Recombinant immunotoxin anti-c-Met/PE38KDEL inhibits proliferation and promotes apoptosis of gastric cancer cells
© Wei et al; licensee BioMed Central Ltd. 2011
Received: 16 May 2011
Accepted: 7 July 2011
Published: 7 July 2011
Our study aims to evaluate the anti-growth effects of recombinant immunotoxin (IT) anti-c-Met/PE38KDEL on gastric cancer cells, and its mechnisms.
Gastric cancer cells were treated with increasing doses of IT and c-Met protein was quantified by Western blotting. Cell proliferation was determined by Cell Counting Kit-8 assay (CCK). [3H]-leucine incorporation assay was used to evaluate IT inhibition of protein synthesis. Cell apoptosis was quantified by flow cytometry. Caspase activities were measured using colorimetric protease assays.
Cell growth and protein synthesis of the gastric cancer cell lines were suppressed by IT in a dose- and time-dependent manner. IT also induced apoptosis in a dose-dependent manner. The apoptosis rates of gastric cancer cell lines MKN-45 and SGC7901 were 19.19% and 27.37%, respectively when treated with 50 ng/ml of IT. There were significant increase ofcaspase-3 activity at 24 hr of IT treatment (100 ng/ml) (P < 0.01) in these gastric cancer cell lines.
IT anti-c-Met/PE38KDEL has anti-growth effects on the gastric cancer cell lines in vitro, and it provides an experimental basis for c-Met-targeted therapy towards in vivo testing.
Gastric carcinoma (GC) is one of the most common and lethal malignant cancers . Despite the improving surgical techniques and new chemotherapeutic treatment regimens, the patient survival rate remains dismal , and effective alternative treatment approach is in vital need. GC has been shown to harbor multiple somatic mutations as well as over-expressions of oncoproteins. Identification of these GC-associated biomarkers may entail possible discovery of new therapeutic targets . Among various GC-associated biomarkers, c-MET gene is frequently found gnomically-amplified and over-expressed in GC cell lines . The proto-oncogene c-MET, a receptor of hepatocyte growth factor (HGF, also known as scatter factor), encodes a 190 kDa heterodimeric transmembrane tyrosine kinase. HGF binding to c-Met triggers tyrosine kinase domain auto-phosphorylation and induces pleiotropic responses such as proliferation, motility, morphogenesis and angiogenesis in many cell types including normal and tumor cells . c-MET amplification has been identified in nearly 74% of human GC specimens . HGF and c-MET both play important roles in the progression and metastasis of GC . Thus, c-Met has been considered as a promising therapeutic target for various cancers.
Immunotoxins (ITs) are fusion proteins composed of a toxin fused to an antibody or growth factor with distinct target specificity . IT exerts its anti-growth effect by inhibiting protein synthesis and promoting apoptosis . IT anti-c-Met/PE38KDEL (anti-c-Met Fab, which resulted from screening and characterization from a natural human Fab phage antibody library; PE38KDEL, which is a modified structure of PE38, lost the function of combining with non-mammalian cells specifically, but retained a complete cytotoxicity after internalization) has shown specific cytotoxic effects against c-Met-positive cancer cells . In this study, we investigated the effects of IT anti-c-Met/PE38KDEL on proliferation and apoptosis of two different c-Met-positive malignant gastric cell lines, MKN-45 and SGC7901 [11, 12], and a normal gastric mucosa cell GES-1 . We found that IT anti-c-Met/PE38KDEL exerts its anti-growth effect primarily through rapid inhibition of protein synthesis.
Materials and Methods
IT anti-c-Met/PE38KDEL was described previously . It induces apoptosis in hepatic carcinoma cells SMMC7721. Cell Counting Kit 8 (CCK8) was purchased from Sigma Chemical. Caspase colorimetric assay kit and anti-caspase-3 antibody were from Biovision. Antibodies against c-Met and β-actin were purchased from Santa Cruz. Protein lysis buffer was from TaKaRa Biotechnology.
GC cells lines, MKN-45 and SGC7901, and normal gastric mucosa cells GES-1 were obtained from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China), and were grown in DMEM (Invitrogen) supplemented with 10% fetal calf serum (FCS) and incubated at 37°C with 5% CO2. All cell lines were routinely tested and found to be free from mycoplasma contamination.
GES-1, MKN-45 and SGC7901 cells grown in 6-well plates were collected in lysis buffer for total cellular protein. Protein concentrations were measured using a Bradford reagent (Bio-Rad). Equal amounts of protein (80 μg/lane) from each cell line were boiled for 5 min, separated by SDS-PAGE, and then transferred on to a nitrocellulose membrane before blocking in 5% non-fat dried milk in Tris-buffered saline (TBS) for 120 min at room temperature. The membranes were then incubated with a primary anti-human c-Met polyclonal antibody (diluted 1:150 in a new batch of the blocking buffer) or a goat polyclonal primary anti-β-actin (diluted 1:1000, Santa Cruz, CA, USA) for 2 hr and followed by incubation with peroxidase-labelled anti-IgG secondary antibody for 1 hr. After washing with TBST for 3 times, the films were developed and the protein bands were quantified by densitometry using ImageJ software (NIH, Bethesda, MD, USA).
To detect the caspase-3 activity, both floating and adherent cells were collected 24 hr following IT treatment. Total cellular protein was prepared as described above. All the experiments were performed at least twice with similar results.
Cell proliferation assay
Cell growth inhibition rate (IR) was determined using a CCK- 8 assay following the manufacturer instructions (Sigma). GES-1, MKN-45 and SGC7901 cells were seeded at a concentration of 1 × 105 cells/90 μl/well in 96-well culture plates. After incubation of cells with the indicated concentrations of IT for 24 hr and 48 hr, 10 μl/well of cell Counting Kit-8 solution was added to the medium and the cells were incubated for an additional 4 hr. The absorbance at 450 nm was then measured in a Microplate Reader. IR was calculated using the following equation: IR = [1-(A value in the treated samples-A value in the blank samples) / (A value in the control samples-A value in the blank samples)] *100%. The assays were performed in triplicates and repeated at least twice .
Protein synthesis inhibition assay
IT-induced inhibition of protein synthesis in GES-1, MKN-45 and SGC7901 cells were evaluated using the [3H]-leucine incorporation assay . Cells were seeded in 48-well plates (1 × 104 per well) and allowed to grow overnight before the addition of IT at different concentrations. After 5 or 24 hr incubation, cells were washed twice with cold phosphate-buffered saline (PBS) containing 0.1% FCS, and then incubated with [3H]-leucine (2 μCi ml-1) in leucine-free medium at 37°C for 45 min. Cells were then washed with 5% trichloroacetic acid (TCA) for 5 and 10 min, respectively, and dissolved in 0.1M KOH for 10-15 min. The resultant solution was transferred to the liquid scintillator. Sample counts were determined in a liquid scintillation counter. Assays were performed in duplicates and repeated at least three times. Counts per minute (cpm) for treated cells were compared to cpm for untreated cells and reported as a percentage of leucine incorporation with the control value set to 100%. The experiment was completed in the isotope laboratory of Nanjing Medical University.
Flow cytometric analysis of cell apoptosis
Apoptosis were determined by flow cytometric analysis. Briefly, cells in triplicates, were incubated with or without various concentrations of IT for 24 hr. Cells were then harvested, washed in cold PBS, and fixed with 1 ml 75% ice-cold ethanol at -20°C until processing. An aliquot (1 ml) of fixed cell suspension containing 1 × 106 cells was washed twice in cold PBS and then treated with fluorochrome DNA staining solution (1 ml) containing 40 μg of propidium iodide and 0.1 mg of RNase A in the dark at room temperature for 0.5 hr. Flow cytometric analysis were performed three times .
Caspase activity assay
Caspase activity was determined in 96-well plates using cell lysates from 1 × 106 cells for each measurement. Caspase-3 and caspase-8 activities were determined using colorimetric assay kits according to the manufacturer's protocol (BioVision). GES-1, MKN-45 and SGC7901 cells were treated with anti-c-Met/PE38KDEL (100 ng/ml) for 24 hr prior to the assay. Cell extracts were incubated with 5 μl of 4 mM tetrapeptide substrates (DEVD, caspase-3; IETD, and caspase-8) at 37°C for 1-2 hr. The reaction was measured at 405 nm in a Microplate Reader. Background readings from cell lysates and buffers were subtracted from the readings of both IT-induced and control samples before calculating the relative change increase in caspase activity in the IT-induced samples compared to that of the control. IT treated samples were normalized to the caspase activity of the untreated sample, which was set to 1.0. Fold of increases in caspase activities were presented.
Statistical analysis was performed with SPSS 13.0 software. Data were presented as mean ± standard deviation. Student's t-test was used to compare two samples, and the single-factor analysis of variance (One-way ANOVA) was used to compare multiple samples. A p-value less than 0.05 is considered statistically significant (*, p < 0.05; **, p < 0.01).
Increased c-Met expression in MKN-45 and SGC7901 cells
IT anti-c-Met/PE38KDEL inhibited cell proliferation and protein synthesis
IT anti-c-Met/PE38KDEL inhibits tumor cell growth through induction of apoptosis
IT anti-c-Met/PE38KDEL activates caspase-3
GC is the second leading cause of cancer mortality in the world . The receptor tyrosine kinase c-Met is constitutively activated in many GCs . Amplifications of c-Met have been associated with human GC progression  C-Met is also related to lymph node metastasis in GC . Therefore, c-Met is considered a promsing therapeutic target for this type of cancer . The aim of this study was to evaluate the effects of recombinant immunotoxin anti-c-Met/PE38KDEL on proliferation and apoptosis of GC cells and explore the mechanism underlying the action of anti-c-Met/PE38KDEL.
SGC7901 was derived from moderately differentiated GC, with a high metastatic potential . MKN-45 was derived from poorly differentiated GC with low metastatic potential . We found that SGC7901 cells expressed high level of c-Met than MKN-45 cells. Normal gastric mucosa cells GES-1 expressed a minimum level of c-Met. Studies have shown that c-Met overexpression in carcinoma cells is associated with liver metastasis of GC . Moreover; c-Met expression can be used as an indicator of liver metastasis for GC patients. It has also been reported that HGF is a lymphangiogenic factor, which can directly or indirectly stimulate lymphangiogenesis and contribute to lymphatic metastasis in GC . Therefore, we hypothesized that IT anti-c-Met/PE38KDEL may be effective in preventing GC's metastasis.
Our data showed that IT decreased GC cell proliferation in a time- and dose-dependent manner. After 48 hr of IT treatment (100 ng/ml), cell inhibition rate in MKN-45 and SGC7901 cells was about 75% and 95%, but only 30% in GES-1 cells, presumably due to low c-Met expression on GES-1 than the two GC cells. IT attenuates cancer cell growth not only by inhibiting protein synthesis but also by inducing apoptosis . We found that IT anti-c-Met/PE38KDEL induced a rapid inhibition of protein synthesis with simultaneous induction of apoptosis in GC cells. Nearly 80% and 100% inhibitions of protein synthesis were observed after 24 hr treatment with IT (100 ng/ml) in the MKN-45 and SGC7901 cells, respectively. The inhibition was much less pronounced in GES-1 cells (35%), suggesting that IT anti-c-Met/PE38KDEL is selective against GC. In addition, IT exerts its anticancer effect mostly via induction of cells apoptosis. The apoptosis rates in three cells were all increased after treatment with IT, more prominent in the two GC cell lines.
Caspases are classified into two functional subgroups-initiator caspases and effector caspases. The initiator caspases are caspase 2, 8, 9 and 10, and the effector caspases are caspase 3, 6 and 7 . Caspases are critical mediators of apoptosis . Activation of caspase is responsible for multiple molecular and structural changes in apoptosis . Caspase-3 is a potent effector of apoptosis in a variety of cells  and plays a central role in both death-receptor and mitochondria-mediated apoptosis. Caspase-8 is the prototypical apoptosis initiator downstream of TNF super-family death receptors. Our data showed that caspase-3 enzyme activity exhibited 3.70, and 5.02 fold increases in IT-treated MKN-45 and SGC7901 cells as compared to the activity of untreated controls (P < 0.01). The increase in caspase-8 enzyme activity was less significant.
Our results demonstrate the time- and dose-dependent anti-growth effects of IT anti-c-Met/PE38KDEL against GC cell lines. The anti-cancer effect of IT occurred primarily through inhibition of protein synthesis, and caspase-3-mediated apoptosis, suggesting the potential value of IT as an anti-c-MET therapeutics for GC.
hepatocyte growth factor
Cell Counting Kit 8
fetal calf serum
sodium dodecyl sulphate
polyacrylamide gel electrophoresis.
Acknowledgements and Funding
This study was funded by nature science founation of jiangsu province (BK2008483).
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