Experimental Study On The Effect Of Bortezomib Or In Combination With Arsenic Trioxide For Nude Mice Bearing HL-60 Cells Xenografts

Background and objective:Leukemia is a kind of clonal malignant disease of hemopoietic stem cell. It is themost common cause of death under the age of 35. In recent years, the incidence ofleukemia is going up. Although the treatment for patients suffered from leukemiahas improved throughout the last decades, the side-effects and relapse still severelydegrade the patients' overall survival rates. So new chemotherapeutic agents areneeded to optimize treatment protocols, minimize side-effects and increase overallsurvival rates. In the last decade, the use of targeted therapy drugs might prove a hugestep forward. Proteasome inhibitor (PI) is a kind of new targeted therapy drug, whichblocks cancer progression by interfering with the degration of regulatory proteins.One of the most described and well-known PIs is bortezomib, a dipeptide boronicacid analogue with a broad antitumor activity in several cell lines and in murine andhuman tumor models. It was approved for the treatment of patients with relapsed orrefractory multiple myeloma by the US Food and Drug Administration and theCommittee for Proprietary Medicinal Products of the European Union. It had alreadybeen shown that human leukemic cell lines and primary leukemia cells expressed abnormally high levels of proteasomes compared with normal peripheral blood cells.Bortezomib successfully induced apoptosis in leukemic cells. When bortezomib incombination with other chemotherapeutics, it had additional effect or synergisticeffect. In the phaseâ… studies, although bortezomib seemed to have biological activity,the clinical benefits were limited when given as a single-drug agent. Whencombinated with other chemotherapeutics, it is well tolerated and very effective. Inour previous study, bortezomib could effectively induce apoptosis in HL-60 cells.When added arsenic trioxide simultaneously to the cells, it had synergistic effect ininduction of apoptosis. So we design animal experiment to assess the antitumoractivity and the adverse effects of bortezomib or in combination with arsenic trioxidein vivo and investigate the working mechanisms.Methods:1. The tumor cells were injected s.c. into the back of nude mice to form humanleukemia xenografts models. When mean tumor volume was 50-100 mm³, drugadministration was started.2. According to the volume of transplantation tumor, the HL-60- bearing nudemice were randomly divided into four subgrounds: Group A, saline alone i.p., d1-21;Group B, 0.5ms/ks bortezomib i.p., d1,4,8,11; Group C, 5ms/ks As₂O₃ i.p., d1-21;Group D, 0.5ms/ks bortezomib i.p., dl,4,8,11+5ms/ks As₂O₃ i.p., d1-21.3. To assess the antitumor activity of bortezomib or in combination with arsenictrioxide in vivo by comparing the growth situation of transplantation tumors and thesurvival time of each individual mouse. At the same time, the adverse effects of drugsin HL-60-bearing nude mice were observed.4. The specimens of transplantation tumors were stained with HE. Then thechanges of each specimen in different experimental groups were analyzed. After administration, the bone marrows of HL-60- bearing nude mice were observed todetermine whether bone marrow depression and tumor metastasis existed or not. Thetissues of heart, liver, spleen, lung and kidney from the dying HL-60- bearing nudemice were observed to determine whether pathological changes and tumor metastasisexisted or not.5. The in situ TUNEL assay for detection of apoptosis was performed inspecimens from transplantation tumors. The immunohistochemical assay fordetection of proliferation cell nuclear antigen-positive nuclei cell was performed inspecimens from transplantation tumors.6. The results of experiment were analyzed by SPSS10.0. The difference of thevolume of the transplantation tumor before or after administration in each group ofthe HL-60-bearing nude mice were test by repeated measure ANOVA. Thedifferences of relative tumor volume and the survival time of HL-60- bearing nudemice between four experimental groups were test by one-way ANOVA. Ps of lessthan 0.05 were considered significant.Results1. Inoculating HL-60 cells culturing in vitro, tumor growth in 20% of animals,compared with 53% of mice inoculated HL-60 cells culturing in vivo. Tumor grewonly at the sites of cells inoculation. No metastases were found at autopsy. In thetransplantation tumor, the tumor cells were large and primitive, vesicular nucleicontained multiple nucleoli, and it had a few promyelocytes.2. There was an increase of the transplantation tumor's volume as time went on.The tumor volumes had significant difference before or after drug administration (F=330.357, P＜0.001). And it also had significant difference between experimentalgroups (F=4.200, P=0.030). The rate of tumor volume increase in Group A was the fastest, followed by Group B and Group C. And the rate in Group D was the lowest.The relative tumor volume of experimental groups had significant difference sinced7 during administration course. Since then, the relative tumor volume of Group Dwere significant lower than Group A's and Group B's, but there was no significantdifference between Group C and Group D. And from d11 on, the relative tumorvolume of Group C were significant lower than Group A's. The tumor inhibitingratioes were 19%, 29% and 35% in Group B, Group C and Group D.3. There was significant difference in the survival times among experimentalgroups (F=4.171, P=0.047). The survival times of the HL-60- bearing nude micein Group C and Group D were significantly longer than Group A, the P values was0.036 and 0.009 respectively. The survival time prolongation ratioes were 12%, 16%and 22% in Group B, Group C and Group D.4. During administration course, no significant changes of the mental state,locomotor activity, reaction ability and appetite of each HL-60- bearing nude mousewere observed. After administration completed, the bone marrows of HL-60- bearingnude mice didn't exist bone marrow depression. There was an increase in the bodyweight of HL-60- bearing nude mouse along with time went on. The body weight ofeach group had significant difference before or after administration (F=76.709, P＜0.001). But there was no significant difference between each group (F=0.260, P=0.853).5. In pathologic observation, the tumor cells were large, heterogeneous andbasophilic, with high nucleocytoplasmic ratio. In Group A, the tumor cellsdistributed compactly, and a few of apoptosis cells and apoptotic bodies were found.In Group B, more apoptosis cells and apoptotic bodies were found than in Group A.In Group C and Group D, the density of tumor cells decreased, and the quantities of apoptosis cells and apoptotic bodies increased. In the transplantation tumors fromdying nude mice, tumor cells distributed around necrotic tissue, and there was a fewof apoptosis cells and apoptotic bodies.6. The apoptosis indices were 4%, 11%, 16% and 25% in Group A, Group B,Group C and Group D. The proliferation cell nuclear antigen—positive nuclei cellindices were 56%, 45%, 41% and 38% in Group A, Group B, Group C and Group D.Conclusions:1.Both inoculating of HL-60 cells cultured in vitro and in vivo could form themouse metastasis model of human leukemia. Compared with achievement ratio, itwas more effective to use HL-60 cell cultured in vivo than in vitro.2. Both bortezomib and arsenic trioxide had antitumor activity in vivo. Whenbortezomib combinated with arsenic trioxide, it could enhance the antitumor activityin vivo.3. When bortezomib combinated with arsenic trioxide, it was well tolerated andthere were no significant adverse effects.4. Both bortezomib and arsenic trioxide could induce HL-60 cells apoptosis andinhibit the proliferation of HL-60 cells in vivo. And it was more effectively whenbortezomib combinated with arsenic trioxide.