Human Colon Cancer DLD-1 Cells Research Articles

Microvesicle-mediated RNA Molecule Delivery System Using Monocytes/Macrophages

Microvesicles (MVs) and exosomes, which are shed from cells as a cell-to-cell communication tool, are possible vehicles for navigating RNA molecules to body tissues. It is considered that intravenous injection of such MVs or exosomes from patients would not cause severe not-self and toxic reactions. Previously, we found that macrophages take up liposome-entrapped RNA molecules, some of which remain undegraded in the cells. Here, we demonstrate that transfected RNA molecules in human monocytic leukemia THP-1 cells were shed from THP-1 macrophages as contents in MVs during incubation in serum-free medium, which shedding was shown by biochemical analyses such as quantitative reverse transcription (qRT)-PCR, expression of TSG101 (a membrane-associated exosomal protein), and immunoelectron microscopic study. More chemically modified RNA molecules (miR-143BPs) entrapped by MVs (MV-miR-143BPs) were secreted from THP-1 macrophages after miR-143BP transfection compared with the amount after transfection with nonmodified miR-143 transfection. Furthermore, we show that the THP-1 macrophages, which were transfected with the miR-143BP ex vivo, secreted MV-miR-143BPs in xenografted nude mice after intravenous injection, because miR-143 levels were significantly increased in the serum, tumor, and kidney of the host animals. These data suggest that some of the transfected miR-143BPs were secreted from THP-1 macrophages as MV-RNAs both in vitro and in vivo.

6,7,4'-Trihydroxyisoflavone inhibits HCT-116 human colon cancer cell proliferation by targeting CDK1 and CDK2

Colon cancer is a common epithelial malignancies worldwide. Epidemiologic evidence has shown that nutrition and dietary components are important environmental factors involved in the development of this disease. We investigated the biological activity of 6,7,4'-trihydroxyisoflavone (6,7,4'-THIF, a metabolite of daidzein) in in vitro and in vivo models of human colon cancer. 6,7,4'-THIF suppressed anchorage-dependent and -independent growth of HCT-116 and DLD1 human colon cancer cells more effectively than daidzein. In addition, 6,7,4'-THIF induced cell cycle arrest at the S and G2/M phases in HCT-116 human colon cancer cells. Western blot analysis revealed that 6,7,4'-THIF effectively suppressed the expression of cyclin-dependent kinase (CDK) 2, but had no effect on other S- or G2/M-phase regulatory proteins such as cyclin A, cyclin B1 or CDK1. Daidzein did not affect the expression of any of these proteins. In kinase and pull-down assays, 6,7,4'-THIF, but not daidzein, inhibited CDK1 and CDK2 activities in HCT-116 cells by directly interacting with CDK1 and CDK2. In a xenograft mouse model, 6,7,4'-THIF significantly decreased tumor growth, volume and weight of HCT-116 xenografts. 6,7,4'-THIF bound directly to CDK1 and CDK2 in vivo, resulting in the suppression of CDK1 and CDK2 activity in tumors corresponding with our in vitro results. Collectively, these results suggest that CDK1 and CDK2 are potential molecular targets of 6,7,4'-THIF to suppress HCT-116 cell proliferation in vitro and in vivo. These findings provide insight into the biological actions of 6,7,4'-THIF and might establish a molecular basis for the development of new cancer therapeutic agents.

Impact of protein binding on the analytical detectability and anticancer activity of thymoquinone

Thymoquinone (TQ), an active component of Nigella sativa L., is known to have anti-cancer and anti-inflammatory effects; however, no studies on its analytical detection in serum and its protein binding have been published. Using high performance liquid chromatography analysis, we show that the average recovery of TQ from serum is 2.5% at 10μg/ml of TQ and 72% at 100μg/ml. The low recovery of TQ from serum is due to its extensive binding to plasma proteins, as more than 99% of TQ was bound within 30min of incubation. The binding of TQ to the major plasma proteins, bovine serum albumin (BSA) and alpha -1 acid glycoprotein (AGP), was studied and found to be 94.5 ± 1.7% for BSA and 99.1 ± 0.1% for AGP. Mass spectrometric analysis revealed that TQ was bound covalently to BSA, specifically on Cyst-34. Using WST-1 proliferation assay, we showed that BSA plays a protective role against TQ-induced cell death; pre-incubation with BSA prevented TQ from exerting its anti-proliferative effects against DLD-1 and HCT-116 human colon cancer cells. On the other hand, binding of TQ to AGP did not alter its anti-proliferative activity against both cell lines. When TQ was pre-incubated with AGP prior to the addition of BSA, the activity of TQ against DLD-1 was maintained, suggesting that AGP prevented the binding of TQ to BSA. This is the first time the covalent binding and inhibitory effect of BSA on TQ is documented. These data offer new grounds for TQ future pharmacokinetic analysis in vivo. The online version of this article (doi:10.1007/s12154-010-0052-4) contains supplementary material, which is available to authorized users.

Dysregulation of microRNA-34a expression causes drug-resistance to 5-FU in human colon cancer DLD-1 cells

MiR-34a was identified as one of the down-regulated micro-RNAs (miRs) in human colorectal cancer 5-fluorouracil (5-FU)-resistant DLD-1 cells compared with those in the parental DLD-1 cells. Exposure to 5-FU at 30 μM activated phosphoinositide 3-kinase (PI3K)/Akt signaling markedly from 12 h up to 48 h in the 5-FU-resistant cells compared with that in the parental cells and resulted in an overt difference in growth at those times. Furthermore, the expression of miR-34a in the 5-FU-resistant cells was sustained at a low-level, whereas it was up-regulated in the parental cells after the 5-FU treatment. Sirt1, which is one of the target genes for miR-34a and related to drug-resistance, was strikingly up-regulated in the 5-FU-resistant cells. The ectopic expression of miR-34a in the 5-FU-resistant cells inhibited growth, as in the parental cells, and attenuated the resistance to 5-FU through the down-regulation of Sirt1 and E2F3. Moreover, the silencing of Sirt1 significantly canceled the resistance to 5-FU in the 5-FU-resistant cells. These findings suggest that miR-34a targeting the Sirt1 and E2F3 genes could negatively regulate, at least in part, the resistance to 5-FU in human colorectal cancer DLD-1 cells.

Comparative proteomic analysis of the ribosomes in 5-fluorouracil resistance of a human colon cancer cell line using the radical-free and highly reducing method of two-dimensional polyacrylamide gel electrophoresis

Many auxiliary functions of ribosomal proteins (r-proteins) have received considerable attention in recent years. However, human r-proteins have hardly been examined by proteomic analysis. In this study, we isolated ribosomal particles and subsequently compared the proteome of r-proteins between the DLD-1 human colon cancer cell line and its 5-fluorouracil (5-FU)-resistant sub-line, DLD-1/5-FU, using the radical-free and highly reducing method of two-dimensional polyacrylamide gel electrophoresis, which has a superior ability to separate basic proteins, and we discuss the role of r-proteins in 5-FU resistance. Densitometric analysis was performed to quantify modulated proteins, and protein spots showing significant changes were identified by employing matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry. Three basic proteins (L15, L37 and prohibitin) which were significantly modulated between DLD-1 and DLD-1/5-FU were identified. Two proteins, L15 and L37, showed down-regulated expression in DLD-1/5-FU in comparison to DLD-1. Prohibitin, which is not an r-protein and is known to be localized in the mitochondria, showed up-regulated expression in DLD-1/5-FU. These 3 proteins may be related to 5-FU resistance.

Involvement of ST6Gal I in the biosynthesis of a unique human colon cancer biomarker candidate, α2,6-sialylated blood group type 2H (ST2H) antigen

The alpha2,6-sialylated blood group type 2H (ST2H) antigen (Fucalpha1-2(NeuAcalpha2-6)Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc-Cer) is a fucoganglioside found in human colon cancer tissues. To elucidate an enzyme responsible for the ST2H antigen formation, we screened some partially purified candidate enzymes, alpha2,6-sialyltransferases, ST6Gal I and ST6Gal II, and alpha1,2-fucosyltransferases, FUT1 and FUT2 for their activities towards pyridylaminated type 2H (Fucalpha1-2Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc-PA) or LS-tetrasaccharide c (LST-c: NeuAcalpha2-6Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc-PA) as acceptor substrates. Here we show the ST6Gal I transfers NeuAc from the donor CMP-NeuAc to the terminal Gal of PA-type 2H, which formed the ST2H antigen, but the others could not synthesize it. Using a recombinant ST6Gal I, enzymatic reactions with two types of acceptors, PA-type 2H and PA-lacto-N-neotetraose (LNnT), were kinetically analysed. On the basis of catalytic efficiency (V(max)/K(m)), the specificity of ST6Gal I towards the PA-type 2H was estimated to be 42 times lower than that for PA-LNnT. The overexpression of ST6Gal I in human colon cancer DLD-1 cells effectively resulted in the ST2H antigen formation, as judged by LC-ESI-IT-MS. Many lines of evidence suggest the up-regulation of ST6Gal I in human colon cancer specimens. Collectively, these findings indicate that ST6Gal I is responsible for ST2H antigen biosynthesis in human colon cancer cells.

Abstract 293: Regulation of inhibitor of differentiation 1 (Id1) by PKC signaling in the intestinal epithelium

Abstract Increasing evidence supports a role for protein kinase C (PKC) signaling in regulation of intestinal epithelial self-renewal, and alterations in PKC isozyme expression/activity have been broadly linked to intestinal carcinogenesis. PKCα, which exhibits growth inhibitory and tumor suppressor properties in the intestine, is lost early during intestinal tumor development. To gain further insight into the tumor suppressive effects of PKCα signaling, we explored the consequences of restoration of PKCα expression in DLD-1 human colon cancer cells by microarray analysis. The most downregulated known gene in PKCα-expressing DLD-1 cells was inhibitor of differentiation 1 (Id1), a member of the Id family of dominant negative antagonists of bHLH transcription factors that has cell cycle stimulatory functions and has been associated with tumorigenesis in a number of systems. Suppression of Id1 protein by PKCα in colon cancer cells was confirmed by Western blot analysis in a panel of cell lines including DLD-1, HCT116, FET, FET/DNR, and GEO. Notably, activation of PKC/PKCα in non-transformed IEC-18 intestinal crypt cells also resulted in rapid and profound downregulation of Id1 mRNA and protein, pointing to a role for PKCα in Id1 regulation in the normal intestinal epithelium. PKC-induced suppression of Id1 was found to occur via an ERK/MAPK-dependent, PI3K-independent mechanism. Manipulation of Id1 levels in normal and transformed intestinal cells demonstrated its ability to affect two key cell cycle targets of PKCα, the potent mitogen cyclin D1 and the cdk inhibitor p21Waf1/Cip1. Knockdown of Id1 consistently decreased steady-state levels of cyclin D1, while overexpression of the factor resulted in reduced levels of p21Waf1/Cip1. Thus, Id1 appears to control the expression of important cell cycle regulators in intestinal cells. Immunohistochemical analysis of normal intestinal mucosa further demonstrated that PKCα activation, which occurs precisely at the point of growth arrest in the mid/upper crypt, coincides with marked downregulation of Id1 in vivo. Conversely, loss of PKCα in intestinal tumors is associated with Id1 overexpression. Ongoing studies are focused on delineating the link between the tumor suppressor functions of PKCα and its inhibitory effects on Id1 expression in intestinal cells. Supported by NIH grants DK54909, DK60632, CA16506, and CA113048. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 293.

Antitumor effect of extracts from moutan cortex on DLD-1 human colon cancer cells in vitro

Moutan cortex, the root bark of Paeonia suffruticosa Andrews (Paeoniaceae), is a traditional Chinese medicine widely used for the treatment of various diseases. In the present study, we examined the antiproliferative effect and apoptosis-inducing activity of paeonol and the crude total glycosides (CTG) extracted from moutan cortex against DLD-1 human colon cancer cells in vitro. Cell viability was measured using the WST-8 assay. Apoptosis was detected by Hoechst 33258 fluorescence staining and flow cytometry. Paeonol and the CTG significantly reduced cell viability in a dose- and time-dependent manner. The induction of apoptosis in DLD-1 cells was characterized by morphological changes and an increased percentage of hypodiploid cells. After treatment for 48 h with paeonol (400 µg/ml) or CTG (200 µg/ml), the ratio of apoptotic cells reached 34.79 and 48.12%, respectively. The findings obtained indicate that paeonol and the CTG extracted from moutan cortex have a significant growth-inhibitory effect on human colon cancer cells. This effect may be related to the induction of apoptosis.

Regulation of peroxisome proliferator‐activated receptor‐β/δ by the APC/β‐CATENIN pathway and nonsteroidal antiinflammatory drugs

Studies indicate that peroxisome proliferator-activated receptor-beta/delta (PPAR beta/delta) can either attenuate or potentiate colon cancer. One hypothesis suggests that PPAR beta/delta is upregulated by the adenomatous polyposis coli (APC)/beta-CATENIN pathway and a related hypothesis suggests that PPAR beta/delta is downregulated by nonsteroidal antiinflammatory drugs (NSAIDs). The present study examined these possibilities using in vivo and in vitro models. While APC/beta-CATENIN-dependent expression of CYCLIN D1 was observed in vivo and in vitro, expression of PPAR beta/delta was not different in colon or intestinal polyps from wild-type or Apc(min) heterozygous mice or in human colon cancer cell lines with mutations in APC and/or beta-CATENIN. No difference in the level of PPAR beta/delta was found in colon from wild-type or Apc(min) heterozygous mice following treatment with NO-donating aspirin (NO-ASA). NSAIDs inhibited cell growth in RKO (wild-type APC) and DLD1 (mutant APC) human colon cancer cell lines but expression of PPAR beta/delta was not downregulated in these cell lines in response to a broad concentration range of celecoxib, indomethacin, NS-398, or nimesulide. However, indomethacin caused an increase in PPAR beta/delta mRNA and protein that was accompanied with increased expression of a known PPAR beta/delta target gene. Interestingly, expression of PPAR alpha was also increased in the human colon cancer cell lines by several NSAIDs at the highest concentration examined. Results from these studies provide additional evidence indicating that PPAR beta/delta is not upregulated by the APC/beta-CATENIN pathway. Further, these studies suggest that increased PPAR beta/delta and/or PPAR alpha by NSAIDs in human colon cancer cell lines could contribute to the mechanisms underlying the chemopreventive effects of NSAIDs.

Comparative study of antioxidant activity and antiproliferative effect of hot water and ethanol extracts from the mushroom Inonotus obliquus

The medicinal mushroom Inonotus obliquus is a traditional and widely used multi-functional fungus. Hot water (50 degrees C, 70 degrees C, and 80 degrees C) and ethanol crude extracts of I. obliquus were investigated for their antioxidant activity with superoxide dismutase (SOD) and (1,1-diphenyl-2-picryhydrazyl) (DPPH) radical-scavenging activity assays. We also investigated the antiproliferative effects and ability of the extracts to induce apoptosis in human colon cancer DLD-1 cells. Among the four extracts, the ethanol extract (EE) exhibited the strongest SOD-like activity and antiproliferative effect on DLD-1 cells, and exposure to the EE resulted in the induction of apoptosis, whereas no apoptosis was observed in DLD-1 cells exposed to the hot water extracts (HWEs). HWE at 70 degrees C (HWE70) exhibited the strongest DPPH radical-scavenging activity (EC50, 126 microg/ml), whereas the EE showed the weakest activity (EC50, 224 microg/ml). The different biological activities among the four extracts may be attributed to differences in their chemical composition, partially supported by polysaccharide, protein and phenolic content, and the 1H-NMR spectra.

N6‐isopentenyladenosine inhibits cell proliferation and induces apoptosis in a human colon cancer cell line DLD1

N(6)-isopentenyladenosine (i6A) is a modified nucleoside with a pentaatomic isopentenyl derived from mevalonate that induces inhibition of tumor cell proliferation and apoptosis in several tumor cell lines. In this study, we reported that N(6)-isopentenyladenosine inhibited the proliferation and promotes apoptosis in DLD1 human colon cancer cells. It suppressed the proliferation of cells through inhibition of DNA synthesis, causing a cell cycle arrest that correlated with a decrease in the levels of cyclin E, cyclin A and cyclin D1 and with a concomitant increase in the levels of cyclin-dependent kinase inhibitor p21waf and p27kip1. Moreover, it induced apoptosis through an increase in the number of annexin V-positive cells, a downregulation of antiapoptotic products and caspase-3 activation. The apoptotic effects of N(6)-isopentenyladenosine were accompanied by sustained phosphorylation and activation of c-jun N-terminal kinase (JNK) that induced phosphorylation of c-jun. Overall, our data show that JNK, could play an important role in i6A-mediated apoptosis in DLD1 human colon cancer cells.

Early induction of LDL receptor gene expression by genistein in DLD-1 colon cancer cell line

The phytoestrogen genistein has been demonstrated to possess anti-tumor properties by mechanisms not yet clearly established. The present study was designed to investigate the effects of isoflavone genistein exposure at concentrations ranging from 0.01 microM to 50 microM on the LDL receptor and HMGCoA reductase gene expression in the estrogen receptor positive DLD-1 human colon cancer cell line. LDL receptor and HMGCoA reductase gene expressions were evaluated by reverse transcription followed by real-time PCR. Genistein induced an early increase of LDL receptor gene expression and later decreased HMGCoA reductase mRNA expression in DLD-1 cells. These findings provide direct evidence on the role of genistein in regulating LDL receptor and HMGCoA reductase gene expression in colon cancer.

CDK inhibitor enhances the sensitivity to 5-fluorouracil in colorectal cancer cells

Thymidylate synthase (TS) is a dNTP synthetic enzyme and is also a target enzyme of 5-fluorouracil (5-FU). 5-FU is one of the anticancer agents most frequently used for the treatment of colorectal cancers. However, the clinical rate of response to its use as a single agent is not exceptionally high. Therefore, various combination chemotherapies have been devised. The elevated expression of TS in cancer cells is a serious obstacle in the clinical use of 5-FU. In the present study, TS expression was up-regulated by the knockout of the p21WAF1/CIP1 gene in human colorectal cancer HCT116 cells, suggesting that TS expression is mediated through the inhibition of cyclin-dependent kinase (CDK). Based on these findings, we tested whether the CDK inhibitor (CDKI) SU9516, acted as a suppressor of TS. SU9516 effectively reduced the expression of TS in a dose-dependent manner. Furthermore, the reduction of TS expression resulted in enhancement of the sensitivity to 5-FU in human colon cancer DLD-1 cells. Thus, SU9516 might be a promising compound for combination chemotherapy with 5-FU.

Anti-Cancer Effects of Xanthones from Pericarps of Mangosteen

Mangosteen, Garcinia mangostana Linn, is a tree found in South East Asia, and its pericarps have been used as traditional medicine. Phytochemical studies have shown that they contain a variety of secondary metabolites, such as oxygenated and prenylated xanthones. Recent studies revealed that these xanthones exhibited a variety of biological activities containing anti-inflammatory, anti-bacterial, and anti-cancer effects. We previously investigated the anti-proliferative effects of four prenylated xanthones from the pericarps; alpha-mangostin, beta-mangostin, gamma-mangostin, and methoxy-beta-mangostin in various human cancer cells. These xanthones are different in the number of hydroxyl and methoxy groups. Except for methoxy-beta-mangostin, the other three xanthones strongly inhibited cell growth at low concentrations from 5 to 20 microM in human colon cancer DLD-1 cells. Our recent study focused on the mechanism of alpha-mangostin-induced growth inhibition in DLD-1 cells. It was shown that the anti-proliferative effects of the xanthones were associated with cell-cycle arrest by affecting the expression of cyclins, cdc2, and p27; G1 arrest by alpha-mangostin and beta-Mangostin, and S arrest by gamma-mangostin. alpha-Mangostin found to induce apoptosis through the activation of intrinsic pathway following the down-regulation of signaling cascades involving MAP kinases and the serine/threonine kinase Akt. Synergistic effects by the combined treatment of alpha-mangostin and anti-cancer drug 5-FU was to be noted. alpha-Mangostin was found to have a cancer preventive effect in rat carcinogenesis bioassay and the extract from pericarps, which contains mainly alpha-mangostin and gamma-mangostin, exhibited an enhancement of NK cell activity in a mouse model. These findings could provide a relevant basis for the development of xanthones as an agent for cancer prevention and the combination therapy with anti-cancer drugs.

Effect of genistein on cholesterol metabolism-related genes in a colon cancer cell line

The major soy-derived isoflavones such as genistein has been demonstrated to possess anticarcinogenic activity in animal model systems. The present study was designed to investigate the effects of isoflavone genistein exposure at concentrations ranging from 0.01 to 50 muM on the LDL receptor and HMG-CoA reductase gene expression in the estrogen receptor positive DLD-1 human colon cancer cell line. LDL receptor and HMG-CoA reductase gene expressions were evaluated by reverse transcription followed by real-time PCR. Genistein induced an increase of LDL receptor gene expression and later decrease of HMG-CoA reductase mRNA expression in DLD-1 cells. These findings provide direct evidence on the role of genistein in regulating LDL receptor and HMG-CoA reductase gene expression in colon cancer.

Radiosensitization by 2-benzoyl-3-phenyl-6,7-dichloroquinoxaline 1,4-dioxide under oxia and hypoxia in human colon cancer cells

BackgroundThe sensitizing effects of 2-benzoyl-3-phenyl-6,7-dichloroquinoxaline 1,4-dioxide (DCQ) and ionizing radiation (IR) were determined in four colon cancer cells and in FHs74Int normal intestinal cells.MethodsCell cycle modulation, TUNEL assay, clonogenic survival and DNA damage were examined under oxia or hypoxia. Effects on apoptotic molecules and on p-Akt and Cox-2 protein expression were investigated.ResultsThe four cell lines responded differently to DCQ+IR; HT-29 cells were most resistant. Combination treatment caused significant increases in preG1 (apoptosis) in HCT-116, while G2/M arrest occurred in DLD-1. DCQ potentiated IR effects more so under hypoxia than oxia. Pre-exposure of DLD-1 to hypoxia induced 30% apoptosis, and G2/M arrest in oxia. The survival rate was 50% lower in DCQ+IR than DCQ alone and this rate further decreased under hypoxia. FHs74Int normal intestinal cells were more resistant to DCQ+IR than cancer cells.Greater ssDNA damage occurred in DLD-1 exposed to DCQ+IR under hypoxia than oxia. In oxia, p-Akt protein expression increased upon IR exposure and drug pre-treatment inhibited this increase. In contrast, in hypoxia, exposure to IR reduced p-Akt protein and DCQ restored its expression to the untreated control. Apoptosis induced in hypoxic DLD-1 cells was independent of p53-p21 modulation but was associated with an increase in Bax/Bcl-2 ratio and the inhibition of the Cox-2 protein.ConclusionDCQ is a hypoxic cell radiosensitizer in DLD-1 human colon cancer cells.

Downregulation of microRNAs‐143 and ‐145 in B‐cell malignancies

Recently, it has been found that inappropriate expression of microRNAs (miRNAs) is strongly associated with carcinogenesis. In this study, we demonstrated that the expression of miRNAs (miRs) -143 and -145, the levels of which were previously shown to be reduced in colon cancers and various kinds of established cancer cell lines, was also decreased in most of the B-cell malignancies examined, including chronic lymphocytic leukemias (CLL), B-cell lymphomas, Epstein-Barr virus (EBV)-transformed B-cell lines, and Burkitt lymphoma cell lines. All samples from 13 CLL patients and eight of nine B-cell lymphoma ones tested exhibited an extremely low expression of miRs-143 and -145. The expression levels of miRs-143 and -145 were consistently low in human Burkitt lymphoma cell lines and were inversely associated with the cell proliferation observed in the EBV-transformed B-cell lines. Moreover, the introduction of either precursor or mature miR-143 and -145 into Raji cells resulted in a significant growth inhibition that occurred in a dose-dependent manner and the target gene of miRNA-143 was determined to be ERK5, as previously reported in human colon cancer DLD-1 cells. Taken together, these findings suggest that miRs-143 and -145 may be useful as biomarkers that differentiate B-cell malignant cells from normal cells and contribute to carcinogenesis in B-cell malignancies by a newly defined mechanism.

Characterized mechanism of α-mangostin-induced cell death: Caspase-independent apoptosis with release of endonuclease-G from mitochondria and increased miR-143 expression in human colorectal cancer DLD-1 cells

α-Mangostin, a xanthone from the pericarps of mangosteen ( Garcinia mangostana Linn.), was evaluated for in vitro cytotoxicity against human colon cancer DLD-1 cells. The number of viable cells was consistently decreased by the treatment with α-mangostin at more than 20 μM. The cytotoxic effect of 20 μM α-mangostin was found to be mainly due to apoptosis, as indicated by morphological findings. Western blotting, the results of an apoptosis inhibition assay using caspase inhibitors, and the examination of caspase activity did not demonstrate the activation of any of the caspases tested. However, endonuclease-G released from mitochondria with the decreased mitochondrial membrane potential was shown. The levels of phospho-Erk1/2 were increased in the early phase until 1 h after the start of treatment and thereafter decreased, and increased again in the late phase. On the other hand, the level of phospho-Akt was sharply reduced with the process of apoptosis after 6 h of treatment. Interestingly, the level of microRNA-143, which negatively regulates Erk5 at translation, gradually increased until 24 h following the start of treatment. We also examined the synergistic growth suppression in DLD-1 cells by the combined treatment of the cells with α-mangostin and 5-FU which is one of the most effective chemotherapeutic agents for colorectal adenocarcinoma. The co-treatment with α-mangostin and 5-FU, both at 2.5 μM, augmented growth inhibition compared with the treatment with 5 μM of α-mangostin or 5 μM 5-FU alone. These findings indicate unique mechanisms of α-mangostin-induced apoptosis and its action as an effective chemosensitizer.

Induction of Apoptosis in DLD-1 Human Colon Cancer Cells by Peridinin Isolated from the Dinoflagellate,Heterocapsa triquetra

Peridinin, which is uniquely present in dinoflagellates, is one of the most abundant carotenoids found in nature. We evaluated the apoptotic effect of peridinin on DLD-1 human colorectal cancer cells. Peridinin significantly reduced the cell viability in a dose-dependent manner (0-20 microM) and induced apoptosis by activating both caspase-8 and caspase-9. Our findings could be important for the high-performance utilization of marine bioproducts.

High-Throughput Bioluminescence Screening of Ubiquitin-Proteasome Pathway Inhibitors from Chemical and Natural Sources

To discover original inhibitors of the ubiquitin-proteasome pathway, the authors have developed a cell-based bioluminescent assay and used it to screen collections of plant extracts and chemical compounds. They first established a DLD-1 human colon cancer cell line that stably expresses a 4Ubiquitin-Luciferase (4Ub-Luc) reporter protein, efficiently targeted to the ubiquitin-proteasome degradation pathway. The assay was then adapted to 96- and 384-well plate formats and calibrated with reference proteasome inhibitors. Assay robustness was carefully assessed, particularly cell toxicity, and the statistical Z factor value was calculated to 0.83, demonstrating a good performance level of the assay. A total of 18,239 molecules and 15,744 plant extracts and fractions thereof were screened for their capacity to increase the luciferase activity in DLD-1 4Ub-Luc cells, and 21 molecules and 66 extracts inhibiting the ubiquitin-proteasome pathway were identified. The fractionation of an active methanol extract of Physalis angulata L. aerial parts was performed to isolate 2 secosteroids known as physalin B and C. In a cell-based Western blot assay, the ubiquitinated protein accumulation was confirmed after a physalin treatment confirming the accuracy of the screening process. The method reported here thus provides a robust approach to identify novel ubiquitin-proteasome pathway inhibitors in large collections of chemical compounds and natural products.