Metagenomic next-generation sequencing, instead of procalcitonin, could guide antibiotic usage in patients with febrile acute necrotizing pancreatitis: a multicenter, prospective cohort study.

BACKGROUNDS: The effectiveness of procalcitonin-based algorithms in guiding antibiotic usage for febrile acute necrotizing pancreatitis (ANP) remains controversial. Metagenomic next-generation sequencing (mNGS) has been applied to diagnose infectious diseases. The authors aimed to evaluate the effectiveness of blood mNGS in guiding antibiotic stewardship for febrile ANP. MATERIALS AND METHODS: The prospective multicenter clinical trial was conducted at seven hospitals in China. Blood samples were collected during fever (T ≥38.5°C) from ANP patients. The effectiveness of blood mNGS, procalcitonin, and blood culture in diagnosing pancreatic infection was evaluated and compared. Additionally, the real-world utilization of antibiotics and the potential mNGS-guided antimicrobial strategy in febrile ANP were also analyzed. RESULTS: From May 2023 to October 2023, a total of 78 patients with febrile ANP were enrolled and 30 patients (38.5%) were confirmed infected pancreatic necrosis (IPN). Compared with procalcitonin and blood culture, mNGS showed a significantly higher sensitivity rate (86.7% vs. 56.7% vs. 26.7%, P <0.001). Moreover, mNGS outperformed procalcitonin (89.5 vs. 61.4%, P <0.01) and blood culture (89.5 vs. 69.0%, P <0.01) in terms of negative predictive value. Blood mNGS exhibited the highest accuracy (85.7%) in diagnosing IPN and sterile pancreatic necrosis, significantly superior to both procalcitonin (65.7%) and blood culture (61.4%). In the multivariate analysis, positive blood mNGS (OR=60.2, P <0.001) and lower fibrinogen level (OR=2.0, P <0.05) were identified as independent predictors associated with IPN, whereas procalcitonin was not associated with IPN, but with increased mortality (Odds ratio=11.7, P =0.006). Overall, the rate of correct use of antibiotics in the cohort was only 18.6% (13/70) and would be improved to 81.4% (57/70) if adjusted according to the mNGS results. CONCLUSION: Blood mNGS represents important progress in the early diagnosis of IPN, with particular importance in guiding antibiotic usage for patients with febrile ANP.

Is Contemporary Open Pancreatic Necrosectomy Still Useful In The Minimally Invasive Era?

BACKGROUND: Previous studies have shown that open pancreatic necrosectomy for infected pancreatic necrosis was associated with high morbidity and mortality. However, these results were mostly concluded from historical cohorts with traditional early necrosectomy in the absence of a minimally invasive step-up approach. OBJECTIVE: To explore the value of contemporary open pancreatic necrosectomy for infected pancreatic necrosis in the minimally invasive era. METHODS: A post hoc analysis was performed in a prospective maintained database of 320 patients with infected pancreatic necrosis from January 2011 to December 2022 at a large Chinese tertiary hospital. RESULTS: A total of 320 patients with infected pancreatic necrosis received either a minimally invasive step-up approach (245, 76.6%) or open pancreatic necrosectomy (75, 23.4%), which included upfront open pancreatic necrosectomy (32, 10.0%) and salvage open pancreatic necrosectomy (43, 13.4%). Upfront open pancreatic necrosectomy was associated with similar morbidity and mortality rates but fewer surgical interventions compared with a minimally invasive step-up approach. However, salvage open pancreatic necrosectomy was associated with significantly higher mortality (48.8% vs 18.8%, P = .007), gastrointestinal fistula (44.2% vs 18.8%, P = .021), hemorrhage (48.8% vs 15.6%, P = .003), and intensive care unit stay (25 vs 7 days, P = .040) compared with upfront open pancreatic necrosectomy. Multivariate analysis suggested that multiple organ failure (hazard ratio = 5.1; 95% confidence interval, 1.4-18.2, P = .013) and synchronous critical acute pancreatitis (hazard ratio = 3.0; 95% confidence interval, 1.1-8.6, P = .040) were 2 independent risk factors of death for patients who received open pancreatic necrosectomy. CONCLUSION: Patients undergoing upfront open pancreatic necrosectomy received fewer surgical interventions with comparable efficacy compared to the minimally invasive step-up approach. Salvage open pancreatic necrosectomy was potentially lifesaving, though it carried high morbidity and mortality. Multiple organ failure and synchronous critical acute pancreatitis were 2 independent risk factors of death for patients who received open pancreatic necrosectomy.

Prediction of survival in patients with infected pancreatic necrosis: a prospective cohort study.

BACKGROUND: Infected pancreatic necrosis (IPN) is a severe complication of acute pancreatitis, with mortality rates ranging from 15 to 35%. However, limited studies exist to predict the survival of IPN patients and nomogram has never been built. This study aimed to identify predictors of mortality, estimate conditional survival (CS), and develop a CS nomogram and logistic regression nomogram for real-time prediction of survival in IPN patients. METHODS: A prospective cohort study was performed in 335 IPN patients consecutively enrolled at a large Chinese tertiary hospital from January 2011 to December 2022. The random survival forest method was first employed to identify the most significant predictors and capture clinically relevant nonlinear threshold effects. Instantaneous death risk and CS was first utilized to reveal the dynamic changes in the survival of IPN patients. A Cox model-based nomogram incorporating CS and a logistic regression-based nomogram were first developed and internally validated with a bootstrap method. RESULTS: The random survival forest model identified seven foremost predictors of mortality, including the number of organ failures, duration of organ failure, age, time from onset to first intervention, hemorrhage, bloodstream infection, and severity classification. Duration of organ failure and time from onset to first intervention showed distinct thresholds and nonlinear relationships with mortality. Instantaneous death risk reduced progressively within the first 30 days, and CS analysis indicated gradual improvement in real-time survival since diagnosis, with 90-day survival rates gradually increasing from 0.778 to 0.838, 0.881, 0.974, and 0.992 after surviving 15, 30, 45, 60, and 75 days, respectively. After further variables selection using step regression, five predictors (age, number of organ failures, hemorrhage, time from onset to first intervention, and bloodstream infection) were utilized to construct both the CS nomogram and logistic regression nomogram, both of which demonstrated excellent performance with 1000 bootstrap. CONCLUSION: Number of organ failures, duration of organ failure, age, time from onset to first intervention, hemorrhage, bloodstream infection, and severity classification were the most crucial predictors of mortality of IPN patients. The CS nomogram and logistic regression nomogram constructed by these predictors could help clinicians to predict real-time survival and optimize clinical decisions.

Novel insights into biomarkers of progression in Desmoid tumor.

Desmoid tumor (DT) is a rare neoplasm characterized by the proliferation of myofibroblastic cells that infiltrates and invades adjacent tissues. Due to its locally aggressive and recurrent nature, DT often causes local symptoms and can be challenging to manage clinically. Therefore, identifying biomarkers that can predict the progression of DT and guide treatment decisions is critical. This review summarizes several biomarkers that have been implicated in active surveillance (AS) and the prediction of postoperative recurrence and attempts to elucidate their underlying mechanisms. Some of these novel markers could provide prognostic value for clinicians, and ultimately help facilitate optimal and accurate therapeutic decisions for DT.

CONSTRUCTION OF SEPSIS DIAGNOSTIC MODELS AND IDENTIFICATION OF MACROPHAGE SUBPOPULATIONS BASED ON PYROPTOSIS-RELATED GENES.

ABSTRACT: Background: Numerous studies have shown that pyroptosis is associated with sepsis progression, which can lead to dysregulated host immune responses and organ dysfunction. Therefore, investigating the potential prognostic and diagnostic values of pyroptosis in patients with sepsis is essential. Methods: We conducted a study using bulk and single-cell RNA sequencing (scRNA-seq) from the Gene Expression Omnibus database to examine the role of pyroptosis in sepsis. Univariate logistic analysis, least absolute shrinkage, and selection operator regression analysis were used to identify pyroptosis-related genes (PRGs), construct a diagnostic risk score model, and evaluate the selected genes' diagnostic value. Consensus clustering analysis was used to identify the PRG-related sepsis subtypes with varying prognoses. Functional and immune infiltration analyses were used to explain the subtypes' distinct prognoses, and scRNA-seq data were used to differentiate immune-infiltrating cells and macrophage subsets and study cell-cell communication. Results: A risk model was established based on 10 key PRGs ( NAIP , ELANE , GSDMB , DHX9 , NLRP3 , CASP8 , GSDMD , CASP4 , APIP , and DPP9 ), of which four ( ELANE , DHX9 , GSDMD , and CASP4 ) were associated with prognosis. Two subtypes with different prognoses were identified based on the key PRG expressions. Functional enrichment analysis revealed diminished nucleotide oligomerization domain-like receptor pathway activity and enhanced neutrophil extracellular trap formation in the subtype with a poor prognosis. Immune infiltration analysis suggested a different immune status between the two sepsis subtypes, with the subtype with a poor prognosis exhibiting stronger immunosuppression. The single-cell analysis identified a macrophage subpopulation characterized by gasdermin D (GSDMD) expression that may be involved in pyroptosis regulation, which was associated with the prognosis of sepsis. Conclusion: We developed and validated a risk score for sepsis identification based on 10 PRGs, four of which also have potential value in the prognosis of sepsis. We identified a subset of gasdermin D macrophages associated with poor prognosis, providing new insights into the role of pyroptosis in sepsis.

Escherichia coli infection indicates favorable outcomes in patients with infected pancreatic necrosis.

Introduction: Infected pancreatic necrosis (IPN) is a severe complication of acute necrotizing pancreatitis with increasing morbidity. Escherichia coli is the most frequently cultured microorganism in IPN. However, the implications of Escherichia coli infection on the outcomes of patients with IPN remain unclear. Therefore, this study aimed to evaluate the clinical impacts of Escherichia coli infection on IPN. Methods: A prospective database with consecutive patients with IPN between January 2010 and April 2022 at a tertiary hospital was post-hoc analyzed. The clinical and microbiological characteristics, surgical management, and follow-up data of patients with and without Escherichia coli infection were compared. Results: A total of 294 IPN patients were enrolled in this cohort. Compared with non-Escherichia coli infection cases (n=80, 27.2%), patients with Escherichia coli infection (n=214, 72.8%) were characterized by more frequent polymicrobial infections (77.5% vs. 65.0%, P=0.04) but a lower occurrence of severe acute pancreatitis (SAP) (42.5% vs. 61.7%, P=0.003). In addition, significantly lower mortality (12.5% vs. 30.4%, p=0.002), fewer step-up surgical interventions (73.8% vs. 85.1%, P=0.025), and a lower rate of multiple organ failure (MOF) (25.0% vs. 40.2%, P=0.016) were also observed in patients with Escherichia coli infection. Multivariate analysis of mortality predictors indicated that MOF (odds ratio [OR], 6.197; 95% confidence interval [CI], 2.373-16.187; P<0.001) and hemorrhage (OR, 3.485; 95% CI, 1.623-7.487; P=0.001) were independent predictors associated with higher mortality in patients with IPN. Escherichia coli infection was significantly associated with a lower mortality (OR, 0.302; 95% CI, 0.121-0.751; P= 0.01). Conclusion: Escherichia coli infection indicates a favorable prognosis in patients with IPN, although the mechanism needs further investigation.

Novel Insights into the Roles and Mechanisms of GLP-1 Receptor Agonists against Aging-Related Diseases.

Aging and aging-related diseases have emerged as increasingly severe health and social problems. Therefore, it is imperative to discover novel and effective therapeutics to delay the aging process and to manage aging-related diseases. Glucagon-like peptide-1 receptor agonists (GLP-1 RAs), one of the classes of antihyperglycemic drugs, have been recommended to manage type 2 diabetes mellitus (T2DM). Moreover, GLP-1 RAs have been shown to protect against oxidative stress, cellular senescence and chronic inflammation, which are widely accepted as the major risk factors of aging. However, their significance in aging or aging-related diseases has not been elucidated. Herein, we explain the underlying mechanisms and protective roles of GLP-1 RAs in aging from a molecular, cellular and phenotypic perspective. We provide novel insights into the broad prospect of GLP-1 RAs in preventing and treating aging-related diseases. Additionally, we highlight the gaps for further studies in clinical applications of GLP-1 RAs in aging-related diseases. This review forms a basis for further studies on the relationship between aging-related diseases and GLP-1 RAs.

Integrated genomic analysis reveals regulatory pathways and dynamic landscapes of the tRNA transcriptome.

tRNAs and tRNA-derived RNA fragments (tRFs) play various roles in many cellular processes outside of protein synthesis. However, comprehensive investigations of tRNA/tRF regulation are rare. In this study, we used new algorithms to extensively analyze the publicly available data from 1332 ChIP-Seq and 42 small-RNA-Seq experiments in human cell lines and tissues to investigate the transcriptional and posttranscriptional regulatory mechanisms of tRNAs. We found that histone acetylation, cAMP, and pluripotency pathways play important roles in the regulation of the tRNA gene transcription in a cell-specific manner. Analysis of RNA-Seq data identified 950 high-confidence tRFs, and the results suggested that tRNA pools are dramatically distinct across the samples in terms of expression profiles and tRF composition. The mismatch analysis identified new potential modification sites and specific modification patterns in tRNA families. The results also show that RNA library preparation technologies have a considerable impact on tRNA profiling and need to be optimized in the future.

BCREval: a computational method to estimate the bisulfite conversion ratio in WGBS.

BACKGROUND: Whole genome bisulfite sequencing (WGBS) also known as BS-seq has been widely used to measure the methylation of whole genome at single-base resolution. One of the key steps in the assay is converting unmethylated cytosines into thymines (BS conversion). Incomplete conversion of unmethylated cytosines can introduce false positive methylation call. Developing a quick method to evaluate bisulfite conversion ratio (BCR) is benefit for both quality control and data analysis of WGBS. RESULTS: Here we provide a computational method named "BCREval" to estimate the unconverted rate (UCR) by using telomeric repetitive DNA as native spike-in control. We tested the method by using public WGBS data and found that it is very stable and most of BS conversion assays can achieve> 99.5% efficiency. The non-CpG DNA methylation at telomere fits a binomial model and may result from a random process with very low possibility (the ratio < 0.4%). And the comparison between BCREval and Bismark (Krueger and Andrews, Bioinformatics 27:1571-1572, 2011), a widely used BCR evaluator, suggests that our algorithm is much faster and more efficient than the latter. CONCLUSION: Our method is a simple but robust method to QC and speculates BCR for WGBS experiments to make sure it achieves acceptable level. It is faster and more efficient than current tools and can be easily integrated into presented WGBS pipelines.

[Research on promoter region methylation status of tumor repressor gene P16 in human hepatocellular carcinoma by using methylation sensitive restriction enzyme and semi-nested PCR assay].

OBJECTIVE: To construct a combination method of methylation sensitive restriction enzyme and semi-nested touch down PCR assay for studying the promoter region methylation status of P16 gene in human hepatocellular carcinoma. METHODS: According to the sequence of CpG rich promoter region of P16 gene, three primers were designed and synthesized for semi-nested touch down PCR assay to examine the promoter region methylation status of P16 gene. 340 bp segment of this region was cloned into vector pMD18-T; the plasmid was transformed into E. coli JM109 to harvest an extended quantity, then the plasmid was treated by CpG methylase M. Sss I, the methylated plasmid was named P16Pm+. This P16Pm+ is validated by digestion of Hpa II and is employed in studying the specificity and sensitivity of this constructed method. After construction, the method was used to examine the promoter region methylation status in P16 gene of 40 DNA samples from human HCCs and three DNA samples from normal human liver tissue. RESULTS: It was confirmed that the specificity and sensitivity of this method are solid and reliable (100 fg). It was found that 12/40 (30%) of hepatocellular carcinoma showed promoter region methylation in P16 gene whereas none (0/3) of the normal tissues was methylated in the promoter region in P16 gene. CONCLUSION: Promoter region methylation in P16 gene may take part in human hepatocellular carcinogenesis. The constructed method is simple, cost-effective and is of high specificity and sensitivity, thus suggesting its potential application to detecting promoter methylation in population-based studies.

[Experimental study on molecular mechanism of nm23-H1 gene transfection reversing the malignant phenotype of human high-metastatic large cell lung cancer cell line].

BACKGROUND: nm23-H1 gene is a well-known tumor metastasis suppression gene. Our previous study has found that transfection of wild type nm23-H1 gene can significantly downregulate the ERK1/2 activity of human high-metastatic large cell lung cancer cell line L9981. The aim of this study is to investigate the influence of nm23-H1 and exogenous ERK1/2 pathway inhibitor U0126 on the extracellular signal-regulated kinase (ERK1/2) of human high-metastatic large cell lung cancer cell line L9981 and its malignant biological behaviors. METHODS: The expressive levels of total-ERK1/2, dually phosphorylated ERK1/2 and ERK1/2 relative activity of the human high-metastatic large cell lung cancer cell lines, L9981 (parent cell line with nm23-H1 gene hetero-deletion), L9981-nm23-H1 (transfected with nm23-H1 gene ) and L9981-PLXSN (transfected with vector) were detected by Western blot and immunoprecipitation technique after treating with U0126 (40µmol/L for 20 minutes). The in vitro proliferative and invasive abilities among the above three lung cancer cell lines were determined by MTT and improved Boyden chamber methods. RESULTS: The phosphorylated ERK1/2 expression level and relative activity in L9981-nm23-H1 lung cancer cell line were remarkably lower than those in L9981 and L9981-PLXSN lung cancer cell lines after being treated with U0126 (P < 0.01), but there was no significant difference between L9981 and L9981-PLXSN lung cancer cell lines. No significant difference of total ERK1/2 expression level was observed among the three lung cancer cell lines (P > 0.05) after being treated with U0126. The in vitro proliferation and invasion of L9981-nm-23H1 lung cancer cell line were remarkably lower than those of L9981 and L9981-PLXSN lung cancer cell lines (P < 0.01 ), but no significant difference was found between L9981 and L9981-PLXSN lung cancer cell lines (P > 0.05 ); U0126 could significantly down-regulate the in vitro proliferation and invasion of L9981 lung cancer cell line (P < 0.01). CONCLUSIONS: Blocking the activity of ERK1/2 in L9981 lung cancer cell line and transfecting the nm23-H1 gene into the L9981 lung cancer cell line may produce similar cell biological behavior changes, namely the significant reduction of in vitro proliferation and invasion of L9981 lung cancer cell line. These results indicate that the molecular mechanism which nm23-H1 gene reverses invasion and proliferation of the human high-metastatic large cell lung cancer cell line may be related to its effects of down-regulating the activity of the key kinase ERK1/2 of Ras-to-MAPK signal transduction pathway.

[Growth inhibition and demethylation by a component of natural drug, CDP, in cancer cell lines].

OBJECTIVE: To investigate the inhibition of tumor cell lines' growth and the demethylation of p16 gene by a component of natural drug, CDP. METHODS: Human liver cancer cell line Huh-7 and breast cancer cell line T47D were treated with the CDP and DNA methyltransferase inhibitor, 5-azacytidine (5-aza-C). The inhibition of growth was assayed by MTT; the methylation status of p16 gene promoter was analyzed by MSP. RESULTS: 1. The CpG islands in promoter of p16 gene were identified as completely methylated in Huh-7 and T47D. 2. CDP and 5-aza-C inhibited the proliferation of Huh-7 and T47D cell lines in a dose, time-dependent mode. 3. Methylation-specific bands of CpG islands in p16 gene' promoter were still existed in Huh-7 and T47D, while unmethylation-specific bands appeared in Huh-7 after the cells being treated with 25, 50, 75 and 100 micromol/ L of CDP for 6 days as well as in T47D after the cells being treated with 50, 75 and 100 micromol/L of CDP for 6 days. 4. After the cells being treated with 50 micromol/L of CDP, the methylation-specific bands of CpG island in p16 gene' promoter were still existed in Huh-7 and T47D; unmethylation-specific bands appeared at 12 h and lasted to 144 h in Huh-7 while at 72 h and lasted to 144 h in T47D. CONCLUSION: CDP has inhibition effect on the cancer cell lines and has the ability to reverse the hypermethylated p16 gene promoter. These results suggest that CDP has great potential in the development of effective anticancer drugs.

[Detection of aberrant promoter CpG islands hypermethylation of GSTP1 in human primary hepatocellular carcinomas].

OBJECTIVE: To detect the aberrant promoter CpG islands hypermethylation status of GSTP1 gene in hepatocellular carcinomas (HCC) and to assess its significant role in hepatocarcinogenesis. METHODS: Surgically resected cancerous and non-cancerous liver tissues of 26 hepatocellular carcinoma patients were obtained from West China Hospital, and 11 peripheral blood samples from healthy donors as negative control were collected. Breast cancer cell line MCF-7 with CpG islands hypermethylation of GSTP1 as positive control was obtained from the Cell Bank of Chinese Academy of Sciences in Shanghai. All genomic DNA were extracted using common phenolchloroform approach, and the 5' CpG islands methylation status of GSTP1 gene was studied by methylation-specific polymerase chain reaction (MSP). RESULTS: GSTP1 gene promoter CpG island hyperthylation was detected in 88.5% (23/26) of cancerous tissues and in 69% (18/26) of corresponding non-cancerous tissues from the 26 HCC patients. None of the 11 control samples were methylation positive. CONCLUSION: The data indicates that the detection of GSTP1 gene methyl-lation may be a valuable biomarker by MSP for HCC early diagnosis and disease monitoring.

[Effect of nm23-H1 on reversing malignant phenotype on human lung cancer cell line L9981].

nm23-H1 is a proven tumor metastasis suppressive gene, tumor metastasis phenotype could be reversed by transfected nm23-H1 cDNA. This study was conducted to transfect nm23-H1 cDNA into L9981 cells and to explore the function of nm23-H1 in reversing the malignant phenotype of L9981 cells. The plasmid of pLXSN-nm23-H1-EGFP was constructed by gene clone technique, and the transfected nm23-H1 cDNA cell lines of L9981-nm23-H1 was established. The protein expression of nm23-H1 was detected by Western blot. The biologic features of L9981-nm23-H1 cells were studied in vitro and in vivo. The results showed that the fusion protein of nm23-H1-EGFP was stable, continuous and expressed with high efficiency in L9981-nm23-H1 cells. The cell proliferation, colon formation and invasive ability are significantly lowered in L9981 cells transfected nm23-H1 cDNA (P < 0.01); the tumorgenesis and the lung metastasis incidence was lower in tranfected nm23-H1 cells than in L9981 and L9981-Plxsn in nude mice (P < 0.01); the rate for inhibiting tumorgenesis of nm23 -H1 was 82.56%. These data suggest that the malignant phenotype could be reversed by wild nm23-H1 gene in L9981 cells.

[Molecular mechanism of reversing metastatic phenotype in human high-metastatic large cell lung cancer cell line L9981 by nm23-H1].

BACKGROUND & OBJECTIVE: nm23-H1, a tumor metastasis suppressive gene, can reverse tumor metastasis phenotype. But the molecular mechanism of nm23-H1 in inhibiting or reversing metastasis of lung cancer is unclear. This study was to explore the molecular mechanism of nm23-H1 in reversing metastasis phenotype of lung cancer. METHODS: nm23-H1 gene and pLXSN were separately transfected into human lung cancer cell line L9981. Proliferation of L9981, L9981-pLXSN, and L9981-nm23-H1 cells was detected by MTT assay, cell invasive ability was detected by modified Boyden chamber. Tumorigenesis and experimental lung metastasis were determined in vivo. mRNA and protein levels of beta-catenin, E-Cadherin, CD44S, CD44V6, matrix metalloproteinase-2 (MMP-2), tissue inhibitor of metalloproteinase-1 (TIMP-1), and vascular endothelial growth factor (VEGF) were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. RESULTS: (1)Cell proliferation, clone formation, and invasive ability were significantly lower in L9981-nm23-H1 cells than in L9981 cells [(19.5+/-2.9)% vs. 100%, 10.3+/-0.7 vs. 21.7+/-1.3, 31.0+/-3.0 vs. 151.0+/-6.3, P<0.01]. (2) The inhibitory rate of tumorigenesis of nude mice was significantly higher in L8891-nm23-H1 group than in L9981 group (85.6% vs. 0%, P<0.001)u the lung metastatic rate was significantly lower in L9981-nm23-H1 group than in L9981 group (0% vs. 100%, P<0.001). (3)nm23-H1 up-regulated mRNA and protein levels of beta-catenin, E-Cadherin, and TIMP-1, and down-regulated levels of MMP-2, CD44V6, and VEGF (P<0.01). (4) nm23-H1 up-regulated mRNA level of CD44s, protein level of CD44s didn't change (P>0.05). CONCLUSION: nm23-H1 gene can reverse malignant and metastatic phenotype of L9981 cells through regulating the expressions of lung cancer metastasis-related genes.

[Transcription expression of endostatin mRNA in non-small cell lung cancer tissues].

BACKGROUND: It has been proved that tumor development and metastasis are dependent on angiogenesis. Suppression of tumor angiogenesis can inhibit tumor growth and metastasis. Collagen X VIII/endostatin is one of the most effective inhibitors of angiogenesis at present. The aim of this study is to study the relationship between transcription expression of endostatin mRNA and clinical and pathophysiological characteristics in non-small cell lung cancer (NSCLC). METHODS: The transcription expression of endostatin mRNA was detected in 46 lung cancer tissues and paracancerous lung tissues, 14 benign pulmonary lesion tissues as control by RT-PCR method. RESULTS: (1)The transcription expression of endostatin mRNA in lung cancer tissues (0.872±0.071) was significantly higher than that in paracancerous tissues (0.717±0.073) and benign pulmonary lesion tissues (0.611±0.026) (P < 0.001).(2)The transcription expression of endostatin mRNA in lung cancer tissues was closely related to P-TNM stages, distant metastasis, grade of cell differentiation and size of the primary tumors (P < 0.05), but not to location of tumor, lymph node status, histological classification, age and sex of the patients and smoking or not (P > 0.05). CONCLUSIONS: The transcription expression of endostatin mRNA in NSCLC tissues is significantly higher than that in paracancerous tissues and benign pulmonary lesion tissues, and is closely related to P-TNM stages, distant metastasis, grade of cell differentiation and size of the primary tumors, hence it might be helpful to evaluate the biological behavior of lung cancer.

[Effects of targeting inhibition of ERK1/2 signal transduction pathway on malignant biological behaviors of human high-metastatic large cell lung cancer cell line L9981].

BACKGROUND: It has been known that oncogenesis and development of lung can- cer is a complex process regulated many genes and involved in multistages. Recent studies have demonstrated that signal transduction abnormality may play a very important role in these procedures. The aim of this study is to investigate the influence of exogenous MEK1/2 pathway inhibitor U0126 on the expression and activity of extracellular signal-regulated kinase (ERK1/2) of human high-metastatic large cell lung cancer cell line L9981 and its malignant biological behaviors. METHODS: The expressive levels of total-ERK1/2, dually phosphorylated ERK1/2 and ERK1/2 relative activity of the human high-metastatic large cell lung cancer cell line L9981 (parent cell line with nm23-H1 gene hetero-deletion ) were detected by Western blot and immuno-precipitation technique after treating with different doses of U0126. The in vitro proliferative and invasive abilities of the lung cancer cell line were determined by MTT and modified Boyden chamber methods. RESULTS: The ERK1/2 relative activity of L9981 gradually decreased accompanied with increase of U0126 doses, and a highly significant difference of phosphorylated ERK1/2 expression level existed among the different concentration groups of U0126 (P < 0.01), but no significant difference of total ERK1/2 expression was found among the different concentration groups of U0126 (P=0.387). After treatment with same concentration of U0126 for different time, the ERK1/2 relative activity of L9981 gradually decreased as the treatment time of U0126 prolonging, and a highly significant difference of phosphorylated ERK1/2 expression level was observed among different treatment time groups (P < 0.01). But no significant difference of total ERK1/2 expression level was found among different time groups (P=0.689). The inhibition of ERK1/2 pathway by MEK1/2 specific inhibitor U0126 targeting the ERK1/2 pathway of L9981 was dose- and time-dependent. After treatment with different concentration of U0126, the proliferation of L9981 gradually decreased accompanied with increase of U0126 concentration and a highly significant difference existed among different groups of U0126 concentration (P < 0.001). The in vitro invasion of L9981 gradually decreased accompanied with increase of U0126 concentration. No significant difference of in vitro invasion of L9981 was found among 0, 10 and 20µmol/l of U0126 (P > 0.05). A highly significant difference was observed when U0126 concentration increased to 40 and 60µmol/l compared with 0, 10 and 20µmol/l of U0126 (P < 0.001). CONCLUSIONS: The inhibition of Ras-to-MAPK pathway by Ras-to-MAPK specific inhibitor U0126 targeting the Ras-to-MAPK pathway of the human high-metastatic large cell lung cancer cell line L9981 is dose-dependent and time-dependent. Suppressing or blocking of Ras-to-MAPK signal transduction pathway can reverse the invasive and metastatic phenotype of the human high-metastatic large cell lung cancer cell line L9981. These results suggest that the key kinase MEK1/2 of the ERK1/2 pathway may be a potent therapeutic target for human lung cancer.

[Research on the growth inhibition of T47D cell and reversion of p16 hypermethylation by CDP].

OBJECTIVE: To inquire into the mechanism of CDP (a purified component of Chinese crude drug) in inhibiting the breast cancer T47D cell growth. METHODS: T47D cells were treated with different concentration of 5-azacytidine (5-aza-CR) and CDP for several days. The growth rate was assessed by cell proliferation experiment (MTT colorimetric assay). The changes in apoptic peak and cell cycle distribution were detected by flow cytometry (FCM). The levels of methylation and unmethylation status of p16 were detected by methylation specific PCR (MSP). RESULTS: After treatment with the two drugs, the cell growth rate decreased in a dose and time dependent manner (P<0.05). The cell cycle was influenced by the well-chosen concentration of 5-aza-CR (2 micromol/L) and CDP (50 micromol/L): the cell number increased from 65.1% to 71.3%, 84.3% in G0/G1 phase and decreased from 19.4% to 14.3%, 7.2% in S phase. Demethylation on p16 gene occurred after treatment with any of the two drugs for 6 days. CONCLUSION: CDP can reverse p16 hypermethylation and may hence inhibit the proliferation of tumor cells.

[The imprinting status and expression of insulin-like growth factor 2 gene in human hepatocellular carcinoma].

OBJECTIVE: To study the imprinting status and expression level of insulin-like growth factor 2 (IGF2) gene in hepatocellular carcinoma and to provide a clue for elucidating the mechanism of carcinogenesis of hepatocellular carcinoma. METHODS: The heterozygote status of IGF2 gene was detected by restriction fragment length polymorphism. The imprinting status and expression level of IGF2 were evaluated by semi-quantitative reverse transcription-polymerase chain reaction. RESULTS: It was found that 10/18 (55.6%) of hepatocellular carcinoma showed the gain of imprinting (GOI), with 6/10 (60%) adjacent cirrhosis of liver tissues also displaying GOI of IGF2. Overexpression of IGF2 in cancer tissues was detected in 9/18 (50%) samples, but no significant difference was observed among each imprinting status. CONCLUSION: GOI of IGF2 may take part in human hepatocellular carcinogenesis.