c from the mitochondrial intermembrane space into the cytosol. Therefore, we investigated whether LAC117 increases the release of cytochrome c through loss of mitochondria membrane potential. For this experiment, HepG2 and Huh7 cells were treated with LAC117 (100 μg/mL) for 6 h and then observed cytochrome c release into the cytosol by double staining with Mitotracker (green) and antibody against cytochrome c (red) in HCC cells. In this study, we observed that LAC117 significantly increased cytochrome c release which triggers the apoptotic process through caspase activation (Fig. 3a). We also investigated whether LAC117 could increase cleaved caspases 3/PARP, leading to apoptosis in HCC cells. When treated with LAC117 (100 μg/mL) for 24 h, the cells showed morphological features of apoptotic cells, such as DNA fragmentation by TUNEL staining. Also, the percentages of TUNEL-positive cells were increased in the LAC117-treated groups (Fig. 3b). We further found in western blotting study that treatment with LAC117 significantly increased the expression of apoptotic proteins (cleaved PARP and cleaved caspase-3), whereas the expression of anti-apoptotic protein (XIAP) was decreased in both cell lines, compared with control (Fig. 3c). These results indicate that LAC117 could induce mitochondria-mediated apoptosis in HCC cells.

Deregulated PI3K/AKT/mTOR signaling pathways are commonly found in HCC [20]. To understand the mechanism underlying the enhanced ant-cancer effect of LAC117, we investigated the inhibition of the PI3K/AKT signaling pathway in Huh7 and HepG2 cells after LAC117 (100 μg/mL) treatment for 1 h. LAC117 was observed to decrease the phosphorylation of AKT, mTOR, and GSK3β in Huh7 cells (Fig. 4).

To further determine the apoptotic effect of LAC117, an ex vivo organotypic spheroid culture was established using xenograft-derived tumors in the Balb/c nude mice (Fig. 5a). As observed by H&E staining, apoptotic cells were significantly increased in the LAC117-treated group compared to that of the control group (Fig. 5b). Moreover, LAC117 treatment decreased the number of proliferating cells (PCNA-positive cells), but increased the number of apoptotic cells (cleaved caspase-3 positive cells, Fig. 5b). Taken together, our results show that LAC117 exhibit potent anti-cancer activity by inhibiting cell proliferation and inducing apoptosis.

To further assess whether LAC117 suppresses tumor growth in vivo, HCC xenograft models were used in Balb/c nude mice. After inoculation with HepG2 cells, mice were injected intraperitoneally with 100 mg/kg LAC117 for 15 days. As shown in Figs. 5c and 6a, LAC117 significantly reduced tumor volume and weight after 15 days. Moreover, no significant changes in body weight were observed in animals treated with LAC117 (Fig. 6b), showing that LAC117 was minimally toxic to mice at the curative dose. In histopathological analysis by H&E staining, we observed that there was a greater degree of tumor apoptosis and necrosis in the LAC117-treated group compared with the control group (Fig. 7). Also, LAC117 markedly decreased the expression of PCNA, a cell proliferation marker and increased that of cleaved caspase-3. In addition, LAC117 decreased the expression of p-AKT and p- mTOR, downstream of the PI3K/AKT pathways in tumor tissues. Experimental raw data were presented in Additional file 1.