Contributed equally
The existence of cancer stem cells (CSCs) or cancer stem-like cells (CSLCs) is regarded as the cause of tumor formation and recurrence. Matrine has been reported to exhibit antitumor effects in cancer cells. In the present study, a preliminary study was performed on the mechanisms of matrine on hepatocellular carcinoma (HCC) stem-like cells. The HCC SMMC-7721 cell line was cultured in tumor stem cell-specific medium to form spheres, and different concentrations (1, 2 and 5 mg/kg) of cisplatin were then used in order to purify the most drug-resistant cells, which were used as CSLCs. An MTT assay was performed to detect the inhibitory effects of matrine against CSLC proliferation. Quantitative polymerase chain reaction (qPCR) and western blot analysis were used to detect changes in cell adhesion regulating gene (CAR), E-cadherin, laminin and fibronectin. As a result, using tryptose sulfite cycloserine medium culture and cisplatin-resistance screening, CSLCs were successfully isolated from the SMMC-7721 cell line. Matrine inhibited the proliferation of CSLCs
Current evidence demonstrates that cancer stem cells (CSCs) or cancer stem like cells (CSLCs) are present in tumors (
Matrine, as an alkaloid, has a wide spectrum of biological activities including immune regulation (
In the present study, CSLCs were isolated and purified from a human liver cancer cell line, and the positive antitumor effects of matrine were observed. Furthermore, the associated mechanism was studied
The human liver cancer SMMC-7721 cell line was purchased from American Type Culture Collection (Manassas, VA, USA). BALB/c nude mice (40 males, aged between 4 and 6 weeks and weighing between 16 and 18 g) were purchased from Shanghai Experimental Animal Center of Chinese Academy of Sciences (Shanghai, China). The nude mice were caged individually under specific-pathogen free conditions in the Laboratory Animal Research Center of Ningbo University at a temperature of 22±2°C, a relative humidity between 40 and 60% and artificially illuminated on an ~12-h light/dark cycle. The air exchange rate was about 18 times/h. All of the nude mice were provided with sterilized normal pellet food and sterile water
The human hepatocellular carcinoma SMMC-7721 cell line was cultured in DMEM containing 10% fetal bovine serum at 37°C in a 5% CO2 atmosphere. At the logarithmic growth phase, cells were harvested, resuspended in tumor stem cell enrichment medium (DMEM serum-free culture medium + 10 ng/ml EGF + 10 ng/ml bFGF + 10 ng/ml noggin + 1,000 µ/ml leukemia inhibitory factor) and then plated into polyhydroxy ehtyl methacrylate pretreatment flasks for two weeks. Subsequent to the formation of clones, cells were digested, centrifuged (1,000 × g for 10 min) at 4°C and resuspended in PBS. The cell suspension (~5×105 cells) was inoculated into the back of nude mice to form a solid tumor. After two weeks, 1, 2 and 5 mg/kg of cisplatin was injected into mice to select the strongest resistance of tumor stem cells. Subsequently, the tumor tissue within the most notable effects of cisplatin intervention was irrigated, trimmed, cut broken, repeatedly digested into single cells and then cultured in tryptose sulfite cycloserine (TSC) medium at 37°C in a 5% CO2 atmosphere. During this time period, the adherent cells were continually discarded. The suspended cells were continuously collected, repeatedly triturated into a single cell suspension and reinoculated in TSC medium.
The expression of CD24 was firstly analyzed using immunofluorescence microscopy. Liver CSC clones from the SMMC-7721 cell line were digested into single cells with trypsin (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and incubated in a blocking solution consisting of 10% normal fetal bovine serum and 0.1% Triton X-100 (Sigma-Aldrich; Merck KGaA) in 0.1 M PBS for 1 h at room temperature. Then, rat anti-human CD24 (cat. no. 563545; dilution, 1:1,000; BD Pharmingen; BD Biosciences, Franklin Lakes, NJ, USA) were added to the cells and incubated at room temperature for 1 h. FITC-conjugated anti-rat IgM monoclonal antibody (cat. no. 553887; dilution, 1:500; BD Pharmingen; BD Biosciences) were applied at 4°C overnight for visualization. Following antibody staining, cells were fixed with 2% paraformaldehyde at room temperature for 15 min and mounted with DAPI-fluoromount-G (SouthernBiotech, Birmingham, AL, USA). Fluorescent micrographs were obtained using Leica DM IL LED inverted fluorescent microscope (magnification, ×100; Leica Microsystems, Inc.).
MTT (Sigma-Aldrich; Merck KGaA) was applied to evaluate the effects on proliferation and viability of liver stem cells. Liver CSC clones were digested into single cells and plated in triplicate at 5,000 cells per well onto 96-well plates, and cultured with different concentrations of matrine (0, 5, 25, 50, 100 and 200 µg/ml). Following incubation for 72 h, 10 µl of MTT solution (5 µg/ml) was added to each well. The plates were then incubated for 4 h at 37°C. Intracellular formazan crystals were dissolved by the addition of 100 µl of isopropanol-hydrochloric acid-sodium dodecyl sulfate solution (10%) to each well. Following overnight incubation at 37°C, the optical density of the samples was determined at 570 nm. Rate of inhibition was calculated using the equation: Cell inhibition rate (%)=(treated group-control group)/control group ×100.
The total RNA of hepatocellular carcinoma SMMC-7721 cells (SMMC-7721), hepatocellular carcinoma stem cell SMMC-7721-sphere (SMMC-7721-sphere) and hepatocellular carcinoma stem cell SMMC-7721-sphere with 50 µg/ml matrine treatment (SMMC-7721-sphere-Matrine) were extracted using the TRIzol® Reagent (Invitrogen; Thermo Fisher Scientific, Inc.) Reverse transcription was performed using 2 µg of total RNA, according to the manufacturer's protocol for Verso 1-Step RT-PCR kit's (Thermo Fisher Scientific, Inc.). Surface marker expression of octamer-binding transcription factor-4 (OCT-4), NANOG, Notch, cluster of differentiation (CD)133, CD24, sex determining region Y-box-2 (SOX-2) and CD90 in tumor stem-like cells was detected using RT-qPCR. Furthermore, the association between mRNA levels of CAR, E-cadherin, laminin and fibronectin and cell invasion and metastasis was also determined. β-actin was used as an internal control. The primers used for amplification are shown in
Collected cells were lysed immediately in RIPA buffer (150 mM NaCl, 50 mM Tris, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, pH 7.4), supplemented with protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland), Protein concentration was determined using the Micro BCA kit (Pierce; Thermo Fisher Scientific, Inc.). Equal amounts of protein (60 µg) were boiled for 5 min, separated by SDS-PAGE and electro-blotted to a nitrocellulose membrane. Following blocking, the blots were incubated with an appropriate dilution of specific rabbit antibodies against E-cadherin (catalog no. 3195S; dilution, 1:1,000; Cell Signaling Technology Inc., Dnavers, MA, USA), laminin (catalog no. ab11575; dilution, 1:1,000; Abcam, Cambridge, UK), fibronectin (catalog no. ab2413; dilution, 1:1,000; Abcam) for 1 h at room temperature. The blots were washed by TBST reagent (50 mM Tris, pH 7.4, 150 mM NaCl, 0.05% Tween-20) three times and then incubated with a 1:2,000 dilution of horseradish peroxidase-conjugated secondary antibody (catalog no. sc-2492; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) for 1 h at room temperature. The blots were washed three times and then developed using a chemiluminescence assay. Finally, β-actin (Cell Signaling Technology, Inc., Danvers, MA, USA) was used as a loading control.
Statistical analysis was performed using SPSS 16.0 (SPSS, Inc., Chicago, IL, USA). All measurement data was analyzed by one-way analysis of variance test, followed by Tukey's test. For data without a normal distribution, Wilcoxon test were used to analyze the difference between the same cell groups exposed to different treatments. P<0.05 was considered to indicate a statistically significant difference. Dose-dependent associations were determined by multiple linear regressions. All experiments were repeated at least twice (with duplicate assays).
Morphological observation revealed that the cultured SMMC-7721 cells possessed the typical characteristics of epithelial cells, and adhered to the bottom of the dish in a monolayer (
qPCR revealed that the expression levels of
MTT results revealed that matrine inhibited the growth of liver cancer stem cells
To explore the mechanisms underlying matrine-induced apoptosis, the expression of CAR, E-cadherin, laminin and fibronectin was detected by RT-qPCR. As shown in
Furthermore, western blot analysis results (
For decades, matrine, as a traditional Chinese herbal medicine, has proved to possess cytoprotective effects and biological safety, which has been used for a number of treatments, including hepatic fibrosis, atherosclerosis, arrhythmias and infectious diseases (
In the present study, tumor stem-like cells were isolated and purified from the human HCC SMMC-7721 cell line, which overexpresses the tumor stem cell specific marker CD24 (
CAR performs a role in the process of tumor recurrence and metastasis. Yamamoto
The present study was supported by Ningbo Medical Project Foundation (grant no. 2011B05) and the Major Science and Technology Planning Program of Ningbo (grant no. 2012C5013).
SMMC-7721 cells cultured in TSC medium formed clones after two weeks. (A) SMMC-7721 cells cultured in Dulbecco's modified Eagle's medium. (B) Following transfer to TSC medium for two weeks, cell spheres were formed. The cell imaging was performed using the Leica DM IL LED microscope (magnification, ×100); scale bar, 20 µm. TSC, tryptose sulfite cycloserine.
Cancer stem cell specific markers were highly expressed in SMMC-7721-sphere and SMMC-7721-sphere-cisplatin cells isolated and purified from the SMMC-7721 cell line. *P<0.05; **P<0.01. CD, cluster of differentiation; SOX-2, sex determining region Y-box-2; OCT-4, octamer binding transcription factor-4.
Comparison of CD24 expression in (A) SMMC-7721-sphere, (B) SMMC-7721-sphere-cisplatin and (C) and SMMC-7721, identified using immunofluorescence. The nucleus was stained with DAPI (blue). CD24 expression examined by primary antibodies followed by fluorescein isothiocyanate-conjugated secondary antibodies (red). Fluorescent micrographs were obtained using the Leica DM IL LED inverted fluorescent microscope (magnification, ×100). CD, cluster of differentiation.
Matrine inhibited the proliferation of liver stem like cells purified from the SMMC-7721 cell line in a dose-dependent manner.
Matrine induced the expression of CAR, E-cadherin, laminin and fibronectin in liver stem like cells (SMMC-7721-sphere-matrine) compared with the SMMC-7721 cell line. *P<0.05; **P<0.01. CAR, cell adhesion regulating gene.
Western blot analysis of the expression of E-cadherin, laminin and fibronectin in different cell lines following matrine treatment for 72 h: (A) SMMC-7721; (B) SMMC-7721-sphere; and (C) SMMC-7721-sphere-matrine.
PCR amplification primers.
Genes | Primers |
---|---|
OCT-4 | F: 5′-CTGGGTTGATCCTCGGACCT-3′ |
R: 5′-CCATCGGAGTTGCTCTCCA-3′ | |
NANOG | F: 5′-TTTGTGGGCCTGAAGAAAACT-3′ |
R: 5′-AGGGCTGTCCTGAATAAGCAG-3′ | |
Notch | F: 5′-GCACTTTCTGTGAGGAGGACAT-3′ |
R: 5′-AGCAGGAGCTCTCTGTGCAGT-3′ | |
CD133 | F: 5′-TCGGAAACTGGCAGATAGCAA-3′ |
R: 5′-GTGAACGCCTTGTCCT-3′ | |
CD24 | F: 5′-TTTGACTAGATGATGAATGCCAAT-3′ |
R: 5′-GGATGTTGCCTCTCCTTCAT-3′ | |
SOX-2 | F: 5′-AAGAGAACACCAATCCCATCCA-3′ |
R: 5′-AGTCCCCCAAAAAGAAGTCCA-3′ | |
CD90 | F: 5′-GTTAGGCTGGTCACCTTCTG-3′ |
R: 5′-GAGATCCCAGAACCATGAACC-3′ | |
CAR | F: 5′-TGTTCATGCCGACGCTTGCA-3′ |
R: 5′-TTCCAACTACACAGTTTATT-3′ | |
E-cadherin | F: 5′-GGTCTCCTCATGGCTTTGCC-3′ |
R: 5′-CACAGTTCTCAAAGCACAGCG-3′ | |
Laminin | F: 5′-CAGGCCCGCAAACAAGCAGC-3′ |
R: 5′-TCCAAGCGTGTGGACCCGGA-3′ | |
Fibronectin | F: 5′-GCCGCCACGTGCCAGGATTA-3′ |
R: 5′-ACCAGTTGGGGAAGCTCGTCTG-3′ | |
β-actin | F: 5′-CTGTCTGGCGGCACCACCAT-3′ |
R: 5′-GCAACTAAGTCATAGTCCGC-3′ |
F, forward; R, reverse; CD, cluster of differentiation; SOX-2, sex determining region Y-box 2; OCT-4, octamer binding transcription factor-4; CAR, cell adhesion regulating gene.
Tumor size in nude mice inoculated with different concentrations of cisplatin (mm2).
Time, days | Control | 1 mg/kg | 2 mg/kg | 5 mg/kg |
---|---|---|---|---|
3 | 520.20±3.36 | 519.70±5.21 | 517.90±7.08 | 510.30±6.09 |
6 | 699.90±5.53 | 650.20±5.71 | 599.90±5.61 |
529.90±5.72 |
9 | 1,000.00±6.99 | 971.10±6.67 | 739.80±5.77 |
560.20±5.71 |
12 | 1,502.00±10.09 | 1,299.10±7.37 | 799.30±5.30 |
529.80±5.14 |
15 | 1,601.00±8.39 | 1,502.30±6.42 | 829.60±4.55 |
520.20±3.35 |
18 | 1,700.50±8.24 | 1,600.90±8.53 | 799.50±5.40 |
499.90±6.72 |
P<0.05
P<0.01.