Inhibitory Effect of Curcumin on Proliferation of Human Pterygium Fibroblasts

In order to investigate the effect of curcumin on proliferation and apoptosis of human pterygium fibroblasts (HPF) in culture and search for a new method to prevent the recurrence after pterygium surgery, HPF was incubated with 0-160 μmol/L curcumin for 24-96 h. The MTT method was used to assay the biologic activities of curcumin at different time points and different doses. The expression of proliferating cell nuclear antigen (PCNA) in each group was detected by immunohistochemistry. The cell cycle distribution was detected by flow cytometry (FCM). Admini- stration of 20-80 μmol/L curcumin for 24-72 h could significantly inhibit HPF proliferation in a dose- and time-dependent manner (P<0.05). After treatment with curcumin at different concentrations of 20, 40, 80 and 160 μmol/L for 24 h, FCM revealed there was a significant sub-G1 peak at each concentration. The number of HPF in G0/G1 phase was increased, while in S phase, it was decreased (P<0.05). At the concentration of 20-80 μmol/L, curcumin, in a dose-dependent manner (P<0.05), could inhibit the expression of PCNA in HPF. It was suggesterd that curcumin could significantly in- hibit the proliferation of HPF, make HPF arrest in G0/G1 phase and induce the apoptosis of HPF in a dose- and time-dependent manner.

EFFECT OF TNF-a AND IFN-g ON THE EXPRESSION OF INDUCIBLE NITRIC OXIDE SYNTHASE GENE AND PROLIFERATION INHIBITION OF HUMAN COLON CANCER CELL LINE

Objective: To study the expression of the inducible nitric oxide synthase (iNOS) gene and the effects of tumor necrosis factor-α(TNF-a) and interferon-γ(IFN-g)on proliferation of the continuous cultured human colon cancer cell line CCL229. Methods: Using the molecular and biochemical techniques and electron microscopy to analyze the expression of iNOS, production of NO and growth characteristics of human colon cancer cells. Results: cytokine treatment can induce expression of the iNOS gene and production of nitric oxide was significantly higher after treatment of CCL229 cells with TNF-αor IFN-γ. Treatment with either cytokine or a combination of both significantly increased levels of Malondialdehyde (MDA) over control. Furthermore, cytokine treatment increased the proliferation inhibition rate as assessed in vitro and decreased the cell proliferation index on flow cytometry. Electron microscopy showed that cells treated with cytokines had fewer pseudopodia or cell processes than control cells and that cytokine treated cells had dilatation of the mitochondria and endoplasmic reticulum and dilated vesicular or tubular cisternae. Conclusion: Our findings indicate that TNF-α and IFN-γ induce the expression of iNOS gene in CCL229 cells, which increases the production of nitric oxide, inhibits proliferation, causes lipid peroxidation, and results in ultrastructural changes.

Inhibiting Smooth Muscle Cell Proliferation via Immobilization of Heparin/Fibronectin Complexes on Titanium Surfaces

The aim of this study was to investigate the inhibitory effect of heparin/fibronectin （Hep/Fn） complexes on neointimal hyperplasia following endovascular intervention. Hep/Fn complexes were immobilized onto titanium （Ti） surfaces, with subsequent X-ray photoelectron spectroscopy （XPS）, Toluidine Blue 0 （TBO） and immunohistochemistry methods were used to characterize surface properties. Smooth muscle cell （SMC） cultures were used to evaluate the effect of Hep/Fn complexes on SMC proliferation. Results showed that Hep/Fn complexes successfully immobilized onto Ti surfaces and resulted in an inhibition of SMC proliferation. This study suggests that Hep/Fn surface-immobilized biomaterials develop as a new generation of biomaterials to prevent neointimal hyperplasia, particularly for use in cardiovascular implants.

Adenovirus-mediated NDRG2 inhibits the proliferation of human renal cell carcinoma cell line OS-RC-2 in vitro

Objective:To investigate the inhibitory effects of adenovirus-mediated NDRG2 on the proliferation of human renal cell carcinoma cell line OS-RC-2 in vitro.Methods:NDRG2 was harvested by RT-PCR,confirmed by DNA sequencing,and then cloned into the eukaryotic expression vector pIRES2-EGFP,which encodes green fluorescent protein(GFP),to construct p1RES2-EGFP-NDRG2 plasmid.OS-RC-2 cells with NDRG2 negative expression were transfected with p1RES2-EGFP-NDRG2 plasmid.The growth of transfected OS-RC-2 cells was observed under light and fluorescence microscopes.After colony-forming cell assays,cell proliferation detection and MTT assays,the growth curves of cells in each group were plotted to investigate the inhibitory effects of adenovirus-mediated NDRG2 on the proliferation of OS-RC-2 cells.Cell cycle was determined by flow cytometry.Confocal laser scanning microscopy showed that NDRG2 protein was specifically located on subcellular organelle.Results:A eukaryotic expression vector p1RES2-EGFP-NDRG2 was successfully constructed.After NDRG2 transfection,the growth of OS-RC-2 cells was inhibited.Flow cytometry showed that cells were arrested in S phase but the peak of cell apoptosis was not present,and confocal laser scanning microscopy showed that NDRG2 protein was located in mitochondrion.Conclusions:NDRG2 can significantly inhibit the proliferation of OS-RC-2 cells in vitro and its protein is specifically expressed in the mitochondrion.

CREG inhibits VSMCs proliferation by modulating the internalization of IGFII

Background To study the molecular mechanisms of CREG(the cellular repressor of E1A-stimulated gene) on proliferation of VSMCs in vitro.Methods The pRc/CMV-CREG plasmid or the pSM2-siCREG plasmid was transferred into human vascular smooth muscle cells(hVSMCs) to produce the cell clone that over-expression or down-expression of CREG respectively.BrdU assay and FACS cell cycle analysis were used to detect the proliferation of cells.Western blotting and immunocytochemistry show the expression and localization of IGF2R in hVSMCs.RT-PCR and ELISA assay determined the expression and secretion of IGFII factor. Alex488-labeled rhIGFII was used to investigate the endocysis of cells.And the blockade of IGFII internalization by treatment both the neutralized antibody of anti-IGF2R and rsIGF2R detected the effect of IGFII on VSMCs growth. Furthermore,Western blotting and signal pathway inhibitor were used to analysis the activation of PI3K/AKT and ERK on VSMCs proliferation.Results Western blotting identified that the expression of CREG in hVSMCs-CREG cells increased compared to control cell,and the decreased obviously in hVSMCs-siCREG cells.Meanwhile,the overexpression of CREG in cells was detected to inhibit the proliferation of VSMCs and to enhance the distribution of IGF2R in cellular membrane.Furthermore,overexpression of CREG also accelerated the endocysis of IGFII in hVSMCs-CREG,and attenuate the secretion of IGFII into cell medium by ELISA analysis and Alex488 labeled IGFII analysis.Blockade experiments both neutralized antibody of IGF2R and rhIGF2R fragment determined that enhancement of IGFII secretion promoted the VSMCs proliferation,and PI3K/AKT and ERK signal pathway mediated the effect of IGFII on VSMCs.Conclusions Altogether, these data indicate that CREG inhibits the proliferation of hVSMCs through interfering into the internalization pathway of IGF2R-IGFII.

Discovery of Chrysoeriol,a PI3K-AKT-mTOR Pathway Inhibitor with Potent Antitumor Activity against Human Multiple Myeloma Cells in vitro

This study was designed to determine the impact of chrysoeriol on proliferation and cell cycle progression in the human multiple myeloma cell lines RPMI 8226 and KM3,and its related molecular mechanisms.Chryseoriol was identified by using the phosphorylated AKT-specific cytoblot high throughput assay.CCK-8 assay was employed to examine the growth inhibition rate and IC 50 (48 h) in peripheral blood mononuclear cells (PBMNCs),RPMI 8226 and KM3 cells treated with chrysoeriol at various concentrations.Cells were labeled with 5-6-carboxyfluorescein diacetate succinimidyl ester (CFSE),and the proliferation dynamics was detected by flow cytometry and analyzed with ModFit software.The cell cycles of RPMI 8226 and KM3 cells were measured by flow cytometry when the IC 50 concentration of chrysoeriol was adopted.The alterations in cell-cycle related proteins (Cyclin B1,Cyclin D1,p21) and proteins in PI3K-AKT-mTOR pathway were determined by Western blot analysis.The results showed the proliferation of multiple myeloma cells was significantly inhibited by chrysoeriol,resulting in cell cycle arrest in G 2 /M phase.Chrysoeriol could significantly reduce the expression of p-AKT (s473) and p-4eBP1 (t37/46) protein,meanwhile enhanced Cyclin B1 and p21 protein expression.Similar effects were not observed in PBMNCs from normal donors.It was concluded that chrysoeriol was a selective PI3K-AKT-mTOR pathway inhibitor.It restrained the proliferation of human multiple myeloma cells,but didn’t affect proliferation of PBMNCs from normal donors.It might exhibit the cell cycle regulatory effect via the inhibition of PI3K-AKT-mTOR signal pathway.

Inhibitory Effects of Cordyceps militaris( Bombyx mori )Extract against Respiratory Syncytial Virus

[ Objective] This study aimed to investigate the inhibitory effects of 95%, 70% ethanol extracts and water extract of Cordyceps militaris （Bombyx mori）, on cells infected with respiratory syncytial virus （RSV）. [Method] Human embryonic kidney cells （HEK293, RSV virus-sensitive cells） were infected with respiratory syncytial virus and incubated with tissue cell culture method, to analyze the inhibitory effects of different extracts of Cordyceps militaris （Bombyx mori） on proliferation of RSV-infected cells by cytopathic effect （CPE） observation and tetrazolium salt reduction assay （ MTF method）. [ Result] Different extracts of Cordyceps militaris （Bombyx mori） were non-toxic to HEK293 cells. CPE occurred successively in various sample groups after 48 h, and the degree of CPE was reduced gradually with the increasing concentration. To be specific, 95 % ethanol extract （100 μg/ml）, 70% ethanol extract （100, 25 μg/ml） and water extract （100, 25 μg/ml） of Cordyceps militaris （BombTx mori） exhibited inhibitory effects against RSV subtype A; 70% ethanol extract （100, 25 μg/ml） and water extract （100, 25 μg/ml） of Cordyceps militaris （Bombyx mori） exhibited inhibitory effects against RSV subtype B; the effects of water extract （100 μg/ml） of Cordyceps militaris （Bombyx mori） on RSV subtypes A and B were similar to positive control group. However, different extracts of Cordyceps militaris （Bombyx mot/） with low concentrations （6.25, 1.56 μg/ml） exhibited no inhibitory effects against respiratory syncytial virus. [ Conclusion ] Cordyceps militaris （Bombyx mori） exhibited significant inhibitory effects against syncytial virus, and the inhibitory effects were related to the water soluble components.

Proliferation-Inhibiting and Apoptosis-lnducing Effects of Ursolic Acid and Oleanolic Acid on Multi-Drug Resistance Cancer Cells in Vitro

Objective：To investigate the proliferation-inhibiting and apoptosis-inducing effects of ursolic acid（UA） and oleanolic acid（OA） on multi-drug resistance（MDR） cancer cells in vitro.Methods：UA and OA in different concentrations（0-100μmol/L） were added separately to cultures of different cancer cell lines, including the human colon cancer cell lines SW480 and SW620,human acute myelocytic leukemia cancer cell lines HL60 and HL60/ADR,human chronic myelogenous leukemia cell lines K562 and K562/ADR,and the human breast cancer cell lines MCF-7 and MCF-7/ADR.Effects of UA and OA on cell proliferation were detected by 3-（4,5-dimethyl-2-thiazole）-2-5-biphenly-tetrazole bromide（MTT） method and effects on cell apoptosis were tested by flow cytometry（FCM） and Western blot at 24,48,and 72 h after treatment.Results：Both UA and OA showed significant inhibition on parent and MDR cell lines in a time- and concentration-dependent manner;the drug-resistant multiple of them on K562 and K562/ADR as well as on HL60 and HL60/ADR was 1;the effects of UA were better than those of OA in inhibiting cell growth of solid colonic cancer and breast cancer.After SW480 cells were treated by UA at the concentrations of 0-40μmol/L for 48 h,FCM showed that annexin V （AV） positive cells and hypodiploid peak ratio increased along with the increase in the drug＇s concentrations; and Western blot found that expressions of Bcl-2,Bcl-xL and survivin decreased in a concentration-dependent manner.Conclusions：Both UA and OA have antitumor effects on cancer cells with MDR,and the optimal effect is shown by UA on colonic cancer cells.Also,UA shows cell apoptosis-inducing effect on SW480,possibly by way of down-regulating the expressions of apoptosis antagonistic proteins,Bcl-2,Bcl-xL,and survivin.

Xiaoji Decoction(消积饮) Inhibited Cell Proliferation and Induced Apoptosis through Akt Signaling Pathway in Human Lung Cancer A549 Cells

Objective： To investigate the inhibitive effect and the underlying mechanism of Xiaoji Decoction （消极饮 XJD） in human lung cancer A549 cells. Methods： A549 cells in logarithmic proliferation were cultivated in RPMI-1640 containing 10% low, medium or high dosages of XJD serum. The inhibitive effect of XJD in A549 cell proliferation was assessed by methylthiazolyldiphenyl-tetrazolium bromide （MTT） assay. The pro-apoptotic effect of XJD in A549 cells was observed by fluorescence microscope via Hoechst 33258 staining. The role of the Akt signaling pathway was observed by examining the presence of p-Akt protein by Western blot and the mRNA expression of downstream proteins such as Bcl-2/BcI-XL-associated death promoter （BAD） and caspase-9 by real time polymerase chain reaction. Results： MTT assay revealed that XJD could inhibit A549 proliferation in a dose- and time-dependent manner. Hoechst 33258 staining showed that XJD induced the typical nuclear apoptotic morphology after XJD treatment. Moreover, XJD could reduce the phosphorylation of Akt and increase the mRNA expression of BAD and caspase-9. Conclusions： XJD can inhibit the proliferation of A549 cells in a dose- and time-dependent manner through signaling Akt pathway via up-regulating the expression of BAD and caspase-9. XJD may provide a novel therapeutic model for lung cancer and deserve further study.

Inhibition of vascular smooth muscle cell proliferation by in troduction of retinoblastoma gene via a recombinant adenovirus vector

This study was supported in part by grant from National Natural Science Foundation of China (No. 39570775). Objective To investigate the vascular smooth muscle cell (SMC) growth suppression by recombinant adenovirus vector expressing a retinoblastoma (Rb) protein and to explore a gene therapy approach for vascular proliferative disorders including atherosclerosis and artery restenosis. Methods A replication deficient adenovirus vector encoding a wild type Rb and AdCMVRb, was constructed and transfected into cultured rabbit aortic SMC. The efficiency of gene transfection and expression was detected by immunochemical staining and polymerase chain reaction. The role of Rb in regulating vascular SMC proliferation was observed by cell counting, thymidine incorporation, and flow cytometry. Results Wild type Rb gene transfected effectively into the cultured SMC with AdCMVRb can suppress growth factor stimulated cell proliferation through regulation of DNA synthesis and cell cycle progression. Conclusion The results demonstrate the potential of adenovirus mediated Rb gene therapy for atherosclerosis and artery restenosis after balloon angioplasty.

Anti-Proliferative Effects Induced by Anti-CD4 Human/Murine Chimeric Antibody and Murine Anti-CD4 Monoclonal Antibody

Summary: The effects of chimeric anti CD4 human/murine chimeric antibody and murine anti CD4 monoclonal antibody (McAb) on the proliferation induced by anti CD3 McAb, phytohemagglutinin (PHA), IL 2, and allogeneic cells were studied. The results showed that chimeric anti CD4 antibody and murine anti CD4 McAb could inhibit the proliferation induced by the above inducers and the inhibitory effects were related to the dosage of the antibodies.

Role of Extracelluar Regulated Protein Kinases in FTY720-induced Apoptosis of Leukemia Cell Lines HL-60 and U937

The effects of a novel immunosuppressive agent FTY720 on proliferation inhibition and apoptosis of acute leukemia cell lines HL 60 and U937, and the role of extracelluar regulated protein kinase (ERK) in the course of proliferation inhibition and apoptosis induced by FTY720 were studied. The proliferation inhibition rate of HL 60 and U937 cells by various concentrations of FTY720 was detected by MTT assay. Cell apoptosis was detected by DNA fragment analysis and flow cytometry. The phosphorylated ERK1/2 protein expression was observed by Western blotting. The change of intracellular distribution of ERK1/2 protein was identified by SP immunohistochemical staining. The results showed that FTY720 could inhibit the growth of HL 60 and U937 cells effectively in a dose dependent manner. After incubation with FTY720 for 24 h, apoptosis was observed in HL 60 and U937 cells. The intracellular expression of phosphorylated ERK1/2 protein was also down regulated and the distribution of ERK1/2 protein in cell nuclear was reduced during FTY720 induced apoptosis. So, that FTY720 inhibited ERK1/2 phosphorylation might mediate the role of FTY720 induced apoptosis and proliferation inhibition of leukemia cells.

ANTI-CANCER EFFECT OF PSP PURIFIED PRODUCTS ON HUMAN TUMOR CELL LINES IN VITRO

The anti-cancer effect of PSP purified products, PSP-A, PSP-B, PSP-C and crude product PSP-Cr was compared on four human tumor cell lines in vitro. It was found that the inhibition rate of cell proliferation of PSP-A was higher than that of PSP-Cr (P<0. 05). On SPC cells, the inhibition rate of PSP-A at a dosage of 1000μg/ml was 62. 7%, being the highest as compared with those on the other three cell lines. Morphological changes were seen in all the four cell lines, especially in SPC cells after PSP-A treatment.

Anticancer effects of Chinese herbal medicines on esophageal carcinoma: a literature research-based review

Background:Esophageal carcinoma is one of the most common malignant tumors worldwide,with China the hardest hit area accounts for nearly 50 percent of the morbidity and mortality each year.Despite the deepening of the research and treatment of esophageal carcinoma,its five-year survival rate is still less than 20%,partly because of the formation of drug resistance and after-effects with conventional treatment.Recent years,accumulating evidence suggests that natural medicines have remarkable effects on the aspect of anticancer without or with mild side effects,therefore,the anticancer drugs development on esophageal carcinoma has transitioned from chemical synthesis to natural medicines.China has the tradition of using Chinese herbal medicines for various diseases curing for thousands of years and has been proved to be effective,including esophageal carcinoma.With almost 10,000 Chinese herbal medicines have been excavated,the studies on Chinese herbal medicines for esophageal carcinoma is growing increasingly.Every Chinese herbal medicine is comprised of kinds of chemical compositions,exerts anticancer effect by targeting multiple targets via multiple signaling pathways with lower incidence rate of drug resistance and almost no side effects,which has caught increasingly attention.In this review,we summarized some monomers,extracts and composite of CHMs with anticancer activity on esophageal carcinoma by inhibiting proliferation,anti-metastasis,et al.,and elaborate their mechanisms of action,hoping this will be valuable for esophageal carcinoma treatment and drug development.

Enzyme-triggered deep tumor penetration of a dual-drug nanomedicine enables an enhanced cancer combination therapy

Cancer cells could be eradicated by promoting generation of excessive intracellular reactive oxygen species(ROS)via emerging nanomedicines.However,tumor heterogeneity and poor penetration of nanomedicines often lead to diverse levels of ROS production in the tumor site,and ROS at a low level promote tumor cell growth,thus diminishing the therapeutic effect of these nanomedicines.Herein,we construct an amphiphilic and block polymer-dendron conjugate-derived nanomedicine(Lap@pOEGMA-b-p(GFLG-Dendron-Ppa),GFLG-DP/Lap NPs)that incorporates a photosensitizer,Pyropheophorbide a(Ppa),for ROS therapy and Lapatinib(Lap)for molecular targeted therapy.Lap,an epidermal growth factor receptor(EGFR)inhibitor that plays a role in inhibiting cell growth and proliferation,is hypothesized to synergize with ROS therapy for effectively killing cancer cells.Our results suggest that the enzyme-sensitive polymeric conjugate,pOEGMA-b-p(GFLG-Dendron-Ppa)(GFLG-DP),releases in response to cathepsin B(CTSB)after entering the tumor tissue.Dendritic-Ppa has a strong adsorption capacity to tumor cell membranes,which promotes efficient penetration and long-term retention.Lap can also be efficiently delivered to internal tumor cells to play its role due to the increased vesicle activity.Laser irradiation of Ppa-containing tumor cells results in production of intracellular ROS that is sufficient for inducing cell apoptosis.Meanwhile,Lap efficiently inhibits proliferation of remaining viable cells even in deep tumor regions,thus generating a significant synergistic anti-tumor therapeutic effect.This novel strategy can be extended to the development of efficient membrane lipid-based therapies to effectively combat tumors.

Studies on New Steroidal Saponins from Allii macrostemonis Bulbus and Their Antitumor Activities

Two new steroidal saponms（2, 3） and two known compounds（1, 4） were isolated from 75% ethanol extract ofAllii macrostemonis bulbus by various spectral methods. The two new steroidal saponins were respectively elucidated as （25S）-26-O-β-D-glucopyranosyl-5α-furostanol-2α,3β,22,26-tetrol-3-O-β-D-glucopyranosyl（1→2）-[β-D- glucopyranosyl（1→3）]-β-D-glucopyranosyl（1→4 ）-β-D-galactopyranoside（2 ） and 26-O-β-D-glucopyranosyl-5α- furostanol-25（ 27）-ene-3β,22,26-tetrol-3-O-β-D-glucopyranosyl（ 1→2 ）-[β-D-glucopyranosyl（ 1→3 ）] -β-D-glucopyranosyl- （1→4）-β-D-galactopyranoside（3）. Compound 4 was obtained from this medical plant for the first time. The activity experiments of proliferation inhibition on tumor cells were performed by cell counting kit-8（CCK-8） method for compound 2（25S-configuration） and its isomer（compound 1, 25R-configttration）. The results show that compounds 1 and 2 have weak inhibition on lung cancer A549 cells, melanoma A375 cells and breast cancer MCF-7 cells as well as certain inhibitory effect on liver cancer HepG2 cells, and the inhibitory effect of compound 1 is stronger than that of compound 2.

Slow degrading Mg-based materials induce tumor cell dormancy on an osteosarcoma-fibroblast coculture model

Osteosarcoma is one of the most common cancers in young adults and is commonly treated using surgery and chemotherapy.During the past years,these therapy approaches improved but failed to ameliorate the outcomes.Therefore,novel,targeted therapeutic approaches should be established to enhance treatment success while preserving patient’s quality of life.Recent studies suggest the application of degradable magnesium(Mg)alloys as orthopedic implants bearing a potential antitumor activity.Here,we examined the influence of Mg-based materials on an osteosarcoma-fibroblast coculture.Both,Mg and Mg-6Ag did not lead to tumor cell apoptosis at low degradation rates.Instead,the Mg-based materials induced cellular dormancy in the cancer cells indicated by a lower number of Ki-67 positive cancer cells and a higher p38 expression.This dormancy-like state could be reversed by reseeding on non-degrading glass slides but could not be provoked by inhibition of the protein kinase R-like endoplasmic reticulum kinase.By investigating the influence of the disjunct surface-near effects of the Mg degradation on cell proliferation,an increased pH was found to be a main initiator of Mg degradation-dependent tumor cell proliferation inhibition.