Downregulation of the Notch signaling pathway inhibits hepatocellular carcinoma cell invasion by inactivation of matrix metalloproteinase-2 and -9 and vascular endothelial growth factor

  • Authors:
    • Liang Zhou
    • De-Sheng Wang
    • Qing-Jun Li
    • Wei Sun
    • Yong Zhang
    • Ke-Feng Dou
  • View Affiliations

  • Published online on: June 20, 2012     https://doi.org/10.3892/or.2012.1880
  • Pages: 874-882
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Abstract

Hepatocellular carcinoma (HCC) is one of the most common malignancies. The main cause of death in HCC patients is tumor progression with invasion and metastasis. However, the underlying mechanisms of HCC invasion and metastasis are still not fully understood. Some studies show that the Notch signaling pathway may participate in tumor invasion and metastasis. However, the mechanisms by which the Notch signaling pathway mediates tumor cell invasion, especially in hepatocellular carcinoma, are not yet known. In the current study, we investigated the anti-invasion effect of the downregulation of the Notch signaling pathway by DAPT in HCC cells. The Notch signaling pathway inhibitor could suppress invasion of HCC cells via the extracellular signal-regulated kinases 1 and 2 (ERK1/2) signaling pathways, resulting in the downregulation of matrix metalloproteinase-2 and -9 (MMP-2 and -9) and vascular endothelial growth factor (VEGF). These observations suggested that inhibition of the Notch signaling pathway by DAPT would be useful for devising novel preventive and therapeutic strategies targeting invasion of HCC.

Introduction

Hepatocellular carcinoma (HCC) is the seventh most common malignancy and the third leading cause of cancer-related deaths worldwide (1). Despite improvements in detection and clinical treatment strategies, the 5-year survival rate for HCC is still very low (2). The main cause of death in HCC patients is tumor progression with invasion and metastasis. However, the underlying mechanisms of HCC invasion and metastasis are still not fully understood (3). Thus, the discovery and subsequent development of novel agents to block HCC invasion and metastasis are primary research objectives for HCC.

Tumor metastasis occurs by a series of steps, including cell invasion, degradation of basement membranes, and the stromal extracellular matrix, ultimately leading to tumor cell invasion and metastasis. Matrix metalloproteinases (MMPs) are a family of related enzymes that degrade the extracellular matrix (ECM) and activation of these enzymes allow tumor cells access to the vasculature, migration, and invasion into target organs, and the development of tumor metastasis (4). Among the previously reported human MMPs, MMP-2 and MMP-9 play the most important role in tumor invasion and metastasis because of their specificity for type IV collagen which is the principal component of the basement membrane (5,6). Angiogenesis plays an important role in tumor metastasis from the initial stage of carcinogenesis to the end stage of metastatic disease (7). The development of neovasculature in the tumor provides essential functions for growth, invasion and metastasis. VEGF is one of the isolated angiogenic peptides, and is the most well studied angiogenic factor so far. Moreover, VEGF is known to play a vital role in tumor-associated invasion (8,9).

The Notch signaling pathway includes Notch ligands, receptors, negative and positive modifiers, and Notch target transcription factors. As an important signaling pathway, Notch is not only involved in cell development and fate determination, but also plays an important role in tumor development (10,11). The Notch signaling pathway is aberrantly activated in a variety of human tumors, including T-cell acute lymphoblastic leukemia, lung, colorectal, prostate, and breast carcinomas (1215). In contrast to its tumor-facilitative role, the Notch signaling pathway has been identified in B-cell malignancies (16), neural crest tumors (17) and skin cancer (18). Therefore, the Notch signaling pathway seems to function as an oncogene or a tumor suppressor, depending on the tissue type. Pharmacologic manipulation of the Notch signaling pathway is becoming a new strategy for human tumors. γ-secretase inhibitors (GSI) can inhibit the proteolytic processing of Notch receptors by γ-secretase, which is essential for Notch activation (19), and are being investigated clinically in T-cell leukemia and breast cancer.

However, knowledge of the role of the Notch signaling pathway in invasion of HCC is still limited. Therefore, in this study, we investigated the role and mechanism of Notch signaling pathway inhibition by DAPT. DAPT, a GSI, resulted in inhibiting HCC cells invasion in vitro. Our results suggest that the inhibition of Notch signaling pathway caused decreases of MMP-2, MMP-9 and VEGF, thus resulting in the inhibition of HCC cell invasion, mediated through the inactivation of extracellular signal-regulated kinase (ERK) phosphorylation.

Materials and methods

Cell culture and reagents

The human liver non-tumor cell line (HL-7702) and the HCC cell lines (HepG2, HuH-7, SMMC-7721 and MHCC97H) were cultivated in DMEM medium supplemented with 10% fetal calf serum (Sigma Chemicals Co., St. Louis, MO). The liver non-tumor cell and HCC cells were seeded into 6-well cell culture plates at a density of 1×105 cells/well. All experiments were carried out using confluent cultures. To attain normoxic condition, cultures were maintained at 37°C in a humidified incubator containing 20% O2, 5% CO2, and 75% N2. Primary antibodies for MMP-2, MMP-9 and VEGF were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Primary antibodies for the Notch1 intracellular domain (NICD) and ERK1/2 were purchased from Abcam (Cambridge, UK). All secondary antibodies were obtained from Pierce (Rockford, IL). MMP-2 small interfering RNA (siRNA), MMP-9 siRNA, VEGF siRNA, and siRNA control were obtained from Santa Cruz Biotechnology. Lipofectamine 2000 was purchased from Invitrogen (Carlsbad, CA, USA). To suppress the Notch signaling pathway, DAPT in DMSO was used at different doses. To inhibit ERK1/2, PD98059 (Calbiochem, San Diego, CA) in DMSO was used at 10 μmol/l. All other chemicals and solutions were purchased from Sigma-Aldrich, unless otherwise indicated.

Growth curves and cell growth

HepG2 and MHCC97H cells treated with different doses of DAPT were seeded onto 6-well cell culture plates at a density of 5×103 cells/well and were grown for up to 4 days. Each day, we used a hemocytometer to determine the number of cells. We used the relative density of the cells (vs. the density of the primary cells at 100%) to establish the growth curve. Each experiment included six replications and was repeated three times. The data are summarized as means ± SDs.

MTT assay

The HepG2 and MHCC97H cells treated with different doses of DAPT were seeded into 6-well cell culture plates at a density of 1×104 cells/well and were grown for up to 4 days. Cell viability was assessed using the 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay (Sigma Chemicals Co.) in accordance with the manufacturer’s protocol. Each experiment included six replications and was repeated three times. The data are summarized as means ± SDs.

Small interfering RNA transfection

According to the protocol of Lipofectamine 2000, the HepG2 and MHCC97H cells were transfected with MMP-2 siRNA, MMP-9 siRNA, VEGF siRNA and siRNA control respectively. Cells transfected with siRNA were seeded into 6-well cell culture plates at a density of 1×105 cells/well. The cells were allowed to grow further for 24 h and were then harvested for further analysis.

Real-time reverse transcription-PCR analysis for gene expression

Total RNA from different cells was isolated by TRIzol (Invitrogen, Carlsbad, CA, USA) and purified using an RNeasy Mini kit and RNase-free DNase Set (Qiagen, Valencia, CA) according to the protocols of the manufacturer. Total RNA (1 μg) from each sample was subjected to first-strand cDNA synthesis using TaqMan reverse transcription reagent kit (Applied Biosystems, Foster City, CA) in a total volume of 50 μl, including 6.25 units MultiScribe reverse transcriptase and 25 pmol random hexamers. The reverse transcription reaction was performed at 25°C for 10 min followed by 48°C for 30 min and 95°C for 5 min. The primers used in the PCR reaction are as follows: Hes1, forward primer (5′-AGGCGGA CATTCTGGAAATG-3′) and reverse primer (5′-TCGTTCA TGCACTCGCTGA-3′); Hes5, forward primer (5′-ACCGCAT CAACAGCAGCATT-3′) and reverse primer (5′-AGGCTTT GCTGTGCTTCAGGT-3′); Hey1, forward primer (5′-AACTG TTGGTGGCCTGAATC-3′) and reverse primer (5′-GCGGT AAATGCAGGCGTAT-3′); GAPDH, forward primer (5′-AA ATCCCATCACCATCTTCC-3′) and reverse primer (5′-TCA CACCCATGACGAACA-3′). The primers were checked by running a virtual PCR and a primer concentration was optimized to avoid primer dimer formation. Also, dissociation curves were checked in order to avoid a non-specific amplification. Real-time PCR amplifications were undertaken in a M×4000 Multiplex QPCR System (Stratagene, La Jolla, CA) using 2X SYBR-Green PCR Master mix (Applied Biosystems). One microliter of reverse transcription reaction was used for a total volume of 25 μl quantitative PCR reactions. The thermal profile for SYBR real-time PCR was 95°C for 10 min followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min. Data were analyzed according to the comparative Ct method and were normalized by GAPDH expression in each sample.

Protein extraction and western blotting

The cells were lysed in lysis buffer [50 mmol/l Tris (pH 7.5), 100 mmol/l NaCl, 1 mmol/l EDTA, 0.5% NP40, 0.5% Triton X-100, 2.5 mmol/l sodium orthovanadate, 10 μl/ml protease inhibitor cocktail, and 1 mmol/l PMSF] by incubating for 20 min at 4°C. The protein concentration was determined using the Bio-Rad assay system (Bio-Rad Laboratories, Hercules, CA). Total proteins were fractionated using SDS-PAGE and were transferred onto nitrocellulose membranes. The membranes were blocked with 5% non-fat dried milk or bovine serum albumin in 1X TBS buffer containing 0.1% Tween-20 and then were incubated with the appropriate primary antibodies. Horseradish peroxidase-conjugated anti-rabbit or anti-goat IgG was used as the secondary antibody, and the protein bands were detected using the enhanced chemiluminescence detection system (Amersham Pharmacia Biotech, Amersham, UK). Quantification of western blots was performed using laser densitometry, and relative protein expression was then normalized to GAPDH levels in each sample. The results are presented as the means of three independent experiments with error bars representing SDs. For reprobing, membranes were incubated for 30 min at 50°C in a buffer containing 2% SDS, 62.5 mmol/l Tris (pH 6.7), and 100 mmol/l 2-mercaptoethanol, washed and incubated with the desired primary antibody.

Invasion assays

The cell invasion capacity was analyzed by using Matrigel-coated Transwell cell culture chambers (8 μm pore size) (Millipore, Billerica, MA, USA). Briefly, treated cells (5×104 cells/well) were serum-starved for 24 h and were plated in the upper insert of a 24-well culture plates in serum-free medium. Medium containing 10% serum as a chemoattractant was added to the well. The cells were incubated under normoxic conditions for 24 h. Non-invading cells were removed from the upper surface by scrubbing with a cotton swab, after which the membrane was fixed with 4% formaldehyde for 10 min at room temperature and was stained with 0.5% crystal violet for 10 min. Finally, invasive cells were counted at ×200 magnification from 10 different fields of each filter. For treatment with DAPT and PD98059, the cells were pretreated for 2–4 h, and the treatment continued during the invasion experiment.

ELISA assay

Enzyme-linked immunosorbent assay (ELISA) technique (Amersham Pharmacia Biotech) was used to quantify the activity of individual MMP-2, MMP-9, VEGF and ERK1/2. The samples were thawed on ice, and all reagents were equilibrated to room temperature. Assays were carried out according to the manufacturer’s instructions.

Statistical analysis

Each experiment was repeated at least three times. All data were summarized and are presented as means ± SDs. The differences among means were statistically analyzed using a t-test. All statistical analyses were performed using the SPSS 13.0 software (SPSS Inc., Chicago, IL, USA). P<0.05 was considered as statistically significant.

Results

Expression of Notch signaling pathway increased in HCC cells

As illustrated in Fig. 1A, the HCC cell lines showed higher levels of penetration through Transwell cell culture chambers, with Matrigel-coating vs. the liver non-tumor cell. In HCC cell lines, the level of penetration was the lowest in HepG2 cells and was the highest in MHCC97H cells. These results demonstrated that HCC cells had higher invasion capabilities than the liver non-tumor cells. In HCC cells, HepG2 cells had the lowest invasion capability and the MHCC97H cells had the highest invasion capability.

Next, we examined the expression of the Notch signaling pathway in the liver non-tumor and HCC cells. RT-PCR analysis showed that HCC cells had higher mRNA levels of the Notch signaling pathway downstream target genes Hes1, Hes5, and Hey1 compared to the liver non-tumor cells (Fig. 1B-D). NICD also exhibited similar increased tendencies regarding the protein levels (Fig. 1E). These results may illustrate that the expression of the Notch signaling pathway is upregulated in HCC cells. The expression of Hes1, Hes5, Hey1 and NICD were the lowest in HepG2 cells and were the highest in MHCC97H cells. This indicates that the upregulated levels of the Notch signaling pathway may have a correlation with the invasion capability.

DAPT can efficiently downregulate the Notch signaling pathway and inhibit invasion in HCC cells

Invasion capacity was the lowest in the HepG2 cell and the highest in the MHCC97H cells. Therefore, we only used HepG2 and MHCC97H cells for the next experiment. We further investigated the role of DAPT on the expression of the Notch signaling pathway in HepG2 and MHCC97H cells. The cells were treated with DAPT at different doses (1 and 5 μmol/l) and the cells not-treated or treated with DMSO were used as controls. The mRNA expression levels of Hes1, Hes5, and Hey1 were measured by RT-PCR and protein expression of NICD was measured by western blotting. As shown in Fig. 2A-D, HepG2 and MHCC97H cells treated with DAPT reduced the mRNA expression levels of Hes1, Hes5, Hey1, and the protein expression of NICD in a dose-dependent manner. We next sought to determine whether DAPT affected the invasion capabilities of HepG2 and MHCC97H cells. As shown in Fig. 2E and F, the invasion capabilities of HepG2 and MHCC97H cells were strongly inhibited by DAPT in a dose-dependent manner. As shown in Fig. 3, the indicated concentrations of DAPT (1 or 5 μmol/l) had no effect on the growth and viability of HepG2 and MHCC97H cells. These results indicate that the inhibitory effects of DAPT (1 or 5 μmol/l) on cell invasion were independent of cellular cytotoxicity.

MMP-2, MMP-9 and VEGF may participate in HCC cell invasion

MMP-2, MMP-9 and VEGF are associated with enhanced invasion of tumor cells. With western blotting, the protein expressions of MMP-2, MMP-9 and VEGF were upregulated in the HCC cells compared with the liver non-tumor cells at the protein level (Fig. 4A). The proteolytic activities of MMP-2, MMP-9 and VEGF exhibited similar increased tendencies in HCC cells (Fig. 4B-D). The protein expression levels and proteolytic activities of MMP-2, MMP-9 and VEGF were the lowest in the HepG2 cells and the highest in the MHCC97H cells. These results also showed upregulated levels of MMP-2, MMP-9 and VEGF may correlate with invasion capability. The HepG2 and MHCC97H cells were transfected with human MMP-2 siRNA, MMP-9 siRNA, and VEGF siRNA. The cells non-transfected and transfected with control siRNA were as control. siRNA can efficiently downregulate the expression of MMP-2, MMP-9 and VEGF (Fig. 4E-G). Cells transfected with MMP-2 siRNA or MMP-9 siRNA or VEGF siRNA showed a lower level of penetration through the membrane, compared with control siRNA-transfected cells (Fig. 4H and I). These results indicated that MMP-2, MMP-9 and VEGF may participate in HCC cell invasion.

Inhibition of Notch signaling pathway by DAPT decreased the protein expression and proteolytic activities of MMP-2, MMP-9 or VEGF

We determined whether inhibition of Notch signaling pathway by different doses of DAPT could have effects on MMP-2, MMP-9 and VEGF. Western blotting and ELISA assay were used to analyze the protein expression and proteolytic activity. DAPT was able to effectively inhibit the protein expressions of MMP-2, MMP-9 and VEGF in a dose-dependent manner in HepG2 and MHCC97H cells (Fig. 5A). Using ELISA assay (Fig. 5B-G), we found that DAPT also could effectively inhibit the proteolytic activities of MMP-2, MMP-9 and VEGF. These results indicated that the Notch signaling pathway may regulate MMP-2, MMP-9 and VEGF in HCC cells.

Downregulation of the Notch signaling pathway by DAPT inhibited ERK1/2 activity resulting in inhibition of the invasion of HCC cells

ERK1/2 is known to play a major role in signaling pathways concerning invasion, and regulates the expression of MMPs and VEGF. We investigated whether the antagonistic effects of DAPT on the upregulation of MMP-2, MMP-9 and VEGF expression and decreased invasion in HCC cells could be attributed to the inhibition of ERK1/2. As shown in Fig. 6A, using western blot analysis, we found that increasing doses of DAPT abolished ERK1/2 phosphorylation in a dose-dependent manner. We further confirmed that DAPT inhibited ERK1/2 activity by ELISA assay (Fig. 6B and C). The results indicated that DAPT significantly inhibited the activity of ERK1/2 in HCC cells. To further study the relationship between ERK1/2 activity and the Notch signaling pathway in the control of tumor invasion, HepG2 and MHCC97H cells were treated with 10 μmol/l PD98059 and 5 μmol/l DAPT to block ERK1/2 activity and the Notch signaling pathway, respectively (Fig. 6D and E). Treatment with PD98059 or DAPT alone reduced HepG2 and MHCC97H cell invasion. However, treatment with PD98059 in combination with DAPT did not block these biological functions of HCC cells to a greater extent than treatment with PD98059 or DAPT alone. These results suggest that the Notch signaling pathway may modulate HCC cell invasion through ERK1/2 regulating MMP-2, MMP-9 and VEGF.

Discussion

The high recurrence rate of intrahepatic and distant metastasis is a major obstacle in improving the survival rate of patients with HCC (20). If the mechanisms regulating HCC invasion can be clearly defined, there are likely to be key elements that can be exploited therapeutically, reducing metastasis and improving survival. There has been increasing evidence indicating that the Notch signaling pathway increases the metastatic potential of tumor cells by increasing processes such as invasion (21,22). Our present studies investigated the role of the Notch signaling pathway on HCC cell invasion. Our findings indicate that the inhibition of the Notch signaling pathway decreased the protein expression and proteolytic activities of MMP-2, MMP-9, and VEGF by inhibiting ERK1/2 activity and suppressed HCC cell invasion. Taken together, the data indicate that inhibition of the Notch signaling pathway should be further evaluated in HCC invasion therapy.

Invasion and metastasis are the processes by which tumor spreads from the place it first appears as a primary tumor to distant locations in the body. This includes a series of sequential steps, including tumor-induced angiogenesis, tumor invasion, and the establishment of metastatic foci at the secondary site involving various molecules (23,24). The MMPs family proteins are the proteolytic enzymes in ECM that contribute to tumor invasion, angiogenesis and metastasis (25). Among the previously reported human MMPs, MMP-2 and MMP-9 have been implicated in invasion and metastasis because of their role in the degradation of basement membrane collagen (26,27). It was reported that MMP-2 and MMP-9 are correlated with an aggressive, invasive or metastatic tumor phenotype (28,29). It is well known that the MMP inhibitors block endothelial cell activities which are essential for new vessel development leading to proliferation and invasion (30). Therefore, MMP-2 and MMP-9 are considered therapeutic targets of anticancer drugs based on the degrading action of both enzymes on gelatins which are major components of the basement membrane. Another important molecule involved in tumor cell invasion and metastasis is VEGF. The expression of VEGF is commonly found to be upregulated in tumors and there was a trend toward an association between the expression of VEGF and distant metastasis. Investigations by other laboratories have shown that VEGF promotes migration and invasion of tumor cells (31,32). Here, we showed that the protein expressions and proteolytic activities of MMP-2, MMP-9 and VEGF were higher in HCC cells. The inhibition of MMP-2, MMP-9 and VEGF by siRNA can also decrease the invasion capabilities of HCC cells. These results indicate that MMP-2, MMP-9 and VEGF may participate in HCC cell invasion.

The Notch signaling pathway is involved in the carcinogenesis, progress, invasion and neurovascular formation of many malignant tumors (22,3335). The Notch signaling pathway can regulate MMP-2, MMP-9 and VEGF (22,3640), which are important in the processes of invasion and metastasis of tumor. In the current study, the invasion capabilities of HepG2 and MHCC97H cells treated with DAPT decreased. We showed that an increase in the DAPT dose in response to Notch signaling pathway inhibition resulted in suppression of MMP-2, MMP-9 and VEGF. These results suggest that the inhibitory effect of DAPT on HCC cell invasion can be partially attributable to the downregulation of MMP-2, MMP-9 and VEGF. Some studies have shown that the Notch signaling pathway regulates MMPs and VEGF partly due to activation of the NF-γB pathway in cell invasion in pancreatic cancer cells (22). However, the potential mechanisms between Notch signaling pathway, MMPs and VEGF in HCC invasion are poorly understood.

Extracellular signal-regulated kinase 1 and 2 (ERK1/2) belongs to the family of mitogen-activated protein kinases (MAPKs) which play a major role in the signaling pathways concerning scattering/motility, invasion, proliferation and survival (4143). ERK1/2 activation has also been reported to regulate the expression of a variety of important genes in some cellular responses, including metastasis related genes, such as VEGF and MMP-2 and -9 (4447). Because ERK1/2 plays an important role in many cellular processes, studies on the interaction of ERK1/2 activation with other cell signal transduction pathways, including the Notch signaling pathway, has received increased attention in recent years. The Notch signaling pathway has also been reported to crosstalk with the ERK1/2 pathway (48). In the present study, we showed that the downregulation of the Notch signaling pathway by DAPT reduced ERK1/2 activity and concomitantly inhibited the protein expression and proteolytic activities of MMP-2, MMP-9 and VEGF. We also found that the inhibited Notch signaling pathway and/or ERK1/2 has the same role in suppressing invasion of HCC cells. Thus, the downregulation of the Notch signaling pathway results in lower ERK1/2 activity and its downstream targets (MMP-2 and -9 and VEGF). Therefore, it is possible that Notch signaling pathway induced HCC cell invasion is partly due to the activation of the ERK1/2 pathway.

Taken together, our data showed that the Notch signaling pathway inhibitor could suppress invasion of HCC cells via ERK1/2 signaling pathways, resulting in the downregulation of MMP-2, MMP-9 and VEGF. Inhibition of Notch signaling pathway could be useful as a therapeutic target for inhibiting HCC invasion. Further studies will elucidate the mechanism of the Notch signaling pathway and ERK1/2 interaction.

Acknowledgements

We are grateful to Fuqin Zhang who provided technical support. This study was supported by grants from the National Natural Science Foundation of China (grants no. 30872480) and the Major Program of the National Natural Science Foundation of China (grants no. 81030010/H0318).

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September 2012
Volume 28 Issue 3

Print ISSN: 1021-335X
Online ISSN:1791-2431

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Spandidos Publications style
Zhou L, Wang D, Li Q, Sun W, Zhang Y and Dou K: Downregulation of the Notch signaling pathway inhibits hepatocellular carcinoma cell invasion by inactivation of matrix metalloproteinase-2 and -9 and vascular endothelial growth factor. Oncol Rep 28: 874-882, 2012
APA
Zhou, L., Wang, D., Li, Q., Sun, W., Zhang, Y., & Dou, K. (2012). Downregulation of the Notch signaling pathway inhibits hepatocellular carcinoma cell invasion by inactivation of matrix metalloproteinase-2 and -9 and vascular endothelial growth factor. Oncology Reports, 28, 874-882. https://doi.org/10.3892/or.2012.1880
MLA
Zhou, L., Wang, D., Li, Q., Sun, W., Zhang, Y., Dou, K."Downregulation of the Notch signaling pathway inhibits hepatocellular carcinoma cell invasion by inactivation of matrix metalloproteinase-2 and -9 and vascular endothelial growth factor". Oncology Reports 28.3 (2012): 874-882.
Chicago
Zhou, L., Wang, D., Li, Q., Sun, W., Zhang, Y., Dou, K."Downregulation of the Notch signaling pathway inhibits hepatocellular carcinoma cell invasion by inactivation of matrix metalloproteinase-2 and -9 and vascular endothelial growth factor". Oncology Reports 28, no. 3 (2012): 874-882. https://doi.org/10.3892/or.2012.1880