Molecular Cancer Therapeutics Genome- No Abstract
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zhang, S.-Z.
Right arrow Articles by Li, C.-J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zhang, S.-Z.
Right arrow Articles by Li, C.-J.
Mol Cancer Ther. 2005;4:1577-1584
© 2005 American Association for Cancer Research

Knockdown of c-Met by adenovirus-delivered small interfering RNA inhibits hepatocellular carcinoma growth in vitro and in vivo

Sheng-Zhou Zhang1,2, Fei-Yan Pan1, Jian-Feng Xu1, Jun Yuan1, Shi-Ying Guo1, Gu Dai1, Bin Xue1, Wei-Gan Shen1, Chuan-Jun Wen1, Dong-Hong Zhao1 and Chao-Jun Li1

1 Jiangsu Key Laboratory for Molecular and Medical Biotechnology, Nanjing Normal University, Nanjing, PR China and 2 College of Life Sciences, Anhui Normal University, Wuhu, PR China

Requests for reprints: Chao-Jun Li, Jiangsu Key Laboratory for Molecular and Medical Biotechnology, Nanjing Normal University, Nanjing 210097, PR China. Phone: 86-25-8359-8812; Fax: 86-25-8359-8812. E-mail: licj{at}njnu.edu.cn


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
c-Met is highly expressed and constitutively activated in various human tumors. We employed adenovirus-mediated RNA interference technique to knock down c-Met expression in hepatocellular carcinoma cells and observed its effects on hepatocellular carcinoma cell growth in vitro and in vivo. Among the five hepatocellular carcinoma and one normal human liver cell lines we analyzed, c-Met was highly expressed and constitutively tyrosine phosphorylated in only MHCC97-L and HCCLM3 hepatocellular carcinoma cells. Knockdown of c-Met could inhibit MHCC97-L cells proliferation by arresting cells at G0-G1 phase. Soft agar colony formation assay indicated that the colony forming ability of MHCC97-L cells decreased by ~70% after adenovirus AdH1-small interfering RNA (siRNA)/met infection. In vivo experiments showed that adenovirus AdH1-siRNA/met inhibited the tumorigenicity of MHCC97-L cells and significantly suppressed tumor growth when injected directly into tumors. These results suggest that knockdown of c-Met by adenovirus-delivered siRNA may be a potential therapeutic strategy for treatment of hepatocellular carcinoma in which c-Met is overexpressed.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
c-Met is a transmembrane tyrosine kinase receptor for hepatocyte growth factor/scatter factor encoded by the c-met proto-oncogene (1–3). It is composed of a 50 kDa extracellular {alpha}-subunit and a 145 kDa transmembrane ß-subunit with an intracellular tyrosine kinase domain (4, 5). On hepatocyte growth factor binding, c-Met is tyrosine phosphorylated and initiates a range of signals that lead to the activation of cellular behaviors including cell survival, proliferation, migration, and morphogenesis (6–10). Signaling by c-Met has also been shown to be essential for normal embryologic development (11, 12) and is believed to play an important role in tissue growth and regeneration (13), wound healing (14), and angiogenesis (15).

In addition to regulating normal cell functions, c-Met is implicated in the process of tumorigenesis. Aberrant c-Met signaling has been described in a variety of human cancers (16, 17). c-Met mutations, c-Met amplification/overexpression, and acquisition of autonomous growth control through autocrine signaling loops can all contribute to tumorigenesis (16–18).

c-Met expression is elevated and constitutively activated in various human tumors (16–19). It is found to be overexpressed at both mRNA and protein levels in hepatocellular carcinoma (20–22). Overexpression of wild-type Met in hepatocytes of mice enables a ligand-independent activation that leads to hepatocellular carcinoma with a high penetrance (23). Although substantial evidence implicates c-Met overexpression with hepatocarcinogenesis, no studies have assessed the effects of reducing c-Met expression on tumorigenic growth of hepatocellular carcinoma in vitro and in vivo.

In this study, we established that c-Met is highly expressed and constitutively tyrosine phosphorylated in MHCC97-L hepatocellular carcinoma cells. Then, we employed the newly developed adenovirus-delivered small interfering RNA (siRNA) technique (24) to study the effects of knockdown of c-Met on hepatocellular carcinoma cell growth in vitro and in vivo.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tissue Samples, Cell Lines, and Cell Culture Conditions
Twenty hepatocellular carcinoma and surrounding nontumor liver samples were obtained from patients who had undergone surgery for the removal of their tumors in Nanjing Gulou Hospital, Nanjing, China. Informed consent was obtained from each patient and the present study conformed to the ethical guidelines of China.

BEL7402, SMMC7721, HepG2, MHCC97-L, and HCCLM3 cells were all derived from patients with human hepatocellular carcinoma. BEL7402, SMMC7721, and LO2 (normal human liver cells) were purchased from the cell bank of Shanghai Institute of Cell Biology; HepG2 and 293 (Ad5 E1-transformed human embryo kidney cell line) cells were purchased from American Type Culture Collection (Manassas, VA); MHCC97-L and HCCLM3 hepatocellular carcinoma cell lines, which have low and high lung metastatic ability, respectively, were established in the Shanghai Cancer Institute (25). All cells were maintained in DMEM (Life Technologies, Inc., Invitrogen, Carsbad, CA) plus 10% heat-inactivated fetal bovine serum at 37°C in a humidified incubator with 5% CO2.

Recombinant Adenovirus Generation
The cDNA sequence of c-Met was obtained from GenBank3 (accession number NM_000245). The potential target sequences for RNA interference were scanned with the siRNA Target Finder and Design Tool available at the Ambion, Inc. website.4 The target sequence selected, 5'-AACATGGCTCTAGTTGTCGAC-3' (sense), corresponds to a region 349 to 369 bp after the c-met start codon. The target sequence was subcloned into pShuttle-H1 according to the method of Shen et al. (24). The pShuttle-H1-siRNA/Met was recombined with backbone pAdEasy-1 in BJ5183 bacteria. Adenovirus generation, amplification, and titer were done according to the simplified system described by He et al. (26). Viral particles were purified by cesium chloride density gradient centrifugation.

Adenovirus Infection
Cells were incubated with adenovirus in a small volume of serum-free medium at 37°C. After adsorption for 2 hours, fresh complete growth medium was added and cells were placed in the incubator for additional time as indicated for the following experiments. We used a green fluorescent protein–expressing recombinant adenovirus (AdCMV/GFP, stored in our lab) as a control to determine the transfection efficiency.

Reverse Transcription-PCR Analysis
Total RNA was extracted using TRIZOL reagent (Invitrogen, Carsbad, CA) according to the protocol of the manufacturer. Two micrograms of RNA were subjected to reverse transcription. The PCR primers used were as follows: for c-Met, 5'-TGCAAGGGAGAAGACTCCTA-3' (forward), 5'-TGTGTCCACCTCATCATCAG-3' (reverse); for Ron, 5'-GCATGAGATGAATGTGCGTC-3' (forward), 5'-GTCACTGCTGAGTCCACTGT-3' (reverse); for cyclin D1, 5'-GTGGCCTCTAAGATGAAGGA-3' (forward), 5'-GGTCACACTTGATCACTCTG-3' (reverse); and for ß-actin, 5'-TCCTGTGGCATCCACGAAACT-3' (forward), 5'-GAAGCATTTGCGGTGGACGAT-3' (reverse). PCR products were separated on a 1% agarose gel, visualized, and photographed under UV light.

Immunoprecipitation and Western blotting
Cells were extracted using a lysis buffer that contained 50 mmol/L Tris, 150 mmol/L NaCl, 0.1% SDS, 1% NP40, 1 mmol/L Na3VO4, 2 mmol/L NaF, 100 µg/mL phenylmethylsulfonyl fluoride, 1 µg/mL leupeptin, and 1 µg/mL aprotinin for 30 minutes on ice. The protein concentration was quantified using the Bradford method. For immunoprecipitation, a total of 200 µg protein was incubated with l µg of anti-pTyr antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and the complex was recovered with 30 µL of protein A/G beads (Roche, Mannhein, Germany) and rotation at 4°C overnight. For Western blotting, equal amounts of protein were loaded and separated by 10% SDS-PAGE and then electroblotted onto polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA). The membranes were incubated with anti–c-Met antibodies (Zymed Laboratories, South San Francisco, CA). Bound antibody was then visualized using alkaline phosphatase–conjugated secondary antibodies.

Cell Viability, Proliferation, and Anchorage-Independent Growth Assay
Cell viability was examined by routine 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay at 3 days after virus infection at the indicated multiplicity of infection (MOI). A cell proliferation assay was done by counting cell number. MHCC97-L cells were plated at a density of 5.0 x 104 cells/mL in 24-well plates in triplicate and infected with virus at an MOI of 150. Cells were harvested daily and counted using a hemocytometer. A soft agar colony formation assay was used to assess the anchorage-independent growth ability of cells. Specifically, MHCC97-L cells were infected with virus for 24 hours at an MOI of 150. Cells were then plated on a 0.6% agarose base in six-well plates (1.0 x 104 per well) in 1 mL of DMEM medium containing 10% fetal bovine serum and 0.3% agarose. Colonies >50 µm were counted 15 days after plating.

Cell Cycle Assay
Three and five days after infection, MHCC97-L cells were collected and fixed. After incubation in RNase A (1 µg/mL) for 30 minutes at 37°C, the cells were stained with propidium iodide (50 µg/mL). Flow cytometric analysis was done using a FACScan instrument (Becton Dickinson, Mountain view, CA) and CellQuest software.

In vivo Tumorigenicity Assay
Six- to eight-week-old athymic BALB/c-nu/nu mice were obtained from Animal Center of Chinese Academy of Science (Shanghai, China) and housed in laminar flow cabinets under specific pathogen-free conditions. Adenovirus-infected MHCC97-L cells were injected into the flanks of mice in a total volume of 100 µL (5.0 x 106 cells). The tumor-bearing mice were sacrificed 42 days after inoculation and the tumors were removed and weighed.

Gene Therapy Studies
MHCC97-L cells were injected into the flanks of BALB/c nude mice in a total volume of 100 µL (5.0 x 106 cells). Tumors were allowed to grow in vivo for 15 days, reaching an average size of 0.5 cm in diameter. Before therapy, the animals were randomized and regrouped by tumor size (eight mice per group). Intratumoral and peritumoral injections of 100 µL of AdH1-siRNA/met suspension, AdH1-null virus suspension, or mock injection were then done (using 5.0 x 1010 particles of virus/dose) every 5 days until animals received a total of six injections. Tumor sizes were measured every 5 days. Tumor volumes (in cubic millimeter) were calculated as length x width2 x 0.52 (27).

Immunohistochemistry
Tissue samples were fixed in 10% buffered formalin solution and embedded in paraffin. For c-Met immunostaining, rabbit anti–c-Met polyclonal antibody (Zymed Laboratories) was used at 1:300 dilution. For proliferating cell nuclear antigen (PCNA) immunostaining, rabbit anti-PCNA polyclonal antibody (Calbiochem, San Diego, CA) was used at 1:500 dilution. The tumor sections were visualized using a streptavidin peroxidase kit (Beijing Zhongshan Company, Beijing, China).

Statistical Analysis
All data are expressed as mean ± SD. Statistical significance was determined by Student's t test or by ANOVA in the case of comparison of multiple groups. P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
c-Met Expression Is Elevated in ~45% of Hepatocellular Carcinoma Patient Samples
c-Met is overexpressed in various human tumors including hepatocellular carcinomas (16–22). Here, we investigated the expression of c-Met in hepatocellular carcinoma patient samples in China. Our results indicated that c-Met is located in both the cell membrane and the cytoplasm (Fig. 1A); 9 of the 20 hepatocellular carcinomas exhibited c-Met overexpression, with an increase ranging between 2- and 7-fold when compared by densitometry with the surrounding nontumor liver. By contrast, in the remaining 11 cases, c-Met expression was almost identical to that of the surrounding nontumoral liver tissue. Tumorigenesis is associated with multiple factors and c-Met overexpression may be an important factor in hepatocellular carcinoma; thus, we hypothesize that knockdown of c-Met expression may inhibit the growth of hepatocellular carcinoma in which c-Met is overexpressed.



View larger version (48K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 1. A, immunohistochemical staining for c-Met in nontumor liver samples (left) and hepatocellular carcinoma tissues (right); original magnification, x 400. B, c-Met is highly expressed in MHCC97-L and HCCLM3 hepatocellular carcinoma cells. The indicated cells were lysed and subjected to Western blot analysis with anti–c-Met or anti–ß-tubulin antibodies; ß-tubulin levels were used as internal controls. Results of densitometric quantification of c-Met/ß-tubulin ratios. c-Met expression in MHCC97-L and HCCLM3 hepatocellular carcinoma cells is 7- to 8-fold higher than that of LO2, BEL7402, SMMC7721, and HepG2 cells. C, c-Met is constitutively tyrosine phosphorylated in MHCC97-L and HCCLM3 hepatocellular carcinoma cells. The cell lysate was immunoprecipitated using anti-pTyr antibodies, then subjected to Western blot analysis with anti–c-Met antibodies.

 
c-Met Is Highly Expressed and Constitutively Tyrosine Phosphorylated in MHCC97-L and HCCLM3 Hepatocellular Carcinoma Cells
To confirm our hypothesis, we analyzed c-Met status in several established hepatocellular carcinoma cell lines to identify cell lines in which c-Met was overexpressed. Among the five hepatocellular carcinoma lines and one normal human liver cell line analyzed, MHCC97-L and HCCLM3 highly expressed c-Met. c-Met protein level was 7- to 8-fold higher in MHCC97-L and HCCLM3 than in LO2, BEL7402, SMMC7721, and HepG2 cell lines (Fig. 1B). Furthermore, c-Met was tyrosine autophosphorylated even without hepatocyte growth factor stimulation (Fig. 1C).

Construction of Recombinant Adenovirus AdH1-siRNA/met and Its Effects on c-Met Expression
To knock down c-Met expression, we employed the newly developed adenovirus-delivered siRNA technique and constructed recombinant adenoviruses, AdH1-siRNA/met expressing siRNA targeting c-Met and AdH1-null (control) viruses.

Reverse transcription-PCR analysis indicated that c-Met mRNA expression in MHCC97-L cells was dramatically reduced after infected with AdH1-siRNA/met, and in the same cells expression of Ron, a c-Met related tyrosine kinase (17), was unaffected (Fig. 2A). Thus, adenovirus AdH1-siRNA/met could effectively knock down c-Met expression in a sequence-specific manner. Western blot analysis showed that AdH1-siRNA/met decreased c-Met expression in a MOI-dependent manner. When the AdH1-siRNA/met MOI was as high as 150, c-Met expression was knocked down by over 90% whereas AdH1-null infection did not affect c-Met expression (Fig. 2B).



View larger version (38K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 2. A, analysis of c-Met mRNA expression by reverse transcription-PCR. c-Met mRNA expression in AdH1-siRNA/met–infected MHCC97-L cells was dramatically reduced compared with either AdH1-null– or mock-infected cells. Expression levels of Ron, a c-Met related tyrosine kinase, and the control ß-actin did not show any changes. B, Western blot analysis of c-Met protein expression with anti–c-Met antibody; ß-tubulin levels were used as internal controls. Results of densitometric quantification of c-Met/ß-tubulin ratios. Infection with AdH1-siRNA/met caused c-Met expression to decrease in a MOI-dependent manner. Similar results were obtained in three independent experiments and only one was shown here.

 
MHCC97-L Cell Proliferation Is Inhibited by AdH1-siRNA/met
Given that AdH1-siRNA/met could effectively decrease c-Met expression, its effects on the proliferation of hepatocellular carcinoma cells in vitro were investigated. As shown in Fig. 3A, compared with AdH1-null or mock infection, AdH1-siRNA/met decreased the viability of MHCC97-L cells in a MOI-dependent manner. A significant inhibition was shown at MOIs of ≥50 (P < 0.01). When the MOI was 150, the viability of MHCC97-L cells was significantly reduced by 35% (P < 0.01). Cell count experiments showed that the cell proliferation of AdH1-siRNA/met–infected MHCC97-L cells was significantly inhibited at 3 days of infection (P < 0.01) and the average proliferation inhibition rates at 3, 4, and 5 days were 29%, 35%, and 38%, respectively (Fig. 3B). Furthermore, AdH1-siRNA/met reduced MHCC97-L cell colony formation by ~70% (P < 0.01; Fig. 3C).



View larger version (16K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 3. A, the viability of MHCC97-L cells was decreased after infection with adenovirus AdH1-siRNA/met. Analysis of cell viability was done by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Compared with AdH1-null or mock infection, AdH1-siRNA/met decreased the viability of MHCC97-L cells in a MOI-dependent manner. A significant inhibition was shown at MOIs of ≥50 (P < 0.01). B, cell growth curve of MHCC97-L cells. Cell proliferation of AdH1-siRNA/met–infected MHCC97-L cells was significantly inhibited on the third day of infection (P < 0.01) and the average proliferation inhibition rates at 3, 4, and 5 d were 29%, 35%, and 38%, respectively. C, adenovirus AdH1-siRNA/met inhibits MHCC97-L cell colony formation in soft agar. MHCC97-L cells infected for 24 h with AdH1-siRNA/met, with AdH1-null at an MOI of 150, or mock infected were subjected to colony formation assay as described in Materials and Methods. The adenovirus AdH1-siRNA/met reduced MHCC97-L cell colony formation by ~70%. D, cell viability of LO2, BEL7402, SMMC7721, HepG2, and HCCLM3 cells after infection with adenovirus was analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Columns, mean (n = 3); bars, SD. *, P < 0.01 compared with control.

 
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay indicated that AdH1-siRNA/met could also inhibit HCCLM3 cell growth by over 30% at 3 days of infection (P < 0.01) but it could only inhibit BEL7402, SMMC7721, and HepG2 cell growths by 8% to 10% and did not significantly affect the growth of LO2 cells (Fig. 3D).

AdH1-siRNA/met Arrests MHCC97-L Cells at G1-G0 Phase
To analyze the mechanisms by which AdH1-siRNA/met inhibits cell proliferation, flow cytometric analysis was applied to analyze the cell cycle of MHCC97-L cells after infection with adenovirus for 3 and 5 days. As shown in Fig. 4A, the percentage of cells at G1-G0 phase was increased from 52.83% (AdH1-null) to 63.41% (AdH1-siRNA/met) and the S-phase cells were decreased from 31.71% to 22.89% after 3 days of infection. Similar results were observed after infection for 5 days. No apoptosis peak was detected before G1-G0 phase. Cyclin D1 was a critical regulator of the G1-S transition (28). Reverse transcription-PCR analysis indicated that cyclin D1 expression in MHCC97-L cells was reduced by ~40% after infection with AdH1-siRNA/met (Fig. 4B). These data show that knockdown of c-Met can arrest cells at G1-G0 phase.



View larger version (31K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 4. A, flow cytometric analysis of the cell cycle of MHCC97-L cells. MHCC97-L cells infected with AdH1-siRNA/met, with AdH1-null, or mock infected for 3 and 5 d were collected for flow cytometric analysis. More cells were arrested at G1-G0 phase after AdH1-siRNA/met infection compared with AdH1-null or mock infection. B, analysis of cyclin D1 mRNA expression by reverse transcription-PCR. Cyclin D1 mRNA expression was reduced by ~40% in AdH1-siRNA/met–infected MHCC97-L cells. Results of densitometric quantification of cyclin D1/ß-actin ratios.

 
AdH1-siRNA/met Inhibits Tumorigenicity In vivo
Next, we checked the tumorigenicity of MHCC97-L cells after knockdown of c-Met in vivo. All mice (eight of eight) developed tumors from mock-infected or AdH1-null–infected MHCC97-L cells (control) without significant difference in tumor weight. In contrast, only five of eight mice developed tumors from AdH1-siRNA/met–infected MHCC97-L cells and the tumors were significantly smaller than those of control mice (Fig. 5). These results suggest that c-Met plays an important role in tumorigenicity.



View larger version (54K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 5. Adenovirus AdH1-siRNA/met inhibits tumorigenicity in vivo. Ex vivo assay was done as described in Materials and Methods. Six weeks after injection, tumors were harvested and analyzed. Results of tumor weight. Tumors from AdH1-siRNA/met–infected MHCC97-L cells were significantly smaller than those of control mice. Columns, mean (n = 5); bars, SD. *, P < 0.01 compared with control.

 
AdH1-siRNA/met Inhibits Tumor Growth In vivo
We further investigated the possibility to use c-met as a target gene of hepatocellular carcinoma therapy. As shown in Fig. 6A, intratumoral and peritumoral injections of AdH1-siRNA/met could inhibit tumor growth; significant inhibition was shown on the third injection (P < 0.05). Tumors in AdH1-siRNA/met–injected mice exhibited ~61% growth inhibition relative to control (AdH1-null) after being injected six times (P < 0.01). Immunostaining and Western blotting showed that c-Met expression was significantly reduced after AdH1-siRNA/met treatment (Fig. 6B and C). PCNA-positive cells in AdH1-siRNA/met–treated tumors were also significantly decreased (P < 0.05; Fig. 6B); the proliferative indices of mock-, AdH1-null–, and AdH1-siRNA/met–injected tumors were 72.8%, 69.8%, and 23.4%, respectively (Fig. 6D). These results suggest that knockdown of c-Met has therapeutic potential application for hepatocellular carcinoma.



View larger version (67K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 6. Adenovirus AdH1-siRNA/met inhibits tumor growth in vivo. A, tumor growth curve after injection with AdH1-siRNA/met, AdH1-null, or mock injection every 5 d for a total of six times. Intratumoral and peritumoral injections of AdH1-siRNA/met could inhibit tumor growth; a significant inhibition was shown on the third injection. Points, mean (n = 8); bars, SD. *, P < 0.05; **, P < 0.01, compared with controls. B, left to right, tumor sections obtained from mock-, AdH1-null–, and AdH1-siRNA/met–injected tumors, respectively, were immunostained using anti–c-Met (top) and anti-PCNA (bottom) antibodies. In AdH1-siRNA/met–treated tumors, c-Met expression was significantly reduced and PCNA-positive cells were significantly decreased. C, c-Met expression of the tumors after injection with AdH1-siRNA/met, AdH1-null, or mock injection was analyzed by Western blotting. D, the proliferative index was obtained by counting the average number of PCNA-positive cells relative to the total number of cells on the photomicrographs. The proliferative index of AdH1-siRNA/met–injected tumors was significantly lower than that of AdH1-null– or mock-injected tumors. Columns, mean (n = 5); bars, SD. *, P < 0.01 compared with controls.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Hepatocellular carcinoma is the fourth most common cause of death from cancer and China alone accounts for 53% of all liver cancer deaths worldwide (29). With the expectation of increasing therapeutic efficacy, gene therapy is being investigated as a new treatment modality (30, 31). c-Met has been considered a very important target molecule for cancer therapy because it plays a pivotal role in tumorigenesis (17, 18). In this study, we detected that c-Met expression is elevated in ~45% of hepatocellular carcinoma patient samples in China, and consequently, we hypothesized that overexpression of c-Met may be associated with hepatocarcinogenesis and knockdown of c-Met may inhibit tumorigenic growth of hepatocellular carcinoma in which c-Met is overexpressed. We then screened an established hepatocellular carcinoma cell line, MHCC97-L, in which c-Met is highly expressed and constitutively tyrosine phosphorylated to confirm our hypothesis.

RNA interference is a newly developed technique to knock down specific gene expressions and has potential application in cancer gene therapy studies (32–34). Several vectors have been developed to deliver siRNA. Advantages such as the availability of high virus titer, infection of a broad spectrum of cell types and nondependence on active cell division, and efficient delivery of siRNA both in vitro and in vivo make adenovirus the choice for siRNA delivery in cancer gene therapy studies (24). We employed this technique and constructed a recombinant adenovirus, AdH1-siRNA/met targeting c-Met. Our results showed that this adenovirus could specifically decrease c-Met expression of hepatocellular carcinoma cells by over 90%. Recently, Shinomiya et al. (35) reported RNA interference targeting Met could reduce Met expression by 62% to 100% with different efficiencies in different cell lines; hepatocellular carcinoma cell lines were not included in their studies. Our results indicated that the adenovirus-delivered siRNA technique was effective in knocking down c-Met expression in hepatocellular carcinoma cells in vitro and in vivo.

c-Met antisense oligonucleotides and ribozymes have also been used to reduce c-Met expression (36–42). Using antisense oligonucleotide strategies, Kaji et al. (36) and Stabile et al. (37) reported respectively that knockdown of c-Met could inhibit proliferation or invasiveness of human gastric cancer cells and suppress growth of human non–small-cell lung tumors. Abounader el al. (38, 39) designed an U1snRNA/ribozyme for targeting c-Met and reported that it reversed the malignancy of glioma cells, inhibited their growth, and promoted apoptosis. Ribozyme targeting of c-Met could also inhibit growth or invasion of human breast cancer cells, colorectal carcinoma cells, and prostate tumor cells (40–42). In most of these experiments, c-Met expression was decreased by 30% to 50%. In colon tumor cells, Herynk et al. (41) have shown that reductions of only ~30% in c-Met expression are sufficient to decrease the ability of these cells to form tumors in the livers of nude mice. Adenovirus-delivered siRNA represents an alternative therapeutic strategy to suppress c-Met expression. Our in vitro experiments indicated that adenovirus AdH1-siRNA/met inhibited MHCC97-L hepatocellular carcinoma cell growth in a MOI-dependent manner at MOIs of ≥25 and a significant inhibition was seen at an MOI of 50 (P < 0.01), which caused c-Met expression to be reduced by ~70%. In vivo experiments showed that AdH1-siRNA/met inhibited tumorigenicity of MHCC97-L cells and suppressed tumor growth significantly when injected directly into tumors.

Sustained activity of an oncogene is required for maintenance of tumors (43). There are at least three possible results when the oncogenes are inactivated: First, malignant cells might differentiate into normal cells. Felsher and his coworkers (44) discovered that some tumor cells differentiate into hepatocytes and some differentiate into hepatocyte stem cells when they turn Myc of hepatocellular carcinoma cells off. Second, as most oncogenes are mitogens, their inactivation might lead to proliferative arrest. Third, tumor cells undergo apoptosis. Our in vitro and in vivo experiments showed that the proliferation of hepatocellular carcinoma cells was significantly inhibited after knockdown of c-Met by adenovirus-delivered siRNA, which supported the second mechanism. p42/p44 mitogen-activated protein kinase is constitutively activated in tumor cells with constitutive phosphorylation of Met and Met RNA interference could suppress the activation of p42/p44 mitogen-activated protein kinase completely (35). Cyclin D1, a downstream molecule of p42/p44 mitogen-activated protein kinase (45), is a critical regulator of the G1-S transition (28). Our results indicated that cyclin D1 expression in MHCC97-L cells was reduced and the cells were arrested at G1-G0 phase after infection with AdH1-siRNA/met. Shinomiya et al. (35) reported that RNA interference targeting Met could promote apoptosis in several tumor types. Here, we did not detect apoptosis of AdH1-siRNA/met–infected MHCC97-L cells by flow cytometric analysis in our experiments and we also could not find apoptotic cells by terminal deoxyribonucleotidyl transferase–mediated dUTP nick end labeling assay and morphologic observation under microscopes; thus, knockdown of c-Met caused hepatocellular carcinoma cell growth arrest by a mechanism that does not depend on increased apoptosis, which may be associated with cell type–specific effects. It has been reported that c-Met and Fas formed a complex on the cell surface; when c-Met parts from Fas, cell apoptosis was initiated by the Fas ligand/Fas signaling pathway (46). Here, we found that no Fas is expressed in MHCC97-L cells by Western blotting; thus, it is understandable that MHCC97-L cells did not undergo apoptosis after c-Met silencing.

Because c-Met is implicated in the process of tumorigenesis, it has been an attractive target for tumor gene therapy (17, 18). Our in vitro experiments indicated that adenovirus AdH1-siRNA/met did not affect growth of LO2 cells significantly. Huh et al. showed that loss of c-met did not seem to be detrimental to hepatocyte function under physiologic conditions (47). We also examined the effects of AdH1-siRNA/met on the growth of BEL7402, SMMC7721, and HepG2 cells in which c-Met expression is almost identical to that of LO2 cells, and only 8% to 10% of growth inhibition rates were observed. AdH1-siRNA/met could significantly inhibit HCCLM3 cell growth. Therefore, our studies suggest that knockdown of c-Met by adenovirus-delivered siRNA may be a potential therapeutic strategy in the treatment of hepatocellular carcinoma in which c-Met is overexpressed.


   Acknowledgments
 
We thank C. Shen and S.N. Reske for the pShuttle-H1 vector and T.C. He and B. Vogelstein for the plasmid pAdEasy-1.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

3 http://www.ncbi.nlm.nih.gov/entrez Back

4 http://www.ambion.com/techlib/misc/siRNA_finder.html Back

Received 4/ 6/05; revised 7/22/05; accepted 8/10/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Cooper CS, Park M, Blair DG, et al. Molecular cloning of a new transforming gene from a chemically transformed human cell line. Nature 1984;311:29–33.[CrossRef][Medline]
  2. Bottaro DP, Rubin JS, Faletto DL, et al. Identification of the hepatocyte growth factor receptor as the c-met proto-oncogene product. Science 1991;251:802–4.[Abstract/Free Full Text]
  3. Naldini L, Weidner KM, Vigna E, et al. Scatter factor and hepatocyte growth factor are indistinguishable ligands for the MET receptor. EMBO J 1991;10:2867–78.[Medline]
  4. Gonzatti-Haces M, Seth A, Park M, Copeland T, Oroszlan S, vande Woude GF. Characterization of the TPR-MET oncogene p65 and the MET proto-oncogene p140 protein-tyrosine kinases. Proc Natl Acad Sci U S A 1988;85:21–5.[Abstract/Free Full Text]
  5. Giordano S, Di Renzo MF, Narsimhan RP, Cooper CS, Rosa C, Comoglio PM. Biosynthesis of the protein encoded by the c-met proto-oncogene. Oncogene 1989;4:1383–8.[Medline]
  6. Naldini L, Vigna E, Ferracini R, et al. The tyrosine kinase encoded by the MET proto-oncogene is activated by autophosphorylation. Mol Cell Biol 1991;11:1793–803.[Abstract/Free Full Text]
  7. Ponzetto C, Bardelli A, Zhen Z, et al. A multifunctional docking site mediates signaling and transformation by the hepatocyte growth factor/scatter factor receptor family. Cell 1994;77:261–71.[CrossRef][Medline]
  8. Gherardi E, Stoker M. Hepatocyte growth factor-scatter factor: mitogen, motogen, and met. Cancer Cells 1991;3:227–32.[Medline]
  9. Taher TE, Derksen PW, de Boer OJ, et al. Hepatocyte growth factor triggers signaling cascades mediating vascular smooth muscle cell migration. Biochem Biophys Res Commun 2002;298:80–6.[CrossRef][Medline]
  10. Bussolino F, Di Renzo MF, Ziche M, et al. Hepatocyte growth factor is a potent angiogenic factor which stimulates endothelial cell motility and growth. J Cell Biol 1992;119:629–41.[Abstract/Free Full Text]
  11. Uehara Y, Minowa O, Mori C, et al. Placental defect and embryonic lethality in mice lacking hepatocyte growth factor/scatter factor. Nature 1995;373:702–5.[CrossRef][Medline]
  12. Schmidt C, Bladt F, Goedecke S, et al. Scatter factor/hepatocyte growth factor is essential for liver development. Nature 1995;373:699–702.[CrossRef][Medline]
  13. Michalopoulos GK, DeFrances MC. Liver regeneration. Science 1997;276:60–6.[Abstract/Free Full Text]
  14. Neuss S, Becher E, Woltje M, Tietze L, Jahnen-Dechent W. Functional expression of HGF and HGF receptor/c-met in adult human mesenchymal stem cells suggests a role in cell mobilization, tissue repair, and wound healing. Stem Cells 2004;22:405–14.[Abstract/Free Full Text]
  15. Grant DS, Kleinman HK, Goldberg ID, et al. Scatter factor induces blood vessel formation in vivo. Proc Natl Acad Sci U S A 1993;90:1937–41.[Abstract/Free Full Text]
  16. Stuart KA, Riordan SM, Lidder S, Crostella L, Williams R, Skouteris GG. Hepatocyte growth factor/scatter factor-induced intracellular signaling. Int J Exp Pathol 2000;81:17–30.[CrossRef][Medline]
  17. Maulik G, Shrikhande A, Kijima T, Ma PC, Morrison PT, Salgia R. Role of the hepatocyte growth factor receptor, c-Met, in oncogenesis and potential for therapeutic inhibition. Cytokine Growth Factor Rev 2002;13:41–59.[CrossRef][Medline]
  18. Danilkovitch-Miagkova A, Zbar B. Dysregulation of Met receptor tyrosine kinase activity in invasive tumors. J Clin Invest 2002;109:863–7.[CrossRef][Medline]
  19. Inoue T, Kataoka H, Goto K, et al. Activation of c-Met (hepatocyte growth factor receptor) in human gastric cancer tissue. Cancer Sci 2004;95:803–8.[Medline]
  20. Boix L, Rosa JL, Ventura F, et al. C-met mRNA overexpression in human hepatocellular carcinoma. Hepatology 1994;19:88–91.[CrossRef][Medline]
  21. Grigioni WF, Fiorentino M, D'Errico A, et al. Overexpression of c-met proto-oncogene product and raised Ki67 index in hepatocellular carcinomas with respect to benign liver conditions. Hepatology 1995;21:1543–6.[CrossRef][Medline]
  22. Ueki T, Fujimoto J, Suzuki T, Yamamoto H, Okamoto E. Expression of hepatocyte growth factor and its receptor c-met proto-oncogene in hepatocellular carcinoma. Hepatology 1997;25:862–6.[CrossRef][Medline]
  23. Wang R, Ferrell LD, Faouzi S, Maher JJ, Bishop JM. Activation of the Met receptor by cell attachment induces and sustains hepatocellular carcinomas in transgenic mice. J Cell Biol 2001;153:1023–34.[Abstract/Free Full Text]
  24. Shen C, Buck AK, Liu X, Winkler M, Reske SN. Gene silencing by adenovirus-delivered siRNA. FEBS Lett 2003;539:111–4.[CrossRef][Medline]
  25. Li Y, Tang ZY, Ye SL, et al. Establishment of cell clones with different metastatic potential from the metastatic hepatocellular carcinoma cell line MHCC97. World J Gastroenterol 2001;7:630–6.[Medline]
  26. He TC, Zhou S, da Costa LT, Yu J, Kinzler KW, Vogelstein B. A simplified system for generating recombinant adenoviruses. Proc Natl Acad Sci U S A 1998;95:2509–14.[Abstract/Free Full Text]
  27. Fahmy RG, Dass CR, Sun LQ, Chesterman CN, Khachigian LM. Transcription factor Egr-1 supports FGF-dependent angiogenesis during neovascularization and tumor growth. Nat Med 2003;9:1026–32.[CrossRef][Medline]
  28. Baldin V, Lukas J, Marcote MJ, Pagano M, Draetta G. Cyclin D1 is a nuclear protein required for cell cycle progression in G1. Genes Dev 1993;7:812–21.[Abstract/Free Full Text]
  29. Pisani P, Parkin DM, Bray F, Ferlay J. Estimates of the worldwide mortality from 25 cancers in 1990. Int J Cancer 1999;83:18–29.[Medline]
  30. Friedmann T. Progress toward human gene therapy. Science 1989;244:1275–81.[Abstract/Free Full Text]
  31. Strauss M. Liver-directed gene therapy: prospects and problems. Gene Ther 1994;1:156–64.[Medline]
  32. McManus MT, Sharp PA. Gene silencing in mammals by small interfering RNAs. Nat Rev Genet 2002;3:737–47.[CrossRef][Medline]
  33. Cheng JC, Moore TB, Sakamoto KM. RNA interference and human disease. Mol Genet Metab 2003;80:121–8.[CrossRef][Medline]
  34. Duxbury MS, Whang EE. RNA interference: a practical approach. J Surg Res 2004;117:339–44.[CrossRef][Medline]
  35. Shinomiya N, Gao CF, Xie Q, et al. RNA interference reveals that ligand-independent met activity is required for tumor cell signaling and survival. Cancer Res 2004;64:7962–70.[Abstract/Free Full Text]
  36. Kaji M, Yonemura Y, Harada S, Liu X, Terada I, Yamamoto H. Participation of c-met in the progression of human gastric cancers: anti-c-met oligonucleotides inhibit proliferation or invasiveness of gastric cancer cells. Cancer Gene Ther 1996;3:393–404.[Medline]
  37. Stabile LP, Lyker JS, Huang L, Siegfried JM. Inhibition of human non-small cell lung tumors by a c-Met antisense/U6 expression plasmid strategy. Gene Ther 2004;11:325–35.[CrossRef][Medline]
  38. Abounader R, Ranganathan S, Lal B, et al. Reversion of human glioblastoma malignancy by U1 small nuclear RNA/ribozyme targeting of scatter factor/hepatocyte growth factor and c-met expression. J Natl Cancer Inst 1999;91:1548–56.[Abstract/Free Full Text]
  39. Abounader R, Lal B, Luddy C, et al. In vivo targeting of SF/HGF and c-met expression via U1snRNA/ribozymes inhibits glioma growth and angiogenesis and promotes apoptosis. FASEB J 2002;16:108–10.[Abstract/Free Full Text]
  40. Jiang WG, Grimshaw D, Lane J, et al. A hammerhead ribozyme suppresses expression of hepatocyte growth factor/scatter factor receptor c-MET and reduces migration and invasiveness of breast cancer cells. Clin Cancer Res 2001;7:2555–62.[Abstract/Free Full Text]
  41. Herynk MH, Stoeltzing O, Reinmuth N, et al. Down-regulation of c-Met inhibits growth in the liver of human colorectal carcinoma cells. Cancer Res 2003;63:2990–6.[Abstract/Free Full Text]
  42. Kim SJ, Johnson M, Koterba K, Herynk MH, Uehara H, Gallick GE. Reduced c-Met expression by an adenovirus expressing a c-Met ribozyme inhibits tumorigenic growth and lymph node metastases of PC3-LN4 prostate tumor cells in an orthotopic nude mouse model. Clin Cancer Res 2003;19:5161–70.
  43. Chin L, DePinho RA. Flipping the oncogene switch: illumination of tumor maintenance and regression. Trends Genet 2000;16:147–50.[CrossRef][Medline]
  44. Goodman L. Targeting oncogenes. J Clin Invest 2004;114:1362.[Medline]
  45. Lavoie JN, L'Allemain G, Brunet A, Muller R, Pouyssegur J. Cyclin D1 expression is regulated positively by the p42/p44MAPK and negatively by the p38/HOGMAPK pathway. J Biol Chem 1996;271:20608–16.[Abstract/Free Full Text]
  46. Wang X, DeFrances MC, Dai Y, et al. Mechanism of cell survival: sequestration of Fas by the HGF receptor Met. Mol Cell 2002;9:411–21.[CrossRef][Medline]
  47. Huh CG, Factor VM, Sanchez A, Uchida K, Conner EA, Thorgeirsson SS. Hepatocyte growth factor/c-met signaling pathway is required for efficient liver regeneration and repair. Proc Natl Acad Sci U S A 2004;101:4477–82.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Clin. Cancer Res.Home page
W. Wang, F. Wu, F. Fang, Y. Tao, and L. Yang
Inhibition of Invasion and Metastasis of Hepatocellular Carcinoma Cells via Targeting RhoC In vitro and In vivo
Clin. Cancer Res., November 1, 2008; 14(21): 6804 - 6812.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zhang, S.-Z.
Right arrow Articles by Li, C.-J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zhang, S.-Z.
Right arrow Articles by Li, C.-J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online