Effective Mechanisms Of The CXCR4 In Carcinomatosis Of Gastric Cancer

Objective: The peritoneal seeding metastasis after gastric cancer radical dissection is one of the important causes of death in patients of gastric cancer. In advanced gastric cancer, peritoneal metastasis is the most frequent (50%) type of recurrence after curative resection, as well as the death cause of most patients. The mechanism of peritoneal seeding of gastric cancer has not been identified, so effective prevent and cure methods are not available. CXC chemokine receptor4(CXCR4) is one of the most common chemokine receptors in tissues and plays important roles in malignant tumors growth, migration and metastasis. The abnormal activation of CXCR4 had been observed in a variety of human malignancies development, including breast cancers, prostatic carcinomas, lung cancers and so on, but whether the activated CXCR4 could affect peritoneal seeding and metastasis of gastric cancer or not, and what downstream genes regulated by CXCR4 could induce the appearance of cancer cells invasive phenotype are not known. So, clinical samples of gastric cancer patients were collected. The expressions of CXCR4 in those samples were detected, and the relationships between the expression level and pathologic parameters were observed firstly. Secondly, the CXCR4-siRNA Lentivirus expression vectors were transfected into gastric cancer cells MKN-45 to block the activated CXCR4 protein especially, and the repressive effects were observed. Finally, after the gastric cancer animal model of peritoneal implantation had been established in nude mice, changes of implantation capacity and the expressions of relative regulative factors in gastric cancer cells were observed in order to reveal the mechanism of CXCR4 in the course of gastric cancer peritoneal seeding and provide new strategy for tumor prevention and treatment.Methods:1. 90 clinical samples of tumor patients and 30 adjacent noncancerous specimens of human gastric cancer were collected. Peritoneal lavage cytology was used to exam the exfoliated cancer cells, and nested Immuohistochemistry(SP)was employed to detect the expression of CXCR4, SDF-1, and VEGF-C expression and relative clinical pathological parameters were observed.2. CXCR4-siRNA Lentivirus expression vectors were constructed and transfected into MKN-45 cells. There were 6 groups in our study, normal control group (SDF-1- negative), normal control group(SDF-1-positive), keno-viral vector group (SDF-1- negative), keno-viral vector group(SDF-1-positive), CXCR4-siRNA experimental group (SDF-1- negative), CXCR4-siRNA experimental group (SDF-1- positive). The changes of cell proliferation, apoptosis, cell cycle and chemotactic activity,and expressive changes of VEGF-C,NRP-1 were observed by MTT assay, flow cytometry, chemotaxis assay, RT-PCR and Western blot respectively. The molecular mechanisms of CXCR4-siRNA transfection on inhibiting peritoneal invasion and metastasis were investigated.3. The tumor animal model of peritoneal implantation was established in nude mice. The capability of cancer cell's peritoneal seeding in nude mice was observed, and changes of cellular proliferation activity, cellular affinity and the invasive and metastatic potentials were observed in vivo, after CXCR4-siRNA Lentivirus expression vectors were transfected.Results:1. Immunohistochemistry illustrated that the expression of CXCR4 was on the membrane or in the cytoplasm of gastric cancer cell. There was no CXCR4 expression in normal gastric mucosa cell .The expression rate of CXCR4 in primary tumor, lymph mode and peritoneal metastatic tumor was 83.33%, 90.14% and 100% respectively(P<0.01 or P<0.05). The expression of VEGF-C was mainly in the cytoplasm of gastric cancer cell. The expression rate of VEGF-C in normal gastric mucosa cell, primary tumor, lymph mode and peritoneal metastatic tumor was 6.67%, 80.00%, 91.55% and 100% respectively (P <0.01 or P< 0.05). The expression of SDF-1was on the membrane and in the cytoplasm of gastric cancer cell. SDF-1 could be expressed in normal gastric mucosa cell(93.33%). The expression rate of SDF-1 in primary tumor, lymph mode and peritoneal metastatic tumor was 68.89%, 42.25% and 11.11% respectively (P<0.01 or P<0.05). The expressions of CXCR4, SDF-1, and VEGF-C in primary tumor were correlated with tumor stage, depth of tumor invasion, pathological differentiation and lymph node metastasis(r=0.337, P<0.05), but not with age of patients, gender, location of tumor and tumor size(P>0.05).2. CXCR4-siRNA Lentivirus expression vectors were constructed successfully. After the vector was transfected into gastric cancer cells, tumor cells` proliferation was inhibited in a time-dependent manner. Among the distribution of cell cycle, the ratio of G0/G1 increased, and the S stage and ratio of G2/M decreased. Meanwhile, the proliferation index decreased(P<0.05) and apoptotic index increased(P < 0.05), with significantly decreased expressions of VEGF-C and NRP-1(P<0.05). 3. The gastric cancer model of peritoneal seeding was established. There were significant differences in ascitic volume, tumor max diameter, max weight, quantity of tumor, survival time, survival rate, prolonged survival ratio between the CXCR4-siRNA group, the keno-viral vector group and the control group(P<0.05, P<0.01). Among the distribution of cell cycle, the rate of G0/G1 increased ,and the S stage and rate of G2/M decreased. The proliferation index decreased(P<0.05), and apoptotic index also increased obviously(P<0.05). The migrative and invasive abilities of transfected MKN45 cells in vivo decreased in some degree (P<0.05 or P<0.01), but the distribution of cell cycle was no difference between the control group and the keno-viral vector group(P>0.05).Conclusions:1. As close correlations existed between the expression of CXCR4 and the peritoneal implantation of gastric cancer, it could act as a standard for judging the invasive and metastatic states of gastric cancer and forecasting the prognostic of patients. Laparoscopic radical gastrectomy does not facilitate the peritoneal implantation of gastric cancer cells.2. Positive correlations existed between the expressive intensity of CXCR4 and the implantation and metastatic potentials of gastric cancer cells.3. Restraint of CXCR4 effects in nucleus could directly result in the impairment of implantation and metastatic potentials of gastric cancer cells both in vitro and in vivo.