Propolis Enhances 5-Fluorouracil Mediated Antitumor Efficacy and Reduces Side Effects in Colorectal Cancer: An in Vitro and in Vivo Study

In this study, we investigated the combined treatment of 5-fluorouracil (5-FU) and Anatolian propolis extract (PE) on colorectal cancer (CRC)using in vitro and in vivo studies. We exposed luciferase-transfected (Lovo-Luc CRC) cells and healthy colon cells (CCD-18Co) to varying concentrations of 5-FU and PE to assess their genotoxic, apoptotic, and cytotoxic effects, as well as their intracellular reactive oxygen species (iROS) levels. We also developed a xenograft model in nude mice and evaluated the anti-tumor effects of PE and 5-FU using various methods. Our findings showed that the combination of PE and 5-FU had selectivity against cancer cells, particularly at higher doses, and enhanced the anti-tumor effectiveness of 5-FU against colon CRC. The results suggest that PE can reduce side effects and increase the effectiveness of 5-FU through iROS generation in a dose-dependent manner.     

The Interplay between Chondrocyte Redifferentiation Pellet Size and Oxygen Concentration

Chondrocytes dedifferentiate during ex vivo expansion on 2-dimensional surfaces. Aggregation of the expanded cells into 3-dimensional pellets, in the presence of induction factors, facilitates their redifferentiation and restoration of the chondrogenic phenotype. Typically 1×10⁵–5×10⁵ chondrocytes are aggregated, resulting in “macro” pellets having diameters ranging from 1–2 mm. These macropellets are commonly used to study redifferentiation, and recently macropellets of autologous chondrocytes have been implanted directly into articular cartilage defects to facilitate their repair. However, diffusion of metabolites over the 1–2 mm pellet length-scales is inefficient, resulting in radial tissue heterogeneity. Herein we demonstrate that the aggregation of 2×10⁵ human chondrocytes into micropellets of 166 cells each, rather than into larger single macropellets, enhances chondrogenic redifferentiation. In this study, we describe the development of a cost effective fabrication strategy to manufacture a microwell surface for the large-scale production of micropellets. The thousands of micropellets were manufactured using the microwell platform, which is an array of 360×360 µm microwells cast into polydimethylsiloxane (PDMS), that has been surface modified with an electrostatic multilayer of hyaluronic acid and chitosan to enhance micropellet formation. Such surface modification was essential to prevent chondrocyte spreading on the PDMS. Sulfated glycosaminoglycan (sGAG) production and collagen II gene expression in chondrocyte micropellets increased significantly relative to macropellet controls, and redifferentiation was enhanced in both macro and micropellets with the provision of a hypoxic atmosphere (2% O₂). Once micropellet formation had been optimized, we demonstrated that micropellets could be assembled into larger cartilage tissues. Our results indicate that micropellet amalgamation efficiency is inversely related to the time cultured as discreet microtissues. In summary, we describe a micropellet production platform that represents an efficient tool for studying chondrocyte redifferentiation and demonstrate that the micropellets could be assembled into larger tissues, potentially useful in cartilage defect repair.     

Morphological parallelism of sympatric cave-dwelling microsnails of the genus Georissa at Mount Silabur,Borneo (Gastropoda,Neritimorpha, Hydrocenidae)

Parallel evolution in phenotype may result when closely related taxa are adapting in the face of similar ecological pressures. Here, we discuss possible parallelism in shell morphology in the context of the microgeographic phylogeography of two conchologically distinct sympatric hydrocenid snails inhabiting a limestone outcrop and its cave system, Georissa pyrrhoderma and Georissa silaburensis, respectively, at Mount Silabur in Sarawak, Malaysian Borneo. Our results show a certain degree of morphological parallelism of a third, possibly new, cryptic Georissa species within the same cave that strongly diverged from its above-ground sister species, G. pyrrhoderma. We found that both sympatric cave species have shifted from a more sculptured, conical shell toward a broader, less sculptured form.     

Combination of AT-101/cisplatin overcomes chemoresistance by inducing apoptosis and modulating epigenetics in human ovarian cancer cells

We investigated the effects of AT-101/cisplatin combination treatment on the expression levels of apoptotic proteins and epigenetic events such as DNA methyltransferase (DNMT) and histone deacetylase (HDAC) enzyme activities in OVCAR-3 and MDAH-2774 ovarian cancer cells. XTT cell viability assay was used to evaluate cytotoxicity. For showing apoptosis, both DNA Fragmentation and caspase 3/7 activity measurements were performed. The expression levels of apoptotic proteins were assessed by human apoptosis antibody array. DNMT and HDAC activities were evaluated by ELISA assay and mRNA levels of DNMT1 and HDAC1 genes were quantified by qRT-PCR. Combination of AT-101/cisplatin resulted in strong synergistic cytotoxicity and apoptosis in human ovarian cancer cells. Combination treatment reduced some pivotal anti-apoptotic proteins such as Bcl-2, HIF-1A, cIAP-1, XIAP in OVCAR-3 cells, whereas p21, Bcl-2, cIAP-1, HSP27, Clusterin and XIAP in MDAH-2774 cells. Among the pro-apoptotic proteins, Bad, Bax, Fas, phospho-p53 (S46), Cleaved caspase-3, SMAC/Diablo, TNFR1 and Cytochrome c were induced in OVCAR-3 cells, whereas, Bax, TRAILR2, FADD, p27, phospho-p53 (S46), Cleaved caspase-3, Cytochrome c, SMAC/Diablo and TNFR1 were induced in MDAH-2774 cells. Combination treatment also inhibited both DNMT and HDAC activities and also mRNA levels in both ovarian cancer cells. AT-101 exhibits great potential in sensitization of human ovarian cancer cells to cisplatin treatment in vitro, suggesting that the combination of AT-101 with cisplatin may hold great promise for development as a novel chemotherapeutic approach to overcome platinum-resistance in human ovarian cancer.     

Acute adriamycin-induced cardiotoxicity is exacerbated by angiotension II

Adriamycin (ADR) increases the production of reactive oxygen species (ROS), which diminishes mitochondrial function. Angiotensin-II stimulates mitochondrial ROS generation. The aim of the study was to examine whether angiotensin converting enzyme (ACE) or renin inhibitors protect against ADR-induced mitochondrial function impairment. Rats were divided into five groups as control, ADR, co-treatment ADR with captopril, co-treatment ADR with aliskiren, co-treatment ADR with both captopril and aliskiren. Left ventricular function and blood pressures were assessed at the end of treatment period. Mitochondrial membrane potential (MMP) and ATP levels were determined. ADR treatment decreased the left ventricular pressure and increased the left ventricular end-diastolic pressure. ADR decreased MMP and ATP levels in myocyte mitochondria due to increasing oxidative stress. ADR decreased MMP and ATP levels due to increased oxidative stress in the heart. Inhibitors of ACE and renin caused the elevation of the decreased of MMP and ATP levels. The pathologic changes in electrocardiogram, blood pressure and left ventricular function were decreased by inhibition of Ang-II production. We concluded that inhibitors of angiotensin II are effective against ADR cardiotoxicity via the restoration of MMP and ATP production and prevention of mitochondrial damage in vivo.     

Anti-inflammatory effects of leptin and cholecystokinin on acetic acid-induced colitis in rats: role of capsaicin-sensitive vagal afferent fibers

Leptin and cholecystokinin (CCK) have a synergistic interaction in the suppression of food intake, and afford similar gastroprotective activity. The present study was designed to investigate the putative protective effects of CCK and leptin on acute colonic inflammation. Leptin or CCK-8s was injected to rats intraperitoneally immediately before and 6 h after the induction of colitis with acetic acid. CCK-A receptor antagonist (L-364,718) or CCK-B receptor antagonist (L-365,260) was injected intraperitoneally 15 min before leptin or CCK treatments. In a group of rats, vagal afferent fibers were denervated by topical application of capsaicin on the cervical vagi. Rats were decapitated at 24 h, and the distal 8 cm of the colon were removed for macroscopic scoring, determination of tissue wet weight index (WWI), histologic assessment and tissue myeloperoxidase (MPO) activity. All inflammation parameters were increased by acetic acid-induced colitis compared to control group. Leptin or CCK-8s treatment reduced these parameters in a similar manner, while co-administration of leptin and CCK was found to be more effective in reducing the macroscopic score and WWI. CCK-8s-induced reduction in the score and WWI was prevented by CCK-A, but not by CCK-B receptor antagonist, whereas neither antagonist altered the inhibitory effect of leptin on colitis-induced injury. On the other hand, perivagal capsaicin prevented the protective effects of both CCK-8s and leptin on colitis. Our results indicate that leptin and CCK have anti-inflammatory effects on acetic acid-induced colitis in rats, which appear to be mediated by capsaicin-sensitive vagal afferent fibers involving the reduction in colonic neutrophil infiltration.     

Endothelin-1-induced PMN infiltration and mucosal dysfunction in the rat small intestine

The objectives of this study were to characterize the effects of endothelin (ET)-1 on intestinal mucosal parameters and to assess the contribution of polymorphonuclear leukocytes (PMNs), intercellular adhesion molecule-1 (ICAM-1), and a platelet-activating factor (PAF) to the mucosal dysfunction induced by ET-1. Different concentrations of ET-1 (100, 200, and 400 pmol/kg) were infused into the superior mesenteric artery for 10 min, and tissue samples were obtained 30 min after terminating the infusion. ET-1 administration significantly elevated tissue myeloperoxidase activity, plasma carbonyl content, and tissue chemiluminescence intensity, indicating that ET-1 produces PMN infiltration and oxidant stress. Blood-to-lumen clearance of (51)Cr-EDTA significantly increased after ET-1 infusion (400 pmol/kg). Monoclonal antibodies against ICAM-1 (1A29, 2 mg/kg), antineutrophil serum, and PAF antagonist (WEB-2086, 10 mg/kg) attenuated the mucosal barrier dysfunction induced by ET-1. Overall, our data indicate that ET-1 causes PMN accumulation, oxidant stress, and mucosal dysfunction in the rat small intestine and that ET-1-induced mucosal dysfunction involves a mechanism that includes a role for PMNs, ICAM-1, and PAF.     

Packed Bed Bioreactor for the Isolation and Expansion of Placental-Derived Mesenchymal Stromal Cells

Large numbers of Mesenchymal stem/stromal cells (MSCs) are required for clinical relevant doses to treat a number of diseases. To economically manufacture these MSCs, an automated bioreactor system will be required. Herein we describe the development of a scalable closed-system, packed bed bioreactor suitable for large-scale MSCs expansion. The packed bed was formed from fused polystyrene pellets that were air plasma treated to endow them with a surface chemistry similar to traditional tissue culture plastic. The packed bed was encased within a gas permeable shell to decouple the medium nutrient supply and gas exchange. This enabled a significant reduction in medium flow rates, thus reducing shear and even facilitating single pass medium exchange. The system was optimised in a small-scale bioreactor format (160 cm²) with murine-derived green fluorescent protein-expressing MSCs, and then scaled-up to a 2800 cm² format. We demonstrated that placental derived MSCs could be isolated directly within the bioreactor and subsequently expanded. Our results demonstrate that the closed system large-scale packed bed bioreactor is an effective and scalable tool for large-scale isolation and expansion of MSCs.     

Purification and Characterisation of Rat Kidney Glutathione Reductase

Glutathione reductase [GR, E.C.1.8.1.7] catalyses NADPH dependent reduction of glutathione disulfide (GSSG) to reduced glutathione (GSH). Thus, it is the crucial enzyme to maintain high [GSH]/[GSSG] ratio and physiological redox status in cells. Kidney and liver tissues were considered as a rich source of GR. In this study, rat kidney GR was purified and some of its properties were investigated. The enzyme was purified 2,356 fold with a yield of 16% by using heat-denaturation and Sephadex G25 gel filtration, 2′,5′-ADP Agarose 4B, PBE94 column chromatographies. The purified enzyme had a specific activity (Vm) of 250 U/mg protein and the ratio of absorbances at wavelengths of A ₂₇₃/A _463, A ₂₈₀/A ₄₆₀, A ₃₆₅/A ₄₆₀, and A ₃₇₉/A ₄₆₃, were 7.1, 6.8, 1.2 and 1.0, respectively. Each mol of GR subunit bound 0.97 mol of FAD. NADH was used as a coenzyme by rat kidney GR but with a lower efficiency (32.7%) than NADPH. Its subunit molecular weight was estimated as 53 kDa. An optimum pH of 6.5 and optimum temperature of 65 °C were found for rat kidney GR. Its activation energy (Ea) and temperature coefficient (Q₁₀) were calculated as 7.02 kcal/mol and 1.42, respectively. The Km_(NADPH) and kcat/Km _(NADPH) values were found to be 15.3 ± 1.4 μM and 1.68 × 10⁷ M⁻¹ s⁻¹ for the concentration range of 10-200 μM NADPH and when GSSG is the variable substrate, the Km_(GSSG) and the kcat/Km_(GSSG) values of 53.1 ± 3.4 μM and 4.85 × 10⁶ M⁻¹ s⁻¹ were calculated for the concentration range of 20–1,200 μM GSSG.     

Melatonin treatment for prevention of oxidative stress: involving histopathological changes

This study was undertaken to test the effect of irradiation on the histopathology of the hypothalamus and cerebral cortex. In addition, the probable effects of radiotherapy on the activities of antioxidant enzymes and levels of nitric oxide (NO) in the plasma were investigated as well. The effects of melatonin treatment on radiotherapy-based central nervous system (CNS) damage were also studied. For this purpose, the rats were randomized into four groups. The first group was the control group (sham-exposed group), the second group received only melatonin, the third group was irradiated and the fourth group received both melatonin and irradiation. Plasma samples of rats were collected for measuring the activities of superoxide dismutase (SOD), catalase (CAT) and the levels of NO. 24 h after the interventions, tissue samples were obtained from the hypothalamus and the cerebral cortex for the light microscopic investigations. These tissues were mostly affected by radiation. The results indicated that the application of radiation significantly enhanced the levels of plasma SOD and NO. On the other hand, melatonin pretreatment prevented the decrease in plasma CAT activity induced by irradiation. It was found that the application of melatonin could significantly prevent the irradiation-induced damages. Light microscopic results revealed that the damage of the CNS by radiation was prevented by the application of melatonin.