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The Therapeutic Effect and the Influence on the Expression of NF-κBp65 of DFO and DFO in Combination with Arsenic Trioxide on Transplanted Tumor of HL-60 Cells in Nube Mice

Author: YuRunHong
Tutor: LiuYuFeng
School: Zhengzhou University
Course: Pediatrics
Keywords: Deferoxamine Arsenic Trioxide NF-kappaB Leukemia Nude Mice
CLC: R733.7
Type: Master's thesis
Year: 2010
Downloads: 53
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Abstract

ObjectiveTo observe the therapeutic effect and the influence on NF-KBp65 expression level of transplanted tumors of deferoxamine and deferoxamine(DFO) in combination with arsenic trioxide (As2O3)by establishing a model of implanted tumor of human high tumorigenic leukemia cell line HL-60 in nude mice,and investigate the mechanism,so as to provide the experimental evidence for treatment or auxiliary treatment clinically for leukemia of iron chelators.Methods1.Xenograft tumor model of human high tumorigenic leukemia cell line HL-60 in nude mice was established by means of inoculating subcutaneously,then the tumor-bearing mice were randomly divided into four groups:50mg/kg DFO group, combination group(50mg/kg DFO+1.5mg/kg As2O3),3mg/kg As2O3 group, normal saline control group. The tumor-bearing mice were given drugs from that day of inoculating subcutaneously,and drug administration continued for 10 days continuously through intraperitoneal injection.The experimental protocol was approved by the ethical committee of First Clinical Medical College of Zhengzhou University.2.Observing closely general conditions and adverse drug effects of the tumor-bearing mice,time of forming tumor, growth characteristic of transplanted tumor during administration of drugs.The maximum longitudinal(a) and transverse(b) diameters of transplanted tumor were measured with slide callipers every day. Tumor volumes were calculated according to the equation(volume=ab2/2),and then transplanted tumor growth curves were drawn.3.The next day The tumor-bearing mice were executed after administration of drugs.Then tumor weights and tumor inhibitory rate of transplanted tumors were calculated.4.After the tumor-bearing mice were executed, the specimens of transplanted tumors were stained with HE.Routine pathological examination were performed on transplanted tumors to observe the changes of tissue morphology. Then the changes of each specimen in different experimental groups were analyzed. The bone marrows of the tumor-bearing mice were observed to determine whether bone marrow depression and infiltration of leukemic cells existed or not. The tissues of heart,liver,spleen,lung and kidney of the tumor-bearing mice were observed to determine whether pathological changes and tumor metastasis existed or not.5.NF-κBp65 expression levels of transplanted tumors were detected by two-step Immunohistochemistry detection(PowerVisionTM),and then integrate optical density(IOD) was detected by using professional software Biosens Digital Imaging System (v1.6).Mean integrate optical density value of the positive area was used for representing the expression levels of NF-κBp65.6.The results of experiment were analyzed by SPSS16.0 for Windows.Sample averages were expressed by X±S.The differences of the weights of transplanted tumors between treatment groups and control group were test by t test.The differences of the NF-κBp65 expression levels of transplanted tumors between four experimental groups were test by one-way ANOVA.Statistic test were two-sided test of probability(α=0.05).Probability of less than 0.05 were considered significant.Results1.General conditions of forming tumor, growth characteristic of transplanted tumor after inoculating:Inoculating human high tumorigenic leukemia cell line HL-60 (about 1×106 cells/each mouse) culturing in vitro,tumor grew in 100% of animals.From the seventh day to the eighth day, macroscopic tumors were observed 0.5cm～1.0cm in diameter. Tumors which were orbicular or elliptic grew only at the sites of cells inoculation and expansive grew outward with rapid growth at the time of early growth.After that, with the enlargement of tumor size, tumors became irregular gradually and multinodular confluent on the surface.From the outside watching, clear boundary, no adhesion with the skin, good mobility, having peplos apparently and epidermis rich in blood vessels were observed,but the mobility of tumor decreased gradually with the progressive increase of tumor volume. When the tumors were bigger than two centimeters in diameter, brown necrotic foci which had irregular margins were observed on the central Epidermis of tumors,and necrotic area continued to expand with time.The transplanted tumor tissues were easily stripped with layer of intact peplos rich in blood vessels.The section of tumors looks like fish.The tissues were crispy and easily broken.2.General conditions of the tumor-bearing mice during administration course:A good mental state, normal activities,agile response,eating and drinking normally, normal skin color, normal stool and no abnormal situation were observed in all the tumor-bearing mice from the first day to the seventh day of administration of drugs.With the enlargement of tumor size, normal activities of the tumor-bearing mice were influenced gradually from the eighth day of administration of drugs,for example,a slowing of movement and reduced eating and drinking.When the tumors were bigger than two centimeters in diameter, cachexia was observed in the tumor-bearing mice.Skin color gradually changed from pink to light cyan,and the weight of the tumor-bearing mice increased gradually with the enlargement of tumor size from the eighth day. Ascites were observed in two tumor-bearing mice of 3mg/kg As2O3 group,and metastatic tumor were observed in a tumor-bearing mice of control group. Apart from this,there was no significant difference between treatment groups and control group in general conditions of the tumor-bearing mice.3.There was an increase of the transplanted tumor’s volume as time went on. The rate of tumor volume increase in control group was the fastest, followed by 50mg/kg DFO group and 3mg/kg As2O3 group.And the rate in combination group(50mg/kg DFO+1.5mg/kg As2O3)was the lowest.No tumour grew from the first day to the fifth day of administration of drugs. From the sixth day mean volumes of tumors in control group were higher than them in treatment groups.And the differences of mean volumes of tumors between treatment groups and control group were further expanded as time went on.On the eleventh day the tumor volume of control group was the largest, followed by 50mg/kg DFO group, and 3mg/kg As2O3 group is next. The tumor volume of combination group(50mg/kg DFO+1.5mg/kg As2O3) was the smallest.So we can see that different drugs affected the trend of transplanted tumor’s volume over time.4.The tumor inhibitory ratioes of 50mg/kg DFO group and 3mg/kg As2O3 group were 2.56% and 10.67% respectively.The transplanted tumor weights of 50mg/kg DFO group and 3mg/kg As2O3 group were(2.55±0.82)g and(2.34±0.79)g respectively.There was no significant difference between DFO group and the control group(F=1.290,P=0.866>0.05),and there was no significant difference between As2O3 group and the control group (F=1.714, P=0.474>0.05).The tumor inhibitory ratio of combination group(50mg/kg DFO +1.5mg/kg As2O3)was 25.49%, The transplanted tumor weight of combination group was(1.95±0.39)g,and significantly lower than that of control group(F=0.028,P=0.019<0.05).It indicated that DFO combined with As2O3 had the synergistic anti-tumor effect.5.After the tumor-bearing mice were executed, hemorrhage, necrosis and black scab were observed on the tumor tissue through macroscopic observation.By light microscope the tumor cells in control group were large, orbicular or elliptic, heterogeneous and basophilic,with high nucleocytoplasmic ratio,and distributed compactly. A few of apoptosis cells and apoptotic bodies were found in control group,in which areas of focal necrosis scattered. The number of tumor cells in tissue of treatment groups were smaller than that of normal saline control group.Small tumor cells with hyperchromatic nuclei,pycnosis, Some remaining nucleus,irregular necrosis,hemorrhage, flaky eosinophilic Structure,apoptosis cells and apoptotic bodies with a small flaky distribution were found in treatment groups.especially in combination group.6.By light microscope,Complete structure, no obvious pathological changes except of congestion in different degrees and no metastases were found in the tissues of heart,liver,spleen,lung and kidney of the tumor-bearing mice.7.After administration completed, bone marrow depression and infiltration of leukemic cells HL-60 were not observed in the bone marrows of the tumor-bearing mice,and the active proliferation of bone marrow karyocyte and normal myeloid erythroid ratio were observed in bone marrows.8.NF-κBp65 expression levels of transplanted tumors were detected by two-step Immunohistochemistry detection(PowerVisionTM),and then the statistical results showed that there was significant difference in average value of integrate optic density(IOD) among experimental groups(P<0.05).The average value of IOD of control group was the largest, followed by 3mg/kg As2O3 group, and 50mg/kg DFO group is next. The average value of IOD of combination group(50mg/kg DFO+1.5mg/kg As2O3) was the smallest. So both DFO and As2O3 could lower NF-icBp65 expression levels of transplanted tumors, and especially DFO combined with As2O3 had a stronger effect.Conclusion1.The xenograft model of the human leukemia cell line HL-60 in nude mice was successfully established by Inoculating human high tumorigenic leukemia cell line HL-60 (about 1×106 cells/each mouse).Transplanted tumor grew in 100% of animals,and grew rapidly after forming tumours.From the seventh day to the eighth day after inoculating, macroscopic tumors were observed 0.5cm～1.0cm in diameter.2.Both DFO and As2O3 could inhibit the growth of transplanted tumors,and DFO combined with As2O3 had a obvious and significant effect on inhibiting the growth of transplanted tumors.3.When DFO combined with As2O3,it was well tolerated and there were no significant adverse effects.DFO not only lowered the dosage of As2O3,but also increased the killing effect of As2O3 on tumor.4.Both DFO and As2O3 could lower NF-κBp65 expression levels of transplanted tumors, and especially DFO combinated with As2O3 had an obvious effect.

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