Molecular and Functional Characterization of Choline Transporter-Like Proteins in Esophageal Cancer Cells and Potential Therapeutic Targets

In this study, we examined the molecular and functional characterization of choline uptake in the human esophageal cancer cells. In addition, we examined the influence of various drugs on the transport of [3H]choline, and explored the possible correlation between the inhibition of choline uptake and apoptotic cell death. We found that both choline transporter-like protein 1 (CTL1) and CTL2 mRNAs and proteins were highly expressed in esophageal cancer cell lines (KYSE series). CTL1 and CTL2 were located in the plasma membrane and mitochondria, respectively. Choline uptake was saturable and mediated by a single transport system, which is both Na+-independent and pH-dependent. Choline uptake and cell viability were inhibited by various cationic drugs. Furthermore, a correlation analysis of the potencies of 47 drugs for the inhibition of choline uptake and cell viability showed a strong correlation. Choline uptake inhibitors and choline deficiency each inhibited cell viability and increased caspase-3/7 activity. We conclude that extracellular choline is mainly transported via a CTL1. The functional inhibition of CTL1 by cationic drugs could promote apoptotic cell death. Furthermore, CTL2 may be involved in choline uptake in mitochondria, which is the rate-limiting step in S-adenosylmethionine (SAM) synthesis and DNA methylation. Identification of this CTL1- and CTL2-mediated choline transport system provides a potential new target for esophageal cancer therapy.

Fucoidan attenuates 6-hydroxydopamine-induced neurotoxicity by exerting anti-oxidative and anti-apoptotic actions in SH-SY5Y cells

Parkinson's disease (PD) is an irreversible neurological disorder with related locomotor dysfunction and is haracterized by the selective loss of nigral neurons. PD can be experimentally induced by 6-hydroxydopamine (6-OHDA). It has been reported that reactive oxygen species, which deplete endogenous glutathione (GSH) levels, may play important roles in the dopaminergic cell death characteristic of PD. Fucoidan, a sulfated algal polysaccharide, exhibits anti-inflammatory and anti-oxidant actions. In this study, we investigated whether fucoidan can protect against 6-OHDA-mediated cytotoxicity in SH-SY5Y cells. Cytotoxicity was evaluated by using MTT and LDH assays. Fucoidan alleviated cell damage evoked by 6-OHDA dose-dependently. Fucoidan reduced the number of apoptotic nuclei and the extent of annexin-V-associated apoptosis, as revealed by DAPI staining and flow cytometry. Elevation of lipid peroxidation and caspase-3/7 activities induced by 6-OHDA was attenuated by fucoidan, which also protected against cytotoxicity evoked by buthionine-sulfoximine-mediated GSH depletion. Reduction in the glutathione/glutathione disulfide ratio induced by 6-OHDA was reversed by fucoidan, which also inhibited 6-OHDA-induced disruption of mitochondrial membrane potential. The results indicate that fucoidan may have protective action against 6-OHDA-mediated neurotoxicity by modulating oxidative injury and apoptosis through GSH depletion.

Grapevine fruit extract protects against radiation-induced oxidative stress and apoptosis in human lymphocyte.

Ionizing radiation (IR) causes oxidative stress through overwhelming generation of reactive oxygen species (ROS) in the living cells leading the oxidative damage further to biomolecules. Grapevine (Vitis vinifera L.) posses several bioactive phytochemicals and is the richest source of antioxidants. In this study, we investigated V. vinifera for its phytochemical content, enzymes profile and, ROS- and oxidant-scavenging activities. We have also studied the fruit extract of four different grapevine viz., Thompson seedless, Flame seedless, Kishmish chorni and Red globe for their radioprotective actions in human lymphocytes. The activities of ascorbic acid oxidase and catalase significantly (P <0.01) differed among extracts within the same cultivar, while that of peroxidase and polyphenol oxidase did not differ significantly. The superoxide radical-scavenging activity was higher in the seed as compared to the skin or pulp of the same cultivar. Pretreatment with grape extracts attenuated the oxidative stress induced by 4 Gy γ-radiation in human lymphocytes in vitro. Further, γ-radiation-induced increase in caspase 3/7 activity was significantly attenuated by grape extracts. These results suggest that grape extract serve as a potential source of natural antioxidants against the IR-induced oxidative stress and also inhibit apoptosis. Furthermore, the protective action of grape depends on the source of extract (seed, skin or pulp) and type of the cultivars.

Effects of hydroquinone on retinal and vascular cells in vitro.

Aim: To explore the molecular pathophysiology that might explain the epidemiologic association between cigarette smoke and age-related macular degeneration (AMD) by examining the effects of hydroquinone (HQ), a toxic compound present in high concentration in cigarette smoke-related tar, on human retinal pigment epithelial cells (ARPE-19), rat retinal neurosensory cells (R-28), and human microvascular endothelial cells (HMVEC). Materials and Methods: ARPE-19, R-28, and HMVEC were treated for 24 h with four different concentrations of HQ (500 μM, 200 μM, 100 μM, 50 μM). Cell viability, caspase-3/7 activation, DNA laddering patterns, and lactate dehydrogenase (LDH) levels were analyzed. Results: At 50 μM HQ, R-28 cells showed a significant decrease in cell viability compared with the dimethyl sulfoxide (DMSO)-treated controls. At the 100–500 μM concentrations, all three cell lines showed significant cell death (P < 0.001). In the ARPE-19, R-28, and HMVEC cultures, the caspase-3/7 activities were not increased at any of the HQ concentration. Conclusion: Our findings suggest that the mechanism of cell death in all three cell lines was through non-apoptotic pathway. In addition, neuroretinal R-28 cells were more sensitive to HQ than the ARPE-19 and HMVEC cultures.

Effects of Honokiol on Proliferation and Apoptosis on U937 Cells / 中山大学学报(医学科学版)

[Objective] To investigate the anti-proliferative and apoptosis effect induced by Honokiol (HNK) on human myeloid leukemia cell line U937 cells in vitro.[Methods] After treated with different concentration of HNK,Hoechst33342 fluorescent staining was used to detect cell apoptosis;the growth inhibition ration of U937 cells and PBMCs were analyzed by MTT assay;the apoptosis ration was detected by flow cytometry;mitochondrial membrane potential was explored by rhodamine 123 stain;Caspase3/7 protein activity kit was used to test the Caspase3/7 activity;the Caspase-3 and Caspase-7 mRNA levels were detected by real-time fluorescent relative-quantification reverse transcriptional PCR (FQ-PCR).[Results] Honokiol could significantly inhibit the proliferation of U937 cells in terms of the indexes of IC50/U937 11.8 μg/mL and IC50/PBMCs 40.3 μg/mL,and the anti-proliferative effect was in a time and concentration dependent manner;Flow cytometry analysis manifested that Honokiol could induce U937cells apoptosis by Annexin V/PI double Annexin V/PI fluorescein stain;Honokiol significantly inhibited the mitochondrial membrane potential of U937 cells and enhanced the ability of Caspase3/7 and the mRNA expression levels,but not the PBMCs.[Conclusion] HNK can inhibit U937 cells proliferation and induce cells apoptosis via activating Caspase 3/7.

RNAi inhibits Survivin expression and increases cell apoptosis / 基础医学与临床

Objective To investigate the effect of down regulation of SURVIVIN on cell and subsequent apoptosis in cervical cancer cells HeLa.Methods(1)The U6 promoter was obtained by PCR from liver of mouse and ligated into TA vector.RNAi vector(psSURVIVIN) and psN(control vector) containing of the U6 promoter was established.(2)Using HeLa cells as a model system,two groups were set stably up transfected with RNAi control plamid and psSURVIVIN(SURVIVIN RNAi plasmid),respectively.The expression of SURVIVIN in HeLa cells was measured by Western blot and immunofluorescence methods.(3)The activity of caspase 3/7 was detected using caspase-Glo(tm) 3/7 assay reagent.Results(1)Cell apoptotic rate was significantly increased by transfection with RNAi targeting plasmid,and cell was arrested at G0/G1(P