Huaxing Luo

Autophagy Protects against Oxaliplatin-Induced Cell Death via ER Stress and ROS in Caco-2 Cells

Oxaliplatin is included in a number of effective combination regimens used as first and subsequent lines of therapy for metastatic colorectal cancer. Accumulating evidence indicates that autophagy plays a significant role in response to cancer therapy. However, the role of autophagy in oxaliplatin-induced cell death remains to be clarified. In this study, we showed that oxaliplatin induced cell death and autophagy in Caco-2 colorectal cancer cells. The suppression of autophagy using either pharmacologic inhibitors (3-methyladenine, bafilomycin A1) or RNA interference in essential autophagy genes (ATG5 or Beclin1) enhanced the cell death and reactive oxygen species (ROS) production induced by oxaliplatin in Caco-2 cells. Blocking oxaliplatin-induced ROS production by using ROS scavengers (NAC or Tiron) decreased autophagy. Furthermore, numerous dilated endoplasmic reticula (ER) were present in oxaliplatin-treated Caco-2 cells, and blocking ER stress by RNA interference against candidate of metastasis-1 (P8) and C/EBP-homologous protein (CHOP) decreased autophagy and ROS production. Taken together, these data indicate that oxaliplatin activates autophagy as a cytoprotective response via ER stress and ROS in human colorectal cancer cells.

Aberrant expression of Cx43 is associated with the peritoneal metastasis of gastric cancer and Cx43-mediated gap junction enhances gastric cancer cell diapedesis from peritoneal mesothelium.

The process of peritoneal metastasis involves the diapedesis of intra-abdominal exfoliated gastric cancer cells through the mesothelial cell monolayers; however, the related molecular mechanisms for this process are still unclear. Heterocellular gap-junctional intercellular communication (GJIC) between gastric cancer cells and mesothelial cells may play an active role during diapedesis. In this study we detected the expression of connexin 43 (Cx43) in primary gastric cancer tissues, intra-abdominal exfoliated cancer cells, and matched metastatic peritoneal tissues. We found that the expression of Cx43 in primary gastric cancer tissues was significantly decreased; the intra-abdominal exfoliated cancer cells and matched metastatic peritoneal tissues exhibited increasing expression compared with primary gastric cancer tissues. BGC-823 and SGC-7901 human gastric cancer cells were engineered to express Cx43 or Cx43T154A (a mutant protein that only couples gap junctions but provides no intercellular communication) and were co-cultured with human peritoneal mesothelial cells (HPMCs). Heterocellular GJIC and diapedesis through HPMC monolayers on matrigel-coated coverslips were investigated. We found that BGC-823 and SGC-7901 gastric cancer cells expressing Cx43 formed functional heterocellular gap junctions with HPMC monolayers within one hour. A significant increase in diapedesis was observed in engineered Cx43-expressing cells compared with Cx43T154A and control group cells, which suggested that the observed upregulation of diapedesis in Cx43-expressing cells required heterocellular GJIC. Further study revealed that the gastric cancer cells transmigrated through the intercellular space between the mesothelial cells via a paracellular route. Our results suggest that the abnormal expression of Cx43 plays an essential role in peritoneal metastasis and that Cx43-mediated heterocellular GJIC between gastric cancer cells and mesothelial cells may be an important regulatory step during metastasis. Finally, we observed that the diapedesis of exfoliated gastric cancer cells through mesothelial barriers is a viable route of paracellular migration.

Mouse forestomach carcinoma cells immunosuppress macrophages through transforming growth factor-β1.

Peritoneal implantation metastasis of gastric cancer cells is associated with poor prognosis. Peritoneal macrophages are the most important immune cells in the abdominal cavity to control tumor metastasis. In the present study, the immunosuppressive effects of mouse forestomach cells on macrophages were examined. Conditioned medium from mouse forestomach cell cultures were used to treat isolated peritoneal macrophages. A colorimetry-based phagocytosis assay was performed to investigate the functional change of macrophages. The alteration of cytokine secretion by macrophages was measured by ELISA assay. Specific markers of macrophage polarization were analyzed by real-time RT-PCR. TGF-β1 signaling was evaluated by western blotting. Neutralization experiments were performed using an anti-TGF-β1 antibody. Conditioned medium reduced the phagocytotic capability of macrophages. Lower TNF-α and IL-1β levels and higher IL-10 and VEGF levels were observed. Real-time RT-PCR showed increased mRNA levels of M2 macrophage markers. Further study revealed that TGF-β1 was significantly elevated in the conditioned medium and TGF-β1 signaling was activated in the macrophages by the treatment of conditioned medium. Neutralization of TGF-β1 reversed the immunosuppressive effects on macrophages. Immunosuppressive macrophages can be induced by conditioned medium from mouse forestomach cell cultures. These effects appeared to occur through the production of TGF-β1 by the tumor cells. Targeted TGF-β1 intervention may help to control peritoneal metastasis of gastric cancers.

Suppression of vascular endothelial growth factor abrogates the immunosuppressive capability of murine gastric cancer cells and elicits antitumor immunity

The mechanisms underlying immune evasion by gastric cancer have not been well described due to a lack of gastric tumor models in immunocompetent mice. In the current study, we found that supernatants from MFC cells, a murine gastric cancer line, inhibited the lipopolysaccharide (LPS) induced maturation and cross‐presentation of bone‐marrow‐derived dendritic cells (BMDCs). Moreover, MFC tumor‐derived factors markedly altered the cytokine profiles of BMDCs, leading to a trend of increased levels of interleukin 4 (IL4), IL6, IL23 and transforming growth factor β, as well as decreased levels of tumor necrosis factor α. qPCR and ELISA revealed that MFC cells expressed a high level of vascular endothelial growth factor (VEGF). Downregulating VEGF expression abrogated the inhibitory effect of MFC‐derived factors on the maturation and cross‐presentation of BMDCs. In addition, VEGF knockdown greatly impaired the tumorigenicity of MFC cells in immunocompetent mice. Compared with parental MFC tumors, VEGF‐low MFC tumors grew much more slowly and the survival of tumor‐inoculated mice was significantly improved. More importantly, mice rejecting inoculated VEGF‐low MFC tumor cells gained resistance to re‐challenged parental tumors, which was attributed to an antitumor immunity response against parental MFC tumors. These results reveal an immunosuppressive role for VEGF in murine gastric cancer.

Short-term surgical outcomes of a randomized controlled trial comparing laparoscopic versus open gastrectomy with D2 lymph node dissection for advanced gastric cancer

BackgroundLaparoscopy-assisted gastrectomy (LAG) has gained acceptance as one of the best treatments for early gastric cancer. However, the application of LAG with D2 lymph node dissection in patients with locally advanced gastric cancer (AGC) remains controversial.MethodsWe launched a prospective randomized controlled trial comparing laparoscopic and open gastrectomy with D2 lymph node dissection for locally AGC to evaluate technical safety and oncologic feasibility. The postoperative morbidity and mortality rates were based on the modified intention-to-treat analysis.ResultsBetween January 2010 and June 2012, a total of 328 patients with preoperative clinical stage T2–3N0–3M0 gastric cancer were enrolled in the trial. Six patients with unresected AGC were excluded, and the remaining 322 patients were randomized to the laparoscopic group (162 patients) or the open group (160 patients) for radical surgery. All patients underwent D2 lymph node dissection including 18 (5.59%) proximal gastrectomies, 196 (60.87%) distal gastrectomies, and 108 (33.54%) total gastrectomies. Six patients (3.70%) in the LAG group were converted to open procedures. The overall complication rate was 11.72% in the LAG group and 14.38% in the open group (P = 0.512). No mortality occurred in either group.ConclusionsThe short-term results of the current study suggest that LAG with D2 lymph node dissection is a safe and feasible procedure in treating patients with locally AGC in experienced centers.

Novel method for esophagojejunal anastomosis after laparoscopic total gastrectomy: Semi-end-to-end anastomosis

AIM To test a new safe and simple technique for circular-stapled esophagojejunostomy in laparoscopic total gastrectomy (LATG). METHODS We selected 26 patients with gastric cancer who underwent LATG and Roux-en-Y gastrointestinal reconstruction with semi-end-to-end esophagojejunal anastomosis. RESULTS LATG with semi-end-to-end esophagojejunal anastomosis was successfully performed in all 26 patients. The average operation time was 257 ± 36 min, with an average anastomosis time of 51 ± 17 min and an average intraoperative blood loss of 88 ± 46 mL. The average postoperative hospital stay was 8 ± 3 d. There were no complications and no mortality in this series. CONCLUSION The application of semi-end-to-end esophagojejunal anastomosis after LATG is a safe and feasible procedure, which can be easily performed and has a short operation time in terms of anastomosis.

Effects of CO2 Pneumoperitoneum on Peritoneal Macrophage Function and Peritoneal Metastasis in Mice with Gastric Cancer

Background: Whether laparoscopy with CO2 pneumoperitoneum affects the peritoneal metastasis of gastric cancer is a pressing question. In light of the important impact change in peritoneal macrophage function has on the peritoneal metastasis of gastric cancer, this study investigated the change in peritoneal macrophage function in gastric cancer in the CO2 pneumoperitoneum environment, as well as its effect on the peritoneal metastasis of gastric cancer. Methods: An orthotopic transplantation model of murine forestomach carcinoma was established using the 615 mouse line. The mice bearing tumors were randomly divided into four groups (30 mice each group): anesthesia alone, laparotomy, mini-laparotomy, and CO2 insufflation. After the operation, peritoneal macrophages were collected from 6 mice in each group and cultured. The phagocytosis of neutral red by macrophages and the levels of NO, TNF-α, IL-10, and VEGF produced by macrophages were measured after 12, 24, 48, and 72 h of culture. The remaining mice were observed after 2 weeks for the rate of peritoneal metastasis of forestomach carcinoma cells and the total weight of implanted nodules. Results: In the laparotomy group, 4 mice died intraoperatively and 2 died in the CO2 insufflation group. The uptake of neutral red by peritoneal macrophages and the levels of NO, TNF-α, IL-10, and VEGF secreted by peritoneal macrophages in the laparotomy group and mini-laparotomy group after 12 h of culture were all significantly higher than those in the anesthesia-alone group (p < 0.05). The corresponding levels in the CO2 insufflation group after 12 h were all significantly lower than those in the anesthesia-alone group (p < 0.05). There were no significant differences among the four groups at 24, 48, and 72 h after culture. Comparing with those in the laparotomy group, the uptake of neutral red by peritoneal macrophages and the levels of NO, TNF-α, IL-10, and VEGF secreted by peritoneal macrophages in the CO2 insufflation group were all significantly lower after 12 h of culture (p < 0.05), but did not differ significantly at 24, 48, and 72 h of culture (p > 0.05), and did not differ significantly in the mini-laparotomy group at all the time (p > 0.05). The rate of peritoneal metastasis of mouse forestomach carcinoma was 50% in the laparotomy group, 45.83% in the mini-laparotomy group, and 45.45% in the CO2 insufflation group; this difference was not statistically significant (p > 0.05). The total weight of implanted nodules of mouse forestomach carcinoma was 1.02 ± 0.38 g in the laparotomy group, 0.97 ± 0.41 g in the mini-laparotomy group, and 0.93 ± 0.45 g in the CO2 insufflation group, which was not a statistically significant difference (p > 0.05). Conclusion: CO2 pneumoperitoneum neither significantly changes the phagocytosis and cytokine secretion functions of peritoneal macrophages in gastric cancer-bearing mice nor significantly promotes peritoneal metastasis of gastric cancer.