Significant Increase In Caspase-3 Expression Research Articles

The Possible Protective Role of Resveratrol against Zinc Oxide Nanoparticles Induced Hepatic Tissue Injury in Adult Albino Rat

Abstract Background Zinc oxide nanoparticles (ZnO NPs) are widely used in industries and medications. This may elevate the probability of their existence in the environment, which may have harmful side effects on different body organs, especially on the liver. So it became mandatory to search for natural hepatoprotective elements such as Resveratrol. Aim of the Work The aim of the present work was to determine the effect of zinc oxide nanoparticles on the histological structure and biochemical markers of the liver of adult male albino rats and to evaluate the possible protective role of Resveratrol. Material and Methods 40 adult male albino rats aged 3-6 months, were divided randomly into four groups; Group I (Control group), which consisted of 10 male albino rats with free access to food (rat chew) and water; Group II (ZnO NPs group), which included 10 male albino rats, which received a dose of 50 mg/kg body weight ZnO NPs through oral gavage daily for 4 weeks; Group III (Resveratrol group) was composed of 10 male albino rats which received a dose of 20 mg/kg body weight Resveratrol through oral gavage daily for 4 weeks, and Group IV (ZnO NPs + Resveratrol group) included 10 male albino rats which were co-administered with a combination of ZnO NPs at a dose of 50mg/kg body weight and Resveratrol at a dose of 20 mg/kg body weight dose through oral gavage daily for 4 weeks. At the end of the experiment, blood samples were collected for serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP). All the animals were sacrificed, and the liver was extracted and processed into paraffin blocks for light microscopic examination. A morphometric study and a statistical analysis were done. Results The present work demonstrated that liver sections obtained from adult male albino rats that received ZnO NPs showed disorganized hepatic architecture, severe affection of the hepatocytes, which had small darkly stained nuclei, and deep acidophilic cytoplasm. The hepatocytes sometimes showed extensive cytoplasmic vacuolations. Most hepatocytes had small apoptotic nuclei and were surrounded by clear halos. While some hepatocytes lost their cell boundaries and coalesced together. Central veins were dilated, distorted, and congested with areas of epithelial lining detachment. Most of the blood sinusoids appeared congested with marked inflammatory cell infiltration. The portal tracts showed vascular congestion, dilatation, and proliferation of the bile ducts. There was a marked increase in the collagen deposition around the portal tracts, central veins, and in-between hepatocytes. Marked depletion of glycogen content with sporadic positive PAS reactions within the cytoplasm of the hepatocytes was also noticed. Hepatocyte apoptosis was evident by a significant increase in caspase-3 expression compared to the control group. On the other hand, ZnO NP +Resveratrol group showed the restoration of normal hepatic lobules with few areas of subcapsular haemorrhage. Most hepatocytes appeared polygonal in shape, with acidophilic cytoplasm and central rounded vesicular nuclei. While few hepatocytes appeared degenerated with dark stained pyknotic nuclei and cytoplasm showed hydropic degeneration. Most of the blood sinusoids returned to their normal sizes with flattened endothelial cells. However, sometimes they were congested, dilated, and contained Kupffer cells. Some central veins appeared congested. The portal tract also returned to its normal size with a marked decrease in inflammatory cell infiltration and showed apparent minimal collagen fiber deposition. Another finding in the present work was that Resveratrol restored the glycogen content of the hepatocytes which were depleted in ZnO NPs group. This was marked by a strong PAS positive reaction in the cytoplasm of most hepatocytes, with few hepatocytes with weak PAS reaction. Resveratrol co-treatment with ZnO NPs significantly reduced the degeneration and necrosis of hepatocytes. This was evident in our study by the apparent negative Caspase-3 reaction in the cytoplasm of most hepatocytes. Clear deterioration of liver function tests was observed in ZnO NPs group compared with the control, with marked improvement in the group treated with ZnO NPs and Resveratrol together. Conclusion The present study demonstrated the degenerative and hepatotoxic effects of ZnO NPs on the histological structure and function of the liver and the hepatoprotective role of Resveratrol against these effects.

Silver Nanoparticles Phytofabricated through Azadirachta indica: Anticancer, Apoptotic, and Wound-Healing Properties.

Silver nanoparticles (AgNPs) have unlocked numerous novel disciplines in nanobiotechnological protocols due to their larger surface area-to-volume ratios, which are attributed to the marked reactivity of nanosilver, and due to their extremely small size, which enables AgNPs to enter cells, interact with organelles, and yield distinct biological effects. AgNPs are capable of bypassing immune cells, staying in the system for longer periods and with a higher distribution, reaching target tissues at higher concentrations, avoiding diffusion to adjacent tissues, releasing therapeutic agents or drugs for specific stimuli to achieve a longer duration at a specific rate, and yielding desired effects. The phytofabrication of AgNPs is a cost-effective, one-step, environmentally friendly, and easy method that harnesses sustainable resources and naturally available components of plant extracts (PEs). In addition, it processes various catalytic activities for the degradation of various organic pollutants. For the phytofabrication of AgNPs, plant products can be used in a multifunctional manner as a reducing agent, a stabilizing agent, and a functionalizing agent. In addition, they can be used to curtail the requirements for any additional stabilizing agents and to help the reaction stages subside. Azadirachta indica, a very common and prominent medicinal plant grown throughout the Indian subcontinent, possesses free radical scavenging and other pharmaceutical properties via the regulation of proinflammatory enzymes, such as COX and TOX. It also demonstrates anticancer activities through cell-signaling pathways, modulating tumor-suppressing genes such as p53 and pTEN, transcriptional factors, angiogenesis, and apoptosis via bcl2 and bax. In addition, it possesses antibacterial activities. Phytofabricated AgNPs have been applied in the areas of drug delivery, bioimaging, biosensing, cancer treatment, cosmetics, and cell biology. Such pharmaceutical and biological activities of phytofabricated AgNPs are attributed to more than 300 phytochemicals found in Azadirachta indica, and are especially abundant in flavonoids, polyphenols, diterpenoids, triterpenoids, limonoids, tannins, coumarin, nimbolide, azadirachtin, azadirone, azadiradione, and gedunin. Parts of Azadirachta indica, including the leaves in various forms, have been used for wound healing or as a repellent. This study was aimed at examining previously biosynthesized (from Azadirachta indica) AgNPs for anticancer, wound-healing, and antimicrobial actions (through MTT reduction assay, scratch assay, and microbroth dilution methods, respectively). Additionally, apoptosis in cancer cells and the antibiofilm capabilities of AgNPs were examined through caspase-3 expression, dentine block, and crystal violet methods. We found that biogenic silver nanoparticles are capable of inducing cytotoxicity in HCT-116 colon carcinoma cells (IC50 of 744.23 µg/mL, R2: 0.94), but are ineffective against MCF-7 breast cancer cells (IC50 >> 1000 µg/mL, R2: 0.86). AgNPs (IC50 value) induced a significant increase in caspase-3 expression (a 1.5-fold increase) in HCT-116, as compared with control cells. FITC-MFI was 1936 in HCT-116-treated cells, as compared to being 4551 in cisplatin and 1297 in untreated cells. AgNPs (6.26 µg/mL and 62.5 µg/mL) induced the cellular migration (40.2% and 33.23%, respectively) of V79 Chinese hamster lung fibroblasts; however, the improvement in wound healing was not significant as it was for the controls. AgNPs (MIC of 10 µg/mL) were very effective against MDR Enterococcus faecalis in the planktonic mode as well as in the biofilm mode. AgNPs (10 µg/mL and 320 µg/mL) reduced the E. faecalis biofilm by >50% and >80%, respectively. Natural products, such as Syzygium aromaticum (clove) oil (MIC of 312.5 µg/mL) and eugenol (MIC of 625 µg/mL), showed significant antimicrobial effects against A. indica. Our findings indicate that A. indica-functionalized AgNPs are effective against cancer cells and can induce apoptosis in HCT-116 colon carcinoma cells; however, the anticancer properties of AgNPs can also be upgraded through active targeting (functionalized with enzymes, antibiotics, photosensitizers, or antibodies) in immunotherapy, photothermal therapy, and photodynamic therapy. Our findings also suggest that functionalized AgNPs could be pivotal in the development of a novel, non-cytotoxic, biocompatible therapeutic agent for infected chronic wounds, ulcers, and skin lesions involving MDR pathogens via their incorporation into scaffolds, composites, patches, microgels, or formulations for microneedles, dressings, bandages, gels, or other drug-delivery systems.

Possible ameliorative effect of human placental extract on methotrexate-induced nephrotoxicity in albino rats

BackgroundMethotrexate (MTX) is one of chemotherapeutic drugs that induce several side effects. The present study aimed to investigate the ameliorative effect of human placental extract (HPE) against MTX-induced nephrotoxicity in rats. In this study, forty adult male albino rats were equally divided into four groups. Control group: rats were daily injected intraperitoneally with physiological saline (0.5 ml for each rat) for 5 days, HPE group: rats were subcutaneously injected with HPE at a dose level of 10.08 mg/Kg b.w/day for 2 weeks, MTX group: rats were intraperitoneally injected with MTX at a dose level of 5 mg/Kg b.w/day for 5 consecutive days, MTX and HPE group: rats were intraperitoneally injected with MTX (at the same dosage of MTX group) for 5 days and at the same time they were subcutaneously injected with HPE (at an exact dosage of HPE group), daily for 2 weeks. Twenty-four hours after the last dose for each treatment, rats were killed and blood samples were collected for determination of urea, creatinine, sodium (Na+) and potassium (K+) levels. Kidney tissues were taken for histological examination and immunohistochemical staining of both cysteine-aspartic protease-3 (caspase-3) and proliferating antigen Ki-67 (Ki-67) expressions.ResultsFrom the obtained data, MTX induced nephrotoxicity through a highly significant increase in urea, creatinine, Na+ and K+ levels compared with the control group. In addition to massive histological alterations, a highly significant increase in caspase-3 expression and a significant decrease in Ki-67 expression were observed. On the other hand, injection with HPE ameliorated urea, creatinine, Na+ and K+ levels comparing to MTX group. Moreover, HPE markedly improved the histological and immunohistochemical changes resulted from MTX treatment.ConclusionsIt is concluded that HPE ameliorated the nephrotoxicity induced by MTX.

Alpha-lipoic acid-role in improving both reserpine toxicity and paroxetine treatment in the cerebral cortex of albino rats; histological, ultrastructural, immunohistohemical and biochemical studies

BackgroundReserpine is a monoamine depletory drug cause oxidative damage and used to induce depression-like features in rodent model. Paroxetine is an antidepressant drug that exerts its effects by inhibiting dopaminergic neurons although it may exert much pathological damage. Alpha-lipoic acid (ALA) is an endogenous antioxidant co-factor of important enzymatic complexes. The present study was aimed to elucidate the possible protective effect of ALA in the improvement of the deleterious cerebral cortex injury after reserpine and paroxetine treatment. Forty adult male albino rats were equally divided into 5 groups. Group I served as control group orally treated with saline solution all the experiment period. Group II animals orally treated with ALA (200 mg/kg/day) for six weeks. The induction of depression-like features occurred when the rest of animals were intraperitoneally treated with 25 mg/kg of reserpine once daily for consecutive 14 day. Then these animals were divided into; Group III (reserpine group) animals in this group were sacrificed on 15th day. Group IV; reserpine-treated animals were treated with paroxetine (20 mg/kg) daily for 6 weeks. Group V, animals in this group were received paroxetine and ALA daily for 6 weeks.ResultsReserpine-treated rats showed disorganized layers of cerebral cortex with degenerative, apoptotic and necrotic changes. Ultrastructure changes include both pyramidal and granule cells with severe degenerative, necrotic and apoptotic features. The nuclei appeared pyknotic; irregular with chromatin condensation as well as the cytoplasm of these cells contained many degenerated organelles. In addition, a significant increase in total oxidative stress and decrease in total antioxidant capacity, norepinephrine, dopamine and serotonin levels were recorded. The same treatment showed significant decrease in proliferating cell nuclear antigen (PCNA) expression and significant increase in caspase-3 expression in the granule and pyramidal cells. After paroxetine-treatment these parameters were more or less similar to those observed in reserpine-treated ones. While an obvious improvement was appeared when animal treated with both paroxetine and ALA and; all parameters restored its normal features.ConclusionsThis study concluded that; ALA treatment attenuated the cerebral injury induced by reserpine and improved the effects of paroxetine in rats due to its anti-inflammatory, anti-apoptotic and antioxidant activities.

Protective effects of Zingiber officinale extract on myocardium and placenta against labetalol-induced histopathological, immune-histochemical, and ultrastructural alterations in pregnant rats

BackgroundLabetalol is an antihypertensive drug commonly used in obstetrics for both long-term treatment and the acute management of severe maternal hypertension. However, there have not been published articles about the effects of labetalol on the myocardium and the placenta. This study aimed to estimate the histological, immune-histochemical, and ultrastructural cardio- and placental-toxicity of labetalol administration and the effectiveness of ginger against this toxicity in pregnant rats. Labetalol was daily administrated orally with or without ginger at a dose of 300 mg/kg and 200 mg/kg, respectively, during the gestation days 6 to 20.ResultsIn the labetalol-administrated group, the myocardium displayed histological and ultrastructure destructive changes and a significant increase in caspase-3 expression. Labetalol also decreased the placental weight compared with the control group, caused marked degeneration and disorganization of their architecture, and increased caspase-3 expression. Co-administration of ginger after labetalol highly ameliorates the adverse effect of labetalol on both cardiac and placental tissues.ConclusionsIt is concluded that ginger can mitigate cardiac and placental toxicity induced by labetalol administration into pregnant rats.

Toxicity and postwithdrawal effects of ketamine on the reproductive function of male albino rats: Hormonal, histological, and immunohistochemical study

Ketamine is increasingly used in clinical practice, and ketamine addiction is common in young individuals. There are limited reviews on the chronic effects of ketamine on the testes. Three groups of rats received saline or ketamine 50 mg/kg/day intraperitoneally for 6 weeks with or without a subsequent 4-week drug-free period. Serum follicle-stimulating hormone, luteinizing hormone, prolactin, and testosterone levels, as well as testicular malondialdehyde concentrations, were measured. Epididymal sperm parameters were assessed. Testicular tissues were examined by hematoxylin and eosin staining and immunohistochemical staining using caspase-3 and vimentin antibodies. Chronic ketamine injection significantly decreased the levels of the examined hormones and adversely affected sperm parameters. Testicular tissue showed a significant increase in caspase-3 expression. In addition, Sertoli cell shape and position were disrupted. These effects disappeared 4 weeks after drug withdrawal. Chronic ketamine treatment has revisable hazardous effects on the rat reproductive function. There is a need to increase the knowledge of physicians and the public regarding these harmful effects of ketamine.

Intracellular mechanisms and behavioral changes in mouse model of attention deficit hyperactivity disorder: Importance of age-specific NMDA receptor blockade

Exposure of NMDA receptor antagonists during developmental stages leads to behavioral consequences like attention deficit hyperactivity disorder (ADHD). However, the underlying molecular mechanisms have remained poorly understood. Herein, we studied the phosphorylated Akt (pAkt) and caspase-3, the key regulators of neuronal cell survival/death, as the probable downstream targets of MK-801 often used to engender ADHD-like condition. Swiss albino mice at postnatal days (PND) 7, 14 or 21 were injected with a single dose of MK-801 and evaluated for hyperactivity (open field test) and memory deficit at adolescence (PND 30) and adult stages (PND 60). PND 7 or 14 treatment groups (but not PND 21) consistently showed hyperactivity at the adolescence stage. A significant increase in working and reference memory errors in radial arm maze was noted at the adolescence age. PND 7 group continued to display the symptoms even in adulthood. All the treatment groups showed a significant decrease in the percent alterations (Y-maze) and discrimination index (novel object recognition test) at adolescence age. A significant increase in caspase-3 expression was noted in the prefrontal cortex (PFC) and hippocampus, whereas increased pAkt was noticed only in the hippocampus, following a single injection of MK-801 at PND 7. Concurrently, PND 7 treatment group showed significantly decreased neuronal nuclei (NeuN) expression (a marker for mature neurons) in the dentate gyrus, cornu ammonis-3 and PFC, but not in cornu ammonis-1, at adolescence age. We suggest that the observed symptoms of ADHD at adolescence and adulthood stages may be linked to alteration in pAkt and caspase-3 followed MK-801 treatment at PND 7.

Effect of Hydroxyprogesterone (17-OHPC) on Placenta in a Rat Model of Preeclampsia: Histological and Immunohistochemical Study

Background: Preeclampsia (PE) is a leading cause of maternal and fetal morbidity and mortality. Patients with severe PE exhibit significantly lower serum progesterone concentrations. There is limited information about the use of progesterone to manage or treat PE.Aim of the work: To investigate the possible protective and/or therapeutic effects of 17-hydroxyprogesterone caproate (17-OHPC) therapy in a rat model of PE and the possibly involved mechanisms that monitored biochemically, histologically, and immunohistochemically.Methods: Twenty-four pregnant female albino rats were randomly divided into 4 groups (6 rats/each): The control group, preeclampsia group (PE-group), prophylactic group, and treated group. The mean arterial blood pressure (MAP) and 24-hour protein in urine were determined. Rats were sacrificed at day 22 of gestation and placentae were processed for paraffin.Results: The MAP and proteinuria in the PE-group were significantly higher compared to the control group. The prophylactic and treated groups showed significant decrease in MAP and proteinuria as compared to PE-group. In the treated group, they nearly returned to the normal levels. The histological examination of the PE-group showed dilated maternal blood sinuses, depositions of hemosiderin granules and numerous phagocytic trophoblastic cells containing cytoplasmic hemosiderin granules. Fetal blood vessels showed homogenous acidophilic material occluding their lumen, edema of the extra-embryonic fetal membranes and intra-villous tissue, and numerous nucleated RBCs. The prophylactic group showed some improvement while the treated group showed more or less normal maternal blood sinuses and interhemal membrane with few hemosiderin granules and few nucleated RBCs. There was a significant increase in caspase-3 expression and a significant decrease in the eNOS expression in PE-group compared to the control group. While the prophylactic and the treated groups had a significant decrease in caspase-3 expression and a significant increase in the eNOS expression compared to PE-group.Conclusion: The biochemical, morphological and morphometric findings suggested that the administration of 17-OHPC to preeclamptic rat decreased blood pressure, proteinuria, inflammation, apoptosis, and improved vascular eNOS expression in placenta. The 17-OHPC possessed curative effect on L-NAME induced PE changes in rat placenta which was more obvious than its protective effect.

Reduction of miR-21 induces SK-N-SH cell apoptosis and inhibits proliferation via PTEN/PDCD4.

MicroRNA (miR/miRNA)-21 is a well-known oncogenic miRNA that is overexpressed in various types of tumors. The tumor-suppressor genes programmed cell death 4 (PDCD4) and phosphatase tensin homologue (PTEN), are targets of miR-21, and are underexpressed in several types of cancer. However, the expression of miR-21 and its target genes in neuroblastoma (NB) remains unclear. In the present study, a miR-21 inhibitor oligonucleotide was transfected into the SK-N-SH cell line, and the expression of miR-21, PTEN and PDCD4 was detected through quantitative polymerase chain reaction analysis. Western blotting was used to examine levels of PTEN, PDCD4 and caspase-3 proteins. The expression of PTEN and PDCD4 in the SK-N-SH cell line transfected with the miR-21 inhibitor was significantly increased compared with untransfected SK-N-SH and negative control-transfected cells. Cell proliferation was inhibited and the apoptotic ratio was significantly increased in miR-21 inhibitor-transfected cells compared with untransfected SK-N-SH and negative control-transfected cells. Western blot analysis revealed a significant increase in caspase-3 expression compared with untransfected SK-N-SH and negative control-transfected cells. The results of the present study indicate that miR-21 may serve an oncogenic role in the cellular processes underlying NB development and thus may be a novel therapeutic target for the treatment of patients with NB.

Young Barley Indicates Antitumor Effects in Experimental Breast Cancer In Vivo and In Vitro

ABSTRACTThe effect of dietary administered young barley containing a mixture of phytochemicals to female rats for the prevention of N-methyl-N-nitrosourea-induced mammary carcinogenesis was evaluated. After carcinogen administration (14 wk), mammary tumors were removed and prepared for histopathological and immunohistochemical analysis. Moreover, in vitro evaluation of possible mechanisms in MCF-7 breast cancer cell line was performed. Barley (0.3%) demonstrated mild antitumor effect in mammary carcinogenesis, yet 3% barley did not further improve this effect. Immunohistochemical analysis of rat tumor cells in treated groups showed significant increase in caspase-3 expression and significant reduction in Ki67 expression. In addition, 3% barley significantly decreased dityrosine levels versus control. Barley in higher dose significantly decreased serum low-density lipoprotein-cholesterol in rats. In vitro studies showed that barley significantly decreased survival of MCF-7 cells in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and significantly decreased 5-bromo-20-deoxyuridine incorporation versus control. Barley prevented cell cycle progression and extended incubation with barley showed significant increase in the percentage of annexin V/propidium iodide-positive MCF-7 cells. Our results propose an antitumor effect for the mixture of phytochemicals present in young barley in a breast cancer model.

Can Malachite Green Concentrations Cause Hepatic Toxic Effects in Adult Albino Rats: Histological, Biochemical and Immunohistochemical

Malachite green (MG) has been widely used as the most efficacious antifungal agent in the fish farming industry. The aim of this study is to evaluate hepatotoxicity of different MG concentrations in adult albino rats. Forty five adult male albino rats were equally divided into 3 groups, each contains 15 rats. Group I (control), Group II (received daily dose of 3ug/kg b.wt of MG by oral gavage): divided into three subgroup equally according to time of taken sample and Group III (received daily gavage dose of 60ug/kg b.wt of MG by oral gavage): divided into three subgroup equally according to time of taken sample (Samples were taken 2 and 4 weeks from the start of the experiment and 2 weeks after drug stoppage (wash out). Hepatotoxicity was evaluated by biochemical, histopathological and immunohistochemical study. MG caused an increase of alanine aminotransferase, aspartate amino transferase (ALT and AST), alkaline phosphatase (ALP) levels, and decrease in total protein in animals treated with MG as compared to control. These biochemical alterations were confirmed by the presence of different histological changes as degeneration of hepatocytes with congested dilated central vein , mononuclear inflammatory cells infiltration with significant increase in caspase-3 expression with decrease in survivin expression and this changes were highly significant in rats treated with higher doses than other groups and persist even after 2 weeks from MG stoppage .We can conclude that MG and its residues in the edible tissue of fish could not be ignored due to their suspected hepatotoxicity with apoptotic changes even at lower doses. This leads us to put strict limitations on its use in the fish farming industry in Egypt.

Effect of feeding a high-fat diet independently of caloric intake on reproductive function in diet-induced obese female rats.

IntroductionGlobally, the prevalence of overweight and obesity is increasing, predisposing females to health hazards including compromised reproductive capacity. Our objective was to investigate the effect of ad libitum, isocalorically and hypocalorically restricted high-fat diet (HFD) feeding on reproductive function in diet-induced obese female rats.Material and methodsTwenty female albino Sprague Dawley rats were used; 5 rats were kept on a standard pellet animal diet to serve as a control group (A) and 15 rats were fed a HFD for 9 weeks to induce obesity. The HFD fed animals were equally divided into three groups: an ad libitum HFD group (B), an isocalorically restricted HFD group (C), and a hypocalorically restricted HFD group (D). Estrous cyclicity, hormonal levels, ovarian histopathology and caspase-3 immunoreactivity were evaluated.ResultsThe HFD-fed rats in groups B, C and D had significant irregularity in estrous cyclicity Vs group A (p = 0.001, 0.003 and 0.034 respectively). Groups C and D had significant reduction in serum progesterone level (p = 0.006 and 0.018 Vs A). Isocaloric restriction of HFD feeding significantly increased serum LH. Groups B and C had a significant increase in caspase-3 expression in the ovary (p < 0.001).ConclusionsAd libitum HFD interfered with the normal estrous cycle and enhanced apoptosis of luteal cells in obese female rats. The HFD restriction interfered with the normal estrous cycle and caused functional insufficiency of the corpus luteum in obese female rats. These results suggest that HFD feeding determinately affects female reproductive function independently of caloric intake.

Comparative evaluation of PLGA nanoparticle delivery system for 5-fluorouracil and curcumin on squamous cell carcinoma

PurposeThe purpose of this study is to assess the effect of 5-fluorouracil nanoparticles and curcumin naoparticles on cell proliferation and the expression of the apoptotic marker (caspase 3) in squamous cell carcinoma cell line. Material and methodsPLGA 5-fluorouracil nanopartciles and PLGA curcumin nanoparticles were prepared and applied for 24 and 48h on human laryngeal squamous carcinoma cell line (Hep-2) as regard IC 50 concentration. MTT assay was used for evaluation of cytotoxicity of prepared nanoparticles. Quantitaive reverse transcriptase polymerase chain reaction (QRT-PCR) was used for the assessment of caspase-3 expression in the treated cell line. ResultsThe drug release rate profiles was dependent upon polymer to drug ratio, noting that the higher PLGA polymer ratio to 5-fluprouracil or curcumin drug showed faster release rates. On the other hand, the least PLGA polymer ratio to 5-fluprouracil or curcumin drug showed the slowest release rates. MTT assay revelaed that 5-fluorouracil nanoparticels or curcumin nanoparticels showed a clear cytotoxic effect on Hep-2 cell line compared to non treated cancer cells. The RT-PCR assessment of caspase-3 expression revealed that there was a significant increase in caspase-3 expression in Hep-2 cell line treated with 5-fluorouracil nanoparticles or curcumin compared to non treated cancer cells. ConclusionCurcumin nanoparticles could be more active in inducing apoptosis in short term assays (24h) than long term assays (48h) due to differential cellular uptake. While 5-fluorouracil nanoparticles induced higher significant apoptosis in long term (48h) compared to curcumin group.

Fruit peel polyphenols demonstrate substantial anti-tumour effects in the model of breast cancer.

Fruit and vegetable intake is inversely correlated with cancer; thus, it is proposed that an extract of phytochemicals as present in whole fruits, vegetables, or grains may have anti-carcinogenic properties. Thus, the anti-tumour effects of fruit peel polyphenols (Flavin7) in the chemoprevention of N-methyl-N-nitrosourea-induced mammary carcinogenesis in female rats were evaluated. Lyophilized substance of Flavin7 (F7) was administered at two concentrations of 0.3 and 3 % through diet. The experiment was terminated 14 weeks after carcinogen administration, and mammary tumours were removed and prepared for histopathological and immunohistochemical analysis. In addition, using an in vitro cytotoxicity assay, apoptosis and proliferation after F7 treatment in human breast adenocarcinoma (MCF-7) cells were performed. High-dose F7 suppressed tumour frequency by 58 % (P < 0.001), tumour incidence by 24 % (P < 0.05), and lengthened latency by 8 days (P > 0.05) in comparison with the control rats, whereas lower dose of F7 was less effective. Histopathological analysis of tumours showed significant decrease in the ratio of high-/low-grade carcinomas after high-dose F7 treatment. Immunohistochemical analysis of rat carcinoma cells in vivo found a significant increase in caspase-3 expression and significant decrease in Bcl-2, Ki67, and VEGFR-2 expression in the high-dose group. Both doses demonstrated significant positive effects on plasma lipid metabolism in rats. F7 significantly decreased survival of MCF-7 cells in vitro in MTT assay by dose- and time-dependent manner compared to control. F7 prevented cell cycle progression by significant enrichment in G1 cell populations. Incubation with F7 showed significant increase in the percentage of annexin V-/PI-positive MCF-7 cells and DNA fragmentation. Our results reveal a substantial tumour-suppressive effect of F7 in the breast cancer model. We propose that the effects of phytochemicals present in this fruit extract are responsible for observed potent anti-cancer activities.

Combination of Pitavastatin and melatonin shows partial antineoplastic effects in a rat breast carcinoma model

Our previous results indicated significant tumor-suppressive effects of different statins in rat mammary carcinogenesis. The purpose of this experiment was to examine the chemopreventive effects of Pitavastatin alone and in combination with the pineal hormone melatonin in the model of N-methyl-N-nitrosourea-induced mammary carcinogenesis in female Sprague-Dawley rats. Pitavastatin was administered dietary (10mg/kg) and melatonin in an aqueous solution (20μg/ml). Chemoprevention began 7 days prior to carcinogen administration and subsequently continued for 15 weeks until autopsy. At autopsy, mammary tumors were removed and prepared for histopathological and immunohistochemical analysis. Compared to controls, Pitavastatin alone reduced average tumor volume by 58% and lengthened latency by 8 days; on the other hand, the drug increased tumor frequency by 23%. Combined administration of Pitavastatin with melatonin decreased tumor frequency by 23%, tumor volume by 44% and lengthened tumor latency by 5.5 days compared to control animals. The analysis of carcinoma cells showed significant increase in caspase-3 expression in both treated groups and a tendency of increased caspase-7 expression after Pitavastatin treatment alone. Significant expression decrease of Ki67 was found in carcinoma cells from both treated groups. Compared to control carcinoma cells, Pitavastatin alone increased VEGF expression by 41%, however melatonin totally reversed its undesirable effect. Pitavastatin combined with melatonin significantly increased femur compact bone thickness in animals. Pitavastatin alone decreased plasma triglycerides and total cholesterol levels, however it significantly increased levels of glucose. In summary, our results show a partial antineoplastic effect of Pitavastatin combined with melatonin in the rat mammary gland carcinoma model.

Phytic acid decreases deoxynivalenol and fumonisin B1-induced changes on swine jejunal explants

The purpose of the present study was to investigate the effects of phytic acid (IP6) on morphological and immunohistochemical parameters on intestinal explants exposed to deoxynivalenol (DON) and fumonisin B1 (FB1). The jejunal explants were exposed for 4 h to different treatments: control, DON (10 μM), DON plus 2.5 mM or 5 mM IP6, FB1 (70 μM), and FB1 plus 2.5 mM or 5 mM IP6. Both mycotoxins induced significant intestinal lesions and decreased villi height. The presence of 2.5 mM and 5 mM IP6 significantly inhibited the morphological changes caused by the mycotoxins. DON induced a significant increase in caspase-3 (83%) and cyclooxygenase-2 (71.3%) expression compared with the control. The presence of 5 mM IP6 induced a significant decrease in caspase-3 (43.7%) and Cox-2 (48%) expression compared with the DON group. FB1 induced a significant increase in caspase-3 expression (47%) compared to the control, whereas IP6 induced no significant change in this expression. A significant decrease in cell proliferation was observed when explants were exposed to 5 mM of IP6 in comparison with the DON and FB1 groups. The present data provide evidence that phytic acid modulates the toxic effects induced by DON and FB1 on intestinal tissue.

The prophylactic role ofd-saccharic acid-1,4-lactone against hyperglycemia-induced hepatic apoptosis via inhibition of both extrinsic and intrinsic pathways in diabetic rats

Sustained hyperglycemia and increased oxidative stress play major roles in the development of secondary complications in diabetes including liver injury. Dietary supplement of antioxidants is effective in preventing oxidative stress mediated tissue damage in diabetic pathophysiology. D-Saccharic acid 1,4-lactone (DSL), a derivative of D-glucaric acid, is present in many dietary plants and is known for its detoxifying and antioxidant properties. Our early investigation showed that DSL can ameliorate alloxan (ALX) induced diabetes mellitus and oxidative stress in rats by inhibiting pancreatic β-cell apoptosis. In the present study we investigated the protective role of DSL against hepatic dysfunction in ALX induced diabetic rats. ALX exposure elevated the blood glucose, serum ALP and ALT levels, the production of reactive oxygen species (ROS), and disturbed the intra-cellular antioxidant machineries. Oral administration of DSL restored all these alterations close to normal. By investigating the mechanism of its protective activity, we observed that DSL prevented hyperglycemia induced hepatic apoptosis by inhibiting both extrinsic and intrinsic pathways. Results showed that in the liver tissue, diabetes promoted a significant increase of TNF-α/TNF-R1 and led to the activation of caspase-8 and t-Bid. In addition, ALX exposure reciprocally regulated Bcl-2 family protein expression, disturbed mitochondrial membrane potential, and subsequently released cytochrome c from mitochondria to cytosol. As a consequence, a significant increase in caspase-3 expression was observed in the liver of diabetic animals. However, treatment of diabetic rats with DSL counteracted these changes, making it a promising approach in lessening diabetes mediated tissue damage.

Increased caspase-3 and altered expression of apoptosis-associated proteins, Bcl-2 and Bax in lichen planus

Lichen planus (LP) is a chronic inflammatory disease of probable immune-based aetiology. The pathogenesis of LP is unclear, but apoptotic changes in epidermal (epithelial) cells have been reported. To evaluate apoptosis in LP through studying caspase-3 expression and to determine whether the apoptosis-associated proteins Bcl-2 and Bax are significantly involved in the pathogenesis of LP. In total, 25 lesional biopsy specimens [15 cutaneous LP (CLP) and 10 oral LP (OLP)] and 10 control specimens [5 normal skin and 5 normal oral mucosa] were studied using immunochemical methods for the expression of caspase-3, Bcl-2 and Bax proteins. Compared with controls, a significant increase in caspase-3 and Bax protein expressions were found in LP lesions. Basal cell expression of caspase-3 was positive in 14 cases (56%), and 12 cases (48%) showed mild expression. Caspase-3 expression in inflammatory infiltrate was positive in 13 cases (52%). Of these, 12 cases (48%) showed mild positivity. Bax was localized mostly to the upper prickle layer. Basal cell expression of Bcl-2 was negative in 18 (72%) cases, with no significant difference between patients with LP and controls. Bcl-2 was expressed in the inflammatory infiltrate in 15 cases of LP (60%), showing mild expression in 12 cases (48%). Compared with CLP, there was a significant increase in caspase-3 expression in OLP, despite the nonsignificant difference in Bcl-2 and Bax protein expressions by the epithelial cells. Increased caspase-3 and altered expression of Bcl-2 and Bax were found in LP, indicating the possible involvement of these proteins in the pathogenesis of the disease. The observed increase in apoptosis in OLP compared with CLP might explain the difference in clinical behaviour that distinguishes these LP variants.

Progesterone and Caspase‐3 Activation in Equine Cyclic Corpora Lutea

Soon after ovulation, the newly formed corpus luteum (CL) starts secreting progesterone (P(4)), necessary for implantation. The CL, an ovarian transient endocrine organ, undergoes growth and regression throughout its life span. The objective of this study was to evaluate if caspase-3 mediates cell death in the equine cyclic luteal structures and relate it to luteal endocrine function. Blood and luteal tissue were collected during the breeding season after slaughter from 38 randomly assigned cycling mares. Luteal tissues were classified as corpora haemorrhagica (CH; n = 7); mid luteal phase corpora lutea (Mid-CL; n = 17); late or regressing corpora lutea (Late-CL; n = 9) and corpora albicans (CA; n = 5). Plasma P(4) concentration, determined by radioimmunoassay, showed a significant increase from CH to Mid-CL (p < 0.001), followed by a decrease to Late-CL (p < 0.001) and CA (p < 0.001). Caspase-3 processing and poly (ADP) ribose polymerase (PARP) degradation were assessed by western blotting. Active caspase-3 was twofold increased in Mid-CL, Late-CL and CA as compared with CH (p < 0.05). Immunocytochemistry also showed a significant increase in caspase-3 expression in large luteal cells in all structures when compared with CH (p < 0.05). Consistently, the endogenous caspase-3 substrate, PARP, was markedly degraded from CH to CA (p < 0.05). In fact, the ratio of full-length to degraded PARP showed a significant decrease from CH to Mid-CL, Late-CL and CA (p < 0.05). Finally, the decrease in P(4) from Mid- to Late-CL coincided with no further increases in apoptosis. In conclusion, these results suggest that the effector caspase-3 of apoptosis, might play an important role during luteal tissue involution in the mare, even though its relationship with P(4) remains to be elucidated.

E2f-2 Regulates Caspase-3 Expression and Mitotic Checkpoint Control during End-Stage Erythroid Maturation.

E2f-2 is a member of the E2F superfamily of transcription factors that plays multiple roles in regulating cell cycle progression, cell cycle exit and checkpoint control [1]. While over-expression studies have ascribed a role for E2f-2 as a transcriptional activator of cell cycle genes, our loss of function studies performed in vivo have revealed a novel role for E2f-2 in repressing cellular proliferation and mediating differentiation. Our expression analyses demonstrate that E2f-2 is up-regulated in hematopoietic tissues, and is most strongly expressed in differentiating erythroblasts. Furthermore, E2f-2 is the major pRb-associated E2F in hematopoietic tissues, suggesting E2f-2 can function as a co-repressor of cell cycle genes during end-stage differentiation. Compound Rb−/−;E2f2−/− mice were generated to understand how deregulated E2f-2 activity contributes to the erythroid defects in Rb null mice. Loss of E2f-2 restored end-stage erythroid differentiation and enucleation to Rb null erythroblasts and extended survival of Rb null mice. Loss of E2f-2 was sufficient to bypass the mitotic arrest observed in Rb null erythroblasts [2] and was associated with reduced expression of known E2f target genes involved in M phase checkpoint control, such as Aurora B and Survivin. In addition, we observed a significant increase in caspase-3 expression in E2f-2 null erythroblasts and demonstrate, for the first time, direct regulation of caspase-3 expression by E2f-2. Given known roles for caspases in promoting erythroid differentiation and enucleation, we have over-expressed caspase-3 in Rb null erythroblasts and are currently analyzing its effects on cell cycle and maturation. Thus, our work identifies caspase-3 as a key downstream target of E2f-2 during end-stage erythroid differentiation whose expression may alleviate enucleation and mitotic defects in Rb null erythroblasts. Future work aims to understand which E2f-2 target genes ensure mitotic checkpoint control, and the molecular mechanism by which caspase-3 coordinates cell cycle control with erythroid differentiation.