Human Colorectal Cancer SW480 Cells Research Articles

Overexpression of secretagogin promotes cell apoptosis and inhibits migration and invasion of human SW480 human colorectal cancer cells

ObjectiveIn order to investigate the effect of secretagogin (SCGN) on colorectal cancer (CRC) cells apoptosis, invasion and migration in vitro. MethodsExpression of SCGN in CRC tissues and the paired adjacent non-tumorous tissues (n = 36) and four human CRC cell lines (HT29, HCT116, SW480 and SW620) were detected. SW480 cells were transfected with the SCGN overexpression plasmid (eGFP-SCGN), si-SCGN-773, and the corresponding negative controls (NCs). Then, cell-cycle distribution, cell apoptosis, migration, invasion and expression of apoptosis- and metastasis-related proteins were detected. ResultsSCGN was significantly downregulated in CRC tissues as compared with the adjacent non-tumorous tissues. The expression of SCGN in HT29 and SW480 cells were lower than those in HT116 and SW620 cells. We transfected SW480 cells with SCGN overexpression plasmid eGFP-SCGN and found the increased cell apoptosis, with cell arresting at G0/G1 phase. SW480 cells with SCGN overexpression showed wider wound width and fewer invaded cells than control and blank cells, with upregulated Bax, cleaved Caspase 3 and E-cadherin, and downregulated Bcl-2 and Vimentin. We also transfected SW480 cells with si-SCGN-773 and found si-SCGN increased cell migration and invasion, but did not affect cell apoptosis and expression of related proteins. ConclusionWe concluded that the overexpression of SCGN in SW480 cells promoted cell apoptosis and inhibited cell migration and invasion.

Caveolin-1 Inhibits Proliferation, Migration, and Invasion of Human Colorectal Cancer Cells by Suppressing Phosphorylation of Epidermal Growth Factor Receptor.

BackgroundAlthough downregulation of caveolin-1 (Cav-1), which is a key constituent of membrane caveolae and a regulator of cellular processes, is associated with colorectal cancer (CRC), its involvement in the disease progression is largely unknown. This study aimed to explore the role of Cav-1 in CRC and the associated mechanisms.Material/MethodsFresh tissues from patients with CRC and human CRC SW480 cells were used to evaluate Cav-1 and Ki-67 expression using immunohistochemistry and Western blotting. The MTS and Transwell assays were performed to determine the effects of Cav-1 overexpression via pcDNA3.1/Cav-1 plasmid on cell proliferation and metastasis. The effect of Cav-1 on the epidermal growth factor receptor (EGFR) activation was evaluated by Western blotting. The correlation of Cav-1 expression with clinicopathological factors was statistically analyzed.ResultsOverexpression of Cav-1 significantly reduced proliferation, migration, and invasion of SW480 cancer cells in vitro. The EGF-induced phosphorylation of EGFR and activations of the RAF-MEK-ERK and PI3K-AKT pathways were adversely regulated by Cav-1 overexpression in vitro. In 76 cases of CRC patients with EGFR expression, a negative correlation was observed between the level of Cav-1 and tumor-node-metastasis stage, lymph node metastasis, and distant metastasis (All p<0.05). Finally, the expression level of Cav-1 was negatively correlated with that of Ki-67.ConclusionsThis report is the first to show that overexpression of Cav-1significantly inhibits the proliferation, migration, and invasion potential of SW480 cells, possibly through reducing EGF-induced EGFR activation. High Cav-1 expression level may be a predictor of positive outcomes in patients with colorectal cancer.

Abstract A187: Exploring isoform-dependent functions of the mediator kinases in colorectal cancer

Abstract Introduction: CDK8 and its paralogue CDK19 are highly similar cyclin-dependent kinases that function as components of the kinase module of the Mediator complex. Despite their structural similarity, emerging data suggest that these proteins may have distinct functions in the regulation of transcription. Recently, we identified two chemical series of CDK8/19 ligands (1, 2); however, as these ligands have similar binding affinities for CDK8 and CDK19 it has not been possible, until now, to determine if the biologic effects observed were driven by inhibition of one or both kinases. Here, we describe a functional genomics approach that utilizes these ligands, and others described in the literature, to identify substrates of CDK8/19 and isoform-dependent functions of these kinases. Materials and Methods: Experiments were conducted on COLO205 and SW620 human colorectal cancer cell lines and SW620 cells harbouring CRISPR knockouts of CDK8 and/or CDK19. Transcription factor reporter assays were carried out to define signalling pathways and biologic processes modulated by CDK8/19 ligands. Cells were reverse-transfected with both an inducible transcription-factor-responsive construct encoding firefly luciferase under the control of a basal promoter element, and a construct constitutively expressing the Renilla luciferase. At 42 h post-transfection, cells were treated with one of our CDK8/19 ligands, CCT251545 or MSC2530818, or the CDK8/19 inhibitors Senexin B (3) or Selvita 120-34 (4), at a 10 x EC50 concentration. Reporter activity was quantified following 6 h continuous exposure. In addition, gene set enrichment analysis (GSEA) was employed to examine changes in gene expression resulting from ligand treatment or CDK8/19 knockout, including changes in the expression of those genes regulated by transcription factors identified in the pathway reporter assay. Results: The pathway reporter assay identified transcription factor activity that was modulated by compound treatment and/or CDK8/19 knockout. Enriched transcription factors included key players in Notch signalling, Wnt signalling, the interferon gamma response, the cellular stress response, and MAPK/Jnk signalling, all of which are known to be modulated directly or indirectly by CDK8. Some of the transcription factors were differentially regulated by the test compounds, as well as by CDK8, CDK19 and CDK8/CDK19 knockout. Data from the CRISPR-mediated CDK8/19 knockout line demonstrated that the majority of altered gene expression, and corresponding decreased phosphorylation of STAT1SER727, was driven by loss of CDK8 with some limited compensatory activity from CDK19. These observations suggest that there may be some functional differences between the two kinases and that the main biologic effects of ligand treatment result from CDK8 inhibition. Conclusions: These experiments have identified potential substrates of CDK8/19 and determined that the majority of the effects on gene expression or biomarker phosphorylation observed in colorectal cancer cell lines after treatment with CDK8/19 ligands are driven by the inhibition of CDK8.

Short interfering RNA-mediated silencing of actin-related protein 2/3 complex subunit 4 inhibits the migration of SW620 human colorectal cancer cells.

Actin-related protein 2/3 complex subunit 4 (ARPC4) acts as an actin nucleator in actin cytoskeleton branching and contributes to cell migration. ARPC4 has previously been demonstrated to be abnormally expressed in various colorectal carcinoma cell lines, particularly SW620 cells. The present study explored the biological action and the possible mechanisms underlying the function of ARPC4 in the progression of carcinoma. The proliferation and migration of SW620 cells transfected with ARPC4-specific short interfering (si)RNAs were assessed using western blot, cell counting, flow cytometry and transwell assays. SW620 cells exhibited the highest ARPC4 expression of the cell lines investigated, and siRNA538 was the most effective of the siRNAs considered. The results of the present study demonstrated that ARPC4-silencing exhibited a significant effect on the capacity of cells for migration, but did not affect their proliferative ability. ARPC4-silencing inhibited human SW620 cell migration, but not proliferation, in vitro, suggesting that ARPC4 may be a putative therapeutic target for colorectal carcinoma.

Quercetrin from Toona sinensis leaves induces cell cycle arrest and apoptosis via enhancement of oxidative stress in human colorectal cancer SW620 cells.

Finding effective strategies against colorectal cancer (CRC) is still an emergent health problem. In the present study, we investigated the anticancer activity of quercetrin from Toona sinensis leaves (QTL) and explored the underlying mechanism in human CRC cell line SW620. The cells were treated with various concentrations of QTL and the cytotoxic effects of QTL were determined using the MTT assay. Apoptosis and cell cycle status were detected by flow cytometry. Reactive oxygen species (ROS) levels and mitochondrial membrane potential (∆Ψm) were assessed using DCF-DA and JC-1 fluorescence spectrophotometry, respectively. Western blot analysis was used to quantify the expression of apoptosis-related proteins. RT-PCR was applied to determine the mRNA levels of glutathione peroxidase (GPx) and catalase (CAT). QTL exhibited growth inhibitory effects and caused cell cycle arrest in the G2/M phase, which was accompanied by increased expression of p53 and p21 proteins. QTL promoted apoptosis which was consistent with the upregulated expression of Bax, cytochrome c, caspase-9, Apaf-1 and caspase-3. In addition, QTL induced the loss of mitochondrial membrane potential and triggered ROS generation, as revealed by the downregulated mRNA expression and enzymatic activity of GPx and CAT. Furthermore, both N-acetyl cysteine (NAC) and GSH attenuated the QTL-induced growth inhibition observed in SW620 cells along with the increase of ROS levels. These findings revealed that QTL inhibited the growth of CRC cells and facilitated apoptosis by enhancing oxidative stress. QTL may therefore have potential for use in CRC chemotherapy.

Molecular mechanism of cardol, isolated from Trigona incisa stingless bee propolis, induced apoptosis in the SW620 human colorectal cancer cell line.

BackgroundCardol is a major bioactive constituent in the Trigona incisa propolis from Indonesia, with a strong in vitro antiproliferative activity against the SW620 colorectal adenocarcinoma cell line (IC50 of 4.51 ± 0.76 μg/mL). Cardol induced G0/G1 cell cycle arrest and apoptotic cell death. The present study was designed to reveal the mechanism of cardol’s antiproliferative effect and induction of apoptosis.MethodsChanges in cell morphology were observed by light microscopy. To determine whether the mitochondrial apoptotic pathway was involved in cell death, caspase-3 and caspase-9 activities, western blot analysis, mitochondrial membrane potential, and intracellular reactive oxygen species (ROS) levels were assayed.ResultsChanges in the cell morphology and the significantly increased caspase-3 and caspase-9 activities, plus the cleavage of pro-caspase-3, pro-caspase-9 and PARP, supported that cardol caused apoptosis in SW620 cells within 2 h after treatment by cardol. In addition, cardol decreased the mitochondrial membrane potential while increasing the intracellular ROS levels in a time- and dose-dependent manner. Antioxidant treatment supported that the cardol-induced cell death was dependent on ROS production.ConclusionCardol induced cell death in SW620 cells was mediated by oxidative stress elevation and the mitochondrial apoptotic pathway, and these could be the potential molecular mechanism for the antiproliferative effect of cardol.

Inhibitory effect of hyperoside isolated from Zanthoxylumbungeanum leaves on SW620 human colorectal cancer cells via induction of the p53 signaling pathway and apoptosis.

The present study aimed to demonstrate the antiproliferative effect of hyperoside from Zanthoxylum bungeanum leaves (HZL) and explain the underlying molecular mechanisms in the SW620 human colorectal cancer cell line. The cytotoxic effects of HZL were determined using a3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide assay. Apoptosis and cell cycle were detected using flow cytometry. Reactive oxygen species (ROS) levels and mitochondrial membrane potential (∆Ψm) were assessed using 2′,7′-dichlorofluorescin diacetate and 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolyl carbocyanine iodide fluorescence spectrophotometry, respectively. Western blot analysis was used to quantify the expression levels of apoptosis-associated proteins. Reverse transcription-quantitative polymerase chain reaction analysis was used to determine the mRNA expression of glutathione peroxidase (GSH-Px) and catalase (CAT). HZL had a marked anti-proliferative effect on the SW620 human colorectal cancer cells by inducing cell cycle G2/M phase arrest and apoptosis, which was associated with an increase in the expression of p53 and p21. Further mechanistic investigations revealed that the induction of apoptosis was associated with increased generation of ROS, reduced ∆Ψm, and upregulation of B-cell lymphoma 2-associated X protein, cytochrome c, caspase-9, apoptotic protease activating factor 1 and caspase-3. The antitumor potency of HZL was also attributed to inhibition of the mRNA expression levels of GSH-Px and CAT. These data indicated that HZL may be involved in the pro-apoptotic signaling of SW620 human colorectal cancer cells via induction of the caspase-dependent apoptosis and p53 signaling pathways.

Anti-inflammatory effect of lycopene in SW480 human colorectal cancer cells.

BACKGROUND/OBJECTIVESAlthough the antioxidative effects of lycopene are generally known, the molecular mechanisms underlying the anti-inflammatory properties of lycopene are not fully elucidated. This study aimed to examine the role and mechanism of lycopene as an inhibitor of inflammation.METHODS/MATERIALSLipopolysaccharide (LPS)-stimulated SW 480 human colorectal cancer cells were treated with 0, 10, 20, and 30 µM lycopene. The MTT assay was performed to determine the effects of lycopene on cell proliferation. Western blotting was performed to observe the expression of inflammation-related proteins, including nuclear factor-kappa B (NF-κB), inhibitor kappa B (IκB), mitogen-activated protein kinase (MAPK), extracellular signal-related kinase (ERK), c-jun NH2-terminal kinase (JNK), and p38 (p38 MAP kinase). Real-time polymerase chain reaction was performed to investigate the mRNA expression of tumor necrosis factor α (TNF-α), interleukin-1 beta (IL-1β), interleukin-6 (IL-6), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2). Concentrations of nitric oxide (NO) and prostaglandin E2 (PGE2) were determined via enzyme-linked immunosorbent assays.RESULTSIn cells treated with lycopene and LPS, the mRNA expression of TNF-α, IL-1β, IL-6, iNOS, and COX-2 were decreased significantly in a dose-dependent manner (P < 0.05). The concentrations of PGE2 and NO decreased according to the lycopene concentration (P < 0.05). The protein expressions of NF-κB and JNK were decreased significantly according to lycopene concertation (P < 0.05).CONCLUSIONSLycopene restrains NF-κB and JNK activation, which causes inflammation, and suppresses the expression of TNF-α, IL-1β, IL-6, COX-2, and iNOS in SW480 human colorectal cancer cells.

Methyl jasmonate induces the apoptosis of human colorectal cancer cells via downregulation of EZH2 expression by microRNA‑101.

Methyl jasmonate is found universally in the plant kingdom and functions to regulate plant growth and development, as well as in stress responses through signal transduction pathways. The present study aimed to investigate the anticancer effect of methyl jasmonate on SW620 human colorectal cancer cells and its potential underlying mechanism. SW620 cells were treated with 0, 0.5, 0.75, 1.5 and 2.0mM methyl jasmonate for 12, 24 and 48h. Methyl jasmonate was shown to be able to inhibit cell growth and induce apoptosis of SW620 cells in a concentration and time‑dependent manner, whilst promoting an increase in caspase‑3 protein expression. Compared with control, the anticancer effect of methyl jasmonate inhibited Enhancer of zeste homolog 2 (EZH2) protein expression and activated microRNA (miR)‑101 expression in SW620 cells. However, knockdown of miR‑101 suppresses methyl jasmonate‑induced cell growth inhibition, activation of caspase‑3 expression and inhibition of EZH2 expression in SW620 cells. These results demonstrate that methyl jasmonate induced the apoptosis of human colorectal cancer cells via downregulation of EZH2 expression by miR‑101.

Phosphoproteome Characterization of Human Colorectal Cancer SW620 Cell-Derived Exosomes and New Phosphosite Discovery for C-HPP.

Identification of all phosphorylation forms of known proteins is a major goal of the Chromosome-Centric Human Proteome Project (C-HPP). Recent studies have found that certain phosphoproteins can be encapsulated in exosomes and function as key regulators in tumor microenvironment, but no deep coverage phosphoproteome of human exosomes has been reported to date, which makes the exosome a potential source for the new phosphosite discovery. In this study, we performed highly optimized MS analyses on the exosomal and cellular proteins isolated from human colorectal cancer SW620 cells. With stringent data quality control, 313 phosphoproteins with 1091 phosphosites were confidently identified from the SW620 exosome, from which 202 new phosphosites were detected. Exosomal phosphoproteins were significantly enriched in the 11q12.1-13.5 region of chromosome 11 and had a remarkably high level of tyrosine-phosphorylated proteins (6.4%), which were functionally relevant to ephrin signaling pathway-directed cytoskeleton remodeling. In conclusion, we here report the first high-coverage phosphoproteome of human cell-secreted exosomes, which leads to the identification of new phosphosites for C-HPP. Our findings provide insights into the exosomal phosphoprotein systems that help to understand the signaling language being delivered by exosomes in cell-cell communications. The mass spectrometry proteomics data have been deposited to the ProteomeXchange consortium with the data set identifier PXD004079, and iProX database (accession number: IPX00076800).

Chemopreventive Activity of Naphthoquinones from Alkanna tinctoria (L.) Tausch in Human Colorectal Cancer Cells

Botanically derived natural products have recently become an attractive source of new chemotherapeutic agents. To explore active anti-colorectal cancer compounds, we performed phytochemical studies on Alkanna tinctoria and isolated eight quinone compounds. Using spectroscopical and physicochemical methods including high-resolution mass spectrometry and NMR spectra, compounds 1, 2, 3, 5 and 6 were identified as alkannin, acetylalkannin, angelylalkannin, dimethylacryl alkannin and arnebifuranone, respectively. Novel compounds 4, 7, and 8 are named as 5-methoxyangenylalkannin, alkanfuranol and alkandiol, respectively, and structurally elucidated based on extensive spectroscopic evidence. The anti-proliferative effects of these eight compounds on HCT-116 and SW-480 human colorectal cancer cells were determined by the MTS method. Cell cycle and apoptosis were determined using flow cytometry. Enzymatic activities of caspases were determined by colorimetric assay, and interactions of compound 4 and caspase 9 were explored by docking analysis. Among the eight compounds, alkannin (1), angelylalkannin (3), and 5-methoxyangenylalkannin (4) showed strong anti-proliferative effects, while compound 4 showed the most potent effects. Compound 4 arrested cancer cells in the S and G2/M phases, and significantly induced cell apoptosis. The apoptotic effects of compound 4 were supported by caspase assay and docking analysis.

Physcion inhibits the metastatic potential of human colorectal cancer SW620 cells in vitro by suppressing the transcription factor SOX2.

Physcion, an anthraquinone derivative, exhibits hepatoprotective, anti-inflammatory, anti-microbial and anti-cancer activities. In this study we examined whether and how physcion inhibited metastatic potential of human colorectal cancer cells in vitro. Human colorectal cancer cell line SW620 was tested. Cell migration and invasion were assessed using a wound healing and Transwell assay, respectively. The expression levels of transcription factor SOX2 in the cells were modulated with shRNA targeting SOX2 and SOX2 overexpressing plasmid. The expression of target molecules involved in epithelial-mesenchymal transition (EMT) process and the signaling pathways was determined with Western blots or qRT-PCR. ROS levels were measured using DCF-DA. Physcion (2.5, 5 mol/L) did not affect the cell viability, but dose-dependently inhibited the cell adhesion, migration and invasion. Physcion also inhibited the EMT process in the cells, as evidenced by the increased epithelial marker E-cadherin expression, and by decreased expression of mesenchymal markers N-cadherin, vimentin, fibronectin and α-SMA, as well as transcriptional repressors Snail, Slug and Twist. Physcion suppressed the expression of SOX2, whereas overexpression of SOX2 abrogated the inhibition of physcion on metastatic behaviors. Physcion markedly increased ROS production and phosphorylation of AMPK and GSK3β in the cells, whereas the AMPK inhibitor compound C or the ROS inhibitor NAC abolished the inhibition of physcion on metastatic behaviors. Physcion inhibits the metastatic potential of human colorectal cancer cells in vitro via activating ROS/AMPK/GSK3β signaling pathways and suppressing SOX2.

Antitumor Activity of Total Flavonoids from Daphne genkwa in Colorectal Cancer.

Daphne genkwa Sieb.et Zucc. is a well-known medicinal plant. This study was designed to investigate the anticancer effects of total flavonoids in D. genkwa (TFDG) in vitro and in vivo. HT-29 and SW-480 human colorectal cancer cells were cultured to investigate the anticancer activity of TFDG. In addition, the Apc(Min/+) mouse model was applied in the in vivo experiment. Results of the cell experiment revealed that TFDG possessed significant inhibitory effects on HT-29 and SW-480 human colorectal cancer cells (both p < 0.01). Furthermore, our in vivo data showed that after treatment with TFDG, there was a significant increase in life span (both p < 0.01) and tumor numbers were reduced in the colon (both p < 0.01), which was supported by the data of tumor distribution, body weight changes and organ index. Our results also indicated that expressions of interleukin (IL)-1α, IL-1β, IL-6, granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor in gut tissue were downregulated by treatments of TFDG, and immunity cytokine secretions in the serum were regulated after oral administration of TFDG. Taken together, these findings suggested that TFDG has a potential clinical utility in colorectal cancer therapeutics, and TFDG's action is likely linked to its ability to regulate immune function and inhibit the production of inflammatory cytokines.

Insulin-Like Growth Factor-2 Is Induced Following 5-Aminolevulinic Acid-Mediated Photodynamic Therapy in SW620 Human Colon Cancer Cell Line.

The IGF system is a family of polypeptide growth factors, which plays a significant role in the development and growth of many cells. Dysregulation of insulin-like growth factors and their pathway components has been connected with essential tumor properties, such as tumor cell proliferation, antiapoptotic properties, invasive behavior and chemotherapy resistance. However, the effects of photodynamic therapy (PDT), one of the cancer treatment methods for the regulation of the IGF signaling pathway, are still unclear. The aim of this study was to investigate the expression of IGF-2 after 5-aminolevulinic acid (5-ALA)-mediated-PDT in SW620 human colorectal cancer cells with evaluation of cell proliferation and apoptosis and to determine the effects of PDT on the IGF-2 receptor (IGF-2R), IGF-2 binding protein-1 (IGF-2BP-1) and the proapoptotic protein, BAX. Cells were treated with 5-aminolevulinic acid and its methyl ester. Changes of the expression and concentration of IGF-2 before and after treatment were assayed by immunocytochemistry, Western blot and ELISA. We found that IGF-2 was significantly overexpressed in the SW620 cell line, while its receptor and binding protein-1 were not significantly changed. Within this study, we would like to suggest that IGF-2 contributes to the effects of PDT and that its expression will influence post-PDT efficacy.

Antitumor and immunomodulatory activity of genkwanin on colorectal cancer in the APCMin/+ mice

Colorectal cancer is the third most common malignant tumor with high morbidity and mortality. To evaluate the antitumor effect of genkwanin on colorectal cancer enhanced by western high-fat diet, we investigated the activity of genkwanin on HT-29 and SW-480 human colorectal cancer lines in vitro and on the APCMin/+ mice in vivo. In a cell culture system, six different inflammatory cytokines obviously stimulated two cancer cells growth in a concentration-dependent manner, while genkwanin significantly inhibited HT-29 and SW-480 human colorectal cancer cells proliferation and inflammatory cytokine IL-8 secretion. In the APCMin/+ mice, the body weights, spleen and thymus indexes and immunity cytokine secretions were significantly improved after oral administration 12.5 and 25mg/kg/day of genkwanin. Besides, the tumor multiplicity changes and inflammatory cytokine levels were markedly reduced in two genkwanin-treated groups. The dysplastic adenomatous changes were also obviously ameliorated in gut histopathology. Taken together, our results indicated that genkwanin had a better antitumor activity partly via enhancing host immunity and decreasing the inflammatory cytokine levels. Genkwanin may be an effective chemotherapeutic agent for the treatment of colorectal cancer.

Targeted inhibition of p38MAPK-enhanced autophagy in SW620 cells resistant to photodynamic therapy-induced apoptosis

Photodynamic therapy (PDT) is a promising and noninvasive treatment that can induce apoptosis, autophagy, or both depending on the cell phenotype. In this work, chlorin e6 (Ce6) was used to photosensitize human colorectal cancer SW620 cells. In cells, apparent autophagy and apoptosis with dependence on intracellular reactive oxygen species (ROS) generation were detected. p38MAPK activation followed by ROS generation might be a core component in Ce6 mediate PDT (Ce6-PDT)-induced autophagy and apoptosis signaling pathway. By using p38MAPK siRNA, the results showed a marked enhancement on cell apoptosis in Ce6-PDT with increased annexin (+) apoptotic cells, nuclear condensation, caspase-3, and PARP cleavage. Besides, impairment of p38MAPK also promoted the autophagic response to photodamage as indicated by conversion of LC3 and monodansyl cadaverine (MDC) labeling patterns. It appears that Ce6-PDT induced ROS production involving activation of p38MAPK, probably to prevent SW620 cells from photodamage. Moreover, autophagy inhibitor 3-methyladenine/bafilomycin A1 greatly aggravated Ce6-PDT-induced apoptosis in SW620 cells with knockdown of p38MAPK. Taken together, this study suggests that autophagy could represent a promising field in cancer treatment and p38MAPK may be a potential therapeutic target to enhance the efficacy on clinical evaluation for the treatment of colorectal cancer.

Apple Polysaccharide Reduces Nf-Kb Mediated Colitis-Associated Colon Carcinogenesis

Nuclear factor-kappa B (NF-κB) is an important molecule in mediating inflammatory colitis, which can lead to colorectal cancer (CRC). The aim of this study was to evaluate the chemopreventive efficacy of apple polysaccharide extract (AP) in inhibiting NF-κB-mediated inflammation pathways in CRC. We evaluated AP in vitro in HT-29 and SW620 human CRC cells. We also used the azoxymethane and dextran sodium sulphate (AOM/DSS) model to induce colon carcinogenesis in vivo. The chemoprotective effects of AP were assessed using Western blot, immunofluorescence assay, real-time PCR, electrophoretic mobility shift assay, and flow cytometry. AP reduced AOM/DSS-associated toxicities, prevented carcinogenesis, and decreased the expression of TLR4, MD2, MyD88, TRAM, TRIF-related adapter molecule, interferon-β, tumor necrosis factor-α, and interleukin-6. The protective effects of AP may be related to the inhibition of TLR4/MD2-mediated signaling, including MyD88 and TRIF, as well as the inhibition of NF-κB-mediated inflammatory signaling pathways. Therefore, AP could be used in combination therapy for the prevention of colitis-associated colon cancer.

Secretion of small/microRNAs including miR-638 into extracellular spaces by sphingomyelin phosphodiesterase 3.

A recent study demonstrated that intracellular small/microRNAs are released from cells, and some of these extracellular RNAs are embedded in vesicles, such as ceramide-rich exosomes, on lipid-bilayer membranes. In the present study, we examined the effects of sphingomyelin phosphodiesterase 3 (SMPD3), which generates ceramide from sphingomyelin, on the release of small/microRNAs from intracellular to extracellular spaces. In these experiments, SW480 human colorectal and HuH-7 human hepatocellular cancer cells were cultured for 48 h in serum-free media. Culture supernatants were then collected, and floating cells and debris were removed by centrifugation and filtration through a 0.22-μm filter. Extracellular small RNAs in purified culture supernatants were stable for 4 weeks at room temperature, after 20 freeze-thaw cycles and exposure to pH 2.0, and were resistant to ribonuclease A degradation. Amino acid sequence analyses of SMPD3 showed high homology between mammals, indicating evolutionary conservation. Therefore, to investigate the mechanisms of cellular small/microRNA export, SW480 and HuH-7 cells were treated with the SMPD3 inhibitor GW4869 in serum-free media. Culture supernatants were collected for microarray and/or reverse transcription quantitative polymerase chain reaction (RT-qPCR) experiments. The number of microRNAs in culture supernatants was decreased following treatment with GW4869. Among these, extracellular and intracellular miR-638 were dose-dependently decreased and increased, respectively. These data suggest that SMPD3 plays an important role in the release of microRNAs into extracellular spaces.

Abstract 3798: Shift of Cx43 isoform expression by PQ1 in SW480 human colorectal cancer cells

Abstract Colorectal cancer is one of the most common cancers in men and women in the United States. On average, the risk of developing colorectal cancer is about 1 in 20 over an individuals' lifetime. Cancer cells exhibit many deficiencies in cell-to-cell communication, particularly gap junctional intercellular communication (GJIC). GJIC has been reported to diminish as cancer cells progress. Gap junctions are intercellular channels composed of connexin proteins, which mediate the direct passage of small molecules from one cell to the next. They are involved in the regulation of cell cycle, cell differentiation, and cell signaling. Previously, a class of substituted quinolines, known as PQs, demonstrated to increase GJIC in cancer cells and subsequently attenuated tumor growth. The goal of this study is to determine whether the mode of PQ1 is by directly regulating the connexin protein. The results show that the low expression of connexin 43 (Cx43) in SW480 human colorectal cancer cells remains constant in the presence of PQ1. However, PQ1 caused a change in isoform expression of Cx43 in which there was a shift of Cx43 expression from P0 to P1 and P2 isoforms. This suggests that the trafficking of Cx43 from the Golgi to the membrane occurs in the presence of PQ1. Furthermore, the results show that Cx43 interacts with PKCα in SW480 cells; however, PQ1 does not affect the interaction of Cx43 and PKCα, suggesting that the shift in Cx43 isoforms is not due to PKCα phosphorylation. These findings provide in part that the increase of GJIC by PQ1 is due to the shift in Cx43 from P0 to P1 and P2 isoforms in SW480 colorectal cancer cells. Citation Format: Kristina M. Bigelow, Thu Annelise Nguyen. Shift of Cx43 isoform expression by PQ1 in SW480 human colorectal cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3798. doi:10.1158/1538-7445.AM2014-3798

Activation of JNK/p38 pathway is responsible for α-methyl-n-butylshikonin induced mitochondria-dependent apoptosis in SW620 human colorectal cancer cells.

α-Methyl-n-butylshikonin (MBS), one of the active components in the root extracts of Lithospermum erythrorhizon, posses antitumor activity. In this study, we assess the molecular mechanisms of MBS in causing apoptosis of SW620 cells. MBS reduced the cell viability of SW620 cells in a dose-and time-dependent manner and induced cell apoptosis. Treatment of SW620 cells with MBS down-regulated the expression of Bcl-2 and up-regulated the expression of Bak and caused the loss of mitochondrial membrane potential. Additionally, MBS treatment led to activation of caspase-9, caspase-8 and caspase-3, and cleavage of PARP, which was abolished by pretreatment with the pan-caspase inhibitor Z-VAD-FMK. MBS also induced significant elevation in the phosphorylation of JNK and p38. Pretreatment of SW620 cells with specific inhibitors of JNK (SP600125) and p38 (SB203580) abrogated MBS-induced apoptosis. Our results demonstrated that MBS inhibited growth of colorectal cancer SW620 cells by inducing JNK and p38 signaling pathway, and provided a clue for preclinical and clinical evaluation of MBS for colorectal cancer therapy.