| Colon carcinonma is the one of the most common malignant tumors, has only to lung cancer, stomach cancer,is the third cause of cancer mortality in china,And gradually increased morbidity and mortality, is a threat to human life. Surgical resection is a commonly used therapy for patients with colon carcinoma However,tumor metastasis and recurrence after colon cancer surgery often occurs, which is the leading cause of deaths in patients with colon cancer.Growth of tumor cells consists a series of complex process, an imbalance between multi-gene during tumor progression. Molecular targeted therapy brings new opportunities, the study of molecular targeted therapy for cancer molecular and cytological become a new hot. High-mobility group box1(HMGB1)has been associated with many human cancers, which is a kind of tumor growth,inwasion,metastasis and angiogenesis are closed related to chromatin proteins.The proteins were first purified from nuclei in the1970s and termed high mobility group (HMG) proteins to reflect their rapid mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. HMGB1gene located on human chromosome13q12, a relative molecular weight of30×103, of a total of215amino acids. It is extensively modified posttranslationally in several ways by glycosylation, acetylation, methylation, and phosphorylation.Acetylation and phosphorylation induce translocation of HMGB1from the nucleus to the cytoplasm. HMG proteins are small DNA-binding proteins that play an important role in transcriptional regulation, The HMGB1sequence is highly conserved among species:murine HMGB1differs from human HMGB1by only two amino acids. HMGB1consists of two tandem L-shaped domains, HMGB boxes A and B, each～75amino acids in length, and a highly acidic carboxyl terminus30amino acids in length. A box can replace full-length HMGB1binds to the receptor but does not play a biological role, B boxes are caused by inflammation of the specific structure, while A box can antagonism B box, C major regulator ends with DNA affinity. HMGB1is widely present in lymphoid tissue, brain, liver, lung, heart, spleen and kidney and other tissues. Various different distribution of HMGB1in the liver and brain tissues are mainly distributed in the cytoplasm, while other organizations are mostly found in the nucleus. Overexpression of HMGB1has been observed in most tumor cells including Liver cancer, stomach cancer, colorectal cancer, prostate cancer, pancreatic cancer, bladder cancer, oral cancer, nasopharyngeal cancer, and breast cancer. Compared to the normal tissue, it is weakly positive expression in colon cancer adjacent tissues, but a high level of expression in colorectal adenocarcinomas. HMGB1is overexpressed in colorectal cancer cells (HCT116, HT-29, SW480, and DLD-1derived from primary lesions), and Lovo and SW620cells (derived from metastatic lymph nodes). HMGB1overexpression promotes cell motility, invasiveness, proliferation, and angiogenesis in cancer progression. Targeting the HMGB1ligand or its receptor represents an important potential application in cancer therapeutics. The main mechanism may be the HMGB1binding RAGE receptor and activation of P38, JNK,ERK1/2pathway,etc, which in turn activates matrix metalloproteinase-9(MMP-9), matrix metalloproteinase-2(MMP-2). The interaction between HMGB1and RAGE receptor can activate NF-κB, regulation of apoptosis-related factors, the cells to evade apoptosis. HMGB1can also serve as a pro-angiogenic growth factors and binding with receptor RAGE, TLR2, TLR4, then increased leukocyte adhesion molecules, thereby inducing endothelial and hematopoietic cells to promote vascular endothelial growth factor (VEGF) is maintained angiogenesis. HMGB1can promote the activation of immune cells, endothelial cell activation, inducing the release of inflammatory factors.Many studies indicate that HMGB1promotes the development of tumor cells, invasion and metastasis,HMGBl could become a new molecular diagnostic cancer markers and a potential target for cancer therapy. UTI is a urinary trypsin inhibitor, can inhibit the activity of a variety of proteases and has the capable of inhibiting trypsin, hyaluronidase, elastase, plasmin, urokinase-type plasminogen activator, matrix metalloproteinase etc, which can suppress tumor progression. The role of UTI in cancer therapy get attentioned. The current study is the role of UTI for UPA (fiber protease activating factor), CD44, MMP-9, MMP-2, VEGF, etc. Ryo Tanaka has found that UTI was possible suppress the generation of HMGB1in the inflammatory process. In recents, studies have not been at home and abroad ulinastatin effect on colon cancer. In this study, we observed the effect of ulinastatin in cultured human colon lovo cells, and the following two studies:1explore ulinastatin on human colon lovo cell proliferation, invasion, apoptosis aspects;2. Investigate the effects of ulinastatin on human colon cancer lovo cells HMGB1, the expression of NF-κB and HMGB1distribution.Method1.Drug assayWe divided the cells into four groups for the drug assay:control (no UTI added), UTI1(400U/mL UTI), UTI2(800U/mL UTI), and UTI3(1600U/mL UTI).2.Cell growthThe lovo4×104cells/ml, seeded in96-well plates, and processed according to the packet, the total amount of medium per well200μl. Cells (2000cells/well) were seeded in96-well tissue culture plates and cultured for12h in regular medium. Cells were washed twice with phosphate-buffered saline (PBS) and treated with UTI. Cell growth was monitored after24h,48h and72h with3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Each well was added20μl MTT solution (5mg/ml), incubated4h; suction hole after the culture medium in each well was added150μl DMSO, at room temperature, the plates were placed on a shaker10 min, the crystal was dissolved. Measured by enzyme-linked immunosorbent assay at the wavelength of490nm absorbance of each well, and is calculated as the rate of cell proliferation. Inhibition rate (%)=(1-experimental A490/control A490) x100%. All experiments were repeated three times under the same conditions.3.Lovo cell invasion assayThe transwell chamber paved plastic spare.At24hours after the human colon cancer cells were treated in accordance with the grouping, UTI-treated Lovo cells (1x105) were resuspended in complete medium (200μl) and seeded in the upper chamber. DMEM medium with10%FBS to join the lower chamber, set37℃,5%CO2incubator for24hours. Remove the small room, PBS light wash chamber with pipette discard liquid, wet swab graze remove cells and Matrigel, a small chamber chamber; moved in advance plus a good4%poly-fixed30minutes formaldehyde hole, PBS liquid light wash several times. Add500ul0.1%crystal violet solution, the membrane was immersed in a chamber wherein incubated at37℃5%CO2incubator for30min, photographed under a microscope (×200), counting the number of cells, the experiment was repeated three times.4.Caspase activity assayAfter the cancer cells were treated24hours in accordance with the groups, according to the the caspase-3Fluorometric Protease Assay Kit (BioVision, CA, USA) according to the manufacturer’s protocol.5.Effects of UTI on HMGBlmRNA expressionAfter the colon lovo cells were treated in accordance with the grouping24hours, using a semi-quantitative reverse transcription polymerase chain reaction (reverse transeriptase a polymeraseehainreaetion, RT-PCR) to detect the expression of HMGB1mRNA.6.The expression of HMGB1, NF-κBproteinAfter the colon lovo cells were treated in accordance with the grouping24hours,using western blotting to detect the expression of HMGB1and NF-κB protein.7.Immunofluorescence imaging analysis. Cells were cultured in96-well plates,after the colon lovo cells were treated in accordance with the grouping24hours, fixed in4%paraformaldehyde in PBS for30min at room temperature. The cells were washed with PBS and incubated for10min at4℃with permeabilization buffer containing0.1%Triton X-100in PBS. Samples were blocked with10%normal serum blocking in PBS for10min and incubated with rabbit anti-HMGB1(1:100) for4degrees overnight.After washing three times with0.2%BSA in PBS, Alexa Fluor488secondary antibody(1:50) was added. Mounting medium containing Hoechst (1:50)was used. Fluorescent-labeled cells were observed using a inverted microscope; images were processed using XV Image processing software.Data analysis was performed by SPSS13.0software. Data were expressed as mean土standard deviation (SD). The expression of HMGB1and NF-κB were assessed by using one-way analysis of variance (ANOVA). LSD test (equal variance) or Dunnett’s T3test (heterogeneity of variance) was used for post hoc comparisons. P<0.05was considered statistically significant.Results1.Lovo cell proliferationCompared with the control group, the treatment with ulinastatin can inhabit the proliferation of lovo cells (P<0.05). Between any two different treatment groups, except there was no significant difference between UTI dose group (U2) and UTI high dose group (U3) outside (P>0.05), the remaining pairwise comparison of different groups were significant differences (P<0.05).And with the extension of time, the more obvious inhibition.2.1n vitro invasionAt24h after treatment, the number of Ul cells that had invaded the membrane was significantly lower (95±6cells/well) than that of untreated cells (153±11cells/well, P=0.000). The number of invading U2cells was significantly lower (38±5cells/well) than that of untreated cells(153±11cells/well, P=0.000). The number of invading U3cells was significantly lower (28±3cells/well) than that of untreated cells (153±11cells/well, P=0.000).The UTI could reduced the number of mesangial cells and decreased the invasion of colon cancer lovo cells. Between any two different treatment groups, except there was no significant difference between UTI dose group (U2) and UTI high dose group (U3) outside (P>0.05), the remaining pairwise comparison of different groups were significant differences (P<0.05).3.Caspase-3activityAt24h after treatment, UTI effectively enhanced caspase-3activity, and there was a significant difference (P<0.05).With the increase in the concentration of ulinastatin, caspase-3activity increased more significantly, the difference is statistically significance (P<0.05).4.Effects of UTI on HMGB1expressionLovo cells were exposed to UTI for24h, and HMGB1mRNA and protein expression were examined using RT-PCR and immunoblotting. HMGB1mRNA levels were reduced after UTI treatment in comparison with the control group. And with the increase in the concentration of ulinastatin, a decrease of approximately significant difference between treatment groups was statistically significant (P <0.05).Compared with the control, the expression of HMGB1was also reduced in UTI treatments. And as the increase in the concentration of ulinastatin, a decrease of approximately significant difference between treatment groups was statistically significant (P<0.05).5.Effects of UTI on NF-κB protein expressionLovo cells were exposed to UTI for24h,NF-κB protein expression was examined using immunoblotting,and which shows UTI significantly inhibited the protein expression of NF-κB compared with the control group. And as the increase in the concentration of ulinastatin, a decrease of approximately significant difference between treatment groups was statistically significant (P<0.05).6.1mmunofluorescence imagingCytoplasmic HMGB1was observed in the control cells. In the UTI-treated cells, HMGB1was present mostly in the nucleus, indicating that the distribution of HMGB1had been altered. All data are representative of two or three experiments; Due to differences between the treatment groups Ulinastatin obvious, so just take ulinastatin treatment group400U/ml to.ConclusionUTI can inhibit the proliferation, invasion of colon cancer lovo cells, increase the activity of caspase-3, the possible mechanism is that Ulinastatin could inhibiting the expression of HMGB1and NF-κB, and the inhibition of HMGB1from the nucleus to the cytoplasm. |
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