Transformation to small cell lung cancer is irrespective of EGFR and accelerated by SMAD4-mediated ASCL1 transcription independently of RB1 in non-small cell lung cancer.

OBJECTIVES: Histological transformation to small cell lung cancer (SCLC) has been identified as a mechanism of TKIs resistance in EGFR-mutant non-small cell lung cancer (NSCLC). We aim to explore the prevalence of transformation in EGFR-wildtype NSCLC and the mechanism of SCLC transformation, which are rarely understood. METHODS: We reviewed 1474 NSCLC patients to investigate the NSCLC-to-SCLC transformed cases and the basic clinical characteristics, driver gene status and disease course of them. To explore the potential functional genes in SCLC transformation, we obtained pre- and post-transformation specimens and subjected them to a multigene NGS panel involving 416 cancer-related genes. To validate the putative gene function, we established knocked-out models by CRISPR-Cas 9 in HCC827 and A549-TP53-/- cells and investigated the effects on tumor growth, drug sensitivity and neuroendocrine phenotype in vitro and in vivo. We also detected the expression level of protein and mRNA to explore the molecular mechanism involved. RESULTS: We firstly reported an incidence rate of 9.73% (11/113) of SCLC transformation in EGFR-wildtype NSCLC and demonstrated that SCLC transformation is irrespective of EGFR mutation status (P = 0.16). We sequenced 8 paired tumors and identified a series of mutant genes specially in transformed SCLC such as SMAD4, RICTOR and RET. We firstly demonstrated that SMAD4 deficiency can accelerate SCLC transition by inducing neuroendocrine phenotype regardless of RB1 status in TP53-deficient NSCLC cells. Further mechanical experiments identified the SMAD4 can regulate ASCL1 transcription competitively with Myc in NSCLC cells and Myc inhibitor acts as a potential subsequent treatment agent. CONCLUSIONS: Transformation to SCLC is irrespective of EFGR status and can be accelerated by SMAD4 in non-small cell lung cancer. Myc inhibitor acts as a potential therapeutic drug for SMAD4-mediated resistant lung cancer. Video Abstract.

PPM1D exerts its oncogenic properties in human pancreatic cancer through multiple mechanisms.

Protein phosphatase, Mg(2+)/Mn(2+) dependent, 1D (PPM1D) is emerging as an oncogene by virtue of its negative control on several tumor suppressor pathways. However, the clinical significance of PPM1D in pancreatic cancer (PC) has not been defined. In this study, we determined PPM1D expression in human PC tissues and cell lines and their irrespective noncancerous controls. We subsequently investigated the functional role of PPM1D in the migration, invasion, and apoptosis of MIA PaCa-2 and PANC-1 PC cells in vitro and explored the signaling pathways involved. Furthermore, we examined the role of PPM1D in PC tumorigenesis in vivo. Our results showed that PPM1D is overexpressed in human PC tissues and cell lines and significantly correlated with tumor growth and metastasis. PPM1D promotes PC cell migration and invasion via potentiation of the Wnt/ß-catenin pathway through downregulation of apoptosis-stimulating of p53 protein 2 (ASPP2). In contrast to PPM1D, our results showed that ASPP2 is downregulated in PC tissues. Additionally, PPM1D suppresses PC cell apoptosis via inhibition of the p38 MAPK/p53 pathway through both dephosphorylation of p38 MAPK and downregulation of ASPP2. Furthermore, PPM1D promotes PC tumor growth in vivo. Our results demonstrated that PPM1D is an oncogene in PC.

Different expression of S100A8 in malignant and benign gallbladder diseases.

BACKGROUND: Proteomic analysis is a powerful tool for complete establishment of protein expression. Comparative proteomic analysis of human bile from malignant and benign gallbladder diseases may be helpful in research into gallbladder cancer. AIMS: Our objective was to establish biliary protein content for gallbladder cancer, gallbladder adenoma, and chronic calculous cholecystitis for comparative proteomic analysis. METHODS: Bile samples were collected from patients with gallbladder cancer, gallbladder adenoma, and chronic calculous cholecystitis. Peptides of biliary proteins were separated by two-dimensional liquid chromatography then identified by tandem mass spectrometry. RESULTS: Up to 544, 221, and 495 unique proteins were identified in bile samples from gallbladder cancer, gallbladder adenoma, and chronic calculous cholecystitis. Forty-three, 16, and 28 proteins with more than one unique peptide, respectively, were identified in the three groups. Among these, 30 proteins including S100A8 were overexpressed in gallbladder cancer, compared with benign gallbladder diseases. We also confirmed, by immunohistochemical analysis, that S100A8 is more abundant in tumor-infiltrating immune cells in cancerous tissue. CONCLUSIONS: Compared with benign gallbladder diseases, consistently elevated S100A8 levels in malignant gallbladder bile and tissue indicate that gallbladder cancer is an inflammation-associated cancer. S100A8 may be a biomarker for gallbladder cancer.

Decreasing pancreatic leak after distal pancreatectomy: bovine pericardium wrapping stump in a porcine model.

BACKGROUND: Despite marked improvements in pancreatic surgery, the high incidence and morbidity of pancreatic leak after resection has remained unchanged. The current study investigated the safety and efficacy of bovine pericardium wrapping stump after distal pancreatectomy in a porcine model. METHODS: Thirty-two swine were randomly assigned to control and experiment groups to undergo conventional scalpel transection with single hand-sewn closure of the pancreatic remnant (control) or bovine pericardium wrapping stump combined with hand-sewn closure (experiment). Closed-suction drainage was collected and measured daily. Animals were necropsied at 3 weeks postoperatively, and the pancreatic remnants were examined for histology. Primary end points were the development of a pancreatic fistula defined as greater than threefold drain/serum amylase after the 3rd postoperative day, and the presence of undrained amylase-rich fluid collections/abscess. RESULTS: The incidence of pancreatic leak in the wrapping group was 6.3 versus 46.7% in the control group (p < 0.05). The amount of drainage fluid was higher in the control group than the experiment group during the postoperative days. There were no differences in operative time or other clinical parameters measured. No other significant differences were found in macroscopic changes between groups at reexploration. Histological examination demonstrated focal, chronic inflammation with necrosis in all animals. CONCLUSIONS: Bovine pericardium wrapping stump effectively reduced the incidence of pancreatic leakage after the distal pancreatectomy.

Knockdown of RAD51AP1 suppressed cell proliferation and invasion in esophageal squamous cell carcinoma.

BACKGROUND: Esophageal cancer is a common malignant tumor of digestive tract with esophageal squamous cell carcinoma (ESCC) being the main histological subtype. This study aimed to identify potential hub gene associated with the pathophysiology of ESCC through bioinformatics analysis and experiment validation. METHODS: Three microarray datasets were obtained from the Gene Expression Omnibus (GEO) database. The overlapping differentially expressed genes (DEGs) were analyzed by GEO2R tool. Gene Ontology (GO) and Kyoto Encyclopedia of Genes (KEGG) pathway analyses were performed to predict the potential functions of DEGs. Nine hub genes were identified using protein-protein interaction (PPI) network and Cytoscape software. We selected RAD51-associated protein 1 (RAD51AP1) for further research because of its poor prognosis and it has not been sufficiently studied in ESCC. The effects of RAD51AP1 on proliferation, apoptosis, migration and invasion of ESCC cells were determined by in vitro functional assays. RESULTS: RAD51AP1 expression was significantly upregulated in ESCC tissues compared with normal tissues by using The Cancer Genome Atlas (TCGA) database. High expression of RAD51AP1 was associated with worse survival in ESCC patients. RAD51AP1 expression was positively associated with the enrichment of Th2 cells and T helper cells. Furthermore, CCK-8 and colony formation assays showed knockdown of RAD51AP1 inhibited the proliferation of ESCC cells. Flow cytometry analysis indicated knockdown of RAD51AP1 induced cell cycle arrest and apoptosis in ESCC cells. Transwell assay revealed knockdown of RAD51AP1 suppressed the migration and invasion of ESCC cells. CONCLUSIONS: Finally, our results demonstrated that RAD51AP1 silencing significantly inhibited cell proliferation and invasion in ESCC, thereby highlighting its potential as a novel target for ESCC treatment.

The role of Med19 in the proliferation and tumorigenesis of human hepatocellular carcinoma cells.

AIM: To explore the role of Med19, a component of the Mediator complex that coactivates DNA-binding transcription factors, in the proliferation and tumorigenesis of human hepatocellular carcinoma cells. METHODS: The human hepatocellular carcinoma cell lines HepG2 and Hep3B were infected with lentiviral vectors encoding interfering RNA (RNAi) targeting the Med19 gene. To further confirm the inhibitory effects of RNAi vectors on Med19 gene expression, quantitative real-time RT-PCR and Western blotting assays were used. The proliferation of HepG2 and Hep3B cells after transduction with the Med19-RNAi-Lentivirus vector was evaluated by MTT conversion, BrdU incorporation, colony formation, and cell-cycle assays in vitro. In addition, the ability of the Med19-RNAi-Lentivirus vector-infected Hep3B cells to form tumors after inoculation into nude mice was determined. RESULTS: Recombinant lentiviral vectors expressing small interfering RNA (siRNA) against Med19 were constructed and were found to efficiently downregulate Med19 mRNA and protein levels in HepG2 and Hep3B cells. Furthermore, the inhibition of Med19 by RNAi dramatically reduced hepatocellular carcinoma cell proliferation, induced cell-cycle arrest in the G(0)/G(1) phase, and suppressed tumor formation. CONCLUSION: These results provide new evidence of an important role for Med19 in the development of hepatocellular carcinomas, suggesting that lentivirus-mediated RNAi to target Med19 is a potential tool for inhibiting cancer cell proliferation and tumorigenesis.

Herbal compound "Songyou Yin" reinforced the ability of interferon-alfa to inhibit the enhanced metastatic potential induced by palliative resection of hepatocellular carcinoma in nude mice.

BACKGROUND: Liver resection is a widely accepted treatment for hepatocellular carcinoma (HCC). Our previous clinical study showed that the rate of palliative resection was 34.0% (1958-2008, 2754 of 8107). However, the influence of palliative resection on tumor metastasis remains controversial. The present study was conducted to evaluate the effect of palliative resection on residual HCC and to explore interventional approaches. METHODS: Palliative resection was done in an orthotopic nude mice model of HCC (MHCC97H) with high metastatic potential. Tumor growth, invasion, metastasis, lifespan, and some molecular alterations were examined in vivo and in vitro. Mice that underwent palliative resection were treated with the Chinese herbal compound "Songyou Yin," interferon-alfa-1b (IFN-α), or their combination to assess their effects. RESULTS: In the palliative resection group, the number of lung metastatic nodules increased markedly as compared to the sham operation group (14.3 ± 4.7 versus 8.7 ± 3.6, P < 0.05); tumor matrix metalloproteinase 2 (MMP2) activity was elevated by 1.4-fold, with up-regulation of vascular endothelial growth factor (VEGF) and down-regulation of tissue inhibitor of metalloproteinase 2 (TIMP2). The sera of mice undergoing palliative resection significantly enhanced cell invasiveness by 1.3-fold. After treatment, tumor volume was 1205.2 ± 581.3 mm3, 724.9 ± 337.6 mm3, 507.6 ± 367.0 mm3, and 245.3 ± 181.2 mm3 in the control, "Songyou Yin," IFN-α, and combination groups, respectively. The combined therapy noticeably decreased the MMP2/TIMP2 ratio and prolonged the lifespan by 42.2%. Moreover, a significant (P < 0.001) reduction of microvessel density was found: 43.6 ± 8.5, 34.5 ± 5.9, 23.5 ± 5.6, and 18.2 ± 8.0 in the control and treatment groups, respectively. CONCLUSION: Palliative resection-stimulated HCC metastasis may occur, in part, by up-regulation of VEGF and MMP2/TIMP2. "Songyou Yin" reinforced the ability of IFN-α to inhibit the metastasis-enhancing potential induced by palliative resection, which indicated its potential postoperative use in patients with HCC.

Gemcitabine combined with gum mastic causes potent growth inhibition and apoptosis of pancreatic cancer cells.

AIM: To investigate the antiproliferative and apoptotic effects of gemcitabine combined with gum mastic and the underlying mechanisms in human pancreatic cancer cell lines. METHODS: Cell proliferation and apoptosis were examined using the methyl thiazolyl tetrazolium (MTT) assay and propidium iodine staining, respectively. The expression of Bcl-2, Bax, NF-kappaB p65 subunit, and IkappaBalpha protein was measured using Western blotting. RESULTS: Gemcitabine 0.01-100 microg/mL inhibited cell proliferation and induced apoptosis in both pancreatic cancer BxPC-3 and COLO 357 cells. Gum mastic 40 microg/mL significantly potentiated the antiproliferative and apoptotic effects of gemcitabine 10 microg/mL after 72-h treatment. When cells were treated with gemcitabine in combination with gum mastic, the IkappaBalpha level was increased, whereas NF-kappaB activation was blocked; the expression of Bax protein was substantially increased, but Bcl-2 protein was down-regulated. CONCLUSION: Gemcitabine combined with gum mastic causes potent apoptosis in pancreatic cancer cells. The combination may be an effective therapeutic strategy for pancreatic cancer.

Prognostic significance of S100A4 and vascular endothelial growth factor expression in pancreatic cancer.

AIM: To investigate the expression of vascular endothelial growth factor (VEGF) and calcium-binding protein S100A4 in pancreatic cancer and their relationship to the clinicopathological parameters and prognosis of pancreatic cancer. METHODS: Expression status of VEGF and S100A4 was examined in 62 surgical specimens of primary pancreatic cancer by immunohistochemistry. Correlation between the expression of VEGF and S100A4 and clinicopathological parameters was analyzed. RESULTS: Thirty-eight of 62 (61.3%) specimens of primary pancreatic cancer were positive for S100A4. Thirty-seven (59.7%) specimens showed positive expression of VEGF. The positive correlation between S100A4 and VEGF expression was significant in cancer tissues (P < 0.001). S100A4 expression was significantly correlated with tumor size, TNM stage and poorer prognosis. VEGF expression had a significant correlation with poorer prognosis. The prognosis of 17 S100A4- and VEGF-negative cancer patients was significantly better than that of other patients (P < 0.05). Distant metastasis (P = 0.001), S100A4- (P = 0.008) and VEGF-positive expression (P = 0.016) were significantly independent prognostic predictors (P < 0.05). CONCLUSION: Over-expression of S100A4 and VEGF plays an important role in the development of pancreatic cancer. Combined examination of the two molecules might be useful in evaluating the outcome of patients with pancreatic cancer.

Prokinetic effects of a ghrelin receptor agonist GHRP-6 in diabetic mice.

AIM: To investigate the effects of a ghrelin receptor agonist GHRP-6 on delayed gastrointestinal transit in alloxan-induced diabetic mice. METHODS: A diabetic mouse model was established by intraperitoneal injection with alloxan. Mice were randomized into two main groups: normal mice and diabetic mice treated with GHRP-6 at doses of 0, 20, 50, 100 and 200 microg/kg ip. Gastric emptying (GE), intestinal transit (IT), and colonic transit (CT) were studied in mice after they had a phenol red meal following injection of GHRP-6. Based on the most effective GHRP-6 dosage, atropine was given at 1 mg/kg for 15 min before the GHRP-6 injection for each measurement. The mice in each group were sacrificed 20 min later and the percentages of GE, IT, and CT were calculated. RESULTS: Percentages of GE, IT, and CT were significantly decreased in diabetic mice as compared to control mice. In the diabetic mice, GHRP-6 improved both GE and IT, but not CT. The most effective dose of GHRP-6 was 200 microg/kg and atropine blocked the prokinetic effects of GHRP-6 on GE and IT. CONCLUSION: GHRP-6 accelerates delayed GE and IT, but has no effect on CT in diabetic mice. GHRP-6 may exert its prokinetic effects via the cholinergic pathway in the enteric nervous system, and therefore, has therapeutic potential for diabetic patients with delayed upper gastrointestinal transit.

[Treatment of late-staged pancreatic carcinoma with implantation of 125I seeds in combination with regional arterial infusion chemotherapy].

OBJECTIVE: To evaluate the effect of combined use of implantation of (125)I seeds with regional arterial infusion chemotherapy on late-staged pancreatic carcinoma. METHODS: During operation, (125)I seeds were implanted into tumor parenchyma or operative beds for 7 patients with pathological diagnosis of late-staged pancreatic carcinoma, then chemotherapy pump was implanted into common hepatic artery or celiac artery through left gastric artery or right epigastric artery. Gemcitabine (1000 mg/m(2)) plus fluorouracil (500 mg/m(2)) was used for postoperative adjuvant chemotherapy once a week for 7 consecutive weeks. RESULTS: The mean number of implanted (125)I seeds was 42 and the mean dose was 29.4 mCi. The mean follow-up time was 245 days. Rate of pain relief was 100%, metastasis foci was reduced in 2 cases. CA-19-9 Antigen level decreased to normal in all patients. Pancreatic fistula occurred in one case and was cured with conservative therapy. CONCLUSIONS: The combination therapy is effective for late-staged pancreatic carcinoma, it brings symptom relief and improvement in quality of life.

Impact of Abdominal Shape on Short-Term Surgical Outcome of Laparoscopy-Assisted Distal Gastrectomy for Gastric Cancer.

BACKGROUND: Laparoscopy-assisted distal gastrectomy (LADG) has been widely accepted for the treatment for gastric cancer. The aim of the present study was to explore the impact of abdominal shape parameters on gastric antrum cancer patients' short-term surgical outcomes of LADG with D2 lymph node dissection in both genders, including the number of lymph nodes retrieved and surgical safety index. METHODS: This was a retrospective analysis of 177 gastric antrum cancer patients, who underwent LADG between April 2009 and January 2016. The abdominal shape parameters, including abdominal anterior-posterior diameter (APD), transverse diameter (TD), xiphoid process of the sternum-navel distance (XND), and thickness of subcutaneous fat (SCF) at the umbilicus level, were calculated by preoperative abdominal computed tomography (CT) scans. The effects of abdominal shape parameters on the short-term surgical outcomes of LADG were analyzed. RESULTS: In male patients undergoing LADG and D2 lymph node dissection, the number of retrieved lymph nodes was significantly lower in patients with APD ≥17.3 cm (P = 0.005), TD ≥27.4 cm (P = 0.029), SCF ≥1.2 cm (P = 0.014), and BMI ≥22.2 (P = 0.008), whereas in female patients, these were statistically insignificant (P > 0.05). APD, TD, SCF, and BMI were negatively correlated with the number of retrieved lymph nodes in male patients. There was no significant difference in the number of lymph nodes retrieved between high-XND group and low-XND group in either gender. Operation time was significantly shorter in male patients with XND < 17.0 cm (P = 0.044) and in female patients with SCF < 2.15 cm (P = 0.013). Intraoperative blood loss and postoperative complication rate were not significantly different between high- and low-APD groups, high- and low-TD groups, high- and low-XND groups, and high- and low-SCF groups in either gender. Compared with male patients, SCF and TD were significantly higher in female patients. In addition, a higher incidence rate of hypertension was observed in patients of both genders with large APD and SCF, although statistically significant only in male patients. CONCLUSIONS: LADG with D2 lymph node dissection can effectively achieve the lymph node dissection requirement of radical distal gastrectomy for patients with various abdominal shapes. It is worth noting that APD, TD, and SCF can impact on lymph node dissection of LADG in male patients. Nevertheless, in female patients, abdominal shape do not impact on lymph node dissection of LADG. Moreover, LADG with D2 lymph node dissection is proved to be safe for various abdominal shape in both genders, even for abdominal obese patients.

Expression and prognostic significance of focal adhesion kinase in hepatocellular carcinoma.

AIM: To examine the expressions of focal adhesion kinase (FAK) and its clinical significance in hepatocellular carcinoma (HCC). METHODS: We determined the expression levels of FAK on both steady-state mRNA and protein levels in 50 HCC samples by quantitative real-time PCR and immunohistochemistry, respectively. The correlations between FAK expression and various clinicopathological parameters were analyzed. RESULTS: The expression of FAK on the mRNA level was consistent with that on the protein level. FAK mRNA levels in tumor tissues were significantly higher than in the paratumor tissues (0.229 +/- 0.027 vs. 0.163 +/- 0.019; P < 0.001), but lower than in the macroscopic cancer emboli (0.506 +/- 0.155 vs. 0.377 +/- 0.176; P < 0.05). Compared within the tumor tissues, FAK expressions were significantly higher in those with cancer emboli than those without (0.343 +/- 0.05 vs. 0.165 +/- 0.025; P = 0.003). Univariate and multivariate analyses revealed that FAK expression was an independent prognostic factor for disease-free survival and overall survival. Both 3-year disease-free survival rates and overall survival rates in FAK-negative group were significantly higher than in positive group (52 vs. 20% and 72 vs. 29%, P < 0.001). CONCLUSION: Our results suggest that FAK expression is up-regulated in HCC and its expression is an independent prognostic factor for HCC.

Ghrelin improves delayed gastrointestinal transit in alloxan-induced diabetic mice.

AIM: To investigate the effects of ghrelin on delayed gastrointestinal transit in alloxan-induced diabetic mice. METHODS: A diabetic mouse model was established by intraperitoneal injection with alloxan. Mice were randomized into two main groups: normal mice group and diabetic mice group treated with ghrelin at doses of 0, 20, 50, 100 and 200 mug/kg ip. Gastric emptying (GE), intestinal transit (IT), and colonic transit (CT) were studied in mice after they had a phenol red meal following injection of ghrelin. Based on the most effective ghrelin dosage, atropine was given at 1 mg/kg 15 min before the ghrelin injection for each measurement. The mice in each group were sacrificed 20 min later and their stomachs, intestines, and colons were harvested immediately. The amount of phenol red was measured. Percentages of GE, IT, and CT were calculated. RESULTS: Percentages of GE, IT, and CT were significantly decreased in diabetic mice as compared to control mice (22.9 +/- 1.4 vs 28.1 +/- 1.3, 33.5 +/- 1.2 vs 43.2 +/- 1.9, 29.5 +/- 1.9 vs 36.3 +/- 1.6, P < 0.05). In the diabetic mice, ghrelin improved both GE and IT, but not CT. The most effective dose of ghrelin was 100 mug/kg and atropine blocked the prokinetic effects of ghrelin on GE and IT. CONCLUSION: Ghrelin accelerates delayed GE and IT but has no effect on CT in diabetic mice. Ghrelin may exert its prokinetic effects via the cholinergic pathway in the enteric nervous system, and therefore has therapeutic potential for diabetic patients with delayed upper gastrointestinal transit.