A network pharmacology approach to investigate the anticancer mechanism of cinobufagin against hepatocellular carcinoma via downregulation of EGFR-CDK2 signaling.

Hepatocellular carcinoma (HCC) is one of the deadliest cancers with high mortality and poor prognosis, and the investigation on new approaches and effective drugs for HCC therapy is of great significance. In our study, we demonstrate that treatment with cinobufagin, a natural compound isolated from traditional chinese medicine Chansu, reduces proliferation and the colony formation capacity of the human hepatoma cells in vitro, in addition, cinobufagin induces mitotic arrest in human hepatoma cells. The results of a network pharmacology-based analysis show that EGFR, MAPK1, PTK2, CDK2, MAPK3, ESR1, CDK1, PRKCA, AR, and CSNK2A1 are the key targets involved in the anti-tumor activities of cinobufagin, additionally, several signaling pathways such as proteoglycans in cancer, pathways in cancer, HIF-1 signaling pathway, VEGF signaling pathway, ErbB signaling pathway, and PI3K-AKT signaling pathway are identified as the potential pathways involved in the inhibitory effects of cinobufagin against HCC. Furthermore, at the molecular level, we find that cinobufagin decreases EGFR expression and CDK2 activity in human hepatoma cells. Inhibition of EGFR or CDK2 expression could not only suppress the growth of tumor cells but also enhance the inhibitory effects of cinobufagin on the proliferative potential of human hepatoma cells. We also demonstrate that EGFR positively regulates CDK2 expression. Furthermore, EGFR inhibitor gefitinib or CDK2 inhibitor CVT-313 synergistically enhances anticancer effects of cinobufagin in human hepatoma cells. Taken together, these findings indicate that cinobufagin may exert antitumor effects by suppressing EGFR-CDK2 signaling, and our study suggests that cinobufagin may be a novel, promising anticancer agent for the treatment of HCC.

Autoantibodies against islet cell antigens in children with type 1 diabetes mellitus.

We investigated the prevalence of glutamic acid decarboxylase 65 autoantibody (GADA), insulinoma-associated protein 2 autoantibody (IA2A), and insulin autoantibody (IAA) in 750 children with type 1 diabetes (T1D) living in Taiwan. GADA, IA2A, and IAA were measured by radioimmunoassay. The data were assessed by χ2 test, binary logistic regression, and Spearman rank correlation. Of the 750 T1D patients, 66.3% had GADA, 65.3% IA2A, 35.7% IAA, and 17.2% no autoantibodies. The prevalence of GADA and IA2A significantly decreased along T1D duration. The positivity of either GADA or IA2A was 89.4% within the first year of disease and decreased to 36.7% after 9 years (P = 1.22 × 10-20). Female patients had significantly higher prevalence of GADA compared with male patients (72.3% vs. 59.7%, P = 0.00027). The patients diagnosed before 12 years of age had a positive rate of 92.2% for either GADA or IA2A. Patients diagnosed at age 12 or above had a significantly lower positive rate of 81.6% (P = 0.011). GADA and IA2A significantly correlated with each other (rs = 0.245, P = 1.09 × 10-11). We concluded that autoantibodies were detectable in 89.4% of T1D patients within one year after diagnosis. Their prevalence declined with disease duration. GADA was more prevalent in female patients. GADA and IA2A weakly correlated with each other.

Changes of Atrial Natriuretic Peptides after Defibrillation Threshold Testing Predicted Future Ventricular Arrhythmia Event.

BACKGROUND: We investigated the change of natriuretic peptides during defibrillation threshold (DFT) testing and its relationship with future ventricular arrhythmia (VA) events in patients implanted with an implantable cardioverter defibrillator (ICD). METHODS: Atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and c-type natriuretic peptide (CNP) were measured in 21 patients (mean age 61 ± 13 years; 67% male) undergoing ICD implantation. Blood samples of the patients were drawn at pre-implantation, 30 minutes, 60 minutes, and 24 hours after DFT testing. The patients were followed and divided into two groups according to the occurrence of VA in 18 months. The biomarker levels and their changes were compared in patients with and without further VA. RESULTS: The pre-implantation ANP levels were higher at pre-implantation and increased significantly at 30 minutes after DFT testing (Δ30minANP) among patients with VA events. The BNP and CNP levels did not change significantly after DFT testing in both groups. The area under curve was 0.82 for the change in Δ30minANP determining further ventricular events. The optimal Δ30minANP cutoff value was 0.51 pg/ml, with sensitivity of 0.83 and specificity of 0.68. Multivariable analysis confirmed that patients with Δ30minANP more than 0.51 pg/ml have a higher risk of further ventricular events (hazard ratio 39.8, 95% confidence interval: 2.87-553.01, p = 0.006). The pre-implantation ANP level could not predict future VA events (hazard ratio 1.06, 95% CI: 1.00-1.14, p = 0.06). CONCLUSIONS: The increase of ANP concentration after DFT testing predicted future VA events after ICD implantation while the BNP and CNP levels did not predict future VA events.

Responses of cardiac natriuretic peptides after paroxysmal supraventricular tachycardia: ANP surges faster than BNP and CNP.

Atrial natriuretic peptide (ANP) secretion increases after 30 min of paroxysmal supraventricular tachycardia (PSVT). Whether this phenomenon also applies to brain or C-type natriuretic peptides (BNP or CNP) remains unknown. Blood samples of 18 patients (41 ± 11 yr old; 4 men) with symptomatic PSVT and normal left ventricular systolic function (ejection fraction 65 ± 6%) were collected from the coronary sinus (CS) and the femoral artery (FA) before and 30 min after the induction, and 30 min after the termination of PSVT. The results showed that the ANP levels rose steeply after the PSVT and then reduced at 30 min after the termination (baseline vs. post-PSVT vs. posttermination: CS: 34.0 ± 29.6 vs. 74.1 ± 42.3 vs. 46.1 ± 32.9; FA: 5.9 ± 3.24 vs. 28.2 ± 20.7 vs. 10.0 ± 4.6 pg/ml; all P < 0.05). In contrast, compared with ANP, the increases of BNP and CNP in CS after the PSVT were less sharp, but continued to rise after the termination of tachycardia (BNP, 10.2 ± 6.4 vs. 11.3 ± 7.1 vs. 11.8 ± 7.9; CNP, 4.5 ± 1.2 vs. 4.9 ± 1.4 vs. 5.0 ± 1.4 pg/ml; all P < 0.05). The rise of BNP and CNP in FA was similarly less sharp after the PSVT and remained stationary after the termination. PSVT exerted differential effects on cardiac natriuretic peptide levels. ANP increased greater after a 30-min induced PSVT, but dropped faster after termination of PSVT, compared with BNP and CNP.

Effects of carbenoxolone on flow-mediated vasodilatation in healthy adults.

Gap junctions play a key role in maintaining the functional integrity of the vascular wall. Using carbenoxolone (CBX) as a gap junction blocker, we aimed to assess the contribution of gap junctions in the vascular wall to flow-mediated vasodilatation (FMD) in healthy adults. Percentage FMD (%FMD) and circulating vasoactive molecules/activity, including atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP), aldosterone, cortisol, plasma renin activity (PRA), and endothelin (ET-1), were measured in 25 healthy volunteers (mean age: 30.1 ± 5.4 yr; 14 males) before and after oral administration of CBX (100 mg). %FMD decreased after ingestion of CBX (9.71 ± 3.1 vs. 3.40 ± 2.0%; P < 0.0001). The levels of ANP, BNP, cortisol, and ET-1 remained stationary, while both PRA and aldosterone decreased (P < 0.005) after CBX ingestion. Blood pressure and heart rate were minimally changed by CBX. Inhibition of gap junctional communication by CBX impairs FMD in healthy persons, suggesting that physiologically, vascular gap junctions participate in the maintenance of FMD. CBX does not induce the release of vasoconstricting molecules or enhance vasoconstriction, suggesting that inhibition of gap junctional communication by CBX underlies the impairment of FMD. Therefore, administering CBX in FMD examination can be a way to follow the effect of gap junctions on endothelial function, but further work remains to verify the specificity of CBX effect.

C-type natriuretic peptide in individuals with normal left ventricular systolic function.

OBJECTIVE: The objective of this study was to evaluate production or release of CNP in individuals without CHF. METHODS: Nineteen patients with symptomatic paroxysmal supraventricular tachycardia (PSVT) and normal left ventricular systolic function were enrolled into the study. Blood samples were collected from the coronary sinus (CS), the femoral artery (FA), and the peripheral vein (PV) before pacing, after rapid RA pacing, and post electrophysiological study (EPS) and/or radiofrequency (RF) ablation. RESULTS: The CNP level in the CS, compared to FA and PV, was significantly higher before pacing (CS, 3.2+/-0.8; FA, 2.6+/-0.7; PV, 2.5+/-0.5 pg/ml; CS vs. either FA or PV, both p<0.001), after the pacing (CS, 3.2+/-1.3; FA, 2.4+/-0.6 pg/ml; p=0.004), and post the EPS and/or RF ablation (CS, 3.1+/-0.7; FA, 2.6+/-0.9; PV, 2.5+/-0.8 pg/ml; CS vs. either FA or PV, both p<0.01). CONCLUSION: The myocardium regularly produces or releases CNP in patients with normal LV systolic function. Brief periods of rapid RA pacing, PSVT, or EPS does not change the production and/or release.

Metabolic disorders in children and adolescents with type 2 diabetes mellitus.

BACKGROUND: Type 2 diabetes mellitus (T2DM) in children and adolescents is increasing in incidence worldwide. It is the leading type of newly diagnosed diabetes in Taiwan among school children. T2DM is associated with metabolic syndrome in adults, so we tried to find out if these metabolic disorders are present in children. METHODS: From 1989 to 2003, 22 children and adolescents were diagnosed with T2DM in our hospital. Their ages ranged from 8.8 to 17.0 (11.7+/-2.3) years; 6 of them were boys. We compared their clinical characteristics with those of 42 healthy and 237 obese children and adolescents. Physical examination was performed and plasma glucose and serum cholesterol, triglycerides, uric acid, creatinine, HDL-cholesterol, and insulin levels were measured and LDL-cholesterol was calculated. Demographic and laboratory data were compared among the T2DM, obese and control groups. RESULTS: The female: male ratio among the patients was 2.7: 1; 18% were overweight and 68% obese, and 64% had acanthosis nigricans. There were no significant differences between the T2DM and obese groups in terms of biochemistry profiles except for the higher plasma glucose in the T2DM group. Children with T2DM had higher levels of cholesterol and triglycerides but lower levels of HDL-cholesterol compared with healthy children. Among obese children without T2DM, the levels of glucose, triglycerides, uric acid, insulin, HOMA-IR were higher than in the healthy group, and HDL-cholesterol levels were lower. CONCLUSIONS: Children with T2DM or obesity should be evaluated for metabolic disorders.

Brain natriuretic peptide and C-type natriuretic peptide are differently regulated by age but similarly elevated in coronary artery disease.

OBJECTIVE: We investigated the effect of age and coronary angioplasty on brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP). METHODS AND RESULTS: Serum levels of both peptides immediately before catheterization and at the end of angioplasty in 15 patients (age 68 +/- 8 years, 8 men) with coronary artery disease (CAD) were compared to 12 elderly (65 +/- 7 years, 8 men) and 16 non-elderly (34 +/- 7 years, 10 men) healthy individuals. The results showed that in healthy individuals the levels of both peptides are age-dependent dissimilarly. Compared to the non-elderly, while BNP increases in the elderly (7.81 +/- 1.60 vs. 10.01 +/- 2.06 pg/ml, p = 0.002), CNP decreases (5.39 +/- 1.30 vs. 2.22 +/- 0.80 pg/ml, p < 0.001). On the other hand, compared to the elderly healthy persons, patients with CAD have a marked increase in the baseline levels of BNP (20.02 +/- 17.43 pg/ml, p = 0.03) and CNP (4.41 +/- 1.20 pg/ml, p < 0.001). However, both peptides remain stationary immediately after angioplasty (BNP, 21.02 +/- 16.95; CNP, 4.51 +/- 1.06 pg/ml; both p = 0.4). CONCLUSIONS: BNP and CNP are differentially regulated by age in a healthy state, suggesting that each peptide has a distinct role during the aging process. The elevation of both peptides in CAD but little change shortly after angioplasty may indicate that both peptides respond to a chronic state rather than an acute episode of vascular damage.

Insulin resistance in obese adolescents.

The prevalence of obesity in adolescents is increasing rapidly. Obesity is associated with insulin resistance, which increases the risk for type 2 diabetes. We investigated insulin resistance in obese adolescents and its relationship to acanthosis nigricans. Height, weight and body mass index (BMI) were measured and each was expressed as a standard deviation score (SDS) based on national growth standards. Insulin resistance was estimated by using the homeostasis model assessment (HOMA-IR) score, calculated as fasting insulin (microU/mL) x fasting glucose (mmol/L)/22.5. Obese adolescents had significantly greater weight-SDS, BMI-SDS, fasting insulin levels, and HOMA-IR than did controls with normal weight. Acanthosis nigricans was present in 58.4% of obese adolescents. Fasting insulin levels and BMI-SDS were positively correlated with the degree of acanthosis nigricans (r = 0.383, P<0.0001 and r = 0.164, P < 0.05, respectively). On multiple regression analysis, HOMA-IR and BMI-SDS explained 16.8% of the degree of acanthosis nigricans (P<0.0001). Acanthosis nigricans thus may be a marker of insulin resistance and should be carefully looked for in obese adolescents.

Wolfram (DIDMOAD) syndrome: report of two patients.

We report a girl with Wolfram syndrome who presented with juvenile-onset diabetes mellitus when she was 4 3/12 years old. Optic atrophy and high frequency sensorineural hearing loss were found at 7 and 9 5/12 years of age, respectively. Her younger brother also developed Wolfram syndrome when he was 3 2/12 years old. Wolfram syndrome is also called DIDMOAD (diabetes insipidus, diabetes mellitus, optic atrophy and deafness). This syndrome is transmitted as an autosomal recessive trait and is a progressive neurodegenerative disorder. It should be considered in a diabetic patient with unexplained optic atrophy, hearing loss, or polyuria and polydipsia in the presence of adequate blood glucose control. Visual acuity should be checked annually in patients with juvenile-onset diabetes mellitus. Optic atrophy should be considered if visual acuity is impaired.

Thyroid function in children with newly diagnosed type 1 diabetes mellitus.

Evaluation of thyroid hormone indices was performed in 138 children with newly diagnosed type 1 diabetes, with their siblings serving as controls. The DKA group consisted of 76 children who had diabetic ketoacidosis (DKA) at initial diagnosis. The non-DKA group consisted of 62 children and the control group of 35. The thyroid function tests of the patients were measured within 3 days of the initial diagnosis of diabetes and at least one follow-up test one month to two years after adequate treatment of diabetes. The DKA group had significantly lower levels of T3, T4, free T4 and FTI than did the other two groups (p < 0.0001, p < 0.0001, p < 0.0001, and p < 0.0001, respectively). T3 concentration was lower in non-DKA subjects than in controls (p = 0.0003), but the two groups did not significantly differ in terms of T4, free T4, and FTI. The TSH level did not differ among the three groups. We conclude that DKA changes thyroid function measurements. In the absence of true thyroid disease, abnormal thyroid function tests are reversible after institution of good diabetic control. We suggest that thyroid function tests should be restricted to those patients suspected of having thyroid disorders at the initial diagnosis of type 1 diabetes.