Cell Line LoVo Research Articles

Colorectal cancer cell growth inhibition by linoleic acid is related to fatty acid composition changes

Polyunsaturated fatty acids (PUFAs) possess anti-cancer action both in vitro and in vivo. In the present study, we detected cell viability with methyl thiazolyl tetrazolium (MTT) assay and cell membrane permeability with propidium iodide (PI) fluorescence dyeing, and calculated cell membrane fluidity change as fluorescence anisotropy. Fatty acid content in cells was measured by gas chromatography/mass spectroscopy (GC/MS), and the relationship between fatty acid composition and cell viability was studied. We observed that n-6 PUFA linoleic acid (LA) inhibited tumor cell growth at high concentrations (≥300 µmol/L), while low concentrations (100-200 µmol/L) seemed to promote cell proliferation. Analyses of cell membrane permeability, cell membrane fluidity, and cell fatty acid composition suggested that the anti-cancer action of LA could be related to changes in the ratio of n-6 to n-3 PUFAs. We observed that pre-incubation of cancer cells with 100 µmol/L LA for 24 h enhanced cell sensitivity to the cytotoxic action of LA, whereas undifferentiated cell line LoVo seemed to have a distinct path in LA-induced death. These results showed that one of the mechanisms by which supplementation of LA induces cancer cell death could be altering the ratio of n-6/n-3 PUFAs, and this may be related to cell differentiation status.

Preventive effects of berberine on experimental colon cancer and relationship with cyclooxygenase-2 expression

To investigate the anti-colon cancer effects of berberine and possible relationship with cyclooxygenase-2. Wistar rat colon cancer model was induced by 1-2 dimethylhydrazine (DMH) (40 mg x kg(-1), sc) + 1% dextran sodium sulfate solution (DSS) (freely drinking). All rats were randomly divided into 3 groups: Control (DMH + DSS + solvant), meloxicam (Mel) (DMH + DSS + Mel 1.35 mg x kg(-1)), berberine (Ber) (DMH + DSS + Ber 100 mg x kg(-1)). The drugs were given orally once a day for 5 day per week. The body weight, the number of colon ACFs, the incidence and number of colon cancer in rats, as well as the morphological changes of rat colon tissues were evaluated. Human colon cancer lovo cell line was treated by either Ber or Mel in various concentrations (1 10(-6) mol x L(-1), 1 x 10(-5) mol x L(-1), 1 x 10(-4) mol x L(-1), 1 x 10(-3) mol x L(-1)) for 6, 12 and 24 h, respectively, and the cell growth was assayed by MTT method. RT-PCR and western-blot were used to evaluate the mRNA and protein expressions of COX-2 from lovo cells treated with Ber and Mel. Ber significantly improved the dyscrasia induced by DMH + DSS, the both of body weight and general condition were better than control group. Ber also significantly inhibited ACF and colon cancer incidence in the rats treated by DMH + DSS for 10 weeks or 20 weeks, which was similar to that of Mel. Ber inhibited the proliferation of lovo cells in concentration- and time-dependent manners, and the IC50 values were significantly smaller than that of Mel at 6, 12 and 24 h after lovo cells were treated by either Ber or Mel. Ber also concentration-dependently decreased expressions of COX-2 mRNA and COX-2 protein from lovo cells. Ber can inhibit ACF and tumor formation induced by DMH + DSS, and decrease the lovo cell proliferation index. The anti-tumor effects of Ber may involve in an unknown pathway through which the expressions of COX-2 mRNA and protein were inhibited.

The serine protease inhibitor serpinE2 is a novel target of ERK signaling involved in human colorectal tumorigenesis

BackgroundAmong the most harmful of all genetic abnormalities that appear in colorectal cancer (CRC) development are mutations of KRAS and its downstream effector BRAF as they result in abnormal extracellular signal-related kinase (ERK) signaling. In a previous report, we had shown that expression of a constitutive active mutant of MEK1 (caMEK) in normal rat intestinal epithelial cells (IECs) induced morphological transformation associated with epithelial to mesenchymal transition, growth in soft agar, invasion and metastases in nude mice. Results from microarrays comparing control to caMEK-expressing IECs identified the gene encoding for serpinE2, a serine protease inhibitor, as a potential target of activated MEK1.Results1- RT-PCR and western blot analyses confirmed the strong up-regulation of serpinE2 expression and secretion by IECs expressing oncogenic MEK, Ras or BRAF. 2- Interestingly, serpinE2 mRNA and protein were also markedly enhanced in human CRC cells exhibiting mutation in KRAS and BRAF. 3- RNAi directed against serpinE2 in caMEK-transformed rat IECs or in human CRC cell lines HCT116 and LoVo markedly decreased foci formation, anchorage-independent growth in soft agarose, cell migration and tumor formation in nude mice. 4- Treatment of CRC cell lines with U0126 markedly reduced serpinE2 mRNA levels, indicating that expression of serpinE2 is likely dependent of ERK activity. 5- Finally, Q-PCR analyses demonstrated that mRNA levels of serpinE2 were markedly increased in human adenomas in comparison to healthy adjacent tissues and in colorectal tumors, regardless of tumor stage and grade.ConclusionsOur data indicate that serpinE2 is up-regulated by oncogenic activation of Ras, BRAF and MEK1 and contributes to pro-neoplastic actions of ERK signaling in intestinal epithelial cells. Hence, serpinE2 may be a potential therapeutic target for colorectal cancer treatment.

Imaging analysis of the gliadin direct effect on tight junctions in an in vitro three-dimensional Lovo cell line culture system

Tight junctions play a pivotal role in maintaining the integrity of the intestinal barrier. Their alteration is involved in the pathogenesis of celiac disease. Our aim was to investigate the gliadin effect on the tight junction proteins in an in vitro three-dimensional cell culture model through imaging analyses. Lovo multicellular spheroids were treated with enzymatically digested (PT) gliadin 500 μg/mL and its effect on actin, occludin and zonula occludens-1, was evaluated by means of confocal laser microscopy, transmission electron microscopy and image capture analysis. Compared to untreated spheroids, PT-gliadin-treated ones showed enlargement of the paracellular spaces (9.0 ± 6.9 vs. 6.2 ± 1.7 nm, p < 0.05) at transmission electron microscopy and tight junction protein alterations at confocal microscopy and image analyses. In untreated cell cultures thickness of the fluorescence contour of actin, zonula occludens-1 and occludin appeared significantly larger and more intense than in the treated ones. In occludin planimetric analysis the lengths of the integral uninterrupted cellular contour appeared longer in untreated than in PT-gliadin treated spheroids (71.8 ± 42.8 vs. 23.4 ± 25.9 μm, p < 0.01). Our data demonstrated that tight junction proteins are directly damaged by gliadin as shown by means of quantitative imaging analysis.

Possibility of paclitaxel as an alternative radiosensitizer to 5-fluorouracil for colon cancer

To evaluate the feasibility of paclitaxel (PTX) radiosensitization for colon cancer, we investigated the cytotoxic and G2/M checkpoint protein (Chk1, Wee1, Bub1, MAD2) effects of 5-fluorouracil (5-FU) or PTX combined with radiation in the human colon cancer cell line LoVo. Cytotoxicity and radiocytotoxicity were evaluated for each drug by the WST-8 colorimetric assay. The IC20 for each drug was determined as a cytotoxic concentration from a survival curve. LoVo cells were exposed to the IC20 of each drug for 24 h and then irradiated. Expressions of the G2/M checkpoint proteins were confirmed by Western blot analysis. Cytotoxicity was induced by 5-FU or PTX alone in a time- and dose-dependent manner. The IC20 of PTX caused higher radiosensitivity than the IC20 of 5-FU (P<0.05). Western blot analysis revealed different expression patterns of the G2/M checkpoint proteins between 5-FU and PTX pre-treatments. 5-FU combined with radiation tended to decrease the expressions of all G2/M checkpoint proteins in a time-dependent manner. PTX combined with radiation maintained high expressions of Chk1 and MAD2 proteins for 24 h post-radiation and, in particular, MAD2 protein was strongly induced by PTX with high-dose radiation. PTX showed higher radio-sensitization than 5-FU for the colon cancer cell line LoVo and may be an alternative radiosensitizer to 5-FU in the clinical setting.

The use of protein array to identify targetable receptor tyrosine kinases for treatment of human colon cancer

Several studies have reported that activated receptor tyrosine kinases (RTKs) are highly expressed in colon cancer and may promote tumor growth and survival. However, there is little information available as to the function and signaling of RTKs in colon cancers. In the present study, we performed protein array technology to determine the expression status of various RTKs that are activated in colon cancer compared to normal colonic cells and tissues. Of the 42 different phospho-RTKs, 5 (ErbB2, FGFR1, FGFR2a, FGFR3 and MSPR) were activated in Caco-2, SW480, WiDr, Lovo colon cancer cell lines and cancerous tissues. In order to determine the effect of inhibition of RTKs, especially ErbB2, athymic nude mice bearing xenograft tumors were treated with the ErbB2-targeting drug trastuzumab alone, or in combination with 5-Fluorouracil (5-FU). Similar to the treatment of 5-FU alone, trastuzumab suppressed the growth of colon cancer. Combination therapy of trastuzumab and 5-FU inhibited tumor growth significantly compared to the treatment of 5-FU alone or trastuzumab alone. In addition, xenograft tumors were also analyzed by phospho-MAPK protein array. The activity of Akt3/PKBgamma was inhibited with 5-FU alone and trastuzumab, indicating that trastuzumab may inhibit colon cancer growth through ErbB2-Akt3/PKBgamma signaling. These data demonstrate that ErbB2 could be an important candidate for colon cancer therapy and the addition of trastuzumab to 5-FU therapy might augment the clinical response in colon cancer patients. Therefore, the analysis of phospho-RTK expression by protein array as a useful tool might identify novel therapies for individual patients with colon cancer.

MicroRNA-195 promotes apoptosis and suppresses tumorigenicity of human colorectal cancer cells

Deregulated microRNAs and their roles in cancer development have attracted much attention. In the present study, we analyzed the roles of miR-195 in colorectal cancer pathogenesis, as its participation in some other types of cancer has been suggested by previous reports. By comparing miR-195 expression in 81 human colorectal cancer tissues and matched non-neoplastic mucosa tissues, we found that miR-195 was downregulated in cancer tissues. And restoration of miR-195 in colorectal cancer cell lines HT29 and LoVo could reduce cell viability, promote cell apoptosis and suppress tumorigenicity. Moreover, important antiapoptotic Bcl-2 was identified to be directly targeted by miR-195, and miR-195 was further suggested to exert its proapoptotic function mainly through targeting Bcl-2 expression. Taken together, our study provides important roles of miR-195 in colorectal cancer pathogenesis and implicates its potential application in cancer therapy.

Two new triterpenoids from the roots of Actinidia chinensis

Two new triterpenoids ( 1, 2), together with one flavonoid glycoside and thirteen known triterpenoids were isolated from the roots of Actinidia chinensis Planch (Actinidiaceae). The structures of the new constituents were elucidated as 12α-chloro-2α, 3β, 13β, 23-tetrahydroxyolean-28-oic acid-13-lactone ( 1), 2α, 3α, 19α, 23, 24-pentahydroxyurs-12-en-28-oic acid ( 2). Structure elucidation was accomplished by 1D, 2D NMR spectra (HMQC, HMBC, 1H- 1H COSY, TOCSY, and NOESY) and mass spectrometry (ESIMS). Moreover, two known triterpenoids showed positive cytotoxic activity against LOVO and HepG2 cell lines.

Functional characterisation of cell cycle-related kinase (CCRK) in colorectal cancer carcinogenesis

Cell cycle-related kinase (CCRK) is a newly identified protein kinase homologous to Cdk7. We have previously shown that CCRK is a candidate oncogene in human glioblastoma. However, whether CCRK is a bona fide oncogene remains to be tested. The aim of this study was to investigate the role of CCRK in human colorectal cancer carcinogenesis. By Western blotting, we analysed the expression profile of CCRK protein in 10 colorectal cancer tissue samples and their adjacent normal colon tissues and in seven colorectal cancer cell lines. CCRK protein expression was also investigated by immunohistochemistry in a colorectal tissue microarray, which contained 120 cases of primary colorectal cancer and adjacent normal colorectal mucosa. The effects of CCRK knock-down on cell cycle profile and proliferation of colorectal cancer cells were examined by transfecting LoVo and DLD1 human colorectal cancer cell lines by either short-hairpin RNA (shCCRK) or small interfering RNA targeting CCRK (siCCRK). We found that CCRK protein levels were elevated by more than 1.5-fold in 70% of colorectal cancer patient samples examined and CCRK was detectable in all seven colorectal cancer cell lines tested. Colorectal tissue microarray indicated that overexpression of CCRK was detected in 62/109 (56.9%) of informative colorectal cancer cases and was significantly associated with the tumour pT and pN status ( p < 0.05). Suppression of CCRK by siCCRK led to G1 phase cell cycle arrest and reduced cell growth. Consistently, stable clones of LoVo and DLD1 cells expressing shCCRK exhibited decreased cell proliferation rates. Furthermore, we showed that CCRK is required for the phosphorylation of Cdk2 (on Thr-160) and Rb (on Ser-795) and the expression of cyclin E. These results suggest for the first time that CCRK is involved in colorectal cancer carcinogenesis and G1/S cell cycle transition by regulating Cdk2, cyclin E and Rb.

Evaluation of Cytotoxic Activity ofSchisandra chinensisLignans

Using exhaustive chromatographic separation we have isolated (-)-tigloyl-deangeloyl-gomisin F as a novel dibenzocyclooctadiene lignan from schisandra chinensis. With the help of HPLC, we further isolated (+)-schisandrin, (+)-deoxyschisandrin, (+)-γ-schisandrin, (-)-gomisin J, (+)-gomisin A, (-)-gomisin N, (-)-tigloyl-gomisin P, (-)-wuweizisu C, (-)-gomisin D, rubrisandrin A, (-)-gomisin G, (+)-gomisin K (3) and (-)-schisantherin C. A full NMR description of (-)-schisantherin C was carried out with the aim to confirm previous reports of its structure. Compounds isolated were identified on the basis of UV, IR, (1)H- and (13)C-NMR and MS. The cytotoxicity of lignans was tested for the BY-2 cell line alone and as a synergistic effect with the cytotoxic agent camptothecin. Lignans showed various toxicity and synergistic and antagonistic effects on camptothecin-induced cytotoxicity. Cytotoxicity against colon cancer cell line LoVo was also tested.

GABA-receptor agonist, propofol inhibits invasion of colon carcinoma cells

Propofol (2,6-diisopropylphenol), one of the most commonly used intravenous anaesthetic agents during cancer resection surgery, has been reported to have the ability of influencing the invasion of human cancer cells. In the present study, using the human colon carcinoma cell line LOVO, we demonstrated that propofol stimulation significantly decreased the expression of MMP-2 and -9 and subsequently decreased the invasive activity of the cancer cells. Because MAPK signaling is one of the key regulators of MMP expression, we further evaluated MAPK signaling after stimulation with propofol. It was found that propofol stimulation inhibited the phosphorylation of MAPKs, including ERK1/2, JNK, and p38. Deactivation of ERK1/2 phosphorylation was sustained for up to 12 h, while deactivation of phosphorylation of JNK and p38 returned to the endogenous level by 30 min. It was noteworthy that the ras/raf/MEK/ERK pathway inhibitor PD98059 attenuated the down-regulation of propofol-induced MMP-9 expression of LOVO cells. We also demonstrated that the propofol-induced decrease in invasive ability via ERK1/2 down-regulation was mediated mainly through the GABA-A receptor. These results indicate that propofol stimulation inhibits cancer cell invasion and that the effect is partly due to ERK1/2-dependent down-regulation of MMPs.

Development of a magnetite-gene complex for gene transfection

The key to successful gene therapy is to find a suitable method and carrier for transfection to allow a gene to be transferred into a cell and integrated into the target gene. The aim of this study was to determine whether biomagnetic material could be combined with the nucleic acid for gene transfection. Dextran-coated iron oxide nanoparticles (DCIONPs) were prepared and mixed with the plasmid pGenesil-1 containing the test gene, which expresses enhanced green fluorescent protein (eGFP). PGenesil-1 empty vector was used as a control. The binding ability was assessed by electrophoresis of the DNA on agarose gels and quantification using BANDSCAN software. Using different gene carriers, Lipofectamine 2000, Sofast, and DCIONPs, the large intestine cancer (Lovo) cell line was transfected in vitro with or without a magnetic field. The expression of eGFP was observed by fluorescence microscopy, and the transfection efficiency was compared. The results showed there was a rapid increase in combining rate when the quality ratio of DCIONPs and pGenesil-1 ascended from 1:1 to 5:1. However, the combining rate increased less rapidly as the quality ratio continued ascending. The expression of eGFP showed that the early transfection rate could be improved by applying a magnetic field. In conclusion, the DCIONPs we synthesized are able to carry plasmid DNA and enhance the early transfection efficiency when using a magnetic field.

Abstract 2546: IGR-1R pathway role in oxaliplatin acquired resistance in colon cancer cell lines and its regulation by MMP-7 and IGFBP-2

Abstract Background: IGF-1R pathway and MMPs have been implicated in chemotherapy resistance in colorectal cancer. This receptor could be activated by IGF1 or IGF2 ligands, which bioavailability is regulated by IGFBPs, what could be degraded by MMPs. Objectives: To determine the basal levels and the modulation by chemotherapy (oxaliplatin), of the levels of IGF-1R, IGFBPs and MMPs in colon cancer cell lines (HT-29 (IC50: 6,46 uM) and LoVo (IC50: 0,25 uM) and their oxaliplatin resistant cell lines (HT-29OXAR3 (IC50: 29,86 uM) and LoVOXAR3 (2,45 uM)). To analyze the differences in apoptosis between parental and oxaliplatin resistant cell lines when are treated with oxaliplatin. Methods: MMP-7 levels were analyzed by RT-PCR, Western Blot and ELISA. Levels of IGF-1R, activated IGF-1R, IGFBP-2, and IGFBP-3 were determined by Western Blot and/or ELISA. Differences in cell viability were analyzed by MTT, and intrinsic (bcl-2, bax) and extrinsic (Fas, caspase 8) apoptotic pathways, by Western Blot, in parental and oxaliplatin resistant cell lines with dose-response experiments. Results: LoVo and LoVOXAR3 express low levels of MMP-7 and IGF-1R and high levels of IGFBP-2 compared with HT-29 and HT-29OXAR3. In addition, HT-29OXAR3 cells showed increased MMP-7, IGF-1R and pIGF-1R levels compared with HT-29 and lower levels of IGFBP-2. We also observed after oxaliplatin treatment in HT-29 cells a decrement of IGF-1R and IGFBP-2 and an increment in MMP-7 levels. In apoptotic experiments, FAS and caspase 8 (extrinsic pathway) were induced by oxaliplatin in LOVO (0.2 uM) and in LOVOXAR3 (20uM). Bcl-2 and bax (intrinsic pathway) were induced or decremented respectively in LOVO (2uM) and LOVOXAR3 (20uM). No changes in these extrinsic apoptotic proteins were observed in HT29 and HT29OXR3. In HT-29OXAR3 cell line higher doses of oxaliplatin (20 uM instead of 2uM) were needed to induce intrinsic apoptotic (bax) and antiapoptotic proteins (bcl-2) Conclusions: IGF-1R signalling is implicated in acquired oxaliplatin resistance and it is regulated by MMP-7 and IGFBP-2 in a different manner in our two models of cancer colon cell lines. The two apoptotic pathways were implicated in LoVo cell lines, but only intrinsic pathway regulates apoptosis in HT-29 cell lines. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2546.

Abstract 5442: In vitro and in vivo upregulation of thymidine phosphorylase expression in colon cancer cells by HDAC inhibitor vorinostat resulted in synergistic antitumor effect in combination with capecitabine

Abstract The oral fluoropyrimidine capecitabine is converted to the active compound 5-florouracil (5-FU) in several steps, the last of which is the conversion of 5′-deoxy-5-fluorouridine (5′DFUR) to 5-FU by thymidine phosphorylase (TP). Capecitabine was designed to take advantage of the increased levels of TP observed in tumors as opposed to normal tissues, potentially allowing for selective toxicity in tumors. In this study we found that the antiproliferative effect induced by the histone deacetylase inhibitor vorinostat in colorectal cancer cells in vitro and in vivo xenograft models, was paralleled by downregulation of thymidilate synthase, an essential enzyme for DNA synthesis and the target of 5-FU, and by upregulation of TP. These effects were evident at the mRNA and at the protein level and were not observed in peripheral blood lymphocyte (PBL) from healthy donors treated ex vivo by vorinostat. On the basis of these results, we have evaluated in vitro the effects of the combination between vorinostat and the capecitabine metabolite 5′-DFUR on the growth of human LoVo, LS174T and SW620 colon cancer cell lines, showing that simultaneous exposure of equipotent doses of the two agents, for 96 hours, resulted in synergistic antiproliferative effect in all cell lines as demonstrated by calculating combination indexes. We also demonstrated that vorinostat and 5′-DFUR in combination, compared to single agents treatment, increased apoptotic cell death, demonstrated by flow cytometry analysis of DNA content after propidium iodide staining, by the induction of BAX protein expression and by the cleavage of PARP. The observed apoptotic effect could be, at least in part, related to the increase in the ROS content induced by vorinostat and 5′-DFUR combination compared to single agents treatment. In order to verify in vivo the synergistic effects on tumor cell growth demonstrated in vitro, we evaluated vorinostat in combination with capecitabine in SW620 colon cancer cells xenograft model. A marked inhibition of tumor growth was observed in mice group treated with vorinostat (100 mg/kg p.o. daily) plus capecitabine (359 mg/Kg p.o. daily) as compared to single agents treatment, with a consequent increase by 2.4-fold of tumor growth delay and a substantial increase in survival. Analysis of xenografts tumor sections by immunohistochemistry showed a significant increase in apoptotic cells in vorinostat plus capecitabine treated group compared with control, or single agent treated groups, as well as the down-regulation TS and upregulation TP proteins expression in mice treated with vorinostat alone or in combination with capecitabine. Overall these data suggested that the association of vorinostat plus capecitabine could be clinically explored in colon cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5442.

Abstract 3367: CD133- colorectal cancer cells have higher chemo-resistance ability than CD133+cells

Abstract (Background and purpose) Recently, many reports suggest that a limited population of tumor cells have the capability of tumorigenesis. These cells, called cancer stem cells (CSCs) or tumor initiating cells, are suggested to be involved in tumor recurrence after long interval as well as in chemo-resistance. There are several CSC markers, including CD44 and CD133. CD133, originally a hematopoietic stem cell marker, was identified also as a stem cell marker for various tumor types. In the present study, we aimed to compare the characteristics of CD133+ and CD133- colorectal cancer cells to test the hypothesis of CD133 being a potential specific marker for colorectal CSCs. (Methods) Using auto magnetic activated cell sorting (MACS), the human colon cancer cell line LoVo was separated into CD133+ and CD133- cell subpopulations, and their sensitivity to 5-FU, specially focusing on apoptosis and autophagy inductions were investigated. The sensitivity of CD133+ and - cells to 5-FU was tested by the MTS assay. And the induction of apoptosis was analyzed by flow-cytometry, after double-staining with annexin V-FITC/PI. The expression of β1-integrins was analyzed by flow-cytometry, and their ability to bind extracellular matrix (ECM) proteins was evaluated by the adhesion assay. Induction of autophagy was investigated by flow-cytometry after staining with acridine orange, and by Western blot for the expressions of the LC3 protein. (Results) After MACS isolation, CD133+ and CD133- cells were separately cultured. The purity of the obtained cell populations was more than 98%. After several days of culture, the proportion of CD133+ and CD133- cells was not changed in both cell populations during about a month. CD133- cells showed higher resistance to 5-FU than CD133+cells, in vitro. It was dependent on the higher induction of apoptosis in CD133+ cells. CD133+ cells had lower expression of β1-integrins compared with CD133- cells, and consequently, their ability to adhere ECM proteins was weaker. Additionally, AVOS formation was weaker in CD133+ cells, as well as the expression of LC3-II. (Discussion) CD133+ cells were more sensitive to 5-FU than CD133- cells, and it may be dependent on the higher ability of CD133- cells to induce autophagy in response to 5-FU. Additionally, CD133- cells had higher ability to adhere to ECM proteins through β1-integrins, and it may be another mechanism of chemo-resistance. Taking these results, higher ability to adhere to ECM proteins may confer resistance to 5-FU by improving the ability of cells to induce autophagy as a cell survival mechanism. Presently, there is controversy regarding CD133 as a specific marker of CSCs, but from our results, CD133- cells are more resistant to chemotherapy, one typical feature of CSCs. Therefore, we concluded that CD133 may not be used as a single CSC marker, but on the other hand, it may be a potential marker for the prediction of chemo-sensitivity in colorectal cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3367.

A new antitumor arabinopyranoside from Laurencia majuscula induces G2/M cell cycle arrest

A new arabinopyranoside was isolated from the alga Laurencia majuscula (Harvey) Lucas, collected from the Xisha Islands in the South China Sea. Its structure was elucidated as hexadecyl-1-O-α-L-arabinopyranoside by spectroscopic analysis. It was found that arabinopyranoside had significant antitumor activity in LOVO and Bel-7402 cell lines. Flow cytometric analysis showed that arabinopyranoside arrested the cell cycle in the G2/M phase. Western blotting demonstrated that the protein expression of CDK1 and cyclin A related to the G2/M phase decreased markedly with arabinopyranoside treatment, with slight changes in cyclin B1 expression. Taken together, the findings identify a potential new antitumor therapeutic arabinopyranoside isolated from red alga Laurencia majuscula.

Free radical scavenging, angiotensin I-converting enzyme (ACE) inhibitory, and in vitro anticancer activities of ramie (Boehmeria nivea) leaves extracts

The objectives of this study were to evaluate the antioxidant activity, angiotensin I-converting enzyme (ACE) inhibitory activity, and anticancer activity of ramie (Boehmeria nivea) leaves (RL). The RL was extracted with 70%(v/v) ethanol (RLE) and fractionated with the solvents of hexane, chloroform, ethylacetate, and aqueous. The ethylacetate fraction (EF) contained the highest phenolic contents of 651.65 mg/g, followed by RLE of 148.72 mg/g. The EF showed strong 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity, superoxide radical scavenging activity, and hydroxyl radical scavenging with a 50% inhibition concentration (IC50) of 0.097, 0.129, and 0.191 mg/mL, respectively. The ACE-inhibitory activity of EF was 80.32% at a concentration of 0.1 mg/mL. The EF showed growth-inhibitory effect of 67.21% at 0.25 mg/mL on the LoVo cell line, and 56.08% at 0.25 mg/mL on the NCI-H460 cell line, respectively. Therefore, it was suggested that the RL were potential materials for use as functional food and medicine.

Thioredxin reductase inhibitor ethaselen increases the drug sensitivity of the colon cancer cell line LoVo towards cisplatin via regulation of G1 phase and reversal of G2/M phase arrest

We evaluated the combination treatment of ethaselen (BBSKE) as a thioredoxin reductase (TrxR) inhibitor plus cisplatin (CDDP) on the human colon adenocarcinoma cell line LoVo. Therapeutic effects ranging from nearly additive to clearly synergistic demonstrated an effective combination, i.e., the cytostatic dose of CDDP could be reduced without a loss in efficacy. To further investigate the cellular response mechanisms of these favorable outcomes, we analyzed the cell-cycle profiles, mRNA expression patterns, and protein levels of several key genes after incubation with BBSKE or CDDP separately and in combination. In appropriate conditions, CDDP induced arrest at the G2/M phase accompanied by the enhanced inhibitory phosphorylation of Cdk1 and the elevated protein expression of cyclin B1. BBSKE downregulated expression of cyclin D1 by increasing mRNA and protein levels of p21, and thus induced G1 phase arrest. BBSKE returned Cdk1 to an activated state, and reduced the protein level of cyclin B1 after incubation in combination with CDDP, which was consistent with the reduction in the percentage of cells in G2/M identified by flow cytometry. By regulating the G1 phase and reversing CDDP-induced G2/M phase arrest, BBSKE increases drug sensitivity of LoVo cells toward CDDP, and probably provides a meaningful anticancer strategy for further clinical studies.

MicroRNAs targeting mutant K-ras by electrotransfer inhibit human colorectal adenocarcinoma cell growth in vitro and in vivo

Mutations of K-ras have been found in 30-60% of colorectal carcinomas and are believed to be associated with tumor initiation, tumor progression and metastasis formation. Therefore, silencing of mutant K-ras expression has become an attractive therapeutic strategy for colorectal cancer treatment. The aim of our study was to investigate the effect of microRNA (miRNA) molecules directed against K-ras (miRNA-K-ras) on K-ras expression level and the growth of colorectal carcinoma cell line LoVo in vitro and in vivo. In addition, we evaluated electroporation as a gene delivery method for transfection of LoVo cells and tumors with plasmid DNA encoding miRNA-K-ras (pmiRNA-K-ras). Results of our study indicated that miRNAs targeting K-ras efficiently reduced K-ras expression and cell survival after in vitro electrotransfection of LoVo cells with pmiRNA-K-ras. In vivo, electroporation has proven to be a simple and efficient delivery method for local administration of pmiRNA-K-ras molecules into LoVo tumors. This therapy shows pronounced antitumor effectiveness and has no side effects. The obtained results demonstrate that electrogene therapy with miRNA-K-ras molecules can be potential therapeutic strategy for treatment of colorectal cancers harboring K-ras mutations.

Invasiveness of and Drug Sensitivity to Various Anti-cancer Regimens in Five Colorectal Cancer Cell Lines

Purpose: Colorectal cancer (CRC) is one of the leading causes of cancer death in South Korea. Angiogenesis has been associated with invasion and metastasis of tumors and with the secretion of various growth factors. Bevacizumab is a humanized monoclonal antibody that recognizes and blocks vascular endothelial growth factor (VEGF) and that targets integrin aVb3 and matrix metalloproteinases (MMPs) as angiogensis inhibitors. The aims of this study were identification of the mechanism of target molecules related to angiogenesis and demonstration of identifiable invasion by using chemotherapeutic regimens in vitro. Methods: The five colorectal cancer cell lines were treated with bevacizumab using standard or combined regimens. The expression of integrin aVb3 was detected and the investigation of apoptosis was done by using flow cytometry. The activations of MMP-2 and MMP-9 were measured by using gelatin zymography. Results: The apoptotic cell death was significantly increased for the combined regimens, especially for FOLFOX (5-FU, leucovorin, and oxaliplatin) with bevacizumab. Bevacizumab inhibited the expression of integrin aVb3 in the HT29 (59%), LoVo (67%), and SW480 (17%) cell lines, but did not in the AMC5 and the RKO cell lines. The activations of MMP-2 and MMP-9 were significantly reduced by treatment with bevacizumab in the HT29 and the LoVo cell lines. In the HT29 and the LoVo cell lines, thus, bevacizumab inhibited invasion and metastasis activity through down-regulation of integrin aVb3 and MMPs. Conclusion: Our results provide biological evidence of potent angiogenic activity and indicate that angiogenesis is a complex process that involves multiple factors, including VEGF, integrin aVb3, and MMPs.