Colo205 Cells Research Articles

Design and synthesis of N-(4-aminopyridin-2-yl)amides as B-RafV600E inhibitors

B-RafV600E was an effective target for the treatment of human cancers. Based on a pan-Raf inhibitor TAK-632, a series of N-(4-aminopyridin-2-yl)amide derivatives were designed as novel B-RafV600E inhibitors. Detailed structure–activity studies of the compounds revealed that most of the compounds displayed potent enzymatic activity against B-RafV600E, and good selectivity over B-RafWT. One of the most promising compound 4l exhibited potent inhibitory activity with an IC50 value of 38nM for B-rafV600E, and displayed antiproliferative activities against colo205 and HT29 cells with IC50 values of 0.136 and 0.094μM, respectively. It also displayed good selectivity on both enzymatic and cellular assays over B-RafWT. These inhibitors may serve as lead compounds for further developing novel B-RafV600E inhibitors as anticancer drugs.

Upregulation of the Multidrug Resistance P‐glycoprotein ABCB1 by Transcription Factor Pituitary Homeobox 2 (Pitx2) in Human Colon and Kidney Cancers

BackgroundPituitary homeobox 2 (Pitx2) is a master regulator of organ asymmetry during development but is reactivated in neoplastic cells, contributing to cell proliferation, tumor cell invasion and cancer progression. Upregulation of the multidrug resistance P‐glycoprotein ABCB1 confers chemotherapeutic drug resistance to tumor cells, hampering effective treatment. We have previously shown that Pitx2 is upregulated in a multidrug resistant (MDR) kidney cancer cell line and serves as a positive transcriptional regulator of ABCB1 thereby increasing resistance to the anthracycline chemotherapeutic drug, doxorubicin (DOX) (Lee, W.‐K. et al. Int J Cancer 113, 556–67, 2013). The objective of this study was to determine if ABCB1 regulation by Pitx2 is also in engaged by other cancer types that exhibit intrinsic MDR.MethodsExpression of Pitx2 and ABCB1 was determined by immunofluorescence microscopy, immunoblotting and immunohistochemistry. Cell viability was assessed by MTT and clonogenic survival assays. Pitx2 transcriptional activity was measured by luciferase reporter gene assay. Isolation of nuclei was performed by Tenbroeck homogenization in hypoosmotic buffer and a discontinuous sucrose gradient.ResultsIn human colorectal adenocarcinoma Colo320DM cells, which exhibit MDR and harbor augmented ABCB1 expression, nuclear Pitx2 expression and Pitx2 reporter gene activity were significantly increased compared to less drug resistant Colo205 cells. The ABCB1 inhibitor PSC833 (0.1 μM) reversed drug resistance to 1 μM DOX after 24 h in Colo320DM cells. Further, knockdown of Pitx2 by RNAi in Colo320DM cells resulted in decreased ABCB1 protein by almost 3‐fold and diminished resistance to DOX resulting in attenuated cell survival. Moreover, the Pitx2 isoforms that are involved in regulation of ABCB1 in colon and kidney cancer cells were investigated. Both Pitx2A and Pitx2B were found to be present in the nuclear fraction of Colo320DM and MDR A498 kidney carcinoma cells. Analysis of human kidney and colon cancer tissue arrays by co‐immunostaining suggests a causal relationship between nuclear Pitx2 and ABCB1 expression.ConclusionPitx2 is a transcriptional regulator of ABCB1 in cancers of the colon and kidney suggesting a contributory role to the MDR phenotype.Support or Funding InformationFunded by an Internal Research Grant (UW/H) and ZBAF

Effects of curcumin metabolites on human colon adenocarcinoma cells

Stepwise cancer development involves metastatic migration as well as uncontrolled cell growth. Cancer cells undergo migration and invasion which allow them to translocate within tissues. Tetrahydrocurcumin (THC), a major metabolite of curcumin, exhibits anti‐carcinogenesis and antioxidation in vivo. In the present study, we found that THC and two other metabolites of curcumin, tetrahydrodemethoxycurcumin (THDC) and tetrahydrobisdemethoxycurcumin (THBC), could reduce the survival ratio of human colon adenocarcinoma cells, COLO205, HCT116, and HT29, in a dose‐dependent manner. The results also indicated that the fewer the methoxy groups of the curcumim metabolites, the more the inhibitory effects. However, curcumin metabolites did not trigger apoptotic cell death in COLO205 cells significantly, whereas curcumin did. Our data also strongly suggest a role of THBC in inhibiting cellular migration in HCT116 cells. Taken together, these results demonstrated the anticancer efficacy of curcumin metabolites which provides a potential application basis for the prevention of human colon cancer.Support or Funding InformationThis study was supported by NSC102‐2320‐B‐309‐001‐MY3 to M‐F Lee and NSC100‐2313‐B‐309‐003 to A‐C Cheng.

Triazole tethered C5-curcuminoid-coumarin based molecular hybrids as novel antitubulin agents: Design, synthesis, biological investigation and docking studies

Keeping in view the confines allied with presently accessible antitumor agents and success of C5-curcuminoid based bifunctional hybrids as novel antitubulin agnets, molecular hybrids of C5-curcuminoid and coumarin tethered by triazole ring have been synthesized and investigated for in-vitro cytotoxicity against THP-1, COLO-205, HCT-116 and PC-3 human tumor cell lines. The results revealed that the compounds A-2 to A-9, B-2, B-3, B-7 showed significant cytotoxic potential against THP-1, COLO-205 and HCT-116 cell lines, while the PC-3 cell line among these was found to be almost resistant. Structure activity relationship revealed that the nature of Ring X and the length of carbon-bridge (n) connecting triazole ring with coumarin moiety considerably influence the activity. Methoxy substituted phenyl ring as Ring X and two carbon-bridges were found to be the ideal structural features. The most potent compounds (A-2, A-3 and A-7) were further tested for tubulin polymerization inhibition. Compound A-2 was found to significantly inhibit the tubulin polymerization (IC50 = 0.82 μM in THP-1 tumor cells). The significant cytotoxicity and tubulin polymerization inhibition by A-2 was further rationalized by docking studies where it was docked at the curcumin binding site of tubulin.

The anti-oxidative transcription factor Nuclear factor E2 related factor-2 (Nrf2) counteracts TGF-β1 mediated growth inhibition of pancreatic ductal epithelial cells -Nrf2 as determinant of pro-tumorigenic functions of TGF-β1.

BackgroundNuclear factor E2 related factor-2 (Nrf2) is an oxidative stress inducible transcription factor being essential in regulating cell homeostasis. Thus, acute induction of Nrf2 in epithelial cells exposed to inflammation confers protection from oxidative cell damage and mutagenesis supporting an anti-tumorigenic role for Nrf2. However, pancreatic ductal adenocarcinoma (PDAC) is characterized by persistent Nrf2 activity conferring therapy resistance which points to a pro-tumorigenic role of Nrf2. A similar dichotomous role in tumorigenesis is described for the Transforming Growth Factor-beta 1 (TGF-β1). The present study therefore aimed at elucidating whether the switch of Nrf2 function towards a tumor promoting one relates to the modulation of TGF-β1 induced cell responses and whether this might occur early in PDAC development.MethodsIn situ analysis comprised immunohistochemical stainings of activated (phosphorylated) Nrf2 and Ki67 in pancreatic tissues containing normal ducts and pancreatic intraepithelial neoplasia (PanINs). In vitro, Nrf2 levels in benign (H6c7-pBp), premalignant (H6c7-kras) and malignant (Colo357) pancreatic ductal epithelial cells were modulated by Nrf2 specific siRNA or Nrf2 overexpression. Then, the effect of Nrf2 alone and in combination with TGF-β1 on cell growth and survival was investigated by cell counting, Ki67 staining and apoptosis assays. The underlying cell signaling was investigated by western blotting. Statistical analysis was performed by Shapiro-Wilk test for normal distribution. Parametric data were analyzed by one-way ANOVA, while non-parametric data were analyzed by Kruskal-Wallis one-way ANOVA on ranks.ResultsSignificantly elevated expression of activated Nrf2 and Ki67 could be detected in PanINs but not in normal pancreatic ductal epithelium. While the effect of Nrf2 on basal cell growth of H6c7-pBp, H6c7-kras and Colo357 cells was minor, it clearly attenuated the growth inhibiting effects of TGF-β1 in all cell lines. This enhanced Nrf2-mediated cell survival was predominantly based on an enhanced proliferative activity. Accordingly, expression of p21 expression along with expression of phospho-p38 and phospho-Smad3 was diminished whereas Erk-phosphorylation was enhanced under these conditions.ConclusionsOverall, our data demonstrate that Nrf2 being elevated in early precursor lesions counteracts the growth inhibiting function of TGF-β1 already in benign and premalignant pancreatic ductal epithelial cells. This could represent one fundamental mechanism underlying the functional switch of both- TGF-β1 and Nrf2 – which may manifest already in early stages of PDAC development.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2191-7) contains supplementary material, which is available to authorized users.

In vitro antiproliferative activity of 2,3-dihydroxy-9,10-anthraquinone induced apoptosis against COLO320 cells through cytochrome c release caspase mediated pathway with PI3K/AKT and COX-2 inhibition

The present study investigated the anticancer activity of 2,3-dihydroxy-9,10-anthraquinone against different cancer cells such as MCF-7, COLO320, HepG-2, Skov-3, MOLM-14, NB-4, CEM, K562, Jurkat, HL-60, U937, IM-9 and Vero. 2,3-dihydroxy-9,10-anthraquinone showed good antiproliferative activity against COLO320 cells when compared to other tested cells. The cytotoxicity results showed 79.8% activity at the dose of 2.07 μM with IC50 value of 0.13 μM at 24 h in COLO320 cells. So we chose COLO320 cells for further anticancer studies. mRNA expression was confirmed by qPCR analysis using SYBR green method. Treatment with 2,3-dihydroxy-9,10-anthraquinone was found to trigger intrinsic apoptotic pathway as indicated by down regulation of Bcl-2, Bcl-xl; up regulation of Bim, Bax, Bad; release of cytochrome c and pro-caspases cleaving to caspases. Furthermore, 2,3-dihydroxy-9,10-anthraquinone stopped at G0/G1 phase with modulation in protein levels of cyclins. On the other hand PI3K/AKT signaling plays an important role in cell metabolism. We found that 2,3-dihydroxy-9,10-anthraquinone inhibits PI3K/AKT activity after treatment. Also, COX-2 enzyme plays a major role in colorectal cancer. Our results showed that the treatment significantly reduced COX-2 enzyme in COLO320 cells. These results indicated antiproliferative activity of 2,3-dihydroxy-9,10-anthraquinone involving apoptotic pathways, mitochondrial functions, cell cycle checkpoint and controlling the over expression genes during the colorectal cancer. Molecular docking studies showed that the compound bound stably to the active sites of Bcl-2, COX-2, PI3K and AKT. This is the first report of anticancer mechanism involving 2,3-dihydroxy-9,10-anthraquinone in COLO320 cells. The present results might provide helpful suggestions for the design of antitumor drugs toward colorectal cancer treatment.

Supporting Data for Multifunctional all-in-one drug delivery systems for tumor targeting and sequential release of three different anti-tumor drugs.

Although nanoparticulate drug delivery systems (NDDSs) can preferentially accumulate in tumors, active targeting by targeting ligands (e.g. monoclonal antibody) is necessary for increasing its targeting efficacy in vivo. We conjugated mAb198.3 on the SiO2@AuNP system surface to make it obtain active targeting efficacy. The FAT1 targeting capability of SiO2@AuNP system is the first issue to be solved. Thus, flow cytometry analysis was attempted to demonstrate that the SiO2@AuNP system could bind to native FAT1 molecules on the surface of Colo205 cells. Also, together with the drug release behavior study of self-decomposable SiO2 NPs, the continuous morphological evolution needed to be clarified. Therefore, to characterize the morphological evolution in vitro, we analyzed the morphology of inner self-decomposable NPs in different time intervals using transmission electron microscopy (TEM). A more comprehensive analysis of this data may be obtained from the article “Multifunctional all-in-one drug delivery systems for tumor targeting and sequential release of three different anti-tumor drugs” in Biomaterials.

Early Prediction of Tumor Response to Treatment: Preclinical Validation of 99mTc-Duramycin.

Noninvasive imaging of cell death can provide an early indication of the efficacy of tumor treatment, aiding clinicians in distinguishing responding patients from nonresponding patients early on. (99m)Tc-duramycin is a SPECT tracer for cell death imaging. In this study, our aim was to validate the use of (99m)Tc-duramycin for imaging the early response of tumors to treatment. An in vitro binding assay was performed on COLO205 cells treated with 5-fluorouracil (3.1, 31, or 310 μM) and oxaliplatin (0.7 or 7 μM) or radiation (2 or 4.5 Gy). (99m)Tc-duramycin cell binding and the levels of cell death were evaluated after treatment. In vivo imaging was performed on 4 groups of CD1-deficient mice bearing COLO205 human colorectal cancer tumors. Each group included 6 tumors. The first group was given irinotecan (100 mg/kg), the second oxaliplatin (5 mg/kg), the third irinotecan (80 mg/kg) plus oxaliplatin (5 mg/kg), and the fourth vehicle (0.9% NaCl and 5% glucose). For radiotherapy studies, COLO205 tumors received 4.5 Gy, 2 fractions of 4.5 Gy in a 24-h interval, pretreatment with an 80 mg/kg dose of irinotecan combined with 2 fractions of 4.5 Gy in a 24-h interval, or no treatment (n = 5-6/group). Therapy response was evaluated by (99m)Tc-duramycin SPECT 24 h after the last dose of therapy. Blocking was used to confirm tracer specificity. Radiotracer uptake in the tumors was validated ex vivo using γ-counting, cleaved caspase-3, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) histology. Chemotherapy and radiotherapy increased (99m)Tc-duramycin binding to COLO205 cells in a concentration/dose- and time-dependent manner, which correlated well with cell death levels (P < 0.05) as analyzed by annexin V and caspase 3/7 activity. In vivo, (99m)Tc-duramycin uptake in COLO205 xenografts was increased 2.3- and 2.8-fold (P < 0.001) in mice treated with irinotecan and combination therapy, respectively. Blocking with unlabeled duramycin demonstrated specific binding of the radiotracer. After tumor irradiation with 4.5 Gy, (99m)Tc-duramycin uptake in tumors increased significantly (1.24 ± 0.07 vs. 0.57 ± 0.08 percentage injected dose per gram in the unirradiated tumors; P < 0.001). γ-counting of radioactivity in the tumors positively correlated with cleaved caspase-3 (r = 0.85, P < 0.001) and TUNEL (r = 0.81, P < 0.001) staining. We demonstrated that (99m)Tc-duramycin can be used to image induction of cell death early after chemotherapy and radiotherapy. It holds potential to be translated into clinical use for early assessment of treatment response.

Isolation and characterization of 2-hydroxy-9,10-anthraquinone from Streptomyces olivochromogenes (ERINLG-261) with antimicrobial and antiproliferative properties

Currently Streptomyces is one of the most important antibiotic producing microorganisms against several diseases. In the present study Streptomyces olivochromogenes ERINLG-261 was isolated from the soil samples of the Mudumalai hills, Western Ghats, India. Morphological, physiological, biochemical and 16S rRNA studies strongly suggested that this isolate belonged to the genus Streptomyces. ERINLG-261 showed good antimicrobial activity against different bacteria and fungi in Micromonospora fermentation medium. The active ethyl acetate extract was packed in column chromatography over silica gel which led to the isolation of 2-hydroxy-9,10-anthraquinone as the active principle. The isolated compound showed good antimicrobial activity against tested bacteria and fungi in minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) studies. The compound showed moderate in vitro antiproliferative activity against A549 and COLO320 cells. The compound was subjected to molecular docking studies for the inhibition of Topoisomerase, TtgR and Beta-lactamase enzymes which are targets for antimicrobials. Docking results of the compound showed low docking energy with these enzymes indicating its usefulness as antimicrobial agent. This is the first report of antimicrobial and antiproliferative activity of 2-hydroxy-9,10-anthraquinone isolated from Streptomyces olivochromogenes along with molecular docking studies.

Spatane diterpinoid from the brown algae, Stoechospermum marginatum induces apoptosis via ROS induced mitochondrial mediated caspase dependent pathway in murine B16F10 melanoma cells.

Spatane diterpinoids isolated from the brown marine algae Stoechospermum marginatum were known to have cytotoxic effects in human cancerous cell lines and murine melanoma cells; the underling apoptotic mechanism of diterpinoids still remains unclear so far. Thus, in the present study, the apoptotic mechanism of a spatane diterpinoid, 5(R), 19-diacetoxy-15,18(R and S), dihydro spata-13, 16(E)-diene (DDSD) was investigated mainly in B16F10 melanoma cells because they were most susceptible to DDSD than THP1, U937, COLO205, and HL60 cells. The treatment of B6F10 cells with DDSD resulted in morphological alterations, nuclear condensation, and DNA fragmentation, which leads to cell growth inhibition in a concentration-dependent manner. Data indicate that DDSD induced the generation of ROS, consequentially caused alteration in Bax/Bcl-2 ratio that disrupted the inner mitochondrial transmembrane potential (ΔΨm) resulting in cytochrome c redistribution to the cytoplasm and activation of caspase-mediated apoptotic pathway. Flow cytometric analysis clearly indicated that the DDSD inducing phosphatidylserine externalization and mediated "S-phase" arrest in cell cycle. In addition, results also found that DDSD induced apoptosis through deregulating PI3K/AKT signaling pathway. The anti-tumor activity of DDSD was evaluated in C57BL/6 mice bearing B16F10 melanoma. It effectively inhibited tumor growth (volume and weight) in a dose dependent manner, yet without apparent toxic effects. Morphology and apoptotic status of tumor tissues in the treated mice were assessed by microscopy and TUNEL assay, respectively. Our study shows a therapeutic potential of DDSD for the treatment of malignant melanoma and a new source of anticancer drugs. © 2016 Wiley Periodicals, Inc.

Tanshinone IIA enhances chemosensitivity of colon cancer cells by suppressing nuclear factor-κB.

The aim of the present study was to investigate the effect and molecular mechanism of tanshinone IIA (TSA) on colon cancer cells. Cell viability was determined using Cell Counting kit-8 assay and the results demonstrated that TSA treatment significantly decreased the cell viability of HCT1116 and COLO205 cells in a dose-dependent manner. TSA treatment also sensitized HCT1116 and COLO205 cells to fluorouracil therapy in a concentration-dependent manner. Western blotting was performed in order to investigate the molecular mechanisms of TSA action and determine the level of phosporylated p65 and nuclear factor-κB (NF-κB)-regulated genes, including vascular endothelial growth factor (VEGF), c-Myc, cyclooxygenase-2 (COX-2) and B-cell lymphoma-2 (Bcl-2). The results revealed that TSA treatment greatly decreased the level of phosphorylated p65 in the nucleus, which indicated the inhibition of NF-κB activation by TSA treatment. TSA also decreased the expression levels of VEGF, c-Myc, COX-2 and Bcl-2. Furthermore, the inhibition of NF-κB activation with the specific inhibitor, pyrrolidine dithiocarbamate, increased the induction of cell death and chemosensitization effect of TSA in colon cancer cells. In conclusion, these results suggest that TSA induces cell death and chemosensitizes colon cancer cells through the suppression of NF-κB signaling.

HsDNA groove binding, photocatalytic activity, and in vitro breast and colon cancer cell reducing function of greener SeNPs.

Selenium nanoparticles (SeNPs) have attracted great attention because of their superior optical properties and wide utilization in biological and biomedical studies. This paper reports an environmentally benign procedure of greener monodispersible SeNP synthesis using the reducing power of Trigonella foenum-graecum extract, characterization and their protective effect against unfolded (Herring sperm DNA) hsDNA. We investigated the anti-cancer activity of SeNPs against MCF-7, MDA MB 435 and COLO-205 cells. The photocatalytic activity of SeNPs was investigated for the degradation of a Sunset Yellow FCF (SYFCF) dye using ultraviolet-B light. The reduction of the Se ion to SeNPs was monitored by ultraviolet-visible spectroscopy (UV-vis). The size and morphology of the SeNPs were characterized by high resolution transmission electron microscopy (HRTEM), X-ray diffraction (XRD), and Dynamic Light Scattering (DLS). The SeNPs were stable, and the diameter was homogeneous at around 5-12 nm. Interactions of various concentrations of SeNPs with hsDNA were systematically investigated by UV-vis, fluorescence, circular dichroism (CD), polarimetry and FTIR spectroscopy under physiological conditions. The results from fluorescence spectroscopy indicated that SeNPs quenched the fluorescence intensity of hsDNA with increasing concentrations. The modified Stern-Volmer quenching rate constant Ksv, binding constant K and binding sites n at different temperatures and the corresponding thermodynamic parameters ΔH°, ΔG° and ΔS° were calculated. Hoechst 33258 and methyl green (MG) site markers, melting experiment (Tm), viscosity measurements and sequence specificity verification by DNA bases clarified that SeNPs bind to hsDNA via a groove site. The rate of photocatalytic degradation of the SYFCF dye in the presence and absence of photocatalysts (SeNPs) was studied using UV-vis, the results showed appreciable degradation of the SYFCF dye. Our results suggested that nano Se can be used as a promising selenium species with potential application in cancer treatment. These nanoparticles were found to be the most active cytotoxic agent prepared in a new green synthesis manner, causing >50% inhibition of MCF-7, MDA MB-435 and COLO-205 cell proliferation at concentrations <10(-7) M. Hence these SeNPs could be recognized as promising materials for biomedical applications.

Acetoxyroyleanone exhibits selective anti-cancer effects and induces apoptosis in human colon carcinoma cells through the mediation of NF-κB and caspase-3 signalling pathways

&lt;p&gt;The objective of the current study was to evaluate the antiproliferative and apoptotic activities of acetoxyroyleanone against various cancer cells along with studying its effect on chromatin condensation, NF-κB and caspase-3 expressions and cell migration. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay was used to evaluate cell viability while as fluorescence microscopy revealed the effects of acetoxyroyleanone on cellular morphology of Colo-205 cells. Western blotting revealed effects on NF-κB and caspase-3 expressions. The results revealed that acetoxyroyleanone induced potent and dose-dependent antiproliferative effects against a range of cancer cell lines with Colo-205 being the most susceptible cell line. However, it required six to eight times higher concentration of acetoxyroyleanone to induce 50% cell death in normal epithelial (fR-2) cell line. Further, following acetoxyroyleanone treatment to Colo-205, it was observed that acetoxy-royleanone induced substantial down-regulation of NF-κB and up-regulation of caspase-3 expressions. In addition, acetoxyroyleanone impairs cell migration, chromatin condensation, cell shrinkage and membrane blebbing.&lt;/p&gt;&lt;p&gt; &lt;/p&gt;

Interleukin-6 increases expression of serine protease inhibitor Kazal type 1 through STAT3 in colorectal adenocarcinoma.

Inflammation promotes colorectal cancer (CRC) tumorigenesis, but the underlying molecular mechanisms are still being uncovered. Proinflammatory cytokine interleukin-6 (IL-6) stimulates survival signaling in CRC; inflammatory signals also regulate production and activity of proteases and their inhibitors. Over-expression of serine protease inhibitor Kazal type 1 (SPINK1) predicts an unfavorable outcome in colon cancer. The SPINK1 gene contains an IL-6 responsive element, suggesting it could act as an acute phase reactant. We assessed the connection between IL-6 and SPINK1, and the function and mechanism of this signaling. Our results show that Colo205 and HT-29 cells express and secrete SPINK1, and both fibroblast-derived and recombinant IL-6 further increased the SPINK1 levels. Concurrently CRC cells augmented the IL-6 production in fibroblasts. In CRC tissues cancer cells were positive for SPINK1, whereas IL-6 was found in stromal cells. In Colo205 cells IL-6 also stimulated the secretion of trypsin-1 and -2, the key targets of SPINK1 protease inhibition, whereas in HT-29 cells trypsin-1 and -2 levels remained constantly low. Functionally, both IL-6 and SPINK1 increased the motility of the CRC cells. Mechanistically, IL-6 activated the canonical STAT3 pathway and inhibition of STAT3 phosphorylation decreased the levels of SPINK1, trypsin-1 and -2. Taken together, our results indicate a novel link between inflammatory signals originating from the tumor microenvironment and increased SPINK1 levels. This finding has potential therapeutic implications for targeted therapy, as it confirms that SPINK1 acts as an acute phase reactant and that it participates in the paracrine crosstalk with the tumor microenvironment of colon cancer. © 2015 Wiley Periodicals, Inc.

G2/M arrest and apoptosis of human colorectal cancer cells induced by water extract from residues of jelly fig achene.

Jelly fig (Ficus awkeotsang) achenes have been utilized to prepare a traditional drink in Taiwan. Herein, we evaluated the effect of water extract from jelly fig seed residues (WERJFA) on cancer cells. WERJFA could inhibit the growth of human colorectal cancer cells, COLO205 and HT29 in both dose- and time-dependent manners. The flow cytometric analysis with propidium iodide (PI) showed that WERJFA primarily arrested COLO205 and HT29 cells at the G2/M phase of cell cycle as the concentration reached to at least 0.5 mg/ml. WERJFA induced apoptosis of these two cell lines, as evidenced by annexin V-FITC/PI and 4', 6-diamidino-2-phenylindole (DAPI) staining using flow cytometry and confocal microscopy, respectively. Reactive oxygen species (ROS) production and the loss of mitochondrial membrane potential in WERJFA-treated cells were detected by flow cytometry with H2DCF-DA and 5,5', 6,6'-Tetrachloro-1, 1', 3,3'-tetraethylbenzimidazolocarbocyanine iodide (JC-1). Our results showed that WERJFA exerted anti-proliferative and apoptotic effects on colorectal cancer cells. WERJFA arrested cell cycle, and caused apoptotic death in these cancer cells possibly via mitochondrial pathway involved with exceeding ROS level.

Abstract C96: Thyroid hormone induced Beta-catenin-dependent proliferation in colorectal cancer cells

Abstract Thyroid hormone has been shown to induce proliferation of a variety of human cancer cell lines via a cell surface binding site on integrin αvβ3. Clinical studies indicate that thyroid hormone may support the growth of brain tumor, breast cancer, head-and-neck cancer, renal cell carcinoma and several other cancers. The effect of microenvironmental thyroid hormone on the growth of colorectal cancer has not been investigated fully. Numerous evidences suggest that the Wnt/β-catenin pathway, which is involved in both normal and oncogenic developments of the gut, is associated with thyroid hormone and its downstream signaling. The study is therefore aimed to look at the role of thyroid hormone, thyroxine (T4) on colon cancer progression through β-catenin activation. Materials and Methods: The effect of T4 in two APC mutant colon cancer cell lines HT-29 and Colo205 cells and one APC wild type colon cancer HCT116 cells were studied. MTT assays were performed to evaluate cell proliferation under different concentrations of T4. Relative mRNA and protein expressions of relevant genes were examined using qPCR and Western blotting, respectively. Results: T4 (10−8 to 10−6 M) induced proliferation in colorectal cancer HT-29 cells, HCT116 cells and Colo205 cells in a concentration-dependent manner. In HT29 and Colo205 cancer cell lines, the expressions of β-catenin and HMGA2 were not significantly altered by the application of T4. Alternatively, T4 increased accumulation of nuclear β-catenin and HMGA2 in HT-29 cells. Nuclear β-catenin and message were elevated by T4 in Colo205 cells. On the other hand, in addition to the cell proliferation and nuclear accumulation of β-catenin and HMGA2, T4 (10−8-10−6 M) induced the expression of β-catenin and HMGA2 in HCT116 cells. The expression of genes involved in proliferation was enhanced by thyroxine treatment. Conclusions: T4 promoted proliferation in colon cancer HT-29, Colo205 and HCT116 cells. The effect of T4 in colon cancer involved nuclear distribution of β-catenin and its activation. However, the different phenotypes of APC affected the accumulation of nuclear β-catenin and HMGA2 induced by T4. Further study concerning detailed mechanisms is required to elucidate the role of thyroid hormone in colon cancer progression. By understanding the mechanisms behind the pathogenic effects of thyroid hormone, we will propose and develop new treatment approaches to colorectal cancers. Citation Format: Hung-Yun Lin. Thyroid hormone induced Beta-catenin-dependent proliferation in colorectal cancer cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C96.

Abstract C175: FASN inhibition studies in preclinical tumor models identify biomarkers that align with in vitro and in vivo sensitivity to TVB-2640

Abstract Tumor cells have an increased dependence on FASN-synthesized palmitate compared to non-tumor cells, which obtain many of their required lipids from the extracellular milieu. Palmitate and palmitate-derived lipids comprise diverse cellular components and function in processes required for tumor cell proliferation and survival. Previously we showed that FASN inhibition results in tumor cell apoptosis in vitro and xenograft tumor growth inhibition in vivo. Our studies demonstrated that diverse tumor types exhibit sensitivity to FASN inhibition and characterized mechanisms of action that associate with the antitumor activity of highly selective small molecule FASN inhibitors. In vitro studies with diverse tumor cell types elucidated a mechanism of action that includes plasma membrane remodeling, signal transduction pathway inhibition, and gene expression reprogramming. TVB-2640 and TVB-3166 belong to a series of orally available, reversible, potent, and selective FASN inhibitors discovered and developed by 3-V Biosciences. Tumor xenograft studies were conducted in rats and mice to examine the in vitro and in vivo relationship of tumor cell sensitivity to FASN inhibition. Pharmacodynamic analyses of tumor and serum samples from these studies characterized the mechanism of action and biomarkers of sensitivity to in vivo FASN inhibition. Once daily oral dosing of TVB-2640 or TVB-3166 caused inhibition of xenograft tumor growth for varied tumor models that included COLO-205 and HCT-116 colon adenocarcinoma cell lines. In vivo sensitivity to FASN inhibition was in agreement with in vitro data. Analysis of lipid, metabolite, protein, and RNA expression in tumor, blood or serum samples showed drug-induced modulation that was consistent with independent in vitro or in vivo studies. Lipid and metabolite changes included decreased palmitate and palmitate-associated lipids as well as increased expression of acylcarnitine species. Decreased expression of pAkt (S473), β-catenin, pβ-catenin (S675), and Myc proteins were found to associate quantitatively with xenograft tumor growth inhibition. Additionally, mRNA expression was modulated in a manner that revealed coordinated changes in the mRNAs from fatty acid, metabolism, cell survival, and cell growth-associated pathways. Expression changes in lipids, metabolites, proteins, and RNA species are leading to the development of a biomarker panel that describes FASN inhibitor target engagement and tumor sensitivity in both in vitro and in vivo studies. These mechanism-based marker panels will be evaluated in current and upcoming clinical studies of TVB-2640. Citation Format: Timothy S. Heuer, Richard Ventura, Kasia Mordec, Julie Lai, Joanna Waszczuk, Claudia Rubio, Marie O' Farrell, Douglas Buckley, George Kemble. FASN inhibition studies in preclinical tumor models identify biomarkers that align with in vitro and in vivo sensitivity to TVB-2640. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C175.

Dasatinib blocks transcriptional and promigratory responses to transforming growth factor-beta in pancreatic adenocarcinoma cells through inhibition of Smad signalling: implications for in vivo mode of action

BackgroundWe have previously shown in pancreatic ductal adenocarcinoma (PDAC) cells that the SRC inhibitors PP2 and PP1 effectively inhibited TGF-β1-mediated cellular responses by blocking the kinase function of the TGF-β type I receptor ALK5 rather than SRC. Here, we investigated the ability of the clinically utilised SRC/ABL inhibitor dasatinib to mimic the PP2/PP1 effect.MethodsThe effect of dasatinib on TGF-β1-dependent Smad2/3 phosphorylation, general transcriptional activity, gene expression, cell motility, and the generation of tumour stem cells was measured in Panc-1 and Colo-357 cells using immunoblotting, reporter gene assays, RT-PCR, impedance-based real-time measurement of cell migration, and colony formation assays, respectively.ResultsIn both PDAC cell lines, dasatinib effectively blocked TGF-β1-induced Smad phosphorylation, activity of 3TPlux and pCAGA(12)-luc reporter genes, cell migration, and expression of individual TGF-β1 target genes associated with epithelial-mesenchymal transition and invasion. Moreover, dasatinib strongly interfered with the TGF-β1-induced generation of tumour stem cells as demonstrated by gene expression analysis and single cell colony formation. Dasatinib also inhibited the high constitutive migratory activity conferred on Panc-1 cells by ectopic expression of kinase-active ALK5.ConclusionsOur data suggest that the clinical efficiency of dasatinib may in part be due to cross-inhibition of tumour-promoting TGF-β signalling. Dasatinib may be useful as a dual TGF-β/SRC inhibitor in experimental and clinical therapeutics to prevent metastatic spread in late-stage PDAC and other tumours.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-015-0468-0) contains supplementary material, which is available to authorized users.

Selenium Dioxide–Mediated Synthesis of Fused 1,2,4-Triazoles as Cytotoxic Agents

A series of fused 1,2,4-triazoles has been prepared by oxidative intramolecular cyclization of heterocyclic hydrazones with selenium dioxide. General applicability of this practical protocol was confirmed by the synthesis of moderate to good yields of 1,2,4-triazolo[4,3-a]pyridines, 1,2,4-triazolo[4,3-a]pyrimidines, 1,2,4-triazolo[4,3-a]pyramidines, and 1,2,4-triazolo-[4,3-a]quinoxa lines. All compounds were tested in vitro for their cytotoxic activity against HCT-116, A549, and Colo-205 cell lines. Two compounds, 3-(4-methoxyphenyl)-7-methyl-[1,2,4]triazolo[4,3-a]pyridine and 1-(4-methoxyphenyl)-[1,2,4]triazolo[4,3-a]quinoxaline, showed potent antiproliferative activity against the three cell lines.

Triazole linked mono carbonyl curcumin-isatin bifunctional hybrids as novel anti tubulin agents: Design, synthesis, biological evaluation and molecular modeling studies

Keeping in view the limitations associated with currently available anticancer drugs, molecular hybrids of mono carbonyl curcumin and isatin tethered by triazole ring have been synthesized and evaluated for in vitro cytotoxicity against THP-1, COLO-205, HCT-116, A549, HeLa, CAKI-I, PC-3, MiaPaca-2 human cancer cell lines. The results revealed that the compounds SA-1 to SA-9, SB-2, SB-3, SB-4, SB-7 and SC-2 showed a good range of IC50 values against THP-1, COLO-205, HCT-116 and PC-3 cell lines, while the other four cell lines among these were found to be almost resistant. Structure activity relationship revealed that the nature of Ring X and substitution at position R influences the activity. Methoxy substituted phenyl ring as Ring X and H as R were found to be the ideal structural features. The most potent compounds (SA-2, SA-3, SA-4, SA-7) were further tested for tubulin inhibition. Compound SA-2 was found to significantly inhibit the tubulin polymerization (IC50=1.2μM against HCT-116). Compound SA-2, moreover, lead to the disruption of microtubules as confirmed by immunofluorescence technique. The significant cytotoxicity and tubulin inhibition by SA-2 was streamlined by molecular modeling studies where it was docked at the curcumin binding site of tubulin.