Mechanism of metformin enhancing the sensitivity of human pancreatic cancer cells to gem-citabine by regulating the PI3K/Akt/mTOR signaling pathway.

OBJECTIVE: To investigate the effect of metformin (MET) on enhancing the sensitivity of human pancreatic cancer cells to gemcitabine (GEM) by regulating the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway. MATERIALS AND METHODS: The GEM-resistant human pancreatic cancer PANC-1/GEM cell line was established, and the proliferation ability of PANC-1 and PANC-1/GEM cell lines was detected using the Cell Counting Kit-8 (CCK-8), which was then detected by flow cytometry after they were labeled by Ki67. Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) and Western blotting were adopted to detect the difference in the mTOR expression between PANC-1 and PANC-1/GEM cell lines. The proliferation ability of PANC-1/GEM/MET and PANC-1/GEM cell lines was determined using CCK-8 after drug-resistant cell lines were treated with 20 mmol/L MET combined with 0.4 µmol/L GEM or 0.4 µmol/L GEM alone for 48 h. Colony formation assay was applied to detect the proliferation ability of cells. The difference in the expression of mTOR/PI3K/Akt between PANC-1/GEM/MET and PANC-1/GEM cell lines was tested via qRT-PCR and Western blotting, respectively. RESULTS: Compared with PANC-1 cells, PANC-1/GEM cells had significantly enhanced proliferation ability (p<0.01). Flow cytometry results showed that the proliferation ability of PANC-1/GEM cells was notably enhanced (p<0.01). The expression level and phosphorylation level of mTOR in drug-resistant cell lines were increased (p<0.01). After the drug-resistant cell lines were treated with 20 mmol/L MET for 48 h, the proliferation ability of PANC-1/GEM/MET cells was evidently decreased compared with that of PANC-1/GEM cells (p<0.01). The messenger ribonucleic acid (mRNA) and protein expression levels of mTOR/PI3K/Akt were markedly down-regulated (p<0.01). CONCLUSIONS: MET can regulate the PI3K/Akt/mTOR signaling pathway to enhance the sensitivity of human pancreatic cancer cells to GEM.

[Value of fibrinogen to albumin ratio on predicting spontaneous recanalization of infarct-related artery in patients with acute ST-segment elevation myocardial infarction].

Objective: To investigate the value of fibrinogen to albumin ratio (FAR) at admission on predicting spontaneous recanalization of infarct-related artery (IRA) in patients with acute ST-segment elevation myocardial infarction (STEMI). Methods: Clinical data from 255 acute STEMI patients ((61.1±11.2) years old, 189 males) who underwent emergency coronary angiography within 12 hours in our hospital from December 2015 to April 2018 were retrospectively analyzed. The acute STEMI patients were divided into non-spontaneous recanalization group (thrombolysis in myocardial infarction (TIMI) flow grade 0-1, 203 cases) and spontaneous recanalization group (TIMI flow grade 2-3, 52 cases). Multivariate logistic regression analysis was used to evaluate related factors of IRA spontaneous recanalization. The receiver operating characteristic (ROC) curve was used to evaluate the value of FAR in predicting spontaneous coronary recanalization. Results: There was no significant difference in age,gender, hypertension, diabetes, smoking,systolic blood pressure,diastolic blood pressure,heart rate, duration of chest pain, type of infarction, infarct-related artery, door-to-balloon time, and drug used before admission between non-spontaneous recanalization group and spontaneous recanalization group (all P>0.05). The FAR and high-sensitivity C-reactive protein levels were significantly lower in the spontaneous recanalization group than in the non-spontaneous recanalization group (8.20±1.85 vs. 11.02±2.75, P<0.001; (6.87±3.36) g/L vs. (8.51±3.72) g/L, P=0.004). Multivariate logistic regression analysis showed that FAR (OR=0.492, 95%CI 0.354-0.686, P<0.001), serum uric acid (OR=0.994, 95%CI 0.989-0.999, P=0.018) and high-sensitivity C-reactive protein (OR=0.774, 95%CI 0.614-0.975, P=0.030) were independent negative correlation with spontaneous recanalization of infarct-related artery in patients with acute STEMI. The ROC curve showed that the area under the curve of FAR predicting spontaneous recanalization of infarct-related artery in patients with acute STEMI was 0.807 (95%CI 0.630-0.758, P<0.001), and the diagnostic threshold was 9.26, the sensitivity was 76.9%, the specificity was 75.9%. Conclusion: The level of admission FAR has certain predictive value for spontaneous recanalization of infarct-related arteries in patients with acute STEMI.

[Application of RapidHIT™ 200 System in Forensic Medicine].

OBJECTIVES: To validate the analysis capability of RapidHIT™ 200 system for four kinds of routine forensic samples and the recyclable capability of template, template DNA and PCR products in the process of twice duplicate detection. METHODS: The buccal swabs underwent the test twice by RapidHIT™ 200 system, and the template DNA and PCR products that arose in the system were also tested for two times. After four kinds of routine forensic samples were detected by RapidHIT™ 200 system, the follow-up tests of the template, template DNA and PCR products that arose in the system were performed. RESULTS: The STR loci could be detected in the buccal swabs by the system for the first time. However, part of the STR loci lost during the second test. And the peak value obtained in the second test was significantly reduced than the one in the first time. The average STR loci detection rates of the template DNA and PCR products were both less than 50% in the second test, which were significantly reduced than that in the first test. In addition, the analysis capability of the system for the tissues and buccal swabs was better than that for the blood and cigarette butts. Compared with the first test, the STR loci detection rate of the tested items, template DNA and PCR products decreased with the numbers of tests. CONCLUSIONS: RapidHIT™ 200 system is more effective in retesting buccal swabs than other samples, whereas the items, DNA template, PCR products obtained in the first and second time cannot be directly used for the further application and study of forensic medicine.

S-phase kinase-associated protein 2 knockdown blocks colorectal cancer growth via regulation of both p27 and p16 expression.

The objective of this study was to determine the role and mechanism of S-phase kinase-associated protein 2 (Skp2) in colorectal cancer cell proliferation and survival both in vitro and in vivo. Adenoviral vector expressing Skp2 short hairpin RNA was transduced into SW480 cells. The effects of Skp2 on cell cycle and survival were assessed by Flow Cytometry. Cell proliferation was analyzed by MTT assay. The expression of cell cycle regulators p16 and p27 were measured by western blot. In vivo, human colorectal cancer was produced by xenograft of cancer cells in nude mouse. Tumor growth inhibitory rate was calculated to generate growth curve. Tumor growth was monitored by examining proliferating cell nuclear antigen expression, whereas tumor cell apoptosis was detected by TdT-mediated dUTP nick-end labeling (TUNEL) staining. Knockdown of Skp2 blocked SW480 tumor cell growth and induced cell apoptosis. Skp2 appeared to be very important for the progression of cell cycle at G1/S phase. In vivo, blockade of Skp2 expression inhibited tumor growth and induced tumor apoptosis. Mechanistically, Skp2 regulated the expression of both p27 and p16 both in vitro and in vivo. The conclusion that we derive from this study is that Skp2 regulates colorectal cancer cell growth by inhibiting the expression of cell cycle regulator p27 and p16.

Prognostic significance of cyclinD1 amplification and the co-alteration of cyclinD1/pRb/ppRb in patients with esophageal squamous cell carcinoma.

CyclinD1/pRb/ppRb is one of the most important pathways regulating the cell cycle, and related with the development of many cancers. However, the co-alteration of CyclinD1/pRb/ppRb in esophageal squamous cell carcinomas is less understood. This study aims to analyze the combined prognostic significance of cyclinD1 (CCND1) DNA amplification and the co-alteration of CCND1/pRb/ppRB in patients with esophageal squamous cell carcinoma. CCND1 DNA amplification and the protein expression of CCND1, pRb, and ppRb on 100 tumor specimens and 11 normal tissues were detected using real-time quantitative reverse transcription polymerase chain reaction and immunohistochemistry, respectively. Their prognosis significance was analyzed by Kaplan-Meier method. We found that 41% of the patients had CCND1 DNA amplification, which had a short survival time compared with the patients without CCND1 amplification (25.63 months vs. not reached, P=0.007). The patients with the co-alternation of CCND1(+) /pRb(-) /ppRb(+) protein expression levels have a poorer overall survival than the others (11.4 vs. 43.4 months, P=0.001). Cox regression analysis showed that the co-alternation of CCND1/pRb/ppRb and CyclinD1 amplification were the two most independent prognosis factors of patients with esophageal cancer. These findings suggested that CCND1 amplification and co-alternation of CCND1(+) /pRb(-) /ppRb(+) may play a crucial role in the prognostic evaluation of patients with esophageal cancer, and the patients with CCND1(+) /pRb(-) /ppRb(+) have the worst prognosis in all the patients. The results also indicated that the patients with CCND1 amplification or co-alternation of CyclinD1(+) /pRb(-) /ppRb(+) might be the preponderant people for therapy targeting the CCND1/pRb/ppRb pathway in the future.

Ablative therapies for colorectal liver metastases: a systematic review.

AIM: The standard treatment for colorectal liver metastases (CRLM) is surgical resection. Only 20-30% of patients are deemed suitable for surgery. Recently, much attention has focused on ablative therapies either to treat unresectable CRLM or to extend the margins of resectability. This review aims to assess the long-term outcome and complication rates of various ablative therapies used in the management of CRLM. METHOD: A literature search was performed of electronic databases including Medline, Cochrane Collaboration Library and the National Library of Medicine's ClinicalTrials.gov. Inclusion criteria were ablation for CRLM with minimum 1 year follow-up and >10 patients, published between January 1994 and January 2010. RESULTS: In all, 226 potentially relevant studies were identified, of which 75 met the inclusion criteria. Cryotherapy (26 studies) had local recurrence rates of 12-39%, with mean 1-, 3- and 5-year survival rates of 84%, 37% and 17%. The major complication rate ranged from 7% to 66%. Microwave ablation (13 studies) had a local recurrence rate of 5-13%, with a mean 1-, 3- and 5-year survival of 73%, 30% and 16%, and a major complication rate ranging from 3% to 16%. Radiofrequency ablation (36 studies) had a local recurrence rate of 10-31%, with a mean 1-, 3- and 5-year survival of 85%, 36% and 24%, with major complication rate ranging from 0% to 33%. CONCLUSION: Ablative therapies offer significantly improved survival compared with palliative chemotherapy alone with 5-year survival rates of 17-24%. Complication rates amongst commonly used techniques are low.

Hepatic steatosis, body mass index and long term outcome in patients undergoing hepatectomy for colorectal liver metastases.

BACKGROUND: Hepatic steatosis (HS) is as an independent risk factor for morbidity and mortality post-hepatectomy. Recent studies report significant correlation between chemotherapy (now frequently employed pre-hepatectomy for colorectal liver metastases (CRLM)), HS and steatohepatitis. Furthermore, raised body mass index (BMI) predisposes to HS. However, no previous study has analysed the effect of HS on long-term survival. METHOD: A retrospective analysis of a prospective consecutive cohort of 102 patients undergoing hepatectomy with 60 months follow-up data was performed. Resection specimens were examined histologically and the degree of steatosis graded accordingly. The data was compared to BMIs and other clinical characteristics. Statistical analyses included log-rank, contingency, logistic regression and Fisher's exact tests. RESULTS: No detectable fatty change in 27 patients; 1 patient had cirrhosis; 57 had HS: 26 graded mild; 10 moderate, 21 severe and 17 not graded. 1 patient (BMI 29.5 kg/m(2)) had steatohepatitis but survived surgery. No significant difference in median survival between patients with and without HS (28.6 vs. 32.3 months, log-rank p>0.05). Results were similar between patients with BMI<25 and BMI>or=25 (32.3 vs. 36.8 months, log-rank p>0.05). Analyses of BMI against steatosis grade showed that patients with a higher BMI were at an increased risk of having a more severe HS (logistic regression, p<0.01; Fisher's exact, p<0.01). Contingency analyses on the influence of diabetes, chemotherapy and increasing number of risk factors on the likelihood of obtaining HS were insignificant (Fisher's exact, all p>0.05). CONCLUSION: While patients with higher body mass index values are at increased risk of having more severe hepatic steatosis, neither BMI nor hepatic steatosis significantly influences long-term survival. We conclude therefore that neither obesity nor hepatic steatosis has significant prognostic relevance on long-term survival of CRLM patients undergoing hepatectomy.

Dental caries patterns and oral health behaviors in Arizona infants and toddlers.

OBJECTIVES: The aim of this study was to use tooth eruption sequence, and a tooth- and surface-specific caries analysis method to determine: (1) the temporal relationship between tooth eruption and caries onset; (2) the validity of pre-existing concepts of caries progression; and (3) the relationship of certain putative health behaviors with caries prevalence. METHODS: A total of 2,428 Arizona children aged 6-36-months, who were recruited from WIC programs (a federal program for low-income children at nutritional risk), health fairs and private day care centers, received visual dental caries examinations. Additionally, an oral health behavior survey was administered to the parents of the 1,529 children recruited from the WIC programs. RESULTS: Dental caries was detected soon after tooth eruption, and by 34-36 months of age 25% of this population had caries. Maxillary anterior caries developed as early as 10-12 months of age. Fissure caries of the molars, either by itself or with maxillary anterior caries, was seen as early as 13-15 months of age. Posterior proximal caries was seen as early at 19-21 months, and only was present in conjunction with the other patterns. Over 40% of the 13-36-month-old children whose parents completed the survey still used a bottle. Night-time bottle use was associated with maxillary anterior caries in 24-36-month-old children, but no association was found in younger children or with posterior caries patterns. Survey responses also showed that fewer than 15% of these children reported having had a dental visit. CONCLUSIONS: Dental caries was a significant health issue for these children under 3 years of age, and factors other than bottle feeding may play an important role in its etiology. Prevention of dental caries in children under age three will depend on a better understanding of the etiology as well as improved access to care.

Umbilical cord blood lead levels in Shanghai, China.

This study was designed to determine the cord blood lead (BPb) levels of babies born in one urban area of Shanghai, and to preliminarily identify the demographic, social environment and prenatal factors which have an effect on the cord BPb concentrations. From August to November 1993, umbilical cord blood samples were obtained from 605 live newborns in the Yangpu Maternal and Child Hospital. 257 samples were excluded from measurement because of clotting. In 348 cord samples, the geometric mean of cord BPb levels was 9.2 micrograms/dl, with a 95% confidence interval of the mean 8.86-9.54 (micrograms/dl). 142 babies (40.8%) had cord BPb levels of 10 micrograms/dl or greater. As a result of this high percentage of newborns with BPb levels equal to or greater than 10 micrograms/dl, we estimate that each year in the Shanghai City about 60,000 newborns are at risk for developing neuropsychological deficiencies caused by maternal lead exposure during pregnancy. To investigate the factors affecting cord blood levels, the subjects with levels greater than the 70th percentile (10.7 micrograms/dl) (n = 104) and less than the 30th percentile (7.4 micrograms/dl) (n = 104) were selected to compare the demographic, environment and prenatal medical history. Increased BPb levels at birth were associated with maternal passive smoking, a family member being occupationally exposed to lead, proximity to major traffic way, household coal combustion, neighborhood coal combustion, low level of maternal occupations, and the increasing occurrence of having the high lead foodstuff pidan (preserved duck egg) during pregnancy. We conclude that prenatal lead exposure has become an important health issue for young children in Shanghai.

A calcium conducting channel akin to a calcium pump.

Calcium conducting channels were studied in blebs of sarcoplasmic reticulum described by Stein & Palade (1988). The calcium channels had at least three conductance states (70 pS, 50 pS and 37 pS) and were weakly selective for calcium ions, with a permeability ratio Ca2+ to K+ of about 3.4. The open probability of the channel was strongly voltage dependent, decreasing at positive membrane voltages. 10 microM ryanodine and 5 microM ruthenium red had no effect on this channel; neither did millimolar concentrations of ATP, Mg2+, caffeine, and Ca2+, implying that the calcium conducting channels are not ryanodine receptors. Several calcium pump inhibitors--namely, vanadate, AlF4-, reactive red 120, and cyclopiazonic acid--had obvious effects on the calcium conducting channels, suggesting that the calcium conducting channel of SR membrane blebs is some form of the SR calcium pump.

Immunoglobulin G-induced single ionic channels in human alveolar macrophage membranes.

While it is well known that the engagement of IgG Fc receptors on the macrophage surface triggers a number of cellular responses, including particle ingestion, secretion, and respiratory burst activity, the mechanism of signal transmission following ligand binding remains poorly understood. To acquire more data in this area, we studied the electrical properties of the macrophage membrane and its response to oligomeric immunoglobulin G (IgG) using the patch-clamp technique on human alveolar macrophages that were obtained by bronchoalveolar lavage and maintained in short-term tissue culture. The results showed that cell resting potentials, as determined from whole-cell tight seal recordings, increased from -15 mV on the day of plating to -56 mV after the first day in culture and remained stable at this hyperpolarized level. Macrophages revealed an input resistance of 3.3 G omega, independent of age in culture. Extracellular application of heat-aggregated human IgG to cells voltage-clamped at -70 mV resulted in peak inward currents of approximately 470 pA. We identified an IgG-dependent, nonselective channel in both cell-attached and isolated membrane patches, with a unitary conductance of approximately 350 pS and a predominant subconductance level of 235 pS in symmetrical NaCl solutions. Single channel open times were observed to be in the range of seconds and, in addition, were dependent upon membrane voltage. Channel opening involved transitions between a number of kinetic states and subconductance levels. Channel events recorded in cell-attached patches showed characteristic exponential relaxations, which implied a variation in membrane potential as a result of a single ion channel opening. These data suggest that the IgG-dependent nonselective cation channel that we have characterized may provide the link between Fc receptor engagement and subsequent cellular activation.

Single-channel recordings of apical membrane chloride conductance in A6 epithelial cells.

The apical membrane of epithelial cells from the A6 cell line grown on impermeable substrata was studied using the patch-clamp technique. We defined the apical membrane as that membrane in contact with the growth medium. In about 50% of the patches, channels with single-unit conductances of 360 +/- 45 pS in symmetrical 105 mM NaCl solutions, and characteristic voltage-dependent inactivation were observed. Using excised membrane patches and varying the ionic composition of the bathing medium, we determined that the channels were anion selective, with a permeability ratio for Cl- over Na+ of about 9:1, calculated from the reversal potential using the constant-field equation. The channel was most active at membrane potentials between +/- 20 mV and inactivated, usually within a few seconds, at higher potentials of either polarity. Reactivation from this inactivation was slow, sometimes requiring minutes. In addition to its fully open state, the channel could also enter a flickering state, which appeared to involve rapid transitions to one or more submaximal conductance levels. The channel was inhibited by the disulfonic stilbene SITS in a manner characteristic of reversible open-channel blockers.