Colo205 Cells Research Articles

RETRACTED ARTICLE: Long intergenic non-protein coding RNA 960 regulates cancer cell viability, migration and invasion through modulating miR-146a-5p/interleukin 1 receptor associated kinase 1 axis in pancreatic ductal adenocarcinoma

ABSTRACT Long non-coding RNAs (lncRNAs) are considered as crucial regulatory factors in cancer biology. However, the biological function of long intergenic non-protein coding RNA 960 (LINC00960) in the tumorigenesis of pancreatic ductal adenocarcinoma (PDAC) is still unknown. The goal of this study is to investigate the role of LINC00960 in PDAC. Quantitative real‐time polymerase chain reaction (qRT-PCR) was used to examine the expression levels of LINC00960 in PDAC tissues and cell lines. After transfection, the loss-of-function models of LINC00960 or interleukin 1 receptor-associated kinase 1 (IRAK1) were established with BxPC-3 cells and Colo357 cells, and the malignant phenotypes of BxPC-3 cells and Colo357 cells were detected by CCK-8 assay, BrdU assay and Transwell assay, respectively. The interactions among LINC00960, miR-146a-5p and IRAK1 were predicted by bioinformatics analysis, and verified by luciferase reporter assay, RNA immunoprecipitation assay and RNA pull-down assay. The regulatory functions of LINC00960 and miR-146a-5p on IRAK1 were detected by Western blot. We demonstrated that the LINC00960 expression was increased in PDAC tissues and cell lines. Knocking down LINC00960 or IRAK1 could repress the viability, migration, and invasion of BxPC-3 and Colo357 cells. LINC00960 functioned as a molecular sponge for miR-146a-5p, and IRAK1 was verified as a target gene of miR-146a-5p. Additionally, LINC00960 could up-regulate IRAK1 expression via repressing miR-146a-5p, and the oncogenic properties of LINC00960 were partly reversed by miR-146a-5p. Our findings reveal that LINC00960 is a promoter of PDAC progression through regulating miR-146a-5p/IRAK1axis.

BRAF paradox breakers PLX8394, PLX7904 are more effective against BRAFV600Ε CRC cells compared with the BRAF inhibitor PLX4720 and shown by detailed pathway analysis

PLX7904 and PLX8394 are novel BRAFV600E inhibitors-BRAFi that are designed to evade the paradoxical MAPK activation, a trait for the name “paradox breakers”-PB. Current FDA approved inhibitors (Vemurafenib, Dabrafenib, Encorafenib) although improved progression-free survival of mtBRAF melanoma patients suffer from this treatment related side effect. mtBRAF Colorectal Cancer (CRC) is resistant to the approved BRAF inhibitors, although combinatorial treatment co-targeting BRAF and EGFR/MEK is offering a promising prospect. In an effort to explore the potential of the novel BRAF inhibitors-PB to impede CRC cell proliferation, they were tested on RKO, HT29 and Colo-205 cells, bearing the BRAFV600E mutation. This study shows that the BRAF paradox breakers PLX7904 and PLX8394 cause a more prolonged MAPK pathway inhibition and achieve a stronger blockage of proliferation and reduced viability than PLX4720, the sister compound of Vemurafenib. In some treatment conditions, cells can undergo apoptosis. Genomic analysis on the more resistant RKO cells treated with PLX7904, PLX8394 and PLX4720 showed similar gene expression pattern, but the alterations imposed by the PB were more intense. Bioinformatic analysis resulted in a short list of genes representing potential master regulators of the cellular response to BRAF inhibitors' treatments. From our results, it is clear that the BRAF paradox breakers present a notable differential regulation of major pathways, like MAPK signalling, apoptosis, cell cycle, or developmental signalling pathways. Combinatorial treatments of BRAFi with Mcl-1 and Notch modulators show a better effect than mono-treatments. Additional pathways could be further exploited in novel efficient combinatorial treatment protocols with BRAFi.

Novel 1,2,3-Triazole-functionalized pyrido[3',2':4,5]furo[3,2-d]pyrimidin- 4(3H)-one Derivatives: Synthesis, Anticancer Activity, CoMFA and CoMSIA Studies

A series of novel triazole functionalized pyrido [3',2':4,5] furo[3,2-d] pyrimidin-4 (3H)-one derivatives 7a-p were prepared from ethyl furo[2,3-b]pyridine-2-carboxylate 3 on reaction with ammonia to afford furo[2,3-b]pyridine-2-carboxamide 4. This compound, on reaction with triethyl orthoformate TEOF, gave compound 5. Compound 5 on propargylation, followed by a reaction with substituted aryl azides under Sharpless reaction conditions, furnished triazole tagged pyrido [3',2':4,5]furo[3,2-d] pyrimidin-4(3H)-one derivatives. All the products 7a-p were screened against four human cancer cell lines, such as HeLa - Cervical cancer (CCL-2), COLO 205- Colon cancer (CCL-222), HepG2- Liver cancer (HB-8065), and MCF7 - Breast cancer (HTB-22) and one normal cell line (HEK 293). Compounds 7b, 7n, 7o and 7p, which showed promising anticancer activity, were identified and found to be non-toxic to normal cell line. Studies for HeLa, COLO205, HepG2, and MCF-7 using CoMFA and CoMSIA were carried out . Models from 3D-QSAR provided a strong basis for future rational design of more active and selective HeLa, COLO205, HepG2, and MCF-7 cell line inhibitors.

Design, synthesis and biological evaluation of sulphonamide derivatives of benzofuran-imidazopyridines as anticancer agents

Abstract A novel library of sulphonamide derivatives of benzofuran-imidazopyridines (9a-j) were designed, synthesized and screened for their anticancer activity against four human cancer cell lines such as breast cancer (MCF-7), lung cancer (A549), colon cancer (Colo-205) and ovarian cancer (A2780) by employing MTT assay. The results are expressed with IC50 µM, which indicated that all of the compounds showed good to moderate activity on tested cell lines. Among them, compound 9c showed potent anticancer activities against MCF-7, A549, Colo-205 and A2780 cell lines with IC50 values of 0.011 ± 0.0075 µM, 0.073 ± 0.0012 µM, 0.10 ± 0.19 µM and 0.034 ± 0.0041 µM respectively.

Boswellia sacra essential oil manages colon cancer stem cells proliferation and apoptosis: a new perspective for cure

ABSTRACT Boswellia sacra Flueck essential oil is obtained from frankincense obtained from Boswellia sacra trees. The aim of this study was to investigate the in vitro anti-cancer activity of Boswellia sacra (BS) essential oil in CD133+ and CD133- Colo-320 cells. Essential oil was analyzed by GC-MS. CD133+ cells, cancer stem cells, were obtained from the Colo-320 by the MiniMACS system. Cytotoxicity were evaluated with MTT assay. Anti-cancer activity of BS essential oil was investigated by immunocytochemistry. 41 compounds were identified in the essential oil. 1:2 dilution BS essential oil was found to be active against all the cells for 24 h incubation. β-catenin immunoreactivity was significantly decreased in both BS essential oil-treated CD133+ and CD133- Colo-320 cells. Caspase-3 immunoreactivity significantly increased in BS essential oil-treated CD133- Colo-320 cells. We conclude that BS essential oil decreased β-catenin signaling molecules which play a crucial role in cancer cell proliferation. BS essential oil promoted apoptosis in CD133- Colo-320 cells.

99mTc-labelled and pH-awakened microbeads entrapping surface-modified lipid nanoparticles for the augmented effect of oxaliplatin in the therapy of colorectal cancer

Aim This study was aimed to develop Eudragit S100–coated, pH-awakened microbeads (MBs) encapsulating folic acid (FA)-modified tristearin solid lipid nanoparticles (SLNs) loaded with oxaliplatin (OP). Afterward, these formulations were evaluated (in vitro and in vivo) for their potential against colorectal cancer (CRC). Methods The SLNs were synthesised by employing the solvent diffusion technique and then they were entrapped in the MBs. The prepared uncoupled and coupled SLNs (SLN-OP and FA-SLN-OP, respectively) were examined for in vitro cytotoxicity effect against COLO-205. Gamma-scintigraphy study was used for determining biodistribution (in vivo) of drug in different organs through MBs. Results Outcomes for FA-SLN-OP revealed more cytotoxicity (50% inhibitory concentration [IC50] = 6.8 µg/ml) against COLO-205 cells (in vitro) than OP solution (IC50 = 8.0 µg/ml) and SLN-OP (IC50= 7.5 µg/ml). MBs were also investigated in vivo using Gamma-scintigraphy study. After 48 h study, 99mTc-EuB-FA-SLN-OP confirmed an elevated level of drug in the colonic tumour, which was found significantly (p< 0.0001) higher than that of 99mTc-EuB-SLN-OP. Conclusions In conclusion, developed MBs formulation (99mTc-EuB-FA-SLN-OP) suggested promising results against therapy of CRC using dual targeting (i.e. ligand-directed and pH-awakened) approach.

Synthesis and biological evaluation of chalcone derivatives of 1,2,4-thiadiazol-benzo[d]imidazol-2-yl)quinolin-2(1H)-one as anticancer agents

Abstract A new series of chalcone incorporated 2-quinoline-benzimidazole-1,2,4-thiadiazoles (9a-j) were designed, synthesized and examined for their anticancer activities against four different human cancer cell lines like MCF-7 (breast cancer), A549 (Lung cancer), Colo-205 (Colon cancer) and A2780 (Ovarian cancer) and compared with clinical drug candidate as etoposide. Among them, compound 9d showed potent anticancer activities against MCF-7, A549, Colo-205 and A2780 cell lines with IC50 values of 0.012±0.007 µM, 0.074±0.004 µM, 1.46±0.73 µM and 0.083±0.002 µM, respectively.

Overexpression of RNF126 Promotes the Development of Colorectal Cancer via Enhancing p53 Ubiquitination and Degradation.

BackgroundRING finger protein 126 (RNF126), as a novel E3 ubiquitin ligase, plays an oncogenic role in several solid cancers. But its potential role in colorectal cancer (CRC) that harbored 50% mutant p53, to our knowledge, is rarely reported.Materials and MethodsWe investigated the clinical significance and relationship of RNF126 and p53 in CRC tissues and cells. Meanwhile, WB, qRT-PCR, co-IP, MTT, and transwell were used to investigate the function and molecular mechanism of RNF126 in regulating malignant biology in vitro.ResultsRNF126 was overexpressed in human CRC specimens, which was tightly associated with tumor size (P=0.021), T stage (P=0.030), lymph node metastasis (P=0.006), TNM stage (P=0.001), and the poor survival (P=0.003) of CRC patients. RNF126 had no association with p53 mutation in CRC specimens, and in p53 mutant Colo-205 and SW620 cells. However, in p53 wildtype HCT116 and HCT-8 cells, RNF126 silencing upregulated p53 and p21 but inhibited Rb phosphorylation at Serine 780 (pRb), which was inhibited by p53siRNA. Conversely, RNF126 overexpression downregulated p53 and p21 but promoted pRb expression, which was reversed by a classic proteasome inhibitor, MG132. However, the mRNA levels of above target genes were unchanged, implying a ubiquitination dependent post-translational modification involving in above regulation. Meanwhile, RNF126 was co-immunoprecipitated with p53 and p21 to form a triple complex. RNF126 silencing and overexpression inhibited and promoted p53 ubiquitination and degradation in vitro, respectively. In addition, p53siRNA reversed RNF126 silencing-inhibited cell proliferation, drug resistance, and cell mobility in HCT116 cells. Conversely, MG132 inhibited RNF126 overexpression-promoted above cell biology in HCT-8 cells.ConclusionOverexpression of RNF126 was remarkably associated with multiple advanced clinical characters of CRC patients independent of mutant p53. RNF126 promotes cell proliferation, mobility, and drug resistance in CRC via enhancing p53 ubiquitination and degradation.

Design, synthesis, anti-cancer evaluation and binding mode studies of benzimidazole/benzoxazole linked β-carboline derivatives

A new series of benzimidazole/benzoxazole linked β-carbolines (9a-j) were rationally designed and synthesized by combining two different anti-cancer fragments. The new hybrid β-carbolines are subjected to anti-cancer screening against four different human cancer cell lines such as MCF-7 (breast), A549 (lung), Colo-205 (colon) and A2780 (ovarian) by using standard MTT assay. These hybrid β-carbolines exhibited significant and high fold anti-cancer activity against MCF-7 cell lines than reference standard. They are also proved to be effective against A549 and Colo-205 cell lines. Further, compound 9b, 9c from benzimidazole and 9i from benzoxazole series have exhibited maximum anti-cancer activity among these hybrid β-carbolines. Later, all of the hybrid β-carbolines were subjected to molecular interaction analysis against a few selected kinase targets such as cdc-like kinase (CLK-1 to CLK-4), epidermal growth factor reductase (EGFR) kinase, protein (ATR) kinase along with APC-Asef interface. The violin plot of binding energies of 9a-9j have suggested them as good kinase binders. Result interpretation suggested hybrid β-carbolines as promising CLK binders. The anti-cancer data of new hybrid β-carbolines against MCF-7 cell lines are in agreement with parent β-carboline skeleton.

Ambrisentan, an endothelin receptor type A-selective antagonist, inhibits cancer cell migration, invasion, and metastasis

Several studies reported a central role of the endothelin type A receptor (ETAR) in tumor progression leading to the formation of metastasis. Here, we investigated the in vitro and in vivo anti-tumor effects of the FDA-approved ETAR antagonist, Ambrisentan, which is currently used to treat patients with pulmonary arterial hypertension. In vitro, Ambrisentan inhibited both spontaneous and induced migration/invasion capacity of different tumor cells (COLO-357 metastatic pancreatic adenocarcinoma, OvCar3 ovarian carcinoma, MDA-MB-231 breast adenocarcinoma, and HL-60 promyelocytic leukemia). Whole transcriptome analysis using RNAseq indicated Ambrisentan’s inhibitory effects on the whole transcriptome of resting and PAR2-activated COLO-357 cells, which tended to normalize to an unstimulated profile. Finally, in a pre-clinical murine model of metastatic breast cancer, treatment with Ambrisentan was effective in decreasing metastasis into the lungs and liver. Importantly, this was associated with a significant enhancement in animal survival. Taken together, our work suggests a new therapeutic application for Ambrisentan in the treatment of cancer metastasis.

Leaf Anatomical-Histophytochemical Study and Evaluation of the Cytotoxicity of Ottonia frutescens Trel (Piperaceae)

To investigate the leaf histochemistry and micromorphology of Ottonia frutescens Schltdl Trel (Piperaceae), the antioxidant potential, and in vitro cytotoxic bioactivity of the leaf extract of this species against human leukemia and colorectal cells. Leaf micromorphology and histoanatomy were detailed and recorded by optical and transmission and scanning electron microscopy. The histochemical results reveal classes of secondary phytometabolites such as alkaloids, anthraquinones, coumarins, polyphenols, and saponins. Cardiotonics, flavonoids, and triterpenes compounds were not detected. All concentrations of leaf extracts showed a significant dose-dependent and time-dependent apoptotic effect with more than 90% apoptosis of U937 cells and about 60% apoptosis of COLO-205 cells in 24 h of culture under 200 μg/mL of leaf extract. In addition, the phenolic compounds present in the fractions of the sample contribute to its strong antioxidant capacity. Ottonia frutescens exhibit anatomical and histochemical characteristics similar to other Piperaceae, and for the first time, provides a new source for secondary metabolite classes that exhibit high antioxidant potential and cytotoxic bioactivity, reduce cell viability and induce apoptosis in U937 leukemic cells and COLO-205 colorectal cells.

Abstract 2238: Synergistic effect of sunitinib and PD-1 inhibitor nivolumab on colorectal cancer in vitro and in vivo

Abstract Colorectal cancer (CRC) is a common cause of oncological deaths worldwide. In clinical practice, a main concerning for choosing immune checkpoints inhibitors (ICI) is the low response rate, despite multiple predictive biomarkers have been adopted for patient selection. Recent clinical trials showed that ICI could effectively induce tumor regression in combination therapy with anti-angiogenetic treatment because the latter damages blood vessel and reprograms the tumor immune microenvironment. Here we tested sunitinib in combination with anti-PD-1 (nivolumab and CD279) in both in vitro and in vivo CRC models in order to assess the antitumor potential and shid light on cellular mechanisms underpinning drugs combination effects. To this purpose we utilized CaCo-2, Colo205, LoVo and HT-29 CRC cell lines for in vitro testing and CT-26 cell line for generating the syngenic in vivo model. For in vitro study the cells were challenged with scalar concentrations of sunitinib and nivolumab alone and in combination, for cytotoxicity evaluation by MTT and the combination index (CI) was calculated to determine the pharmacological interaction. The effects on cell cycle progression and apoptosis induction were determined by propidium iodide and Annexin V staining, respectively, followed by FACS analysis. Cell targets modulation was evaluated by western blotting. The in vivo study was performer in CT-26 syngenic mouse model. To treat established tumours (∼100 mm3), from day 8 after cell inoculation sunitinib and the anti-PD-1 CD279 were administered intraperitoneally to mice as follows: group (1) vehicle alone (control group); group (2) Sunitinib 1 mg/mouse for 7 consecutive days; group (3) on day 8, CD279 250 µg/mouse, three times at 2-day intervals; group (4) combination treatment of Sunitinib 1 mg/mouse for 7 consecutive days, followed by CD279 250 µg/mouse, three times at 2-day intervals. After treatment, the mice were observed for 12 days and then killed by an anaesthetic overdose. Moreover, the tumor rate has been calculated. In in vitro characterization, the combination resulted in a synergistic inhibition of cells proliferation with a CI ranging between 0.6-0.8 and the study of cell cycle progression evidenced a significant arrest at G0/G1 phase of cell cycle. Sunitinib strongly induced apoptosis, however the combination didn't lead to a further increase compared to sunitinib alone. Of note both sunitinib and drugs combination led to the LC3 A/B authophagic marker activation. Furthermore sunitinib reduced the levels of VEGF released by cells to the same extent of drugs combination. The in vivo study showed a time dependent synergistic effect of drugs combination compared to each drug alone with an improved overall survival of mice. Our study provides a rationale to further develop combination strategies with anti-angiogenic drug and PD-1 inhibitors for the management of CRC patients. Citation Format: Letizia Porcelli, Roberta Di Fonte, Domenica Rea, Claudio Arra, Amalia Azzariti, Angelo V. Paradiso. Synergistic effect of sunitinib and PD-1 inhibitor nivolumab on colorectal cancer in vitro and in vivo [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2238.

Using Two Peptide Isotopologues as Internal Standards for the Streamlined Quantification of Low-Abundance Proteins by Immuno-MRM and Immuno-MALDI.

Mass spectrometry (MS), particularly targeted proteomics, is increasingly being used for quantifying specific proteins and peptides in clinical specimens. The coupling of immuno-enrichment of proteotypic peptides with MS [e.g., immuno-multiple reaction monitoring (MRM) and immuno-matrix-assisted laser desorption ionization (MALDI)] enables the development of highly sensitive and specific assays for low-abundance signaling proteins. By incorporating stable isotope-labeled standards, these workflows allow the determination of endogenous protein concentrations. This is typically achieved through external calibration, often using surrogate matrices, which has inherent limitations for the analysis of clinical specimens as there are often substantial variations in the sample matrix, and sample amounts are typically limited. We have previously introduced the use of two peptide isotopologues for generating external calibration curves in plasma. Here, we present a two-point internal calibration (2-PIC) strategy using two isotopologues for immuno-MS assays and demonstrate its flexibility and robustness. Quantification of the tumor suppressor PTEN in Colo-205 cells by immuno-MRM and immuno-MALDI using 2-PIC and external calibration yielded very similar results (relative standard deviation between 2-PIC and external calibration: 4.9% for immuno-MRM; 1.1% for immuno-MALDI), without the need for a surrogate matrix or additional patient material for calibration, while concurrently reducing the instrument time and cost. Although our PTEN immuno-MRM and immuno-MALDI assays can be considered to be orthogonal as they utilized entirely different sample preparation and MS analysis workflows, targeted different PTEN peptides, and were performed in different laboratories, the endogenous Colo-205 PTEN levels determined with 2-PIC showed a good correlation (r2 = 0.9966) and good agreement (0.48 ± 0.01 and 0.29 ± 0.02 fmol/μg of total protein) between immuno-MRM and immuno-MALDI.

Pomegranate (Punica granatum) Fruit Extract Suppresses Cancer Progression and Tumor Angiogenesis of Pancreatic and Colon Cancer in Chick Chorioallantoic Membrane Model

Pomegranate fruit extract contains many polyphenols and flavonoids of diverse biological importance including anticancer potential. In cancer, the angiogenesis process facilitates solid cancer growth and metastasis. Here, the antiangiogenic effect of pomegranate fruit extract against human pancreatic cancer (Suit-2) and colon (colo205) cell lines in the chick chorioallantoic membrane (CAM) model was studied along with the effect of pomegranate fruit extract on fibroblast growth factor (FGF2). Pomegranate fruit extract significantly reduced the tumor weight and hemoglobin content in CAM models of pancreatic Suit-2 and colon colo205.

Placenta-specific protein 1 enhances liver metastatic potential and is associated with the PI3K/AKT/NF-κB signaling pathway in colorectal cancer.

To better explore the underlying mechanism of liver metastatic formation by placenta-specific protein 1 (PLAC1) in human colorectal cancer, we investigated the proliferation, invasion and angiogenic capabilities of human colorectal cancer cells with different liver metastatic potentials as well as the mechanism of action of PLAC1 in the metastatic process. The expression of PLAC1 was detected by reverse transcriptase PCR, western blot, and real-time PCR. The effect of PLAC1 on metastatic potential was determined by proliferation, invasion, and angiogenesis assays, including an in-vitro coculture system consisting of cancer cells and vascular endothelial cells that were used to detect the relationship between cancer cells and angiogenesis. In addition, we also determined PLAC1 downstream targets that preferentially contribute to the metastatic process. PLAC1 was expressed in HT-29, WiDr, and CaCo-2 colorectal cancer cells but not in Colo320 colorectal cancer cells. PLAC1 not only enhanced significantly the proliferation of CoLo320 and human umbilical vein endothelial cells (HUVECs) but also promoted the invasion of CoLo320 cells. The angiogenesis of HUVECs was enhanced by PLAC1 in a dose-dependent manner. In cocultured systems, angiogenesis was significantly increased by coculture with HT-29 cells. In addition, PLAC1 could promote angiogenesis in coculture with HT-29 cells. Furthermore, PLAC1-enhanced metastatic potential of colorectal cancer cells was dependent on the activation of the PI3K/Akt/NF-κB pathway. The activation of PI3K/Akt/NF-κB signaling by PLAC1 may be critical for metastasis of colorectal cancer cells. According to our results, we suggest that modification of PLAC1 function might be a promising new therapeutic approach to inhibit the aggressive spread of colorectal cancer.

Effectiveness of a Controlled 5-FU Delivery Based on FZD10 Antibody-Conjugated Liposomes in Colorectal Cancer In vitro Models.

The use of controlled delivery therapy in colorectal cancer (CRC) reduces toxicity and side effects. Recently, we have suggested that the Frizzled 10 (FZD10) protein, a cell surface receptor belonging to the FZD protein family that is overexpressed in CRC cells, is a novel candidate for targeting and treatment of CRC. Here, the anticancer effect of novel immuno-liposomes loaded with 5-Fluorouracil (5-FU), decorated with an antibody against FZD10 (anti-FZD10/5-FU/LPs), was evaluated in vitro on two different CRC cell lines, namely metastatic CoLo-205 and nonmetastatic CaCo-2 cells, that were found to overexpress FZD10. The anti-FZD10/5-FU/LPs obtained were extensively characterized and their preclinical therapeutic efficacy was evaluated with the MTS cell proliferation assay based on reduction of tetrazolium compound, scratch test, Field Emission Scanning Electron Microscopes (FE-SEM) investigation and immunofluorescence analysis. The results highlighted that the cytotoxic activity of 5-FU was enhanced when encapsulated in the anti-FZD10 /5-FU/LPs at the lowest tested concentrations, as compared to the free 5-FU counterparts. The immuno-liposomes proposed herein possess a great potential for selective treatment of CRC because, in future clinical applications, they can be encapsulated in gastro-resistant capsules or suppositories for oral or rectal delivery, thereby successfully reaching the intestinal tract in a minimally invasive manner.

Quercetin-Mediated Apoptosis and Cellular Senescence in Human Colon Cancer.

Quercetin is a flavonol from the flavonoid group of polyphenols, which positively affects human health due to its anti-cancer, anti-inflammatory, anti-microbial and cardioprotective effects. The effects of phenolic compounds, including quercetin, on programmed cell death and cellular senescence, have been the subject of research in recent years. In this study, we aimed to investigate the effects of quercetin on cell viability, apoptosis and cellular senescence in primary (Colo-320) and metastatic (Colo-741) colon adenocarcinoma cell lines. Cytotoxicity was analyzed via MTT assay in Colo-320 and Colo-741 cell lines. After quercetin treatment, cell ularsenescence and apoptosis were evaluated by TUNEL staining, X-Gal staining and indirect peroxidase technique for immunocytochemical analysis of related proteins such as Bax, Bcl-2, caspase-3, Hsp27, Lamin B1, p16, cyclin B1. The effective dose for inhibition of cell growth in both cell lines was determined to be 25μg/ml quercetin for 48 hours. Increased Baximmunoreactivityfollowingquercetin treatment was significant in both Colo-320 and Colo-741 cell lines, but decreased Bcl-2 immunoreactivitywas significant only in theColo-320 primary cell line. In addition, after quercetin administration, the number of TUNEL positive cells and, immunoreactivities for p16, Lamin B1 and cyclin B1 in both Colo-320 and Colo-741 cells increased. Our results suggest that quercetin may only induce apoptosis in primary colon cancer cells. Furthermore, quercetin also triggered senescence in colon cancer cells, but some cells remained alive, suggesting that colon cancer cells might have escaped from senescence.

Neopapillarine, an Unusual Coumarino-Alkaloid from the Root Extract of Neocryptodiscus papillaris with Cytotoxic Activity on Renal Cancer Cells.

Several simple and prenylated coumarin derivatives were isolated from the dichloromethane extract of the root of Neocryptodiscus papillaris based on moderate cytotoxic activity of the extract in COLO205, KM12 and MCF7 cancer cells. While the major prenylated furanocoumarin derivatives and osthol isolated from the dichloromethane extract were responsible for the activity in the colon and breast cancer cell lines, the 4′-acylated osthol derivatives including a novel coumarino-alkaloid; neopapillarine) demonstrated selective cytotoxic activity in A498 and UO31 renal cancer cell lines.

An Animal Model of Colorectal Cancer Liver Metastasis With a High Metastasis Rate and Clonal Dynamics.

Various animal models have been introduced into the study of liver metastasis of colorectal cancer, but they have not been compared under the same conditions. The aim of this study was to identify an optimized mouse model that showed a high rate of hepatic metastasis and expression of clonal dynamics. Athymic nude mice (n=30) were divided into two equal groups for the creation of a splenic injection model (SIM) and surgically orthotopic implantation model (SOIM) of liver metastasis of colorectal cancer using HCT116 cells. Hepatic metastasis was confirmed by gross and microscopic examinations. Expression of MET transcriptional regulator MACC1 (MACC1) in colon cancer cell lines and metastatic tumors in the group with a higher liver metastasis rate was confirmed by quantitative reverse-transcription-polymerase chain reaction. The observation time was significantly shorter for SIM than for SOIM (33.0±6.8 vs. 41.2±7.2 days, p<0.001). The rate of hepatic metastasis was significantly higher in SIM than in SOIM (76.9% vs. 38.4%, p=0.038). MACC1 was expressed in Colo201, HCT116, HT29, LS513, SW620, and WiDr cells but not in SW480 cells. All hepatic metastases in SIM mice expressed MACC1, and metastatic HCT116 cells had significantly greater expression than did the original HCT116 cells (p<0.001). With a higher rate of hepatic metastasis with clonal dynamics in a shorter observation time than the SOIM, SIM appears to be a good animal model for identifying new targets and in drug development for colorectal cancer liver metastasis. SOIM should also be considered for the study of the full steps of metastasis.

Erratum to: Identification of Tn antigen O-GalNAc-expressing glycoproteins in human carcinomas using novel anti-Tn recombinant antibodies.

The Tn antigen is a neoantigen abnormally expressed in many human carcinomas and expression correlates with metastasis and poor survival. To explore its biomarker potential, new antibodies are needed that specifically recognize this antigen in tumors. Here we generated two recombinant antibodies to the Tn antigen, Remab6 as a chimeric human IgG1 antibody and ReBaGs6 as a murine IgM antibody and characterized their specificities using multiple biochemical and biological approaches. Both Remab6 and ReBaGs6 recognize clustered Tn structures, but most importantly do not recognize glycoforms of human IgA1 that contain potential cross-reactive Tn antigen structures. In flow cytometry and immunofluorescence analyses, Remab6 recognizes human cancer cell lines expressing the Tn antigen, but not their Tn-negative counterparts. In immunohistochemistry (IHC), Remab6 stains many human cancers in tissue array format but rarely stains normal tissues and then mostly intracellularly. We used these antibodies to identify several unique Tn-containing glycoproteins in Tn-positive Colo205 cells, indicating their utility for glycoproteomics in future biomarker studies. Thus, recombinant Remab6 and ReBaGs6 are useful for biochemical characterization of cancer cells and IHC of tumors and represent promising tools for Tn biomarker discovery independently of recognition of IgA1.