Yingxue Hao

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.

Cumulative summation analysis of learning curve for robot-assisted gastrectomy in gastric cancer

The purpose of this study was to determine the learning curve for robot‐assisted gastrectomy using the Cumulative Summation (CUSUM) technique.

Chloride intracellular channel 1 regulates colon cancer cell migration and invasion through ROS/ERK pathway

AIM To investigate the mechanisms of chloride intracellular channel 1 (CLIC1) in the metastasis of colon cancer under hypoxia-reoxygenation (H-R) conditions. METHODS Fluorescent probes were used to detect reactive oxygen species (ROS) in LOVO cells. Wound healing assay and transwell assay were performed to examine the migration and invasion of LOVO cells. Expression of CLIC1 mRNA and protein, p-ERK, MMP-2 and MMP-9 proteins was analyzed by reverse transcription-polymerase chain reaction and Western blot. METHODS H-R treatment increased the intracellular ROS level in LOVO cells. The mRNA and protein expression of CLIC1 was elevated under H-R conditions. Functional inhibition of CLIC1 markedly decreased the H-R-enhanced ROS generation, cell migration, invasion and phosphorylation of ERK in treated LOVO cells. Additionally, the expression of MMP-2 and MMP-9 could be regulated by CLIC1-mediated ROS/ERK pathway. CONCLUSION Our results suggest that CLIC1 protein is involved in the metastasis of colon cancer LOVO cells via regulating the ROS/ERK pathway in the H-R process.

HIF-1α induces the epithelial-mesenchymal transition in gastric cancer stem cells through the Snail pathway

Substantial evidence suggests that the epithelial-mesenchymal transition (EMT) phenotype is associated with the invasive characteristics of cancer stem cells (CSCs),which possess an EMT phenotype that may predominate in tumor invasion and metastasis. However, the mechanisms for the generation and regulation of these CSCs have not been clearly defined. As hypoxia and EMT-related factors may have important functions in EMT-like CSCs, the aim of this study was to investigate the effects of hypoxia on these cells. CSCs were established from the gastric cancer cell lines MGC-803 and SGC7901, and the relationship between hypoxia and EMT-like CSCs was investigated in gastric cancer. After hypoxia treatment, some gastric CSCs exhibited a marked increase in hypoxia-inducible factor-1α (HIF-1α)expression and increased migration and invasion capabilities compared with the normoxic control. These CSCs were defined by activation of the mesenchymal cell marker Vimentin and by inhibition of the epithelial cell marker E-cadherin. Our analyses also show that HIF-1α was responsible for activating EMT via increased expression of the transcription factor Snail in gastric CSCs. Moreover, inhibition of Snail by shRNA reduced HIF-1α-induced EMT in gastric CSCs. The results demonstrated that hypoxia-induced EMT-like CSCs rely on HIF-1αto activate Snail, which may result in recurrence and metastasis of gastric cancer.

Comparison of laparoscopy-assisted and open radical gastrectomy for advanced gastric cancer: A retrospective study in a single minimally invasive surgery center

AbstractLaparoscopy-assisted gastrectomy (LAG) has gained international acceptance for the treatment of early gastric cancer (EGC). However, the use of laparoscopic surgery in the management of advanced gastric cancer (AGC) has not attained widespread acceptance. This retrospective large-scale patient study in a single center for minimally invasive surgery assessed the feasibility and safety of LAG for T2 and T3 stage AGC. A total of 628 patients underwent LAG and 579 patients underwent open gastrectomy (OG) from Jan 2004 to Dec 2011. All cases underwent radical lymph node (LN) dissection from D1 to D2+. This study compared short- and long-term results between the 2 groups after stratifying by pTNM stages, including the mean operation time, volume of blood loss, number of harvested LNs, average days of postoperative hospital stay, mean gastrointestinal function recovery time, intra- and post-operative complications, recurrence rate, recurrence site, and 5-year survival curve. Thirty-five patients (5.57%) converted to open procedures in the LAG group. There were no significant differences in retrieved LN number (30.4 ± 13.4 vs 28.1 ± 17.2, P = 0.43), proximal resection margin (PRM) (6.15 ± 1.63 vs 6.09 ± 1.91, P = 0.56), or distal resection margin (DRM) (5.46 ± 1.74 vs 5.40 ± 1.95, P = 0.57) between the LAG and OG groups, respectively. The mean volume of blood loss (154.5 ± 102.6 vs 311.2 ± 118.9 mL, P < 0.001), mean postoperative hospital stay (7.6 ± 2.5 vs 10.7 ± 3.6 days, P < 0.001), mean time for gastrointestinal function recovery (3.3 ± 1.4 vs 3.9 ± 1.5 days, P < 0.001), and postoperative complications rate (6.4% vs 10.5%, P = 0.01) were clearly lower in the LAG group compared to the OG group. However, the recurrence pattern and site were not different between the 2 groups, even they were stratified by the TNM stage. The 5-year overall survival (OS) rates were 85.38%, 79.70%, 57.81%, 34.60% and 88.31%, 75.49%, 56.84%, 33.08% in patients with stage Ib, IIa, IIb, and IIIa, respectively, in the LAG and OG groups. There were no statistically significant differences in the OS rate for patients with the same TNM stage between the 2 groups. LAG with radical LN dissection is a safe and technically feasible procedure for the treatment of AGC staged below T3.

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.

Influence of Laparoscopic Gastrectomy on the Detection Rate of Free Gastric Cancer Cells in the Peritoneal Cavity

BackgroundWhether laparoscopic gastrectomy affects the number of gastric cancer cells exfoliated from the cancer-invaded serosa remains unclear. This study aimed to compare the detection rate of free gastric cancer cells in the peritoneal cavity during laparoscopic and open gastrectomy.MethodsIntraoperative peritoneal washings were collected from 83 gastric cancer patients undergoing laparoscopic gastrectomy and 81 patients undergoing open surgery. Hematoxylin and eosin (H&E) staining and real-time reverse-transcription polymerase chain reaction (RT-PCR) were used to examine the free cancer cells.ResultsThe postoperative positive rates of free cancer cells detected by cytological and real-time RT-PCR were 39.76 and 43.20% in the laparoscopic and open groups, respectively. Depth of tumor invasion, area of invaded serosa, regional lymph node involvement, and higher pathological staging were significantly associated with presence of free cancer cells.ConclusionThe laparoscopic techniques used in gastric cancer surgery did not increase the detection rate of free cancer cells in the peritoneal cavity compared with conventional techniques.

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.

Comparison of laparoscopic and open gastrectomy on cancer cells exfoliating from the cancer-invaded serosa.

Background Whether laparoscopic gastrectomy may reduce the frequency of gastric cancer cells exfoliating from the cancer-invaded serosa remains unclear. This study aimed to compare the detection of free gastric cancer cells in the peritoneal cavity during laparoscopic and open gastrectomy. Methods Intraoperative peritoneal washings were collected from 63 gastric cancer patients undergoing laparoscopic gastrectomy and 61 patients undergoing open surgery. Hematoxylin and eosin staining and real time reverse transcription-polymerase chain reaction were used to examine the free cancer cells. Results The postoperative positive rates of free cancer cells detected by cytologic and real time reverse transcription-polymerase chain reaction were 39.68% and 44.26% in the laparoscopic and open groups, respectively. The depth of tumor invasion, area of invaded serosa, regional lymph node involvement, and higher tumor node metastasis staging were significantly associated with the presence of free cancer cells. Conclusions The laparoscopic techniques used in gastric cancer surgery were not associated with a greater risk for the intraperitoneal dissemination of cancer cells than conventional techniques.