Novel management indications for conservative treatment of chylous ascites after gastric cancer surgery.

BACKGROUND: Chylous ascites (CA) presents a challenge as a relatively common postoperative complication in gastric cancer (GC). Primary conservative therapy involved total parenteral nutrition, continuous low-pressure drainage, somatostatin, and a low-fat diet. Drainage tube (DT) clamping has been presented as a potential alternative conservative treatment for GC patients with CA. AIM: To propose novel conservative treatment strategies for CA following GC surgery. METHODS: The data of patients with CA after GC surgery performed at the Fudan University Shanghai Cancer Center between 2006 and 2021 were evaluated retrospectively. RESULTS: 53 patients underwent surgery for GC and exhibited postoperative CA during the study period. Postoperative hospitalization and time of DT removal showed a significant positive association (R 2 = 0.979, P < 0.001). We further observed that delayed DT removal significantly extended the total and postoperative hospitalization, antibiotic usage duration, and hospitalization cost (postoperative hospitalization: 25.8 d vs 15.5 d, P < 0.001; total hospitalization: 33.2 d vs 24.7 d, P < 0.01; antibiotic usage duration: 10.8 d vs 6.2 d, P < 0.01; hospitalization cost: ¥9.2 × 104 vs ¥6.5 × 104, P < 0.01). Multivariate analysis demonstrated that postoperative infection and antibiotic usage were independent factors for delayed DT removal. Furthermore, DT removal times were shorter in seven patients who underwent DT clamping (clamped DT vs normal group, 11.8 d vs 13.6 d, P = 0.047; clamped DT vs delayed group, 13.6 d vs 27.4 d, P < 0.001). In addition, our results indicated that removal of the DT may be possible after three consecutive days of drainage volumes less than 300 mL in GC patients with CA. CONCLUSION: Infection and antibiotic usage were vital independent factors that influenced delayed DT removal in patients with CA. Appropriate standards for DT removal can significantly reduce the duration of hospitalization. Furthermore, DT clamping might be a recommended option for conservative treatment of postoperative CA.

LncRNA lncAY is upregulated by sulfatide via Myb/MEF2C acetylation to promote the tumorigenicity of hepatocellular carcinoma cells.

LncRNA (long noncoding RNA) is often dysregulated in tumors especially hepatocellular carcinoma (HCC). However, the dysregulation mechanism of lncRNAs is largely unknown. Here, we showed that lncRNA lncAY expression was stimulated in HCC by either endogenous or exogenous sulfatide. Elevated lncAY promoted HCC cell migration or angiogenesis, whereas lncAY silence suppressed HCC cell migration and proliferation. Interestingly, the activity of lncAY gene promoter was enhanced by sulfatide. Then Myb and MEF2C were identified as the transcription factors responsible for the stimulation of lncAY promoter activity and transcription by sulfatide. Both Myb and MEF2C enrichment on lncAY promoter was further confirmed, and their occupancy on lncAY promoter was strengthened by sulfatide for Myb or MEF2C was acetylated. Mutant Myb-K456A exhibited reduced acetylation and weak stimulation for lncAY transcription. However, Myb mutation K456/503A prevented Myb from acetylation induced by sulfatide. The mutant Myb K456/503A further was unable to occupy lncAY promoter and enhance lncAY transcription. In conclusion, this study demonstrated lncAY transcription was abnormally upregulated by sulfatide in HCC through Myb/MEF2C to promote HCC progression.

Preoperative apolipoprotein B/apolipoprotein A1 ratio: a novel prognostic factor for gastric cancer.

BACKGROUND: The correlations between lipid profile (lipid molecules and their derivative indexes) and clinical outcome have been widely testified in many carcinomas, but its prognostic value remains unknown in gastric cancer (GC). Our purpose in the study was to comprehensively evaluate the clinical significance of lipid profile in GC. METHODS: We retrospectively collected clinical information of 1,201 GC patients who received surgery at Sun Yat-sen University Cancer Center from 2005 to 2010. Kaplan-Meier analysis and Cox proportional hazards regression model were performed to determine its prognostic significance. RESULTS: Lipid profile including cholesterol, triglyceride, high-density lipoprotein-cholesterol (HDL-C), low-density lipoprotein-cholesterol (LDL-C), apolipoprotein A1 (ApoA1), apolipoprotein B (ApoB), LDL-C/HDL-C ratio, and ApoB/ApoA1 ratio were analyzed. For the first time, we found ApoB/ApoA1 ratio showed the biggest prognostic potency among all lipid-related variables and could act as an independent prognostic factor in GC. Patients with a high ApoB/ApoA1 ratio (≥1) had a shorter overall survival (hazard ratio: 1.373, 95% confidence interval: 1.123-1.68; P=0.002). CONCLUSION: Preoperative serum ApoB/ApoA1 ratio might be used as a novel prognostic indicator of GC.

Advanced gastrointestinal stromal tumor patients benefit from palliative surgery after tyrosine kinase inhibitors therapy.

The role of palliative surgery is controversial in advanced gastrointestinal stromal tumors (GIST) after tyrosine kinase inhibitors (TKIs) therapy.We evaluated safety and clinical outcomes in a single institution series of advanced GIST patients from January 2002 to December 2008.One hundred and fifty-six patients had been recruited, including 87 patients underwent surgical resection and 69 patients kept on TKIs treatment. Four patients had major surgical complications. Median follow-up was 38.3 months, the overall survival (OS) and progression-free survival (PFS) of the patients in surgical group were longer than the nonsurgical group, PFS: 46.1 versus 33.8 months (P < .01), OS: 54.8 versus 40.4 months. In the subgroup analysis for the patients received surgery, the median PFS for patients with progression disease, stable disease, and partial response was 33.3, 51.5, and 83.0 months, respectively (P < .01). Median OS was 68.0 months in those with only liver or peritoneal metastases, and 45.3 months in those with both metastases. Median PFS of patients underwent R0/R1 resection was 73.6 months compared with 35.8 months in R2 resection patients (P < .01).Patients with advanced GISTs have prolonged OS after debulking procedures. Surgery for patients who have responsive disease after TKIs treatment should be considered.

Long Noncoding RNA GCASPC, a Target of miR-17-3p, Negatively Regulates Pyruvate Carboxylase-Dependent Cell Proliferation in Gallbladder Cancer.

Long noncoding RNAs (lncRNA) are being implicated in the development of many cancers. Here, we report the discovery of a critical role for the lncRNA GCASPC in determining the progression of gallbladder cancer. Differentially expressed lncRNAs and mRNAs between gallbladder cancer specimens and paired adjacent nontumor tissues from five patients were identified and validated by an expression microarray analysis. Quantitative real-time PCR was used to measure GCASPC levels in tissues from 42 gallbladder cancer patients, and levels of GCASPC were confirmed further in a separate cohort of 89 gallbladder cancer patients. GCASPC was overexpressed or silenced in several gallbladder cancer cell lines where molecular and biological analyses were performed. GCASPC levels were significantly lower in gallbladder cancer than adjacent nontumor tissues and were associated with tumor size, American Joint Committee on Cancer tumor stage, and patient outcomes. GCASPC overexpression suppressed cell proliferation in vitro and in vivo, whereas GCASPC silencing had opposite effects. By RNA pull-down and mass spectrometry, we identified pyruvate carboxylase as an RNA-binding protein that associated with GCASPC. Because GCASPC is a target of miR-17-3p, we confirmed that both miR-17-3p and GCASPC downregulated pyruvate carboxylase level and activity by limiting protein stability. Taken together, our results defined a novel mechanism of lncRNA-regulated cell proliferation in gallbladder cancer, illuminating a new basis for understanding its pathogenicity. Cancer Res; 76(18); 5361-71. ©2016 AACR.

Xc- inhibitor sulfasalazine sensitizes colorectal cancer to cisplatin by a GSH-dependent mechanism.

Sulfasalazine (SSZ) is an anti-inflammatory drug that has been demonstrated to induce apoptosis and tumor regression through inhibition of plasma membrane cystine transporter xc(-). Cysteine is a rate-limiting precursor for intracellular glutathione (GSH) synthesis, which is vital for compound detoxification and maintaining redox balance. Platinum-based chemotherapy is an important regimen used in clinics for various cancers including colorectal cancer (CRC). We hypothesized that targeting xc(-) transporter by SSZ may annihilate cellular detoxification through interruption of GSH synthesis and may enhance the anti-cancer activity of cisplatin (CDDP) by increasing drug transport. In the present study, we revealed that xCT, the active subunit of xc(-), is highly expressed in CRC cell lines and human colorectal carcinoma tissues compared with their normal counterparts. SSZ effectively depleted cellular GSH, leading to significant accumulation of reactive oxygen species and growth inhibition in CRC cells. In contrast, the normal epithelial cells of colon origin were less sensitive to SSZ, showing a moderate ROS elevation. Importantly, SSZ effectively enhanced the intracellular platinum level and cytotoxicity of CDDP in CRC cells. The synergistic effect of SSZ and CDDP was reversed by antioxidant N-acetyl-L-cysteine (NAC). Together, these results suggest that SSZ, a relatively non-toxic drug that targets cystine transporter, may, in combination with CDDP, have effective therapy for colorectal cancer.

Micro-RNA-155 is induced by K-Ras oncogenic signal and promotes ROS stress in pancreatic cancer.

The oncogenic K-Ras can transform various mammalian cells and plays a critical role in development of pancreatic cancer. MicroRNAs (miRNA) have been shown to contribute to tumorigenic progression. However, the nature of miRNAs involved in K-Ras transformation remains to be investigated. Here, by using microarray we identified miR-155 as the most upregulated miRNA after both acute and prolonged activation of K-Ras in a doxycyline-inducible system. Pharmacological inhibition of MAPK and NF-κB pathway blocked the induction of miR-155 in response to K-Ras activation. Overexpression of miR-155 caused inhibition of Foxo3a, leading to decrease of major antioxidants including SOD2 and catalase, and enhanced pancreatic cell proliferation induced by ROS generation. Importantly, correlations of K-Ras, miR-155 and Foxo3a were also validated in human pancreatic cancer tissues. Therefore, we propose that miR-155 plays an important role in oncogenic K-Ras transformation mediated by cellular redox regulation.

Identification of NDUFAF1 in mediating K-Ras induced mitochondrial dysfunction by a proteomic screening approach.

Increase in aerobic glycolysis and mitochondrial dysfunction are important biochemical features observed in human cancers. Recent studies suggest oncogenic K-Ras can cause suppression of mitochondrial respiration and up-regulation of glycolytic activity through a yet unknown mechanism. Here we employed proteomic approach and used a K-RasG12V inducible cell system to investigate the impact of oncogenic K-Ras on mitochondria and cell metabolism. Mitochondria isolated from cells before and after K-Ras induction were subjected to protein analysis using stable isotope labeling with amino acids (SILAC) and liquid chromatography coupled with mass spectrometry (LC-MS). 70 mitochondrial proteins with significant expression alteration after K-Ras induction were identified. A majority of these proteins were involved in energy metabolism. Five proteins with significant decrease belong to mitochondrial respiratory chain complex I. NADH dehydrogenase 1 alpha subcomplex assembly factor 1 (NDUFAF1) showed most significant decrease by 50%. Such decrease was validated in primary human pancreatic cancer tissues. Knockdown of NDUFAF1 by siRNA caused mitochondrial respiration deficiency, accumulation of NADH and subsequent increase of glycolytic activity. Our study revealed that oncogenic K-Ras is able to induce significant alterations in mitochondrial protein expression, and identified NDUFAF1 as an important molecule whose low expression contributes to mitochondrial dysfunction induced by K-Ras.

Long non-coding RNA-LET is a positive prognostic factor and exhibits tumor-suppressive activity in gallbladder cancer.

The identification of cancer-associated long non-coding RNAs (lncRNAs) and the investigation of their molecular and biological functions are vital for understanding the molecular biology and progression of cancer. The lncRNA-LET, a newly identified lncRNA, was demonstrated to be down-regulated in hepatocellular cancer. However, little is known about its role in gallbladder cancer. In the present study, an obvious down-regulation of lncRNA-LET was observed in gallbladder cancer compared to their adjacent normal tissues. Meanwhile, patients with low expression of lncRNA-LET have significantly poorer prognosis than those with high expression. We confirmed that hypoxia decreased lncRNA-LET levels in gallbladder cancer cells. Moreover, lncRNA-LET overexpression was further validated to inhibit the invasion of gallbladder cancer cells under hypoxic or normoxic conditions in vitro. We demonstrated that lncRNA-LET overexpression conferred a proliferative advantage to tumor cells under hypoxic conditions. The ectopic expression of lncRNA-LET led to the promotion of cell cycle arrest at G0/G1 phase and to the induction of apoptosis under hypoxic conditions. Ectopic expression of LncRNA-LET also suppressed gallbladder tumor growth in vivo. Our findings indicate that lncRNA-LET may represent a prognostic marker and a potential therapeutic target for gallbladder cancer.

Differentiation therapy: sesamin as an effective agent in targeting cancer stem-like side population cells of human gallbladder carcinoma.

BACKGROUND: Recent studies have demonstrated that side population (SP) cells isolated from various cancer cell lines and primary tumors possess stem cell-like properties. Sesamin, a food-derived agent, possesses anti-cancer activities both in vitro and in vivo. The present study was designed to determine whether sesamin also have effects on cancer stem-like SP cells from gallbladder cancer (GBC). METHODS: In this study, we sorted SP cells by flow cytometry. SP cells were cultured and treated with sesamin. Tumor-sphere formation, colony formation, Matrigel invasion and tumorigenic potential were determined. Expression of nuclear NF-κB, IL-6, p-Stat3, Twist, E-cadherin and Vimentin was measured by Western blot, immunofluorescence staining or RT-PCR analysis. Nuclear NF-κB activity and IL-6 protein level were assessed with ELISA. Xenograft tumors were generated in nude mice. RESULTS: After treated with sesamin, SP cells differentiated into cells expressing the epithelial marker (E-cadherin). Sesamin effectively affected SP cells stem cell-like characteristics (i.e., tumor-sphere formation, colony-formation, Matrigel invasion), weakened the drug-resistance of SP cells and inhibited tumor growth both in vitro and in vivo. Treatment with sesamin significantly reduced the expression of nuclear NF-κB, IL-6, p-Stat3, Twist and Vimentin (a mesenchymal marker) in SP cells. Nuclear NF-κB activity and IL-6 level were also decreased after treatment with sesamin. CONCLUSION: Food-derived sesamin directs the epithelial differentiation of cancer stem-like SP cells from GBC, which is associated with attenuation of NF-κB-IL-6-Stat3-Twist signal pathway.

Long non-coding RNA HOTAIR, a c-Myc activated driver of malignancy, negatively regulates miRNA-130a in gallbladder cancer.

BACKGROUND: Protein coding genes account for only about 2% of the human genome, whereas the vast majority of transcripts are non-coding RNAs including long non-coding RNAs. A growing volume of literature has proposed that lncRNAs are important players in cancer. HOTAIR was previously shown to be an oncogene and negative prognostic factor in a variety of cancers. However, the factors that contribute to its upregulation and the interaction between HOTAIR and miRNAs are largely unknown. METHODS: A computational screen of HOTAIR promoter was conducted to search for transcription-factor-binding sites. HOTAIR promoter activities were examined by luciferase reporter assay. The function of the c-Myc binding site in the HOTAIR promoter region was tested by a promoter assay with nucleotide substitutions in the putative E-box. The association of c-Myc with the HOTAIR promoter in vivo was confirmed by chromatin immunoprecipitation assay and Electrophoretic mobility shift assay. A search for miRNAs with complementary base paring with HOTAIR was performed utilizing online software program. Gain and loss of function approaches were employed to investigate the expression changes of HOTAIR or miRNA-130a. The expression levels of HOTAIR, c-Myc and miRNA-130a were examined in 65 matched pairs of gallbladder cancer tissues. The effects of HOTAIR and miRNA-130a on gallbladder cancer cell invasion and proliferation was tested using in vitro cell invasion and flow cytometric assays. RESULTS: We demonstrate that HOTAIR is a direct target of c-Myc through interaction with putative c-Myc target response element (RE) in the upstream region of HOTAIR in gallbladder cancer cells. A positive correlation between c-Myc and HOTAIR mRNA levels was observed in gallbladder cancer tissues. We predicted that HOTAIR harbors a miRNA-130a binding site. Our data showed that this binding site is vital for the regulation of miRNA-130a by HOTAIR. Moreover, a negative correlation between HOTAIR and miRNA-130a was observed in gallbladder cancer tissues. Finally, we demonstrate that the oncogenic activity of HOTAIR is in part through its negative regulation of miRNA-130a. CONCLUSION: Together, these results suggest that HOTAIR is a c-Myc-activated driver of malignancy, which acts in part through repression of miRNA-130a.

Candidate microRNA biomarkers of pancreatic ductal adenocarcinoma: meta-analysis, experimental validation and clinical significance.

BACKGROUND: The diagnostic and prognostic value of microRNA (miRNA) expression aberrations in pancreatic ductal adenocarcinoma (PDAC) has been studied extensively in recent years. However, differences in measurement platforms and lab protocols as well as small sample sizes can render gene expression levels incomparable. METHODS: A comprehensive meta-review of published studies in PDAC that compared the miRNA expression profiles of PDAC tissues and paired neighbouring noncancerous pancreatic tissues was performed to determine candidate miRNA biomarkers for PDAC. Both a miRNA vote-counting strategy and a recently published Robust Rank Aggregation method were employed. In this review, a total of 538 tumour and 206 noncancerous control samples were included. RESULTS: We identified a statistically significant miRNA meta-signature of seven up- and three down-regulated miRNAs. The experimental validation results showed that the miRNA expression levels were in accordance with the meta-signature. The results from the vote-counting strategy were consistent with those from the Robust Rank Aggregation method. The experimental validation confirmed that the statistically unique profiles identified by the meta-review approach could discriminate PDAC tissues from paired nonmalignant pancreatic tissues. In a cohort of 70 patients, the high expression of miR-21 (p=0.018, HR=2.610; 95% CI=1.179-5.777) and miR-31 (p=0.039, HR=2.735; 95% CI=1.317-6.426), the low expression of miR-375 (p=0.022, HR=2.337; 95% CI=1.431-5.066) were associated with poor overall survival following resection, independent of clinical covariates. CONCLUSIONS: The identified miRNAs may be used to develop a panel of diagnostic and prognostic biomarkers for PDAC with sufficient sensitivity and specificity for use in a clinical setting.

Microencapsulated tumor assay: evaluation of the nude mouse model of pancreatic cancer.

AIM: To establish a more stable and accurate nude mouse model of pancreatic cancer using cancer cell microencapsulation. METHODS: The assay is based on microencapsulation technology, wherein human tumor cells are encapsulated in small microcapsules (approximately 420 µm in diameter) constructed of semipermeable membranes. We implemented two kinds of subcutaneous implantation models in nude mice using the injection of single tumor cells and encapsulated pancreatic tumor cells. The size of subcutaneously implanted tumors was observed on a weekly basis using two methods, and growth curves were generated from these data. The growth and metastasis of orthotopically injected single tumor cells and encapsulated pancreatic tumor cells were evaluated at four and eight weeks postimplantation by positron emission tomography-computed tomography scan and necropsy. The pancreatic tumor samples obtained from each method were then sent for pathological examination. We evaluated differences in the rates of tumor incidence and the presence of metastasis and variations in tumor volume and tumor weight in the cancer microcapsules vs single-cell suspensions. RESULTS: Sequential in vitro observations of the microcapsules showed that the cancer cells in microcapsules proliferated well and formed spheroids at days 4 to 6. Further in vitro culture resulted in bursting of the membrane of the microcapsules and cells deviated outward and continued to grow in flasks. The optimum injection time was found to be 5 d after tumor encapsulation. In the subcutaneous implantation model, there were no significant differences in terms of tumor volume between the encapsulated pancreatic tumor cells and cells alone and rate of tumor incidence. There was a significant difference in the rate of successful implantation between the cancer cell microencapsulation group and the single tumor-cell suspension group (100% vs 71.43%, respectively, P = 0.0489) in the orthotropic implantation model. The former method displayed an obvious advantage in tumor mass (4th wk: 0.0461 ± 0.0399 vs 0.0313 ± 0.021, t = -0.81, P = 0.4379; 8th wk: 0.1284 ± 0.0284 vs 0.0943 ± 0.0571, t = -2.28, respectively, P = 0.0457) compared with the latter in the orthotopic implantation model. CONCLUSION: Encapsulation of pancreatic tumor cells is a reliable method for establishing a pancreatic tumor animal model.