Pioglitazone Use and Risk of Bladder Cancer: an In Vitro Study.

Aims: Whether pioglitazone (PIO), a peroxisome proliferator-activated receptor-gamma agonist, increases the risk of developing bladder cancer has been debated for several years. The aim of this study was to investigate the in vitro effects of PIO on normal urothelial transitional epithelium (NUTE) cells and bladder cancer (J82) cells to further evaluate the risk. Methods: NUTE cells were obtained from Sprague-Dawley rats. NUTE and J82 cells were treated with different concentrations of PIO for various time periods. Cell proliferation was tested by the MTT assay. Cell apoptosis was evaluated by flow cytometry. The expressions of p53, cyclin D1, Bcl-2, and Bax were determined by qRT-PCR and western blots. Results: After 24 hours, the treatment of NUTE cells with 10 µmol/L PIO led to morphological changes, without changes in J82 cells. Moreover, PIO inhibited the proliferation and induced apoptosis of NUTE cells, but not J82 cells, in a time- and dose-dependent manner. However, PIO did not alter the growth of cells from other tissues. In addition, treatment with PIO for up to 72 hours did not result in changes in the expressions of p53, cyclin D1, Bcl-2, and Bax in NUTE cells and J82 cells. Interestingly, PIO significantly downregulated the protein levels of p53 and cyclin D1 in J82 cells, but not NUTE cells after more than 192 hours of treatment. Conclusions: PIO did not promote malignant alterations of NUTE cells or stimulate proliferation of J82 cells. PIO decreased the expression of p53 and cyclin D1 in J82 cells after long-term culture, which suggested that PIO may be helpful for diabetic patients with bladder cancer.

[The safety and effectiveness of once daily detemir in patients with type 2 diabetes previously failing oral agents: the Chinese cohort from SOLVE(TM) observational study].

OBJECTIVE: Study of Once-daily LeVEmir(®) (SOLVE(TM)) was a 24-week international observational study to evaluate the safety and effectiveness of initiating once-daily insulin detemir (Levemir) as add-on therapy in patients with type 2 diabetes mellitus (T2DM) who failed treatment of oral anti-diabetic drugs (OAD). METHODS: The present study was derived from the data of Chinese cohort. A total of 3272 patients with T2DM failing OAD were enrolled in the study. Determir were prescribed to the patients by the decision of the physician. Clinical data were collected at baseline, week 12 and week 24 to evaluate the safety and effectiveness of detemir. RESULTS: The age of the patients was (56.2 ± 10.8) years with a diabetes duration of (7.1 ± 5.2) years. Their BMI was (25.3 ± 3.3) kg/m(2). No patient experienced any major or nocturnal hypoglycaemic event during the study. After 24 weeks of treatment, the glycosylated hemoglobin A1c (HbA1c) decreased from (8.33 ± 1.69)% to (7.16 ± 1.18)% with a mean change of -1.17%, the fasting plasma glucose decreased from (9.52 ± 2.59) mmol/L to (6.84 ± 1.42) mmol/L with a mean change of -2.7 mmol/L, and the 7-point blood glucose profile improved overall. Totally 49.1% of patients achieved HbA1c < 7%. The mean body weight decreased by 0.15 kg. CONCLUSIONS: Insulin detemir administered once daily as add-on therapy in patients with T2DM failing OAD regimen significantly reduces the risk of major hypoglycemia, improves glycemic control, increases the percentage of patients achieving treatment target with neutral effect on body weight.

Assessment of carotid atherosclerosis in type 2 diabetes mellitus patients with microalbuminuria by high-frequency ultrasonography.

The aim of this study is to evaluate carotid atherosclerosis in patients of type 2 diabetes mellitus with microalbuminuria (MA) by high-frequency ultrasonography. Two hundred and fifty patients of type 2 diabetes mellitus were divided into two groups according to urinary albumin excretion rate (UAER): normoalbuminuria group (130 cases) and microalbuminuria group (120 cases). The intimal-medial thickness (IMT) and the atherosclerotic plaques of carotid artery were observed in both groups by high-frequency ultrasound. Fasting blood glucose (FBG), hemoglobin A1c, and lipid profiles were measured. The values of IMT of microalbuminuria group were significantly higher than those of normoalbuminuria group (P < 0.05). In univariate analysis, IMT was positively and significantly associated with age (r = 0.265, P < 0.05), waist circumference (r = 0.263, P < 0.05), body mass index (r = 0.285, P < 0.05), systolic blood pressure (r = 0.276, P < 0.05), UAER (r = 0.359, P < 0.05), HbA1c (r = 0.462, P < 0.05) and, duration of diabetes (r = 0.370, P < 0.05). In multivariate linear regression analysis, UAER and HbA1c were independent predictors of IMT (P < 0.05 for all). In the two groups, the rate of soft plaques was higher than that of dense plaques and calcified plaques. In conclusion, there is a significant association between microalbuminuria and IMT which is regarded as the early sign of carotid atherosclerosis in type 2 diabetic patients.

Assessment of carotid arterial wall elasticity in type 2 diabetes mellitus patients with microalbuminuria by real-time ultrasound elastography.

The aim of this study was to evaluate carotid arterial wall elasticity in type 2 diabetes mellitus (T2DM) with microalbuminuria by real-time ultrasound elastography. Two hundred and ten T2DM patients were divided into two groups according to levels of urinary albumin excretion (UAE): T2DM without microalbuminuria (T2DM1 group, 120) and T2DM with microalbuminuria (T2DM2 group, 90). The right common carotid arteries were examined by real-time ultrasound elastography. The strain ratio (SR, blood to arterial wall strain ratio) was calculated by dividing the strain value of the blood by that of the carotid arterial wall. The correlation between SR and general data was analyzed. The mean SR value ± SD of T2DM2 group was significantly higher than that of T2DM1 group (P < 0.05). SR was positively and significantly correlated with UAE, HbA1c, and systolic blood pressure (r = 0.456,0.435,0.235, P < 0.05 for all). The mean value ± SD of UAE, HbA1c, 2hPG, BMI, and TC of T2DM2 group was significantly higher than that of T2DM1 group (P < 0.05 for all). In conclusion, there is an association between microalbuminuria and carotid arterial wall elasticity in T2DM patients.

[The baseline characteristics of patients with type 2 diabetes initiating insulin detemir : the Chinese cohort from the SOLVE(TM) study].

OBJECTIVE: To characterize the baseline status of Chinese diabetic patients based on data derived from Chinese cohort from SOLVE(TM) study. METHODS: Patients with type 2 diabetes initiating basal insulin detemir at the decision of the physician were eligible for the study. Data on demographics, medical history, glycemic profile and treatment regimen at baseline were collected by physicians. RESULTS: A total of 3272 patients [female 42%, male 58%, mean age (56.2 ± 10.8) years] were included in the study. Their BMI was (25.3 ± 3.3) kg/m(2). The duration of diabetes was 4.0 (0.1 - 27.0) years, and the duration of treatment with oral antidiabetic drugs (OADs) was 3.0 (0.0 - 20.2) years. The proportions of subjects with diabetic macro- and micro-vascular complications were 15.8% (515 cases) and 27.1% (866 cases), respectively. The hemoglobin A1c (HbA1c) at baseline was (8.33 ± 1.70)%, and the fasting blood glucose (FPG) was (9.5 ± 2.6) mmol/L. CONCLUSIONS: A large proportion of patients with type 2 diabetes remain in poor glycemic control, and the prevalence of diabetic complications is high, which requires optimal therapeutic strategy for the patients with suboptimal glycemic control.

Construction of antisense Bmi-1 expression plasmid and its inhibitory effect on K562 cells proliferation.

BACKGROUND: Bmi-1 gene determines the proliferative capacity of normal and leukemia stem cells. Expression of Bmi-1 has been found in all types of myeloid leukemia cells in both humans and mice. This study aimed at assessing the effect of antisense Bmi-1 expression on K562 cells proliferation and p16 protein (p16) expression. METHODS: A transcriptional repressor, Bmi-1 cDNA was cloned by reverse transcriptase polymerase chain reaction (RT-PCR) of its mRNA from K562 cells. A plasmid expressing antisense Bmi-1 mRNA was then constructed by reverse design of PCR primers and cloned to the plasmid pLNCX2; G418 was added to the medium after the plasmid was successfully introduced in K562 cells by lipofectin-mediated DNA transfection. The effects of the antisense expression on the proliferation of K562 cells were analyzed by using microculture tetrazolium and colony forming. Cell cycle was analyzed by using flow cytometry. The p16 expression of K562 cells was observed by immunofluorescence histochemical stain. RESULTS: K562 cells transfected with antisense Bmi-1 plasmid grew significantly slower than that of controls (the parental K562 and cells transfected with empty plasmid). The colony forming ability of antisense Bmi-1 plasmid transfected cells decreased significantly (P < 0.01) compared with controls. The p16 expression of cells transfected with antisense Bmi-1 was upgraded more apparently than that of controls. CONCLUSION: The antisense Bmi-1 gene can inhibit the growth of K562 cell and upgrade expression of p16 in K562 cells.

A single tetracycline-regulated vector devised for controlled insulin gene expression.

OBJECTIVE: To construct a single plasmid vector mediating doxycycline-inducible recombined human insulin gene expression in myotube cell line. METHODS: An expression cassette of rtTAnls driven by promoter of human cytomegalovirus and a furin-cuttable recombined human insulin expression cassette driven by a reverse poly-tetO DNA motif were cloned into a single plasmid vector (prTR-tetO-mINS). The prTR-tetO-mINS and pLNCX were co-transfected into a myotube cell line (C2C12) and pLNCX vector were used as a control. After selection with G418, the transfected cells were induced with doxycycline at concentrations of 0, 2, and 10 microg/mL. RT-PCR was used to determine expression levels of recombinant insulin mRNA at the 5th day. Insulin production in cell cultures medium (at different incubation time) and cell extracts (at the 7th day) were analyzed with human pro/insulin RIA kits. RESULTS: Immune reactive insulin (IRI) level in cell medium was found increased at 24 hours of doxycycline incubation, and still increased at the 5th day. After withdrawn of doxycycline, IRI decreased sharply and was at baseline three days later. IRI and human insulin mRNA levels were positively related to different levels of doxycycline. A 25-fold increase in IRI was found against background expression at the 7th day. CONCLUSION: Human insulin expression can be successfully regulated by doxycycline and the background was very low. This single tet-on insulin expression system may provide a new approach to a controlled insulin gene therapy in skeletal muscle.

[Construction of antisense telomerase hTERT and its effect on K562 cells].

OBJECTIVES: To investigate whether antisense human telomerase reverse transcriptase (hTERT) could inhibit the activity of telomerase and the proliferation of K562 cells. METHODS: The antisense plasmid was constructed by reverse insertion of hTERT PCR product into plasmid pLNCX-neo. Then the constructed plasmid was introduced into K562 cells by liposomes-mediated DNA transfection. The inhibition effects of telomerase on the proliferation of K562 cells were analyzed by MTT and colony formation assay, the telomerase activity of K562 cells by TRAP-PCR ELISA methods. RESULTS: The growth rate of antisense hTERT transfected K562 cells was significantly lower than those of the controls, and the colony formation capacity of the transfected cells decreased significantly (P < 0.01), the colony number is (100.33 +/- 7.57)/10(3) cells, (92.67 +/- 5.86)/10(3) cells and (50.33 +/- 6.11)/10(3) cells for control K562 cells, K562 neo cells and antisense hTERT transfected HL60 cells, respectively. The telomerase activity of antisense hTERT transfected K562 cells was significantly inhibited. CONCLUSION: The expression of an antisense sequence to the mRNA sequence of telomerase protein subunit can inhibit the activity of telomerase, slow the cell growth and inhibit the capacity of colony formation of K562 cells.