Cell Line LoVo Research Articles

Two new vinblastine-type N-oxide alkaloids from Catharanthus roseus

Two new vinblastine-type N-oxide alkaloids, 17-desacetoxyvinblastine -oxide (1) and 20′-deoxyvinblastine -oxide (2), were isolated from the leaves of Catharanthus roseus. The structures of 1 and 2 were established by the analysis of their nuclear magnetic resonance and HR-ESI-MS spectroscopic data. All alkaloids were evaluated for their cytotoxic activities against the human hepatocellular carcinoma (HepG2) cell line, human colorectal carcinoma (Lovo) cell line and human breast carcinoma (MCF-7) cell line by the MTT method in vitro, respectively. The results showed that cytotoxic activities of alkaloids 1 and 2 exhibited moderate inhibitory activity on the proliferation of three cancer cells.

Design, synthesis, quantum chemical studies and biological activity evaluation of pyrazole–benzimidazole derivatives as potent Aurora A/B kinase inhibitors

Novel pyrazole–benzimidazole derivatives have been designed and synthesized. The entire target compounds were determined against cancer cell lines U937, K562, A549, LoVo and HT29 and were screened for Aurora A/B kinase inhibitory activity in vitro. The compounds 7a, 7b, 7i, 7k and 7l demonstrated significant cancer cell lines and Aurora A/B kinase inhibitory activities. Molecular modeling studies suggested the derivatives have bound in the active site of Aurora A kinase through the formation of four hydrogen bonds. Quantum chemical studies were carried out on these compounds to understand the structural features essential for activity. The cellular activity of 7k was also tested by immunofluorescence.

Abstract 3263: Evaluation of the activity of rigosertib, an inhibitor of PI3K and MAPK pathways, in a preclinical model of colorectal cancer.

Abstract Background: Rigosertib is an inhibitor of the PI3K and MAPK kinase pathways and is currently being evaluated in clinical trials for pancreatic cancer and hematologic diseases. The PI3K/AKT and MAPK (RAS/RAF/MEK/ERK) signaling pathways have been identified to play a central role in the development and progression of CRC by modulating many different downstream targets that are essential for enhancing cellular survival and proliferation. Mutations in the PI3K (PIK3CA) and MAPK (RAS) pathways have been identified and their importance is well-established in oncogenesis and as drivers of tumor growth. The objective of this study is to determine the anti-proliferative and anti-tumor effects of rigosertib in a preclinical model of colorectal cancer (CRC). Experimental Design: We used colorectal cancer cell lines and a patient-derived explant model to investigate the growth inhibitory activity of rigosertib. We treated 25 CRC cell lines with rigosertib for 72 hours and measured the effect on proliferation by a sulforhodamine B (SRB) assay. We treated 5 patient-derived CRC explants with rigosertib (250 mg/kg/day-daily) for 28 days to determine anti-tumor activity in vivo. A tumor growth inhibition inhibition (TGI) ≥ 50% was considered sensitive. Results: Twenty-four CRC cell lines were sensitive to rigosertib with an IC50 > 1μM. The cell lines MIP-101, Lovo, and Caco-2 were the most sensitive reaching an IC50 at 0.0625 μM. The Colo678 cell line was resistant to rigosertib with an IC50 > 1μM. There was no association between mutational status in PIK3CA or RAS and sensitivity to rigosertib. Zero out of 5 CRC explants showed sensitivity to rigosertib. Conclusion: Rigosertib is a potent growth inhibitor in vitro of the majority of CRC cell lines tested with an IC50 < 1μM. Our upcoming studies will test the inhibitory effects of rigosertib on an additional 15 CRC explants in an attempt to develop biomarkers of sensitivity or resistance. Acknowledgements: The work was supported by a grant from Onconova Therapeutics® Inc. Citation Format: Alicia Purkey, Eun-Kee Song, Kevin Quackenbush, Stacey Bagby, John J. Arcaroli, Antonio Jimeno, Wells Messersmith. Evaluation of the activity of rigosertib, an inhibitor of PI3K and MAPK pathways, in a preclinical model of colorectal cancer. [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 3263. doi:10.1158/1538-7445.AM2013-3263

Abstract 1499: Role of Twist for maintenance of EMT in colon cancer cell .

Abstract Epithelial-mesenchymal transition or transformation (EMT) is a process characterized by loss of cell adhesion, repression of E-cadherin expression, and increased cell motility. Epithelial-mesenchymal transition (EMT) confers tumor cells with metastatic dissemination, cancer stem cell-like characters and drug resistance. However, the molecular mechanism for metastatic dissemination remains unclear. Initiation of metastasis involves invasion, which has many phenotypic similarities to EMT, including a loss of cell-cell adhesion mediated by E-cadherin repression and an increase in cell mobility. In this study, we induced EMT in colon cancer cell line LoVo with expression of Twist, a key transcriptional factor of EMT. The metastasis associated with EMT was determined E-cadherin, vimentin and β-catenin protein expression by western blot and morphological changes associated with EMT in these cells. We found that expression of Twist induced a morphological change associated with EMT in these cells. We also found that the E-cadherin was significantly decreased in Twist-overexpressing cells LoVo. Interestingly, we showed that β-catenin was activated in these Twist-overexpressing cells. Our results indicate that activation of β-catenin is required for the sustention of EMT-associated metastasis dissemination. Citation Format: Hye Ryeon Kim, SoYoung Kim, Young Ae Baik, Hye Kyung Hong, Woo Yong Lee, Yong Beom Cho. Role of Twist for maintenance of EMT in colon cancer cell . [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 1499. doi:10.1158/1538-7445.AM2013-1499

Nuclear localization of P-glycoprotein is responsible for protection of the nucleus from doxorubicin in the resistant LoVo cell line

The high expression of P-glycoprotein (P-gp) belongs to one of the most important factors causing multidrug-resistant (MDR) of cancer cells. P-gp is primarily associated with plasma membrane; however, small fraction of that protein is present in the nuclear envelope. Such phenomenon is observed in cancer cells and may result in the selection of MDR cells as the secondary tumor and/or resistant metastasis that significantly shorten patient survival rate. Here, we confirmed nuclear localization of P-gp in resistant LoVo cells and demonstrated its impact on doxorubicin efflux from the nucleus to cytoplasm. Furthermore, we showed that P-gp located at the nuclear envelope might have a different glycoside chain when compared to the form located in the cytoplasm. It suggests that the glycoside chain plays a role in the intracellular trafficking of P-gp and may decide about the destination place in the cell.

Effect of thymidine phosphorylase cDNA transfection on the inhibition of human colon carcinoma cell line by 5'-deoxy-5-fluorouridine

To investigate the inhibiting impact of 5'-deoxy-5-fluorouridine (5'-DFUR) on human colon carcinoma cell line LOVO after transfection of thymidine phosphorylase (TP) cDNA. TP cDNA was transfected into human colon carcinoma cell line LOVO with lentiviral vector pLenti6.3_MCS_IRES2-EGFP, and the transfection efficiency was analyzed by flow cytometry. TP mRNA and protein expressions were detected by RT-PCR and Western blotting respectively. The IC50 of 5'-DFUR on TP-transfected LOVO and parental cell were evaluated by MTT assay. The volumes of 5-FU converted from 5'-DFUR in media, where TP-transfected and parental LOVO were cultured, were detected by HPLC. The stable transfectants passed 5 generations were obtained and the transfection rate was 95%. Compared with parental cell, the RQ values of mRNA expression in TP-transfected LOVO was (282.5±86.8) folds higher significantly (P<0.01), also the TP protein expression of TP-transfected LOVO was obviously up-regulated as compared to parental cells. The IC50 value of 5'-DFUR of TP-transfectants was (1087.7±89.1) μmol/L, less than (1607.3±56.8) μmol/L of parental cells significantly (P<0.01), while there was no significant difference between parental cells and vector-transfectants [(1699.5±38.7) μmol/L, P>0.05]. HPLC revealed that when medium was added with 0, 500, 1000, and 2000 μmol/L of 5'-DFUR respectively, 0, 2.10, 3.13, and 7.19 μmol/L of 5-FU was found in the parental cells culture, while 0, 22.16, 30.94 and 40.02 μmol/L of 5-FU was found in TP-transfectants culture, but no 5-FU was found in the vector-transfectants culture. TP cDNA transfection into LOVO can up-regulate the TP mRNA and protein expressions, increase the 5-FU converted from 5'-DFUR, and enhance the cytotoxic effect of 5'-DFUR on the LOVO cells.

Study of critical role of c-Met and its inhibitor SU11274 in colorectal carcinoma

c-Met plays an important role in colorectal tumorigenesis and disease progression and thus is believed to be an attractive inhibitory target for receptor molecular therapeutic. SU11274 was identified as a small molecule, ATP competitive inhibitor of the catalytic activity of the c-Met kinase. Our study had investigated the relationship between the high expression of c-Met and colorectal carcinoma and the effect of c-Met inhibitor SU11274 in colorectal carcinoma in vitro and vivo. Immunohistochemistry was used to detect the expression of c-Met in 60 patients with colorectal cancer and 20 patients with benign adenoma and surrounding normal colon tissues. The effect of SU11274 on human colorectal carcinoma LoVo cells was detected by Western blot and MTT. And the influence of SU11274 on cell cycle was determined by flow cytometry. In addition, LoVo cell-transplanted tumor growth and expression of c-Met in nude mice was examined for inhibition of SU11274 in vivo. We found c-Met had high expression and was closely related to lymph node metastasis and TNM stage in colorectal carcinoma tissues. SU11274 significantly suppressed the phosphorylation of c-Met as well as the survival and proliferation of LoVo cell lines. G1-phase arrest was also induced by SU11274. SU11274 apparently restrained the growth of the xenograft tumor in nude mice. Our data suggest developing therapies that specifically inhibit the activation of c-Met may represent a novel therapeutic modality for patients with colorectal carcinoma expressing high levels of c-Met.

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.

Croton lechleri Müll. Arg. (Euphorbiaceae) stem bark essential oil as possible mutagen-protective food ingredient against heterocyclic amines from cooked food

The Amazonian Croton lechleri stem bark essential oil was tested for its anti-mutagenic potential by performing the Ames test against heterocyclic amines (HCAs), in continuing research on applicative functional profile of this phytocomplex as food ingredient (Rossi et al., 2011). Salmonella typhimurium strain TA98 was used with and without metabolic activation (S9 mix). The anti-mutagenic properties was assayed with the following HCAs: 2-amino-3-methylimidazo-[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo-[4,5-f]quinoline (MeIQ), 2-amino-3,8-dimethylimidazo-[4,5-f]quinoxaline (MeIQx), the imidazoles 2-amino-6-methyldipyrido-[1,2-a:3′,2′-d]imidazole (Glu-P-1) and 2-aminodipirydo-[1,2-a:3′,2′-d]imidazole (Glu-P-2). All HCAs with S9 induced mutagenicity at 10−10mol/plate. Without S9, IQ and MeIQ showed mutagenicity at 10−8mol/plate, MeIQx and Glu-P-1 at 10−5mol/plate, while Glu-P-2 was inactive. In presence of HACs (10−9mol/plate), C. lechleri essential oil was tested for mutagen-protective properties (concentration range: 0.01–0.10mg/plate) taking the Highest Uneffective Dose (HUD) as threshold reference. With S9 mix, C. lechleri essential oil displayed a significant reduction of revertants at 0.05mg/plate, from 21% to 34%. The essential oil showed mutagen-protective efficacy against IQ and MeIQ tested as direct mutagens (10−7mol/plate), with a revertants percentage reduction of 39% and 40%, respectively. No anti-mutagen capacity was noted for MeIQx and Glu-P-1 (10−5mol/plate). Since HACs are known as possible colon and liver cancer inducers, C. lechleri essential oil was tested for its cytotoxicity and anti-proliferative capacity against LoVo and HepG2 cancer cell lines showing IC50 of 74.95±0.05μg/ml (LoVo) and 82.28±0.03μg/ml (HepG2), displaying a promising role of this essential oil as a functional food ingredient with interesting mutagen preventing properties.

Peroxisome Proliferator-activated Receptor γ Agonists Induce Cell Cycle Arrest through Transcriptional Regulation of Krüppel-like Factor 4 (KLF4)

Peroxisome proliferator-activated receptor γ (PPARγ), a subgroup of ligand-activated nuclear receptors, plays critical roles in cell cycle regulation, differentiation, apoptosis, and invasion. PPARγ is involved in tumorigenesis and is a potent target for cancer therapy. PPARγ transactivation of KLF4 has been demonstrated in various studies; however, how PPARγ regulates KLF4 expression is not clear. In this study, we reveal that PPARγ regulates the expression of KLF4 by binding directly to the PPAR response element (PPRE) within the KLF4 promoter. The PPRE resides at -1657 to -1669 bp upstream of the KLF4 ATG codon, which is essential for the transactivation of troglitazone-induced KLF4 expression. Furthermore, we found that stable silencing of KLF4 obviously suppressed the G(1)/S arrest and anti-proliferation effects induced by PPARγ ligands. Taken together, our data indicate that up-regulation of KLF4 upon PPARγ activation is mediated through the PPRE in the KLF4 promoter, thus providing further insights into the PPARγ signal transduction pathway as well as a novel cancer therapeutic strategy.

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.

Soluble FAS in the prediction of benefit from cetuximab and irinotecan for patients with advanced colorectal cancer

The FAS/FASL system, comprising membrane-bound (mFAS and mFASL) and soluble forms (sFAS and sFASL), has been related to apoptosis driven by chemotherapy administration. In vitro experiments show chemotherapy upregulating membrane-bound forms, leading to an increase of receptor availability (at 24-72 h) and favoring apoptosis. The regulatory effect of chemotherapy on sFAS in patients has never been explored prospectively in advanced colorectal cancer (ACRC). We performed a pharmacodynamic study to address sFAS/sFASL variation. A prospective phase II translational multicenter study was designed to evaluate progression-free rate (PFR) in patients with ACRC treated with irinotecan and cetuximab in third-line therapy. The effect of sFAS was studied in vitro in colorectal cancer cell lines. Our results showed that statistically significant changes were observed in sFAS at 24-72 h compared to baseline levels in the pharmacodynamic study. Of the 93 patients enrolled in the prospective study in third-line therapy with cetuximab-irinotecan, 85 were evaluated for sFAS/sFASL changes at 48 h. There was no difference in PFR at 4 months between patients with sFAS and sFASL changes. In vitro analysis showed that although LoVo cell lines were sensitive to oxaliplatin and fluorouracil due to modulation of sFAS and FAS, HT29 lines were not. In summary, chemotherapy regulates FAS soluble fractions in vitro and in vivo, but does not predict PFR in ACRC patients undergoing third-line therapy with the combination of cetuximab and irinotecan.

Tridentate bipyrazole compounds with a side-arm as a new class of antitumor agents

Eight tridentate bipyrazole derivatives with different side arms have been prepared in one step and with good yields. The products were screened for their cytotoxic activity against three tumor cell lines—human breast cancer cell line MDA-MB231, human prostate cancer cell line PC3, and human colorectal cell line LoVo, by use of colorimetric MTT assay. Structure–activity relationships reflected the effect of substituted drugs. Among this series, two compounds had remarkable in-vitro antiproliferative activity against the LoVo cell line with IC50 values ranging from 2.6 to 2.7 μg ml−1. All the compounds had suitable drug-like characteristics according to Lipinski’s rule.

MicroRNA-7 is a novel inhibitor of YY1 contributing to colorectal tumorigenesis

Using microRNA (miRNA) expression array, we identified that miR-7 was deregulated in colorectal cancer (CRC). We studied the biological role and molecular target of miR-7 in CRC. miR-7 was downregulated in six out of seven colon cancer cell lines. Ectopic expression of miR-7 suppressed colon cancer cell proliferation (P<0.05), induced apoptosis (P<0.05) and caused cell-cycle arrest in G1 phase (P<0.05). The tumor suppressive function of miR-7 was further confirmed in nude mice (P<0.05). The 3'-untranslated region (3'UTR) of Yin Yang 1 (YY1) mRNA contains an evolutionarily conserved miR-7 binding site using in silico searches, luciferase reporter assay and western blot analysis confirmed that miR-7 directly bound to YY1 3'UTR to negatively regulate the protein expression of YY1 in colon cancer cell lines HCT116 and LOVO. Intriguingly, knock-down of YY1 in three colon cancer cell lines (HCT116, LOVO and DLD1) consistently suppressed cell proliferation (P<0.01) and induced apoptosis (P<0.01), indicating the opposite functions of miR-7 and YY1 in CRC. Consistent with these data, ectopic expression of YY1 promoted cell growth by increasing proliferation (P<0.01) and suppressing apoptosis (P<0.001). The tumorigenic ability of YY1 was further confirmed in vivo in xenograft-nude mouse model (P<0.01). In addition, pathway analyses revealed that the oncogenic effect by YY1 was associated with inhibiting p53 and modulating its downstream effectors p15, caspase cascades and C-Jun, and activating Wnt signaling pathway through activating β-catenin, anti-apoptotic survivin and fibroblast growth factor 4. Furthermore, multivariate analysis revealed that patients with YY1 protein high expression had a significant decrease in overall survival, and Kaplan-Meier survival curves showed that these patients had significantly shorter survival than others (P<0.0001). In conclusion, MiR-7 is a novel miRNA with tumor suppressive function in colon cancer by targeting oncogenic YY1. YY1 promotes colon cancer growth through inhibiting p53 and promoting Wnt signaling pathways and serves as an independent prognostic biomarker for CRC patients.

Systems Analysis of Cancer Cell Heterogeneity in Caspase-dependent Apoptosis Subsequent to Mitochondrial Outer Membrane Permeabilization

Deregulation of apoptosis is a hallmark of carcinogenesis. We here combine live cell imaging and systems modeling to investigate caspase-dependent apoptosis execution subsequent to mitochondrial outer membrane permeabilization (MOMP) in several cancer cell lines. We demonstrate that, although most cell lines that underwent MOMP also showed robust and fast activation of executioner caspases and apoptosis, the colorectal cancer cell lines LoVo and HCT-116 Smac(-/-), similar to X-linked inhibitor of apoptosis protein (XIAP)-overexpressing HeLa (HeLa XIAP(Adv)) cells, only showed delayed and often no caspase activation, suggesting apoptosis impairment subsequent to MOMP. Employing APOPTO-CELL, a recently established model of apoptosis subsequent to MOMP, this impairment could be understood by studying the systemic interaction of five proteins that are present in the apoptosis pathway subsequent to MOMP. Using APOPTO-CELL as a tool to study detailed molecular mechanisms during apoptosis execution in individual cell lines, we demonstrate that caspase-9 was the most important regulator in DLD-1, HCT-116, and HeLa cells and identified additional cell line-specific co-regulators. Developing and applying a computational workflow for parameter screening, systems modeling identified that apoptosis execution kinetics are more robust against changes in reaction kinetics in HCT-116 and HeLa than in DLD-1 cells. Our systems modeling study is the first to draw attention to the variability in cell specific protein levels and reaction rates and to the emergent effects of such variability on the efficiency of apoptosis execution and on apoptosis impairment subsequent to MOMP.

Croton lechleri sap and isolated alkaloid taspine exhibit inhibition against human melanoma SK23 and colon cancer HT29 cell lines

Ethnopharmacological relevanceCroton lechleri Mull. Arg. (Euphorbiaceae) is a traditional medicinal plant which produces a red sap, traditionally known as “Sangre de Drago”; it is used in folk medicine externally for wounds, fractures, and haemorrhoids, internally for intestinal and stomach ulcers and also for the empirical cure of cancers. Materials and methodsWe investigated the effects of Croton lechleri sap and taspine in comparison with taxol and vinblastine on the growth of human cancer cell lines of SK23 (melanoma), LoVo and HT29 (colorectal cancer) using MTT and Trypan blue assays. Further, we studied cell cycle by flow cytometry and detected acetylated-α-tubulin by confocal microscope. ResultsCroton lechleri inhibited cell proliferation starting from 1μg/mL in SK23 cells, whereas 10 times higher concentrations were required for growth inhibition of HT-29 and LoVo cell lines. Also taspine (0.1μg/mL) inhibited the SK23 and HT29 cell proliferation. Further, assay was assessed on SK23 and HT29 cell lines with 24–48h treatment with sap and taspine. Both sap and taspine inhibited cancer cell proliferation; taspine showed higher activity on SK23 cells, which was significantly increased after 48h of SK23 treatment. Using confocal microscopy we observed that Croton lechleri (1μg/mL) caused a loss of microtubule structure, whereas taspine (0.5μg/mL) caused an increase in acetylated α-tubulin and a modification of cellular morphology, mainly in SK23 cells. Croton lechleri sap 10 and 50μg/mL influence cell cycle; 50μg/mL sap caused a dramatic reduction of cells in G1/G0 and S phases with a great increase of subG0 cells. ConclusionsThe data showed that Croton lechleri and taspine could inhibit cell proliferation with higher potency against melanoma SK23 cells, supporting the empirical use of the sap as anticancer in ethnomedicine and taspine as a possible anticancer agent.

Scutellarin-cyclodextrin conjugates: Synthesis, characterization and anticancer activity

A series of scutellarin-cyclodextrin conjugates (SCU-CD conjugates), in which scutellarin was covalently bound to one of the primary hydroxyl groups of β-CD, were prepared, and their structures were determined using NMR and MS. These conjugates were further characterized by XRD and TG. The results showed that the aqueous solubility of the conjugates was much higher than that of scutellarin, and the conjugates could hardly be hydrolyzed to scutellarin in aqueous solutions. The cytotoxicity of SCU-CD conjugates on human colon cancer cell lines HT-29, SW480, Lovo and HTC116 indicated that the antitumor activities of the conjugates were better than that of scutellarin. This high antitumor activity, along with the satisfactory aqueous solubility and high stability of the conjugates, will be potentially useful for their application on human colon cancer chemotherapies.

Effect of overexpresion of ALDH1A2 gene on LoVo cell invasion and metastasis

Objective To evaluate the effects of ALDH1A2 gene over-expression on human LoVo cell line proliferation,invadesion and adhesion.Methods Human eukaryotic expression plasmid pEGFP-N1-ALDH1A2 were transfected to LoVo cell line.Reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting were used to detect the changes of ALDH1A2 gene expression,A methyl thiazol tetrazolium (MTT) assay,Matrigel invasion assay,plate clony-forming test and adhesion test were performed to assess the LoVo cells' proliferative activity,invadsion ability,clony-forming capability and adhesive ability before and after transfection.Results RT-PCR and Western blotting analysis showed that LoVo cells with pEGFP-N1-ALDH1A2 transfected increased the ALDH1A2 gene expression compared with control cells.ALDH1A2 over-expression can reduce LoVo cells proliferation,invadsion ability,clony-forming capability and adhesive ability (P ＜ 0.05 ).Conclusion ALDH1A2 over-expression can inhibit the proliferation and clony-forming ability of human LoVo cell. Key words: Colon carcinoma; ALDH1A2; Clone; Invasion; Adhesion

Hypoxia enhances colon cancer migration and invasion through promotion of epithelial-mesenchymal transition

BackgroundA hypoxic environment exists in most solid tumors because in rapidly growing tumors, the development of angiogenic vasculature is heterogenous, usually not enough to overcome the necessary oxygen supply. In an ischemic condition, cancer cells develop escape mechanisms to survive and leave the unfavorable environment. That result in the acquisition of increased potential for local invasion and evasion to distant organs. However, the escape mechanisms of cancer cells from hypoxic stress have not been fully characterized. Materials and methodsThe human colon cancer cell line LoVo was cultured in hypoxia, and the adhesive and migratory properties were analyzed. The expression of cell surface and cytoplasmic molecules was also investigated. ResultsUnder hypoxic conditions, cells developed epithelial-mesenchymal transition. The expression levels of α2, α5, and β1 integrins were significantly upregulated and, as a consequence, the ability to adhere to and migrate on collagen and fibronectin was increased. On the other hand, the expression of 67-kDa laminin receptor and the abilities to adhere to and migrate on laminin were decreased. Additionally, the expression of CXCR4 was significantly increased on cells cultured in hypoxia, and the chemotactic activity to stromal cell–derived factor 1α was remarkably increased. ConclusionsHypoxic stress induced active epithelial-mesenchymal transition in colon cancer cells, with the typical morphologic and functional changes. These morphologic and functional changes of β1 integrins, the 67-kDa laminin receptor, and CXCR4 may be essential for the acquisition of the invasive and metastatic features in colorectal cancer.

CARMA3 is overexpressed in colon cancer and regulates NF-κB activity and cyclin D1 expression

CARMA3 was recently reported to be overexpressed in cancers and associated with the malignant behavior of cancer cells. However, the expression of CARMA3 and its biological roles in colon cancer have not been reported. In the present study, we analyzed the expression pattern of CARMA3 in colon cancer tissues and found that CARMA3 was overexpressed in 30.8% of colon cancer specimens. There was a significant association between CARMA3 overexpression and TNM stage (p=0.0383), lymph node metastasis (p=0.0091) and Ki67 proliferation index (p=0.0035). Furthermore, knockdown of CARMA3 expression in HT29 and HCT116 cells with high endogenous expression decreased cell proliferation and cell cycle progression while overexpression of CARMA3 in LoVo cell line promoted cell proliferation and facilitated cell cycle transition. Further analysis showed that CARMA3 knockdown downregulated and its overexpression upregulated cyclin D1 expression and phospho-Rb levels. In addition, we found that CARMA3 depletion inhibited p-IκB levels and NF-κB activity and its overexpression increased p-IκB expression and NF-κB activity. NF-κB inhibitor BAY 11-7082 reversed the role of CARMA3 on cyclin D1 upregulation. In conclusion, our study found that CARMA3 is overexpressed in colon cancers and contributes to malignant cell growth by facilitating cell cycle progression through NF-κB mediated upregulation of cyclin D1.