| Objective Radionuclide-labeled somatostatin analogues selectively target somatostatin receptor (SSTR)-expressing tumors as a basis for diagnosis and treatment of SSTR positive tumors,but not for SSTR negative tumor. To treat non-somatostatin receptor expressing tumors with a radionuclide-labeled somatostatin analogue, the hSSTR2 gene was transfected into somatostatin receptor-negative lung adenocarcinoma A549 cells, and a stable cell line was established (pcDNA3-hSSTR2 A549)for further radionuclide-labeled RC-160 selectively target treatment study. The binding characteristics of 188Re labeled RC-160 with pcDNA3-hSSTR2 A549 cells as well as its radiotherapeutic effect was evaluated. For further improve the efficacy of the treatment, a bicistronic adenoviral vector encoding the hSSTR2, PEG-3 and hcaspase-3(Ad-PEG-3/caspase-3- hSSTR2)was constructed, and the growth of A549 cell infected by Ad-PEG-3/caspase-3- hSSTR2 was observered. Method1.RC-160 was labeled directed by 188Re;In vitro radioligand binding and internalization assay with 188Re-RC-160 for the pcDNA3-hSSTR2 A549 cell and the pcDNA3 A549 cell was studied.2.A corresponding animal tumor model was established. Expression of the hSSTR2 reporter gene was imaged by 188Re-RC-160 recognition. Tumors were frozen and evaluated for somatostatin receptor expression by immunohistochemistry.3.The distribution of 188Re-RC-160 in the animal tumor model was measured and the inhibitory effects of 188Re-RC-160 were evaluated by measurement of tumor growth,HE staining and apoptosis respectively.4.The efficiency of adenovirus-mediated gene transfer and the inhibitory effect of Ad-PEG-3/caspase-3- hSSTR2 on the A549 cells were detected by X-gal staining and MTT assay in vitro, respectively.Results1.The labeling rate of 188Re—RC-160 was more than 95%;188Re-RC-160 was shown to have high affinity for the hSSTR2 receptor (Kd= 5nmol/L, IC50=5.01nM) by binding assay and was rapidly internalized by the membrane receptor of pcDNA3-hSSTR2 A549 cell ( 4h 9.3%).2.Biodistribution studies demonstrated that 188Re-RC-160 was highly uptook and retented in hSSTR2-expressing tumor tissue and has rapid overall clearance properties from non-target tissues. In vivo radioimaging revealed specific targeting of RC-160 to tumors derived from pcDNA3-hsstr2 A549 cells (T1),compared to those from pcDNA3 A549 cells (T2) ( T1/ T2 = 3.44, p <0.05). The study on immunohistochemistry showed a markedly higher level of hSSTR2 expression in a pcDNA3-hSSTR2 A549 tumor, compared to no visible hSSTR2 expression in a control pcDNA3 A549 tumor. 3.pcDNA3- hSSTR2 A549 tumor growth inhibition was significantly higher in the single 7.4MBq 188Re-RC-160 treatment group than in the 2×7.4MBq 188Re group, RC-160 group, control group, and pcDNA3 A549 tumors (p <0.05). Treatment fractionation in two doses, 48 h apart, induced significantly increased tumor-growth inhibition compared with mice given a single dose (p < 0.05), moreover, the apoptosis was remarkable in immunohistochemistry of hSSTR2 and TUNEL test. HE stain showed that large or less severe but visible areas bulk necrosis occurred in 2×7.4MBq 188Re-RC-160 or 2×7.4MBq 188Re-RC-160 treatment group. However, no obvious necrosis was observed in 188Re, RC-160,control groups and any pcDNA3 A549 tumor.4.The infection rate of the Ad-PEG-3/caspase-3- hSSTR2(MOI=100)was 99% which can inhibit specifically A549 cells in vitro.Conclusions1.188Re-RC-160 was stabled in vivo and in vitro;188Re-RC-160 have high affinity and internalized ability for the pcDNA3-hSSTR2 A549 cell.2.188Re-RC-160 offered a experimental base for target radionuclide therapy of tumors by hSSTR2 mediated exogenously.3.188Re-RC-160 inhibited obviously growth of tumor with pcDNA3-hSSTR2 A549, 188Re-RC-160 have allied therapeutic effection for the tumor with hSSTR2.4.The growth of A549 cell transfected with Ad-PEG-3/caspase-3-hSSTR2 was inhibited obviously by apoptosis.
|
PDF Full Text Request