[Effects of Vitamin D Receptor on Mucosal Barrier Proteins in Colon Cells under Hypoxic Environment].

To investigate the expressions of mucosal barrier proteins in colon cell line DLD-1 under hypoxic environment in vitro and its mechanism. Methods After DLD-1 cells were treated separately with hypoxia(l% O2),vitamin D(100 nmol/L),or vitamin D plus hypoxia for 48 hours,the expressions of vitamin D receptor(VDR),tight junction proteins zonula occludens-1(ZO-1),occludin,Claudin-1,and adherent junction protein(E-cadherin)were determined by Western blot.Stable VDR knock-down(Sh-VDR)DLD-1 cell line and control DLD-1 cell line were established by lentivirus package technology and the protein expressions after hypoxia treatment were detected. Results Compared with control group,the expressions of occludin,Claudin-1,and VDR increased significantly after hypoxia treatment(all P<0.001).In addition to the protein expressions of occludin,Claudin-1 and VDR,the expressions of ZO-1 and E-cadherin were also obviously higher in vitamin D plus hypoxia group than in single vitamin D treatment group(all P<0.001).After hypoxia treatment,Sh-VDR cell line showed significantly decreased expressions of ZO-1(P<0.001),occludin(P<0.05),Claudin-1(P<0.01)and E-cadherin(P<0.001)when compared with untreated Sh-VDR cell line. Conclusion VDR acts as a regulator for the expressions of intestinal mucosal barrier proteins under hypoxia environment in DLD-1 colon cell line,indicating that VDR pathway may be another important protective mechanism for gut barrier in low-oxygen environment.

A Panel of Serum Noncoding RNAs for the Diagnosis and Monitoring of Response to Therapy in Patients with Breast Cancer.

BACKGROUND The aim of this study was to identify a panel of serum noncoding RNAs (ncRNAs) as potential diagnostic and prognostic biomarkers for breast cancer. MATERIAL AND METHODS Patients with breast cancer (n=30), and normal controls (n=30) were included in the 'training set.' A 'validation set' included cases of breast cancer (n=128) and controls (n=77). All cases provided blood samples for serum analysis. All cases of breast cancer were confirmed histologically and were staged. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) was used to detect the expression of 11 candidate ncRNAs, including long noncoding RNAs (lncRNAs) and microRNAs (miRNAs), in the serum. The expression of the panel of ncRNAs was further analyzed following surgery or chemotherapy. RESULTS The four ncRNAs identified in the serum of patients with breast cancer included let-7a, miR-155, miR-574-5p, and metastasis-associated lung adenocarcinoma transcript 1 (MALAT1). Analysis based on the risk score showed that the panel of these four ncRNAs could effectively distinguish between patients with breast cancer and the control group. For the training set and the validation set, analysis of the receiver-operating characteristic (ROC) curve showed that the areas under the curve (AUCs) were 0.960 and 0.968, respectively. Also, the serum expression levels of the four ncRNAs differed in the pre-treatment and the post-treatment patients with breast cancer, with levels of miR-155 showing a significant decrease following chemotherapy. CONCLUSIONS A panel of serum ncRNAs, including let-7a, miR-155, miR-574-5p, and MALAT1, was shown to be present in patients with breast cancer.

The de-ubiquitinase UCHL1 promotes gastric cancer metastasis via the Akt and Erk1/2 pathways.

Ubiquitin C-terminal hydrolase-L1 (UCHL1) is a de-ubiquitinating enzyme, which enzymatic activity relies on the C90 site. The function of UCHL1 is controversial in different types of cancer, and its role in gastric cancer progression remains unclear. In this study, immunohistochemistry staining was applied to detect the expression of UCHL1 in primary gastric cancer and liver metastases from gastric cancer. MKN45 and BGC823 cell lines with stable expression of de-ubiquitinase active UCHL1 or inactive UCHL1-variant C90S were established by lentiviral infection. The effect of UCHL1 on cell proliferation was evaluated by MTT and colony formation assays. The abilities of cell migration and invasion were determined by transwell assay. Protein expression levels were determined by Western blot. The results indicated that UCHL1 had a significantly higher positive expression rate in liver metastases from gastric cancer compared with primary gastric cancer. Overexpression of UCHL1 in MKN45 and BGC823 cells promoted cell proliferation, migration, and invasion depending on its de-ubiquitinase activity. UCHL1 activated Akt and Erk1/2, which process also required enzymatic activity and was necessary for mediating cell migration and invasion. These findings demonstrated that UCHL1 promoted cell proliferation, migration, and invasion depending on its de-ubiquitinase activity by activating Akt and Erk1/2, which may account for its higher positive expression rate in liver metastases from gastric cancer. UCHL1 could be a candidate biomarker and a therapeutic target for gastric cancer metastasis.

Genetic variants in the calcium signaling pathway participate in the pathogenesis of colorectal cancer through the tumor microenvironment.

Background: Cancer risk is influenced by calcium signaling in intracellular and intercellular signaling pathways. However, the relationship between the calcium signaling pathway and colorectal cancer risk remains unknown. We aim to evaluate the role of genetic variants in calcium signaling pathway genes in colorectal cancer risk through the tumor microenvironment. Methods: An analysis of genetic variants in the calcium signaling pathway was conducted using a case-control study that included 1150 colorectal cancer patients and 1342 non-cancer patients. Using the regression model, we assessed whether single-nucleotide polymorphisms (SNPs) increase the risk of colorectal cancer. We also performed a dual luciferase reporter gene assay using HCT116 cell lines and DLD1 cell lines to demonstrate the regulatory relationship between SNP and candidate risk gene. We evaluated the expression of candidate risk gene in different populations. In addition, we also evaluated candidate risk gene and 22 immune cells correlation studies. Results: There was a significant association between the PDE1C rs12538364 T allele and colorectal cancer risk [odds ratio (OR) = 1.57, 95% confidence interval (CI) = 1.30 - 1.90, P = 3.07 × 10-6, P FDR = 0.004]. Mutation of intron region rs1538364 C to T locus reduces promoter activity of PDE1C in DLD1 and HCT116 cell lines (P < 0.05). We identified that PDE1C is significantly down-regulated in colorectal cancer, closely associated with 22 immune cells. Finally, we found that PDE1C could be the biomarker for individual immunotherapy of colorectal cancer. Conclusion: According to our findings, PDE1C may be a key factor contributing to colorectal cancer, thus improving individual immunotherapy for the disease. The potential mechanism by which polymorphisms in the calcium signaling pathway genes may participate in the pathogenesis of colorectal cancer through the tumor microenvironment.

PD-1 inhibitor in combination with fruquintinib therapy for initial unresectable colorectal cancer: A case report.

BACKGROUND: PD-1 inhibitors in combination with fruquintinib have not previously been reported as neoadjuvant therapy for patients with colorectal cancer. In this case report, the combination of a PD-1 inhibitor and fruquintinib demonstrated good efficacy in patients with MSI-H colorectal cancer. CASE SUMMARY: The patient was a young man in his 30s who had MSI-H type colon cancer. The patient underwent four cycles of neoadjuvant therapy with a PD-1 inhibitor combined with fruquintinib before surgery, resulting in regression of the mass and a successful surgery. CONCLUSION: Some patients with colorectal cancer have the MSI-H type, and the first-line chemotherapy regimen is not effective. However, PD-1 monoclonal antibody immunotherapy has a good therapeutic effect, which can be improved by combination therapy with fruquintinib. We recommend that patients with a history of colon or rectal cancer receive universal MSI testing; then, neoadjuvant therapy should be used.

N7-methylguanosine-related lncRNAs: Predicting the prognosis and diagnosis of colorectal cancer in the cold and hot tumors.

Background: 7-Methylguanosine(m7G) contributes greatly to its pathogenesis and progression in colorectal cancer. We proposed building a prognostic model of m7G-related LncRNAs. Our prognostic model was used to identify differences between hot and cold tumors. Methods: The study included 647 colorectal cancer patients (51 cancer-free patients and 647 cancer patients) from The Cancer Genome Atlas (TCGA). We identified m7G-related prognostic lncRNAs by employing the univariate Cox regression method. Assessments were conducted using univariate Cox regression, multivariate Cox regression, receiver operating characteristics (ROC), nomogram, calibration curves, and Kaplan-Meier analysis. All of these procedures were used with the aim of confirming the validity and stability of the model. Besides these two analyses, we also conducted half-maximal inhibitory concentration (IC50), immune analysis, principal component analysis (PCA), and gene set enrichment analysis (GSEA). The entire set of m7G-related (lncRNAs) with respect to cold and hot tumors has been divided into two clusters for further discussion of immunotherapy. Results: The risk model was constructed with 17 m7G-related lncRNAs. A good correlation was found between the calibration plots and the prognosis prediction in the model. By assessing IC50 in a significant way across risk groups, systemic treatment can be guided. By using clusters, it may be possible to distinguish hot and cold tumors effectively and to aid in specific therapeutic interventions. Cluster 1 was identified as having the highest response to immunotherapy drugs and thus was identified as the hot tumor. Conclusion: This study shows that 17 m7G-related lncRNA can be used in clinical settings to predict prognosis and use them to determine whether a tumor is cold or hot in colorectal cancer and improve the individualization of treatment.

Overexpression of LINC00673 Promotes the Proliferation of Cervical Cancer Cells.

OBJECTIVE: To study the expression of LINC00673 in cervical cancer and cervical intraepithelial neoplasia (CIN) and to explore the role of LINC00673 in the development of cervical cancer. METHODS: The expression of LINC00673 in serum from cervical cancer patients, CIN patients, and healthy participants was detected by RT-qPCR. The function of LINC00673 in cervical cancer cells was analyzed using in vitro and in vivo experiments. RESULTS: Our results revealed that serum LINC00673 levels were highest in cervical cancer patients, followed by patients with CIN and healthy controls. In vitro experiments demonstrated that overexpression of LINC00673 enhanced the proliferation and cell cycle progression of HeLa and SiHa cells. In vivo experiments showed that the tumor weight and volume of nude mice subcutaneously injected with LINC00673-overexpressing HeLa cells were larger than those of nude mice injected with control cells (P < 0.05). Western blotting showed that cell cycle-related proteins cyclin A2 and cyclin E and interstitial-associated proteins Snail and N-cadherin were upregulated and p53 signaling pathway-related proteins were downregulated in LINC00673-overexpressing HeLa and SiHa cells. CONCLUSION: LINC00673 plays an important role in the development of cervical cancer and may serve as a new therapeutic target for cervical cancer.

Overexpression of PIP5KL1 suppresses cell proliferation and migration in human gastric cancer cells.

Phosphatidylinositol-4-phosphate 5-kinase-like 1 (PIP5KL1), the forth member of phosphatidylinositol-4-phosphate 5-kinases (PIPKs) type I, acts as a scaffold for localization and activation of PIPKs, which mediates numerous cellular processes. However, the role of PIP5KL1 in the development of human cancer is still lacking. We therefore examined the expression of PIP5KL1 in human normal and cancer tissues by tissue microarrays (TMAs). Reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence imaging analysis were used to testify the mRNA and protein levels of PIP5KL1 in human gastric cancer cell line (BGC823). The cell proliferation was investigated with 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assay. Both wound healing and transwell migration assay were performed to study the cell migration. The phosphorylation of v-akt murine thymoma viral oncogene homolog 1 (AKT1) was determined by western immunoblot analysis. Immunostaining of gastric cancer tissue microarrays revealed a negative correlation between PIP5KL1 overexpression and gastric cancer in situ. Transient transfection PIP5KL1 induced a significant increase expression at both transcriptional and translational levels and consequent robust inhibition of proliferation (P < 0.05) and migration (P < 0.05) of BGC823 cells. Overexpression of PIP5KL1 markedly inhibited (P < 0.05) serum-induced phosphorylation of AKT1. Taken together, these studies indicate a functional negative correlation between elevated levels of PIP5KL1 and the development of human gastric cancer, suggesting that PIP5KL1 overexpression may suppress gastric cancer formation.

Serum lncRNAs (CCAT2, LINC01133, LINC00511) with Squamous Cell Carcinoma Antigen Panel as Novel Non-Invasive Biomarkers for Detection of Cervical Squamous Carcinoma.

BACKGROUND: We screened long non-coding RNAs (lncRNAs) specifically expressed in the serum of cervical squamous carcinoma (CESC) patient samples and investigated the role of these specific lncRNAs in the diagnosis of CESC and cervical intraepithelial neoplasia (CIN). METHODS: The expression levels of the lncRNAs CCAT2, LINC01133, and LINC00511 in the serum of normal controls and patient with CESC and CIN were measured using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Next, we analyzed the correlation between the serum lncRNAs levels and the clinical characteristics of CESC. Thereafter, we estimated their combined diagnostic value by receiver operating characteristic (ROC) curve analysis. RESULTS: The results showed that CCAT2, LINC01133, and LINC00511 were highly expressed in the serum of patients with CESC. When these lncRNAs and squamous cell carcinoma (SCC) antigen were combined, the area under the ROC curve (AUC) value reached 0.94. We also found that the AUC value of the diagnostic model combining CCAT2 and LINC01133 reached 0.894. CONCLUSION: The serum lncRNAs (CCAT2, LINC01133, and LINC00511) and SCC may be new non-invasive biomarkers for the diagnosis of CESC.

Ubiquitin proteasome system research in gastrointestinal cancer.

The ubiquitin proteasome system (UPS) is important for the degradation of proteins in eukaryotic cells. It is involved in nearly every cellular process and plays an important role in maintaining body homeostasis. An increasing body of evidence has linked alterations in the UPS to gastrointestinal malignancies, including esophageal, gastric and colorectal cancers. Here, we summarize the current literature detailing the involvement of the UPS in gastrointestinal cancer, highlighting its role in tumor occurrence and development, providing information for therapeutic targets research and anti-gastrointestinal tumor drug design.

A circulating non-coding RNA panel as an early detection predictor of non-small cell lung cancer.

AIMS: Early non-small cell lung cancer (NSCLC) diagnosis is generally poor due to the lack of convenient and noninvasive tools. MicroRNAs (miRNAs) and the long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) are non-coding RNAs, that have attracted increased attention for their use as NSCLC tumor diagnostic markers. MAIN METHODS: We constructed a serum miRNA and MALAT1 non-coding RNA panel and tested its diagnostic performance as an NSCLC biomarker. We tested the expression of 11 candidate miRNAs and MALAT1 in a training set (36 NSCLCs vs. 36 controls) by quantitative reverse transcription polymerase chain reactions. The serum non-coding RNA panel's diagnostic efficiency was tested and validated in a second validation sample set (120 NSCLCs and 71 controls) by receiver operating characteristic (ROC) curve analyses. KEY FINDINGS: In the training set, the expression of the four non-coding RNAs (miR-1254, miR-485-5p, miR-574-5p, and MALAT1) was obviously different between the NSCLC patients and healthy controls. Risk score analysis revealed that the four non-coding RNA panel can distinguish NSCLC patient samples from controls. The ROC curve results revealed areas under the curves (AUCs) of 0.861 (95% confidence interval (CI) 0.771-0.952) and 0.844 (95% CI0.778-0.910) for the training set and validation set, respectively. SIGNIFICANCE: The four non-coding RNA risk scores were also associated with NSCLC progression, and its diagnostic efficiency was relatively high for stages I/II/III. In conclusion, these data indicate that the four non-coding RNA panel can serve as a convenient tool for early NSCLC diagnosis.

Mitogen-activated protein kinase signaling pathway and invasion and metastasis of gastric cancer.

The mortality rate of gastric cancer worldwide is as high as 70%, despite the development of novel therapeutic strategies. One reason for the high mortality is the rapid and uninhibited spread of the disease, such that the majority of patients are diagnosed at a stage when efficient therapeutic treatment is not available. Therefore, in-depth research is needed to investigate the mechanism of gastric cancer metastasis and invasion to improve outcomes and provide biomarkers for early diagnosis. The mitogen-activated protein kinase (MAPK) signaling pathway is widely expressed in multicellular organisms, with critical roles in multiple biological processes, such as cell proliferation, death, differentiation, migration, and invasion. The MAPK pathway typically responds to extracellular stimulation. However, the MAPK pathway is often involved in the occurrence and progression of cancer when abnormally regulated. Many studies have researched the relationship between the MAPK signaling pathway and cancer metastasis and invasion, but little is known about the important roles that the MAPK signaling pathway plays in gastric cancer. Based on an analysis of published data, this review aims to summarize the important role that the MAP kinases play in the invasion and metastasis of gastric cancer and attempts to provide potential directions for further research and clinical treatment.

Calcitonin Gene-Related Peptide-Induced Calcium Alginate Gel Combined with Adipose-Derived Stem Cells Differentiating to Osteoblasts.

Calcitonin gene-related peptide (CGRP) has been confirmed with induction osteoblastic differentiation, but if it can make the three-dimensional culture of adipose-derived stem cells (ADSCs) to the osteoblastic differentiation, thus constructing tissue-engineered bone rare reports. To investigate the feasibility of exogenous CGRP-induced calcium alginate gel combined with ADSCs from rabbits in three-dimensional condition to construct tissue-engineered bone. ADSCs were obtained by collagenase I digestion of the subcutaneous adipose tissue of inguinal region of New Zealand rabbits. At the third passage, cells were mixed with sodium alginate to prepare calcium alginate gel, and the cells were assigned into two-group cultivates in 24 orifice plates. ADSCs in the control group were treated with DMEM/F-12 medium supplemented with 10(-2) mol/L ß-glycerophosphate sodium, 10(-7)mol/L dexamethasone, 50 mg/L ascorbic acid, 0.1 % volume fraction of fetal bovine serum. ADSCs in the experimental group were incubated with the same medium as above, and in addition 1.5 µg/L CGRP was added. The cells proliferation and the mRNA expressions of collagen I and osteocalcin were detected by MTT and RT-PCR assays, respectively and alkaline phosphatase(ALP)and calcium concentration at different induction time were detected. The cell proliferation curves were S shaped. The OD values of experimental group were higher than those of control group at 1, 3, 5, 7, 14, and 21 days after osteogenic induction (P < 0.05). ALP and alizarin red stains of ADSCs were all positive, but golden round nodes became bigger and more in the experimental group compared with the control group after 2 weeks. At 7 and 14 days, collagen I and osteocalcin mRNA expression were greater in the experimental group than the control group. ALP and calcium concentration of experimental group were higher than that of control group at 1, 2, 3, 4 weeks after osteogenic induction (P < 0.05). Thus, these results show that the CGRP-induced ADSCs combined with calcium alginate gel to osteoblasts differentiation.

[Over-articular external fixator combined with limited internal fixation for the treatment of high-energy Pilon fractures].

OBJECTIVE: To study clinical effects of the over-articular external fixator combined with limited internal fixation for the treatment of Pilon fractures caused by high energy. METHODS: From September 2003 to April 2011, 36 patients with Pilon fractures caused by high energy were treated with the over-articular external fixator combined with limited internal fixator. There were 25 males and 11 females, ranging in age from 16 to 72 years old,with an average of 38 years old. The diagnoses of all patients were determined by conventional X-ray examination or three-dimensional spiral CT examination. The AOFAS scoring criteria was used to evaluate the therapeutic effects. The patients with comminuted fractures were treated with screw or Kirschner wire fixation without uncovering periost so as to enhance stability between fracture end and bone blocks,followed by the fixation with over-articular external fixators. RESULTS: All the patients were followed up, and the duration ranged from 4 to 27 months, with an average of 13 months. Thirty-two patients got wound healing at the first stage. And the bone union duration ranged from 2 to 6 months, with a mean of 3 months. According to the AOFAS ankle-hindfoot subjective scoring standard, 13 patients got an excellent result, 20 good and 3 fair, with an score of 88.2 +/- 3.6. Twelve patients had infections at pinhole, 5 patients had pinhole pain. One patient had the fixator broken induced by over loading, who was cured after treatment. There were no complications such as nerve or vascular injuries, or osteomyelitis. CONCLUSION: The over-articular external fixation combined with limited internal fixation for the treatment of Pilon fractures caused by high energy is an ideal method, which has such advantages as reliable fixation, simple operation, coincidence with principles of biomechanical fixation, and benefit for fracture healing.

Correlation analysis of peripheral blood T cell subgroups, immunoglobulin and prognosis of early hepatocellular carcinoma after hepatectomy.

To investigate the prediction value of preoperative changes in the immunological function for the prognosis of hepatocellular carcinoma (HCC) after hepatectomy, 158 cases of HCC patients who received liver resection in our hospital from 2009 to 2010 were enrolled in this study. Immune indices [CD3(+), CD4(+), CD8(+), CD19(+) and natural killer (NK), IgA, IgM, IgG], AFP level were calculated. The differences between preoperation and postoperation group, exclude were not both statistically significant (both P > 0.05), whereas IgA group was not (P < 0.05),The follow-up data showed that the 3-year recurrence rates of high level group on HBsAg, CD3, CD4, CD8, CD19 and IgGwerelarger than that of low level group,while the 3-year recurrence rates of high level group on AFP, NK, IgA and IgM smaller than that of low level group, and we found that CD3(+), CD4(+), CD8(+) and NK(+), CD19(+), HBsAg, IgA and IgG above were statistically different (P < 0.05), AFP and IgM were no difference with prognosis of HCC (P > 0.05).The markers level were not both statistically significant with age, gender, grade, tumor size (P > 0.05). Multiple logistic regression analysis indicated that CD4(+), CD8(+) and NK(+) were closely correlated with HCC postoperative survival rate (P < 0.05) (Y = 1.262×CD4(+)+1.448×CD8(+)-0.646×NK(+)). Construction of Cox's proportional hazards regression model showed IgA and IgG were correlated with HCC postoperative survival rate at a trend (P = 0.079) (Y = 127.9×IgG-28.7×IgM). Preoperative Immune indices (CD4(+), CD8(+), NK(+), IgA, IgG) were closely correlated with HCC survival rate and these were considerable predictive value for the malignantfeature and prognosis of HCC.

Identification of a circulating microRNA signature for colorectal cancer detection.

Prognosis of patients with colorectal cancer (CRC) is generally poor because of the lack of simple, convenient, and noninvasive tools for CRC detection at the early stage. The discovery of microRNAs (miRNAs) and their different expression profiles among different kinds of diseases has opened a new avenue for tumor diagnosis. We built a serum microRNA expression profile signature and tested its specificity and sensitivity as a biomarker in the diagnosis of CRC. We also studied its possible role in monitoring the progression of CRC. We conducted a two phase case-control test to identify serum miRNAs as biomarkers for CRC diagnosis. Using quantitative reverse transcription polymerase chain reactions, we tested ten candidate miRNAs in a training set (30 CRCs vs 30 controls). Risk score analysis was used to evaluate the diagnostic value of the serum miRNA profiling system. Other independent samples, including 83 CRCs and 59 controls, were used to validate the diagnostic model. In the training set, six serum miRNAs (miR-21, let-7g, miR-31, miR-92a, miR-181b, and miR-203) had significantly different expression levels between the CRCs and healthy controls. Risk score analysis demonstrated that the six-miRNA-based biomarker signature had high sensitivity and specificity for distinguishing the CRC samples from cancer-free controls. The areas under the receiver operating characteristic (ROC) curve of the six-miRNA signature profiles were 0.900 and 0.923 for the two sets of serum samples, respectively. However, for the same serum samples, the areas under the ROC curve used by the tumor markers carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA19-9) were only 0.649 and 0.598, respectively. The expression levels of the six serum miRNAs were also correlated with CRC progression. Thus, the identified six-miRNA signature can be used as a noninvasive biomarker for the diagnosis of CRC, with relatively high sensitivity and specificity.

[Effects of NAIF1 on biological behavior of esophageal carcinoma cell line EC9706 by using pTet-off system].

AIM: To investigate the effects of NAIF1 on biological behavior of esophageal carcinoma cell line EC9706 by using pTet-off system. METHODS: Constructe the pTRE2-NAIF1 plasmid and co-transfected with PTK-Hyg in human inducable esophageal carcinoma cell line TF93 (derive from EC9706 cell line).After Hygromycin B selection and RT-PCR, Western blot identification we got stable inducable cell clone TF93-pTRE2-NAIF1.By using this clone, we used MTT to test the proliferation rate, flow cytometry to test the cell cycle, Transwell chamber to test the migration ability and tested the adhesion ability of EC9706 under the effect of NAIF1. RESULTS: First we got the stable inducable cell clone TF93-pTRE2-NAIF1, then we found NAIF1 could inhibit the cell proliferation, cause G0/G1 cell arrest and inhibit the migration and adhesion ability of EC9706. CONCLUSION: NAIF1 can inhibit the malignant phenotype of esophageal carcinoma cell line EC9706.

[Effects of hyperthermic chemotherapy on gastrointestinal cancer cells in vitro].

BACKGROUND & OBJECTIVE: Little evidence is known from experimental research for intraoperative hyperthermic intraperitoneal chemotherapy. This study was to investigate the effect of hyperthermic chemotherapy on gastrointestinal cancer cells in vitro and explore the possible factors which may affect this method. METHODS: Gastric cancer cell line MGC-803 and intestinal cancer cell line HCT-116 were chosen. Cells were treated with different drugs, temperatures and duration. Cell viability and growth were measured by MTS-PMS assay. The morphology of the cells was observed under a microscope. RESULTS: Significant synergistic effect was observed when the two cancer cell lines were treated with 5-FU, MMC, DDP and THP in combination with elevated hyperthermia from 41 degrees C to 45 degrees C compared with control group (P<0.01). The strongest effect was achieved at 45 degrees C, which the inhibitory effects of these four drugs were 61.7%, 79.2%, 88.7%, 94.7% on MGC-803 and 76.4%, 78.7%, 77.8%, 91.7 on HCT-1116, respectively. The inhibitory effect demonstrated a time and dose-dependent manner in HCT-116 cells within a certain period of time. The effect revealed a flat curve after 90 min when HCT-116 was treated with 43 centigrade. THP had the strongest effect at any conditions among all tested drugs (P<0.01). Either simple thermotherapy or chemohyperthermia displayed considerable killing effects on cancer cells which were confirmed by microscope observation. And a great deal of dead cells were observed when treated with chemohyperthermia. CONCLUSIONS: DDP or MMC reveals relatively satisfactory antitumor effects with the optimal temperature of 43-45 degrees C. Taken practical application into consideration, 60 min may be selected in clinical use. Synergistic antitumor effects of THP in combination with hyperthermia were prior to 5-FU, DDP or MMC, which deserve further clinical research.

[Inhibitory effect of N-terminal of p55PIK--regulatory subunit of phosphoinositide-3 kinase--on proliferation of gastric cancer cell line MGC803 and its mechanism].

BACKGROUND & OBJECTIVE: p55PIK is one of the regulatory subunits of phosphoinositide-3 kinase (PI3K). The unique 24 amino acids at N-terminal of p55PIK can bind Rb, and their ectopic expression may inhibit cell cycle progression. This study was to observe the effects of ectopic expression of the 24 amino acids at N-terminal of p55PIK (N24p55PIK) on cell proliferation and tumor growth of gastric cancer, and explore possible mechanism. METHODS: Plasmid pEGFPN24 was transfected into gastric cancer cell line MGC803 (MGC803/GFP-N24); pEGFPC1 was transfected into MGC803 cells as control (MGC803/pEGFPC1). Transient expression of GFP-N24 fusion protein was confirmed by Western blot. The growth of cell clones was determined by MTT assay. Effect of N24p55PIK overexpression on cell clonogenic ability was detected by colony formation assay. Tumorigenic capacity of MGC803/GFR-N24 cells was tested by tumorigenicity assay in nude mice. Influence of N24p55PIK on the expression of cell cycle protein Cyclin D1 was analyzed by Western blot. RESULTS: N24p55PIK was efficiently expressed in MGC803 cells, but the level of GFP-N24 fusion protein in MGC803/GFP-N24 cells was much lower than that of GFP in MGC803/pEGFPC1 cells. Compared with MGC803/pEGFPC1 cells, the growth of MGC803/GFP-N24 cells was suppressed and the cell doubling time was prolonged. The volume of MGC803/GFP-N24 cell colonies was smaller than that of MGC803/pEGFPC1 cell colonies. The tumorigenic capacity of MGC803 cells was decreased after transfection of pEGFPN24 in nude mice. The tumor weight and volume were (0.398+/-0.244) g and (408+/-268) mm(3) in MGC803/pEGFPN24 group, and were (0.763+/-0.193) g and (829+/-271) mm(3) in MGC803/pEGFPC1 group (P<0.05). The expression of Cyclin D1 was down-regulated in MGC803/GFP-N24 cells. CONCLUSIONS: Ectopic expression of N24p55PIK might inhibit tumor cell growth both in vitro and in vivo through decreasing the expression of Cyclin D1. The N24 peptide, derived from PI3K regulatory subunit p55PIK, may be a potential drug in antitumor treatment.

[Efficient expression of exogenous human gp96 gene in tumor cells].

BACKGROUND & OBJECTIVE: Immunization of mice with preparations of heat shock protein(HSP) gp96 isolated from cancer cells has been shown to elicit specific protective cytotoxic T lymphocyte response against cells from which gp96 originate. This phenomenon exploits a new practicable pathway for cancer immunotherapy. But gp96 is generally expressed at low level in cells. Gp96 preparations from limited cells or tissue are difficult to meet the needs of study. So the current study aims to acquire minoclonal cell lines expressing gp96 at high level in order to prepare enough gp96 with high quality. METHODS: The recombinant plasmid pcDNA-hgp96 of human gp96 cDNA was constructed by ligating the fragment of gp96 cDNA into the pcDNA3 plasmid, a eucaryotic expressing vector. Then the recombinant plasmid was transfected into Hela cells and the stable transfectants were selected with G418. The expression level of gp96 of the positive monoclonal cells was detected by Western blot analysis. RESULTS: The recombinant expression plasmid of human gp96 cDNA was successfully constructed. The monoclonal cell lines with stable transfection were obtained. A monoclonal cell line expressing gp96 on high level was selected out. CONCLUSIONS: The monoclonal cell line expressing gp96 at high level has been successfully established, which lays the groundwork for the study of its antitumor immunity.