H-RAS V12-induced radioresistance in HCT116 colon carcinoma cells is heregulin dependent

The abilities of mutated active K-RAS and H-RAS proteins, in an isogenic human carcinoma cell system, to modulate the activity of signaling pathways following exposure to ionizing radiation is unknown. Loss of K-RAS D13 expression in HCT116 colorectal carcinoma cells blunted basal extracellular signal-regulated kinase 1/2 (ERK1/2), AKT, and c-Jun NH2-terminal kinase 1/2 activity. Deletion of the allele to express K-RAS D13 also enhanced expression of ERBB1, ERBB3, and heregulin but nearly abolished radiation-induced activation of all signaling pathways. Expression of H-RAS V12 in HCT116 cells lacking an activated RAS molecule (H-RAS V12 cells) restored basal ERK1/2 and AKT activity to that observed in parental cells but did not restore or alter basal c-jun NH2-terminal kinase 1/2 activity. In parental cells, radiation caused stronger ERK1/2 pathway activation compared with that of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway, which correlated with constitutive translocation of Raf-1 into the plasma membrane of parental cells. Inhibition of mitogen-activated protein kinase/ERK1/2, but not PI3K, radiosensitized parental cells. In H-RAS V12 cells, radiation caused stronger PI3K/AKT pathway activation compared with that of the ERK1/2 pathway, which correlated with H-RAS V12-dependent translocation of PI3K into the plasma membrane. Inhibition of PI3K, but not mitogen-activated protein kinase/ERK1/2, radiosensitized H-RAS V12 cells. Radiation-induced activation of the PI3K/AKT pathway in H-RAS V12 cells 2 to 24 hours after exposure was dependent on heregulin-stimulated ERBB3 association with membrane-localized PI3K. Neutralization of heregulin function abolished radiation-induced AKT activation and reverted the radiosensitivity of H-RAS V12 cells to those levels found in cells lacking expression of any active RAS protein. These findings show that H-RAS V12 and K-RAS D13 differentially regulate radiation-induced signaling pathway function. In HCT116 cells expressing H-RAS V12, PI3K-dependent radioresistance is mediated by both H-RAS-dependent translocation of PI3K into the plasma membrane and heregulin-induced activation of membrane-localized PI3K via ERBB3.     

The DNA minor groove-alkylating cyclopropylpyrroloindole drugs adozelesin and bizelesin induce different DNA damage response pathways in human colon carcinoma HCT116 cells

As members of the cyclopropylpyrroloindole family, adozelesin and bizelesin cause genomic DNA lesions by alkylating DNA. Adozelesin induces single-strand DNA lesions, whereas bizelesin induces both single-strand lesions and double-strand DNA cross-links. At equivalent cytotoxic concentrations, these agents caused different biological responses. Low adozelesin concentrations (e.g., 0.5 nM) induced a transient S-phase block and cell cycle arrest in G(2)-M, as well as increased induction of p53 and p21, whereas a high drug concentration (e.g., 2.5 nM) caused apoptosis but no p21 induction. In contrast, both low and high bizelesin concentrations enhanced p53 and p21 induction and triggered G(2)-M cell cycle arrest and eventual senescence without significant apoptotic cell death. However, in cells lacking p21, bizelesin, as well as adozelesin, triggered apoptosis, indicating that p21 was crucial to sustained bizelesin-induced G(2)-M arrest. Thus, despite similar abilities to alkylate DNA, the chemotherapeutic agents adozelesin and bizelesin caused a decrease in HCT116 tumor cell proliferation by different pathways (i.e., adozelesin induced apoptosis, and bizelesin induced senescence).     

Mithramycin SK modulates polyploidy and cell death in colon carcinoma cells

During a normal cell cycle, polyploidy and aneuploidy can be prevented by several checkpoints, which are mainly p53 dependent. Here, we show that treatment of HCT-116 (p53(+/+)) colon carcinoma cells with the novel antitumor antibiotic mithramycin SK (MSK) results in polyploidization and mitotic catastrophe, which occurs after a transient halt in G(1) phase followed by the overtaking of the G(2)-M checkpoint when treated cells are incubated in a fresh drug-free medium. Cells reentering aberrant mitosis mainly died by necrosis, although active caspase-3 was observed. Our results indicate that a decrease in p53 RNA and protein levels, together with concomitant changes in the expression of other proteins such as p21(WAF1), were involved in MSK-induced polyploidy. Furthermore, the effects of MSK on HCT-116 (p53(+/+)) cells cannot be attributed exclusively to the down-regulation of p53 by MSK, because these effects differed from those observed in MSK-treated HCT-116 (p53(-/-)) cells. The p53(-/-) cells died mainly from G(2)-M through early p53-independent apoptosis, which appeared to be mediated by caspase-2, although secondary necrosis was also observed. [Mol Cancer Ther 2008;7(9):2988-97].     

双氢青蒿素抗人结肠癌HCT116细胞作用的研究

目的:研究双氢青蒿素(dihydroartemisinin,DHA)对人结肠癌HCT116细胞增殖及凋亡的影响,观察人结肠癌HCT116细胞中肿瘤转移相关因子尿激酶型纤溶酶原激活剂(urokinase-type plasminogen activator,uPA)表达的变化。方法:MTT法观察不同浓度和时间DHA对HCT116细胞增殖抑制作用;AO/EB双染法及流式细胞仪AnnexinⅤ-FITC法检测HCT116细胞凋亡率;免疫组化及图像分析法观察uPA蛋白的表达的动态变化。结果:MTT显示与对照组相比,各实验组DHA可以显著抑制HCT116细胞增殖(P<0.05);AO/EB双染法可观察到各实验组典型的凋亡细胞,流式细胞仪AnnexinⅤ-FITC法显示DHA可以诱导HCT116细胞凋亡具有浓度依赖性;免疫组化及图像分析显示uPA蛋白阳性单位明显下降。结论:DHA能够有效抑制HCT116细胞增殖并诱导其凋亡,DHA能够下调HCT116细胞中uPA蛋白的表达。     

吲哚美辛对结肠癌细胞HCT116的影响及机制探讨

目的:研究吲哚美辛对结肠癌细胞株HCT116增殖及体外侵袭的影响并对其机制进行探讨。方法:应用WST-8法和体外侵袭小室测定吲哚美辛对结肠癌细胞株HCT116增殖和侵袭能力的影响;Westen-blotting方法检测吲哚美辛作用后HCT116细胞β-连环素(β-Catenin)、细胞周期素D1(Cyclin D1)、基质金属蛋白酶7(matrix metalloproteinase-7,MMP-7)蛋白的表达。结果:吲哚美辛能够明显抑制结肠癌细胞株HCT116的增殖,呈时间-剂量依赖性,作用24、48、72h的IC50值分别为440.36、323.90、254.37μmol/L;吲哚美辛能够明显抑制结肠癌细胞株HCT116的体外侵袭,呈剂量依赖性;吲哚美辛作用24、48、72h后,β-Catenin、CyclinD1、MMP-7蛋白表达均有下降。结论:吲哚美辛能抑制结肠癌细胞株HCT116的增殖与迁移,其作用机制之一可能是通过Wnt/β-catenin信号通路。     

Transcriptome analysis reveals GA induced apoptosis in HCT116 human colon cancer cells through calcium and p53 signal pathways

Gallic acid (GA) is a polyphenol widely found in numerous fruits and vegetables that has been reported to exert anticancer effects, including apoptosis, against cancer cell lines. However, little is known about the induction of apoptotic effects and the underlying mechanism. We used RNA-seq to examine differentially expressed genes in human colon cancer HCT116 cells after 12 h and 24 h exposure to GA. A total of 792 and 911 genes with known functions showed significantly different expression levels in 12 h and 24 h GA-treated HCT116 cells, respectively. KEGG enrichment analysis showed that the identified genes were involved in pathways such as cholinergic synapse, circadian entrainment, calcium signal processing and transport, arachidonic acid metabolism and the p53 signal pathway. Real-time quantitative PCR was used to validate the reliability of the results obtained by RNA-seq. The results of this study indicate that GA triggers apoptosis in HCT116 cells through obstructing the growth of cells in the early phase treatment by down-regulation of calcium channels and then up-regulation of the intrinsic p53 signal pathway through activation of apoptosis caspases, finally leading to the mitochondrial apoptosis pathway.

Gallic acid (GA) is a polyphenol widely found in plants that induced apoptosis in human colon cancer cells through calcium and p53 signal pathways.     

Efficiency of brown seaweed (Sargassum longifolium) polysaccharides encapsulated in nanoemulsion and nanostructured lipid carrier against colon cancer cell lines HCT 116

Bioactive polysaccharides extracted from brown seaweeds have potent antioxidant, antitumor, antibacterial, antiviral, anti-inflammatory activities and nanomedicine applications. In the present study, we have made an attempt to overcome the instability and bioavailability problem of exopolysaccharides extracted from brown seaweed (Sargassum longifolium) by nanoencapsulation technology to enhance its therapeutic applications. Exopolysaccharides was encapsulated in orange oil nanoemulsion (NE) prepared by ultra-sonication method and nanostructured lipid carrier (NLC) prepared by hot solvent diffusion method. The encapsulation efficiency of nanoemulsion was 67.29% and of nanostructured lipid carrier was 78.7%. The prepared nano carriers have particle size 178 nm (NE), 153 nm (NLC) and zeta potential −43.9 mV (NE), −60 mV (NLC). In vitro release kinetics of exopolysaccharides from NE (80%) and NLC (95%) was found to be slow and sustained release which indicates increase in bioavailability. The cytotoxic effect of seaweed polysaccharide, nanocarriers loaded with seaweed polysaccharide was analyzed by MTT method in colon cancer (HCT 116) cell lines with the results revealing that seaweed polysaccharide encapsulated with NLC (80%) was superior to that encapsulated with orange oil nanoemulsion (70%). This is the first report demonstrating the potential of brown seaweed exopolysaccharide encapsulated in orange oil nanoemulsion and nanostructured lipid carrier for its biomedical application.

Bioactive polysaccharides extracted from brown seaweeds have potent antioxidant, antitumor, antibacterial, antiviral, anti-inflammatory activities and nanomedicine applications.     

The antipsychotic drug trifluoperazine inhibits DNA repair and sensitizes non small cell lung carcinoma cells to DNA double-strand break induced cell death

Trifluoperazine (TFP), a member of the phenothiazine class of antipsychotic drugs, has been shown to augment the cytotoxicity of the DNA-damaging agent bleomycin. In the present study, we investigated the effect of trifluoperazine on (a) survival of bleomycin-treated human non-small cell lung carcinoma U1810 cells, (b) induction and repair of bleomycin-induced DNA strand breaks, and (c) nonhomologous end-joining (NHEJ), the major DNA double-strand break (DSB) repair pathway in mammalian cells. By using a clonogenic survival assay, we show here that concomitant administration of trifluoperazine at a subtoxic concentration enhances the cytotoxicity of bleomycin. Moreover, trifluoperazine also increases the longevity of bleomycin-induced DNA strand breaks in U1810 cells, as shown by both comet assay and fraction of activity released (FAR)-assay. This action seems to be related to suppression of cellular DNA DSB repair activities because NHEJ-mediated rejoining of DSBs occurs with significantly lower efficiency in the presence of trifluoperazine. We propose that TFP might be capable of inhibiting one or more elements of the DNA DSB repair machinery, thereby increasing the cytotoxicity of bleomycin in lung cancer cells.     

Anticoagulant properties and cytotoxic effect against HCT116 human colon cell line of sulfated glycosaminoglycans isolated from the Norway lobster (Nephrops norvegicus) shell

Sulfated glycosaminoglycans (SGNL) were extracted for the first time from Norway lobster (Nephrops norvegicus) shell. The monosaccharide composition analysed by GC/MS revealed the presence of galacturonic acid, glucuronic acid, N-acetylgalactosamine and N-acetylglucosamine. The analysis of SGNL with acetate cellulose electrophoresis in Zn-acetate revealed the presence of heparan sulfate (HS) and dermatan sulfate (DS). SGNL were evaluated for their anticoagulant activities using activated partial thromboplastin time (aPTT), thrombin time (TT) and prothrombine time (PT) tests. After 21 h incubation, HCT116 cell proliferation was inhibited (p < 0.05) between 39.7 and 54.8% at 1.5–7.5 mg/mL of SGNL. SGNL don’t show hemolytic activity towards bovine erythrocytes and no cytotoxicity against the normal lymphocytes. The antiproliferative efficacy of these lobster glycosaminoglycans were probably related with the higher sulfate content. SGNL demonstrated promising antiproliferative and anticoagulant potential, which may be used as a novel, effective and promising antithrombotic agent.     

Ultrasound facilitates transduction of naked plasmid DNA into colon carcinoma cells in vitro and in vivo

One approach to improve the efficacy of in vivo gene therapy, with the aim at enhancing expression of the transgene, involves utilization of mechanical forces to facilitate transduction of DNA into cells. In this study, we evaluated the feasibility of mechanical insonation in gene transfers with naked DNA plasmid loading both in vitro and in vivo. We used an ultrasound probe, which can focus the ultrasonic beam in the exit zone of the probe. The reporter pcDNA3-lacZ plasmid, containing Escherichia coli lacZ or the beta-galactosidase gene (beta-gal), and the neomycin 3'-phosphotransferase gene (neo), was used for evaluation of transfer efficiency. Expression of beta-gal in MC38 murine colon carcinoma cells was measured after insonation of 20 W/cm2 with continuous 1.0-MHz wave exposure. In a transient assay, significant numbers of cells were transduced with the beta-galactosidase gene. After cells were treated with geneticin, we also observed a difference in colonogenicity between noninsonated and insonated groups. When MC38 cells were implanted in syngeneic mice and plasmid was injected, the insonation that followed facilitated beta-galactosidase expression. These results indicate that insonation represents a potential approach for gene therapy when combined with naked DNA plasmid injection.     

Green-synthesized gold nanoparticles from black tea extract enhance the chemosensitivity of doxorubicin in HCT116 cells via a ROS-dependent pathway

Green gold nanoparticles (GNPs) were prepared from black tea extract (BTE) and used to examine the chemosensitivity of doxorubicin in colon cancer cell line HCT116. BTE-GNPs were prepared by a single-step method and characterized by UV-Vis spectroscopy, FTIR spectroscopy, SEM, DLS and zeta-potential. The MTT assay was performed to determine the cytotoxicity of HCT116 cells and also normal kidney cells HEK293. Apoptosis and ROS generation were investigated by flow cytometry. The inhibition of ROS levels by the inhibitor NAC was determined by both spectrofluorimetry and confocal microscopy. Expression levels of pro- and anti-apoptotic proteins were determined by a western blot technique. BTE-GNPs significantly enhanced the cytotoxic effect of DOX with its co-treatment in HCT116 cells. The cytotoxic effect of BTE-GNP + DOX was involved in apoptosis via a ROS-dependent pathway by enhancing the pro-apoptotic protein expression. Therefore, our results indicated that green gold nanoparticles of black tea extract (BTE-GNP) may be potent chemosensitizers of doxorubicin.

Green gold nanoparticles (GNPs) were prepared from black tea extract (BTE) and used to examine the chemosensitivity of doxorubicin in colon cancer cell line HCT116.     

Extensive cell death in thymocytes in colon 26-induced cachectic mice

Extensive atrophy has been reported to occur in the thymus in a cancer-burden state but the mechanisms of this atrophy have not been fully elucidated. We investigated changes in the thymus in tumour-bearing mice inoculated with two subclones of the murine colon 26 adenocarcinoma cell line: clone 5 (non-cachectic) and clone 20 (cachectic). In clone 20 mice, body weights and thymocyte numbers decreased significantly compared with controls. Flow cytometric analysis of the thymocytes demonstrated that the frequency of single positive cells (CD4+ CD8- and CD4- CD8+) was significantly increased and that of double positive cells (CD4+ CD8+) was significantly decreased in clone 20 mice and, to a lesser extent, in clone 5 mice compared with controls. Serum levels of interleukin 6 and granulocyte-macrophage colony-stimulating factor (GM-CSF) were significantly elevated. These results suggested that thymocyte apoptosis was accelerated in the cancer-cachectic state, and increased GM-CSF might be partly responsible for thymic atrophy.     

T cell regulation of B cell activation. T cells independently regulate the responses mediated by distinct B cell subpopulations

The present studies have been carried out to characterize the regulatory influences acting upon defined pathways of T cell-dependent B cell activation. In these studies, it was demonstrated that high concentrations of free carrier strongly inhibited the MHC-restricted in vitro T cell-dependent antibody responses of primed Lyb-5- B cells to the corresponding carrier-hapten conjugate. In contrast, these same concentrations of free carrier failed to inhibit the T cell dependent responses of Lyb-5+ B cells to the same antigen. The inhibition of Lyb-5- B cell responses by free carrier was shown to result from active suppression mediated by carrier-specific primed Lyt-1+2- T cells and to require the additional participation of unprimed Lyt-1-2+ T cells. The activation of this suppression was antigen-specific, but suppression once activated was antigen nonspecific in its effect. These findings thus demonstrate that distinct pathways of B cell activation can be independently regulated by T suppressor network influences, and that these pathways therefore constitute potentially independent components of the immune response to a given antigenic stimulus.     

Endonuclease-induced DNA damage and cell death in oxidant injury to renal tubular epithelial cells.

Hydrogen peroxide (H2O2)-induced DNA damage and cell death have been attributed to the direct cytotoxicity of H2O2 and other oxidant species generated from H2O2. We examined the possibility that oxidants activate endonucleases leading to DNA damage and cell death in renal tubular epithelial cells, similar to that described for apoptosis. Within minutes, H2O2 caused DNA strand breaks in a dose-dependent manner, followed by cell death. DNA fragmentation was demonstrated both by the release of [3H]thymidine in 27,000-g supernatant as well as the occurrence of low molecular weight DNA fragments on agarose gel electrophoresis, characteristic of endonuclease cleavage. Endonuclease inhibitors, aurintricarboxylic acid, Evans blue, and zinc ion prevented H2O2-induced DNA strand breaks, fragmentation, and cell death. Inhibitors of protein or mRNA synthesis had only minor protection against H2O2-induced DNA damage in contrast to complete protection reported in apoptotic thymocytes. Micrococcal endonuclease induced similar DNA strand breaks in LLC-PK1 cells, and the endonuclease inhibitors prevented the events confirming the ability of endonucleases to induce DNA damage. The protective effect of aurintricarboxylic acid was not due to the prevention of the rise in intracellular free calcium. We conclude that endonuclease activation occurs as an early event leading to DNA damage and cell death in renal tubular epithelial cells exposed to oxidant stress and, in contrast to apoptotic thymocytes, does not require macromolecular synthesis.     

PD-L1 has distinct functions in hematopoietic and nonhematopoietic cells in regulating T cell responses during chronic infection in mice

The inhibitory receptor programmed death 1 (PD-1) is upregulated on antigen-specific CD8⁺ T cells during persistent viral infections. Interaction with PD-1 ligand 1 (PD-L1) contributes to functional exhaustion of responding T cells and may limit immunopathology during infection. PD-L1 is expressed on both hematopoietic and nonhematopoietic cells in tissues. However, the exact roles of PD-L1 on hematopoietic versus nonhematopoietic cells in modulating immune responses are unclear. Here we used bone marrow chimeric mice to examine the effects of PD-L1 deficiency in hematopoietic or nonhematopoietic cells during lymphocytic choriomeningitis virus clone 13 (LCMV CL-13) infection. We found that PD-L1 expression on hematopoietic cells inhibited CD8⁺ T cell numbers and function after LCMV CL-13 infection. In contrast, PD-L1 expression on nonhematopoietic cells limited viral clearance and immunopathology in infected tissues. Together, these data demonstrate that there are distinct roles for PD-L1 on hematopoietic and nonhematopoietic cells in regulating CD8⁺ T cell responses and viral clearance during chronic viral infection.     

Asbestos and multi-walled carbon nanotubes generate distinct oxidative responses in inflammatory cells

Asbestos exposure is considered a social burden by causing mesothelioma. Despite the use of synthetic materials, multi-walled carbon nanotubes (MWCNTs) are similar in dimension to asbestos and produce mesothelioma in animals. The role of inflammatory cells in mesothelial carcinogenesis remains unclear. Here, we evaluated the differences in inflammatory cell responses following exposure to these fibrous materials using a luminometer and L-012 (8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4-(2H,3H) dione) to detect reactive oxygen species (ROS). Rat peripheral blood or RAW264.7 cells were used to assess the effects on neutrophils and macrophages, respectively. Crocidolite and amosite induced significant ROS generation by neutrophils with a peak at 10 min, whereas that of chrysotile was ~25% of the crocidolite/amosite response. MWCNTs with different diameters (~15, 50, 115 and 145 nm) and different carcinogenicity did not induce significant ROS in peripheral blood. However, the MWCNTs induced a comparable amount of ROS in RAW264.7 cells to that following asbestos treatment. The peaks for MWCNTs (0.5–1.5 h) were observed earlier than those for asbestos (1–5 h). Apocynin and superoxide dismutase significantly inhibited ROS generation for each fiber, suggesting an involvement of NADPH oxidase and superoxide. Thus, asbestos and MWCNTs induce different oxidative responses in inflammatory cells, indicating the importance of mesothelial cell evaluation for carcinogenesis.     

导入针对survivin基因的siRNA对大肠癌Lovo细胞凋亡和增殖的影响

目的：向大肠癌离体LOVO细胞中导入针对survivin基因的siRNA，并研究其对LOVO细胞凋亡的影响。方法：设计2条特异性针对survivin基因的siRNA，并体外转录法合成，利用脂质体导入LOVO细胞并检测转染后细胞的凋亡和增殖情况。结果：RTPCR检测survivin mRNA表达水平发现两RNA干扰组细胞内survivin mRNA表达量明显低于正常LOVO细胞；细胞凋亡检测结果发现两干扰组细胞凋亡率分别为21、5％和26、28％明显高于正常Lovo细胞；细胞增殖结果显示：两干扰组细胞增殖能力比正常Lovo细胞减弱。结论：针对survivin基因的siRNA能有效降低目的基因的mRNA表达，抑制LOVO细胞生长，细胞凋亡率明显增高。为大肠癌基因治疗的可行性提供了有力的证据。