[Preliminary screening and identification of a peptide that binds specifically to gastric cancers cells with high metastasis to peritoneum].
OBJECTIVE
To screen and identify peptides that binds specifically to gastric cancers cells with high metastasis to peritoneum so as to find appropriate vectors for targeting therapy for cancer.


METHODS
Human gastric cancer cells of the line GC9811 and those with high metastasis to peritoneum of the line GC9811-P were co-incubated with the 12-mer bacteriophage random peptide library. After 3 round of repeated screening, phage clones were collected. Forty internalized phage single-stranded DNA that specifically binding to the GC98112-P cells were sequenced. GC9811 and GC9811-P cells were co-inoculated with 5 peptides with the N end marked with fluorescein isothiocyanate (FITC) and 1 un-related peptide not binding to GC9811 and GC9811-P cells. Fluorescence microscopy, ELISA, and flow cytometry were used to detect the binding activity. BALB/cnu/nu mice were inoculated intraperitoneally with GC9811 and GC9811-P cells and then randomly divided into. 2 equal groups: experimental group, inoculated with the peptide PIII-FITC and control group (inoculated with un-related peptide PC-FITC. Forty-eight hours later the mice were killed and the peritoneum and tumor masses in different organs were collected and under fluorescence microscopy.


RESULTS
DNA sequencing showed that 45% (18/45) of the isolated phages displayed repeated sequence SMSIASPYIALE, and SMSI was defined as a conservative motif. Obvious fluorescence was seen in the GC9811-P cells co-incubated with PIII-FITC and weak fluorescence was seen in the GC9811 cells co-incubated with PIII-FITC. Un-marked un-related peptide PC and PIII-FITC did not influenced the fluorescence staining of the GC9811-P cells, however, no fluorescence could be seen in the GC9811-P cells co-incubated with un-marked PIII and PIII-FITC. The fluorescence positive cell rate was 5.9% in the GC9811 cells co-incubated with PIII-FITC, and was 90.2% in the GC9811-P cells co-incubated with PIII-FITC. The fluorescence positive cell rates of the GC9811 cells and GC9811-P cells co-incubated with PC-FITC were 10.1% and 9.9% respectively 10.1% and 9.9% respectively. The fluorescence strength of the GC9811-P cells co-incubated with PIII-FITC was significantly greater than that of the GC9811-P cells co-incubated with PC-FTIC at any time-point and dose (all P < 0.01), and increased along with the increase of co-incubation time and dose of PIII-FITC. The peritoneal tumor tissues caused by the GC9811-P cells of the mice showed strong fluorescence and those caused by GC9811 cells only showed very weak fluorescence. Weak fluorescence could be seen in the tumor masses in the lymph nodes, liver, and muscle of the mice inoculated with GC9811-P cells and was not seen in the tissues of the mice inoculated with GC9811 cells.


CONCLUSIONS
The sequence SMSIASPYIALE that specifically binds to human gastric cancer cells with high metastasis has been screened that has the potential to be used as a marker and targeting vector in diagnosis and treatment of gastric cancer.
Bai
Fei-hu
FH
Department of Gastrointestinal, Affiliated Hospital of Ningxia Medical College, Yinchuan 750004, China.
Wang
Jun
J
Zhao
Peng-tao
PT
Cao
Shan-shan
SS
Lei
Ting
T
Li
Ying
Y
Wu
Kai-chun
KC
Fan
Dai-ming
DM