Abstract
OBJECTIVE To study the inhibition of adhesion and invasion of SGC7901 cells into the ECM by integrinβl antisense oligodeoxynucleotide asODN.
METHODS asODN and control ODN were transfected into SGC7901 cells using liposomes as vectors. The distribution of the ODN was followed by immunochemistry and changes in the expression of integrinβ1 mRNA and protein were determined by RT-PCR and FCM, respectively. The adhesion and invasion into the ECM were measured by the MTT and Boyden chamber methods, respectively.
RESULTS Integrinβ1 asODN which was transfected into SGC7901 cells distributed evenly in the cytoplasm and nucleus. PCR and FCM revealed a weakened band at 489bp and a left-shift curve, respectively. Adhesion and invasion assays showed decreased activity with an inhibition rate of 54% and 76%. The extent of decrease induced by integrinβ1 asODN was larger than that caused by random control ODN (P<0.001 ).
CONCLUSION Transfection of integrinβ1 asODN into SGC7901 cells induced a decrease in the expression of integrinβ1 mRNA and protein, resulting in a decrease in adhesion and invasion into the ECM, with a greater effect than random control ODN.
keywords
Invasion and metastasis of cancer is complicated course of multi-factors, steps and phases. Integral is a heterogeneitic dimer transmembrane glycoprotein bound by disulfide bonds with a, β-subunits, which are combined with the extracellular matrix components and cell bone. This forms a ligand-integrinpi-cell bone transmembrane information system, which mediates the adhesion and invasion of tumor cells. In this study liposomes were used as a vector to transfect specific integrinβl asODN into the highly metastatic human gastric cancer cell line SGC7901 in order to assess the inhibitory effect of target gene expression and its influence on cancer cell adhesion and invasion.
Materials and Methods
Materials
Cells
Human gastric cancer cell line SGC7901
Reagents
DMEM culture medium (Gibco Co.), Matrigel (Sigma Co.), integrinβ1 monoclonal antibody (Fuzhou Maixin Inc.), FITC-conjugated goat anti-mouse IgG (Beijing Zhongshan Biology Technique Inc.), lipofectin reagent (Life Technologies), RT-PCR reaction reagent kit (Promega Co.)
Integrinβ1 asODN sequences
5’-T*G*C-AGT-AAG-CAT-CCA-T*G*T-3’
5’-T*G*C-AGT-AAG-CAT-CCA-T*G*T-3’-Biotin.
rODN sequence
5’-G*C*A-ACG-AGA-GAG-CCG-T*C*G-3’ (*phosphorothioate insert)
Integrinβl primer sequences
5’-GTGGAGAATGTATACAAGCAGG-3’
5’-CTAATGTAAGGCATCACAGTC-3’
(Synthesizded by Shanghai Shenggong Inc.)
Methods
Partial phosphorothioate oligonucleotide transfection of SGC7901 cells
The procedure was strictly performed according to the suppliers’ instructions.
Cellular distribution after integrinβ asODN transfection of SGC7901 cells
Transfection of biotin-integrinβ1 asODN into the cells was conducted as noted above, followed by incubation of 1, 2, 4, 8, 12 and 24 h. The cells were maintained for 2 h after adding DMEM with 10% FCS, and then 50 μ1 of immediately-prepared streptomycin-avidin dioxygenase solution was added. Then DAB was used to stain the cells.
Integrinβ1 mRNA expression assay
Total RNA was extracted by TRIZOL and cDNA was synthesized by PCR with the following conditions: 42°C 1 h, 99°C 5 min, 4°C 5 min. Then the cDNA was amplified by incubation for 5 min at 95 °C, 95 °C 45 s, 57°C 45 s, 72°C 60 s for 30 cycles, PCR reaction for 7 min at 72°C. The products were analyzed using 1.5% agarose gel electrophoresis.
Integrinβ1 protein expression assay
Transfected SGC7901 cells were fixed at 4 °C with 0.5% paraformaldehyde 20 μ1 of integrinβ 1 monoclonal antibody was added (diluted to 1:200) and the cells resuspended in 100 p.1 PBS. Then 5 μ1 of FITC-conjugated goat anti-mouse IgG (diluted to 1: 50) was added and the cells analyzed using a FAC scan at a wavelength of 488nm.
Adhesion assay
The 96-well plates were coated with 8 μg/well of Matrigel, maintained for 1 h at 37°C and suspended cells (4 ⨯ 105) added to the wells, the cells were incubated for 30 min on a slow swing bed at 37°C after which 150 μl DMEM without serum and 50 p.1 MTT were added. Then the cells were incubated for 4 h at 37 °C and 100 p.1 DMSO added per well. The absorption intensity at 490 nm single wavelength was determined. Percent of cell adhesion = (the amount of adhesive cells/the amount of total cells) ⨯ 100%
Invasion assays
NIH3T3 cell conditioned medium was added to the lower section of a Boyden chamber, and an 8 pm Millipore filter membrane was placed between the upper and lower section. Matrigel was spread on the membrane followed by incubation for 1 h at 37°C. Prepared cancer cells (5 ⨯ 105) were added to the upper section followed by incubation for 6 h, dying with haematoxylin and counting under the microscope the number of cells which had invaded to the back of the membrane. Five microscopic fields of vision were randomly accounted using the mean for the first result.
Statistical analysis
Statistics were produced using the t-test by SPSS10.0 software.
Results
Distribution in cells after integrin β1 asODN transfection of SGC7901
Using an immunohistochemistry technique, we could see light brown particles evenly distributed in the cytoplasm and nucleus. The color of particles became deeper with an extention of time, indicating that liposomes could be used to transfect asODN into the cells. Distribution occurred in the cytoplasm and nucleus(Fig.l).
Light brown particles could be seen in cytoplasm and nucleus, integrinβ1 stained with immunohistochemistry technique ⨯ 200.
Influence of asODN on the expression of integrin β1 mRNA
The results of RT-PCR demonstrated that we could see a specific gene band at 482bp in both the control and test groups, but the gene expression after treatment with asODN was significantly lower than that of the untreated control and rODN. The 320bp gene band in each group appeared as an inner control, β-actin amplified results, indicated that there was no significant difference between each group(Fig.2).
The mRNA expression of asODN group was significantly lower than that of the control and rODN group. 1 Marker; 2 Control group; 3 rODN group; 4 asODN group.
Influence of asODN on the expression of integrin β1 protein
The integrinβ1 protein was expressed on the surface of the SGC7901 cells with the expression curve with FAC analysis obviously moved to the left after integrinβ1 asODN transfection. Therefore the extent of reduction of integrinβ1 expression was larger than that seen with rODN transfection (Fig.3).
The protein expression of asODN group was obviously lower than that of the control and rODN group.
Influence on the adhesion and invasion of SGC7901 cells after different ODN transfection
The results of adhesion and invasion assays demonstrated that the ability of cellular adhesion and invasion decreased remarkably after integrinβ1 asODN transfection of the SGC7901 cells. The degree of inhibition was 54% and 76% respectively, obviously greater than the group treated with rODN (P<0.001)(Table 1).
Discussion
Peritoneal metastases are the most common form of relapse following advanced gastric cancer radical resection, resulting in the major cause of death. Early in 1986, Liotta proposed the theory involving 3 steps of tumor-cell invasion of the ECM: adhesion, degradation and movement. This theory proposed that interaction between tumor cells and the ECM, mediated by many adhesive molecules, was a precondition for inducing invasion and metastasis. CD44 and integrinβl are the main and independent factors among the adhesive molecules mediating gastric cancer invasion and metastasis.[1] The literature is replete with evidence that gastric cancer tissue highly expresses CD44 and integrinβl. [2-4]
In 1978, Zamur and Stephenson first proposed the antisense nucleotide technique. Since then it has become a focus for development of tumor prevention and therapy, with the hope for a new approach to patient care. In our study using liposomes as a vector, we transfected the artificially-synthesized partial phosphorothioate integrinβl asODN and rODN into SGC7901 cells. Our purpose was to observe the distribution of oligonucleotides in the cells and the effector adhesion and invasion in the ECM. Our results showed that ODNs could easily pass into the cells after 1 h of transfection, and with time were evenly distributed in the cytoplasm and nucleus. The results of RT-PCR and flow cytometry illustrated that integrinβl asODN could decrease the expression of integrinβl mRNA and protein. Integrinβl asODN could effectively inhibit the adhesion and invasion of the cells into the ECM in a manner, superior to the rODN.
The stability of asODN is of key importance. The physical, chemical and biological properties are very similar between phosphothioate oligonucleotides and standard oligonucleotides, they have strong anti-ribozyme action, activate the Rnase-H activity, to degrade RNA chains. Recently the study of liposomes as vectors for drug passage into cells has made considerable progress, especially as the vector for anti-cancer drugs. Liposomes are composed of phosphatidyl choline and phosphatidylserine. Adding cholesterol can improve the membrane stability, as its structure is a mono-layer or multi-layer phospholipid-bilayered membrane. [5] Liposomes carrying ODNs into cells not only have the effect of anti-degradation by ribozymes, but also can alter the distribution of oligonucleotides in cells, and extend the time that the asODN remain in the nucleus.
At the present time there are goals to use asODN in gastric cancer to inhibit the following genes: C-Has, C-myc, DNA polymerase-α, bcl-2, C-erb-B2, PCNA, C-met and HSP90 et al.[6-10] In our study the partial phosphothioate integrinβl asODN transfection using liposomes as a vector had good biological activity and provided an experimental basis to study integrinβl asODN as an anti-metastatic agent.
Footnotes
This work was supported by the 973 State Project of Important Basic Research and the Key Project of the Department of Education, Liaoning Province.(N0.G1998051203, 2001225002)
- Received July 21, 2004.
- Accepted November 28, 2004.
- Copyright © 2005 by Tianjin Medical University Cancer Institute & Hospital and Springer
In this issue
Jump to section
Related Articles
Cited By...
- No citing articles found.