Kazim Husain

Dose-Dependent Protection by Lipoic Acid against Cisplatin-Induced Nephrotoxicity in Rats: Antioxidant Defense System

This study was designed to investigate the role of graded doses of lipoic acid pretreatment against cisplatin-induced nephrotoxicity. Male Wistar rats were divided into six groups and treated as follows: 1) vehicle (saline) control; 2) cisplatin (16 mg/kg, intraperitoneally); 3) lipoic acid (100 mg/kg, intraperitoneally); 4) cisplatin plus lipoic acid (25 mg/kg); 5) cisplatin plus lipoic acid (50 mg/kg) and 6) cisplatin plus lipoic acid (100 mg/kg). Rats were sacrificed three days after treatment, and plasma as well as kidneys were isolated and analyzed. Plasma creatinine increased (677% of control) following cisplatin administration alone which was decreased by lipoic acid in a dose-dependent manner. Cisplatin-treated rats showed a depletion of renal glutathione (GSH), increased oxidized GSH and decreased GSH/GSH oxidized ratio (62%, 166% and 62% of control), respectively which were restored with lipoic acid pretreatment. Renal superoxide dismutase, catalase, glutathione peroxidase (GSH peroxidase) and glutathione reductase activities decreased (62%, 75%, 62% and 80% of control), respectively, and malondialdehyde content increased (204% of control) following cisplatin administration, which were restored with increasing doses of lipoic acid. The renal platinum concentration increased following cisplatin administration, which was possibly decreased by chelation with lipoic acid. The data suggest that the graded doses of lipoic acid effectively prevented a decrease in renal antioxidant defense system and prevented an increase in lipid peroxidation, platinum content and plasma creatinine concentrations in a dose-dependent manner.

Inflammation, oxidative stress and renin angiotensin system in atherosclerosis

Atherosclerosis is a chronic inflammatory disease associated with cardiovascular dysfunction including myocardial infarction, unstable angina, sudden cardiac death, stroke and peripheral thromboses. It has been predicted that atherosclerosis will be the primary cause of death in the world by 2020. Atherogenesis is initiated by endothelial injury due to oxidative stress associated with cardiovascular risk factors including diabetes mellitus, hypertension, cigarette smoking, dyslipidemia, obesity, and metabolic syndrome. The impairment of the endothelium associated with cardiovascular risk factors creates an imbalance between vasodilating and vasoconstricting factors, in particular, an increase in angiotensin II (Ang II) and a decrease in nitric oxide. The renin-angiotensin system (RAS), and its primary mediator Ang II, also have a direct influence on the progression of the atherosclerotic process via effects on endothelial function, inflammation, fibrinolytic balance, and plaque stability. Anti-inflammatory agents [statins, secretory phospholipase A2 inhibitor, lipoprotein-associated phospholipase A2 inhibitor, 5-lipoxygenase activating protein, chemokine motif ligand-2, C-C chemokine motif receptor 2 pathway inhibitors, methotrexate, IL-1 pathway inhibitor and RAS inhibitors (angiotensin-converting enzyme inhibitors)], Ang II receptor blockers and ranin inhibitors may slow inflammatory processes and disease progression. Several studies in human using anti-inflammatory agents and RAS inhibitors revealed vascular benefits and reduced progression of coronary atherosclerosis in patients with stable angina pectoris; decreased vascular inflammatory markers, improved common carotid intima-media thickness and plaque volume in patients with diagnosed atherosclerosis. Recent preclinical studies have demonstrated therapeutic efficacy of vitamin D analogs paricalcitol in ApoE-deficient atherosclerotic mice.

Combination Therapy with Paricalcitol and Enalapril Ameliorates Cardiac Oxidative Injury in Uremic Rats

Aims: This study investigated the protective effect of the angiotensin-converting enzyme inhibitor, enalapril, and the vitamin D analog, paricalcitol, alone or in combination, on cardiac oxidative stress in uremic rats. Methods: Rats were made uremic by 5/6 nephrectomy and treated for 4 months as follows: (1) uremic + vehicle (n = 11); (2) uremic + enalapril (30 mg/l in drinking water, n = 13); (3) uremic + paricalcitol (200 ng 3× week, n = 6); (4) uremic + enalapril + paricalcitol (n = 14), and (5) controls (n = 6). Results: Cardiac NADPH oxidase activity increased by 300% in uremic rats compared to normal controls. Treatment with enalapril, paricalcitol or the combination of the two protected uremic rats from cardiac oxidative stress by inhibiting enzyme activity. Cardiac malondialdehyde (MDA) levels were significantly increased in uremic rats compared to normal controls. Only the combination therapy inhibited the increase in MDA levels in uremic rats. Cardiac glutathione was significantly reduced in uremic rats compared to normal controls. Enalapril, paricalcitol or the two in combination all protected against this reduction in glutathione. Cardiac copper/zinc superoxide dismutase (CuZn-SOD) activity decreased whereas manganese (Mn-SOD) activity increased in uremic rats compared to controls. Both mono and combination therapies ameliorated the alterations in cardiac SOD activity seen in uremic rats. Conclusion: Enalapril, paricalcitol and their combined therapy afford protection against cardiac oxidative stress in uremia.

Effects of Paricalcitol and Enalapril on Atherosclerotic Injury in Mouse Aortas

Aims: This study investigated the protective effect of vitamin D analog paricalcitol combined with angiotensin-converting enzyme inhibitor (enalapril) on aortic oxidative injury in atherosclerotic mice. Methods: Female mice were treated for 16 weeks as follows: (1) ApoE deficient + vehicle, (2) ApoE deficient + paricalcitol (200 ng 3 times a week), (3) ApoE deficient + enalapril (30 mg/l in drinking water), (4) ApoE deficient + paricalcitol + enalapril, and (5) wild-type controls. Results: ApoE-deficient mice developed hypertension which was prevented by enalapril or enalapril + paricalcitol treatment but not by paricalcitol treatment. Histology showed atherosclerotic plaque in the aorta of ApoE-deficient mice which was prevented by paricalcitol, enalapril, and paricalcitol + enalapril treatments. Aortic malondialdehyde levels, NADPH oxidase subunit p22phox, manganese-superoxide dismutase (Mn-SOD), inducible nitric oxide synthase, monocyte chemoattaractant protein-1, tumor necrosis factor (TNF)-α, and cyclooxygenase-2 protein expressions increased, whereas glutathione levels, CuZn-SOD, and endothelial protein expressions decreased in ApoE-deficient mice compared to controls. Treatment with paricalcitol and enalapril alone or in combination protected the inflammatory and oxidative endothelial injury of the aorta in atherosclerotic mice. Conclusion: Combination therapy affords greater protection against aortic inflammatory and oxidative injury in atherosclerosis than monotherapy.

Vitamin E δ-Tocotrienol Augments the Anti-tumor Activity of Gemcitabine and Suppresses Constitutive NF-κB Activation in Pancreatic Cancer

The NF-κB transcription factor functions as a crucial regulator of cell survival and chemoresistance in pancreatic cancer. Recent studies suggest that tocotrienols, which are the unsaturated forms of vitamin E, are a promising class of anticancer compounds that inhibit the growth and survival of many cancer cells, including pancreatic cancer. Here, we show that tocotrienols inhibited NF-κB activity and the survival of human pancreatic cancer cells in vitro and in vivo. Importantly, we found the bioactivity of the four natural tocotrienol compounds (α-, β-, δ-, and γ-tocotrienol) to be directly related to their ability to suppress NF-κB activity in vitro and in vivo. The most bioactive tocotrienol for pancreatic cancer, δ-tocotrienol, significantly enhanced the efficacy of gemcitabine to inhibit pancreatic cancer growth and survival in vitro and in vivo. Moreover, we found that δ-tocotrienol augmentation of gemcitabine activity in pancreatic cancer cells and tumors is associated with significant suppression of NF-κB activity and the expression of NF-κB transcriptional targets (Bcl-XL, X-linked inhibitor of apoptosis, and survivin). Our study represents the first comprehensive preclinical evaluation of the activity of natural vitamin E compounds in pancreatic cancer. Given these results, we are conducting a phase I trial of δ-tocotrienol in patients with pancreatic cancer using pancreatic tumor cell survival and NF-κB signaling components as intermediate biomarkers. Our data also support future clinical investigation of δ-tocotrienol to augment gemcitabine activity in pancreatic cancer. Mol Cancer Ther; 10(12); 2363–72. ©2011 AACR.

Effect of combining an ACE inhibitor and a VDR activator on glomerulosclerosis, proteinuria, and renal oxidative stress in uremic rats.

Angiotensin-converting enzyme (ACE) inhibitors ameliorate the progression of renal disease. In combination with vitamin D receptor activators, they provide additional benefits. In the present study, uremic (U) rats were treated as follows: U+vehicle (UC), U+enalapril (UE; 25 mg/l in drinking water), U+paricalcitol (UP; 0.8 μg/kg ip, 3 × wk), or U+enalapril+paricalcitol (UEP). Despite hypertension in UP rats, proteinuria decreased by 32% vs. UC rats. Enalapril alone, or in combination with paricalcitol, further decreased proteinuria (≈70%). Glomerulosclerosis and interstitial infiltration increased in UC rats. Paricalcitol and enalapril inhibited this. The increase in cardiac atrial natriuretic peptide (ANP) seen in UC rats was significantly decreased by paricalcitol. Enalapril produced a more dramatic reduction in ANP. Renal oxidative stress plays a critical role in inflammation and progression of sclerosis. The marked increase in p22(phox), a subunit of NADPH oxidase, and decrease in endothelial nitric oxide synthase were inhibited in all treated groups. Cotreatment with both compounds inhibited the uremia-induced increase in proinflammatory inducible nitric oxide synthase (iNOS) and glutathione peroxidase activity better than either compound alone. Glutathione reductase was also increased in UE and UP rats vs. UC. Kidney 4-hydroxynonenal was significantly increased in the UC group compared with the normal group. Combined treatment with both compounds significantly blunted this increase, P < 0.05, while either compound alone had no effect. Additionally, the expression of Mn-SOD was increased and CuZn-SOD decreased by uremia. This was ameliorated in all treatment groups. Cotreatment with enalapril and paricalcitol had an additive effect in increasing CuZn-SOD expression. In conclusion, like enalapril, paricalcitol alone can improve proteinuria, glomerulosclerosis, and interstitial infiltration and reduce renal oxidative stress. The effects of paricalcitol may be amplified when an ACE inhibitor is added since cotreatment with both compounds seems to have an additive effect on ameliorating uremia-induced changes in iNOS and CuZn-SOD expression, peroxidase activity, and renal histomorphometry.

Exercise conditioning attenuates the hypertensive effects of nitric oxide synthase inhibitor in rat

Many individuals with cardiovascular diseases undergo periodic exercise conditioning with or with out medication. Therefore, this study investigated the interaction of exercise training and chronic nitric oxide synthase (NOS) inhibitor (Nitro-L-Arginine Methyl Ester, L-NAME) treatment on blood pressure and its correlation with aortic nitric oxide (NO), antioxidant defense system and oxidative stress parameters in rats. Fisher 344 rats were divided into four groups: (1) sedentary control, (2) exercise training (ET) for 8 weeks, (3) L-NAME (10 mg/kg, subcutaneous for 8 weeks) and (4) ET + L-NAME. Blood pressure (BP) was monitored weekly for 8 weeks with tail-cuff method. The animals were sacrificed 24 h after last treatments and thoracic aortic rings were isolated and analyzed. Exercise conditioning resulted in a significant increase in respiratory exchange ratio (RER), aortic NO production, NO synthase activity and inducible iNOS protein expression. Training significantly enhanced aortic GSH levels, GSH/GSSG ratio and up-regulation of aortic CuZn-SOD, Mn-SOD, catalase (CAT) glutathione peroxidase (GSH-Px) activity and protein expression and significantly decreased aortic lipid peroxidation. Chronic L-NAME administration resulted in a significant depletion of aortic NO, NOS activity, endothelial (eNOS) and iNOS protein expression, GSH level, GSH/GSSG ratio, down-regulation of aortic antioxidant enzyme activities and protein expressions. Aortic xanthine oxidase (XO) activity significantly increased with increased lipid peroxidation and protein oxidation after L-NAME administration. The biochemical changes were accompanied by increased in BP. Interaction of training and chronic NOS inhibitor treatment resulted in normalization of BP and aortic antioxidant enzyme activity and protein expression, up-regulation of aortic GSH/GSSG ratio, NO levels, Mn-SOD protein expression, depletion of GSSG, protein oxidation and lipid peroxidation. The data suggest that training attenuated the oxidative injury caused by chronic NOS inhibitor treatment by up-regulating the NO and antioxidant systems and lowering the BP in rats.

Alcohol-induced hypertension: Mechanism and prevention

Epidemiological, preclinical and clinical studies established the association between high alcohol consumption and hypertension. However the mechanism through which alcohol raises blood pressure remains elusive. Several possible mechanisms have been proposed such as an imbalance of the central nervous system, impairment of the baroreceptors, enhanced sympathetic activity, stimulation of the renin-angiotensin-aldosterone system, increased cortisol levels, increased vascular reactivity due to increase in intracellular calcium levels, stimulation of the endothelium to release vasoconstrictors and loss of relaxation due to inflammation and oxidative injury of the endothelium leading to inhibition of endothelium-dependent nitric oxide production. Loss of relaxation due to inflammation and oxidative injury of the endothelium by angiotensin II leading to inhibition of endothelium-dependent nitric oxide production is the major contributors of the alcohol-induced hypertension. For the prevention of alcohol-induced hypertension is to reduce the amount of alcohol intake. Physical conditioning/exercise training is one of the most important strategies to prevent/treat chronic alcohol-induced hypertension on physiological basis. The efficacious pharmacologic treatment includes the angiotensin-converting enzyme (ACE) inhibitors or angiotensin II type 1 receptor blockers (ARBs) which have antioxidant activity and calcium channel blockers. The most effective prevention and treatment of alcohol-induced hypertension is physical exercise and the use of ACE inhibitors or ARBs in the clinic.

Chronic ethanol ingestion induces aortic inflammation/oxidative endothelial injury and hypertension in rats

The study aim was to investigate the relationship of chronic ethanol-induced inflammation leading to vascular endothelial injury and elevation of blood pressure (BP) in a rat model. Male Fisher rats were divided into two groups of six animals each and treated as follows: (1) Control (5% sucrose, orally) daily for 12 weeks and (2) 20% ethanol (4 g kg-1, orally) daily for 12 weeks. The mean arterial blood pressure was recorded every week. The animals were anesthetized with pentobarbital after 12 weeks; thoracic aorta were isolated and analyzed for aortic reactivity response, inflammatory mediators, oxidant/antioxidant enzyme protein expression and endothelial nitric oxide-generating system. The results show that the mean BP was significantly elevated 12 weeks after ethanol ingestion. The increased BP was related to increased aortic inflammation (tumor necrosis factor [TNF]-α; nitric oxide synthase [iNOS], COX-2 and MCP-1 protein expression) and elevated angiotensin II levels in alcohol-treated group compared to control. Aortic Nicotinamide adenine dinucleotide phosphate reduced (NADPH) oxidase activity, membrane and cytosolic subunits p22 phox and p47 phox expression and Mn-SOD activity and protein expression significantly increased, whereas nitric oxide (NO), endothelial NO synthase (eNOS), vascular endothelial growth factor (VEGF)-A and CuZn-SOD activity and protein expression significantly decreased in alcohol-treated group compared to control. The acetylcholine-mediated vasorelaxation response was depressed in the aorta of ethanol-treated rats compared to control. In conclusion, chronic ethanol-induced elevation in BP is related to increased aortic inflammation, elevated angiotensin II levels, induction of NADPH oxidase causing endothelial injury, depletion of CuZn-SOD, down-regulation of endothelial NO generating system and impaired vascular relaxation in rats.

Vitamin E δ-Tocotrienol Induces p27Kip1-Dependent Cell-Cycle Arrest in Pancreatic Cancer Cells via an E2F-1-Dependent Mechanism

Vitamin E δ-tocotrienol has been shown to have antitumor activity, but the precise molecular mechanism by which it inhibits the proliferation of cancer cells remains unclear. Here, we demonstrated that δ-tocotrienol exerted significant cell growth inhibition pancreatic ductal cancer (PDCA) cells without affecting normal human pancreatic ductal epithelial cell growth. We also showed that δ-tocotrienol-induced growth inhibition occurred concomitantly with G1 cell-cycle arrest and increased p27Kip1 nuclear accumulation. This finding is significant considering that loss of nuclear p27Kip1 expression is a well-established adverse prognostic factor in PDCA. Furthermore, δ-tocotrienol inactivated RAF-MEK-ERK signaling, a pathway known to suppress p27Kip1 expression. To determine whether p27Kip1 induction is required for δ-tocotrienol inhibition of PDCA cell proliferation, we stably silenced the CDKN1B gene, encoding p27Kip1, in MIAPaCa-2 PDCA cells and demonstrated that p27Kip1 silencing suppressed cell-cycle arrest induced by δ-tocotrienol. Furthermore, δ-tocotrienol induced p27Kip1 mRNA expression but not its protein degradation. p27Kip1 gene promoter activity was induced by δ-tocotrienol through the promoters E2F-1 binding site, and this activity was attenuated by E2F-1 depletion using E2F-1 small interfering RNA. Finally, decreased proliferation, mediated by Ki67 and p27Kip1 expression by δ-tocotrienol, was confirmed in vivo in a nude mouse xenograft pancreatic cancer model. Our findings reveal a new mechanism, dependent on p27Kip1 induction, by which δ-tocotrienol can inhibit proliferation in PDCA cells, providing a new rationale for p27Kip1 as a biomarker for δ-tocotrienol efficacy in pancreatic cancer prevention and therapy.