DHM (Sigma, United States) was solubilized in 100% dimethyl sulfoxide to prepare a primary solution of 50 mmol/L and stored at -20 °C. Rabbit anti-human primary antibodies against Notch1, Hes1, Bcl-2 and Bax were obtained from Cell Signaling Technology (Beverly, MA, the United States of America). Notch1 siRNA was obtained from Santa Cruz Biotechnology (the United States of America). FITC Annexin V staining Kit was brought from BD Biosciences (the United States of America). MTT [3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] was obtained from Sigma-Aldrich Biotechnology (United States).

All patients were diagnosed with pathological primary HCC. Tissue samples were obtained from these patients during hepatectomy surgery. HCC was confirmed by pathology. All tissue samples were frozen in liquid nitrogen. This study was approved by the Medical Ethics Committee of the Affiliated Hospital of Guangdong Medical University.

The cells (SK-Hep-1, QGY7703, MHCC97L, HepG2, QGY7701 and HL7702) were maintained in DMEM supplemented with 10% fetal bovine serum and antibiotics (penicillin and streptomycin) in a humidified 5% CO₂ incubator. Upon reaching 80% confluence, the cells were treated with various concentrations of DHM (0, 5, 25, 50, 100 or 150 μmol/L) for 12 h, 24 h and 36 h and DMSO treatment served as a control.

The cells were placed into 96-well plates and treated with different concentrations of DHM. DMSO treatment served as a control. Twenty μL of MTT (5 mg/mL) solution was transferred to each well, and then the plate was incubated at 37 °C for 4 h. Next, the cell supernatants were removed, and 150 μL of DMSO was added to each well to solubilize the formed crystals. The optical density values were measured at 570 nm with a spectrophotometer (PerkinElmer, the United States of America).

The HepG2 and QGY7701 cells were seeded in the 6-well plates. Following overnight growth, the cells were pretreated with 50, 100, or 150 μmol/L DHM and incubated for 24 h. Following careful collection and suspension in binding buffer, the HepG2 and QGY7701 cells were incubated with 5 mL of annexin V-FITC and 5 mL of PI for 15 min in darkness prior to flow cytometric analysis.

The tissues protein samples were prepared with Fastprep-24 Sample Preparation equipment (MP, United States). The cells treated with DHM were harvested and lysed in lysis buffer to assess the expression of Notch1, Hes1, Bcl-2 and Bax. Equal amounts of protein samples were loaded per well. Membranes containing protein blots were incubated in blocking buffer (5% non-fat milk) for 1 h at room temperature, and then the membranes were incubated with the primary antibodies (1:1000 dilution) at 4 °C overnight. Following a wash with TBST, membranes were incubated with goat anti-rabbit secondary antibodies for 1 h. Detection was performed using an Odyssey Infrared Imaging System (LI-COR Biosciences Inc., Lincoln, NE, the United States of America).

Frozen tissues were used to isolate messenger RNA (mRNA) using TRIzol reagent (Invitrogen, Guangzhou, China), according to the manufacturer’s protocol. Reverse transcription was performed with 1 μg of RNA using the PrimeScript RT Reagent kit with a gDNA Eraser kit (Takara Bio, Inc, Otsu, Japan). The primer sequences used were as follows: Notch1 forward, 5’-CACCCATGACCACTACCCAGTT-3’ and reverse, 5’-CCTCGGACCAATCAGAGATGTT-3’; Hes1 forward, 5’-AGATCTATGCCAGCTGATAAT-3’and reverse, 5’-AAGCTTCACTTAATACAGCT-3’, and 18s forward, 5’-CGGCGACGACCCATTCGAAC-3’ and reverse, 5’-GAATCGAACCCTGATTCCCCGTC-3’. Data were evaluated using the comparative count method and normalized to the corresponding 18S value.

In situ cell death detection kit-POD (Roche, Switzerland) was used to detect the late-stage apoptosis. Cells were seeded in 96well plates and treated with various concentrations of DHM. Cells were fixed with 4% paraformaldehyde, and then counterstained with 4’,6diamidino2phenylindole for 5 min at room temperature in the dark and observed under a fluorescence microscope (Olympus IX70, Japan) to detect the apoptotic cells.

QGY7701 and HepG2 cells were plated into 6-well plates and transfection procedures were performed according to the manufacturer’s protocol. Three different Notch1 siRNA sequences were tested in both QGY7701 and HepG2 cell lines for screening the efficacy of Notch1 knockdown and selecting the best performing siRNA transcript. Notch1 siRNA-2 showed the best degree of knockdown and was used in subsequent experiments. Before each experiment, we transiently transfected cells using an Notch1 siRNA gene knockdown kit (JIMA Biotechnology Company, Shanghai, China) and the negative control siRNA.

Cells in each logarithmic growth phase were detached from culture flasks using 0.25% trypsin. Then, the cells were percussed into a single cell suspension, and 1000 cells were cultured in each well of a 6-well plate, and humidity was saturated for 1 wk. The cells were stained with crystal violet. Clones were directly counted by the naked eye or counted using a microscope, and data were analyzed using ImageJ software.

The data were obtained from at least three independent experiments, and all results are presented as the mean ± SD. The results were evaluated using Student’s t test, Pearson correlation analyses were used to examine the correlation of two parameters. The significance levels were defined as P < 0.05.

The expression of Notch1 protein by Western blots and found that Notch1 accumulated predominantly in HCC tissues (Figure 1A). In addition, the expression of Notch1 and Hes1 genes in HCC and normal liver tissue samples was analyzed by qRT-PCR (Figure 1B and C). Among the tested samples (n = 64), the expression levels of Notch1 (75% of patients) and Hes1 (79.7% of patients) mRNA in tumor tissues were higher than in the normal liver tissues (Figure 1D). A positive correlation between Notch1 and Hes1 expression suggests that high expression of Notch1 in tumors may result in an upregulation of the target gene Hes1 (Figure 1E). To clarify the role of Notch1 in tumorigenesis and tumor differentiation, we analyzed the association among Notch1 expression, AFP concentration, and the degree of tumor differentiation. There was a negative correlation between the expression of Notch1 and the degree of differentiation and a positive correlation with the AFP concentration (Figure 1F and G). The results showed that Notch1 was highly expressed in tumor cells with higher degrees of malignancy and proliferative activity. Based on the results above, Notch1 was involved in the development and progression of HCC.

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Figure 1 Notch1 expression is activated in hepatocellular carcinoma tissues and reflects differentiation properties. Protein levels were detected by Western blot analysis. A: Notch1 and Hes1 proteins were obviously abundant in most of the tumor tissues; B and C: Gene expression levels were measured with qRT-PCR. Notch1 and Hes1 genes were expressed at higher levels in tumor tissues compared with normal liver tissues; D: Notch1 and Hes1 gene expression levels were higher in 75% and 79.7% of the hepatocellular carcinoma patients, respectively; E: A Pearson correlation analysis revealed a correlation between Notch1 and Hes1 expression (r² = 0.1893, P < 0.001); E and G: Notch1 is positively correlated with the AFP level (r² = 0.2025, P = 0.0015) and negatively correlated with the degree of differentiation (r² = 0.1411, P = 0.0093).

We also screened the Notch1 protein expression profiles in several hepatoma cell lines. Notch1 exhibited higher expression levels in hepatoma cells compared with the immortalized normal liver cell line HL7702 (Figure 2B). Among the hepatoma cell lines, we found that QGY7701 and HepG2 cells highly express Notch1. Therefore, we chose these two lines for the next experiment. MTT assays showed that the proliferation of DHM-treated QGY7701 and HepG2 cells was inhibited. After 12 h, 24 h and 36 h, the viability of HCC cells treated with DHM was significantly inhibited in a dose- and time-dependent manner (Figure 2C and D). As shown in Figure 2, the proliferation of cells was significantly inhibited after treatment with 150 μmol/L and 50 μmol/L DHM for 12 h (P < 0.01), and the cell viability of both QGY7701 and HepG2 cells treated with 50 μmol/L DHM for 48 h and 25 μmol/L DHM for 24 h was significantly inhibited (P < 0.001). The microscopy suggested that the proliferation of the two cell lines was inhibited and partial apoptosis occurred compared with the control group (Figure 2E). A colony formation assay was utilized to further determine the proliferative ability of cells treated with various concentrations of DHM, and similarly, DHM suppressed colony-forming capability in a dose-dependent manner (Figure 2F-H).

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Figure 2 Dihydromyricetin inhibits the proliferation of QGY7701 and HepG2 hepatocellular carcinoma cells. A: Chemical structure of DHM; B: The Notch1 expression profiles of several hepatocellular carcinoma (HCC) cell lines were screened by Western blotting; C and D: MTT assays were used to analyze the rate of cell inhibition (means ± SD) of QGY7701 and HepG2 cells treated with dihydromyricetin (DHM) at different concentrations (5, 10, 25, 50, 100 and 150 μmol/L) at 12 h, 24 h and 36 h; E: The proliferation of QGY7701 and HepG2 cells treated with different concentrations of DHM (0, 50, or 100 μmol/L) for 24 h was observed under microscope (× 200 magnification); F: Colony formation capability assay with different concentrations of DHM in HCC QGY7701 and HepG2 cells; G and H: The clones were quantified, and the data are presented as a statistical figure; Each experiment was repeated at least three times. ^aP < 0.05, ^bP < 0.01 and ^cP < 0.001, vs 0 μmol/L (control) group.

Flow cytometry revealed that apoptosis was induced in HepG2 and QGY7701 cell lines following 24 h of DHM treatment (Figure 3A-C). A TUNEL assay was utilized to detect latestage apoptosis of the cell lines treated with various concentrations of DHM for 24 h. In the fluorescence images, there were cells in late-stage apoptosis in both the HepG2 and QGY7701 cell lines (Figure 3D-F).

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Figure 3 Dihydromyricetin induces apoptosis in QGY7701 and HepG2 hepatocellular carcinoma cells. A: Apoptosis of QGY7701 and HepG2 cells treated with DHM at different concentrations (0, 50, or 100 μmol/L) for 24 h was analyzed by flow cytometry; B and C: Apoptosis was quantified and presented as a statistical figure; D: A TUNEL assay was utilized to examine the apoptosis of QGY7701 and HepG2 cells treated with different concentrations (0, 50, or 100 μmol/L) of DHM for 24 h; E and F: Apoptosis was quantified and presented as a statistical figure; Each experiment was repeated more than three times; ^aP < 0.05, ^bP < 0.01, ^cP < 0.001 vs 0 μmol/L (control) group; ^eP < 0.05, ^dP < 0.01, ^fP < 0.001 vs 50 μmol/L group.

In this study, Notch1, Hes1 and apoptosis-related proteins were detected by Western blotting. The results demonstrated that DHM downregulated the expression of Notch1 and Hes1 in QGY7701 and HepG2 cells. Meanwhile, Bax was upregulated, and Bcl-2 was downregulated. The results suggested that down-regulation of Notch1 activated the mitochondrial apoptotic pathway (Figure 4A and C). It is known that a lower ratio of Bcl-2/Bax leads to apoptosis, and our data showed that the ratio of Bcl-2/Bax was reduced (Figure 4B and D).

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Figure 4 Effects of dihydromyricetin treatment on proteins in the Notch1 pathway and on apoptosis-related proteins in hepatocellular carcinoma cells. A and C: The levels of the Notch1, Hes1, Bcl-2, and Bax proteins in QGY7701 and HepG2 cells treated with DHM at different concentrations (0, 50, or 100 μmol/L) were analyzed by western blot; B and D: The Bcl-2/Bax protein ratios were measured by optical density analysis with ImageJ software.

To further elucidate whether the inhibition of proliferation and the promotion of apoptosis of QGY7701 and HepG2 cells were caused by the down-regulation of Notch1 following DHM treatment, siRNA was used to silence Notch1 expression, and Notch1-silenced cells were treated with DHM. The knockout efficiency of the three different Notch1 siRNA sequences is shown in Figure 5A. The expression levels of Notch1, Hes1 and the apoptotic proteins Bcl-2 and Bax were analyzed by Western blot. Compared with the control group, Bax was upregulated, and Hes1 and Bcl-2 were downregulated after the depletion of Notch1 via siRNA. The results were consistent with the DHM 100 μmol/L treatment group. However, the effect of the Notch1-siRNA and DHM 100 μmol/L combination on protein expression was more obvious (Figure 5B and D). As expected, the ratio of Bcl2/Bax decreased the most in the Notch1-siRNA + DHM 100 μmol/L group (Figure 5C and E). In summary, DHM exerted anti-tumor activity by downregulating the expression of Notch1.

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Figure 5 Dihydromyricetin induced apoptosis in hepatocarcinoma cells by downregulating the Notch1 pathway. A: Knockdown efficiency of 3 different Notch1 siRNA sequences was tested in both QGY7701 and HepG2 cell lines by Western blot; B and D: The effects of different treatment methods (Notch1-siRNA, dihydromyricetin 100 μmol/L, and combined treatment) on Notch1, Hes1, Bcl-2, and Bax protein levels as determined by Western blot; C and E: The Bcl-2/Bax protein ratios were measured by optical density analysis with ImageJ software. si-NC: si-negative control; si-N1: si-Notch1-1; si-N2: si-Notch1-2; si-N3: si-Notch1-3.

Hepatocellular carcinoma (HCC) is a highly malignant type of solid tumor which is a serious threat to human health. It is urgent to develop novel chemotherapeutic drugs and therapeutic targets for the treatment of HCC.

Notch-1 was detected to be overexpressed in HCC; Notch1 is involved in the development and progression of HCC. Dihydromyricetin (DHM) has numerous pharmacological activities, such as antiinflammatory, antioxidation and anticarcinogenic effects. However, whether DHM can induce hepatoma cell apoptosis by Notch1 is not yet known.

This is the first study to demonstrate that DHM inhibits cell proliferation and promotes apoptosis by down-regulating the expression of Notch1.

DHM may be used a potential effective candidate agent for the treatment of HCC.

This article is well written. The authors investigate whether DHM inhibits cell proliferation and promotes apoptosis by downregulating Notch1 expression. These results show that the anti-tumor activity of DHM in the QGY7701 and HepG2 HCC cell lines is partially exerted through inhibition of the Notch1 pathway.