Role Of Laparoscopic-associated Inflammatory Cytokines In The Peritoneal Adhesion Of Gastric Cancer Cells And Its Mechanism

Background and objectives:Gastric cancer is one of the most common digestive malignant tumors and its mortality is in the second rank of the malignant diseases. Peritoneal metastasis is a major route after radical gastrectomy for the treatment of gastric cancer, which is the leading cause of recurrence and death in patients with gastric cancer. However, there still remain unknown about the exact mechanism of peritoneal metastasis. According to"seeds and soil"theory, the mechanism of peritoneal metastasis may include three steps: 1. gastric cancer cell spill from the original organ; 2. free gastric cancer cells in the peritoneal cavity adhere to peritoneal mesothelial cells; 3.gastric cancer cell transmigrate through the peritoneal mesothelial layer, and subsequently colonize in the submesothelial extracellular matrix. Therefore, any pathogenic factors which have an effect on the above process will affect peritoneal metastasis. While under a suitable local environment, tumor spill and subsequent adhesion of these cells in the peritoneal cavity is an important mechanism accounting for tumor metastasis. The association of inflammation and cancer has been well recognized in many types of cancer and inflammation has been regarded as the'seventh hallmark of cancer'.After abdominal surgery, there is a reactive inflammatory response, during which cytokines and growth factors are produced, such as Tumor necrosis factor-alpha (TNF-α) and interleukin-1beta (IL-1β). These inflammatory cytokines are known to be conducive to wound healing by initiating the cellular cascade in the course of which macrophages and leukocytes migrate to the injured site. Meanwhile, they construct the first line of defense within the abdominal cavity to prevent bacterial infection. But, many recent studies have confirmed that these inflammatory mediators are also beneficial for tumor cells proliferation, infiltration and metastasis. TNF-αand IL-1βare both key cytokines involved in inflammation and have multifunction though convergence of the nuclear factor (NF)-kB signaling pathways, and thereby enhance different genes translation by translocation of NF-kB from cytoplasm to nucleus. Meanwhile, several adhesion molecules such as intercellular adhesion molecule-1(ICAM-1) and vascular cell adhesion molecule-1(VCAM-1), have been found that their promoter have the binding site of NF-kB. Therefore TNF-αand IL-1βhave the potential ability to stimulate a sort of adhesion molecules genes translation. While the overproduction or dysfunction of adhesion molecules is associated with tumor cell adhere to other tumor cell, surrounding normal cells and component of extracellular matrix. However whether TNF-αand IL-1βhave the effect on promoting spill gastric cancer cell to mesothelial cells, and therefore may support peritoneal metastasis of gastric cancer are still unknown.At present, distal gastrectomy is still the only way for curing gastric cancer patients. Laparoscopy-assisted distal gastrectomy (LADG) for gastric cancer has the same effect comparing with conventional open distal gastrectomy (CODG). Moreover, LADG for gastric cancer has the advantage of less invasive trauma such as less abdominal scar, reduced blood loss and faster postoperative recovery. Therefore, there is uncontroversial in the treatment of early gastric cancer by laparoscopy-assisted gastrectomy which has been regarded as a standard procedure in Japan. With regard to advanced gastric cancer, one of the greatest concerns is whether performing laparoscopic-assisted gastrectomy will enhance the possibility of peritoneal metastasis, as compared with conventional open gastrectomy. Thus, the role of laparoscopy surgery remains controversial, and this controversy is highlighted by the issue of tumor dissemination and recurrence. TNF-αand IL-1βare the major mediators of the acute phase response in humans. The post-operative levels of these cytokines have been found to correlate with the magnitude of the surgery and the presence of complications. Owing to less impact on the postoperative abdominal regional and systemic immune responses, whether laparoscopic surgery may not only shows clinically relevant advantages but also causes less effect of inflammatory factors on peritoneal metastasis of gastric cancer than conventional operations?Therefore, in this study primary cultured peritoneal mesothelial cells(HPMCs) were used to investigate the effects of the different concentration of inflammatory cytokines, TNF-αor IL-1β, on the interaction between gastric cancer cells and mesothelial cells. Moreover, after incubation with TNF-αor IL-1β, several adhesion molecules mRNA in mesothelial cells were detected by Real-time PCR. In order to further elucidate the influence by adhesion molecules and their ligand on the process of peritoneal metastasis for gastric cancer, immunohistochemistry staining were used to detect these adhesion molecules and ligand in peritoneal or gastric cancer tissue. Finally, ELISA immunoassay were used to evaluate differences in both the peritoneal and systemic cytokine (TNF-αand IL-1β) concentrations after laparoscopic and conventional surgical approaches. Thus, we hope to offer a novel theoretical and experimental basis for clinical prevention and treatment by the consequences of above experiments.Methods:1. A reproducible human in vitro assay was developed to study the adhesion of human gastric cancer cells to monolayers of primary cultured peritoneal mesothelial cells. Tumor cell adhesion to a mesothelial monolayer was assessed after preincubation of the monolayer with TNF-αand IL-1βby using flow cytometry. To inactivate TNF-αand IL-1β, preincubation was performed with anti-human TNF-αor anti-human IL-1βhomeochronous prior to the adhesion assay. In negative control, PBS was used to substitute inflammatory cytokines.2. After incubation with TNF-αor IL-1β, several adhesion moleculesCD44, ICAM-1 amd VCAM-1 mRNA in mesothelial cells were detected by Real-time PCR.3. mRNA of lymphocyte function-associated antigen-1(LFA-1), very late antigen-4(VLA-4), and CD44 in different gastric cancer cell lines were detected by Real-time PCR.4. The expression of adhesion molecules (ICAM-1, VCAM-1, and CD44v6) and their counterparts (LFA-1, VLA-4 and CD44v6) in peritoneal and gastric cancer tissue were investigated by means of immunocytochemical staining.5. 57 patients with gastric cancer in our center were selected as objects. Radical gastrectomy was performed for 32 cases by LADG and 25 cases by CODG. In both groups, the peritoneal fluid was collected at the beginning and at 24 h and 48 h after surgery from a suction drain, while venous blood samples were collected from a central venous catheter after the induction of anesthesia (baseline) and at 2, 4, 24, and 48 h perioperatively. Then the proinflammatory cytokines TNF-αand IL-1βwere measured by enzyme immunoassay.Results: 1. The percentage of gastric cancer cells adhering to mesothelial cells that were preincubated with TNF-αor IL-1βwas significantly higher than that to non-preincubated mesothelial cells (p<0.05). Moreover, this effect was dose-dependent, and maximum stimulation was achieved at 10ng/ml, meanwhile this effect was completed inhibited by stimulation with a hundred-fold excess of anti-TNF-αand. anti-IL-1βrespectively.2. As HPMCs were preincubated with increasing concentrations of TNF-αand IL-1β, the mRNA expression of ICAM-1, VCAM-1, and CD44 increased, and this effect was dose-dependent. This represented a significant difference compared with the group that did not receive preincubation (p<0.01).3. ICAM-1, LFA-1, and VLA-4 mRNA expression in AGS, SGC-7901 and MKN-45 human gastric cancer cells were observed by Real-time reverse transcriptase polymerase chain reaction.4. The results of immunohistochemical staining show that peritoneal tissues stained positive for ICAM-1, VCAM-1, and CD44v6, and the gastric cancer tissues were positive for LFA-1, VLA-4, and CD44v6.5. Peritoneal fluid levels of TNF-αand IL-1βincreased rapidly after operation in both groups, peaking for both at 24 hours. The level of IL-1βin the peritoneal fluid from the CODG group was significantly higher than that in the LADG group (p<0.05), whereas there was no significant difference in TNF-αlevels in the peritoneal fluid in both groups (p>0.05). And then levels of TNF-αand IL-1βgradually decreased. At 48 hours after operations, the level of TNF-αand IL-1βin the peritoneal fluid from the CODG group was significantly higher than that in the LADG group (p<0.05).6. After gastrectomy TNF-αand IL-1βlevels in serum increased rapidly and peaked at 4 h in both groups. IL-1βlevels were significantly higher after CODG than after LADG (p<0.05). TNF-αlevels did not change significantly in both groups (p>0.05).Conclusion:1. The presented results prove that TNF-αand IL-1βare significant stimulating factors in gastric cancer cells adhesion in vitro and may, therefore, partly account for peritoneal metastasis in vivo because they promote several adhesion molecules expression in peritoneal mesothelial cells, including ICAM-1, VCAM-1 and CD44.2. Anti-TNF-αand Anti-IL-1βcan completely inhibit the effect of TNF-αor IL-1βon the adhesion of gastric cancer cell to mesothelial cells respectively and thus may have the ability to prevent peritoneal metastasis after gastrectomy.3. Owing to less impact on the postoperative abdominal regional and systemic inflammatory responses, laparoscopic surgery may not only shows clinically relevant advantages such as less abdominal scar, reduced blood loss and faster postoperative recovery, but also causes less effect of inflammatory factors on peritoneal metastasis of gastric cancer than conventional operations.