Cell Line COLO Research Articles

The expression of epithelial mesenchymal transition related factors in human colorectal cancer cell lines

Purpose: Epithelial mesenchymal transition (EMT) is a well characterized embryological process thought to play a vital role in tumor progression. The purpose of this study was to evaluate the expression of EMT-related factors and then use EMT status to identify high metastatic potential in commonly used human colorectal cancer cell lines. Methods: In 11 commonly used cell lines, total mRNA expression levels were checked for the cell markers E- cadherin, vimentin, N-cadherin, and fibronectin, and the transcription factors snail, slug, twist, and SIP1 using real time PCR. Results: E-cadherin expression was positive in COLO205, DLD1, and SW48 cell lines. Vimentin expression was positive in COLO205, DLD1, HCT15, KM20, and SW480. Three different EMT status groups were defined according to mRNA expression of the cell markers. The epithelial phenotype expressed E-cadherin without vimentin (SW48), incomplete EMT phenotypes either concurrently expressed or did not express both E-cadherin and vimentin (CACO2, COLO205, DLD1, KM12C, KM12SM, LoVo, and RKO), and complete EMT phenotypes expressed vimentin without E-cadherin (HCT15, KM20, and SW480). The mRNA expression of transcription factors had no correlation with expression of vimentin, N-cadherin, and fibronectin, but there was an inverse directional correlation between mRNA expression levels of transcription factors and E-cadherin. There were no statistically significant correlations, however. Conclusion: Ten cell lines among the 11 were included in the incomplete and complete EMT groups. These findings could provide evidence of the high tumorigenesis and metastatic properties. The levels of mRNA expression and EMT status of human colorectal cancer cell lines can be applied to further investigations into cancer progression.

Isolation, Synthesis and Biological Evaluation of Phenylpropanoids from the Rhizomes of Alpania galanga

Bioactivity guided investigation of the DCM: MeOH (1:1) extract from the rhizomes of Alpinia galanga led to the isolation of phenylpropanoids (1-9) and their structures were established by 1H NMR, 13C NMR, IR a nd LC-MS/MS. Th ese compounds have b een evaluated for their in vitro anticanceractivity against the human cancer cell lines A549 (lung cancer), Colo-205 (colon cancer), A431 (skin cancer), NCI H460 (lung cancer), PC-3 (prostate cancer), and HT-29 (colon cancer). Compounds 4 and 9 showed potent anticancer activity (ranging from 1.3-19.7 microg/mL) against all the tested cancer cell lines. In addition, an asymmetric synthesis of acetoxychavicol acetate (1) and trans-p-coumaryl alcohol (4) has been accomplished in six steps starting from readily available p-hydroxybenzaldehyde for the first time. Grignard reaction and Sharpless kinetic resolution reactions were utilized as the key steps to install the basic core.

Overexpression of miR-145 increases the sensitivity of vemurafenib in drug-resistant colo205 cell line

Vemurafenib is a selective and potent small molecule inhibitor of the V600 mutant form of the BRAF protein used in the treatment of melanoma and colorectal cancer. However, vemurafenib has less effect in BRAF mutant colorectal cancer due to the resistance of tumor cell to vemurafenib. To verify whether or not miR-145, a short RNA molecule of microRNA which has been supposed to be a tumor suppressor, is involved in this process, we established vemurafenib-resistant cell line colo205/V and found that the miR-145 expression was significantly downregulated in colo205/V cells compared to normal colo205 cells. Moreover, the overexpression of miR-145 could increase the sensitivity of colo205/V cells to vemurafenib both in vitro and in vivo. In conclusion, miR-145 might be used as a therapeutic target in the treatment of colorectal cancer patients with BRAF V600E mutation.

Enalapril inhibits nuclear factor-κB signaling in intestinal epithelial cells and peritoneal macrophages and attenuates experimental colitis in mice

AimsEnalapril, an angiotensin-converting enzyme (ACE) inhibitor, has pleiotropic effects such as anti-inflammatory effects. This study investigated the effect of enalapril on the nuclear factor-kappa B (NF-κB) pathway and on experimental colitis. Main methodsThe human intestinal epithelial cell (IEC) line COLO 205 and peritoneal macrophages from C57BL/6 wild-type mice and IL-10-deficient (IL-10−/−) mice were prepared and subsequently stimulated with lipopolysaccharide (LPS) alone or LPS plus enalapril. The effect of enalapril on NF-κB signaling was examined by western blotting to detect IκBα phosphorylation/degradation; an electrophoretic mobility shift assay (EMSA) to assess the DNA binding activity of NF-κB; and ELISAs to qualify IL-8, TNF-α, IL-6, and IL-12 production. In in vivo studies, dextran sulfate sodium (DSS)-induced acute colitis in wild-type mice and chronic colitis in IL-10−/− mice were treated with or without enalapril. Colitis was quantified by histologic scoring, and the phosphorylation of IκBα in the colonic mucosa was assessed using immunohistochemistry. Key findingsEnalapril significantly inhibited LPS-induced IκBα phosphorylation/degradation, NF-κB binding activity, and pro-inflammatory cytokine production in both IEC and peritoneal macrophages. The administration of enalapril significantly reduced the severity of colitis, as assessed based on histology in both murine colitis models. Furthermore, in colon tissue, the up-regulation of IκBα phosphorylation with colitis induction was attenuated in enalapril-treated mice. SignificanceEnalapril may block the NF-κB signaling pathway, inhibit the activation of IECs and macrophages, and attenuate experimental murine colitis by down-regulating IκBα phosphorylation. These findings suggest that enalapril is a potential therapeutic agent for inflammatory bowel disease.

Dynamic Switch Between Two Adhesion Phenotypes in Colorectal Cancer Cells

The hematogenous metastatic cascade is mediated by the interaction of cancer cells and the endothelial cell lining of blood vessels. In this work, we examine the colon cancer cell line COLO 205, which grows simultaneously in both adherent and suspended states in culture and can serve as a good model for studying tumor heterogeneity. The two subpopulations of cells have different molecular characteristics despite being from the same parent cell line. We found that the ratio of adherent to suspended cells in culture is maintained at 7:3 (equilibrium ratio). The ratio was maintained even when we separate the two populations and culture them separately. After 8 h in culture the equilibrium was achieved only from either adherent or suspended population. The adherent cells were found to express less E-selectin binding glycans and demonstrated significantly weaker interaction with E-selectin under flow than the suspended cells. Manipulation of the epithelial–mesenchymal transition (EMT) markers β-catenin and E-cadherin expression, either by siRNA knockdown of β-catenin or incubation with E-cadherin antibody-coated microbeads, shifted the ratio of adherent to suspended cells to 9:1. Interestingly, human plasma supplemented media shifted the ratio of adherent to suspended cells in the opposite direction to 1:9, favoring the suspended state. The dynamic COLO 205 population switch presents unique differential phenotypes of their subpopulations and could serve as a good model for studying cell heterogeneity and the EMT process in vitro.

Folate-chitosan-gemcitabine core-shell nanoparticles targeted to pancreatic cancer.

Human pancreatic cancer is one of the most common clinical malignancies. The effect of comprehensive treatment based on surgery is general. The effects of chemotherapy were not obvious mainly because of lack of targeting and chemoresistance in pancreatic cancer. This study aimed to investigate the effects of folate receptor (FR)-mediated gemcitabine FA-Chi-Gem nanoparticles with a core-shell structure by electrostatic spray on pancreatic cancer. In this study, the levels of expression of FR in six human pancreatic cancer cell lines were studied by immunohistochemical analysis. The uptake rate of isothiocyanate-labeled FA-Chi nanoparticles in FR high expression cell line COLO357 was assessed by fluorescence microscope and the inhibition rate of FA-Chi-Gem nanoparticles on COLO357 cells was evaluated by MTT assay. Moreover, the biodistribution of PEG-FA-ICGDER02-Chi in the orthotopic pancreatic tumor model was observed using near-infrared imaging and the human pancreatic cancer orthotopic xenografts were treated with different nanoparticles and normal saline control. The expression of FR in COLO357 was the highest among the six pancreatic cancer cell lines. The FR mainly distributed on cell membrane and fewer in the cytoplasm in pancreatic cancer. Moreover, the absorption rate of the FA-Chi-Gem nanoparticles was more than the Chi nanoparticles without FA modified. The proliferation of COLO357 was significantly inhibited by FA-Chi-Gem nanoparticles. The PEG-FA-ICGDER02-Chi nanoparticles were enriched in tumor tissue in human pancreatic cancer xenografts, while non-targeted nanoparticles were mainly in normal liver tissue. PEG-FA-Gem-Chi significantly inhibited the growth of human pancreatic cancer xenografts (PEG-FA-Gem-Chi vs. Gem, t=22.950, P=0.000). PEG-FA-FITC-Chi nanoparticles might be an effective targeted drug for treating human FR-positive pancreatic cancer.

Phyllanthus emblica L. fruit extract induces chromosomal instability and suppresses necrosis in human colon cancer cells.

Phyllanthus emblica L. (PE) is an edible fruit indigenous to Southeast Asia. It has been considered as a potent functional food due to its numerous pharmacological applications, such as anti-oxidant, antimicrobial, anti-diabetic and protection for multiple organs. The aim of this study was to evaluate the effects of a water extract of PE fruit on genomic damage and cell death in the human colon adenocarcinoma cell line COLO320 using the cytokinesis-block micronucleus cytome assay. Cells were exposed to RPMI-1640 medium containing 0, 20, 40, 80, or 160 μg/mL PE for 24, 48, 72, or 96 hours. The results showed that PE induced a significant decrease in necrosis (p < 0.001) and nuclear division index (NDI) (p < 0.001) in a dose- and time-dependent manner, and there was a highly significant correlation between the reduction of necrosis and NDI (r = 0.820, p < 0.001). Dose- and time-dependent increases (p < 0.001) in the frequency of chromosomal instability (CIN) were observed after PE exposure, and the frequency of CIN was negatively correlated with NDI (r = - 0.640, p < 0.001). PE also significantly increased apoptosis (p < 0.001), and there was a significant correlation of apoptosis with CIN (r = 0.566, p < 0.001). In conclusion, PE suppresses necrosis and delays mitotic progression, which results in massive CIN followed by apoptosis in COLO320 cells.

JAB1 regulates unphosphorylated STAT3 DNA-binding activity through protein–protein interaction in human colon cancer cells

Recent studies have revealed that unphosphorylated STAT3 forms a dimer, translocates to the nucleus, binds to the STAT3 binding site, and activates the transcription of STAT3 target genes, thereby playing an important role in oncogenesis in addition to phosphorylated STAT3. Among signaling steps of unphosphorylated STAT3, nuclear translocation and target DNA-binding are the critical steps for its activation. Therefore, elucidating the regulatory mechanism of these signaling steps of unphosphorylated STAT3 is a potential step in the discovery of a novel cancer drug. However, the mechanism of unphosphorylated STAT3 binding to the promoter of target genes remains unclear. In this study, we focused on Jun activation domain-binding protein 1 (JAB1) as a candidate protein that regulates unphosphorylated STAT3 DNA-binding activity. Initially, we observed that both unphosphorylated STAT3 and JAB1 existed in the nucleus of human colon cancer cell line COLO205 at the basal state (no cytokine stimulation). On the other hand, phosphorylated STAT3 did not exist in the nucleus of COLO205 cells at the basal state. Immunoprecipitation using nuclear extract of COLO205 cells revealed that JAB1 interacted with unphosphorylated STAT3. To investigate the effect of JAB1 on unphosphorylated STAT3 activity, RNAi studies were performed. Although JAB1 knockdown tended to increase nuclear STAT3 expression, it significantly decreased unphosphorylated STAT3 DNA-binding activity. Subsequently, JAB1 knockdown significantly decreased the expression levels of MDR1, NANOG, and VEGF, which are STAT3 target genes. Furthermore, the expression level of nuclear JAB1, but not nuclear STAT3, correlated with unphosphorylated STAT3 DNA-binding activity between COLO205 and LoVo cells. Taken together, these results suggest that nuclear JAB1 positively regulates unphosphorylated STAT3 DNA-binding activity through protein–protein interaction in human colon cancer cell line COLO205.

Expression of ligands of DNAM-1 and NKG2D in colonic cancer

Objective To investigate the expression of ligands of DNAM-1 and NKG2D in the colonic cancer.Methods The colonic cancer tissue and adjacent normal colonic tissues were collected from 42 colonic cancer patients who were admitted to the Tangdu Hospital of Fourth Military Medical University from June 2010 to January 2011 were retrospectively analyzed.The expressions of CD155,CD112 and MICA/B in the colonic cancer tissues and the normal colonic tissues were detected by immunohistochemistry.The expressions of CD155,CD112 and MICA/B in the colonic cell line SWll6,SW480,SW620 and Colo205 in the Duke's A,B,C and D phases were detected by cell cytometry.The relationship of the expressions of the 3 ligands and the clinicopathological parameters was analyzed using the Mann-Whitney U test,chi-square test and Fisher exact probobility.Results Week expression of CD155 was found in the normal colonic tissues,while the expressions of CD112 and MICA/B were not found.In the colonic cancer tissues,the expressions of CD155,CD112 and MICA/B were 81.0％,52.4％ and 47.6％,which were significantly increased.The expressions of CD155,CD112 and MICA/B were not correlated with the gender,tumor differentiation,lymph node metastasis and Duke's staging (P ＞ 0.05).The overall expression rates of CD155,CD112 and MICA/B in the colonic cancer cell line SWll6,SW480,SW620 and Colo205 were 88.9％,67.4％ and 42.3％,respectively.The overall expression of CD155 was significantly higher than CD112 and MICA/B (F =23.17,P ＜ 0.05).Conclusion CD155,CD112 and MICA/B express in the colonic cancer tissues and colonic cancer cell line SW116,SW480,SW620 and Colo205,and the expression of CD155 is the highest. Key words: Colonic neoplasms ; Natural killer cells ; Receptor; Immunotherapy

Design and synthesis of novel 4-(4-oxo-2-arylthiazolidin-3-yl)benzenesulfonamides as selective inhibitors of carbonic anhydrase IX over I and II with potential anticancer activity

The novel 4-(4-oxo-2-arylthiazolidin-3-yl)benzenesulfonamide derivatives were designed and synthesized for selective carbonic anhydrase IX (CA IX) inhibitory activity with anticancer potential. In the CA inhibition assay, 3f was found to be the most potent and selective inhibitor of CA IX with inhibitory constant (KI) value of 2.2 nM. Among the synthesized compounds, 3f showed IC50 values of 5.03 μg/ml (cisplatin: 6.56 μg/ml), 5.81 μg/ml (cisplatin: 5.85 μg/ml), and 23.93 μg/ml (cisplatin: 2.75 μg/ml) against COLO-205, MDA-MB-231, and DU-145 cell lines, respectively. At IC50, 3f caused cell shrinkage, nuclear condensation, and nuclear fragmentation events characteristic to apoptosis in the Hoechst 33258 and acridine orange-ethidium bromide staining studies of COLO-205 cells. In the Dalton's lymphoma ascites (DLA) solid tumor model 3f decreased tumor volume by 64.83% (cisplatin: 71.62%), while increase in mean body weight was found to be only 4.09% (cisplatin: 3.47%).

P-0301 - The Apoptotic and Cytostatic Influence of Maleimide Derivative on Colorectal Adenocarcinoma Cell Line Colo-205

P-0301 - The Apoptotic and Cytostatic Influence of Maleimide Derivative on Colorectal Adenocarcinoma Cell Line Colo-205

Abstract 1462: Development of a multiplexed RT-PCR-coupled liquid bead array assay for the molecular characterization of CTCs.

Abstract Introduction. The presence of circulating tumor cells (CTCs) in patients’ bloodstream is responsible for hematogenous metastases and as a “liquid biopsy” these cells should be considered as a useful tool for the detection and the characterization of micrometastasis. Molecular characterization of CTCs is really helpful for diagnostic and therapeutic purposes. We recently developed a liquid bead array hybridization assay for studying the expression of 6 genes in CTCs in breast cancer (Markou et al, Clin Chem 2011). The aim of the present study was to expand the potential of this assay by adding more genes of clinical interest in the genes panel. Materials and Methods. We designed and evaluated a multiplexed PCR-coupled liquid bead array for studying simultaneously the expression of 14 genes. We firstly designed in silico primers and capture probes for: a) breast cancer stem cell markers: ALDH1, CD24, CD44, b) EMT markers: SNAIL, TWIST-1 and VIM, c) breast cancer markers: ERBB2, mammaglobin A, MAGEA3, PTEN, d) epithelial markers: CK-19, e) housekeeping genes: PBGD, HPRT1, and PDCD4 as a gene related with apoptosis. The assay is based on the following steps: total RNA isolation, cDNA synthesis, multiplex PCR for 14 genes, biotinylation of multiplex PCR products, hybridization of the biotinylated multiplex PCR products to xMAP microspheres, addition of streptavidin-phycoerythrin and measurement in the Luminex® 100 analyzer. Every single step was optimized in terms of analytical specificity, repeatability and reproducibility using the breast cancer cell lines MCF-7, MDA-MB-231 and SK-BR-3. To evaluate the performance of this assay we used cancer cell lines originated from the following tissues: a. human colon (COLO-205), b. human cervix (HeLa) and c. human mammary gland (T-47D). Results. The total duration of the assay, including hands on work, is approximately 6h. The assay is highly specific for each gene in complex multiplexed formats. Intra-assay CVs (n=3) ranged from 0.2% to 15.3% and inter-assay CVs (n=3) ranged from 1.0% to 18.9%. The developed assay was successfully applied for studying gene expression in COLO-205, HeLa, MCF-7, MDA-MB-231, SK-BR-3 and T-47D cancer cell lines. Conclusions. The previously developed liquid bead array has been successfully expanded including 8 additional gene targets of clinical interest. The developed assay could be applied for the molecular characterization of CTCs, since the expression of 14 genes can be measured simultaneously and reliably, thereby saving precious sample and reducing the costs and time of analysis. The developed assay will be further extended to include additional gene targets. Citation Format: Cleo Parisi, Athina Markou, Areti Strati, Evi Lianidou. Development of a multiplexed RT-PCR-coupled liquid bead array assay for the molecular characterization of CTCs. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1462. doi:10.1158/1538-7445.AM2013-1462

Abstract 2337: Efficacy of the RAF/PI3Kα/anti-EGFR triple combination LGX818 + BYL719 + cetuximab in BRAFV600E colorectal tumor models.

Abstract Selective RAF inhibitors have a significant role in the treatment of patients with metastatic melanoma whose tumors express BRAFV600E with the majority of patients experiencing significant tumor regression. However in patients with colorectal cancer (CRC) whose tumors express BRAFV600E, response-rates appear to be much lower, with only a few patients reported to experience a partial tumor response to vemurafenib (Kopetz et al., 2010) or the combination of dabrafenib plus trametinib (Corcoran et al., 2012). LGX818 is a highly potent RAF inhibitor with selective anti-proliferative activity in cells expressing BRAFV600E. A distinguishing feature of LGX818 is its very long dissociation half-life from BRAFV600E which leads to strong and sustained target inhibition even following drug wash-out. In addition, LGX818 has a very wide therapeutic index, with tumor regression observed at doses as low as 3 mg/kg bid and excellent tolerability up to 300 mg/kg bid in melanoma xenograft models. In the CRC cell line Colo205 (BRAFV600E), LGX818 inhibits proliferation with an EC50 = 0.005 μM and in vivo leads to significant tumor regression at doses as low as 20 mg/kg. However, other BRAFV600E CRC cell lines do not appear to be as sensitive (EC50 = 0.018 to &amp;gt;2.7 μM) and this intrinsic resistance translates in vivo in xenograft models generated from these cell lines. Based on clinical and preclinical data, it is clear that combination strategies will be required if RAF inhibitors are going to play a significant role in the treatment of CRC. Recent publications have implicated feedback-mediated activation of EGFR in BRAFV600E CRC cells treated with RAF and MEK inhibitors and this led us to test combinations of LGX818 with anti-EGFR therapies such as cetuximab and erlotinib. In vitro and in vivo studies indicated that the combination of LGX818 with cetuximab or erlotinib can be highly synergistic, leading to enhanced cell killing. In vivo the combination of LGX818 + cetuximab led to complete inhibition of tumor growth at doses where no single-agent activity was observed. Since PIK3CA mutations often co-occur with BRAFV600E in CRC tumors, we also tested the alpha-selective PI3K inhibitor BYL719 in combination with LGX818 and cetuximab as a triple combination. In vitro, this triple combination produced synergistic anti-proliferative effects and in vivo resulted in tumor regression. These preclinical data led to the initiation of an ongoing Phase I/II trial to evaluate the triple combination of LGX818, cetuximab and BYL719 in BRAFV600E CRC. Based on the preclinical results described above it is anticipated that the combination of a RAF inhibitor with anti-EGFR therapy and a PI3K inhibitor will have superior efficacy to single-agent RAF inhibitor in BRAFV600E CRC. Citation Format: Giordano Caponigro, Z A. Cao, Xiaobin Zhang, Hui Q. Wang, Christine M. Fritsch, Darrin D. Stuart. Efficacy of the RAF/PI3Kα/anti-EGFR triple combination LGX818 + BYL719 + cetuximab in BRAFV600E colorectal tumor models. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2337. doi:10.1158/1538-7445.AM2013-2337

Impact of the 3D Microenvironment on Phenotype, Gene Expression, and EGFR Inhibition of Colorectal Cancer Cell Lines

Three-dimensional (3D) tumor cell cultures grown in laminin-rich-extracellular matrix (lrECM) are considered to reflect human tumors more realistic as compared to cells grown as monolayer on plastic. Here, we systematically investigated the impact of ECM on phenotype, gene expression, EGFR signaling pathway, and on EGFR inhibition in commonly used colorectal cancer (CRC) cell lines. LrECM on-top (3D) culture assays were performed with the CRC cell lines SW-480, HT-29, DLD-1, LOVO, CACO-2, COLO-205 and COLO-206F. Morphology of lrECM cultivated CRC cell lines was determined by phase contrast and confocal laser scanning fluorescence microscopy. Proliferation of cells was examined by MTT assay, invasive capacity of the cell lines was assayed using Matrigel-coated Boyden chambers, and migratory activity was determined employing the Fence assay. Differential gene expression was analyzed at the transcriptional level by the Agilent array platform. EGFR was inhibited by using the specific small molecule inhibitor AG1478. A specific spheroid growth pattern was observed for all investigated CRC cell lines. DLD-1, HT-29 and SW-480 and CACO-2 exhibited a clear solid tumor cell formation, while LOVO, COLO-205 and COLO-206F were characterized by forming grape-like structures. Although the occurrence of a spheroid morphology did not correlate with an altered migratory, invasive, or proliferative capacity of CRC cell lines, gene expression was clearly altered in cells grown on lrECM as compared to 2D cultures. Interestingly, in KRAS wild-type cell lines, inhibition of EGFR was less effective in lrECM (3D) cultures as compared to 2D cell cultures. Thus, comparing both 2D and 3D cell culture models, our data support the influence of the ECM on cancer growth. Compared to conventional 2D cell culture, the lrECM (3D) cell culture model offers the opportunity to investigate permanent CRC cell lines under more physiological conditions, i.e. in the context of molecular therapeutic targets and their pharmacological inhibition.

Growth Inhibition of Human Colon Carcinoma Cells by Sesquiterpenoids and Tetralones of Zygogynum calothyrsum

Bioassay-guided phytochemical investigation of Zygogynum calothyrsum using the human colon carcinoma cell lines COLO205 and KM12 led to the isolation of three new drimane-type sesquiterpenoids, 1β-p-hydroxy-E-cinnamoyldrimeninol (1), 1β-p-hydroxy-E-cinnamoyl-5α-hydroxydrimeninol (2), and methyl ether of 1β-p-hydroxy-E-cinnamoyl-12α-methoxydrimeninol (3). Also isolated was the known 1β-p-coumaroyloxypolygodial (4) together with two new tetralones, 3'-deoxyisozygolone A (5) and calothyrlone A (9), three known tetralones, isozygolone A (6), zygolone A (7), and 4'-O-methylzygolone A (8), and a known cinnamolide (10). Compounds 1, 7, and 8 demonstrated higher cytotoxicity against COLO205 (GI50 18, 17, and 11 μM, respectively) and KM12 (GI50 14, 14, and 17 μM, respectively) than the other compounds.

Novel quinolone substituted thiazolidin-4-ones as anti-inflammatory, anticancer agents: Design, synthesis and biological screening

Nuclear factor-kappaB (NF-κB) has been reported to regulate various genes involved in cancer and inflammation. Accordingly, drugs suppressing or inhibiting NF-κB may possess both anti-inflammatory and anticancer properties. A library of quinolone substituted thiazolidin-4-ones was docked into the active site of NF-κB and the top-ranked 31 compounds were synthesized and evaluated for anti-inflammatory and anticancer activity. The best-ranked compound 6b showed highest anti-inflammatory activity in carrageenan-induced paw edema model. In vitro anticancer studies revealed 1a and 16a as most active compounds against BT-549, HeLa, COLO-205 and ACHN human cancer cell lines. Compounds 1a and 16a exhibited NF-κB dependent anticancer properties and apoptosis mediated cell death. In vivo Ehrlich ascites carcinoma study further confirmed the antitumor activity of 1a and 16a.

The BH3 mimetic ABT-263 synergizes with the MEK1/2 inhibitor selumetinib/AZD6244 to promote BIM-dependent tumour cell death and inhibit acquired resistance

Tumour cells typically exhibit a G(1) cell cycle arrest in response to the MEK1/2 [mitogen-activated protein kinase/ERK (extracellular-signal-regulated kinase) kinase 1/2] inhibitor selumetinib, but do not die, and thus they acquire resistance. In the present study we examined the effect of combining selumetinib with the BH3 [BCL2 (B-cell lymphoma 2) homology domain 3]-mimetic BCL2 inhibitor ABT-263. Although either drug alone caused little tumour cell death, the two agents combined to cause substantial caspase-dependent cell death and inhibit long-term clonogenic survival of colorectal cancer and melanoma cell lines with BRAF(V600E) or RAS mutations. This cell death absolutely required BAX (BCL2-associated X protein) and was inhibited by RNAi (RNA interference)-mediated knockdown of BIM (BCL2-interacting mediator of cell death) in the BRAF(V600E)-positive COLO205 cell line. When colorectal cancer cell lines were treated with selumetinib plus ABT-263 we observed a striking reduction in the incidence of cells emerging with acquired resistance to selumetinib. Similar results were observed when we combined ABT-263 with the BRAF(V600E)-selective inhibitor PLX4720, but only in cells expressing BRAF(V600E). Finally, cancer cells in which acquired resistance to selumetinib arises through BRAF(V600E) amplification remained sensitive to ABT-263, whereas selumetinib-resistant HCT116 cells (KRAS(G13D) amplification) were cross-resistant to ABT-263. Thus the combination of a BCL2 inhibitor and an ERK1/2 pathway inhibitor is synthetic lethal in ERK1/2-addicted tumour cells, delays the onset of acquired resistance and in some cases overcomes acquired resistance to selumetinib.

Targeting FLIP and Mcl‐1 using a combination of aspirin and sorafenib sensitizes colon cancer cells to TRAIL

The multikinase inhibitor sorafenib is highly effective against certain types of cancer in the clinic and prevents colon cancer cell proliferation in vitro. Non-steroidal anti-inflammatory drugs, such as acetylsalicylic acid (aspirin), have shown activity against colon cancer cells. The aims of this study were to determine whether the combination of aspirin with sorafenib has enhanced anti-proliferative effects and increases recombinant human tumour necrosis factor-related apoptosis-inducing ligand (rhTRAIL)-induced apoptosis in the human SW948, Lovo, Colo205, Colo320, Caco-2 and HCT116 colon cancer cell lines. In four cell lines, aspirin strongly stimulated the anti-proliferative effects of sorafenib (∼four-fold enhancement) by inducing cell cycle arrest. Furthermore, combining low doses of aspirin (≤ 5 mm) and sorafenib (≤ 2.5 µm) greatly sensitized TRAIL-sensitive and TRAIL-resistant colon cancer cells to rhTRAIL, much more potently than either drug combined with rhTRAIL. The increase in rhTRAIL sensitivity was due to inhibition of FLIP and Mcl-1 protein expression following aspirin and sorafenib co-treatment, as confirmed by knock-down studies. Next, the clinical relevance of targeting FLIP and Mcl-1 in colon cancer was examined. Using immunohistochemistry, we found that Mcl-1 expression was significantly increased in colon adenoma and carcinoma patient material compared to healthy colonic epithelium, similar to the enhanced FLIP expression we recently observed in colon cancer. These results underscore the potential of combining low doses of aspirin with sorafenib to inhibit proliferation and target the anti-apoptotic proteins FLIP and Mcl-1 in colon cancer cells.

Construction of a eukaryotic expression vector expressing FOXQ1 and its expression in colorectal cancer cell line Colo-320

Construction of a eukaryotic expression vector expressing FOXQ1 and its expression in colorectal cancer cell line Colo-320

Photoinactivation of Gram Positive and Gram Negative Bacteria with the Antimicrobial Peptide (KLAKLAK)2 Conjugated to the Hydrophilic Photosensitizer Eosin Y

We test the hypothesis that the antimicrobial peptide (KLAKLAK)(2) enhances the photodynamic activity of the photosensitizer eosin Y upon conjugation. The conjugate eosin-(KLAKLAK)(2) was obtained by solid-phase peptide synthesis. Photoinactivation assays were performed against the Gram-negative bacteria Escherichia coli , Pseudomonas aeruginosa , and multidrug resistant Acinetobacter baumannii AYE, as well as the Gram-positive bacteria Staphylococcus aureus , and Staphylococcus epidermidis . Partitioning assays were performed with E. coli and S. aureus . Photohemolysis and photokilling assays were also performed to assess the photodynamic activity of the conjugate toward mammalian cells. Eosin-(KLAKLAK)(2) photoinactivates 99.999% of 10(8) CFU/mL of most bacteria tested at a concentration of 1 μM or below. In contrast, neither eosin Y nor (KLAKLAK)(2) cause any significant photoinactivation under similar conditions. The increase in photodynamic activity of the photosensitizer conferred by the antimicrobial peptide is in part due to the fact that (KLAKLAK)(2) promotes the association of eosin Y to bacteria. Eosin-(KLAKLAK)(2) does not significantly associate with red blood cells or the cultured mammalian cell lines HaCaT, COS-7, and COLO 316. Consequently, little photodamage or photokilling is observed with these cells under conditions for which bacterial photoinactivation is achieved. The peptide (KLAKLAK)(2) therefore significantly enhances the photodynamic activity of eosin Y toward both Gram-positive and Gram-negative bacteria while interacting minimally with human cells. Overall, our results suggest that antimicrobial peptides such as (KLAKLAK)(2) might serve as attractive agents that can target photosensitizers to bacteria specifically.