Aspertaichamide a, a novel cytotoxic prenylated indole alkaloid possessing a bicyclo[2.2.2]diazaoctane framework from a marine algal-derived endophytic fungus aspergillus taichungensis 299.

Filamentous fungi belonging to the genus Aspergillus are prodigious producers of alkaloids, particularly prenylated indole alkaloids, that often exhibit structurally diversified skeletons and potent biological activities. In this study, five prenylated indole alkaloids possessing a bicyclo[2.2.2]diazaoctane core ring system, including a novel derivative, namely aspertaichamide A (1), as well as four known compounds, (+)-stephacidin A (2), sclerotiamide (3), (-)-versicolamide B (4), and (+)-versicolamide B (5), were isolated and identified from A. taichungensis 299, an endophytic fungus obtained from the marine red alga Gelidium amansii. The chemical structures of the compounds were elucidated by comprehensive NMR and HRESIMS spectroscopic analyses. In addition to the previously reported prenylated indole alkaloids, aspertaichamide A (1) was characterized as having an unusual ring structure with the fusion of a 3-pyrrolidone dimethylbenzopyran to the bicyclo[2.2.2]diazaoctane moiety, which was rare in these kinds of compounds. The absolute configuration of 1 was determined by TDDFT-ECD calculations. In vitro cytotoxic assays revealed that the novel compound 1 possessed selective cytotoxic activity against five human tumor cell lines (A549, HeLa, HepG2, HCT-116, and AGS), with IC50 values of 1.7-48.5 µM. Most importantly, compound 1 decreased the viability of AGS cells in a concentration-dependent manner with an IC50 value of 1.7 µM. Further studies indicated that 1 may induce AGS cells programmed cell death via the apoptotic pathway.

Two novel teleost calreticulins PoCrt-1/2, with bacterial binding and agglutination activity, are involved in antibacterial immunity.

Calreticulin (Crt), a conserved lectin-like pleiotropic protein, plays crucial roles in mammalian immune response. In fish, the immunological function of Crt is limited investigated. Herein, we studied the antibacterial immunity of two type of Crt homologues (i.e. PoCrt-1 and PoCrt-2) in Japanese flounder (Paralichthys olivaceus). PoCrt-1 and PoCrt-2 are composed of 419 and 427 amino acid residues respectively, with 69.09% overall sequence identities with each other. Both PoCrt-1 and PoCrt-2 contain a signal peptide and three functional domains i.e. N-, P- and C-domains. Both PoCrt-1 and PoCrt-2 were constitutively expressed at various tissues with highest expression level in liver, and obviously regulated by Edwardsiella tarda and Vibrio harveyi. Furthermore, recombinant PoCrt-1 and PoCrt-2 (rPoCrt-1 and rPoCrt-2) could bind to different Gram-negative bacteria with highest binding index with E. tarda. At same time, in vitro rPoCrt-1 and rPoCrt-2 could agglutinate E. tarda, V. harveyi, and Vibrio anguillarum, and inhibit the bacterial growth. Similarly, in vivo rPoCrt-1 and rPoCrt-2 could significantly suppress the dissemination of E. tarda. Overall, these observations add new insights into the antibacterial immunity of Crt in P. olivaceus.

Halometabolites isolated from the marine-derived fungi with potent pharmacological activities.

Halometabolites, usually produced in marine environment, are an important group of natural halogenated compounds with rich biological functionality and drugability and thus play a crucial role in pharmaceutical and/or agricultural applications. In the exploration of novel halometabolites from marine microorganisms, the growing number of halogenated compounds makes it necessary to fully present these metabolites with diverse structures and considerable bioactivities. This review particularly focuses on the chemodiversity and bioactivities of halometabolites from marine-derived fungi. As a result, a total of 145 naturally halogenated compounds, including 118 chlorinated, 23 brominated, and four iodinated compounds, were isolated from 17 genera of marine-derived fungi. Interestingly, many of halometabolites, especially for the brominated and iodinated compounds, are generated by the substitution of bromide and iodide ions for the chloride ion in cultivation process. In addition, these compounds possess diverse structural types, which are classified into polyketides (62.7%), phenols (16.6%), alkaloids (14.5%), and terpenoids (6.2%). Their cytotoxic, antibacterial, and anti-inflammatory activities indicate the high potential of these halogenated compounds as lead compounds for drug discovery.

Preventive effect of sanguinarine on intestinal injury in mice exposed to whole abdominal irradiation.

Intestinal injury is one of the major side effects that are induced by medical radiation exposure, and has limited effective therapies. In this study, we investigated the beneficial effects of sanguinarine (SAN) on intestinal injury induced by ionizing radiation (IR) both in vitro and in vivo. Mice were exposed to whole abdominal irradiation (WAI) to mimic clinical scenarios. SAN was injected intraperitoneally to mitigate IR-induced injury. Histological examination was performed to assess the tissue injuries of the spleen and small intestine. A small intestinal epithelial cell line-6 (IEC-6) was analyzed for its viability and apoptosis in vitro under different treatments. Inflammation-related pathways and serum inflammatory cytokines were detected via Western blot analysis and ELISA, respectively. High-throughput sequencing was used to characterize the gut microbiota profile. High-performance liquid chromatography was performed to assess short-chain fatty acid contents in the colon. In vitro, SAN pretreatment protected cell viability and reduced apoptosis in IEC-6 cells. In vivo, SAN pretreatment protected immune organs, alleviated intestinal injury, and promoted intestinal recovery. SAN also reduced the levels of inflammatory cytokines, suppressed high mobility group box 1 (HMGB1)/ Toll-like receptor 4 (TLR4) pathway activation, and modulated gut microbiota composition. Our findings demonstrate that the beneficial properties of SAN alleviated intestinal radiation injury. Thus, SAN represents a therapeutic option for protecting against IR-induced intestinal injury in preclinical settings.

Antimicrobial and cytotoxic phenolic bisabolane sesquiterpenoids from the fungus Aspergillus flavipes 297.

Phenolic bisabolane-type sesquiterpenoids (PBS) represent a rare class of natural products with diverse biological activities. In this study, chemical investigations of the fungus Aspergillus flavipes 297 resulted in the isolation and identification of seven PBS, including a pair of new enantiomers (+)-1a and (-)-1b, a new derivative 2, and five previously reported ones 3-7. The chemical structures of the isolated PBS were determined by extensive NMR and HRESIMS spectroscopic analysis. The absolute configurations of the separated enantiomers (+)-1a and (-)-1b were solved by comparison of the experimental ECD spectra with those of the TDDFT-ECD calculated spectra. The new compounds 1 and 2 represent rare cases of PBS bearing a methylsulfinyl group, which was distinct from the commonly-observed PBS structurally. All the isolated compounds 1-7 were evaluated their antimicrobial and cytotoxic activities. As a result, the tested compounds showed selective antimicrobial activity against several pathogenic bacteria and fungi with the MIC (minimum inhibiting concentrations) values ranging from 2 to 64 µg/mL. Moreover, enantiomers (+)-1a and (-)-1b, together with compound 2, exhibited promising cytotoxicity against MKN-45 and HepG2 cell lines, respectively, indicating that the methylsulfinyl substituent enhanced cytotoxicity to a certain degree.

Why MUC16 mutations lead to a better prognosis: A study based on The Cancer Genome Atlas gastric cancer cohort.

BACKGROUND: MUC16, encoding cancer antigen 125, is a frequently mutated gene in gastric cancer. In addition, MUC16 mutations seem to result in a better prognosis in gastric cancer. However, the mechanisms that lead to a better prognosis by MUC16 mutations have not yet been clarified. AIM: To delve deeper into the underlying mechanisms that explain why MUC16 mutations signal a better prognosis in gastric cancer. METHODS: We used multi-omics data, including mRNA, simple nucleotide variation, copy number variation and methylation data from The Cancer Genome Atlas, to explore the relationship between MUC16 mutations and prognosis. Cox regression and random survival forest algorithms were applied to search for hub genes. Gene set enrichment analysis was used to elucidate the molecular mechanisms. Single-sample gene set enrichment analysis and "EpiDISH" were used to assess immune cells infiltration, and "ESTIMATE" for analysis of the tumor microenvironment. RESULTS: Our study found that compared to the wild-type group, the mutation group had a better prognosis. Additional analysis indicated that the MUC16 mutations appear to activate the DNA repair and p53 pathways to act as an anti-tumor agent. We also identified a key gene, NPY1R (neuropeptide Y receptor Y1), which was significantly more highly expressed in the MUC16 mutations group than in the MUC16 wild-type group. The high expression of NPY1R predicted a poorer prognosis, which was also confirmed in a separate Gene Expression Omnibus cohort. Further susceptibility analysis revealed that NPY1R might be a potential drug target for gastric cancer. Furthermore, in the analysis of the tumor microenvironment, we found that immune cells in the mutation group exhibited higher anti-tumor effects. In addition, the tumor mutation burden and cancer stem cells index were also higher in the mutation group than in the wild-type group. CONCLUSION: We speculated that the MUC16 mutations might activate the p53 pathway and DNA repair pathway: alternatively, the tumor microenvironment may be involved.

Metachronous pulmonary and pancreatic metastases arising from sigmoid colon cancer: A case report.

BACKGROUND: Metachronous pulmonary and pancreatic metastases from colorectal cancer are rare. The diagnosis of pancreatic metastases is difficult and predominantly relies on computed tomography, pathology and immunohistochemistry. Here, we describe the use of next-generation sequencing (NGS) for determination of the origin of metastasis and prognostic prediction of colorectal cancer. CASE SUMMARY: A 59-year-old man was diagnosed with sigmoid adenocarcinoma stage IIA (T3N0M0) and underwent surgery in April 2014, followed by XELOX adjuvant chemotherapy. The patient developed pulmonary metastasis in the right upper lung and underwent surgery in May 2016 without further adjuvant chemotherapy. In May 2018, pancreatic metastasis was found and he underwent pancreaticoduodenectomy. After surgery, he was treated with adjuvant S-1 chemotherapy from June 2018 to March 2019. Histopathological review of the specimens from all three lesions indicated consistent patterns characteristic of colon cancer. Concordant gene mutation profiles were observed across the three lesions that included oncogenic driver mutations most frequently seen in colon cancer (e.g., APC, TP53, KRAS and FBXW7). Blood circulating tumor (ct)DNA before adjuvant chemotherapy was undetectable with NGS, suggesting a favorable response to chemotherapy. The patient was alive and well at the latest follow-up visit, achieving a disease-free survival of 17 mo. CONCLUSION: The genetic profiles of primary tumor, metastases and ctDNA may have clinical value in auxiliary diagnosis, prognosis and therapeutic decision-making.

Signature of gene aberrant alternative splicing events in pancreatic adenocarcinoma prognosis.

Alternative splicing (AS), as an effective and universal mechanism of transcriptional regulation, is involved in the development and progression of cancer. Therefore, systematic analysis of alternative splicing in pancreatic adenocarcinoma (PAAD) is warranted. The corresponding clinical information of the RNA-Seq data and PAAD cohort was downloaded from the TCGA data portal. Then, a java application, SpliceSeq, was used to evaluate the RNA splicing pattern and calculate the splicing percentage index (PSI). Differentially expressed AS events (DEAS) were identified based on PSI values between PAAD cancer samples and normal samples of adjacent tissues. Kaplan-Meier and Cox regression analyses were used to assess the association between DEAS and patient clinical characteristics. Unsupervised cluster analysis used to reveal four clusters with different survival patterns. At the same time, GEO and TCGA combined with GTEx to verify the differential expression of AS gene and splicing factor. After rigorous filtering, a total of 45,313 AS events were identified, 1,546 of which were differentially expressed AS events. Nineteen DEAS were found to be associated with OS with a five-year overall survival rate of 0.946. And the subtype clusters results indicate that there are differences in the nature of individual AS that affect clinical outcomes. Results also identified 15 splicing factors associated with the prognosis of PAAD. And the splicing factors ESRP1 and RBM5 played an important role in the PAAD-associated AS events. The PAAD-associated AS events, splicing networks, and clusters identified in this study are valuable for deciphering the underlying mechanisms of AS in PAAD and may facilitate the establishment of therapeutic goals for further validation.

LncRNA CRNDE promotes the progression and angiogenesis of pancreatic cancer via miR-451a/CDKN2D axis.

BACKGROUND: The lncRNA colorectal neoplasia differentially expressed (lncRNA CRNDE) has been reported to play a pivotal role in various cancers. However, the expression and function of CRNDE in pancreatic cancer remain unclear. The objective of this study was to investigate the effects of CRNDE on pancreatic cancer and the underlying mechanisms. METHODS: The expression of CRNDE in pancreatic cancer tissues and cell lines was determined by RT-qPCR. Proliferation and angiogenesis were detected by MTT, colony formation, transwell and tube formation assays in vitro and in vivo. ELISA assay was used to detect the secretion of VEGFA. IHC was performed to test the expression levels of Ki67 and CD31. The binding sites between CRNDE, CDKN2D and miR-451a were predicted by bioinformatics analysis. Dual luciferase reporter and RNA immunoprecipitation assays were conducted to confirm the interaction with each other. RESULTS: The results showed that CRNDE was significantly up-regulated in pancreatic cancer tissues as well as cell lines. CRNDE overexpression promoted the progression and angiogenesis of pancreatic cancer cells in vitro and in vivo. Moreover, we identified that CRNDE functioned as a sponge for miR-451a and CRNDE overexpression inhibited the expression of miR-451a. Furthermore, we confirmed that miR-451a directly interacted with CDKN2D and negatively regulated CDKN2D expression. In addition, CRNDE was found to positively regulate CDKN2D expression and mediate pancreatic cancer cell proliferation and angiogenesis through miR-451a/CDKN2D axis. CONCLUSION: CRNDE modulates cell proliferation and angiogenesis via miR-451a/CDKN2D axis in pancreatic cancer, which provides a potential therapeutic target for pancreatic cancer treatment.

At What Dose Can Total Body and Whole Abdominal Irradiation Cause Lethal Intestinal Injury Among C57BL/6J Mice?

PURPOSE AND METHODS: To investigate the doses of total body (TBI) and whole abdominal irradiation (WAI) induced lethal intestinal injury, healthy C57BL/6 J mice were divided randomly into 7 groups: control group; 6, 7, and 8 Gy TBI groups; and 5, 10, and 15 Gy WAI groups. The survival length, general conditions, body weight, daily food and water intake of the mice and the histopathological changes of small intestine were observed. RESULTS: Lethal injury among C57BL/6 J mice was caused by ≥6 Gy TBI and 15 Gy WAI. Their body weight and food intake decreased, the structure of their small intestinal villi was destroyed, and the number of surviving crypts per circumference of the jejunum decreased in ≥6 Gy TBI groups and 15 Gy WAI group. The mice in the 10 Gy WAI group significantly lost weight within 5 days but recovered slowly thereafter. They also had poor appetite and reversibly damaged intestinal mucosa. CONCLUSIONS: Nonlethal intestinal injury could be induced by 10 Gy WAI, whereas lethal intestinal injury could be triggered by ≥6 Gy TBI and >15 Gy WAI in mice. Our results provided a basis for establishing radiation-induced intestinal injury models with C57BL/6 J mice.

Novel risk scoring system for prediction of pancreatic fistula after pancreaticoduodenectomy.

BACKGROUND: The available prediction models for clinically relevant postoperative pancreatic fistula (CR-POPF) do not incorporate both preoperative and intraoperative variables. AIM: To construct a new risk scoring system for CR-POPF that includes both preoperative and intraoperative factors. METHODS: This was a retrospective study of patients who underwent pancreaticoduodenectomy (PD) or pylorus-preserving PD (PPPD) between January 2011 and December 2016 at the First Affiliated Hospital of Soochow University. Patients were divided into a study (01/2011 to 12/2014) or validation (01/2015 to 12/2016) group according to the time of admission. POPF severity was classified into three grades: Biochemical leak (grade A) and CR-POPF (grades B and C). Logistic regression was used to create a predictive scoring system. RESULTS: Preoperative serum albumin ≥ 35 g/L [P = 0.032, odds ratio (OR) = 0.92, 95% confidence interval (CI): 0.85-0.99], hard pancreatic texture (P = 0.004, OR = 0.25, 95%CI: 0.10-0.64), pancreatic duct diameter ≥ 3 mm (P = 0.029, OR = 0.50, 95%CI: 0.27-0.93), and intraoperative blood loss ≥ 500 mL (P = 0.006, OR = 1.002, 95%CI: 1.001-1.003) were independently associated with CR-POPF. We established a 10-point risk scoring system to predict CR-POPF. The area under the curve was 0.821 (95%CI: 0.736-0.905) and the cut-off value was 3.5. Including drain amylase levels improved the predictive power of the model. CONCLUSION: This study established a 10-point scoring system to predict CR-POPF after PD/PPPD using preoperative and intraoperative parameters. Ultimately, this system could be used to distinguish between high- and low-risk populations in order to facilitate timely interventions after PD.

A Panel of Three Biomarkers Identified by iTRAQ for the Early Diagnosis of Pancreatic Cancer.

PURPOSE: Due to a lack of early diagnostic markers, pancreatic cancer (PC) remains a lethal disease. Proteomic approaches are now being applied to identify novel PC biomarkers. EXPERIMENTAL DESIGN: In this study, iTRAQ and LC-MS/MS are used to perform comparative analyses of serum from PC patients and healthy controls (HC), to identify specific serum biomarkers for PC. Serum levels of candidate proteins are determined using ELISA. RESULTS: Among 869 proteins identified, 55 are potential biomarkers; Vitamin K-dependent protein Z (PROZ) and tumor necrosis factor receptor superfamily member 6b (TNFRSF6B) are selected for further analysis. Serum levels of PROZ and TNFRSF6B are significantly higher in PC patients than in HC or pancreatic benign controls (BC) (p < 0.01). The AUCs range from 0.816 to 0.971 for PROZ, TNFRSF6B, and carbohydrate antigen 19-9, either individually or in combination, in PC versus HC+BC, and from 0.711 to 0.932 in PC Stage I versus HC+BC. CONCLUSIONS AND CLINICAL RELEVANCE: It is demonstrated that PROZ and TNFRSF6B are novel serum biomarkers for detecting early stage PC, and for distinguishing PC from pancreatic benign tumor and healthy individuals. Additional large cohort studies are needed to develop PROZ and TNFRSF6B as clinical PC biomarkers.

HIF-2α promotes the formation of vasculogenic mimicry in pancreatic cancer by regulating the binding of Twist1 to the VE-cadherin promoter.

Vasculogenic mimicry (VM) is a blood supply modality that occurs independently of endothelial cell angiogenesis. Hypoxia and the epithelial-mesenchymal transition (EMT) induce VM formation by remodeling the extracellular matrix. Our previous study demonstrated that hypoxia-inducible factor-2 alpha (HIF-2α) promotes the progress of EMT in pancreatic cancer; however, whether HIF-2α promotes VM formation in pancreatic cancer remains unknown. In this study, we investigated HIF-2α expression and VM by immunohistochemistry in 70 pancreatic cancer patients as well as the role of Twist1and Twist2 in HIF-2α-induced VM in vitro and in vivo. We found that the overexpression of HIF-2α and VM were correlated with poor tumor differentiation, late clinical stage and lymph node metastasis, and a poor prognosis in pancreatic cancer. Moreover, the upregulation of HIF-2α in SW1990 cells induced VM formation, whereas the opposite results were found after silencing HIF-2α in AsPC-1 cells. A mechanistic study indicated that HIF-2α might regulate the binding of twist1 to vascular endothelial cadherin (VE-cadherin) to promote VM formation in pancreatic cancer cells, and that the P1 (-421bp) and P4 (-2110bp) regions of the Twist1 binding sequences are positive regulatory elements for VE-cadherin. In addition, we confirmed that the overexpression of HIF-2α increased Twist1 expression and promoted tumor growth and VM formation in pancreatic cancer xenografts in nude mice. These findings indicated that HIF-2α might play a critical role in VM and that HIF-2α and the pathway of HIF-2α inducing VM formation are potential therapeutic targets for pancreatic cancer.

Antineoplastic effects and mechanisms of micheliolide in acute myelogenous leukemia stem cells.

Leukemic stem cells (LSCs) greatly contribute to the initiation, relapse, and multidrug resistance of leukemia. Current therapies targeting the cell cycle and rapidly growing leukemic cells, including conventional chemotherapy, have little effect due to the self-renewal and differentiated malignant cells replenishment ability of LSCs despite their scarce supply in the bone marrow. Micheliolide (MCL) is a natural guaianolide sesquiterpene lactone (GSL) which was discovered in michelia compressa and michelia champaca plants, and has been shown to exert selective cytotoxic effects on CD34+CD38- LSCs. In this study, we demonstrate that DMAMCL significantly prolongs the lifespan of a mouse model of human acute myelogenous leukemia (AML). Mechanistic investigations further revealed that MCL exerted its cytotoxic effects via inhibition of NF-κB expression and activity, and by generating intracellular reactive oxygen species (ROS). These results provide valuable insight into the mechanisms underlying MCL-induced cytotoxicity of LSCs, and support further preclinical investigations of MCL-related therapies for the treatment of AML.

[Ex vivo Culture System of Single Human Hematopoietic Stem Cell Used to Screan the Small Molecular Compounds].

OBJECTIVE: To explore an efficient, stable system and method to verify the regulation effect of small molecule compounds on human hematopoietic stem cells (hHSC). METHODS: By using combination of flow cytometry with study results of surface markers on hHSC, and optimation of sorting process for further studying the effect of small molecular compounds on stem property of hHSC, the single hHSC was treated with published small molecular compounds such as SR1 and UM171 which possess the expansion effect. After treating with hHSC for 14 d, the flow cytometric analysis of cell phenotypes and cell morphologic observation were performed, at the same time the hematopoietic function of cultured hHSC was verified by colony-forming cell (CFC) test and cobblestone area forming cell (CAFC) test. RESULTS: The effects of SR1 and UM171 and their compositions in multi-cell culture were consistent with the published data, therefore the useful concentration of compounds were obtained. The results of multiparameter sorting of single cell (CD34+ CD38- CD45RA- CD90+ CD49f+) and ex vivo culture were consistent with the results of bulk cell culture. The results of cell phenotype analysis was in accordance with flow cytometric results. In addition, CFC test and CAFC test revealed that the colony-forming ability in treated group was significantly higher than that in control group (P<0.05). CONCLUSION: The rapid, efficient stably amplified and short-time culture system for single hHSC and method for varifying the effect of small molecular compounds are established, which provides platform for screening small molecular compounds and lays the foundation for further study of hHSC expansion.

Knockdown of angiopoietin-2 suppresses metastasis in human pancreatic carcinoma by reduced matrix metalloproteinase-2.

Angiopoietin-2 (Ang2) has been shown highly expressed in resected human pancreatic carcinoma samples, and has tightly combination with tumor angiogenesis, but the role in metastasis of it is less clear. We were, therefore, interested in exploring the effects of Ang2 silencing on the invasion and metastasis of pancreatic carcinoma. Lentivirus (LV)-mediated Ang2 small hairpin RNA (LV-RNAi) and mock lentivirus (LV-NC) were transfected into pancreatic carcinoma cell line MIA PaCa-2. Groups were designed in this study: the control group (MIA PaCa-2 cells), the LV-NC group (cells transfected with the LV-NC), the LV-RNAi-KD1 group (cells transfected with LV-RNAi of knock down sequence (1) and the LV-RNAi-KD2 group (cells transfected with LV-RNAi of knock down sequence (2). Boyden chamber transwell assay was used to detect the cell invasion change. The protein levels of Ang2, MMP-2, and MMP-9 gene and mRNA level of MMP-2, MMP-9 were detected by Western blot and real-time polymerase chain reaction, respectively. Orthotopic pancreatic carcinoma xenotransplantation model were successfully built with MIA PaCa-2 cells injection. After treatment with intraperitoneal injection of LV-RNAi-KD2 (LV-RNAi), mice growth, liver function test, tumor volume and peritoneal metastatic numbers were observed and counted. Moreover, expression of Ang2, MMP-2, MMP-9 were measured by immunohistochemistry. Ang2 expression were successfully knocked down in two LV-RNAi groups, especially in the LV-RNAi-KD2group. Compared with the control group and the LV-NC group, the mRNA and protein level of MMP-2 gene were downregulated significantly in LV-RNAi groups, also the invasion cell number decreased in boyden chamber transwell assay after LV-RNAi transfection. Meanwhile, no obvious MMP-9 gene expression changes were found among all the groups. LV-RNAi injection inhibited pancreatic carcinoma metastasis and growth in vivo by downregulating the expression of MMP-2 not MMP-9. Most importantly, LV-mediated gene therapy with Ang2 knockdown exhibited almost no toxicity in vivo. These findings demonstrate that Ang2 gene silencing exert an anti-metastasis effect in vitro and in vivo, and Ang2 targeted gene therapy has the potential to serve as a novel way for pancreatic carcinoma treatment.

B7-H3 overexpression in pancreatic cancer promotes tumor progression.

B7-H3, a member of the B7-family molecules, plays an important role in adaptive immune responses. In addition, B7-H3 is also expressed in several types of human cancers and is correlated with the poor outcome of cancer patients. However, its exact role in cancer is not known. In the present study, we compared B7-H3 expression in normal pancreas and pancreatic cancer tissue specimens, and determined the effects of low B7-H3 expression on the human pancreatic cancer cell line Patu8988 using lentivirus-mediated RNA interference. B7-H3 expression in pancreatic specimens was determined by enzyme-linked immunosorbent assay (ELISA). A Patu8988 cell line with low B7-H3 expression was established by lentivirus-mediated RNA interference to investigate the effect of B7-H3 on cell proliferation, migration and invasion in vitro. By establishing subcutaneous transplantation tumor and orthotopic transplantation pancreatic cancer mouse models, the effect of B7-H3 on cell proliferation, migration and invasion was studied in vivo. B7-H3 in tissue samples was significantly higher in the pancreatic cancer group than in the normal pancreas group (mean ± SD, 193.6±9.352 vs. 87.74±7.433 ng/g; P<0.0001). B7-H3 knockdown by RNA interference decreased cell migration and Transwell invasion up to 50% in vitro. No apparent impact was observed on cell proliferation in vitro. In the subcutaneous transplantation tumor mouse model, the tumor growth rate was reduced by the knockdown of B7-H3. In the orthotopic transplantation pancreatic cancer mouse model, the effect of inhibiting metastasis by knocking down B7-H3 was assessed in terms of the average postmortem abdominal visceral metastatic tumor weight. This demonstrated that inhibition of B7-H3 expression reduced pancreatic cancer metastasis in vivo. In conclusion, B7-H3 is aberrantly expressed in pancreatic cancer. In addition to modulating tumor immunity, B7-H3 may have a novel role in regulating pancreatic tumor progression.

Effects of lorglumide on growth and invasion of human pancreatic cancer cell line Mia PaCa-2 in vitro through the cholecystokinin-cholecystokinin-1 receptor pathway.

BACKGROUND: Cholecystokinin (CCK) has been found to be a growth stimulant through its special receptor pathway, especially for gastrointestinal malignancies. Although the CCK-1 receptor has been shown to be highly expressed in resected human pancreatic cancer samples, its role is less clear. OBJECTIVE: The aim of this in vitro study was to investigate the CCK-1 receptor expression and the function of the CCK-CCK-1 receptor pathway in the human pancreatic adenocarcinoma cell line, Mia PaCa-2. METHODS: The expression of the CCK-1 receptor in Mia PaCa-2 cells was detected by reverse-transcriptase polymerase chain reaction and flow cytometry. CCK-1 receptor agonist CCK-8S (the major transmitter form of CCK) and antagonist lorglumide were cultured respectively with Mia PaCa-2. Three groups were created for this study: CCK-8S group (Mia PaCa-2 cells treated with CCK-8S), lorglumide group (Mia PaCa-2 cells treated with lorglumide), and the control group (Mia PaCa-2 cells alone). Investigators were blinded to group designation. 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and flow cytometry were used to detect the cell growth, cell cycle, and apoptosis. Apoptosis index rate was measured by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling. Cell invasion ability was observed by invasion assay. Expression of matrix metalloproteinase-2 (MMP-2) was measured by Western blotting. RESULTS: Mia PaCa-2 cells were found to express the CCK-1 receptor. Compared with the control group (70.2% [1.5%]), CCK-8S was associated with significant mean (SD) cell proliferation (85.1% [1.7%]; P = 0.039), and the ratio in the S stage of the cell cycle increased significantly (50.5% [1.7%] vs 42.2% [1.4%]; P = 0.021). CCK-8S was also associated with increased Mia PaCa-2 cell invasion ability (123.8 [1.7] vs 102.1 [5.8]; P = 0.005 vs control). Compared with the control group, lorglumide was associated with significantly inhibited cell growth (52.1% [1.8%]; P = 0.002) and cell invasion (77.6% [1.2%]; P = 0.003). Lorglumide also induced G0/G1 cell cycle arrest and apoptosis (27.1% [3-5%] vs 3-7% [0.6%]; P = 0.003 vs control). The change of invasion ability appeared to be mediated by MMP-2 expression, which was upregulated by CCK-8S and downregulated by lorglumide. CONCLUSION: The findings of this in vitro study suggest that CCK may exert a trophic action on the Mia PaCa-2 cell line, while lorglumide inhibited the cell growth and invasion.

Effect of recombinant adenovirus vector mediated human interleukin-24 gene transfection on pancreatic carcinoma growth.

BACKGROUND: Pancreatic cancer is a highly malignant tumor affecting an ever increasing number of patients with a mean 5-year survival rate below 4%. Therefore, gene therapy for cancer has become a potential novel therapeutic modality. In this study we sought to determine the inhibitory effects of adenovirus-mediated human interleukin-24 (AdhIL-24) on pancreatic cancer. METHODS: Human interleukin-24 gene was cloned into replication-defective adenovirus specific for patu8988 tumor cells by virus recombination technology. Reverse transcription-polymerase chain reaction and Western blotting analysis were used to determine the expression of human interleukin-24 mRNA in patu8988 cells in vitro. Induction of apoptosis by overexpression of human interleukin-24 in patu8988 cells was determined by flow cytometry. In vivo efficacy of adenoviral delivery of human interleukin-24 was assessed in nude mice (n = 10 for each group) bearing patu8988 pancreatic cancer cell lines by determining inhibition of tumor growth, endothelial growth factor and CD34 expression, and intratumoral microvessel density (MVD). RESULTS: The recombinant adenovirus vector AdVGFP/IL-24 was constructed with a packaged recombinant retrovirus titer of 1.0 x 10(10) pfu/ml and successfully expressed of both mRNA and protein in patu8988 cells. The AdVGFP/IL-24 induced apoptosis of patu8988 tumor cells in vitro and significantly inhibited tumor growth in vivo (P < 0.05). The intratumoral MVD decreased significantly in the treated tumors (P < 0.05). CONCLUSION: The recombinant adenovirus AdGFP/IL-24 can effectively express biologically active human interleukin-24, which results in inhibition of pancreatic cancer growth.