Line HL-60 Research Articles

Effect of silencing RRM2 by specific small interference RNA on biological behaviors of human hepatoma cancer cell line HepG2

Objective To investigate the effect of small interference RNA (siRNA) silencing RRM2 on the biological behaviors of human hepatoma cancer cell line HepG2 and and the molecular mechanisms.Methods RRM2 expression was knocked down in human hepatoma cancer cell line HepG2 by RRM2 siRNA.The expression level of RRM2 was detected in human hepatoma cancer cell line HepG2 and human normal liver cell line HL-7702 at mRNA and protein levels by real-time polymerase chain reaction (PCR) or Western blotting.The cell proliferation was measured by cell counting Kit-8 (CCK-8).The migration was observed by using Transwell cell migration system.The expression levels of B lymphocytes/leukemia-2 (bcl-2),bax and cytochrome C (Cyt-C) proteins were detected by using Western blotting.Ten male BALB/C nude mice were subcutaneously injected with siRNA-RRM2/HepG2,siRNA-NC/HepG2 or NEG/HepG2 cells respectively to study the growth of tumor in each group.Results siRNA-RRM2 could down-regulate the expression of RRM2 in HepG2 remarkably,and siRNA-RRM2 could inhibit both the proliferation [(41.20 ± 2.13) ％ vs (91.35 ± 10.22) ％] and migration ability of HepG2 cells (21.20 ±1.28 vs 132.50 ±7.88) as compared with the negative control group.siRNA-RRM2 could down-regulate the expression of bcl-2 and up-regulate the expression of bax and Cyt-C.Additionally,the volume of xenograft tumors in siRNA-RRM2 group was decreased significantly as compared with control group.The volume of xenograft tumors in siRNA-RRM2 group was (171.13 ± 28.12) mm3 and that in siRNA-NC group was (1487.41 ± 168.20) mm3 at 8th week.Conclusion siRNA-RRM2 can down-regulate the expression of RRM2 in the HepG2 cells and may play an important role in the regulation of biological behaviors of HepG2 cells,and also inhibit the tumor growth of nude mice model. Key words: RNA interference ; RRM2 gene ; Carcinoma,hepatocellular

Hyperphosphorylation of intermediate filament proteins is involved in microcystin-LR-induced toxicity in HL7702 cells

Microcystin-LR (MC-LR) is commonly characterized as a hepatotoxin, which can cause disruption of keratin filaments. Keratins, however, account for only two types of intermediate filaments (IFs), and the potential involvement of other IF proteins in MC-LR-induced toxicity and the underlying mechanisms are still unclear. In this study, the human normal liver cell line HL7702 was used to investigate whether MC-LR can change the transcription, translation, and phosphorylation levels of major IF proteins and to elucidate the underlying mechanisms. The results showed that MC-LR triggered an accumulation of IFs around the nucleus and led to the formation of dense bundles. When the cells were treated with 10μM MC-LR, cell proliferation significantly decreased with an increase in apoptosis and cell cycle arrest. Moreover, the mRNA and protein levels of keratin 18, vimentin and lamin A/C were not changed; however, the phosphorylation of K8/18 and vimentin was significantly increased. Furthermore, we found MC-LR exposure caused phosphoactivation of P38, JNK and ERK1/2 in a concentration-dependent manner, and P38 and ERK1/2 were involved in MC-LR-induced hyperphosphorylation of IF proteins. Taken together, the results of this study suggest that MC-LR exerts its potential hepatotoxicity through MAPK pathway activation, which cause hyperphosphorylation of IF proteins and result in cytoskeletal architecture remodeling and cell survival/death regulation. Since IFs serve as signaling platforms and dozens of IF proteins are involved in different signaling pathways, future studies focus on different IFs may provide helpful insights into the mechanisms of MC-LR toxicity.

257 Role of Epidermal Growth Factor Receptor (EGFR) in Inflammation Induced Colorectal Cancers

cells (HBMPC) to the lung and formation of pre-metastatic niches. These niches would be suitable environments for seeding of CTC and for the development of metastasis. Thus, we hypothesized that the patients with greater risk of developing metastasis are those not only with CTC but also with circulating HBMPC. Before evaluating the presence of these populations in cancer patients, we sought to evaluate the detection viability of these rare populations in peripheral blood (PB). Therefore, the goal of this study was to determine the lower limit of detection of these rare cells populations in PB by flow cytometry. Material and Methods: We tested the detection of rare populations in PB using a model composed by PB of healthy donors and the cell lines MCF-7 and HL-60, representing CTC and HBMPC, respectively. Each cell line was fluorescently labeled and mixed with whole PB in to achieve concentrations from 5% to 0.01% per 10 leukocytes. After lysis of red blood cells, the mixture was analyzed by flow cytometry, and at least 100,000 events per tube were acquired. Results: Rare events such as 0.01% and 0.05% were successfully detected in our model. Using linear regression, we found that the determination coefficient between the expected number of target cells and the number of detected cells was R = 0.99 to CTC and R = 0.98 to HBMPC. Conclusion: Detection of rare populations of cells in PB is feasible and may allow the investigation of CTC and HBMPC in cancer patients. The association of CTC and HBMPC and cancer progression would suggest that cancer is a systemic disease, even if it is not considered disseminated according to current understanding, once it interacts with the whole organism. This concept would provide the establishment of new approaches for the prevention of metastasis.

Evaluation of anti-apoptotic, anti-injury and anti-hepatitis B virus effects of isochlorogenic acid C in vitro

To study anti-hepatitis effect of isochlorogenic acid C from Laggera alata, anti-apoptotic and anti-injury properties of test compound were evaluated using the d-galactosamine (d-GalN)-induced apoptosis/injury model in human hepatocyte line HL-7702 cells. Hepatocyte apoptotic changes were demonstrated by DNA ladder analysis and caspase-3 activation. Hepatocyte injury was assessed by cell viability. Meanwhile, anti-hepatitis B virus (anti-HBV) effect of test compound was evaluated by the HBsAg, HBeAg, HBV DNA, HBV covalently closed circular DNA (HBV cccDNA) and heme oxygenase-1 (HO-1) expressions in HBV-transfected HepG2.2.15 cells. The results showed that test compound at concentrations of 10 to 100 µg/ml significantly reduced the caspase-3 and transformed growth factor β1 (TGFβ1) levels of the d-GalN-challenged hepatocytes. Also, test compound improved markedly cell viability of the d-GalN-injured hepatocytes and produced a maximum protection rate of 47.28% at a concentration of 100 μg/ml. Furthermore, test compound significantly inhibited productions of HBsAg and HBeAg. Its maximum inhibitory rates on the HBsAg and HBeAg expressions were 86.93 and 59.79%, respectively. In addition, test compound significantly induced the HO-1 expression of HepG2.2.15 cells. This study verifies that isochlorogenic acid C possesses potent hepatoprotective and anti-HBV effects. The anti-apoptotic and anti-injury effects of test compound could be achieved by its anti-oxidative properties and interfering the caspase-3 and TGF-β1 expressions. The anti-HBV target of test compound may mainly be at the downstream links of HBV replication process and is probably associated with blocking translation step. Furthermore, HO-1 induction may contribute to anti-hepatitis B effect of test compound. Key words: Laggera alata, isochlorogenic acid C, d-Galactosamine, anti-hepatitis B, covalently closed circular DNA, heme oxygenase-1.

Abstract 464: CXCR7 protein is strongly expressed in B-acute lymphoblastic leukemia (ALL) but not in T-ALL or acute myelogenous leukemia

Abstract The identification of CXCR7 in addition to CXCR4 as a receptor for the chemokine CXCL12/SDF-1 and recent reports that CXCR7 is expressed in many cancers including breast, lung and prostate prompted us to evaluate the role of CXCR7 in hematological malignancies. While CXCR4 is known to be involved in the progression of acute lymphoblastic leukemia (ALL) and acute myelogenous leukemia (AML), the role of CXCR7 in leukemic dissemination remains unknown. Using immunohistochemistry, we evaluated the expression patterns of CXCR7 and CXCR4 in tissue microarrays (TMAs) constructed from bone marrow biopsies obtained from patients diagnosed with pre B- and B-ALL (n=75; median age 5 yrs, range 6 mo-72 yrs), T-ALL (n=11; median age 12 yrs, range 4-29 yrs) and AML (n=140; median age 62 yrs, range 1-83 yrs). CXCR7 expression was also evaluated at the gene and protein levels in peripheral blood mononuclear cells from ALL and AML patients and in B-cell lines (Raji, NC-37, Ramos, Nalm-6, Reh), T-cell lines (Jurkat, Hut-102B, Sez-4, CEM), and myeloid cell lines (KG-1, HL-60, THP-1, U-937, K562, HEL) using RT-PCR and flow cytometry. We found that in B-and preB-ALL TMAs, CXCR7 was immunopositive in 79% of the patients, in contrast to negative immunostaining in 100% of T-ALL and AML samples. Consistently, flow cytometry showed that CXCR7 protein is expressed on the surface of preB-ALL cells, but not on T-ALL, AML or normal controls. CXCR7 surface expression in the cell lines studied showed that three (NC-37, Raji, Reh) out of five B-cell lines expressed CXCR7, but not in any of the T-cell lines, and only weakly in one (KG-1) of six myeloid cell lines studied. Analysis of the functional role of CXCR7 in leukemia revealed that CXCR7 did not mediate chemotaxis of CXCR7-positive NC-37 B-cell line and CXCR7-negative HL-60 myeloid cell line towards an SDF-1 gradient. However, CXCR7 was found to mediate the trans-endothelial migration, adhesion to HUVEC and survival of NC-37 cells, but not the adhesion to HUVEC or survival of HL-60 cells. In conclusion, the selective strong expression of CXCR7 in B-ALL but not in T-ALL or AML suggests a differential role this protein may play in various types of leukemia. As CXCR7 mediates trans-endothelial migration, adhesion and survival of B-cell line, further investigations into the functional role of CXCR7 in primary B-ALL are warranted with the aim of identifying new therapeutic targets. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 464. doi:1538-7445.AM2012-464

Abstract 1771: KOS-2464, a novel CRM1 inhibitor and a potential drug for multiple myeloma and leukemia

Abstract Purpose: Human multiple myeloma (MM) remains an incurable disease despite improved treatments that include bortezomib- and lenalidomide-based therapies and high-dose chemotherapy with autologous stem cell rescue. New therapeutic targets are needed to further improve treatment outcomes. Our purpose was to investigate the use of a small-molecule inhibitor (KOS-2464) of the nuclear export receptor CRM1 and whether this inhibitor sensitized drug-resistant MM to topoisomerase IIα (topo IIα) inhibitors. Experimental Procedures: In previous studies, we have shown that MM cells possess intrinsic drug resistance to topo II inhibitors due to a CRM1-mediated export of topo IIα in high cell density microenvironments, such as found in the diseased bone marrow. Blocking nuclear export of topo IIα with a CRM1 inhibitor may sensitize MM cells to topo II poisons. In this study, we investigated a novel, small molecule (585 MW), analog of the known CRM1 inhibitor LMB. To test the efficacy of this inhibitor, we treated human MM cell lines, RPMI-8226 and NCI-H929 and AML cell lines Kg1a and HL-60, with KOS-2464 +/− the topo IIα inhibitors doxorubicin (dox) or daunomycin. MM cells were placed at high-density drug resistant conditions and were treated for 20 hours with KOS-2464 (at concentrations of 1 nM, 10 nM and 100 nM), followed by treatment with 2 µM dox or daunomycin for four hours. Cells were then assayed for cell viability by an automated CellTiter-Blue (Promega) analysis. In addition, treated cells were assayed for induction of apoptosis by cleaved caspase 3 staining and flow cytometry. To determine if the CRM1 inhibitor blocked CRM1-mediated export, treated cells were separated into nuclear and cytoplasmic fractions and assayed for export of p53 and topo IIα. Immunofluorescent microscopy was used to confirm fractionation results. Results: KOS-2464 was used in combination with the topo IIα drugs dox and daunomycin to overcome intrinsic drug resistance in MM and AML cells. We found that KOS-2464 blocked nuclear export of both p53 and topo IIα as shown by nuclear/cytoplasmic fractionation and also by immunocytochemical staining. Using the CellTiter-Blue assay, we found that KOS-2464 had low IC50 values (2 nM) as single agent and functioned synergistically with dox (combination index 0.268). KOS-2464/dox combination therapy also induced apoptosis in MM cell cultures, as shown by caspase 3 cleavage and flow cytometric analyses. Conclusions: We found that a specific, small-molecule CRM1 inhibitor KOS-2464, sensitized MM and AML cells to dox and daunomycin as shown by apoptosis and cell viability assays. Sensitization was found to be synergistic. In addition, CRM1 inhibiting activity was verified by nuclear/cytoplasmic fractionation and immunocytochemical staining in MM cells. This compound may be a potential candidate for future clinical trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1771. doi:1538-7445.AM2012-1771

Abstract 2396: Key factors for filtration of tumor cells from whole blood

Abstract Introduction: Circulating tumor cells (CTC) in patients with metastatic carcinomas are associated with poor survival. The current standard CTC enrichment method employs antibodies, which misses cells not expressing the target antigen. Several alternative methods enrich CTC by filtration, exploiting differences in size between blood cells and CTC. Between different filtration methods large variations in material, pore size, operating pressure and sample fixation exist. As their influence for CTC enrichment is poorly understood we have investigated these parameters. Methods: Track etched polycarbonate filters with pore diameters of: 5, 8 and 10 µm and microfabricated silicon nitride filters with pore diameters of 5, 6, 7, 8, 9 and 10 µm were used. 300 cells from the cell lines SKBR3, PC3-9, MDA-231, MDA-468, MCF-7, SW480, HL-60 or K-586 were spiked in 1 ml of blood from healthy volunteers. Samples were filtered while both pressure across the filter membrane and the flow speed was measured. The pressure across the filter provides the force driving a cell through a filter pore. Cells were identified on the filters by fluorescence microscopy. Results: MDA-231, a 15 µm sized epithelial cell line, can readily pass through a 5 µm pore at low pressure (< 50 mbar). We show that the number of red and white blood cells contribute most to the pressure across a filter and at least 10e5 8 µm pores are needed to process 0.5 mL of whole blood per minute at 10 mbar of pressure. The influence of buffers such as PBS or serum was found to be negligible. Fixation using paraformaldehyde increases the pressure needed to push white, red and culture cells through a pore by 25 fold and recovery with spiked samples decreases from 85% to 40% at the same flow rate. Different filter types were compared and evaluated for cell line recovery using unfixed blood. Optimal recovery of cell lines was between 80-90% with low WBC contamination for track etch filters with 8 µm pores and 5 µm pores for silicon nitride chips. Only a weak correlation was found between recovery and size for the eight different cell lines. Conclusions: There are big differences in the ease with which different culture cell lines of similar size pass a filter pore; a challenging cell line such as MDA-231 should be selected for evaluation of filtration methods. Pressure across the filter is an important parameter in determining whether a cell passes a filter pore. Enrichment without fixation is best and should be performed at low pressures (< 50 mbar). A sufficiently small pore retains most of the tumor cells, but passes most white and all red cells. The optimal pore size was different for the different filter types evaluated, possibly due to surface of the filter or filter thickness. In the optimal conditions recoveries of at least 80% were found for most cell lines across a wide range of CTC concentrations and blood volumes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2396. doi:1538-7445.AM2012-2396

Cytoplasmic and Nuclear Localization of TCTP in Normal and Cancer Cells

Objective. Intracellular localization of translationally controlled tumour protein (TCTP) was investigated in cancer cells. Methods. The expression and localization of TCTP were detected at 12 h, 24 h, 48 h, 60 h time points in culture of human hepatocarcinoma cell line HepG2, human cervical carcinoma cell line HeLa, and human normal liver cell line HL-7702 by immunofluorescence. Results. TCTP was expressed in both normal and tumor cells, and its localization changes at different time points. TCTP was mainly expressed in cytoplasm from 24 h to 48 h then expressed in both nucleus and cytoplasm at 60 h in HL-7702 cells. While in HepG2 cells, TCTP first localized at cell membrane within 24 h then at both nucleus and cytoplasm from 48 h to 60 h; TCTP localized at both nucleus and cytoplasm from 12 h to 60 h in Hela cells. Conclusion. The translocation of intracellular expression of TCTP in normal and tumor cells at different time points may pave a path to the studying of TCTP role in tumor growth.

Regulation of heat shock protein 27 phosphorylation during microcystin-LR-induced cytoskeletal reorganization in a human liver cell line

Acute exposure to microcystin-LR (MC-LR) can induce the reorganization or disruption of the cytoskeleton, but proteins or enzymes correlated with this stress response have not been fully identified. Here, we report alterations to HSP27 during MC-LR-induced cytoskeletal reorganization in the human liver cell line HL7702. The cells incubated with MC-LR exhibited the rearrangement of filamentous actins and microtubules. The activity of protein phosphatase 2A was greatly decreased by MC-LR exposure. Furthermore, MC-LR markedly increased the level of HSP27 phosphorylation with the enhanced distribution of phosphorylated HSP27 to the cytoskeleton. To further determine the regulation of MC-LR-induced HSP27 phosphorylation, the activation of the MAPK superfamily was assessed. The result showed phospho-activation of p38 MAPK, JNK and ERK1/2 by MC-LR. Increases in HSP27 phosphorylation were suppressed by pretreating cells with SB203580 or SP600125, which are inhibitors of p38 MAPK or JNK, respectively. These data suggest that phosphorylated HSP27 is involved in cytoskeletal reorganization and is regulated by MAPKs, possibly as a consequence of PP2A inhibition. Moreover, the regulation of HSP27 phosphorylation may be important in MC-LR-induced cytoskeleton reassembly, which may provide helpful insights into the mechanism of MC-LR toxicity.

A straightforward method to determine the cytocidal and cytopathic effects of the functional groups of gallic acid

Gallic acid (GA) has well-known antioxidative, antimutagenic, antiviral and anti-inflammatory properties. GA has been used to label gold nanoparticles for bio-analytical applications, but the effects of gold nanoparticles coated with GA on human cell lines have not been studied. We synthesized GA-coated gold nanoparticles (GA-AuNPs) and compared the biochemical effects of the particles to that of free GA on the human hepatocyte cell line HL-7702. Cell proliferation measurements and apoptosis assays indicated that the presence of GA-AuNPs was less cytotoxic and was less likely to induce apoptosis. Synthesis of the labeled nanoparticles resulted in the oxidation of the two phenolic hydroxyls of GA to their quinine forms. Because this was the only chemical change to the molecule, the differences in toxicity between free GA and GA-AuNPs suggested that these phenolic hydroxyls were necessary for the bioactivity of GA molecules. This study might also reveal a new and easier way to illuminate the importance of the phenolic hydroxyls of GA in the cytocidal and cytopathic effects on cells.

Exploration of the variance in exogenous gene expression driven by the different promoters in leukemia cells

To investigate the variance in exogenous gene expression driven by the different promoters in leukemia cells, four GFP reporter lentivirus vectors carrying different promoters, including EF1α, PGK, Ubiquitin and CMV, were selected. Leukemia cell lines NB4, THP1, HL60 and Kasumi were infected with lentivirus produced from these reporter vectors, respectively. Then, fluorescence microscope, flow cytometry and fluorescence quantitative PCR were used to detect the GFP expression strength. The results of this study clearly showed that the expression levels of the reporter genes with four different promoters were significantly different. Among them, EF1α drove the highest level of GFP expression, while CMV promoter induced the lowest level. Our results suggested that promoters should be carefully chosen in order to get the appropriate exogenous expression level in leukemia cells according to the study need.

Study of the antiproliferative potential of seed extracts from Northeastern Brazilian plants

This study assessed the antiproliferative and cytotoxic potential against tumor lines of ethanolic seed extracts of 21 plant species belonging to different families from Northeastern Brazil. In addition, some underlying mechanisms involved in this cytotoxicity were also investigated. Among the 21 extracts tested, the MTT assay after 72 h of incubation demonstrated that only the ethanolic extract obtained from Myracrodruon urundeuva seeds (EEMUS), which has steroids, alkaloids and phenols, showed in vitro cytotoxic activity against human cancer cells, being 2-fold more active on leukemia HL-60 line [IC(50) value of 12.5 (9.5-16.7) μg/mL] than on glioblastoma SF-295 [IC(50) of 25.1 (17.3-36.3) μg/mL] and Sarcoma 180 cells [IC(50) of 38.1 (33.5-43.4) μg/mL]. After 72h exposure, flow cytometric and morphological analyses of HL-60-treated cells showed that EEMUS caused decrease in cell number, volume and viability as well as internucleosomal DNA fragmentation in a dose-dependent way, suggesting that the EEMUS triggers apoptotic pathways of cell death.

The novel compound OSI-461 induces apoptosis and growth arrest in human acute myeloid leukemia cells

Acute myeloid leukemia (AML) is a heterogeneous hematological malignancy. Treatment of patients suffering from high-risk AML as defined by clinical parameters, cytogenetics, and/or molecular analyses is often unsuccessful. OSI-461 is a pro-apoptotic compound that has been proposed as a novel therapeutic option for patients suffering from solid tumors like prostate or colorectal carcinoma. But little is known about its anti-proliferative potential in AML. Hence, we treated bone marrow derived CD34(+) selected blast cells from 20 AML patients and the five AML cell lines KG-1a, THP-1, HL-60, U-937, and MV4-11 with the physiologically achievable concentration of 1μM OSI-461 or equal amounts of DMSO as a control. Following incubation with OSI-461, we found a consistent induction of apoptosis and an accumulation of cells in the G2/M phase of the cell cycle. In addition, we demonstrate that the OSI-461 mediated anti-proliferative effects observed in AML are associated with the induction of the pro-apoptotic cytokine mda-7/IL-24 and activation of the growth arrest and DNA-damage inducible genes (GADD) 45α and 45γ. Furthermore, OSI-461 treated leukemia cells did not regain their proliferative potential for up to 8days after cessation of treatment following the initial 48 h treatment period with 1μM OSI-461. This indicates sufficient targeting of the leukemia-initiating cells in our in vitro experiments through OSI-461. The AML samples tested in this study included samples from patients who were resistant to conventional chemotherapy and/or had FLT3-ITD mutations demonstrating the high potential of OSI-461 in human AML.

Dual effects of interleukin-18: inhibiting hepatitis B virus replication in HepG2.2.15 cells and promoting hepatoma cells metastasis

Interleukin-18 (IL-18) has been reported to inhibit hepatitis B virus (HBV) replication in the liver of HBV transgenic mice; however, the molecular mechanism of its antiviral effect has not been fully understood. In the present study, it was shown that IL-18 and its receptors (IL-18R) were constitutively expressed in hepatoma cell lines HepG2 and HepG2.2.15 as well as normal liver cell line HL-7702. We demonstrated that IL-18 directly inhibited HBV replication in HepG2.2.15 cells via downregulating the activities of HBV core and X gene promoters. The suppressed HBV replication by IL-18 could be rescued by the administration of BAY11-7082, an inhibitor of transcription factor NF-κB. On the other hand, it was of interest that IL-18 promoted HepG2 cell metastasis and migration dose dependently in both wound-healing assays and Transwell assays. The underlying mechanism could be partially attributable to the increased activities of extracellular matrix metalloproteinase (MMP)-9, MMP-3, and MMP-2 by IL-18, which upregulated the mRNA levels of MMP-3 and MMP-9 in a NF-κB-dependent manner. Furthermore, it was confirmed that expression of IL-18/IL-18R and most MMPs were remarkably upregulated in hepatocellular carcinoma (HCC) liver cancer tissue specimens, suggesting that IL-18/IL-18R-triggered signaling pathway was closely related to HCC metastasis in vivo. Therefore, we revealed the dual effects of IL-18 in human hepatocytes: it not only inhibited HBV replication but also promoted hepatoma cells metastasis and migration. NF-κB played a critical role in both effects. Our work contributed to a deeper understanding of the biological function of IL-18 in human hepatocytes.

Icaritin induces cell death in activated hepatic stellate cells through mitochondrial activated apoptosis and ameliorates the development of liver fibrosis in rats

Icaritin is an active ingredient extracted from the plant Herba Epimedium Sagittatum (Sieb. et Zucc.) Maxim. The purpose of this study is to investigate the effects and mechanisms of icaritin-induced cell death in activated hepatic stellate cells (HSCs) and ameliorating the development of liver fibrosis in rats. : In vitro, icaritin-induced cell death rates in HSC-T6 (rat) and LX-2 (human) HSC lines as well as normal hepatocyte cell lines HL-7702 (L02) and WRL-68 were assayed by MTT method, and the apoptotic ratios were detected by both flow cytometry and the Annexin-V-FITC Apoptosis Detection Kit. A Whole Rat Genome Microarray Kit was used to identify expression of interest genes through fold-change screening. In vivo study, experimental liver fibrosis models were built by carbon tetrachloride (CCl(4)) or common bile duct ligation (CBDL) in Wistar rats. Icaritin (1mg/kg/day, three days a week) was administered by gastric gavage for four weeks (n=6 per group). At the end of the study, serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT) as well as the contents of hydroxyproline and collagen I in liver tissues were measured. Histopathological changes and the distribution of activated HSCs were observed in the liver tissues using hematoxyline-eosin (HE) staining and immunohistochemical staining for α-smooth muscle actin (α-SMA). Icaritin induced apoptosis in HSC-T6 and LX-2 in a concentration- and time-dependent manner with little toxicity to normal hepatocyte cell lines. The IC(50) of icaritin in HSC-T6 was 12.83 μM at 48 h. Apoptotic ratio of HSC-T6 treated with 24 μM icaritin was 20.19%, and the G2 phase of the cell cycle did not occur (P<0.05). Gene analysis showed that icaritin up-regulated Bak-1, Bmf and Bax expression while significantly down-regulated Bcl-2 expression (vs. control group, P<0.01). These results suggested that mitochondrial pathway played an important role in icaritin-induced apoptosis in activated HSCs. In vivo results showed that icaritin reduced the number of activated HSCs, and brought the elevated levels of AST, ALT, hydroxyproline and collagen I to normal or near normal values (vs. model group, P<0.05). Icaritin can induce cell death in activated HSCs through mitochondria-mediated apoptosis, ameliorate the progression of hepatic fibrosis in rats, and could be a promising drug for treating liver fibrosis.

The in vitro and in vivo experimental study of long-circulating superparamagnetic iron oxide liposome nanoparticles as novel MR contrast agent

Objective To evaluate pharmacodynamics of prepared long-circulating superparamagnetic iron oxide (SPIO) liposomes. Methods Control and experimental groups were established after adding SPIO or long-circulating SPIO liposomes as agents. (1)Macrophages experiment in vitro: the RAW 264.7 macrophage cell strains were recovered, cultured and seeded in the culture plate at a density of 2.5×105 cells/well until they reached 80%-90% confluence. The intracellular Fe uptake of control and experimental group cells were quantified by Ferrozine assay after incubation with different concentrations of drugs. Factorial design analysis of variance was used as statistics method. Prussian blue staining method was used to detect staining of experimental cells.(2)Drug biodistribution in mice: C57BL/6J(n=6) were classified into blank control group (n=2), control group(n=2) and experimental group(n=2).Saline, SPIO and long circulating SPIO were injected via the tail vein in the blank control group, control group and experimental group respectively. Then distribution of drugs in the body was observed by pathological examination.(3) MR imaging of tumor-bearing nude mice: 20 BALB/c nude mice bearing lung cancer models were established and classified into control group and experimental group. After administration of drugs, all animals underwent MR scanning. Signal intensities of livers and tumors were measured, SNR-time dynamic curves were drew. Covariance analysis was used to compare post-enhanced SNR at the 12th hour. (4)Cytotoxicity studies (MTT): cytotoxicity of both drugs on human liver cell line HL-7702 was studied, and statistically analyzed using factorial design analysis of variance. Results (1) Macrophages experiment in vitro: The nanoparticle uptake by macrophage cells evaluated by ferrozine assay showed the uptake of blank SPIO was higher than long-circulating SPIO liposomes. Compared with the blank control group, there was strong blue staining in the macrophages with Prussian staining in the control group and little blue staining in the experimental group. (2) Drug biodistribution in mice: for blue stained cells composed of iron particles, the amounts in the liver, spleen, lung, kidney of the control group were more than those in the experimental group. (3) MR imaging of tumor-bearing nude mice: the non-enhanced SNR of livers and tumors in the control group and experimental group were 31.47 ± 0.56, 30.89 ± 1.41, 58.41 ± 0.61, 58.44 ± 1.08, respectively. After injecting of contrast agents, SNR of livers and tumors in the control group and experimental group were 17.00 ± 0.96, 22.29 ± 0.73, 58.50 ± 0.63, 52.47 ± 1.18, respectively. The covariance analysis showed that SNR of the livers in the control group after 12 hours was significantly lower than the experimental group (F=167.022, P=0.000); while the SNR of the tumors in the experimental group was significantly lower than the control group (F=266.106, P=0.000).(4) Cytotoxicity of nanoparticles by MTT method: the viability of HL-7702 cells tend to decrease with the increase of Fe concentration. Cytotoxicity in the long-circulating SPIO liposomes was lower than the SPIO(F=2256.204,P=0.000). Conclusions Long-circulating SPIO liposomes we prepared reveal suitable sizes, even distribution, and good anti-macrophage ability in vitro and in vivo. They have long circulation characteristic and T2 negative enhancement effect in the transplanted lung cancers, while they still maintain low cytotoxicity. Key words: Contrast media; Nanotechnology; Magnetic resonance imaging; Animals,laboratory

Enhanced invasiveness in multidrug resistant leukemic cells is associated with overexpression of P-glycoprotein and cellular inhibitor of apoptosis protein

Multidrug resistance (MDR) and multi-organ infiltration are the major obstacles to the successful treatment of leukemia. It is known that the drug efflux protein, P-glycoprotein (P-gp), and inhibitors of apoptosis proteins (IAPs) are involved in the MDR of leukemic cells, but their roles in leukemia infiltration have not been clearly elucidated. In this study, leukemic cell lines K562 and HL60 and their MDR variants K562R and HL60R have been used to analyze their infiltrative ability. MDR variants display enhanced invasion compared with parental cells. Results from xenografts in SCID (severe combined immunodeficiancy) mice are consistent with these in vitro observations. Furthermore, P-gp and cIAP are overexpressed and co-localize with protein kinase C-ε (PKC-ε) in MDR variants. Our study shows that overexpression of P-gp and cIAP may enhance the infiltration of leukemic cells.

Role of p38MAPK in apoptosis of human paratumor cirrhosis hepatocellular cell line QSG-7701 induced by cisplatin

To investigate the roles of p38 MAPK in apoptosis of the normal liver cell, the paratumor cirrhosis hepatocellular cell and the hepatocellular carcinoma cell. Three cell lines were adopted (the normal liver cell line HL-7702, the paratumor cirrhosis hepatocellular cell line QSG-7701 and the hepatocellular carcinoma cell line QGY-7703) and treated with Diamminedichloroplatin (DDP, cisplatin) and p38MAPK inhibitor SB203580. The apoptosis and cell cycles were detected by flow cytometry and electromicroscopy. The expressions of p38MAPK, CDC25B, p34cdc2 and cyclinB1 were detected by immunocytochemical staining , confocal microscopy and western blot. The apoptotic rates in all three cell lines pretreated with DDP increased obviously and the rates in normal liver cells and HCC cells increased continuously even after SB203580 treatment, whereas in paratumor cirrhosis cells the rate decreased and the cell cycle stopped at S phase. Cisplatin induces apoptosis in the paratumor cirrhosis hepatocellular cell line QSG-7701 via activation of p38MAPK pathway and it differs in the normal liver cells from the hepatocellular carcinoma cells.

Protective effects of HGF‐MSP chimer (metron factor‐1) on liver ischemia‐reperfusion injury in rat model

It has been reported that metron factor-1 (MF-1), an engineered chimerical factor containing selected functional domains of hepatocyte growth factor and macrophage-stimulating protein (HGF-MSP), could prevent apoptosis and have an anti-inflammatory effect. In this study, we investigate the protective effect of MF-1 on liver ischemia-reperfusion (I/R) injury. Overall 30 Sprague Dawley rats were randomly divided into three groups: the I/R model group (n=12), the MF-1 treatment group (n=12), and the sham-operated group (n=6). Liver I/R injury was induced by clamping the blood supply to the left and median lobes of liver by an atraumatic clamp for 90 min, then removing the clamp and allowing reperfusion. Blood samples were obtained on days 1, 2, 3 and 7 to assess liver biochemistry and the histology of liver tissue. Levels of malondialdehyde (MDA), superoxide dismutase (SOD), nitric oxide (NO), endothelial nitric oxide synthase and inducible nitric oxide synthase were measured. In addition, the anti-oxidative effect of MF-1 on hepatocytes was assessed in vitro. MF-1 treatment improved the rat survival rate significantly (P < 0.05). Liver biochemistry and histological changes were significantly ameliorated. MDA increased and SOD and NO decreased in the liver tissue. In vitro, MF-1 protected the human hepatic cell line HL-7702 from damage of oxidative stress. MF-1 could protect the liver from I/R injury, which might involve the reduction of oxygen free radicals and the increase of NO synthesis in an injured liver.

Casearin X exhibits cytotoxic effects in leukemia cells triggered by apoptosis

Clerodane diterpenes have demonstrated cytotoxic, antiplasmodial and anti-ulcer properties. In the present work, we determined the cytotoxic effect of casearin L (Cas L), O (Cas O) and X (Cas X) and (−)-hardwickiic acid isolated from Casearia sylvestris leaves, and investigated the underlying mechanisms involved in in vitro cell death induced by Cas X in HL-60 leukemia cells (0.7, 1.5 and 3.0 μM). Cytotoxicity tests demonstrated that Cas X was the most active compound studied, showing greater cytotoxic effects against CEM and HL-60 lines (IC 50 of 0.4 μM) and human peripheral blood mononuclear cells (PBMC, IC 50 of 1.2 μM). After 24 h exposure, Cas X caused a decrease in 5-bromo-20-deoxyuridine (BrdU) incorporation (36.6 and 24.5% labeling at 0.7 and 1.5 μM, respectively), reduction in viability, and increase in apoptotic and necrotic leukemia cells in a dose-dependent manner evidenced by the trypan blue and AO/EB (acridine orange/ethidium bromide) assays. Moreover, Cas X-treated cells exhibited nuclear fragmentation and cytoplasmic vacuolization depending on the concentration tested. These characteristics of apoptosis or secondary necrosis were confirmed by flow cytometry which revealed DNA fragmentation, phosphatidylserine externalization, activation of the effector caspases 3/7 and mitochondrial depolarization. We then found evidence that Cas X causes cell death via apoptotic pathways, corroborating the potential of casearins as compounds with promising antitumor-related properties.