Study On The Detection Of Apoptotic Tumor With ～(99)Tc～m-rh-Annexin V

PREFACEThe molecular basis of cancer is now widely believed to involve mutation that leadto deregulated cellular proliferation and suppression mechanisms controllingprogrammed cell death. For instance, mutations of powerful apoptosis inducing mycprotein or the loss of p53 protein function have been shown to play a crucial role incarcinogenesis. Tumor sensitivity to any given therapeutic regimen commonly ismediated by the initiation of programmed cell death via available active apoptoticpathways. Many therapeutically effective anticancer drugs act to interfere with DNAsynthesis and cell division, thereby inducing apoptosis in susceptible target tumors.Thus, it may be possible to determine the effectiveness of a proposes anticancerregimen on a patient-by-patient basis by assessing the degree of apoptosis in targettumors soon after the initial treatment.Recently, in vivo imaging of apoptosis has proven to be feasible by usingradiolabeled Annexinâ…¤. This endogenous human protein has a high affinity formembrane bilayer at an early stage of the apoptotic process. PS then serves as arecognition site foe macrophages that digest and remove apoptotic cells. The objectiveof this research is to determine the effectiveness of imaging with ^99mTc labelledrecombinant human Annexinâ…¤(^99mTc-rh-Annexinâ…¤) as a reflection of the degree ofapoptosis in tumor. MATERIALS1. Animals for experiment: purebred 615 mice, female, weighed 17～24g;allogenic hepatoma cells(Hca-F25), were obtained from the Dalian Medical UniversityAnimal care facility.2. Drug and reagents: recombinant human Annexin V; ^99mTcO₄^-;Cyclophosphamide; Annexin V-FITC/PI flow cytometry Kit; TUNEL(AP) Kit;Bcl-2/Bax immunohistochemistry Kit; survivin/caspase-3 immunohistochemistry Kit.3. Instrument: well-type scintillation counter; SPECT; transmission electronmicroscopies; Olympus microscope; computer image analysis software; linearaccelerator (VARIAN—2300 C/D).METHODS1. Animals grouping: Biodistribution group(n=18), randomly divided into sixgroups(every group has three mice). Experimental group(n=41), randomly into fourgroups; group A, chemotherapy group(n=13); group B, radiotherapy group(n=10);,control group(n=2); group D, blank control group(n=6). Group A and group C weredivided into three subgroups respectively depend on imaging time(A1, 1h, n=3; A2,4h, n=4; A3, 6h, n=6; C1, 1h, n=3; C2, 4h, n=4; C3, 6h, n=5); groupB was divided into three subgroups depend on radiotherapy dose(B1, 2Gy, n=4; B2,5Gy, n=3; B3, 10Gy, n=3); group D, was divided into two subgroups(D1, n=3;D2, n=3)2. Animal model preparation: Eight days after being inoculated with allogenichepatoma cells(Hca-F25) into the subcutaneouly of the fight axillary fossa, the micewere prepared for Experiment. Chemotherapy group received a single dose ofcyclophosphamide(150mg/kg, intraperictoneally); radiotherapy group receiveddifferent dose of radiotherapy respectively (2Gy, 5Gy and 10Gy); blank control groupD1 received same dose of cyclophosphamide, and received same doses of ^99mTcO₄^- without labelled with Annexin V; blank control group D2 only received same doses of^99mTcO₄^- without receiving chemotherapy.Part One: The biodistribution of ^99mTc-rh-Annexin V was determined with awell-type scintillation counter and SPECT. The accumulation of ^99mTc-rh-Annexin Vin the tissues(tumor, blood, muscle) was expressed as the %ID/g(percentage activity ofinjection dose per gram of tissue). Apoptosis cells were detected with Annexin V/PIflow cytometry. The morphological changes of the apoptosis hepatoma cells wereobserved by light and transmission electron microscopies.Part Two: Detecting the accumulation of ^99mTc-rh-Annexin V and the positive cellnumber of TUNEL in every group, and detecting the expression of Bcl-2 and Bax withimmunohistochemical methods at the same time.Part Three: Detecting the accumulation of ^99mTc-rh-Annexin V and the positivecell number of TUNEL in every group, and detecting the expression of survivin andcaspase-3 with immunohistochemical methods at the same time.RESULTSPart One: The labeling rate of directly labelled recombinant human Annexin V(^99mTc-rh-Annexin V) was more than 98%. The kidney had the highest concentration ofradioactivity at all time point. On the other hand the liver uptake was lower and otherorgans showed the least concentration of ^99mTc-rh-Annexin V. After treated bychemotherapy or radiotherapy, the apoptotic cell number increased significantly, aswell as the accumulation of ^99mTc-rh-Annexin V in tumor(P＜0.05), and uptake of^99mTc-rh-Annexin V correlated well with the number of Annexin V/PI-positive cells inthe tumor (P＜0.05).Part Two. After treated by chemotherapy or radiotherapy, the apoptotic cellnumber increased significantly, and uptake of ^99mTc-rh-Annexin V correlated well withthe positive cell number of TUNEL (P＜0.01). The expression of Bcl-2 had nosignificant difference in control group and treated group, but the Bcl-2/Bax was significantly high in control group than treated group (P＜0.01). In control group, theexpression of Bcl-2 and Bax had no relationship with the %ID/g and the positivenumber of TUNEL in tumor. In treated group, the expression of Bax correlated wellwith the %ID/g and the positive number of TUNEL, but the Bcl-2/Bax correlatednegatively with %ID/g and the positive number of TUNEL(P＜0.01).Part Three: After treated by chemotherapy or radiotherapy, the apoptotic cellnumber increased significantly, and uptake of ^99mTc-rh-Annexin V correlated well withthe positive cell number of TUNEL (P＜0.01). In treated group, the expression ofcaspase-3 was increased significantly, but the expression of survivin was significantlyhigh in control group than in treated group (P＜0.01). The expression of survivincorrelated negatively with the expression of caspase-3, and the the distribution of⁹⁹Tc^m-rh-Annexin V correlated positively well with the expression of caspase-3 andnegatively with the expression of survivin in all groups.CONCLUSIONPart One:1. Directly labeled recombinant human Annexin V(^99mTc-rh-Annexin V) has ahigh radiolabeling rate, and stable in environment. The kidney had the highestconcentration of radioactivity at all time point.2. The distribution of ^99mTc-rh-Annexin V can reflect the degree of apoptosis intumor early after treatment.3. The distribution of ^99mTc-rh-Annexin V was stable in tumor 4h after injection,and could have a good imaging at the same time.Part Two:1. In control group, the expression of Bcl-2 and Bax can not reflect the degree ofapoptosis in tumor.2. The expression of Bcl-2 had no relationship with the expression of Bax. Theexpression of Bcl-2 had no significant difference in control group and treated group, but the Bcl-2/Bax was significantly high in control group than in treated group.3. In treated group, the distribution of ^99mTc-rh-Annexin V can reflect theexpression of Bax and Bcl-2/Bax.Part Three:1. After treated by chemotherapy or radiotherapy, the expression of caspase-3 hadincreased but the expression of surviving had decreased significantly.2. In mouse hepatoma tissue, the expression of survivin correlated negatively withthe expression of caspase-3, whether or not treatment.3. In mouse hepatoma tissue, the degree of apoptosis correlated positively wellwith the expression of caspase-3 and negatively with the expression of survivin.