Adipocyte-derived IL-6 and leptin promote breast Cancer metastasis via upregulation of Lysyl Hydroxylase-2 expression.

BACKGROUND: Adipocytes make up the major component of breast tissue, accounting for 90% of stromal tissue. Thus, the crosstalk between adipocytes and breast cancer cells may play a critical role in cancer progression. Adipocyte-breast cancer interactions have been considered important for the promotion of breast cancer metastasis. However, the specific mechanisms underlying these interactions are unclear. In this study, we investigated the mechanisms of adipocyte-mediated breast cancer metastasis. METHODS: Breast cancer cells were cocultured with mature adipocytes for migration and 3D matrix invasion assays. Next, lentivirus-mediated loss-of-function experiments were used to explore the function of lysyl hydroxylase (PLOD2) in breast cancer migration and adipocyte-dependent migration of breast cancer cells. The role of PLOD2 in breast cancer metastasis was further confirmed using orthotopic mammary fat pad xenografts in vivo. Clinical samples were used to confirm that PLOD2 expression is increased in tumor tissue and is associated with poor prognosis of breast cancer patients. Cells were treated with cytokines and pharmacological inhibitors in order to verify which adipokines were responsible for activation of PLOD2 expression and which signaling pathways were activated in vitro. RESULTS: Gene expression profiling and Western blotting analyses revealed that PLOD2 was upregulated in breast cancer cells following coculture with adipocytes; this process was accompanied by enhanced breast cancer cell migration and invasion. Loss-of-function studies indicated that PLOD2 knockdown suppressed cell migration and disrupted the formation of actin stress fibers in breast cancer cells and abrogated the migration induced by following coculture with adipocytes. Moreover, experiments performed in orthotopic mammary fat pad xenografts showed that PLOD2 knockdown could reduce metastasis to the lung and liver. Further, high PLOD2 expression correlated with poor prognosis of breast cancer patients. Mechanistically, adipocyte-derived interleukin-6 (IL-6) and leptin may facilitate PLOD2 upregulation in breast cancer cells and promote breast cancer metastasis in tail vein metastasis assays. Further investigation revealed that adipocyte-derived IL-6 and leptin promoted PLOD2 expression through activation of the JAK/STAT3 and PI3K/AKT signaling pathways. CONCLUSIONS: Our study reveals that adipocyte-derived IL-6 and leptin promote PLOD2 expression by activating the JAK/STAT3 and PI3K/AKT signaling pathways, thus promoting breast cancer metastasis.

Factors influencing delayed gastric emptying after pancreaticoduodenectomy - a meta-analysis.

BACKGROUND/AIMS: Delayed gastric emptying (DGE) is the most frequent postoperative complication after pancreaticoduodenectomy. Not only does it contribute considerably to prolonged hospitalization, but it is also associated with increased postoperative morbidity and mortality. We performed a meta-analysis to assess factors influencing the development of DGE after pancreaticoduodenectomy. METHODOLOGY: We systematically searched for studies that assessed association between peri-operative factors and DGE. We reviewed separately each of the factors, including preservation of pylorus, methods of gastrointestinal reconstruction, postoperative enteral feeding and postoperative complications. We identified 1035 studies published between May 1, 1988 and May 1, 2008. RESULTS: Compared with control subjects, the risk of DGE was 2.35 for preservation of pylorus (95% CI, 0.72 to 7.61), 6.14 for postoperative complications (95% CI, 3.47 to 10.85). There was no significant association between the occurrence of DGE with either PD or PPPD. CONCLUSIONS: Postoperative enteral feeding did not show any advantages in preventing DGE. Postoperative complications were the most important factor associated with DGE's occurrence. Antecolic and BII type gastrojejunostomy seems to suggest an improvement in the incidence of DGE.

A Novel ASCT2 Inhibitor, C118P, Blocks Glutamine Transport and Exhibits Antitumour Efficacy in Breast Cancer.

BACKGROUND: The microtubule protein inhibitor C118P shows excellent anti-breast cancer effects. However, the potential targets and mechanisms of C118P in breast cancer remain unknown. METHODS: Real-time cellular analysis (RTCA) was used to detect cell viability. Apoptosis and the cell cycle were detected by flow cytometry. Computer docking simulations, surface plasmon resonance (SPR) technology, and microscale thermophoresis (MST) were conducted to study the interaction between C118P and alanine-serine-cysteine transporter 2 (ASCT2). Seahorse XF technology was used to measure the basal oxygen consumption rate (OCR). The effect of C118P in the adipose microenvironment was explored using a co-culture model of adipocytes and breast cancer cells and mouse cytokine chip. RESULTS: C118P inhibited proliferation, potentiated apoptosis, and induced G2/M cell cycle arrest in breast cancer cells. Notably, ASCT2 was validated as a C118P target through reverse docking, SPR, and MST. C118P suppressed glutamine metabolism and mediated autophagy via ASCT2. Similar results were obtained in the adipocyte-breast cancer microenvironment. Adipose-derived interleukin-6 (IL-6) promoted the proliferation of breast cancer cells by enhancing glutamine metabolism via ASCT2. C118P inhibited the upregulation of ASCT2 by inhibiting the effect of IL-6 in co-cultures. CONCLUSION: C118P exerts an antitumour effect against breast cancer via the glutamine transporter ASCT2.

[Effects of Alkalinity on Partial Nitrification in a Zeolite Sequencing Batch Reactor].

Stable partial nitrification was successfully achieved in a zeolite sequencing batch reactor (ZSBR) with an excellent nitrite accumulation ratio of over 90.0%, and the effects of four alkalinity concentrations (calculated by CaCO3) on the nitritation in the ZSBR were investigated at an influent ammonia nitrogen concentration of 500 mg·L-1. The results showed that the key for the partial nitrification achieved in ZSBR was that the inhibitory effect of free ammonia (FA) on nitrite oxidizing bacteria (NOB) was much greater than that on ammonia oxidizing bacteria (AOB). Additionally, the wastewater containing ammonia nitrogen converted by this process can be used as the influent for anaerobic ammonium oxidation to further remove ammonia nitrogen and total nitrogen in wastewater. When the dosage of alkalinity was 2500 mg·L-1 in the ZSBR, the best nitritation efficiency was obtained with an average ammonia nitrogen conversion efficiency of 66.7%, nitrite accumulation ratio of 98.1%, and nitrite production rate of 0.74 kg·(m3·d)-1. It was demonstrated by high-throughput sequencing analysis that significant microbial community variations occurred after a long period of operation, and these changes involved an enrichment of AOB and inhibition of NOB in the ZSBR.

Correlation between procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 and breast cancer.

Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2), which affects collagen synthesis, is associated with breast cancer. The purpose of the study is to detect the expression of PLOD2 in breast cancer and to evaluate the correlation between PLOD2 and clinicopathologic characteristics and prognosis of patients with breast cancer. 50 paired samples including breast cancer tissues and adjacent non-tumor tissues were formalin-fixed and evaluated by immunohistochemistry. The results revealed that PLOD2 expression in breast cancer tissues was much higher than that in tissues adjacent to breast cancer. High expression of PLOD2 was positively associated with tumor stage (P = 0.003) and lymph node metastasis (P = 0.001). However, high expression of PLOD2 was negatively related to Ki-67 (P < 0.001) while positively related to progesterone receptor (PR) (P = 0.001). PLOD2 expression was positively related to the metastasis of breast cancer. Therefore, high expression of PLOD2 was identified as a poor prognostic biomarker for patients with breast cancer. These results suggest a novel molecular mechanism in breast cancer tumorigenesis, thus providing a potential therapeutic target of breast cancer.

Relationship between overexpression of NK-1R, NK-2R and intestinal mucosal damage in acute necrotizing pancreatitis.

AIM: To study the expression of neurokinin-1 receptor (NK-1R) and neurokinin-2 receptor (NK-2R) in distal ileum of acute necrotizing pancreatitis (ANP) and to evaluate the relationship between expression of these two receptors and intestinal mucosal damage. METHODS: A total of 130 adult Sprague-Dawley rats were randomly divided into two groups: the rats in ANP group (n=80) were induced by the retrograde intraductal infusion of 30 g.L(-1) sodium taurocholate. And the rats in normal control group (n=50) received laparotomy only. Sacrifices were made 6 h, 12 h, 24 h and 48 h later in ANP and normal control group after induction respectively. Intestinal mucosal permeability was studied by intrajejunal injection of 1.5 mCi radioactive isotope (99m)Tc-diethlene triamine pentacetic acid (DTPA) and the radioactivity of (99m)Tc-DTPA content in urine was measured 6 h, 12 h, 24 h and 48 h after induction. Then the pancreas and intestine were prepared for pathology. Reverse transcription polymerase chain reaction (RT-PCR) was used to determine the mRNA expression of NK-1R and NK-2R, and Western blot was used to investigate the protein level of NK-1R and NK-2R. RESULTS: In ANP rats, serious histologic damages in intestinal mucosa were observed, and the radioactivity of (99m)Tc-DTPA in urine increased significantly in the ANP group. RT-PCR revealed that NK-1R and NK-2R mRNA level was overexpressed in the distal ileum of ANP as compared with the normal control group. Western blot discovered stronger NK-1R (14-fold increase) and NK-2R (9-fold increase) immunoreactivity in the intestinal mucosa of ANP rats. Moreover, the overexpression of NK-1R was associated with mucosal pathological score (r=0.77, P<0.01) and intestinal permeability (r=0.68, P<0.01) in ANP rats. CONCLUSION: NK-1R and NK-2R contribute to disrupted neuropeptides loop balance, deteriorate intestinal damage, and are involved in pathophysiological changes in ANP.

[Gene expression analysis in liver tissue at a single cell level by nested polymerase chain reaction and laser microdissection].

OBJECTIVE: To investigate the measurements of gene expressing at a single hepatocyte level. METHODS: Individual hepatocyte was isolated from cryostat tissue section using laser microdissection technique. To detect the mRNA expressed by single hepatocyte, RNA was extracted, reversely transcribed to cDNA and amplified by nested polymerase chain reaction (PCR). RESULTS: Single cell was microdissected from cryostat tissue using an ultraviolet laser micromanipulator. The RNA could be extracted from the isolated cell(s), and the RT-PCR production could be observed after electrophoresis, whose quantitation was compatible with the number of cells. CONCLUSION: Combining laser microdissection and nested RT-PCR can monitor gene expression at a single cell level in vivo.

[Role of neurokinin-1 receptor in lung injury in rats with acute necrotizing pancreatitis].

OBJECTIVE: To investigate the expression of neurokinin-1 receptor (NK-1R) in the lung tissue, and the relationship between expression of NK-1R and lung injury in rats with acute necrotizing pancreatitis (ANP). METHODS: One hundred and twenty adult Sprague-Dawley rats were randomly divided into ANP and control groups. Animals in group ANP were induced by the retrograde intraductal infusion of 5% sodium taurocholate (0.1 ml/kg), and animals in normal control group received laparotomy only. The accumulation of polymorphonuclear leukocytes in lung tissues was measured with myeloperoxidase (MPO) assay. Lung endothelial barrier destruction was measured by lung capillary permeability (LCP). Reverse transcription polymerase chain reaction (RT-PCR) was used to determine the mRNA expression of NK-1R, western blot analysis was used to determine NK-1R protein expression levels, and immunohistochemistry was used to localize expression site of NK-1R. RESULTS: NK-1R mRNA level was enhanced in the lung of ANP compared with normal control group. Western blot analysis showed overexpression of NK-1R protein level exited in ANP group. Statistical analysis revealed correlation between NK-1R mRNA and MPO (r=0.83, P<0.01) and LCP (r=0.79, P<0.01) respectively. With immunohistochemistry staining, moderate to strong NK-1R immunoreactivity was localized to alveolar membrane, I epithelium, II epithelium and polymorphonuclear leukocytes in the lung of ANP. CONCLUSION: In ANP, overexpression of NK-1R contributes to disturbance of neuropeptides loop, resulting in aggregation of neutrophilic granulocyte and promoting deterioration of lung injury.

Expression of pancreatic regenerating gene in lung and intestinal tissue correlates with the severity of disease in rats with acute necrotizing pancreatitis.

The aim of this study was to investigate the expression pattern of regenerating gene I (Reg I) in the lung and intestinal tissues of rats with acute necrotizing pancreatitis (ANP), as well as the correlation of Reg I expression with lung and intestinal injury. Sprague-Dawley rats were randomly allocated to control (n=40) and ANP (n=80) groups. The rats in the control group received laparotomy only. In the ANP group, 3% sodium taurocholate was injected into the pancreatic duct to develop the ANP model. Reverse transcription-polymerase chain reaction (RT-PCR) was performed to detect the Reg I mRNA levels in the pancreas, intestine and lung. The pathological changes in the pancreas, intestine and lung were observed and the serum amylase levels, the wet/dry weight ratio of the lung and the permeability of the intestinal mucosa were measured. The measured parameters were found to correlate with the Reg I mRNA levels. Reg I mRNA was more highly expressed in the pancreas, intestine and lung in the ANP rats than in the control group. The Reg I expression levels were positively correlated with the pathological scores, serum amylase levels, lung pathological scores, lung tissue wet/dry ratios, intestinal pathological scores and intestinal permeability. The levels of Reg I were increased in the lung and intestinal tissue of the ANP rats and the expression levels of Reg I correlated closely with the severity of the lung and intestinal injury.

[Laser microdissection of a single cell from colon tissue for gene analysis].

AIM: To investigate the method of detecting gene expression in colon tissue at a single cell level. METHODS: Individual cell(s) were picked up from colon frozen section using laser microdissection. RNA was extracted, reverse transcribed to complementary DNA (cDNA). cDNA was then analyzed by nested reverse transcription polymerase chain reaction (nested RT-PCR) using two pairs of primers. RESULTS: Single cell(s) were selectively picked up using an ultraviolet laser micromanipulator. RNA was extracted, reverse transcribed and used for nested RT-PCR. Amplification products of cDNA from down to a single cell could be clearly visualized in the agarose gel. CONCLUSION: The combined utilization of laser microdissection and nested RT-PCR provides an opportunity to analyze gene expression at single cell(s) level in colon tissue.