POTENTIAL GENOTOXICITY AND HISTOPATHOLOGICAL ALTERATION EVALUATION OF HEPTEX®

Objective: The present study was performed in order to evaluate potential genotoxicity and the histopathological alteration of a traditional herbal prescription Heptex that used in the treatment of liver disease.Methods: The genotoxicity were evaluated using the in vivo chromosome aberration and micronucleus assays in bone marrow cells of male and female Sprague-Dawley rats. In addition, in vitro chromosome aberration assay in Chinese hamster ovary (CHO) cells and bacterial reverse mutations assay in Salmonella typhimurium strains and Escherichia coli (WP2-uvrA/) with and without metabolic activation system (S9 mix) were performed. Histopathological study was conducted in liver, ovary and testis tissue of Sprague-Dawley rats.Results: The genotoxicity assessment showed that Heptex did not significantly increase the number of chromosomal aberrations and frequencies of micro nucleated polychromatic erythrocytes in bone marrow cells of both male and female rats. In addition, there were no increases in the number of revertant colonies at any concentrations of Heptex used in the study. Heptex did not produce any structural aberration in CHO cells in the presence or absence of S9 mix. In addition, there were no histopathological changes induced by Heptex in rat liver, ovary and testis.Conclusion: Based on abovementioned findings, we can conclude that Heptex is generally non-toxic and does not exhibit genotoxicity or histopathological alteration.Â

Maximization of the in vitro transcorneal release and the in vivo IOP-lowering effects of Latanoprost ophthalmic gel formulations using Azone as a penetration enhancer and Carbopol-974® as a mucoadhesive.

The objective of this study was to maximize the in vitro transcorneal release, the intraocular pressure (IOP) lowering effect and the duration of action, of the Latanoprost acid (LAT) ophthalmic gels. The in vitro transcorneal release of LAT from a first set of gel formulations containing different concentrations of Azone (as enhancer) with a fixed concentration of C-974® (as mucoadhesive) was studied. The formulation that showed the greatest permeability at the lowest Azone concentration was selected for the preparation of a second set of ocular gels containing different C-974® concentrations. Their in vitro permeabilities were evaluated, and the C-974® concentration yielding the greatest in vitro permeability was chosen. The in vivo IOPlowering efficacy study for the scaled-up formulations from both sets of the test formulations was performed using a Tono-pen Avia® tonometer in rabbits for 4 consecutive days. To determine the duration of action, the most effective formulations were used for a single-dose study, and the IOP was measured at predetermined intervals until the IOP base-line was reestablished. The majority of the tested formulations showed significant but varied augmentations in both the in vitro and in vivo permeability results. The formulations GAZ-4 and GC-4 showed the greatest IOP lowering effects, i.e., 7.8±1.8 mmHg and 6.5±2.1 mm Hg, respectively. It is particularly noteworthy that for both formulations the IOP lowering effect continued for 24 hours. Their duration of action in the single-dose study were 47±2.25 hours and 48±1.5 hours, respectively. It was concluded that the in vitro release, onset, magnitude and duration of action of the LAT gels were increased and extended for up to 2 days for the two gel formulations

Syzygium aqueum: A polyphenol- rich leaf extract exhibits antioxidant, hepatoprotective, pain-killing and anti-inflammatory activities in animal models

Syzygium aqueum is widely used in folk medicine. A polyphenol-rich extract from its leaves demonstrated a plethora of substantial pharmacological properties. The extract showed solid antioxidant properties in vitro and protected human keratinocytes (HaCaT cells) against UVA damage. The extract also reduced the elevated levels of ALT, AST, total bilirubin (TB), total cholesterol (TC) and triglycerides (TG) in rats with acute CCl4 intoxication. In addition to reducing the high MDA level, the extract noticeably restored GSH and SOD to the normal control levels in liver tissue homogenates and counteracted the deleterious histopathologic changes in liver after CCl4 injection. Additionally, the extract exhibited promising anti-inflammatory activities in vitro where it inhibited LOX, COX-1, and COX-2 with a higher COX-2 selectivity than that of indomethacin and diclofenac and reduced the extent of lysis of erythrocytes upon incubation with hypotonic buffer solution. S. aqueum extract also markedly reduced leukocyte numbers with similar activities to diclofenac in rats challenged with carrageenan. Additionally, administration of the extract abolished writhes induced by acetic acid in mice and prolonged the response latency in hot plate test. Meanwhile, the identified polyphenolics from the extract showed a certain affinity for the active pockets of 5-lipoxygenase (5-LOX), cyclooxygenase-1 (COX-1) and cyclooxygenase- 2 (COX-2) explaining the observed anti-inflammatory activities. Finally, 87 secondary metabolites (mostly phenolics) were tentatively identified in the extract based on LCMS/ MS analyses. Syzygium aqueum displays good protection against oxidative stress, free radicals, and could be a good candidate for treating oxidative stress related diseases

Rutin Isolated from Chrozophora tinctoria

Osteoporosis is a chronic disease in which the skeleton loses a weighty proportion of its mineralized mass and mechanical pliability. Currently available antiosteoporotic agents suffer adverse effects that include elevated risk of thrombosis and cancer. Phytochemicals may constitute a safer and effective option. In the current work, six flavonoids were obtained from Chrozophora tinctoria and identified as amentoflavone (1), apigenin-7-O-β-D-glucopyranoside (2), apigenin-7-O-6′′-E-p-coumaroyl-β-d-glucopyranoside (3), acacetin-7-O-β-d-[α-l-rhamnosyl(1→6)]3′′-E-p-coumaroyl glucopyranoside (4), apigenin-7-O-(6′′-Z-p-coumaroyl)-β-d-glucopyranoside (5), and rutin (6). An extensive review of the literature as well as NMR and mass spectral techniques was employed in order to elucidate the compound structures. Proliferation was enhanced in MCF7, MG-63, and SAOS-2 cells after exposure to subcytotoxic levels of the tested flavonoids. Rutin was chosen for subsequent studies in SAOS-2 cells. Rutin was not found to cause any alteration in the index of proliferation of these cells, when examining the cell cycle distribution by DNA flowcytometric analysis. Rutin was, however, found to increase osteocyte and osteoblast-related gene expression and lower the expression of RUNX suppressor and osteoclast genes. When examining the influence of rutin on vitamin D levels and the activity of alkaline phosphatase enzyme, it was found to enhance both, while decreasing acid phosphatase which is a marker of osteoporosis. Thus, rutin enhances proliferation and ossification markers in bone cells

Effects of the CB1 receptor antagonists AM6545 and AM4113 on metabolic syndrome-induced prostatic hyperplasia in rats

Metabolic syndrome (MetS) is a combination of metabolic disorders that can predispose individuals to benign prostatic hyperplasia (BPH). The inhibition of the cannabinoid 1 (CB1) receptor has been used to treat metabolic disorders in animal models. This study reports the use of a peripherally restricted CB1 antagonist (AM6545) and a neutral CB1 antagonist (AM4113) to improve MetS-related BPH in rats. Animals were divided into three control groups to receive either a normal rodent diet, AM6545, or AM4113. MetS was induced in the fourth, fifth, and sixth groups using a concentrated fructose solution and high-salt diet delivered as food pellets for eight weeks. The fifth and sixth groups were further given AM6545 or AM4113 for additional four weeks. Body and prostate weights were measured and prostate sections were stained with hematoxylin eosin. Cyclin D1, markers of oxidative stress and inflammation, and levels of the endocannabinoids were recorded. BPH in rats with MetS was confirmed through increased prostate weight and index, as well as histopathology. Treatment with either AM6545 or AM4113 significantly decreased prostate weight, improved prostate histology, and reduced cyclin D1 expression compared with the MetS group. Groups treated with CB1 antagonists experienced reduced lipid peroxidation, recovered glutathione depletion, restored catalase activity, and had lower inflammatory markers interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α). MetS rats treated with either AM6545 or AM4113 showed reduced concentrations of anandamide (AEA) and 2-arachidonoylglycerol (2-AG) in the prostate compared with the MetS group. In conclusion, the CB1 antagonists AM6545 and AM4113 protect against MetS-induced BPH through their anti-proliferative, antioxidant, and anti-inflammatory effects

Induction of Apoptosis Coupled to Endoplasmic Reticulum Stress in Human Prostate Cancer Cells by n-butylidenephthalide

BACKGROUND: N-butylidenephthalide (BP) exhibits antitumor effect in a variety of cancer cell lines. The objective of this study was to obtain additional insights into the mechanisms involved in BP induced cell death in human prostate cancer cells. METHODS/PRINCIPAL FINDINGS: Two human prostate cancer cell lines, PC-3 and LNCaP, were treated with BP, and subsequently evaluated for their viability and cell cycle profiles. BP caused cell cycle arrest and cell death in both cell lines. The G0/G1 phase arrest was correlated with increase levels of CDK inhibitors (p16, p21 and p27) and decrease of the checkpoint proteins. To determine the mechanisms of BP-induced growth arrest and cell death in prostate cancer cell lines, we performed a microarray study to identify alterations in gene expression induced by BP in the LNCaP cells. Several BP-induced genes, including the GADD153/CHOP, an endoplasmic reticulum stress (ER stress)-regulated gene, were identified. BP-induced ER stress was evidenced by increased expression of the downstream molecules GRP78/BiP, IRE1-α and GADD153/CHOP in both cell lines. Blockage of IRE1-α or GADD153/CHOP expression by siRNA significantly reduced BP-induced cell death in LNCaP cells. Furthermore, blockage of JNK1/2 signaling by JNK siRNA resulted in decreased expression of IRE1-α and GADD153/CHOP genes, implicating that BP-induced ER stress may be elicited via JNK1/2 signaling in prostate cancer cells. BP also suppressed LNCaP xenograft tumor growth in NOD-SCID mice. It caused 68% reduction in tumor volume after 18 days of treatment. CONCLUSIONS: Our results suggest that BP can cause G0/G1 phase arrest in prostate cancer cells and its cytotoxicity is mediated by ER stress induction. Thus, BP may serve as an anticancer agent by inducing ER stress in prostate cancer

Inhibition of Intestinal Adenoma Formation in APCMin/+ Mice by Riccardin D, a Natural Product Derived from Liverwort Plant Dumortiera hirsuta

BACKGROUND: Mutation of tumor suppressor gene, adenomatous polyposis coli (APC), is the primary molecular event in the development of most intestinal carcinomas. Animal model with APC gene mutation is an effective tool for study of preventive approaches against intestinal carcinomas. We aimed to evaluate the effect of Riccardin D, a macrocyclic bisbibenzyl compound, as a chemopreventive agent against intestinal adenoma formation in APC(Min/+) mice. METHODS: APC(Min/+) mice were given Riccardin D by p.o. gavage for 7 weeks. Mice were sacrificed, and the number, size and histopathology of intestinal polyps were examined under a microscope. We performed immunohistochemical staining, western blotting, reverse transcriptase-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) in intestinal polyps to investigate the mechanism of chemopreventive effect of Riccardin D. RESULTS: Riccardin D treatment resulted in a significant inhibition of intestinal adenoma formation, showing a reduction of polyp number by 41.7%, 31.1% and 44.4%, respectively, in proximal, middle and distal portions of small intestine. The activity of Riccardin D against polyp formation was more profound in colon, wherein Riccardin D decreased polyp number by 79.3%. Size distribution analysis revealed a significant reduction in large-size polyps (2-3 mm) by 40.0%, 42.5% and 33.3%, respectively, in proximal, middle and distal portions of small intestine, and 77.8% in colon. Histopathological analysis of the intestinal polyps revealed mostly hyperplastic morphology without obvious dysplasia in Riccardin D-treated mice. Molecular analyses of the polyps suggested that the inhibitory effect of Riccardin D on intestinal adenoma formation was associated with its abilities of reduction in cell proliferation, induction of apoptosis, antiangiogenesis, inhibition of the Wnt signaling pathway and suppression of inflammatory mediators in polyps. CONCLUSIONS: Our results suggested that Riccardin D exerts its chemopreventive effect against intestinal adenoma formation through multiple mechanisms including anti-proliferative, apoptotic, anti-angiogenic and anti-inflammatory activity

Chemotherapeutic Sensitization of Leptomycin B Resistant Lung Cancer Cells by Pretreatment with Doxorubicin

The development of novel targeted therapies has become an important research focus for lung cancer treatment. Our previous study has shown leptomycin B (LMB) significantly inhibited proliferation of lung cancer cells; however, p53 wild type lung cancer cells were resistant to LMB. Therefore, the objective of this study was to develop and evaluate a novel therapeutic strategy to sensitize LMB-resistant lung cancer cells by combining LMB and doxorubicin (DOX). Among the different treatment regimens, pretreatment with DOX (pre-DOX) and subsequent treatment with LMB to A549 cells significantly decreased the 50% inhibitory concentration (IC50) as compared to that of LMB alone (4.4 nM vs. 10.6 nM, P<0.05). Analysis of cell cycle and apoptosis by flow cytometry further confirmed the cytotoxic data. To investigate molecular mechanisms for this drug combination effects, p53 pathways were analyzed by Western blot, and nuclear proteome was evaluated by two dimensional-difference gel electrophoresis (2D-DIGE) and mass spectrometry. In comparison with control groups, the levels of p53, phospho-p53 (ser15), and p21 proteins were significantly increased while phospho-p53 (Thr55) and survivin were significantly decreased after treatments of pre-DOX and LMB (P<0.05). The 2D-DIGE/MS analysis identified that sequestosome 1 (SQSTM1/p62) had a significant increase in pre-DOX and LMB-treated cells (P<0.05). In conclusion, our results suggest that drug-resistant lung cancer cells with p53 wild type could be sensitized to cell death by scheduled combination treatment of DOX and LMB through activating and restoring p53 as well as potentially other signaling pathway(s) involving sequestosome 1