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Nitidine chloride inhibits hepatic cancer growth via modulation of multiple signaling pathways
© Lin et al.; licensee BioMed Central Ltd. 2014
Received: 31 March 2014
Accepted: 26 September 2014
Published: 30 September 2014
The development of hepatic cancer is tightly regulated by multiple intracellular signaling pathways. Therefore, most currently-used anti-tumor agents, which typically target single intracellular pathway, might not always be therapeutically effective. Additionally, long-term use of these agents probably generates drug resistance and unacceptable adverse effects. These problems increase the necessity for the development of new chemotherapeutic approaches. Nitidine chloride (NC), a natural benzophenanthridine alkaloid, has been shown to inhibit cancer growth via induction of cell apoptosis and suppression of cancer angiogenesis. But the precise mechanisms of its tumorcidal activity are not well understood.
To further elucidate the precise mechanisms of its anti-tumor activity, using a hepatic cancer mouse xenograft model, the human hepatic cancer cell lines (HepG2, HCCLM3, Huh7), and umbilical vein endothelial cells (HUVEC), here we evaluate the effect of NC on tumor growth in vivo and in vitro and investigated the underlying molecular mechanisms.
We found that NC treatment resulted in significant decrease in tumor volume and tumor weight respectively, but didn’t affect body weight changes. Additionally, NC treatment dose- and time-dependently reduced the cell viability of all three hepatic cell lines. Moreover, NC suppressed the activation of STAT3, ERK and SHH pathways; and altered the expression of critical target genes including Bcl-2, Bax, Cyclin D1, CDK4, VEGF-A and VEGFR2. These molecular effects resulted in the promotion of apoptosis, inhibition of cell proliferation and tumor angiogenesis.
Our findings suggest that NC possesses a broad range of anti-cancer activities due to its ability to affect multiple intracellular targets, suggesting that NC could be a novel multi-potent therapeutic agent for the treatment of hepatic cancer and other cancers.
Primary hepatic cancer or liver cancer is the sixth most commom cancer globally and the second leading cause of cancer-related death [1–5]. The most frequent hepatic cancer is hepatocellular carcinoma (HCC), accounting for approximately 75% of all primary liver cancers. Another type of liver cancer is hepatoblastoma (HBL), which is specifically formed by immature liver cells and primarily develops in children. To date, chemotherapy remains one of the major non-surgical therapeutic approaches for patients with advanced hepatic cancer . However, due to drug resistance, systemic chemotherapy produces a disappointing low response rate, ranging between 10%-15% . Moreover, many currently used anti-cancer agents have potent cytotoxic effects in normal cells . These problems limit the effectiveness of current HCC chemotherapy, thus increasing the necessity for the development of new chemotherapeutic approaches.
The mechanisms underlying pathogenesis and development of HCC are complex and heterogeneous, involving multiple cellular signaling pathways including signal transducer and activator of transcription 3 (STAT3), Sonic Hedgehog (SHH) and extracellular regulated protein kinases (ERK). STAT3 plays an essential role in cell survival, proliferation and angiogenesis . After activation via phosphorylation, STAT3 proteins in the cytoplasm dimerize and translocate to the nucleus where they regulate the expression of critical genes involved in cancer progression. Constitutive activation of STAT3 is strongly associated with cancer development and commonly suggests a poor prognosis [10, 11]. Aberrant activation of SHH is highly correlated with various human cancers [12–14]. SHH signaling activation is initiated at the cell surface by binding of SHH ligand to the transmembrane receptor Patched (Ptc), resulting in the release of Ptc-mediated suppression of Smoothened (Smo). Smo subsequently activates the Gli family of transcription factors that regulate the expression of various HH target genes [15–17]. Extracellularsignal regulated kinase (ERK) signaling is one of the major cell-survival and proliferation pathways. As a major subfamily member of Mitogen-activated protein kinases (MAPKs), activation of ERK is regulated by a central three-tiered kinase core consisting of MAPK kinase kinase (e.g., Raf), MAPK kinase (e.g., MEK), and MAPK (e.g., ERK); wherein Raf phosphorylates MEK which in turn phosphorylates and activates ERK . By altering the levels and activities of transcription factors, activation of ERK pathway regulates the expression of various genes mediating cell apoptosis, proliferation and angiogenesis [19, 20]. These molecular pathways described above modulate the expression of key genes involved in the regulation of cell proliferation, apoptosis, and angiogenesis and are participants in the processes of induction, progression, and metastasis of hepatic cancer. Thus, each serves as a potential target for novel chemotherapeutics.
Natural products have received recent interest in discovery of novel anti-cancer therapeutic agents as they have relatively few side effects and have long been used as alternative remedies for a variety of diseases including cancer [21, 22]. Therefore, identifying naturally occurring agents is a promising approach for anticancer treatment. Nitidine chloride, a natural benzophenanthridine alkaloid, is a major active compound present in a well-known traditional Chinese medicinal herb Zanthoxylum nitidum (Roxb) DC. Previous studies found that NC has antifungal, anti-inflammatory and analgesic activities [23, 24]. Recently it has been shown that NC inhibits the growth of many human cancer cells via induction of cell apoptosis . Moreover, Chen et al. reported that NC can suppress gastric cancer angiogenesis by inhibition of STAT3 pathway , and we previously reported that the NC is able to inhibit hepoatocellular carcinoma growth via modulation of JAK1/STAT3 pathway . In order to further elucidate the mechanism of tumorcidal activity of NC, in the present study we evaluated its effect on hepatic cancer growth in vivo and in vitro, and investigated the underlying molecular mechanisms.
Materials and reagents
Nitidine Chloride (NC, purity >98%) was provided from Institute of Sichuan Xianxin Biochemical Technology (Sichuan, China). Matrigel was provided by Becton Dickinson (San Jose, CA, USA). Roswell Park Memorial Institute Medium 1640 (RPMI 1640), Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin, trypsin-EDTA, 5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethyl-benzimidazol-carbocyanine iodide (JC-1), were purchased from Invitrogen (Grand Island, NY, USA). The In Vitro Angiogenesis Assay Kit was purchased from Millipore (Billerica, MA, USA). A fluorescein isothiocyanate (FITC)-conjugated annexin V apoptosis detection kit was provided by Becton Dickinson (San Jose, CA, USA). TUNEL assay kit (TumorTACS in situ) was purchased from R&D Systems (Minneapolis, MN, USA). All antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). BCA Protein Assay Kit was purchased from Tiangen Biotech Co., Ltd. (Beijing, China). Cignal STAT3 Reporter (luc) Kit was obtained from SABiosciences, QIAGEN company (Hilden, Germany). All other chemicals, unless otherwise stated, were obtained from Sigma-Aldrich (St. Louis, MO, USA).
Human hepatic cancer cell lines (HepG2, HCCLM3 and Huh7) and human umbilical vein endothelial cells (HUVECs) were purchased from Xiangya Cell Center (Hunan, China). HepG2 cells and HUVECs were grown in DMEM and RPMI 1640, respectively. Both DMEM and RPMI 1640 were supplemented with 10% (v/v) FBS, 100 units/ml penicillin, and 100 μg/ml streptomycin.
Male BALB/C athymic nude mice (with an initial body weight of 20–22 g) were obtained from Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China) and housed under pathogen-free conditions with controlled temperature (22°C), humidity, and a 12 hour light/dark cycle. Food and water were given ad libitum throughout the experiment. All animal treatments were performed strictly in accordance with international ethical guidelines and the National Institutes of Health Guide concerning the Care and Use of Laboratory Animals. The experiments were approved by the Institutional Animal Care and Use Committee of Fujian University of Traditional Chinese Medicine.
In vivo nude mice xenograft study
Hepatic cancer xenograft mice were produced with HepG2 cells. The cells were grown in culture and then detached by trypsinization, washed, and resuspended in serum-free DMEM. Resuspended cells (5 × 106) mixed with Matrigel (1:1) were subcutaneously injected into the right flank of mice to initiate tumor growth. At 5 days following xenograft implantation (tumor size approximately 3 mm in diameter), mice were randomized into two groups (n = 10) and treated with 4.5 mg/kg of NC (dissolved in saline) or saline daily by intraperitoneal injection, 6 days a week for 18 days. Body weight and tumor size were measured. Tumor size was determined by measuring the major (L) and minor (W) diameter with a caliper. The tumor volume was calculated according to the following formula: tumor volume = π/6 × L × W2. At the end of the experiment, the animals were anaesthetized and tumors were excised and weighed.
Cell viability evaluation by MTT assay
NC was dissolved in DMSO and diluted to working concentrations with culture medium. The final concentration of DMSO in the medium for all cell-based experiments was 0.1%. Cells (HepG2, HCCLM3, Huh7 cells or HUVECs) were seeded into 96-well plates at a density of 1.0 × 104 cells/well in 0.1 ml medium. 24 h later, cells were treated with various concentrations of NC for different time periods. After NC treatment, 10 μl MTT (5 mg/ml in phosphate buffered saline (PBS)) were added to each well, and the samples were incubated for an additional 4 h at 37°C. The purple-blue MTT formazan precipitate was dissolved in 100 μl DMSO. Absorbance was measured at 570 nm using an ELISA reader (BioTek, Model EXL800, USA).
Colony formation assay
HepG2 cells from different treated groups were seeded in 6-well plates with a density of 200 cells per well for 7 days. The medium was discarded and each well was washed twice with PBS carefully. The colonies were fixed in methanol for 20 min and then stained with Giemsa staining solution. The number of colonies with ≥50 cells was counted and colony forming efficiency was calculated (Percentage of colonies = Number of colonies formed/Number of cells inoculated × 100%).
Cell cycle analysis
Cell cycle analysis was carried out by flow cytometry using FACS analysis with propidium iodide (PI) staining. HepG2 cells were treated with various concentrations of NC for 24 h, harvested, adjusted to a concentration of 1 × 106 cells/ml, and fixed in 70% ethanol at 4°C overnight. The fixed cells were washed twice with cold PBS, and incubated for 30 min with RNase (8 μg/ml) and PI (10 μg/ml). The fluorescent signal was detected through the FL2 channel and the proportion of DNA in different phases was analyzed using ModfitLT Version 3.0 (Verity Software House, Topsham).
Apoptosis detection in HepG2 cells by flow cytometry analysis with annexin V/PI staining
After incubation with various concentrations of NC, apoptosis of HepG2 cells were determined by flow cytometry using a fluorescence-activated cell sorting (FACS) caliber (Becton Dickinson, CA, USA) and Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) kit (Becton Dickinson). Staining was performed according to the manufacturer’s instructions. The percentage of cells in early apoptosis was calculated by Annexin V-positivity and PI-negativity, and the percentage of cells in late apoptosis was calculated by Annexin V-positivity and PI-positivity.
Measurement of mitochonrial membrane potential (Δψm) by flow cytometry analysis with JC-1 staining
JC-1 is a cationic dye that exhibits potential mitochondria-dependent accumulation, indicated by a fluorescence emission shift from green to red, which thus can be used as an indicator of mitochondrial potential. In this experiment, 1×106 treated HepG2 cells were resuspended after trypsinization in 1 ml of medium and incubated with 10 μg/ml of JC-1 (Invitrogen) at 37°C, 5% CO2, for 30 min. Both red and green fluorescence emissions were analyzed by flow cytometry after JC-1 staining.
Apoptosis detection in hepatic tumor tissues by TUNEL staining
Six tumors were randomly selected from NC-treatment or control groups. Tumor tissues were fixed in 10% formaldehyde for 12 h, paraffin-embedded and then sectioned into 4-μm-thick slides. Samples were analyzed by TUNEL staining using TumorTACS in situ kit (R&D Systems). Apoptotic cells were counted as DAB-positive cells (brown stained) at five arbitrarily selected microscopic fields at a magnification of 400×. TUNEL-positive cells were counted as a percentage of the total cells.
Immunohistochemistical analysis of hepatic tumor tissues
Six tumors were randomly selected from NC-treatment or control groups. Tumor tissues were fixed in 10% formaldehyde for 12 h, paraffin-embedded, sectioned, and placed on slides. The slides were subjected to antigen retrieval and endogenous peroxidase activity was quenched with hydrogen peroxide. Non-specific binding was blocked with normal serum in PBS (0.1% Tween 20). Rabbit polyclonal antibodies against Ki-67, CD31, Shh and Gli-1 (all in 1:200 dilution, Santa Cruz Biotechnology) were used to detect the relevant proteins. The binding of the primary antibody was demonstrated with a biotinylated secondary antibody, horseradish peroxidase (HRP)-conjugated streptavidin (Dako), and diamino-benzidine (DAB) as the chromogen. The tissues were counterstained with diluted Harris hematoxylin. After staining, five high-power fields (at magnification of 400×) were randomly selected in each slide. The proportion of positive cells in each field was determined using the true color multi-functional cell image analysis management system (Image-Pro Plus, Media Cybernetics, USA). To control for nonspecific staining, PBS was used to replace the primary antibody as a negative control.
Tube formation assay of HUVECs
HUVEC tube formation was examined using the ECMatrix assay kit (Millipore) following the manufacturer’s instructions. Briefly, confluent HUVECs were harvested and diluted (1 × 104 cells) in 50 μl of medium containing various concentrations of NC. The harvested cells were seeded with ECMatrix gel (1:1 v/v) into 96-well plates and incubated for 9 h at 37°C. The cells were photographed using phase-contrast inverted microscopy at a magnification of 100 ×.
Total RNA was isolated from tumor tissues (three tumors were randomly selected from NC-treatment or control groups) or HepG2 cells and HUVECs with TriZol Reagent (Invitrogen). Oligo (dT)-primed RNA (1 μg) was reverse-transcribed with SuperScript II reverse transcriptase (Promega) according to the manufacturer’s instructions. The obtained cDNA was used to determine the mRNA amount of Cyclin D1, CDK4, Bcl-2, Bax, SHH, Gli-1, VEGF and VEGFR2 by PCR with Taq DNA polymerase (Fermentas). GAPDH was used as an internal control. Samples were analyzed by gel electrophoresis (1.5% agarose). The DNA bands were examined using a Gel Documentation System (BioRad, Model Gel Doc XR+, USA).
Western blotting analysis
Three tumors were randomly selected from NC-treatment or control groups. Tumor tissues were homogenized in nondenaturing lysis buffer and centrifuged at 14,000 × g for 15 min. Protein concentrations of the clarified supernatants were determined by BCA protein assay. HepG2 cells or HUVECs (2.5 × 105) in 5 ml medium were seeded into 25 cm2 flasks and treated with the indicated concentrations of NC for 24 h. Treated cells were lysed in mammalian cell lysis buffer (M-PER, Thermo Scientific, Rockford, IL, USA) containing protease (EMD Biosciences) and phosphatase inhibitor (Sigma-Aldrich) cocktails, and centrifuged at 14,000 × g for 15 min. Protein concentrations in cell lysate supernatants were determined by BCA protein assay. Equal amounts of protein from each tumor or cell lysate were resolved on 12% Tris-glycine gels and transferred onto PVDF membranes. The membranes were blocked for 2 h with 5% nonfat dry milk and incubated with the desired primary antibody directed against STAT3, pSTAT3, ERK, pERK, Cyclin D1, CDK4, Bcl-2, Bax, VEGF, VEGFR2 or β-actin (all in 1:1000 dilutions) overnight at 4°C. Appropriate HRP-conjugated secondary antibodies with chemiluminescence detection were used to image the antibody-detected proteins.
Luciferase gene reporter assay
HepG cells were seeded into 96-well plates at a density of 1 × 104 cells/well in 0.1 ml complete DMEM until about 50% confluency and then continuously cultured in FBS- and antibiotics-free medium overnight. Cells were transfected with a mixture of inducible STAT3-responsive firefly luciferase construct and constitutively expressing Renilla luciferase construct using Lipofectamine™ LTX with PLUS™ Reagent. 6 h after transfection the medium was changed back into DMEM complete with FBS, penicillin and streptomycin. After 24 hours of transfection, cells were treated with various contractions of NC for 1 h followed by IL-6 for another 24 h. Cell extracts were prepared and analyzed using Promega Dural Luciferase Reporter Assay System according to the manufacturer’s instruction. The measured firefly luciferase activity was normalized to the activity of Renilla luciferase in the same well.
Data were presented as mean ± SD for the indicated number of independently performed experiments. Statistical analysis was carried out with Student’s t-test and ANOVA. Differences with P < 0.05 were considered to be statistically significant.
Results and discussion
NC inhibits hepatic cancer growth in vitro and in vivo
NC inhibits hepatic cancer cell proliferation via G1/S cell cycle arrest
NC induces hepatic cancer cell apoptosis via activation of the mitochondrion-dependent pathway
NC inhibits hepatic tumor angiogenesis via suppressing the expression of VEGF-A and VEGFR2
NC suppresses STAT3, ERK and SHH pathways
In summary, here for the first time we demonstrate that Nitidine chloride possesses a broad range of anti-cancer activities due to its ability to affect multiple intracellular targets. Our findings suggest that NC could be a novel therapeutic agent for the treatment of hepatic cancer and other malignancies. However, it remains unclear how NC interacts with STAT3, ERK or hedgehog signaling and deactivate it, although we demonstrate these multiple signaling pathways are affected by NC. It is unknown whether NC is a direct transcriptional suppressor of STAT3, ERK, SHH or Gli. In addition, the dose of NC used in in vitro study is as high as 50 μM. The concentration of NC certainly should be much lower if we try to demonstrate its druggability in pharmaceutical research. These intriguing questions must be addressed in future studies before NC can be further developed as a multi-target drug for cancer therapy.
This work was sponsored by the National Natural Science Foundation of China (81073097 and 81001554).
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