Synergistic Induction of Apoptosis by Quercetin and Curcumin in Chronic Myeloid Leukemia (K562) Cells.

Chronic myeloid leukemia is a major hematopoietic malignancy characterized by expansion of myeloid cells. In this study, we have investigated whether quercetin, curcumin and their combination induce apoptosis and inhibit growth of K562 cells. We have observed that quercetin and curcumin combination induced apoptosis accompanied by increased ROS and decreased GSH levels as well as loss of mitochondrial membrane potential. Our mRNA and protein expression results suggested that cytochrome c was released from mitochondria causing PARP and caspase-9 cleavages, the hallmarks of mitochondrial apoptotic pathway. We believe that triggering of apoptosis is mostly via mitochondrial pathway and ROS generation may induce impairment of mitochondrial membrane potential. The use of quercetin and curcumin combination potentiates individual apoptotic effects of the polyphenols and reduces their effective dose thereby preventing potential toxic effects on normal cells. Additional preclinical studies and clinical trials are certainly required to further validate their usefulness as potent anticancer agents.

Quercetin-Induced Cell Death in Human Papillary Thyroid Cancer (B-CPAP) Cells.

In this study, we have investigated the antiproliferative effect of quercetin on human papillary thyroid cancer cells and determined the apoptotic mechanisms underlying its actions. We have used different concentrations of quercetin to induce apoptosis and measured cell viability. Apoptosis and cell cycle analysis was determined by flow cytometry using Annexin V and propidium iodide. Finally, we have measured changes in caspase-3 and cleaved poly(ADP-ribose) polymerase (PARP) protein expression levels as hallmarks of apoptosis and Hsp90 protein expression level as a marker of proteasome activity in treated and control cells. Quercetin treatment of human papillary thyroid cancer cells resulted in decreased cell proliferation and increased rate of apoptosis by caspase activation. Furthermore, it was demonstrated that quercetin induces cancer cell apoptosis by downregulating the levels of Hsp90. In conclusion, we have shown that quercetin induces downregulation of Hsp90 expression that may be involved in the decrease of chymotrypsin-like proteasome activity which, in order, induces inhibition of growth and causes cell death in thyroid cancer cells. Thus, quercetin appears to be a promising candidate drug for Hsp90 downregulation and apoptosis of thyroid cancer cells.

The effect of resveratrol on signal transduction pathways and the role of pro-apoptotic Bax protein on apoptosis in HCT- 116 colon carcinoma cell lines.

Colon carcinoma is the third among the cancer related deaths. The role of pro-apoptotic Bax protein on the resveratrol related apoptosis, mitochondrial membrane potential and signal pathways has not been identified in colon carcinoma cells. In this direction, HCT-116 bax positive and HCT-116 negative cell lines were utilized to detect the apoptotic effect and I?B, MEK1 and STAT 3 signal transduction pathways of resveratrol. The impact on the cell viability and IC50 value of resveratrol has been determined via WST-1 viability assay. The ratio of apoptosis has been evaluated via flow cytometry following Annexin V/Propidium iodide (PI) double staining. Changes in the mitochondrial membrane potential have been analyzed by flow cytometry and JC-1 fluorometric staining. IkB, MEK1 and STAT3 molecules were measured by Enspire device. Data showed the IC50 value for resveratrol as 50M. According to the flow cytometry, apoptosis ratio has been determined as 29.65% in the experimental group of bax positive cells, as 13.98% in the experimental group of bax negative cells. Changes in the membrane potential has been established as 8.62% in the experimental group of bax positive cells, as 97.98% in the experimental group of bax negative cells. When the obtained data from Enspire device was reviewed; bax positive cells I?B phosphorylations were found as as 5.22 for experimental groups; MEK1 phosphorylations were found as and as 1.15 for experimental groups; STAT 3 phosphorylations were found as and as 2.52 for experimental groups. In HCT-116 bax negative cells, I?B phosphorylation were and 2.71 in experimental groups; MEK1 phosphorylation were 1.18 in experimental groups; STAT 3 phosphorylation were 1.54 in experimental groups. Our data show that Bax protein plays role in the apoptotic effect of resveratrol by altering mitochondrial membrane potential and mitochondrial membrane permeability, signal transduction and the absence of Bax increase the sensitivity of HCT-116 colon carcinoma cells to apoptosis.

The role of HSP90 in Quercetin-induced apoptosis in human papillary thyroid (B-CPAP) cancer cells.

As in many other cancers, genetic alterations that occur in genes encoding for key proteins of major signalling pathways are the driving force for the tumorigenesis of thyroid cancer. HSP90 is an emerging therapeutic target of interest for the treatment of cancer and is responsible for modulating cellular response to stress by maintaining the function of signalling proteins. In this study, we have worked with B-CPAP, a thyroid papillary cancer cell line which is known to have BRAF V600E mutation. We have administered the flavonoid Quercetin, which is known to induce apoptosis by inhibiting HSP production on various cancer cell lines, at different concentrations (between 10 and 75µM) for 24hours. We have measured cell viability using WST-1 assay. We found that the viability decreases in a dose dependent manner. Then, we chose Quercetin concentrations between 10-75µM for 24hours, for the apoptosis and cell cycle analysis in flow cytometry by Annexin V and propidium iodide. We have also applied Hoechst stain to cancer cells for visualizing them on fluorescence microscopy and confirmed apoptosis with the presence of apoptotic signs in nuclei of cancer cells. Finally, we used Western blotting to compare HSP90 and Cleaved-PARP levels in cells treated with Quercetin and the control group. In the light of the obtained data, our results suggest that in future studies it would be useful to investigate the apoptotic mechanisms of Quercetin and use combinational therapies on BCPAP cells. Supported by Marmara University Scientific Research Commission (SAG-C-TUP-130313-0063).

Resveratrol: French paradox revisited.

Resveratrol is a polyphenol that plays a potentially important role in many disorders and has been studied in different diseases. The research on this chemical started through the "French paradox," which describes improved cardiovascular outcomes despite a high-fat diet in French people. Since then, resveratrol has been broadly studied and shown to have antioxidant, anti-inflammatory, anti-proliferative, and anti-angiogenic effects, with those on oxidative stress possibly being most important and underlying some of the others, but many signaling pathways are among the molecular targets of resveratrol. In concert they may be beneficial in many disorders, particularly in diseases where oxidative stress plays an important role. The main focus of this review will be the pathways affected by resveratrol. Based on these mechanistic considerations, the involvement of resveratrol especially in cardiovascular diseases, cancer, neurodegenerative diseases, and possibly in longevity will be is addressed.

Induction of HGF and VEGF in hepatic regeneration after hepatotoxin-induced cirrhosis in mice.

BACKGROUND/AIMS: Liver cirrhosis is the irreversible end-result of fibrous scarring and hepatocellular regeneration, characterized by diffuse disorganization of normal hepatic structure by regenerative nodules and fibrotic tissue. In this study, we elucidated the role of hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF) in liver regeneration. METHODOLOGY: The study was conducted as an experimental laboratory investigation using a mouse model of lethal liver cirrhosis induced by carbon tetrachloride (CCl4), dimethylnitrosamine (DMN) and D-galactosamine (D-gal) administrations. RESULTS: Liver morphology showed fibrosis/cirrhosis in all groups, but to a different extent, as confirmed by the rise in serum transaminase levels. The immunolocalization of VEGF and HGF, and homogenate levels of HGF and serum levels of VEGF, were also analyzed. Liver fibrosis/cirrhosis was more severe in CCl4-treated mice. In cirrhotic livers, immunostaining for HGF was weak and the HGF content of liver tissue was lower. Strong immunoreactivity for VEGF was observed when hepatotoxins were administered, however as cirrhosis became apparent immunoreactivity was reduced. Blood VEGF levels increased gradually. CONCLUSIONS: Our results suggest possible involvement of VEGF in angiogenesis of cirrhotic liver. VEGF might be required for reconstruction of hepatic cells and sequentially participates in liver regeneration by facilitating hepatocyte proliferation. HGF production is supposed to be induced in the necrotic liver during regeneration and severe tissue damage followed by cirrhosis might account for low homogenate HGF levels.

Effects of ACE inhibition and angiotensin II receptor blockade on glomerular basement membrane protein excretion and charge selectivity in type 2 diabetic patients.

Angiotensin-converting enzyme (ACE) inhibitors may reduce urinary albumin excretion (UAE) by decreasing glomerular pressure and increasing glomerular charge selectivity through preservation of glycosaminoglycans. The effect of Angiotensin II antagonism on glomerular charge selectivity remains to be determined. The aim of this study was to compare the effects of an AT1 blocker losartan and an ACE inhibitor (ACE-I) enalapril on UAE, extracellular matrix proteins, glycosaminoglycan excretion (UGAG) and red blood cell anionic charge (RBCCh) which are the indirect markers of glomerular basement membrane anionic content in hypertensive Type 2 diabetic patients. Twenty-four patients were randomised into two groups and received either enalapril (520 mg/d) or losartan (50100 mg/d). All parameters were measured at baseline and after six months of treatment. At the end of six months, systolic and diastolic blood pressures (BP), UAE rates, UGAG excretion and RBCCh were significantly and equally reduced in both treatment groups compared with baseline. RBCCh was negatively correlated with UAE (r=-0.57, p<0.0001) and UGAG excretion (r=-0.57, p<0.0001); UAE was correlated with UGAG excretion (r=0.58, p<0.0001). In conclusion, enalapril and losartan treatment were equally effective in reducing BP, UAE as well as UGAG excretion and preserving RBCCh in hypertensive Type 2 diabetic patients. ACE inhibition and AT1-receptor blockade may have favourable effects on preserving glomerular anionic content in hypertensive diabetic patients.

Effect of zinc supplementation on growth hormone secretion, IGF-I, IGFBP-3, somatomedin generation, alkaline phosphatase, osteocalcin and growth in prepubertal children with idiopathic short stature.

Controversy exists about the effect of zinc on growth and the GH-IGF system. Zinc supplementation has been shown to stimulate linear growth in zinc-deficient children. However the mechanism of this effect has not been well characterized. Furthermore, the effect of zinc supplementation on non-zinc-deficient short children is unknown. We investigated the effect of zinc supplementation on endogenous GH secretion, serum IGF-I and IGFBP-3 concentrations, IGF-I and IGFBP-3 generation in response to exogenous GH, bone formation markers, and linear growth of non-zinc-deficient children with idiopathic short stature. We analyzed prospectively serum zinc, IGF-I, IGFBP-3, alkaline phosphatase, osteocalcin, and GH response to clonidine test, and performed a somatomedin generation test before and 6 weeks after zinc supplementation in 22 (16 M, 6 F) prepubertal children with idiopathic short stature. Serum IGF-I increased from 67.4+/-70.6 to 98.2+/-77.3 ng/ml (p <0.001), IGFBP-3 from 2326+/-770 to 2758+/-826 ng/ml (p <0.001), alkaline phosphatase from 525+/-136 to 666+/-197 U/l (p <0.0001), and osteocalcin from 16.8+/-10.6 to 25.8+/-12.8 ng/ml (p <0.05) after zinc supplementation despite there being no difference in GH response to clonidine after zinc supplementation (peak GH 11.6+/-6.9 vs 13.4+/-7.8 ng/ml, GH area under the curve during clonidine test 689+/-395 vs 761+/-468, NS). Percent change in IGF-I and IGFBP-3 during the somatomedin generation test was not significantly affected by zinc supplementation (118% vs 136% and 57% vs 44%, respectively). There was no significant correlation between percentage increase in zinc levels and percentage increase in parameters tested. Height SDS or weight SDS did not improve significantly in 17 patients who continued on zinc supplementation for at least 6 months (6-12 months) (-2.59 vs -2.53 SDS and -1.80 vs -1.67 SDS, respectively). Zinc supplementation increased basal IGF-I, IGFBP-3, alkaline phosphatase and osteocalcin without changing GH response to clonidine. Zinc supplementation did not affect sensitivity to exogenous GH as tested by IGF-I and IGFBP-3 generation test. These results suggest a direct stimulatory effect of zinc on serum IGF-IGFBP-3, alkaline phosphatase and osteocalcin. Despite improvements in the above parameters, zinc supplementation to children with idiopathic short stature with normal serum zinc levels did not significantly change height or weight SDS during 6-12 months follow-up.

Choline increases serum insulin in rat when injected intraperitoneally and augments basal and stimulated aceylcholine release from the rat minced pancreas in vitro.

Intraperitoneal injection of choline (30-90 mg.kg-1) produced a dose-dependent increase in serum insulin, glucose and choline levels in rats. The increase in serum insulin induced by choline (90 mg.kg-1) was blocked by pretreatment with the muscarinic acetylcholine receptor antagonists, atropine (2 mg.kg-1), pirenzepine (2 mg.kg-1) and 4-diphenylacetoxy-N-methylpiperidine (2 mg.kg-1) or the ganglionic nicotinic receptor antagonist, hexamethonium (15 mg.kg-1). The effect of choline on serum insulin and glucose was enhanced by oral glucose administration (3 g.kg-1). Choline administration was associated with a significant (P < 0.001) increase in the acetylcholine content of pancreatic tissue. Choline (10-130 microm) increased basal and stimulated acetylcholine release but failed to evoke insulin release from the minced pancreas at considerably higher concentrations (0.1-10 mm). Hemicholium-3, a choline uptake inhibitor, attenuated the increase in acetylcholine release induced by choline augmentation. Choline (1-32 mm) inhibited [3H]quinuclidinyl benzilate binding to the muscarinic receptors in the pancreatic homogenates. These data show that choline, a precursor of the neurotransmitter acetylcholine, increases serum insulin by indirectly stimulating peripheral acetylcholine receptors through the enhancement of acetylcholine synthesis and release.

Hyperglycemia induced by intracerebroventricular choline: involvement of the sympatho-adrenal system.

Intracerebroventricular (i.c.v.) injection of choline (75-300 microg) produced a dose-dependent increase in blood glucose levels. Pre-treatment with the nicotinic acetylcholine receptor antagonist, mecamylamine (50 microg, i.c.v.) blocked the hyperglycemia induced by choline (150 microg, i.c.v.), but the response was not affected by pre-treatment with the muscarinic acetylcholine receptor antagonist, atropine (10 microg, i.c.v.). Pre-treatment with the neuronal choline uptake inhibitor, hemicholinium-3 (20 microg, i.c.v.), attenuated the hyperglycemia induced by choline. The hyperglycemic response to choline was associated increased plasma levels of adrenaline and noradrenaline. The hyperglycemia elicited by choline was greatly attenuated by bilateral adrenalectomy, and entirely blocked by either surgical transection of the splanchnic nerves or by pre-treatment with the alpha-adrenoceptor antagonist, phentolamine. These data show that choline, a precursor of acetylcholine, increases blood glucose and this effect is mediated by central nicotinic acetylcholine receptor activation. An increase in sympatho-adrenal activity appears to be involved in the hyperglycemic effect of choline.