Dongzhu Zeng

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.

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.

miR-203 suppression in gastric carcinoma promotes Slug-mediated cancer metastasis.

MicroRNAs (miRNAs) play critical roles in tumorigenesis and cancer metastasis. Recently, miR-203 was reported as a tumor suppressor microRNA silenced in different malignancies including hepatocellular carcinoma, prostate cancer, oral cancer, breast cancer, and hematopoietic malignancy, whereas its role in the carcinogenesis of gastric carcinoma (GC) has not been evaluated. Here, we analyzed the levels of miR-203 and Slug in the GC specimen and studied their correlation. We analyzed the binding of miR-203 to the 3′-UTR of Slug messenger RNA (mRNA) and its effects on Slug translation by bioinformatics analysis and by luciferase-reporter assay, respectively. We modified miR-203 levels in GC cells and studied their effects on the cell invasiveness in transwell cell migration assay. We found that in GC, miR-203 levels were significantly decreased and Slug levels were significantly increased. miR-203 and Slug inversely correlated in patients’ specimen. Bioinformatic analysis predicted that miR-203 may target the 3′-UTR of Slug mRNA to inhibit its translation, which was confirmed by luciferase-reporter assay. Overexpression of miR-203 inhibited Slug and cell invasiveness, while depletion of miR-203 increased Slug and cell invasiveness. These data suggest that miR-203 suppression in GC promotes Slug-mediated cancer metastasis.

Laparoscopic Radical Correction Combined With Extensive Lymphadenectomy and Pelvic Autonomic Nerve Preservation for Mid-to-Low Rectal Cancer

OBJECTIVE The purposes of this study were to confirm the definite metastasis and micrometastasis rate of upward and lateral lymph nodes of mid-to-low rectal cancer at stage II and stage III, and to evaluate the feasibility and safety of laparoscopic radical correction combined with extensive lymphadenectomy and pelvic autonomic nerve preservation (PANP). METHODS The study was performed in 68 patients who were diagnosed with mid-to-low rectal cancer at stage II or stage III and received laparoscopic radical correction combined with extensive lymphadenectomy and PANP from June 2006 to June 2008 in the General Surgery Department of Southwest Hospital. All lymph nodes resected in the surgeries were examined by hematoxylin and eosin (H & E) stain and immunohistochemistry with an antibody against cytokeratin 20 (CK20) to confirm the conditions of metastasis and micrometastasis. We compared the postoperative complications with those of traditional surgeries. RESULTS In 1571 lymph nodes, 16 lymph nodes were found to have definite metastasis in 6 patients (8.8%) and in 41 lymph nodes we found micrometastasis in 12 patients (17.6%). The total metastasis rate of upward and lateral lymph nodes was 19.1%. Compared with traditional surgeries, the new surgery had less blood loss and short convalescence and postoperative complications were not increased. CONCLUSION The total metastasis rate of upward and lateral lymph nodes is 19.1%. The laparoscopic radical correction combined with extensive lymphadenectomy and PANP is feasible and safe.

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.

Single-Tubed Wild-Type Blocking Quantitative PCR Detection Assay for the Sensitive Detection of Codon 12 and 13 KRAS Mutations

The high degree of intra-tumor heterogeneity has meant that it is important to develop sensitive and selective assays to detect low-abundance KRAS mutations in metastatic colorectal carcinoma (mCRC) patients. As a major potential source of tumor DNA in the aforementioned genotyping assays, it was necessary to conduct an analysis on both the quality and quantity of DNA extracted from formalin-fixed paraffin-embedded (FFPE). Therefore, four commercial FFPE DNA extraction kits were initially compared with respect to their ability to facilitate extraction of amplifiable DNA. The results showed that TrimGen kits showed the greatest performance in relation to the quality and quantity of extracted FFPE DNA solutions. Using DNA extracted by TrimGen kits as a template for tumor genotyping, a real-time wild-type blocking PCR (WTB-PCR) assay was subsequently developed to detect the aforementioned KRAS mutations in mCRC patients. The results showed that WTB-PCR facilitated the detection of mutated alleles at a ratio of 1:10,000 (i.e. 0.01%) wild-type alleles. When the assay was subsequently used to test 49 mCRC patients, the results showed that the mutation detection levels of the WTB-PCR assay (61.8%; 30/49) were significantly higher than that of traditional PCR (38.8%; 19/49). Following the use of the real-time WTB-PCR assay, the ΔC q method was used to quantitatively analyze the mutation levels associated with KRAS in each FFPE sample. The results showed that the mutant levels ranged from 53.74 to 0.12% in the patients analyzed. In conclusion, the current real-time WTB-PCR is a rapid, simple, and low-cost method that permits the detection of trace amounts of the mutated KRAS gene.

da Vinci robotic versus laparoscopic surgery in rectal cancer: a meta-analysis of postsurgery complications.

Dear Editor: Laparoscopic surgery (LS) and daVinci robot-assisted surgery (RS) are both used in rectal cancer treatment. Considering the anatomical features of rectal cancer, RS is superior to LS in mid-low set rectal operations and in anus preservation. However, it is unclear which surgical approach has a lower prevalence of postoperative complications and better outcomes. We therefore conducted a meta-analysis of controlled trials to compare postoperative complications and outcomes of LS and RS for resection of rectal cancer. We conducted a worldwide literature search of publications, assembled documents, and related references using the MedLine (PubMed), Embase, Ovid, CNKI, and WanFang databases. The search focused on controlled trials published between 1979 and 2014 comparing the use of RS and LS in rectal cancer surgery. We used the following search terms: da Vinci surgical system, robot, robotic, laparoscopic surgery, total mesorectal excision (TME), and rectal cancer. Two authors independently developed the selection including retrieval and data checks. Disagreements were resolved through discussion and, if necessary, by involving a third independent researcher.We assessed the quality of methods following standards of randomized controlled tests (RCTs) from Cochrane Reviewers Handbook 5.2. Specific evaluation criteria included allocation sequence, allocation concealment, blinding tests of doctors and patients, blinding tests of statistics keepers, incomplete data bias, selective reporting results and other potential factors affecting validity. If the above seven indices were in accordance with quality standards, the quality of the research was deemed high; if not, bias might exist. The inclusion criteria used to screen patients were as follows: (1) definitively diagnosed with rectal cancer and (2) had undergone surgery.We divided patients into two groups: those who underwent RS and those who underwent LS. The exclusion criteria used were as follows: (1) did not exhibit pneumoperitoneum or (2) had a medical history of colorectal surgery. We also excluded studies on nonrectal cancer and TME for nonrectal cancer. The outcomes included operative morbidity, anastomotic leakage, incision infection, intestinal obstruction, abdominal abscess, cleaned lymph node count, and unscheduled reoperation. We performed the meta-analysis using Review Manager (RevMan), version 5.2 (Cochrane Collaboration, London, United Kingdom). Enumeration data were examined using relative risk (RR) and 95 % confidence interval (CI). Overall effects were calculated using the Z test. Statistical significance was considered at P=0.05. For measurement data with the same units, statistical analyses were performed using the weighted mean difference, and for measurement data with different units, analyses were performed using the standardized mean difference. The studies were tested for heterogeneity before being combined. If heterogeneity did not exist, the fixed-effect model was used in the analysis; else the source of clinical heterogeneity was explored. If no clear difference was found, we used the random-effects model. To assess the potential for publication bias, we visually inspected funnel plots. The search identified five studies published in English for inclusion in the meta-analysis. There were 746 patients: 409 were treated with LS and 337 with RS. All studies had complete data records and reported main indices of previous studies. None of the studies lay outside 95 % CIs and there was no * Peiwu Yu yupeiwu01@sina.com