Establishment Of An Animal Model Of Tumor-bearing Mice Cured By A Low Dose Of Chemotherapy And Its Possible Mechanisms

Objective:With the great changes in human lifestyle and environment, the malignant tumor has become one of the most common diseases endangering human health in modern society. According to statistics, about 7 million people are diagnosed and nearly 5 million people die of cancer worldwide each year. In recent years there has been an increasing trend in the incidence and mortality of malignant tumors in china. Because the cause of malignant tumors is not completely clear, at the present time combined treatment methods including surgery, chemotherapy, radiotherapy, treatment with biological agents and so on had been adopted in clinic, and systematic treatment with cytotoxic drugs has been the basis of most effective treatments for patients with disseminated cancers. It is thought that cytotoxic drugs inhibit or kill tumor cells in a dose dependent manner. It was reported that a high dose of chemotherapy could cure gestational trophoblastic tumor in clinic, but five years survival rate of most patients with malignant tumors was not elevated significantly with a high dose chemotherapy scheme. It was found that a high dose of chemotherapy could increase not only the short-term effects on tumor, but also the dose- dependent side effects. And it was not favorable to improving the patients'quality of life and elevating the long-term therapeutic efficacy. With the medical model changes, it was realized living quality and prolonged survival time are of decisive significance for patients with malignant tumors, and tumor-shrinkage is not the primary goal of treatment. Of all anti-cancer drugs used in clinic, they inhibited or killed tumor cells only by ratio. Therefore, factors related to tumor rejection induced by chemotherapy are not clearly understood, and the mechanisms of tumor rejection induced by chemotherapy should be studied further. In order to understand the mechanisms of tumor rejection induced by chemotherapy much better, a tumor-bearing mice model was used and an animal model cured by a single dose of chemotherapy was established. Further, dynamic changes of peripherial blood cells were measured during tumor rejection, and the relationship between chemotherapeutic effects and mouse peripherial blood cells was analyzed. The changes and significance of platelets including platelet count and platelet activating factor (PAF) in chemotherapy-induced tumor rejection was ensured. Using this tumor rejection model induced by chemotherapy, the ultrastructural morphology of tumor tissues 72 hours after chemotherapy treatment was studied by transmission electron microscopy. The mechanisms of chemotherapeutic drugs on tumor cells and tumor stroma cells (especially tumor vascular endothelial cell) will be detected. In order to know whether the injury or death of tumor cells was related to oxidative injury, the concentration of malondialdehyde (MDA), the product of metabolism of lipid peroxidation was measured in tumor tissue within 72 h after chemotherapy. The content of endotoxin and nitric oxide (NO) in plasma of tumor bearing mice within 72 h after chemotherapy were also tested. Possible mechanisms of tumor rejection induced by low-dose CTX chemotherapy would be explained.Methods:Mouse bladder carcinoma tissue was inoculated subcutaneously into BTT739 mice. 7 days later, different doses of cyclophosphamide (CTX) or Mitomycin C (MMC) were used intraperitoneally to treat these tumor- bearing mice and the same volume of NS was used as control. Mice were observed and tumor sizes and body weight were measured and recordered everyday or every 2 to 3 days. Animals were sacrificed once tumors reached a maximal size of 15 mm in diameter. Mice with tumors less than 12 mm in diameter or without tumors were classified as tumor free mice. The minimal effective dose of chemotherapy that could cure most of the tumor-bearing mice was found. Tumor rejection was defined as complete tumor regression after chemotherapy and the absence of recurrent tumor for the entire follow-up period (at least 60 days).Then another 12 tumor-bearing mice were randomly divided into two groups: treatment group and control group. The minimal effective dose of chemotherapy that could cure most of the tumor-bearing mice was given intraperitoneally and the same volume of NS was used as control. Blood samples were obtained from orbital venous sinus at different times after chemotherapy. Complete blood counts were performed and dynamic changes of peripherial blood RBC, Hb, WBC, platelet parameters were obtained. The relationships between the changes of peripheral blood count and tumor shrinkage induced by chemotherapy was analyzed. Establishing a method for testing the level of PAF in plasma by Liquid Chromatography - Mass Spectromete(rLC/MS/MS)Device, the level of plasma PAF in tumor bearing mice was measured 6 h after chemotherapy treatment.Finally, another 18 tumor-bearing mice were randomly divided into two groups: treatment group and control group. The minimal effective dose of chemotherapy that could cure most of the tumor-bearing mice was given intraperitoneally and the same volume of NS was used as control. Blood samples were obtained from orbital venous sinus at 6, 24, 48, 72 h after chemotherapy. Using the BET-16 bacterium endotoxin cryoscope, endo- toxinemia was measured in tumor bearing mice 6 h after CTX treatment by the kinetic turbidimetric limulus assay. Serum nitric oxide (NO) was measured by nitrate reductase method in tumor bearing mice within 72 h after CTX treatment. The mice were killed at different time after CTX treatment. And the content of malondialdehyde (MDA) was measured by the thiobarbituric acid reaction assay (TBARS) in tumor tissue before and different time after CTX treatment. Morphological changes of apoptosis and/or necrosis in tumor tissue 72 h after CTX treatment were observed with transmission electron microscope (TEM).Results:1. Establishment of an animal model of tumor-bearing mice cured by a low dose of CTX Inoculation of 2 mm3 bladder carcinoma tissue could make tumor cell progressive growth in T739 mice. The tumor sizes were 2～4 mm on day 7 after inoculation. Tumor bearing mice were treated with different doses of CTX (15～400 mg/kg) or MMC (1～16 mg/kg). It was found: (1) the higher dose of MMC used in the study could increase not only the speed of tumor shrinkage but also the toxic side effects on tumor bearing mice 1 week after treatment. 2 months after MMC treatment, all mice died in 8～16 mg/kg MMC treatment group and control group; While 25% of mice were alive in 4 mg/kg and 1 mg/kg MMC treatment group respectivelyl; (2) and the higher dose of CTX used for the treatment of these tumor bearing mice could also increase the speed of tumor shrinkage and induce more severe toxic side effects 2 weeks after treatment. 2 months after CTX treatment. All mice died in 100～200 mg/kg CTX treatment group and control group; While 20% of mice were alive in 40 mg/kg CTX treatment group and 60% of mice were alive in 15 mg/kg CTX treatment group. Compared with control, there was a significantly difference in survival rate of tumor bearing mice between 15 mg/kg CTX group and control group (p<0.01). The result indicated that 15 mg/kg CTX was the minimal effective dose of CTX that could cure most of the tumor-bearing T739 mice.2. Changes and the significance of peripheral blood cell count and plasma PAF level in tumor shrinkage induced by a low dose of CTX in T739 mice 15 mg/kg CTX had no obvious inhibitive effects on peripheral blood cell count of tumor bearing mice. The perpherial RBC count, concentration of Hb and WBC count was (9.86±0.64)×10¹²/L, 166.6±15.2 g/L and (12.38±3.96)×10⁹/L respectively at 6 h after 15 mg/kg CTX treatment. Compared with that in control mice (9.0±0.6)×10¹²/L, 148±10 g/L and (10.54±1.74)×10⁹/L, there was no significant difference between them(P>0.05);The perpherial platelet count increased to (1483.4±184.4)×10⁹/L in mice 6 h after CTX treatment. There was significant difference compared with that in mice of control group (1086.6±81.0)×10⁹/L (P<0.01); and there was no significant difference in MPV, PDW and P-LCR between the mice in control group and CTX treatment group (P>0.05). During the 2nd to the 14th day, there was no obvious difference in peripheral RBC count, WBC count and platelet parameters in tumor bearing mice after CTX treatment (P>0.05). Using LC/MS/MS, PAF level in plasma was measured. It was found that the concentration of plasma PAF was 113.98±14.38 ng/ml in tumor bearing mice 6 h after CTX treatment; while only 58.34±13.89 ng/ml in control mice. There was a significant difference in plasma PAF between CTX treatment and control groups (P<0.01). Therefore, not only peripherial PLT count but also plasma PAF concentration increased in tumor bearing mice 6 h after 15 mg/kg CTX treatment. The results indicated that platelet played an important role in the tumor rejection induced by low-dose CTX chemotherapy.3. Possible mechanisms of tumor rejection induced by low-dose CTX chemotherapy 6 h after 15 mg/kg CTX treatment, most of tumor cells in tumor tissue seen under TEM were of classical morphological changes of apoptosis, including decreased cell volume, cytoplasmic condensation, cytoplasmic vacuoles, and condensed nuclear chromatin and so on. Vascular endothelial cells were also of classical morphological changes of apoptosis with decreased the diameter of capillary. Besides these, tumor cells also exhibited typical morphological changes of necrosis. TEM analysis displayed that not only tumor cells but also vascular endothelial cells exhibited typical apoptotic phenotype, and moreover, apoptosis and necrosis of tumor cells coexisted in tumor tissue after low-dose CTX chemotherapy. The MDA content in normal tumor tissue was 16.1±1.35 nmol/mgprot, and 14.69±3.86 nmol/mgprot in tumor tissue after 6 h of 15 mg/kg CTX treatment. There was no significant difference between them (p>0.05). The MDA content decreased to 8.87±2.70, 5.15±2.13 and 8.92±2.94 nmol/mgprot in tumor tissue 24, 48, 72 h after CTX treatment, obviously lower than that in control. The results indicated that there was no lipid peroxidation damage of tumor cell membrane in tumor bearing mice within 72 h after CTX treatment. The plasma concentration of NO in tumor bearing mice 6 h after 15 mg/kg CTX treatment was 60.93±5.59μmol/ml, obviously higher than that 34.88±19.08μmol/ml in control mice (p<0.05). And plasma concentration of NO increased to 81.86±17.33μmol/ml 72 h after CTX treatment, significantly higher that in control mice (p<0.01). The increasing concentration of plasma NO may be related to the increased level of plasma PAF and the injured vascular endothelial cells in tumor bearing mice after CTX treatment. The level of plasma endotoxin was also measured. It was found that level of plasma endotoxin was 0.29±0.38 Eu/ml in mice 6 h after low-dose CTX chemotherapy; significantly lower than that 1.20±0.72 Eu/ml in control mice (p<0.05). The results indicated that there was no endotoxinemia in tumor bearing mice at this time.Conclusions:1. 15 mg/kg CTX administration intraperitoneally could cure most of the tumor-bearing T739 mice. A new mouse tumor rejection model induced by low-dose CTX had been established successfully.2. 15 mg/kg CTX had on obvious inhibitive effects on peripherial blood cells. Peripherial platelet count and the level of plasma PAF was increased significantly in tumor bearing mice 6 h after CTX treatment. The results indicated that platelets played an important role in tumor rejection induced by chemotherapy.3. TEM analysis displayed that low-dose CTX treatment could induce apoptosis not only in tumor cells but also in vascular endothelial cells. The necrosis of tumor cells may be related to the injured vascular endothelial cells in tumors after low-dose CTX chemotherapy. The decreasing content of MDA in tumor tissue within 72 h after CTX treatment indicated that the death of tumor cells had nothing to do with oxidative injury in tumor tissue in vivo. 6 h after CTX treatment,there was no endotoxinemia in tumor bearing mice. The increasing level of plasma NO during 6～72 h after CTX treatment was probably related to the increased plasma PAF level and the injured vascular endothelial cells in tumor bearing mice after CTX treatment...