| Background and ObjectiveMonocyte chemoattractant protein-1(MCP-1) also known as Chemokine (C-C motif) ligand2,(CCL2)) recruits and activates monocytes/macrophages during the inflammatory response via its special receptor, CCR2. More and more studies have demonstrated that MCP-1/CCR2expressed in monocytes, macrophages, smooth muscle cells, endothelial cells, fibroblasts as as well as in breast cancer, lung cancer and prostate cancer.MCP-1has been implicated in the development of multiple inflammatory disorders, such as atherosclerosis and rheumatoid arthritis. The best understanding is its effects in the progression of atherosclerosis. It has been confirmed that the MCP-1secreted by endotheliums can attracted the monocyte expressed CCR2to the subendothelial where the monocytes activated into mcrophages to uptake the lipid bodies. Then the cells turn out to be the foam-like cells which leading to atherosclerosis if the foam cells accumulated. It has been report that MCP-1/CCR2also involved in the progress of angiogensis and act as a direct mediator. With the use of a rat aortic sprouting assay in the absence of leukocytic infiltrates, they ruled out the possibility that the angiogenic effect of MCP-1depended on leukocyte products. Moreover, the direct effect of MCP-1on angiogenesis was consistent with the expression of CCR2, the receptor for MCP-1, on endothelial cells. Taken together, indicate that MCP-1can act as a direct mediator of angiogenesis. Moreover it has been demonstrated that prostate cancer cells themselves express CCR2mRNA and protein. Analysis revealed a positive association between prostate cancer progression and CCR2expression. CCR2mRNA and protein were expressed at higher levels within prostate cancer metastatic tissues as compared with localized prostate cancer and benign prostate tissue. Higher CCR2expression was also associated with higher Gleason score and higher clinical pathologic stages, suggesting that CCR2may be associated with prostate cancer progression. Some studies reported that CCL2acts in a paracrine and autocrine manner to stimulate prostate cancer cell proliferation and migration. This is a new theory about the roles of MCP-1/CCR2axis in the tumorigenesis besides the indirect effect of MCP-1driving immunocyte or stroma cells which can secret some cytokines stimulating tumorigenesis.Although increasing researches about the role of the MCP-1in colorectal cancer indicate that the higher MCP-1levels is associated with the colorectal cancer liver metastasis, little is known about whether CCR2express in colorectal cancer and which, if any, it involve the colorectal cancer invasion. Despite some small studies report that MCP-1expressed in colonic adenoma and colorectal cancer, there is absent the system study of the MCP-1expression in colorectal tissue from adenoma-carcinoma sequence. In addition several small studies have demonstrated that serum CCL2is elevated in cancer patients and associated with tumor stage in patients with breast, ovarian, lung and prostate cancers. But little is known MCP-1expression in serum of colorectal cancer patients. The aim of the present study was to evaluate the role of MCP-1/CCR2axis in the colorectal cancer invasion in vitro and the MCP-1/CCR2axis expression in vivo including the tissue and serum level. Methods and resultsPatients samplesArchival, formalin-fixed, paraffin-embedded tissues were collected from individuals who underwent surgery resection at People’s Hospital of Huizhou City Center from January2003to December2005including177CRCs (male103and the female74, mean age59.0years),65colorectal adenoma (male39and the female26, mean age57.2years) and66normal colorectal mucosa (male41and the female25, mean age56.5years). A total of66serum samples (male33and the female33, mean age60.0years) from patients with CRC who underwent surgery resection and54serum samples from healthy controls(male23and the female31, mean age54.9years) were collected at Hospital of Huizhou City Center from July2009to June2011. Clinical data were obtained from files at the People’s Hospital of Huizhou City Center (huizhou, China). The study was approved by the Institutional Ethical Committee of the Southern Medical University, and informed consent was signed from all the participants in the study.MCP-1and CCR2ImmunohistochemistryArchival, formalin-fixed, paraffin-embedded tissues were obtained from People’s Hospital of Huizhou City Center (huizhou, China). Sections (4μm) were deparaffinized and rehydrated. Endogenous peroxidase activity was blocked by incubating sections in3%hydrogen peroxide. Antigen retrieval was carried out by microwaving in citrate buffer(10mM, pH6.0) at500W for15min for CCR2or hot retrieval in Tris-EDTA Buffer (lOmM Tris Base,1mM EDTA Solution, pH9.0)10min,1200w for MCP-1. And then incubated in isotype serum for30min.The slides were blocked with the primary antibody CCR2(Abeam,2.5μg/ml) or MCP-1(R&D,2.5μg/ml) then incubated at4℃overnight. After three washes with PBST (2000ml PBS+4ml Tween20), the sections were incubated with biotin-conjugated second antibody for10min, and then incubated with streptavidin-peroxidase for10min.3,3’-Diaminobenzidine was used as the chromogen. Slides were counterstained with hematoxylin, dehydrated in a graded series of ethanol, mounted on slides, and cover-slipped. Negative control IHC reactions were performed by omitting the primary antibody.Immunohistochemical evaluationAll slides were assessed by one pathologist and one investigator independently who were blind to the cases. Both the intensity and distribution of IHC staining of MCP-1and CCR2was assessed separately. The intensity of staining was assessed with a semi-quantitative scoring system as follows:0, negative staining;1, weak staining;2, moderate staining;3, strong staining. The distribution of staining was graded by the percentage of stained cells in the region of interest:0, positive cells were less than10%of tumor cells;1, positive cells were10-50%of tumor cells;2, positive cells were50-75%of tumor cells;3, positive cells were more than75%of tumor cells. An overall scores was obtained as the product of the intensity and distribution of positive staining. Cases with0points were considered to be negative (-), cases with a final score of1-3as weakly positive (+), cases with a final score of4-7as moderately positive (++), and cases with a final score of>7as strongly positive (+++).Serum MCP-1measurementThe serum MCP-1concentrations were determined by EL1SA kit (R&D Systems, Minneapolis, MN) according to manufacturer’s instruction. A standard curve was prepared using recombinant human MCP-1. All assays were performed in duplicate. Optical density was determined using a microplate reader (SpectraMax M5) at wave length of450nm and subtracted readings at570nm for MCP-1determination. The concentrations were reported as pg/ml.Statistical analysisData are presented as mean and standard deviation(SD) for continuous variables and as mean rank for categorical variables. MCP-1protein expression was analyzed first as raw levels then in a logarithmic scale, in order to achieve a symmetric distribution. Continuous variables were analyzed using one-way ANOVA, followed by LSD test for multiple comparisons. Differences in categorical variables were determined by the Chi-square or Mann-Whitney U-test or kruskal-wallis H test, as appropriate. Correlations between the expression of MCP-1(IHC scores) with expression of CCR2were evaluated by the Spearman rank-order correlation coefficient. Differences were considered significant if P<0.05. All significance tests were two-tailed. All statistical tests were performed using SPSS software Version13.0(SPSS Inc, Chicago, IL, USA).Material and methodsUsing RT-PCR (reverse transcription-ploymerase chain reaction) to detect the MCP-1and CCR2expression in colorectal cancer cell lines.Human colorectal cancer cells (LoVo, SW1116, HT29, SW620and SW480), obtained from the digestive laboratory of Nanfang Hospital affiliated to Southern Medical University, and were cultured in a50ml/I CO2incubator at37℃in RPMI-1640(GIBCO, USA) supplemented with10%FBS. MCP-1and CCR2mRNA were detected by RT-PCR. The total RNA was extracted from cells using Trizol (invitrogen). RNA samples (3ug) were subjected to reverse transcription using a cDNA synthesis kit (invitrogen).The PCR was initiated by5min incubation at94℃for45s, annealing at56℃and extension72℃for50s, ended after a7min extension at72℃using a PCR kit.Using Western blotting to detect the MCP-1and CCR2expression in colorectal cancer cell lines.Total cell lysates were extracted by RIPA (1%Triton X-100,1%deoxycholate,0.1%SDS) on ice and were centrifuged at13,000rpm at4℃for25min. Concentration of the protein was measured by BCA. Protein (40μg) was mixed with loading buffer with final concentration of1×. After boiled for5min, proteins were separated by running in SDS-polyacrylamide gel. Proteins were then transferred to PVDF membrane. Following transferring, the membranes were blocked for1h at room temperature with5%(w/v) skimmed milk powder in Tris buffered saline Tween20(0.1%by volume, TBS-T). PVDF membrane was incubated with primary antibody overnight at4℃. After washed with TBST for10min three times, PVDF membrane was incubated with secondary antibody for40min followed by washing in with TBST for10min three times. Then the membrane was incubated with Enhanced Chemiluminescence detection system (Millipore, USA) for2min and exposed to film.In vitro cell invasion assayThe selected LoVo cell was evaluated in24well Matrigel invasion chamber (Corning, MA, USA), as previously described. Briefly,200ul serum-deprived2.5×104Lovo cells were added to the upper surface of each migration chamber and500ul RPMI1640containing10%FBS and100ng/ml rhMCP-1(perprotech,USA)was placed in the bottom chamber of24-well dishes. Cells were allowed to migrate to the underside of the membrane for24h, non-invading cells were removed by wiping the upper side of the membrane, and the invaded cells were fixed with4%paraformaldehyde and stained using crystal violet (Sigma, USA). The number of invaded cells was quantified by cell counting at least five random fields (magnification,200×) per filter. In the experiments, LoVo cells transfected with CCR2-siRNA and scrambled sequence (negative control) were applied to the invasion assay.ResultMCP-1/CCR2axis expression increases during human colorectal cancer progression and metastasisBased on the above result that MCP-1and CCR2were overexpressed in CRC cell lines, we set out to investigate if MCP-1and CCR2are also overexpressed in colorectal cancer patients and if MCP-1and CCR2expression is correlated with cancer progression and metastasis. To this end, we examined MCP-1and CCR2expression by IHC in adenomas and CRCs with normal mucosa, lymph node metastasis and distant organ metastasis. Immunoreactivity of MCP-1and CCR2were found in the cytoplasm of CRC and AA. In contrast to normal colon mucosa where the MCP-1and CCR2immunoactivity was either absent or barely detectable, MCP-1and CCR2expression was found upregulated in adenoma (P<0.01). In CRCs, even higher expression of MCP-1and CCR2were found than in adenomas (P<0.01). This suggests that MCP-1and CCR2might regulate CRC metastasis. An increasing expression of MCP-1and CCR2were observed with CRC progression from Dukes A to D and a statistical analysis revealed a significant increase for MCP-1and CCR2expression during CRC development from Dukes A to D (Dukes’ stage A<B<C=D). Furthermore, we also found an increase of MCP-1and CCR2expression in CRCs from well to poorly differentiated tumors(G1<G2=G3)(P<0.01). No significant difference of MCP-1levels was found when compare the gender, age, tumor size, and tumor localization. The Spearman rank-order correlation coefficient were employed to analysis the correlation between MCP-1expression and CCR2in CRC (Rs=0.485, P<0.01)Serum MCP-1levels were elevated in colorectal cancerValues for MCP-1were logarithmically transformed prior to significance testing to achieve a symmetric distribution. The association between CRC1gMCP-1levels (defined as the mean±SD of1gMCP-1) and the clinic pathological variables were analyzed. No significant difference of1gMCP-1levels was found when compare the gender, age, tumor size, and tumor localization.1gMCP-1levels were significantly increased in CRC patients (n=66, mean=2.34, SD=0.23) compared to healthy controls (n=54, mean=2.13,SD=0.21; P<0.01). There was no significant differences between poor tumor differentiation (mean=2.44, SD=0.23) and moderate differentiation (mean=2.38, SD=0.20), but significant differences was observed between moderate differentiation and well differentiation(mean=2.21, SD=0.23; P<0.01). Although there was no significant differences between Dukes’ stage A (1gMCP-1mean=2.23, SD=0.14) and Dukes’ stage B (mean=2.19, SD=0.15) tumors, there were increases in1gMCP-1levels between Dukes’ stage B and C (mean=2.42, SD=0.20; P<0.01), as well as between Dukes’ stage C and D (mean=2.60, SD=0.19; P<0.01). There is an increasing likelihood of more distant disease as the primary tumor invades and, consequently, the Dukes’ stage increases. Therefore, the rise in serum MCP-1levels with increasing tumor stage indicates a relationship to the extent of the tumor. The presence of lymph node metastasis leads to increased1gMCP-1levels compared with nonmetastatic disease (P<0.01), as well as in distant organ metastasis.MCP-1/CCR2axis expression in CRC cellsTotal protein was extracted from six colorectal cancer cell lines (LoVo, SW1116, HT29, SW620and SW480). MCP-1and CCR2protein are differentially expressed in various colorectal cancer cell lines. The expression of MCP-1is consistant with CCR2. MCP-1/CCR2protein was high expressed in LoVo cell lines, moderated in SW1116, SW480, and no or rare expression in HT29and SW620as detected in Western blotting. Expression of CCR2protein decreased significantly in LoVo cells with transfected CCR2siRNA compared to the cells with scramble siRNA, shown by Western blotting analysis. Together, these results suggested that CCR2differentially expressed in colorectal cancer cells, and transfection of the LoVo cells with the CCR2siRNA strongly inhibited the expression of CCR2in these cells, the transfected cells were then used for the invasion assay.CCR2knockdown in colorectal cancer cells abrogate the MCP-1-induced colorectal cancer cell invasionTranswell matrigel invasion assay was used to confirm the role of CCR2in MCP-1induced LoVo cell invasion. The rhMCP-1showed the ability to induce LoVo cell invasion through matrigel-coated membranes, however CCR2-deleted cells with siRNA inhibit their invasion compared with negative control and blank.Conclusion1. MCP-1/CCR2axis play an important role in colorectal cancer progression, maybe involve in the classic cancerous passway of "normal mucosa-adenoma-carcinoma sequence".2. MCP-1/CCR2axis closely related with the tumor invasion and metastasis in vivo, the interaction of them maybe a new mechanism regulating the tumor invasion and metastasis。3. MCP-1/CCR2axis activation closely related with the tumor invasion and metastasis in vitro, the deficiency of CCR2may impair the invasion ability of colorectal cancer cells. |
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