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Tumor-associated macrophages (TAMs) usually adopt a tumor-promoting M2-like phenotype, which largely impedes the immune response and therapeutic efficacy of solid tumors. Repolarizing TAMs from M2 to the antitumor M1 phenotype is crucial for reshaping the tumor immunosuppressive microenvironment (TIME). Herein, we developed self-assembled nanoparticles from the polymeric prodrug of resiquimod (R848) to reprogram the TIME for robust cancer immunotherapy. The polymeric prodrug was constructed by conjugating the R848 derivative to terminal amino groups of the linear dendritic polymer composed of linear poly(ethylene glycol) and lysine dendrimer. The amphiphilic prodrug self-assembled into nanoparticles (PLRS) of around 35 nm with a spherical morphology. PLRS nanoparticles could be internalized by antigen-presenting cells (APCs) in vitro and thus efficiently repolarized macrophages from M2 to M1 and facilitated the maturation of APCs. In addition, PLRS significantly inhibited tumor growth in the 4T1 orthotopic breast cancer model with much lower systemic side effects. Mechanistic studies suggested that PLRS significantly stimulated the TIME by repolarizing TAMs into the M1 phenotype and increased the infiltration of cytotoxic T cells into the tumor. This study provides an effective polymeric prodrug-based strategy to improve the therapeutic efficacy of R848 in cancer immunotherapy.

The development of chemoresistance is a major hurdle for the treatment of colorectal cancer (CRC), which contributes remarkably to the poor clinical prognosis. Nanodrug delivery systems show great potential in overcoming chemoresistance, but limited by the lack of identification of chemoresistance targets from cancer patients. In the present study, we enrolled chemotherapy-resistant or sensitive CRC patients and used the next-generation RNA sequencing to reveal that Asporin (ASPN) is highly expressed in tumor tissues from oxaliplatin (OXA)-resistant patients and closely correlated with a poor prognosis of CRC. Downregulation of ASPN reversed OXA resistance and promoted cell apoptosis both in vitro and in vivo. To overcome ASPN-mediated OXA resistance, we constructed a nanoparticle-based co-delivery system (denoted as PPO-siASPN) for simultaneous delivery of OXA and siRNA targeting ASPN (siASPN). PPO-siASPN not only facilitated the intracellular delivery of OXA through the enhanced cellular uptake, but effectively suppressed ASPN expression for synergistic antitumor activity in vitro and in vivo. In the more clinically relevant patient-derived xenograft (PDX) mouse model, systemic administration of PPO-siASPN achieved a remarkable therapeutic effect. This study uncovered the critical role of ASPN in causing OXA resistance in CRC patients and suggests a promising nanoformulation that may be more effective than current standard-of-care medications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper, (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Pancreatic ductal adenocarcinoma (PDAC) is one of the most malignant tumors with a low survival rate. The therapeutic effect of chemotherapy and immunotherapy for PDAC is disappointing due to the presence of dense tumor stroma and immunosuppressive cells in the tumor microenvironment (TME). Herein, a tumor-penetrating nanoparticle is reported to modulate the deep microenvironment of PDAC for improved chemoimmunotherapy. The tumor pH-sensitive polymer is synthesized by conjugating N,N-dipentylethyl moieties and monomethoxylpoly(ethylene glycol) onto PAMAM dendrimer, into whose cavity a hydrophobic gemcitabine (Gem) prodrug is accommodated. They self-assemble into nanoparticles (denoted as SPN@Pro-Gem) with the size around 120 nm at neutral pH, but switch into small particles (≈8 nm) at tumor site to facilitate deep delivery of Gem into the tumor parenchyma. In addition to killing cancer cells that resided deeply in the tumor tissue, SPN@Pro-Gem could modulate the TME by reducing the abundance of tumor-associated macrophages and myeloid-derived suppressor cells as well as upregulating the expression level of PD-L1 of tumor cells. This collectively facilitates the infiltration of cytotoxic T cells into the tumors and renders checkpoint inhibitors more effective in previously unresponsive PDAC models. This study reveals a promising strategy for improving the chemoimmunotherapy of pancreatic cancer., (© 2021 Wiley-VCH GmbH.)

Activation of the phagocytosis of macrophages to tumor cells is an attractive strategy for cancer immunotherapy, but the effectiveness is limited by the fact that many tumor cells express an increased level of anti-phagocytic signals (e.g., CD47 molecules) on their surface. To promote phagocytosis of macrophages, a pro-phagocytic nanoparticle (SNPA CALR&aCD47 ) that concurrently carries CD47 antibody (aCD47) and a pro-phagocytic molecule calreticulin (CALR) is constructed to simultaneously modulate the phagocytic signals of macrophages. SNPA CALR&aCD47 can achieve targeted delivery to tumor cells by specifically binding to the cell-surface CD47 and block the CD47-SIRPα pathway to inhibit the "don't eat me" signal. Tumor cell-targeted delivery increases the exposure of recombinant CALR on the cell surface and stimulates an "eat me" signal. Simultaneous modulation of the two signals enhances the phagocytosis of 4T1 tumor cells by macrophages, which leads to significantly improved anti-tumor efficacy in vivo. The findings demonstrate that the concurrent blockade of anti-phagocytic signals and activation of pro-phagocytic signals can be effective in macrophage-mediated cancer immunotherapy., (© 2020 Wiley-VCH GmbH.)

Primary central nervous system lymphoma (PCNSL) is a rare brain tumor. Its therapeutic efficacy is much lower than that of traditional lymphoma, largely due to the presence of the blood-brain barrier (BBB), which hinders the effective drug delivery and deposition on the disease site. Angiopep-2 (ANG) can target low-density lipoprotein receptor-related protein (LRP) on the surface of brain capillary endothelial cells (BCECs) and exhibits high BBB transport capability. In this study, we designed an ANG conjugated poly(ethylene glycol)-b-poly(ε-caprolactone) (PEG-b-PCL) (APP) nanoparticle to deliver doxorubicin (DOX) for the treatment of PCNSL. Our data indicated that the targeted APP nanoparticles showed significantly increased cellular uptake by BCECs compared with the control nanoparticles. In the intracranial SU-DHL-2-LUC lymphoma xenograft mice model, APP enhanced drug deposition in tumor tissues, and DOX-loaded APP (APP@DOX) exhibited a better therapeutic effect than free DOX and nontargeted PP@DOX, which significantly prolonged the survival time of mice.

Helicobacter pylori (H. pylori) was reported to be associated with gastric carcinogenesis. Resistin-like molecule beta (RELMβ), a recently described goblet cell-specific protein, was demonstrated to aberrantly express in gastric cancer and correlated with its clinicopathological features. This study aimed to examine the association between H. pylori and RELMβ expression in gastric carcinoma and precursor lesions. H. pylori infection and RELMβ expression were immunohistochemically evaluated in gastric biopsies from 230 patients. The biopsies consisted of normal gastric mucosa (n=20), mucosa with chronic gastritis (n=41), intestinal metaplasia (n=42), dysplasia (n=31), intestinal-type adenocarcinoma (n=56), and diffuse-type adenocarcinoma (n=40). RELMβ expression was measured in gastric biopsies after H. pylori eradication therapy in a subgroup of 32 patients. Cultured gastric cancer cell line SGC-7901 was infected with H. pylori strains, and RELMβ expression was detected by reverse transcription PCR, real-time PCR and Western blotting. Higher RELMβ immunoreactivity was observed in H. pylori-positive intestinal metaplasia (P=0.003), dysplasia (P=0.032), intestinal-type (P=0.037) and diffuse-type adenocarcinomas (P=0.001) than in H. pylori-negative specimens. Expression rates of RELMβ in dysplasia (P=0.005), intestinal-type adenocarcinoma (P<0.001), and diffuse-type adenocarcinoma (P=0.001) were significantly correlated with the grade of H. pylori density. In addition, H. pylori eradication reduced the RELMβ intensity in intestinal metaplasia (P=0.001). Infection of gastric cancer SGC-7901 cells with cag pathogenicity island (PAI)-positive H. pylori TN2, but not with its PAI totally deleted mutant (TN2-ΔPAI) for 4-8 h, resulted in enhanced protein and transcript levels of RELMβ (P<0.05). In summary, our study suggested that H. pylori infection facilitated the expression of RELMβ in gastric garcinoma and precursor lesions.

Certain chemotherapeutics (e.g., oxaliplatin, OXA) can evoke effective antitumor immunity responses by inducing immunogenic cell death (ICD). Unfortunately, tumors always develop multiple immunosuppressive mechanisms, such as the upregulation of immunosuppressive factors, to counteract the effects of immunogenic chemotherapy. Indoleamine 2,3-dioxygenase-1 (IDO1), a tryptophan catabolic enzyme overexpressed in tumor-draining lymph nodes (TDLNs) and tumor tissues, plays a pivotal role in the generation of the immunosuppressive microenvironment. Reversing IDO1-mediated immunosuppression may strengthen the ICD-induced immune response. Herein, we developed a nanoenabled approach for IDO1 pathway interference, which is accomplished by delivering IDO1 siRNA to both TDLNs and tumor tissues with the help of cationic lipid-assisted nanoparticles (CLANs). We demonstrated that the contemporaneous administration of OXA and CLAN si IDO1 could achieve synergetic antitumor effects via promoting dendritic cell maturation, increasing tumor-infiltrating T lymphocytes and decreasing the number of regulatory T cells in a subcutaneous colorectal tumor model. We further proved that this therapeutic strategy is applicable for the treatment of orthotopic pancreatic tumors and offers a strong immunological memory effect, which can provide protection against tumor rechallenge.

Purpose: To report a laparoscopic approach for pediatric inguinal hernia repair using a hybrid single-incision laparoscopic (H-SIL) technique and its clinical outcomes., Materials and Methods: A retrospective study was carried out in inguinal hernia cases treated with the new H-SIL approach using intracorporeal jumping purse-string sutures. The operative time, length of postoperative hospital stay, efficiency of the operation, and complications were analyzed., Results: In total, 157 inguinal high ligations were performed in 106 children (89 boys, 17 girls). The median age was 1.5 years (range, 25 days-11.6 years). The mean operative time was 15.8±3.4 minutes for the single-side procedure and 20.3±2.5 minutes for bilateral procedures. The mean postoperative hospital stay was 0.99±0.52 (range, 0.25-3 days). No postoperative bleeding, abdominal wall emphysema, abdominal viscera injury, or scrotal edema was found, and there were no known cases of postoperative testicular atrophy or hypotrophy. Ninety-three percent of the patients became fully mobile on the first postsurgical day. The median follow-up period was 17 months (range, 9-21 months), with no recurrence, no visible scars on the abdominal wall, and no foreign body felt in the inguinal region., Conclusions: This H-SIL approach is a safe and efficient method for pediatric inguinal hernia repair. The maneuverability is the same as that in the triport laparoscopic technique, and the cosmetic results are similar to those of single-port laparoscopic surgery.

The effects of over-expression of testis-specific expressed gene 1 (TSEG-1) on the viability and apoptosis of cultured spermatogonial GC-1spg cells were investigated, and the immortal spermatogonial cell line GC-1spg (CRL-2053™) was obtained as the cell model in order to explore the function of TSEG-1. We transfected the eukaryotic vector of TSEG-1, named as pEGFP-TSEG-1 into cultured spermatogonial GC-1spg cells. Over-expression of TSEG-1 inhibited the proliferation of GC-1spg cells, and arrested cell cycle slightly at G0/G1 phase. Transfection of TSEG-1 attenuated the transcript levels of Ki-67, PCNA and cyclin D1. In addition, over-expression of TSEG-1 induced early and late apoptosis, and reduced the mitochondrial membrane potential of GC-1spg cells. Moreover, transfection of TSEG-1 significantly enhanced the ratio of Bax/Bcl-2 and transcript levels of caspase 9, and decreased the expression of Fas and caspase 8 in GC-1spg cells. These results indicated over-expression of TSEG-1 suppresses the proliferation and induces the apoptosis of GC-1spg cells, which establishes a basis for further study on the function of TSEG-1.

There is controversy regarding the roles of Ureaplasma urealyticum (U. urealyticum) colonization in the development of bronchopulmonary dysplasia (BPD). This study explored the association between U. urealyticum and bronchopulmonary dysplasia at 36 weeks post-menstrual age (BPD36). Studies published before December 31, 2013 were searched from Medline, Embase, Ovid, Web of Science, and Cochrane databases, with the terms "Ureaplasma urealyticum", "chronic lung disease", or "BPD36" used, and English language as a limit. The association between U. urealyticum colonization and BPD36 was analyzed with RevMan 4.2.10 software, using the odds ratio (OR) and relative risk (RR) for dichotomous variables. Out of the enrolled 81 studies, 11 investigated the BPD36 in total 1193 infants. Pooled studies showed no association between U. urealyticum colonization and subsequent development of BPD36, with the OR and RR being 1.03 (95% CI=0.78-1.37; P=0.84) and 1.01 (95% CI= 0.88-1.16, P=0.84), respectively. These findings indicated no association between U. urealyticum colonization and the development of BPD36.

Background: Neuroblastoma (NB) is the most common extracranial solid tumor in childhood and displays remarkable heterogeneity in clinical behaviors, ranging from spontaneous regression to rapid progression or resistance to multimodal treatment. Recent evidence has shown that microRNAs (miRNAs), a class of small non-coding RNAs, are involved in tumor development and progression. This article aimed to review recent advances in investigating the roles of miRNAs in NB., Methods: We searched the PubMed/MEDLINE database for articles about the expression profile, functions and target genes of miRNAs in NB., Results: We reviewed the most recent evidence regarding the functional roles of oncogenic and tumor suppressive miRNAs in NB and application of novel miRNA-based methods for diagnostic, prognostic and therapeutic purposes., Conclusions: Deregulation of miRNAs is associated with the development and progression of NB, suggesting that miRNAs may serve as novel targets for the treatment of high-risk NB patients. However, their precise functions and underlying mechanisms still warrant further studies.

Aim: To investigate the effects of resistin-like molecule β (RELMβ) over-expression on the invasion, metastasis and angiogenesis of gastric cancer cells., Methods: Human RELMβ encoding expression vector was constructed and transfected into the RELMβ lowly-expressed gastric cancer cell lines SGC-7901 and MKN-45. Gene expression was measured by Western blotting, reverse transcription polymerase chain reaction (PCR) and real-time quantitative PCR. Cell proliferation was measured by 2-(4,5-dimethyltriazol-2-yl)-2,5-diphenyl tetrazolium bromide colorimetry, colony formation and 5-ethynyl-20-deoxyuridine incorporation assays. The in vitro migration, invasion and metastasis of cancer cells were measured by cell adhesion assay, scratch assay and matrigel invasion assay. The angiogenic capabilities of cancer cells were measured by tube formation of endothelial cells., Results: Transfection of RELMβ vector into SGC-7901 and MKN-45 cells resulted in over-expression of RELMβ, which did not influence the cellular proliferation. However, over-expression of RELMβ suppressed the in vitro adhesion, invasion and metastasis of cancer cells, accompanied by decreased expression of matrix metalloproteinase-2 (MMP-2) and MMP-9. Moreover, transfection of RELMβ attenuated the expression of vascular endothelial growth factor and in vitro angiogenic capabilities of cancer cells., Conclusion: Over-expression of RELMβ abolishes the invasion, metastasis and angiogenesis of gastric cancer cells in vitro, suggesting its potentials as a novel therapeutic target for gastric cancer.

Purpose: The aim of this study was to evaluate the clinical outcomes and postoperative anal function in infants with congenital high imperforate anus treated with laparoscopically assisted anorectal pull-through (LAARP)., Methods: From January 2004 to July 2007, 33 patients (28 boys and 5 girls, age ranging from 3 to 10 months) with high imperforate anus underwent LAARP. Clinical data of the LAARP group were retrospectively compared with those treated by posterior sagittal anorectoplasty (PSARP; n = 28) during the same time period. Anorectal function of these patients was evaluated using the following 3 methods: the Kelly score, anorectal vector volume manometry, and magnetic resonance imaging between the ages of 3.1 and 4.4 years., Results: The mean operative time in LAARP and PSARP groups was 112.5 ± 12.4 and 120.4 ± 18.5 minutes (P > .05), respectively. The mean length of hospital stay in the LAARP group was shorter than that of PSARP group (11.3 ± 2.1 vs 14.6 ± 2.3 days, P < .01). No significant difference was observed between LAARP and PSARP groups regarding the Kelly score (3.52 ± 1.42 vs 3.49 ± 0.82). Although magnetic resonance imaging revealed lower malposition rates of rectum in the LAARP group than those of the PSARP group at both I-line (3.0% vs 14.3%) and M-line (3.0% vs 10.7%) levels, this was not statistically different (P > .05). Compared with the PSARP group, lower asymmetric index, larger vector volume, and higher anal canal pressure at rest and during voluntary squeeze were observed in LAARP group (P < .05). However, there were no significant differences in the length of high-pressure zone (15.2 ± 5.8 vs 15.1 ± 6.2 mm) and the presence of rectoanal relaxation reflex (84.8% vs 85.7%)., Conclusions: Satisfactory fecal continence can be achieved in patients with high-type imperforate anus after LAARP. Laparoscopically assisted anorectal pull-through has advantages over PSARP, including shorter hospital stay and better position of rectum. However, long-term follow-up is necessary to compare the benefits of LAARP against PSARP., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Background: This study aimed to present the authors' technique and the intermediate-term outcome for laparoscopic choledochal cyst excision with Roux-en-Y hepatoenterostomy., Methods: This retrospective study investigated 62 children (39 girls and 23 boys) who had undergone laparoscopic resection of choledochal cyst. The average age of the children was 2.3 years. The retrospective data and the following investigations about type of choledochal cyst, surgical technique, conversion rate, morbidity, and mortality were analyzed., Results: Of the 62 patients, 43 (69.4%) showed type 1a choledochal cysts, 16 (25.8%) showed type 1c, 2 (3.2%) showed type 4a, and 1 (1.6%) showed type 4b. Total cyst excision could be performed for 51 of the patients (82.3%). The large cysts were opened on the front wall, then divided circumferentially in 29 cases. The small cysts did not need to be opened before excision in 22 cases. For 11 patients (17.7%), Lilly's (Surg Gynecol Obstet 146:254-256, 1978) technique was adopted, and for 5 patients with a huge cyst, the duodenum together with the head of the pancreas had to be mobilized for visualization of the cyst's lower limit. The hepatic duct was excised, and plastic operation of bile duct was performed for two patients. The mean operative time was 226±41.2 min. Eight patients needed blood transfusion, and conversion was required for one patient. The mean hospital stay was 8±1.5 days, and the mean follow-up period was 38 months. The overall morbidity rate was 8.2% (5/61) including bile leakage (n=1), adhesive small bowel obstruction (n=1), intestinal necrosis (n=1), and cholangitis (n=1). Inflammatory edema anastomotic narrowing occurred in one patient. None of the patients needed surgery due to anastomotic stricture., Conclusions: Laparoscopic choledochal cyst excision, hepaticojejunostomy, and extracorporeal Roux-en-Y anastomosis can be safely and quickly performed for children, with satisfactory intermediate-term results. Extracorporeal Roux-en-Y anastomosis could shorten the operative time.

Background: Surgical correction of the congenital muscular torticollis (CMT) is recommended for patients with unsuccessful conservative treatment. Open operative techniques all leave noticeable scars. We proposed a modified endoscopic 1-trocar transaxillary and neck microincision approach for the treatment of CMT., Methods: Endoscopic release of the sternocleidomastoid (SCM) muscle was performed in 45 infants and children aged 6 months to 15 years. One 5-mm incision was made in the anterior axillary fold, and a subcutaneous tunnel over the clavicular and sternal heads of the SCM muscle was made. A subcutaneous space was established by CO₂ inflation at a pressure of 8 mm Hg and then endoscopically using a 5-mm endoscope. Two additional 1.5- to 2-mm supraclavicular mini-incisions were made beside the SCM muscle for the introduction of miniforceps and electrocautery, respectively. The sternal and clavicular attachments were dissected and divided by electrocautery. Clinical evaluation was performed using the Lee scoring system., Results: The operation was successfully completed endoscopically in all 45 children. The mean operative time was 40 minutes. No injuries of major blood vessels or nerves were encountered. A small bleed was noted in 1 child owing to reoperation. Follow-up for 6 months to 3 years in 42 patients showed complete muscular release and satisfactory cosmetic appearance with no recurrence. The results were classified as excellent in 88.1% (37/42), good in 9.5% (4/42), fair in 2.4% (1/42), and poor in 0 using the Lee scoring system. The neck scars were not visible 1 month after the procedure., Conclusions: The subcutaneous endoscopic transaxillary and micro-neck incision approach for the treatment of CMT is a safe, practical procedure that provides good functional and cosmetic outcomes without vascular or neural injury., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Background: Open colectomy has been preferred for intestinal neuronal dysplasia type B (IND) due to its low morbidity rate and good functional results. The aim of this study was to investigate the feasibility and results of laparoscopic colectomy with transanal Soave pull-through for the treatment of IND in children., Methods: Seventeen infants and children suffering from IND were treated by laparoscopic extensive colectomy with transanal Soave pull-through. The diagnosis of IND was made via anorectal manometry, X-ray contrast enema, suction biopsies, and laparoscopic full-thickness biopsies with hematoxylin-eosin staining. The technique used four or five abdominal ports. The sigmoid, transverse, and right colon up to the last ileal cove were mobilized laparoscopically in the extended form of IND. A modified Soave's anastomosis was performed. The patients' data, surgical procedures, operative data, postoperative complications and clinical outcomes were analyzed., Results: Five patients underwent laparoscopic left colectomy with modified transanal Soave procedures, and the other 12 were treated by laparoscopic subtotal colectomy and required a Deloyers' maneuver for the Soave pull-through. The proximal margin of barium stagnation in patients with left colectomy was restricted to the distal end of the descending colon, sigmoid colon, and that in patients with subtotal colectomy was restricted to the proximal end of the descending colon, transverse colon, hepatic flexure, and ascending colon. Postoperative complications included anastomotic leakage, severe perianal erosions, postoperative enterocolitis, and soiling. During a mean follow-up of 4 years, bowel frequency was 4-10 times per day in 3 months postoperatively in patients with subtotal colectomy. The clinical results were good, with no stool incontinence or constipation., Conclusions: Laparoscopic procedure for left colectomy and subtotal colectomy with transanal Soave pull-through in infants and children with IND is safe, feasible, and effective. The location of barium stagnation in proximal margin may be used as a method to predict initially the proximal margin of the resected bowel segment.

Background: Biliary atresia (BA) is the progressive inflammatory obstruction and fibro-obliteration of all or part of the extrahepatic biliary tree and the intrahepatic bile ducts and has its onset exclusively within the first several months of life. This study was undertaken to present the value of diagnostic laparoscopy in infants with prolonged jaundice and technique for laparoscopic cholangiography., Methods: A 5-mm umbilical trocar was introduced to create a port for a 30-degree laparoscope. If the gallbladder was of good size, the fundus was exteriorized through the right subcostal trocar site and a catheter was inserted into the gallbladder for cholangiography, following partial dissection from the liver bed, if required. If the gallbladder was atretic, the fundus was not exteriorized and a laparotomy was performed and cholangiography was abandoned, because the lumen of an atretic gallbladder was usually not fully patent., Results: At laparoscopy, 12 patients had good-sized gallbladders and minimal-to-mild liver fibrosis. They underwent cholangiography via the exteriorized fundus, and infantile hepatitis syndrome (HIS) or cholestatic syndrome (CS) in 8 cases, BA in 2 cases, and biliary hypoplasia (CBDH) in 2 cases were identified. Five patients' gallbladders dissected from the liver bed underwent cholangiography, and BA in 3 cases and CBDH in 2 cases were identified. The remaining 21 had atretic gallbladders and varying degrees of liver fibrosis, so cholangiography via the exteriorized fundus was abandoned and converted to open Kasai portoenterostomy., Conclusions: Laparoscopy-assisted cholangiography is a simple, accurate, and safe method in the diagnosis of prolonged jaundice in infants and allows the anatomic structure of the biliary tree to be obtained accurately with minimal surgical intervention.

Objective: The study aimed to build a 3-dimensional (3D) reconstruction of pelvic magnetic resonance images and evaluate the clinical value in anorectal malformations (ARMs)., Methods: Magnetic resonance imaging (MRI) examinations were performed on a 1.5-T magnet. Sagittal, coronal, and transverse turbo spin-echo T1-weighted and fast spin-echo T2-weighted images of the pelvic region were obtained in 22 children. A 3D reconstruction was made on a computer and assisted by the 3D-Doctor software (Trial Version, Able Software Corp). The level and type of ARM and the developmental state of the striated muscle complex (SMC) were analyzed with 3D reconstruction image., Results: The 3D images of the pelvic were confirmed in 22 cases. Three-dimensional reconstructed images perfectly displayed the anatomical relationships of the SMC and the rectal atresia in these spaces. The 3D configuration of the SMC was different in each of the high- and low-type cases. The high-type malformation of SMCs differed particularly from the descriptions., Conclusions: Pelvic magnetic resonance 3D reconstructed images were able to show the dimensional anatomical relations of pelvis, bladder, urethra, rectum, and SMC. Both a 3D image and positional information with MRI offers the surgeon a simulated operative profile of the SMC superior to MRI slices alone, which will help in providing morphological data for image diagnosis and operation of the ARM.

Aim: To develop short hairpin RNA (shRNA) against heparanase, and to determine its effects on heparanase expression and the malignant characteristics of gastric cancer cells., Methods: Heparanase-specific shRNA was constructed and transferred into cultured the gastric cancer cell line SGC-7901. Stable subclonal cells were screened by G418 selection. Heparanase expression was measured by reverse transcriptase-polymerase chain reaction (RT-PCR), real-time quantitative PCR and Western blotting. Cell proliferation was detected by 2-(4, 5-dimethyltriazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetry and colony formation assay. The in vitro invasiveness and metastasis of cancer cells were measured by cell adhesion assay, wound healing assay and matrigel invasion assay. The angiogenesis capabilities of cancer cells were measured by tube formation of endothelial cells., Results: Stable transfection of heparanase-specific shRNA, but not of scrambled shRNA and mock vector, resulted in reduced mRNA and protein levels of heparanase. The shRNA-mediated knockdown of heparanase did not affect the cellular proliferation of SGC-7901 cells. However, the in vitro invasiveness and metastasis of cancer cells were decreased after knockdown of heparanase. Moreover, transfection of heparanase-specific shRNA decreased the in vitro angiogenesis capabilities of SGC-7901 cells., Conclusion: Stable knockdown of heparanase can efficiently decrease the invasiveness, metastasis and angiogenesis of human gastric cancer cells. In contrast, stable knockdown of heparanase does not affect the cell proliferation.

Background: The Nuss procedure for repair of pectus excavatum (PE) has been accepted worldwide because of minimal invasiveness and excellent cosmetic results. We summarized our experience with the treatment of 115 patients aged 2.7-18 years., Methods: All the 115 patients underwent the Nuss procedure successfully from July 2003 to February 2008. They were divided into two groups: children group (below 12 years) and adolescents group (aged 12-18 years)., Results: The rate of complications was 14.7% and 37.5% in the children and adolescents groups, respectively (P<0.05). There was significant difference in operation time, length of hospital stay, and analgesic time between the two groups (P<0.05). The initial results of Nuss procedure were excellent., Conclusions: The Nuss procedure can be performed with excellent early results in children. We suggest that children with PE should accept the Nuss procedure as early as possible when they are over 5 years old.

Background: Previous studies indicate that heparanase (HPA), an endoglycosidase involved in tumor angiogenesis and metastasis, is up-regulated in a variety of malignancies. However, the expression of HPA in neuroblastoma (NB), one of the most common extra cranial solid tumors in children, remains unknown. This study was undertaken to explore the expression and clinical significance of HPA in NB., Methods: Immunohistochemical staining was applied to detect the expression of HPA in 42 cases of NB. The relationships among HPA expression, international neuroblastoma staging system (INSS) stages, histopathological classification, and postoperative survival of the NB patients were analyzed., Results: The expression rate of HPA in NB was 61.9% (26/42), mainly in the cytoplasm of neuroblastoma cells. The expression rates of stage 1-2, stage 3-4 and stage 4S were 35.7%, 80.0% and 62.5%, respectively. The differences between stage 1-2 and stage 3-4 were significant (P<0.01). The expression of HPA was significantly higher in the NB cases that had one of the histopathological factors: age more than 1 year (P<0.01), poorer differentiation (P<0.01), and higher mitosis karyorrhexis index (P<0.01). The survival time of HPA-negative patients was significantly longer than that of HPA-positive patients (P<0.05)., Conclusion: Although these results indicate that heparanase might be correlated with development and progression of NB, a larger series of patients with a longer follow-up are probably needed to strengthen its role in assessment of NB prognosis.

Background: Heterotopic pancreas is characterized by pancreatic tissue outside the pancreatic bed. However, duodenal heterotopic pancreas in children is rarely reported so far. We describe herein duodenal heterotopic pancreas in a child who suffered from chronic abdominal pain., Methods: An 8-year-old boy presented with upper abdominal pain and intermittent vomiting, without a history of melena, hematochezia, hematemesis, clay-colored stools, jaundice, hepatitis or dyscrasia. No specific medication or change in position relieved the pain. Based on the elevated serum amylase levels, and the findings of CT, barium meal X-ray examination, magnetic resonance imaging, and upper gastrointestinal endoscopy, a duodenal mass was diagnosed initiatively. Intraoperative frozen section analysis was performed for the diagnosis. The mass was dissected., Results: Intraoperative frozen section analysis and routine pathological examination confirmed the diagnosis of duodenal heterotopic pancreas. The patient had an uneventful recovery and remained asymptomatic postoperatively during a follow-up period of 16 months., Conclusions: Heterotopic pancreas should be considered in children with a duodenal mass and abdominal pain. Intraoperative frozen section analysis is helpful in the diagnosis of the disease. Surgical treatment of the lesion should be performed to prevent bleeding, ulceration, outlet obstruction or malignant degeneration.

Gastric carcinoma with osteoclast-like giant cells (OGCs) is an extremely rare tumor. So far, only six cases have been reported in the literature. Here we report an additional case of this tumor in a Chinese 78-year-old man presented with abdominal pain, vomiting, and hematemesis. Physical examination and gastroscopy revealed a tumor in the gastric antrum. The biopsy and pathological findings indicated a gastric adenocarcinoma with OGCs, which were present in both the tumor and the metastatic lymph nodes. Further immunohistochemical staining indicated that OGCs were reactive with CD68, CD45, and vimentin protein, but not with pancytokeratin, carcinoembryonic antigen, or epithelial membrane antigen, suggesting the monocytic/histiocytic derivation of these OGCs. In situ hybridization for Epstein-Barr virus showed no nuclear positivity in either adenocarcinoma or OGCs. Postoperative follow-up showed that the patient had survived for at least 6 months without recurrence. Further investigation is warranted to clearly define the prognostic significance of OGCs in gastric carcinoma.

Objective: To clone the mouse testis specific gene TSEG-2 via a bioinformatic approach., Methods: The expressed sequence tags (EST) in the normal mouse testis were obtained from the online EST database ZooDDD. Their highly homologous EST sequences were retrieved through the dbEST database to construct contigs and spliced with the biomedical software Biolign. The corresponding exons and introns within the genome sequences were predicted with the software GeneScan. Primers were designed according to the open reading frame. RT-PCR was applied in cloning the cDNA of the novel gene from the mouse testis tissue and analyzing its expression patterns in the undescended testis and various organ tissues as well as in different developmental stages of the mouse testis. The sequencing results of TSEG-2 underwent bioinformatic analyses., Results: The novel mouse testis gene TSEG-2 was successfully cloned, with full-length sequence of 451 bp. The open reading frame was 267 bp, coding a protein of 88 amino acid residues, and demonstrated to be correct by RT-PCR. The expression of TSEG-2 was high in the mouse testis, regular in the testis cDNA samples of different postnatal days, and down-regulated in the cryptorchidism model. No obvious homology with other mouse cDNA was found for TSEG-2. The GenBank accession number EU079025 was achieved. Function prediction showed that mouse TSEG-2 was probably a soluble non-secretary protein located at chromosome 15qE3, or a nucleoprotein with 2 phosphorylation sites of protein kinase C (PKC) and 1 of casein kinase II (CK2)., Conclusion: A novel mouse testis specific gene TSEG-2 was successfully cloned, which could be down-regulated by cryptorchidism-inducible 17-beta estradiol. This has prepared the ground for further researches on the biological function and expression regulation of TSEG-2.

Previous studies have indicated that resistin-like molecule beta (RELM beta), an intestinal goblet cell-specific protein, is markedly increased in the intestinal tumors of min mice and over-expressed in a human colon cancer cell line. We hypothesized that RELM beta might be enhanced in human colon cancer. The aim of this study was to examine the clinical importance of RELM beta expression in colon cancer patients and to correlate its expression with various clinicopathological parameters, upstream regulatory molecule expression, tumor proliferative capacity, and patients' survival. Of the 80 colon cancer patients studied, 65 (81.25%) tested positive for RELM beta, mainly in the cytoplasm of colon mucosa. Contrasting sharply with the strongly RELM beta-positive tumors, normal colon mucous membrane was negative or weakly positive. RELM beta positivity in colon cancer was correlated with histological grade of differentiation and lymph node metastasis, but not with age, gender, tumor location and size, tumor infiltration, Dukes' stage, liver metastasis, and venous invasion. RELM beta expression was significantly correlated with the expression of transcription factor CDX-2 (P < 0.01) but not with that of proliferative index Ki-67 (P > 0.05). The mean postoperative survival time (2.76 years) of RELM beta-positive patients was significantly longer than that (1.26 years) of RELM beta-negative patients (P = 0.032). These findings support evidence of the enhanced RELM beta expression in colon cancer patients and suggest that further investigation is warranted to explore the role of RELM beta in colon cancer.

Aim: Recent evidence has indicated that members of natural jasmonates, a family of plant stress hormones, exhibit anticancer activity. The current study was undertaken to investigate the effects of jasmonates on the in vitro growth of human neuroblastomas, one of the most common solid tumors in children., Methods: Cellular proliferation was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide colorimetry and colony formation assay. Apoptosis was detected by Hoechst 33258 staining and flow cytometry. Western blotting was applied to assay gene expression., Results: The administration of natural jasmonates, methyl jasmonate, cis-jasmone, and jasmonic acid to cultured neuroblastoma cell line SH-SY5Y, resulted in a decrease of cell proliferation in a doseand time-dependent manner. However, the in vitro growth of cultured human embryonic kidney (HEK) cell line HEK 293 was not affected by jasmonates. The cell cycles of jasmonate-treated SH-SY5Y cells were arrested at the G2/M phase. The incubation of SH-SY5Y cells with jasmonates resulted in characteristic changes of apoptosis. The anticancer activities of natural jasmonates on SH-SY5Y cells are as follows: methyl jasmonate>cis-jasmone>jasmonic acid. In addition, the expressions of proliferating cell nuclear antigen and N-myc were downregulated by methyl jasmonate. Moreover, methyl jasmonate decreased the expression of the Xlinked inhibitor of apoptosis protein and survivin, critical members of inhibitors of the apoptosis protein family, in SH-SY5Y cells., Conclusion: Jasmonates suppress the growth of human neuroblastoma cell line SH-SY5Y via inhibiting cell proliferation and inducing apoptosis, which lays the groundwork for further investigation into the anticancer activities and its mechanisms of natural jasmonates on human neuroblastomas.

Recent evidence indicates that methyl jasmonate, a plant stress hormone, exhibits anticancer activity on human cancer cells. Whether methyl jasmonate could inhibit the growth of human neuroblastoma cells still, however, remains largely unknown. In this study, administration of methyl jasmonate to cultured neuroblastoma cell lines, SK-N-SH and BE(2)-C, resulted in a decrease of cell viability in a dose-dependent and time-dependent manner as demonstrated by MTT colorimetry and colony formation assay. The results from RT-PCR indicated that the expression of proliferating cell nuclear antigen, but not of cyclin D1, was downregulated by methyl jasmonate. Accordingly, the cell cycle of methyl jasmonate-treated neuroblastoma cells was arrested at the G0/G1 phase. Moreover, incubation of SK-N-SH and BE(2)-C cells with methyl jasmonate resulted in characteristic changes of apoptosis, as demonstrated by acridine orange-ethidium bromide (AO/EB) staining, Hoechst 33258 staining and flow cytometry. Moreover, methyl jasmonate decreased the expression of the X-linked inhibitor of apoptosis protein and survivin, critical members of the inhibitors of apoptosis protein family, in neuroblastoma cells. These findings indicate that methyl jasmonate suppresses the growth of cultured human neuroblastoma cells associated with downregulation of proliferating cell nuclear antigen, and induces apoptosis accompanied by downregulation of the X-linked inhibitor of apoptosis protein and survivin, which lays the groundwork for further investigation into the mechanisms of methyl jasmonate-mediated anticancer activities.

This study is to explore the inhibitory effect of methyl jasmonate on cell proliferation and expression of XIAP and survivin of human neuroblastoma cell line BE(2)-C. After cultivation of 1 - 2 mmol x L(-1) jasmonates with BE (2) -C cells for 6 - 24 h, the growth inhibiting rates of BE (2) -C cells were studied by MTT colorimetry. Cell proliferation was detected by colony formation assay. Cell cycle phases were assayed by propidium iodide staining flow cytometery. Cell apoptosis was inspected by acridine orange-ethidium bromide fluorescent staining, Hoechst 33258 fluorescent staining, and Annexin V-FITC and propidium iodide staining flow cytometry. Expressions of cyclin D1, XIAP and survivin were determined by RT-PCR and real-time RT-PCR. Methyl jasmonate inhibited the growth of BE(2)-C cells in a dose- and time-dependent manner. After addition of 1, 1.5 and 2 mmol x L(-1) of methyl jasmonate for 24 h, the inhibiting rates of cell growth reached 20.6% - 85.5% (P < 0.01), and the IC50 was 1.35 mmol x L(-1). The cell cycles were arrested at S phase. A part of cells presented the characteristic morphological changes of apoptosis. The early apoptotic rates were 13.51%, 17.32%, 24.59% (P < 0.01) and the cell death rates were 29.36% , 54.73% , 75.52% (P < 0.01), respectively. The expression of XIAP and survivin mRNA were downregulated by 18.5% - 68.9% , 22.4% - 48.7% (P < 0.05), respectively, without change in that of cyclin D1. The results indicated that methyl jasmonate could significantly inhibit the growth of BE(2) -C cells through inducing cell cycle arrest and apoptosis, downregulating the expression of XIAP and survivin might be one of its molecular mechanisms of action.

The expressed sequence tags (ESTs) of normal mouse testis were obtained from online EST database ZooDDD. Their highly homologous EST sequences were found through the dbEST database to construct contigs, and spliced by the biomedical software Biolign. The corresponding exons and introns within genome sequences were predicted by software GeneScan. According to the open reading frame, the primers were designed. RT-PCR was applied in the cloning of novel gene from mouse testis and analyzing its expression pattern in various mouse tissues. The bioinformatics analysis on the sequencing results of TSEG-1 was conducted. Results indicated that a novel gene TSEG-1 was cloned from 1 668-2 011 kb of mouse X chromosome, with full-length sequence of 510 bp. The open reading frame (ORF) is 336 bp in length and en-codes a deduced amino acid sequence of 111 residues. The molecular weight of TSEG-1 protein is 12.84258 kDa, and its pI is 11.4000. RT-PCR demonstrated the correctness of its ORF. TSEG-1 was distinctively expressed in testis, but not in other tissues of mouse. No obvious homology with other mouse cDNA was found for TSEG-1. The GenBank accession number EU079024 was achieved. It was predicted that TSEG-1 is a kind of transmembrane protein, and the transmembrane domain is from 41 amino acid residue to 61 amino acid residue. Blastn analysis revealed its high homology to human testis-specific gene H2AX. Computational prediction of the 5'-untranslated region of TSEG-1 gene revealed a 680 bp-length promoter region. There are four antigen binding sites and two phosphorylation sites of specific protein kinase in TSEG-1 protein, with subcellular localization in mitochondria. The cloning of mouse testis specific gene TSEG-1 laid a foundation for subsequent research of its biological function and expression regulation.

Expressed sequence tags (EST) are short, randomly selected single-pass nucleotide sequence reads derived from cDNA libraries and represent a small part of a gene. Along with the development of bioinformatics and genetic localization, EST has already become a powerful tool for mapping, cloning and expression profiling of genes. Recently, because of the fast distension of EST databases, application of EST in gene mapping and cloning leads to revolutionary change in the strategies for cloning of novel genes. Despite of some insufficiencies, it has been proved that EST could promote the discovery and research of novel genes. In this article, an introduction about EST, especially EST-based strategies for cloning of novel genes will be given in details.

Background: Recent evidences indicate that CD133, a kind of transmembrane protein, can be used as a marker to isolate stem cells from tumors originating from neural crest. This study was undertaken to explore the expression and clinical significance of stem cell marker CD133 in neuroblastoma (NB)., Methods: Immunohistochemical staining was used to detect the expression of CD133 in 32 patients with NB and 8 patients with ganglioneuroblastoma (GNB). The relationships were analyzed among CD133 expression, international neuroblastoma staging system (INSS) stages, pathological classification, and postoperative survival time of NB patients., Results: The expression rates of CD133 in NB and GNB were 46.9% (15/32) and 37.5% (3/8) respectively, mainly in cytoplasm of neuroblastoma cells. The expression rates of stage 1-2, stage 3-4 and stage 4S were 30.7%, 57.9% and 37.5%, respectively. The differences in various stages were significant (P<0.05). The positive rate of CD133 in patients with unfavorable histology (52.4%) was significantly higher than that in patients with favorable histology (36.8%) (P=0.007). The survival time of CD133 negative patients was significantly longer than that of CD133 positive patients (P=0.026)., Conclusions: CD133 which might be correlated with the development and progression of NB can serve as one of the important indicators for prognosis of NB.

Aim: To characterize the molecular mechanisms of nitrofen-induced pulmonary hypoplasia., Methods: After administration of nitrofen to cultured type II A549 pneumocytes, cell proliferation and DNA synthesis were investigated by 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide colorimetry, colony formation assay, flow cytometry and [3H]-thymidine incorporation assay. Apoptosis was measured by terminal transferase-mediated dUTP nick-end-labeling, acridine orange-ethidium bromide staining and flow cytometry. Expression of proliferating cell nuclear antigen (PCNA) and apoptosis-related genes was assayed by immunofluorescence, RT-PCR and Western blot., Results: Nitrofen inhibited the cell proliferation of A549 cells in a dose- and time-dependent manner, accompanied by downregulation of PCNA. As a result, the DNA synthesis of nitrofentreated A549 cells decreased, while cell cycle was arrested at G0/G1 phase. Moreover, nitrofen induced apoptosis of A549 cells, which was not abolished by Z-Val-Ala- Asp(OCH3)- fluoromethylketone. In addition, nitrofen decreased the expression of Bcl-x( L), but not of Bcl-2, Bax, and Bak, resulting in a loss of mitochondrial membrane potential and the nuclear translocation of apoptosis-inducing factor (AIF). Meanwhile, nitrofen strongly activated the p38 mitogen-activated protein kinase (p38-MAPK). Pretreatment of cells with SB203580 (5 micromol/L) blocked nitrofen-induced phosphorylation of p38-MAPK and abolished nitrofen-induced AIF translocation and apoptosis in A549 cells., Conclusion: Nitrofen suppresses the proliferation of cultured type II pneumocytes accompanied by the downregulation of PCNA, and induces mitochondria-mediated apoptosis involving the activation of p38-MAPK.

Objective: To explore the growth inhibition effects and apoptosis inducing mechanisms of curcumin on human ovarian cancer cell line A2780., Methods: After treatment with 10 - 50 micromol/L curcumin for 6 - 24 h, the growth activity of A2780 cancer cells were studied by [4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) colorimetry. Cellular apoptosis was inspected by flow cytometery and acridine orange-ethidium bromide fluorescent staining methods. The fragmentation of cellular chromosome DNA was detected by DNA ladder, the ultrastructural change was observed under a transmission electron microscope, and the protein levels of nuclear factor-kappa B (NF-kappaB, P65) and cysteinyl aspartate specific protease-3 (Caspase-3) in ovarian cancer cells were measured by immunohistochemistry., Results: After treatment with various concentrations of curcumin, the growth inhibition rates of cancer cells reached 62.05% - 89.24%, with sub-G(1) peaks appearing on histogram. Part of the cancer cells showed characteristic morphological changes of apoptosis under fluorescence and electron microscopes, and the rate of apoptosis was 21.5% - 33.5%. The protein expression of NF-kappaB was decreased, while that of Caspase-3 was increased in a time-dependent manner., Conclusion: Curcumin could significantly inhibit the growth of human ovarian cancer cells; inducing apoptosis through up-regulating Caspase-3 and down-regulating gene expression of NF-kappaB is probably one of its molecular mechanisms.

Our aim was to prepare curcumin derivatives and study their apoptosis-inducing effects on bladder cancer cells in order to establish a basis for targeted chemotherapy of cancer. n-Maleoyl-L-valine-curcumin (NVC) and n-maleoyl-glycine-curcumin (NGC) were chemically synthesized. Intracellular esterase activity of the human bladder cancer EJ cell line and renal tubular epithelial (HKC) cells was examined by 6-carboxyfluorescein diacetate fluorometry. After incubation with NVC or NGC for 6-24 h, cell viability was detected by MTT colorimetry. Cell apoptosis and apoptotic rates were measured by acridine orange/ethidium bromide staining, TUNEL labeling and flow cytometry. Intracellular caspase-3 activities were determined by spectrophotometry. The esterase activity within EJ cells was 10.2-fold higher than that of HKC cells, which was abolished by bis-p-nitrophenylphosphate, an esterase inhibitor, resulting in decreases in NVC- and NGC-mediated cell viability arrest. For EJ cells, the IC50 values of NVC (20.1 micromol/l) and NGC (18.7 micromol/l) were close to curcumin (16.5 micromol/l). Meanwhile, their IC50 values on HKC cells were, respectively, 4.06- and 3.23-fold higher than curcumin. Moreover, NVC and NGC induced apoptosis of EJ cells by 10.13-23.36 and 12.42-28.56%, respectively. Administration of these two derivatives resulted in decreased apoptosis of HKC cells compared with curcumin. The caspase-3 activities of EJ cells, but not of HKC cells, were 5.21- and 5.63-fold enhanced by NVC and NGC, respectively. Thus, novel esterase-sensitive curcumin derivatives were synthesized, which induced extensive apoptosis of bladder cancer EJ cells, but not normal cells.

X-linked inhibitor of apoptosis (XIAP) is the most potent member of the inhibitor of apoptosis protein (IAP) gene family in terms of its ability to inhibit caspases and suppress apoptosis. Recent evidence has suggested that XIAP is a key determinant in chemoresistance of cancer cells. To explore a novel approach for ameliorating chemotherapy of gastric cancer, the antisense expression vector for the XIAP gene was constructed and transferred into gastric cancer cell lines, MKN-45 (wild-type p53) and MKN-28 (mutant-type p53). This transfer resulted in significant downregulation of XIAP expression, decreased in vitro cell viabilities, and induced apoptosis. In transferred cells, inactive caspase-3 precursors were cleaved into the active subunits (p20 and p17) during apoptosis induced by downregulation of XIAP. The inhibitory effects of cisplatin and mitomycin C on the growth of XIAP downregulated cancer cells were significantly enhanced. In addition, this process occurred only in wild-type p53 (MKN-45), but not in mutant-type p53 (MKN-28) gastric cancer cells. The data presented suggest that downregulation of XIAP via antisense RNA can lead to apoptosis of gastric cancer cells in vitro, correlating with cellular p53 status and activation of caspase-3. This finding could lead to a potential strategy for improving the efficiency of therapies for gastric cancer.

Aim: To select the optimal antisense accessible sites of survivin, a highly expressed gene in tumor tissues, in order to explore a novel approach to improve biological therapy of gastric cancer., Methods: The 20 mer random oligonucleotide library was synthesized, hybridized with in vitro transcribed total survivin cRNA, then digested by RNase H. After primer extension and autoradiography, the antisense accessible sites (AAS) of survivin were selected. Then RNADraw software was used to analyze and choose the AAS with obvious stem-loop structures, according to which the complementary antisense oligonucleotides (AS-ODNs) were synthesized and transferred into survivin highly- expressing gastric cancer cell line MKN-45. Survivin expression was detected by RT-PCR and Western Blotting. Cellular growth activities were assayed by tetrazolium bromide (MTT) colorimetry. Cellular ultrastructure was observed by electronic microscopy, while apoptosis was detected by annexin V-FITC and propidium iodide staining flow cytometry., Results: Thirteen AAS of survivin were selected in vitro. Four AAS with stem-loop structures were chosen, locating at 207-226 bp, 187-206 bp, 126-145 bp and 44-63 bp of survivin cDNA respectively. When compared with non-tranfection controls, their corresponding AS-ODNs (AS-ODN(1), AS-ODN(2), AS-ODN(3) and AS-ODN(4)) could reduce Survivin mRNA levels in MKN-45 cells by 54.3+/-1.1% (t = 6.12, P<0.01), 86.1+/-1.0% (t = 5.27, P<0.01), 32.2+/-1.3% (t = 7.34, P<0.01) and 56.2+/-0.9% (t = 6.45, P<0.01) respectively, while survivin protein levels were decreased by 42.2+/-2.5% (t = 6.26, P<0.01), 75.4+/-3.1% (t = 7.11, P<0.01), 28.3+/-2.0% (t = 6.04, P<0.01) and 45.8+/-1.2% (t = 6.38, P<0.01) respectively. After transfection with 600 nmol/L AS-ODN1-AS-ODN(4) for 24 h, cell growth was inhibited by 28.12+/-1.54% (t = 7.62, P<0.01), 38.42+/-3.12% (t = 7.75, P<0.01), 21.46+/-2.63% (t = 5.94, P<0.01) and 32.12+/-1.77% (t = 6.17, P<0.01) respectively. Partial cancer cells presented the characteristic morphological changes of apoptosis, with apoptotic rates being 19.31+/-1.16% (t = 7.16, P<0.01), 29.24+/-1.94% (t = 8.15, P<0.01), 11.87+/-0.68% (t = 6.68, P<0.01) and 21.68+/-2.14% (t = 7.53, P<0.01) respectively., Conclusion: The AAS of survivin could be effectively selected in vitro by random oligonucleotide library/RNase H cleavage method combined with computer software analysis, this has important reference values for further studying survivin-targeted therapy strategies for gastric cancer.

Objective: To investigate the effects of overexpression of second mitochondria-derived activator of caspases (Smac) gene on apoptosis of gastric cancer cells., Methods: Under the induction of liposome, MKN-45 cells were transfected by Smac gene and incubated with G418 for subclone selection. Reverse transcriptase-polymerase chain reaction and Western blot were used to determine cellular Smac gene expression. After induction of apoptosis by mitomycin (MMC), cell viabilities were analyzed using trypan blue stain. Apoptosis was measured by electronic microscopy, acridine orange-ethidium bromide fluorescent staining and in situ terminally labelled transferase technique (TUNEL). Western blot and colorimetry were used to assess cellular caspase-3 expression and its activity., Results: The Smac mRNA and protein levels in MKN-45/Smac subclone cells (subclone consistently expressing Smac gene) were significantly higher than those in MKN-45 (P < 0.01). When compared with those in MKN-45, cell viabilities of MKN-45/Smac were reduced by 10.0% to 30.8% (P < 0.01), after treatment with 10 microg/ml MMC for 6 to 24 hours. Some of the MKN-45/Smac cells showed characteristic morphologic changes of apoptosis, their apoptotic rate being increased by 21.2% (P < 0.01). After treatment with MMC, caspase-3 expression and its activity in MKN-45/Smac cells were significantly higher than those in MKN-45 (P < 0.01)., Conclusions: Overexpression of Smac in gastric cancer cell line significantly improves expression and activity levels of caspase-3 after induction by MMC. Such apoptosis-inducing effect establishes a novel strategy for regulating the apoptosis activity of gastric cancer.

Aim: To explore the feasibility of enhancing apoptosis-inducing effects of chemotherapeutic drugs on human gastric cancer cells by stable transfection of extrinsic Smac gene., Methods: After Smac gene was transferred into gastric cancer cell line MKN-45, subclone cells were obtained by persistent G418 selection. Cellular Smac gene expression was determined by RT-PCR and Western blotting. After treatment with mitomycin (MMC) as an apoptotic inducer, in vitro cell growth activities were investigated by trypan blue-staining method and MTT colorimetry. Cell apoptosis and its rates were determined by electronic microscopy, annexin V-FITC and propidium iodide staining flow cytometry. Cellular caspase-3 protein expression and its activities were assayed by Western blotting and colorimetry., Results: When compared with MKN-45 cells, the selected subclone cell line MKN-45/Smac had significantly higher Smac mRNA (3.12+/-0.21 vs 0.82+/-0.14, t = 7.52, P<0.01) and protein levels (4.02+/-0.24 vs 0.98+/-0.11, t = 8.32, P<0.01). After treatment with 10 microg/mL MMC for 6-24 h, growth inhibition rate of MKN-45/Smac (15.8+/-1.2-54.8+/-2.9%) was significantly higher than that of MKN-45 (5.8+/-0.4- 24.0+/-1.5%, t = 6.42, P<0.01). Partial MKN-45/Smac cancer cells presented characteristic morphological changes of apoptosis under the electronic microscope with an apoptosis rate of 36.4+/-2.1%, which was significantly higher than that of MKN-45 (15.2+/-0.8%, t = 9.25, P<0.01). Compared with MKN-45, caspase-3 expression levels in MKN-45/Smac were improved significantly (3.39+/-0.42 vs 0.96+/-0.14, t = 8.63, P<0.01), while its activities were 3.25 times as many as those of MKN-45 (0.364+/-0.010 vs 0.112+/-0.007, t = 6.34, P<0.01)., Conclusion: Stable transfection of extrinsic Smac gene and its over-expression in gastric cancer cell line can significantly enhance cellular caspase-3 expression and activities, ameliorate apoptosis-inducing effects of mitomycin C on cancer cells, which is a novel strategy to improve chemotherapeutic effects on gastric cancer.

Background: Recently, arsenic trioxide (As2O3) was considered as a novel anti-tumor agent. However, it showed severe toxicity effect on normal tissue at the same time. To improve its therapeutic efficacy and decrease its toxicity,we prepared arsenic trioxide-loaded albuminutes immuno-nanospheres [As2O3-(HAS-NS)-BDI-1] targeted with monoclonal antibody (McAb) BDI-1 and tested its specific killing effect against bladder cancer cell., Methods: As2O3-HAS-NS was prepared by chemical cross-linking method. Monoclonal antibody BDI-1 was purified with ammonium sulphate saltingout and chromatography. Albuminutes microspheres were conjugated with McAb by SPDP cross-linking method. Concentration of As in As2O3-(HAS-NS)-BDI-1 and As2O3-HAS-NS was measured by atomic fluometry method. As2O3-(HAS-NS)-BDI-1 and its activity were detected by SDS-PAGE reduction electrophoresis, indirect immunofluorescence test, light microscope and scanning electron microscope observation. Acridine orange staining and tritiated thymidine (3H-TdR) incorporation tests were used to indicate specific killing activity of As2O3-(HAS-NS)-BDI-1 in vitro., Results: In As2O3-(HAS-NS)-BDI-1 groups, we saw two protein bands in SDS-PAGE reduction electrophoresis. Albuminutes immuno-nanospheres were rounded with clear green fluorescence by immunofluorescence test. Under microscope, we observed that BIU-87 cells were covered with the As2O3-(HAS-NS)-BDI-1 and that As2O3-(HAS-NS)-BDI-1 moved with the BIU-87 cells. The albuminutes immuno-nanospheres were tightly junctioned with the BIU-87 cells. Specific killing activity of As2O3-(HAS-NS)-BDI-1 on bladder tumor cells was observed by acridine orange staining and 3H-TdR incorporation assays., Conclusions: As2O3-(HAS-NS)-BDI-1 might bind specifically against BIU-87 cells, thus leading to high activity of killing bladder tumor cells.

Background: BAK (Bcl-2 homologous antagonist/killer) is a novel pro-apoptotic gene of the Bcl-2 family. It has been reported that gastric tumors have reduced BAK levels when compared with the normal mucosa. Moreover, mutations of the BAK gene have been identified in human gastrointestinal cancers, suggesting that a perturbation of BAK-mediated apoptosis may contribute to the pathogenesis of gastric cancer. In this study, we explored the therapeutic effects of gene transfer mediated elevations in BAK expression on human gastric cancer cells in vitro., Methods: Eukaryotic expression vector for the BAK gene was constructed and transferred into gastric cancer cell lines, MKN-45 (wild-type p53) and MKN-28 (mutant-type p53). RT-PCR and Western Blotting detected cellular BAK gene expression. Cell growth activities were detected by MTT colorimetry and flow cytometry, while apoptosis was assayed by electronic microscopy and TUNEL. Western Blotting and colorimetry investigated cellular caspase-3 activities., Results: BAK gene transfer could result in significant BAK overexpression, decreased in vitro growth, cell cycle G0/G1 arrest, and induced apoptosis in gastric cancer cells. In transferred cells, inactive caspase-3 precursor was cleaved into the active subunits p20 and p17, during BAK overexpression-induced apoptosis. In addition, this process occurred equally well in p53 wild-type (MKN-45), or in p53 mutant-type (MKN-28) gastric cancer cells., Conclusions: The data presented suggests that overexpression of the BAK gene can lead to apoptosis of gastric cancer cells in vitro, which does not appear to be dependent on p53 status. The action mechanism of BAK mediated apoptosis correlates with activation of caspase-3. This could be served as a potential strategy for further development of gastric cancer therapies.

Background & Objective: Abnormal apoptosis is one of the key factors for drug resistance of neoplasms. Smac, a novel gene involved in regulation of apoptosis,plays an important role in tumor cell apoptosis induced by chemotherapeutic drugs. This study was designed to explore the effects of overexpressed Smac gene on chemotherapeutic sensitivity of gastric cancer cell line., Methods: By liposome GeneSHUTTLE-40, Smac gene was transfected into gastric cancer cell line MKN-45. Cellular Smac gene expression were determined by reverse transcription-polymerase chain reaction (RT-PCR)and Western blot analysis. Cisplatin (1, 5, 10 microg/ml), mitomycin C (0.1,1,10 microg/ml), and curcumin (10, 20, 40 micromol/L) were selected to treat untransfected and transfected MKN-45 cells. Cellular proliferation activities were assayed by tetrazolium bromide (MTT) colorimetry. The morphological changes of cancer cells were observed under inverted microscope. Cellular apoptosis was determined by Annexin V-FITC and propidium iodide staining flow cytometry., Results: Compared with untransfected control group,Smac mRNA and protein levels in transfected MKN-45 cells were significantly increased (P< 0.01). The growth inhibition rates of MKN-45 cells treated with various concentration of cisplatin, mitomycin C, and curcumin on MKN-45 cells were increased by 10.10%-23.80% (P< 0.01), 10.01%-15.86% (P< 0.01), 11.28%-22.12% (P< 0.01), respectively, with the cells becoming round, obviously refracted, and fragmented. The cellular apoptosis rates were increased by 6.7%-20.2% (P< 0.01), 5.4%-13.2% (P< 0.01), and 10.6%-20.1% (P< 0.01), respectively., Conclusion: Transfection of extrinsic Smac gene results in its over-expression in MKN-45 cells,which could increase the chemotherapeutic sensitivity of MKN-45 cells.

Background & Objective: Curcumin is the major effective component of curcuma, which is a kind of traditional Chinese medicine. It has been paid more attention to curcumin recently for its specific proliferation inhibition and apoptosis inducing effects on tumor cells; however, the involved mechanisms were not clear. This study was designed to explore the apoptosis inducing effects of curcumin on human ovary A2780 cell line and its related molecular mechanisms., Methods: A2780 cancer cells were treated with 10-50 mumol/L curcumin for 6-24 h and the growth inhibition rates of A2780 cancer cells were measured by MTT method. Cell apoptosis was inspected by flow cytometry (FCM) and acridine orange-ethidium bromide fluorescent staining method. The protein levels of NF-kappa B (P65) and Caspase-3 in cancer cells were observed by SP immunohistochemistry., Results: The growth inhibition rates of the cancer cells reached 62.05%-89.24%, with the peak of sub G1 appeared on DNA histogram in FCM. Partial cells presented the characteristic morphological changes of apoptosis under the fluorescent microscope; the apoptosis rates were 21.5%-33.5%. The NF-kappa B (p65) expression was decreased while Caspase-3 expression was increased, which depended on the action time., Conclusions: Curcumin could significantly inhibit the growth of ovary cancer cells; inducing apoptosis through up-regulating Caspase-3 and down-regulating expression of NF-kappa B was probably one of its molecular mechanisms.

Standard microelectrode technique and the whole-cell clamp technique were used to investigate the effects of tetrandrine and rotundine on the action potential of rabbit sinus node dominant pacemaker cell and calcium currents of ventricular myocytes from guinea-pig. The results indicate that: tetrandrine 1-200 mumol.L-1 and rotundine 3-300 mumol.L-1 decreased the action potential amplitude(APA), the maximal upstroke velocity (Vmax), spontaneous depolarizing velocity of phase 4(SP4) and prolonged the sinus circle length(SCL) in a dose-dependent manner. Tetrandrine 0.1 mumol.L-1 in combination with rotundine 0.3 mumol.L-1 was shown to effectively reduce the ICa of ventricular myotytes from guinea-pig, the rate of inhibition is 19.6%. The results suggest that the two drugs have a good synergistic effect on inhibiting calcium current. These results might also put forward a valuable theoretic base for using the two drugs together in treating patients.

The effects of benzyltetrahydropalmatine (BTHP) on delayed rectified K+ currents (Ik) expressed in Xenopus oocytes and Ik of toad (Bufo bufo gargarizans) oocytes were studied. The Ik expressed in Xenopus oocytes was measured after microinjection of mRNA isolated from carp fish (C anratus L.) brains with double -microelectrode voltage clamp technique. The maximum and mean value of Ik expressed in Xenopus oocytes were 600 nA and 360 +/- 104 nA, respectively. BTHP reduced the current amplitude of Ik expressed in Xenopus oocytes in 10-1000 mumol.L-1 dose-dependently, EC50 was 29 mumol.L-1. Also, the reduction of Ik of toad oocytes was 9.1%, 29.1%, 54.7% and 68.6% by BTHP 10, 30, 100 and 1000 mumol.L-1, respectively, EC50 was 33 mumol.L-1. The results showed that BTHP possesses an inhibitory effect on Ik, the main ion mechanism of antiarrhythmic action of BTHP.