Abstract Objective To observe of 5 lipoxygenase activating protein (FLAP) inhibitor MK886 treatment effect on the growth of human colon cancer xenografts, and to explore the possible mechanism of its anti-tumor. HT 29 human colon cancer cells were prepared growth of human colon cancer xenograft model, 15 tumor-bearing nude mice were randomly divided into three groups, the treatment group MK886 dissolved in DMSO dosing, two control groups, respectively, to dimethyl sulfoxide and without any treatment. Tumor growth was observed during the treatment period, the mice were killed after the end of treatment and take the tumor, tumor volume was measured weight using immunohistochemical methods to detect tumor microvessel density and tumor cell apoptosis detected by TUNEL method. Results All 15 nude mice tumor, and that no nude mice died during the experiment; through the measurement of tumor volume, tumor weight results MK886 inhibit human colon tumor growth in nude mice transplanted subcutaneously; experiments also confirmed MK886 on human colon carcinoma transplanted into nude mice to induce tumor cell apoptosis and anti-angiogenic effect. Conclusion MK886 significant treatment effect for the growth of human colon cancer xenografts, MK886 may induce tumor cell apoptosis and inhibit tumor angiogenesis mechanism to control the growth of human colon cancer.
Key words 5 lipoxygenase; inhibitors; nude mice; colon cancer
Efficacy of MK886 in Nude Mouse Model of Human Colonic Cancer
ZHOU Guang jun, ZHU Xiao chao, SHI Kun
Department of General Surgery, Yancheng First Hospital, Yancheng 224001, ChinaAbstract: Objective To investigate the effect of the FLAP inhibitor, MK886 on human colonic cancer xenografts in vivo and to explore its potential anti neoplasm mechanism.Methods Cultured HT 29 human colonic cancer cells were injected into the flanks of fifteen nude mice to develop xenograft models and the tumor bearing nude mice were randomized into three groups to be administered with MK886 dissolved into DMSO, DMSO only, nothing, respectively.The mice were monitored for 4 consecutive weeks for tumor volume changes.Then the tumor tissues was isolated and weighted, followed by pathological examination.The expression of 5 LOX in tumor tissue was detected by immunohistochemistry and apoptosis of colonic cancer cells was also detected by TUNEL.Results None of the 15 nude mice died during the experiment and all nude mice formed in situ mass of colorectal tumor.MK886 inhibited growth of human HT29 colon cancer xenografts in athymic mice, measured as both tumor volume and tumor weight.The expression of 5 LOX in tumor tissue of control group was stronger than that of treated groups.This test also confirmed the induction of apoptosis in colonic cancer cells and the function of inhibiting angiogenesis of tumor by MK886.Conclusion 5 LOX inhibitor, MK886 inhibits the growth of colonic tumor by inducing the apoptosis of human colonic cancer cells and inhibiting the angiogenesis of tumor.
Key words: 5 LOX; Inhibitor; Nude mouse; Colonic cancer
The class of arachidonic acid (arachidonic acid, AA) some metabolites plays an important role in in many tumor development and metastasis. The lipoxygenase (lipoxygenase, LOXs) as a key enzyme in the pathway mediated metabolism of arachidonic acid, which could affect tumor development, the LOX inhibitors more and more into the research and application of the tumor. Since Rygaard in 1969, the first successful human colon cancer in nude mice after people began using nude mice has done a lot of research on human malignancies. Thus, this experiment in nude mice carrier, the choice of the 5 LOX high expression in human colon cancer cell line HT 29 the establishment of a human colon nude model, designed to study the 5 LOX activating protein (FLAP) inhibitor (MK886 ) The inhibitory effect and synergies, and to explore its mechanism.
1 Materials and methods
HT 29 human colon cancer cell line, provided by the Shanghai Institute of Cell Bank.
1.2 in experimental animals
15 SPF level of nude mice, 4-5 weeks old, weighing 16 ~~ 20g are female, provided by the Chinese Academy of Sciences Shanghai Laboratory Animal Center. Are breeding of SPF Laboratory Animal Center at Suzhou University.
1.3 Main reagents and drugs
Purchased from McCoy’5A medium GiBco company; by TUNEL kit provided by Roche; goat anti-human 5 LOX polyclonal antibody by Cayman Chemical Company; dimethyl sulfoxide blood by the First Affiliated Hospital of Suzhou University Research Center ; plug to celecoxib purchased from Pfizer Limited; MK886 were purchased from Cayman Chemical Company.
1.4 Cell culture and passaged
The following operations are carried out in sterile cell culture chamber ultra-clean workbench. Human colon cancer HT 29 cells McCoy’5A medium (containing 10% fetal bovine serum) maintained at 37 ℃, 5% CO2 incubator culture, cells adherent monolayer growth until the adherent cell fusion up to 70% to 80% trypsin (containing 0.02% EDTA) digestion and passage.
1.5 the cells were collected and inoculated in nude mice
Will be in the logarithmic growth phase cells trypsinized with no serum McCoy’5A culture liquid diluted cell suspension was centrifuged, the supernatant was discarded, and then a serum-free McCoy’5A cultivate repeatedly washed and centrifuged 3 times, discard supernatant, no the serum McCoy’5A culture was diluted cells and adjust cell concentration to 3 × 107 / ml; take the cell suspension 0.1ml / only were injected subcutaneously in nude mice right upper limb heel.
1.6 group therapy
Be visible nude mice inoculated with parts of a tumor to grow, 15 mice were randomly divided into three groups (n = 5). Blank control group: do not use any drugs; dimethyl sulfoxide control group: dimethyl sulfoxide 0.5μl of / (g · d) -1, once a day orally; MK886 treatment group: MK886 0.015mg / (g · d) – dissolved in dimethyl sulfoxide 10μl gavage once daily. 4 weeks of continuous treatment.
1.7 observing tumor growth in nude mice generally
After treatment, respectively 1,3,7,14,28 d with a vernier caliper measurement of each tumor size, and repeated measurements three times and take the average tumor volume was calculated by the following formula: V = ab2π/60 a: tumor nodules maximum diameter, b: transverse diameter of the tumor nodules after the last measurements were sacrificed nude mice weighing remove the tumor. During treatment, but also continuous observation of the diet, stool, spiritual activities and emaciated in nude mice.
1.8 produced tumor biopsy
The mice were sacrificed, dissected and remove the complete tumor mass production, with 10% formalin-fixed specimens, biopsy, some sections were HE (hematoxylin – eosin) staining, tumor histological morphology of a conventional light microscope.
1.9 TUNEL assay of tumor cell apoptosis
Paraffin sections were xylene dewaxing graded ethanol hydration, H2O2 closed after 30min PBS (pH7.4) oscillation three times, each time 5min, the proteinase K digestive role in 25 to 30min (37 ° C) and then vibrating washing with PBS 3 times, 5min each incremented by the TUNEL reaction solution, peroxidase-conjugated anti-fluorescein antibody and incubated at 37 ℃, PBS the oscillator washed DAB color, hematoxylin, gradient alcohol dehydration dry, xylene permeabilized , neutral resin Fengpian. Specific steps according to kit instructions. Each batch of staining located negative control (PBS instead of primary antibody) and positive results to determine: positive staining of nuclei stained brown, as apoptotic cells. The count is expressed as a percentage of positive cells per 1000 cells apoptosis index (AI) to high magnification.
The 5 LOX expression of 1.10 immunohistochemical assay of tumor tissue
Paraffin sections conventional xylene dewaxing gradient alcohol hydration 10mmol / L citric acid buffer (pH 6.0), placed in the microwave treatment to fix the antigen, the cooling to room temperature, 1% H2O2 methanol incubated for 15min, was added dropwise 10% monkey serum 15min to close the non-specific antigen, discarded serum added dropwise antibodies (1:100 dilution of goat anti-human 5 LOX polyclonal antibody) 4 ° C, allowed to stand overnight, PBS wash solution three times after the dropwise addition, the biological dropping horseradish peroxidase-labeled avidin biotinylated secondary antibody and incubated at 37 ° C for 60min, PBS solution again washed 3 times, 37 ℃ incubated for 60min, DAB color hematoxylin gradient alcohol dehydration and drying, two toluene permeabilization neutral resin Fengpian. The above operations are carried Reagent Company provided instructions. The 5 LOX expression of the results of determination method: the brown granules to the cytoplasm, nuclear membrane or membrane presents positive cells, only cell nuclei blue is negative.
1.11 Statistical Methods
All data ± s, were statistically analyzed using SPSS 15.1 statistical software.
2.1 rate of tumor
This experiment, nude mice were 15 HT 29 human colon cancer cells were inoculated subcutaneously four days after the inoculation site about 0.4cm size of the tumor nodules, tumor formation was 100%. All mice during treatment were not significant activities to reduce and slow to respond, listlessness, weight loss phenomenon. The end of the experiment, a nude mice died of natural causes.
2.2 tumor growth
2.2.1 the end of treatment tumor volume was measured values in Table 1. Table 1 nude mice tumor volume (cm3) above data using analysis of variance for multiple comparisons (LSD method) results show: two control group, tumor volume was no significant difference (P = 0.729); difference between MK886 treatment group and two control groups were statistically significant (P = 0.000).
2.2.2 each group Comparison of tumor weight, as shown in Table 2. Table 2 tumors in nude mice weight above data using the the LSD method results show that: the MK886 the treatment group compared with the difference between the two control groups were statistically significant (P = 0.000), no statistically significant difference between the control group (P = 0.29) .
2.3 tumor histopathological findings
HE staining of biopsy in the light microscope diffuse distribution of tumor cells, different size, shape, lightly stained cytoplasm, nucleus hypertrophy deeply stained blue, visible mitotic occasionally glandular-like structures, some horizons visible necrotic area line colon adenocarcinoma of Pathology performance, as shown in Figure 1.
2.4 tumor cell apoptosis detection results
Blank control group, the control group of dimethyl sulfoxide, MK886 treatment group AI (3.0438 ± 0.1704) (3.1800 ± 0.1860) (10.7100 ± 1.0193). The above data LSD method: The results showed that MK886 treatment group compared with the two control groups between the differences were statistically significant (P = 0.000) between the control group, the difference was not statistically significant (P = 0.29), as shown in Figure 2 to 4 .
The 5 LOX 5 LOX expression levels of 2.5 tumor tissue blank control group with dimethyl sulfoxide control group tumor tissue showed strong expression of MK886 treatment group showed a weak positive expression, as shown in Figure 5 to 7.
Arachidonic acid metabolism by cyclooxygenase and lipoxygenase completion of two ways, the key enzyme cyclooxygenase and lipoxygenase these two metabolic pathways. Lipoxygenase pathway, 5 LOX and its product of 5 HETE and LTB4, 8 of LOX and its product HETE, 12 LOX and the 12 HETE may promote tumor development, and recent research than more than 5 LOX and its metabolites. Such as the experimental data, were detected in human prostate cancer, colon cancer and other tumor patients leukotriene excessive expression ; Wei Gang Tong six different human pancreatic cancer cells in vitro experiments showed that the six kinds human pancreatic cancer cells were LTB4 receptor expression and LTB4 receptor antagonist LY293111 can inhibit the growth and proliferation of pancreatic cancer cells ; 5 HETE formation and inhibition of human prostate cancer tissue respectively promote and inhibit the growth of prostate cancer cells ; animal experiments also confirmed of LTB4 in receptor antagonist LY293111 can inhibit the growth of colon cancer in nude mice .
MK 886 the 5 LOX activating protein (FLAP) inhibitor. 5 LOX catalyzed metabolism of arachidonic acid to generate LTB4 still need to send LOX activating protein (FLAP) substrates to assist MK 886 energy-specific and FLAP inhibit 5 LOX activity, thereby preventing arachidonic acid 5 LOX metabolic pathways. Experiments show specific FLAP inhibitor MK 886 can effectively inhibit human breast cancer, colon cancer cell growth and proliferation and induce apoptosis [5,6], animal experiments also show that the anti-pancreatic cancer effects of MK 886 [7 ]. The study found that the experiment the the 5 LOX inhibitor MK 886 on human colon cancer transplanted into nude mice therapeutic effect application MK 886 treatment slow tumor growth, measured after the end of treatment, the mean tumor volume of the treatment group , weight were significantly lower than the control group, the difference was statistically significant (P = 0.023, P = 0.025, P = 0.00); TUNEL assay of tumor tissue AI, results showed that the tumor cells with MK 886 treatment group was significantly higher, the difference was statistically significant (P = 0.00); the 5 LOX expression levels Immunohistochemical detection of tumor tissue, the results show that of 5 LOX expression level of MK 886 treatment group was significantly lower than the control group; while the 5 LOX expression level and there is no statistical tumor volume, weight, tumor cells AI difference between the blank control group with dimethyl sulfoxide control group were not statistically significant (P> 0.05), and between the two control groups significance.
Therefore, through this experiment can confirm LOX colon cancer development are closely related, 5 LOX activating protein inhibitor MK 886 can effectively inhibit the growth of human colon cancer and reduce its 5 LOX expression levels, and its anti-cancer mechanisms may induce tumor cell apoptosis, there may be other anticancer mechanism needs further study.