[Corrigendum] Fully human VEGFR2 monoclonal antibody BC001 attenuates tumor angiogenesis and inhibits tumor growth.

Subsequently to the publication of the above paper, an interested reader drew to the authors' attention that, for the scratch wound assay experiments shown in Fig. 1 on p. 2413, the panels showing the '0 h' experiments for the respective incubations with VEGF or BC001 were apparently identical. The authors were able to re­examine their original data files, and realized that this figure had been inadverently assembled incorrectly. The revised version of Fig. 1, containing the correct data for the '0 h / BC001' panel, is shown below. Note that the revisions made to this figure do not affect the overall conclusions reported in the paper. The authors are grateful to the Editor of International Journal of Oncology for allowing them the opportunity to publish this Corrigendum, and apologize to the readership for any inconvenience caused. [International Journal of Oncology 45: 2411­2420, 2014; DOI: 10.3892/ijo.2014.2690].

In Vivo and Mechanistic Studies on Antitumor Lead 7-Methoxy-4-(2-methylquinazolin-4-yl)-3,4-dihydroquinoxalin-2(1H)-one and Its Modification as a Novel Class of Tubulin-Binding Tumor-Vascular Disrupting Agents.

7-Methoxy-4-(2-methylquinazolin-4-yl)-3,4-dihydroquinoxalin-2(1H)-one (2), a promising anticancer lead previously identified by us, inhibited tumor growth by 62% in mice at 1.0 mg/kg without obvious signs of toxicity. Moreover, compound 2 exhibited extremely high antiproliferative activity in the NIH-NCI 60 human tumor cell line panel, with low to sub-nanomolar GI50 values (10-10 M level). It also showed a suitable balance between aqueous solubility and lipophilicity, as well as moderate metabolic stability in vivo. Mechanistic studies using Mayer's hematoxylin and eosin and immunohistochemistry protocols on xenograft tumor tissues showed that 2 inhibited tumor cell proliferation, induced apoptosis, and disrupted tumor vasculature. Moreover, evaluation of new synthetic analogues (6a-6t) of 2 indicated that appropriate 2-substitution on the quinazoline ring could enhance antitumor activity and improve druglike properties. Compound 2 and its analogues with a 4-(2-methylquinazolin-4-yl)-3,4-dihydroquinoxalin-2(1H)-one scaffold thus represent a novel class of tubulin-binding tumor-vascular disrupting agents (tumor-VDAs) that target established blood vessels in tumors.

Prognostic value of angiopoietin-2 in non-small cell lung cancer patients: a meta-analysis.

BACKGROUND: Non-small cell lung cancer (NSCLC) is the most frequent cause of cancer deaths worldwide. The targeted therapy had made important progress in recent years, but few potential predictive biomarkers for prognosis of NSCLC patients were identified. Angiopoietin-2 (Ang-2), a cytokine upregulated in tumor endothelial cells and some tumor cells including NSCLC, is a partial agonist and antagonist of angiopoietin-1 (Ang-1). Ang-1 is another ligand for the tyrosine kinase receptor Tie2; it promotes recruitment of pericytes and smooth muscle cells, stabilizing vascular networks by binding to Tie2. Although many studies mainly considered that Ang-2 correlated with progression and prognosis of NSCLC significantly, there are much conflicting and controversial data. Therefore, we conducted a meta-analysis to assess the relationship between Ang-2 and prognosis, a clinical outcome of NSCLC. METHODS: The search was based on major databases from PubMed, Cochrane Library, EMBASE, and CNKI, and 20 eligible publications (range from 2002 to 2015) are included in our meta-analysis with 2011 NSCLC patients in total. These studies illuminated the correlation between the expression of Ang-2 and NSCLC, based on either prognostic factors or clinicopathological features. Pooled calculations were carried out on the odds ratio (OR) and the corresponding 95 % confidence interval (CI) to perform this meta-analysis, and all statistical analyses were carried out by STATA 12.0 and Review Manager 5.3. RESULTS: According to our results, the expression of Ang-2 in NSCLC tissues was significantly higher than that in normal lung tissues, indicating that Ang-2 over-expression may be a predictive marker (pooled OR = 5.09, corresponding 95 % confidence interval (95 % CI) 3.10-8.36, p = 0.000). In addition, our pooled data showed that Ang-2 expression was positively correlated with tumor stages (pooled OR = 3.58, 95 % CI 2.40-5.35, p = 0.000), differentiation (pooled OR = 0.65, 95 % CI 0.45-0.94, p = 0.02), lymphatic invasion (pooled OR = 3.15, 95 % CI 1.97-5.03, p = 0.000), and poor survival (pooled OR = 1.93, 95 % CI 1.47-2.52, p = 0.000) of NSCLC, but seems to have no significant impact on tumor size (pooled OR = 1.09, 95 % CI 0.59-2.00, p = 0.78). CONCLUSIONS: These results demonstrate that Ang-2 expression significantly correlated with poor prognosis for patients with NSCLC.

Fully human VEGFR2 monoclonal antibody BC001 attenuates tumor angiogenesis and inhibits tumor growth.

The critical role of VEGFR2 in tumor neovascularization and progression has allowed the design of clinically beneficial therapies based on it. Here we show that BC001, a new fully human anti-VEGFR2 monoclonal antibody, inhibits VEGF-stimulated endothelial cell migration, tube formation, and effectively suppressed the transdifferentiation of cancer stem cells into endothelial cells in vitro. Since BC001 exhibited no activity against the mouse VEGFR2 and mouse based study was required to confirm its efficacy in vivo, BC101, the mouse analogue of BC001, was developed. BC101 significantly attenuated angiogenesis according to Matrigel plug assay and resulted in ~80% growth inhibition of mouse B16F10 homograft tumors relative to vehicle control. Similarly, human analogue BC001 suppressed the growth of human xenograft tumors HCT116 and BGC823. Furthermore, immunohistochemical results showed reduced expression of CD31, VEGFR2 and Ki-67, as well as increased expression of Caspase 3 in BC001-treated tumor, which indicated BC001 was able to significantly decrease microvessel density, suppress proliferation and promote apoptosis. These results demonstrate the fully human VEGFR2 monoclonal antibody BC001 can work as an effective inhibitor of tumor angiogenesis and tumor growth both in vitro and in vivo.

Wire-cylinder dielectric barrier discharge induced degradation of aqueous atrazine.

The wire-cylinder dielectric barrier discharge reactor was adopted for removal of aqueous atrazine. The effect of different parameters on the degradation efficiency of atrazine was investigated, and the degradation mechanism of atrazine was studied. The experimental results showed that when the discharge power was 50 W and the air flow rate was 140 L h(-1), 93.7% of atrazine was degraded after 18 min of discharge time. The concentrations of generated O3 and H2O2 increased with increasing discharge time. The pH decreased from 6.80 to 2.50, 12.7% of TOC was removed after 18 min. The concentrations of generated Cl(-) and NO3(-) increased significantly during the degradation process of atrazine, and the decreasing toxicity trend was observed for the treated atrazine solution. The degradation byproducts of atrazine were identified using liquid chromatography-time-of-flight mass spectrometry (LC-TOF-MS), which might be formed mainly in dechlorination hydroxylation, alkyl oxidation, dechlorination hydroxylation combined with alkyl oxidation and demethylation oxidation reactions.

Biodegradation and speciation of roxarsone in an anaerobic granular sludge system and its impacts.

Roxarsone (3-nitro-4-hydroxy benzene arsenic acid) is an organoarsenic feed additive and has been widely used in the poultry industry to prevent coccidiosis and improve feed efficiency. The presence of roxarsone and its degradation products results in the instability of the anaerobic methanogenic process. This study investigated the degradation and speciation of roxarsone in an anaerobic granular sludge (AGS) system and the impacts of roxarsone and its degradation products on the structure of AGS. Roxarsone inhibited methane production, and the added roxarsone was rapidly degraded into 3-amino-4-hydroxyphenylarsonic acid (HAPA). After 240 days of incubation, the distribution of arsenic differed between the aqueous solution and the AGS in the assays of 20 and 350mg/L roxarsone. Species analysis indicated that HAPA was completely degraded in all of the assays with roxarsone addition after 240 days of incubation. Species distribution was affected by the phases and the initial concentration of roxarsone added. The concentration of As(III) was higher than that of As(V) in both the aqueous solution and the AGS in all assays with roxarsone addition. The toxicity of roxarsone and its degradation products resulted in changes in the structure and the microorganism species in the AGS.

Electrochemical stimulation of microbial roxarsone degradation under anaerobic conditions.

Roxarsone (4-hydroxy-3-nitrophenylarsonic acid) has been commonly used in animal feed as an organoarsenic additive, most of which is excreted in manure. Roxarsone is easily biodegraded to 4-hydroxy-3-aminophenylarsonic acid (HAPA) under anaerobic conditions, but HAPA persists for long periods in the environment, increasing the risk of arsenic contamination through diffusion. We investigated the electrochemical stimulation of the microbial degradation of roxarsone under anaerobic conditions. After the carbon sources in the substrate were depleted, HAPA was slowly degraded to form arsenite under anaerobic conditions. The degradation rate of HAPA was significantly increased when 0.5 V was applied without adding a carbon source. The two-cell membrane reactor assays reveal that the HAPA was degraded in the anode chambers, confirming that the anode enhanced the electron transfer process by acting as an electron acceptor. The degradation product formed with electrochemical stimulation was arsenate, which facilitates the removal of arsenic from wastewater. Based on the high performance liquid chromatography-ultraviolet-hydride generation-atomic fluorescence spectrometry (HPLC-UV-HG-AFS) and gas chromatography-mass spectrometry (GC-MS) data, the pathway for the biodegradation of roxarsone and the mechanisms for the electrochemically stimulated degradation are proposed. This method provides a potential solution for the removal of arsenic from organoarsenic-contaminated wastewater.

Preparation, characterization, and pharmacodynamics of thermosensitive liposomes containing docetaxel.

A novel thermosensitive liposome (TL) containing docetaxel (DTX) was designed to enhance DTX-targeted delivery and antitumor effect. TL loading DTX (DTX-TL) were prepared by thin film hydration. The mean particle size of the liposomes was about 100 nm, and the drug entrapment efficiency was more than 95%. The phase transition temperature of liposomes was about 42 °C. In vitro drug release showed that drug released at 37 °C was obviously less than that at 42 °C. For in vivo experiments, the human breast tumor model was established by subcutaneous xenotransplantation on nude mice; liposomes and injection containing DTX were injected i.v. in nude mice, followed by exposure of the tumors to hyperthermia (HT) for 30 min after administration. The tumor inhibition ratio of DTX-TL group was significantly higher than the normal injection group. Combining TL with HT enhanced the delivery of DTX and thereby its antitumor effects. The liposomes reported in this paper could potentially produce viable clinical strategies for improved targeting and delivery of DTX for the treatment of breast cancer.

[Impact factors and degradation mechanism for the ozonation of acetaminophen in aqueous solution].

The effect and mechanism of O3 on the degradation of acetaminophen in aqueous solution were studied by the batch experiment. The results showed that acetaminophen could be degraded effectively by ozone and degradation of acetaminophen fitted well with the pseudo-first-order kinetics model (R2 > 0.992). The degradation of acetaminophen was promoted with the increase of pH, the concentration of bicarbonate and ozone. The results of gas chromatography-mass spectrometry (GC-MS) and ion chromatography analysis showed that degradation products such as hydroquinone and a series of carboxylic acids were firstly formed during ozonation of acetaminophen. Then, the products were further oxidized. The degradation pathways of acetaminophen were also discussed by the identified products. The result of TOC showed that the mineralization of acetaminophen was ultimately lower. When the initial concentration of acetaminophen was 20 mg x L(-1) and the concentration of ozone was 9.10 mg x L(-1), the mineralization was only 16.42% after 130 min.

Preclinical evaluation of herpes simplex virus armed with granulocyte-macrophage colony-stimulating factor in pancreatic carcinoma.

AIM: To investigate the therapeutic efficacy and mechanisms of action of oncolytic-herpes-simplex-virus encoding granulocyte-macrophage colony-stimulating factor (HSV(GM-CSF)) in pancreatic carcinoma. METHODS: Tumor blocks were homogenized in a sterile grinder in saline. The homogenate was injected into the right armpit of each mouse. After vaccination, the mice were randomly assigned into four groups: a control group, a high dose HSV(GM-CSF) group [1 × 107 plaque forming units (pfu)/tumor], a medium dose HSV(GM-CSF) group (5 × 106 pfu/tumor) and a low dose HSV(GM-CSF) group (5 × 105 pfu/tumor). After initiation of drug administration, body weights and tumor diameters were measured every 3 d. Fifteen days later, after decapitation of the animal by cervical dislocation, each tumor was isolated, weighed and stored in 10% formaldehyde solution. The drug effectiveness was evaluated according to the weight, volume and relative volume change of each tumor. Furthermore, GM-CSF protein levels in serum were assayed by enzyme-linked immunosorbent assays at 1, 2, 3 and 4 d after injection of HSV(GM-CSF). RESULTS: Injection of the recombinant mouse HSV encoding GM-CSF resulted in a significant reduction in tumor growth compared to the control group, and dose-dependent effects were observed: the relative tumor proliferation rates of the low dose, medium dose and high dose groups on 15 d after injection were 45.5%, 55.2% and 65.5%, respectively. The inhibition rates of the tumor weights of the low, middle, and high dose groups were 41.4%, 46.7% and 50.5%, respectively. Furthermore, the production of GM-CSF was significantly increased in the mice infected with HSV(GM-CSF). The increase in the GM-CSF level was more pronounced in the high dose group compared to the other two dose groups. CONCLUSION: Our study provides the first evidence that HSV(GM-CSF) could inhibit the growth of pancreatic cancer. The enhanced GM-CSF expression might be responsible for the phenomenon.

[Anaerobic digestion of animal manure contaminated by tetracyclines].

Anaerobic digestion of pig manure spiked with tetracycline (TC) and chlortetracycline (CTC) and the degradation of the two antibiotics during the anaerobic digestion at 35 degrees C were investigated. The results indicate that propionate was the main volatile fatty acid produced during the anaerobic digestion followed by acetate. Compared with the CTC addition, TC + CTC addition showed obvious inhibitory effect on the hydrolysis and acidification of easily digestible organic components of pig manure. The cumulative methane production of TC, CTC, TC + CTC and CK2 during anaerobic digestion was 386.4 mL, 406.0 mL, 412.1 mL and 464.6 mL, respectively. Degradation of TC and CTC followed the first-order kinetic equation. The half-life of TC and CTC was 14-18 days and 10 days, respectively. After the treatment of 45-day anaerobic digestion, the degradation efficiency of TC was 88.6%-91.6% with 97.7%-98.2% of CTC. Therefore, anaerobic digestion shows the benefit on the management of animal manures contaminated by tetracyclines.

Down-regulation of some miRNAs by degrading their precursors contributes to anti-cancer effect of mistletoe lectin-I.

BACKGROUND AND PURPOSE: Mistletoe lectin-I (ML-I), the main anti-cancer component of mistletoe extracts, was originally thought to act exclusively on 28S rRNA. Here, we investigate the down-regulating effect and mechanism of CM-1, an ML-I isolated from Chinese mistletoe, on some miRNAs. EXPERIMENTAL APPROACH: The anti-cancer effects of CM-1 were assessed in vitro and in vivo in colorectal cancer cells. The miRNAs down-regulated by CM-1 were identified by miRNA microarray assay and validated by qRT-PCR analysis. The suppression of host gene transcription or by degradation of precursors was determined by qRT-PCR and enzyme activity assays respectively. The qRT-PCR, Western blot and immunohistochemistry were used to examine the expression of their target gene and related downstream effector. Cell proliferation was assayed in stably transfected HEK-293 cells with different levels of these miRNAs. KEY RESULTS: CM-1 showed prominent anti-neoplastic activity towards CLY and HT-29 cells both in vitro and in vivo. The miR-135a&b were the miRNAs most down-regulated by CM-1. Their host gene transcription was largely up-regulated, while their precursors were degraded directly by CM-1. The expression of their target gene adenomatous polyposis coli and the phosphorylation of related effector ß-catenin were both significantly up-regulated. The IC(50) values of CM-1 on derivative HEK-293 cells with high miR-135a&b levels were 2-4 times lower than that of control cells. CONCLUSIONS AND IMPLICATIONS: CM-1 down-regulated some miRNAs by degrading their precursors, which contributes to its prominent anti-cancer activity.

Production and radioprotective effects of pyrroloquinoline quinone.

Pyrroloquinoline quinone (PQQ) was produced by fermentation of the Methylovorus sp. MP688 strain and purified by ion-exchange chromatography, crystallization and recrystallization. The yield of PQQ reached approximately 125 mg/L and highly pure PQQ was obtained. To determine the optimum dose of PQQ for radioprotection, three doses (2 mg/kg, 4 mg/kg, 8 mg/kg) of PQQ were orally administrated to the experimental animals subjected to a lethal dose of 8.0 Gy in survival test. Survival of mice in the irradiation + PQQ (4 mg/kg) group was found to be significantly higher in comparison with the irradiation and irradiation + nilestriol (10 mg/kg) groups. The numbers of hematocytes and bone marrow cells were measured for 21 days after sublethal 4 Gy gamma-ray irradiation with per os of 4 mg/kg of PQQ. The recovery of white blood cells, reticulocytes and bone marrow cells in the irradiation + PQQ group was faster than that in the irradiation group. Furthermore, the recovery of bone marrow cell in the irradiation + PQQ group was superior to that in irradiation + nilestriol group. Our results clearly indicate favourable effects on survival under higher lethal radiation doses and the ability of pyrroloquinoline quinine to enhance haemopoietic recovery after sublethal radiation exposure.

Microbial transformation of epothilone A by Aspergillus niger AS 3.739.

The microbiological transformation of epothilone A (1) by Aspergillus niger AS 3.739 afforded four main metabolites. Their structures were elucidated by (1)H, (13)C NMR and HSQC, COSY, HMBC, and NOESY spectra as trans-12,13-hydroxylated epothilone A (2), cis-12,13-hydroxylated epothilone A (3), trans-12,15-epoxidated epothilone A (4), and cis-12,15-epoxidated epothilone A (5). All four compounds were firstly found based on their stereochemistry. These new compounds displayed cytotoxicity against human breast carcinoma cells MCF-7 with IC(50) 9.88 microg/ml of 2, 2.52 microg/ml of 3, 9.88 microg/ml of 4, and 5.68 microg/ml of 5.

[Inhibitory effect of Oridonin injection on heterotransplanted gastric adenocarcinoma in nude mice and its mechanism].

OBJECTIVE: To investigate the inhibitory effect of Oridonin injection on heterotransplanted tumors of human gastric adenocarcinoma cell line BGC823 cells in nude mice and explore its mechanism. METHODS: Heterotransplanted models of human gastric adenocarcinoma cell line BGC823 cells in nude mice were established. They were divided at random into three groups as control group, low-dose group and high-dose group. The Oridonin solution at concentration of 37.5 mg x kg(-1 x d(-1) and 75 mg x kg(-1) x d(-1) were injected to the mice in low-dose group and high-dose group, respectively, and 0.9% sodium chloride was injected to the mice of control group per day for 10 days sequentially. The mice of the three groups were sacrificed at 11th day after the first injection of Oridonin. The tumor weight of the sacrificed mice was measured. Morphological and ultrastructural examinations of the tumors were carried out by light and electron microscopy. The expression of bcl-2, Bax, Fas and FasL was detected by immunohistochemistry. RESULTS: Oridonin injection showed a suppressive effect on the growth of heterotransplanted tumors in the nude mice. The tumor growth inhibition rates were 48.5% and 70.7% in the low-dose and high-dose groups, respectively. The morphological study demonstrated that tumor cells displayed a typical appearance of apoptosis. The expression of bcl-2 was down-regulated, while Bax, Fas and FasL were up-regulated. CONCLUSION: Oridonin can markedly inhibit the growth of heterotransplanted human gastric adenocarcinoma in nude mice. It was due, at least in part, to the induction of apoptosis in cancer cells.

Differential sensitivity of colorectal cancer cell lines to artesunate is associated with expression of beta-catenin and E-cadherin.

Artesunate, a remarkable antimalarial agent, also reveals profound cytotoxic activity. In the present investigation, we compared the anticancer effects of artesunate on three colorectal cancer cell lines and analyzed the relationship between drug sensitivity and malignant phenotype of the tumor cells. The findings are as follows: poorly-differentiated was colorectal cancer cell line CLY showing nuclear beta-catenin accumulation and loss of E-cadherin; moderately-differentiated were Lovo cells with cytoplasmic distribution of the two proteins; and well-differentiated were HT-29 cells with membranous localization of them. Also, both in vitro and in vivo, poorly- or moderately-differentiated CLY and Lovo were more susceptible to artesunate treatment than well-differentiated HT-29. Furthermore, the sensitive response of CLY and Lovo to artesunate was associated with membranous translocation of beta-catenin and increased expression of E-cadherin, which indicated the inhibition of hyperactive Wnt signaling pathway and the reversion of the epithelial to mesenchymal transition, respectively. Due to the vital roles of Wnt pathway and the epithelial to mesenchymal transition in tumor differentiation, we thought artesunate could promote the re-differentiation and apoptosis of colorectal cancer cells by inhibition of hyperactive Wnt pathway and reversion of the epithelial to mesenchymal transition.

[Cytotoxic effect of epidermal growth factor receptor tyrosine kinase inhibitor ZD1839 in combination with oxaliplatin on lung cancer cell line A549].

BACKGROUND & OBJECTIVE: ZD1839, a selective epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), has clinical antitumor activity, but its efficacy is low. This study was to assess the effects of ZD1839 in combination with oxaliplatin on lung adenocarcinoma cell line A549, and to provide pre-clinical evidence for optimizing the schedule of oxaliplatin combined with ZD1839. METHODS: Chou and Talalay method was used to analyze the combination effects of sequencing ZD1839 and oxaliplatin on A549 cells. Cell cycle distribution and cell apoptosis were analyzed by flow cytometry. The effects of oxaliplatin combined with ZD1839 on the proliferation of A549 cells in nude mice were also evaluated. RESULTS: Sequential oxaliplatin followed by ZD1839 produced synergistic effect, with a combination index (CI) of 0.51+/-0.01. In contrast, ZD1839 followed by oxaliplatin exhibited antagonist effect, with a CI of 1.56+/-0.03. Compared with other sequences, oxaliplatin followed by ZD1839 induced more cells being arrested in G(2/M) phase (37.9%, P<0.05); the apoptosis rate was 22.3%. The inhibition rate of tumor growth in nude mice was 58.9% when treated with oxaliplatin followed by ZD1839, 52.4% when treated with oxaliplatin and ZD1839 for 24 h and followed by ZD1839 for additional 48 h, and 30.6% when treated with ZD1839 followed by oxaliplatin. CONCLUSION: Sequential oxaliplatin followed by ZD1839 has the maximal inhibitory effect on the proliferation of A549 cells.

Artesunate attenuates the growth of human colorectal carcinoma and inhibits hyperactive Wnt/beta-catenin pathway.

Artesunate (ART), a remarkable antimalarial agent, also inhibited the growth of human colorectal carcinoma. As determined by MTT assay, flow cytometry analysis on apoptosis and indirect immunofluorescence analysis on the proliferation-associated marker Ki67, ART suppressed the proliferation and promoted the apoptosis of colorectal cancer cells in a dose-dependent manner. Furthermore, immunofluorescence analysis on beta-catenin and RT-PCR analysis on Wnt/beta-catenin target genes demonstrated ART translocated beta-catenin from nucleus to adherent junctions of membrane and reduced transcription mediated by beta-catenin. These results suggested the anticancer activity of ART correlated with the inhibition of hyperactive Wnt/beta-catenin signaling pathway. In vivo, ART significantly slowed the growth of colorectal tumor xenografts. Bioluminescent imaging also revealed that ART decreased the physiological activity of tumor xenografts and delayed spontaneous liver metastasis. These antitumor effects were related to the membranous translocation of beta-catenin and the inhibition of the unrestricted activation of Wnt/beta-catenin pathway, which was confirmed by the immunohistochemical staining of tumor tissues. These results and the known low toxicity are clues that ART might be a promising candidate drug for the treatment of colorectal carcinoma.

Establishment and characterization of a novel human colorectal cancer cell line (CLY) metastasizing spontaneously to the liver in nude mice.

Colorectal cancer (CRC) with liver metastasis is a fatal disease with rapid progression and poor patient outcome. However, the molecular mechanism involved in liver metastasis of CRC remains essentially unknown, largely because of the presence of few available CRC cell lines with liver metastasis origin and spontaneous metastatic potentials in nude mice. In this study, we established a novel metastatic CRC cell line, CLY, derived from liver metastasis of a 64-year-old Chinese CRC patient. The cell line was characterized by morphology, growth kinetics, tumorigenicity, spontaneous liver metastatic potential, and cytogenetics. Immunofluorescence analysis of beta-catenin and E-cadherin and methylation-specific PCR (MSP) of the E-cadherin gene (CDH1) promoter were also used to compare CLY with conventional CRC cell lines (HT-29 and Lovo). CLY exhibited compact and polygonal-shaped epithelial cells in vitro and its population doubling time was 29.5 h. Subcutaneous transplantation of CLY into nude mice resulted in subcutaneous tumor formation and spontaneous liver metastasis in all of 10 nude mice. Cytogenetic analysis identified aneuploidy karyotypes with a modal chromosome number of 60. In immunofluorescence analysis, CLY exhibited a low expression but high restricted nuclear localization of beta-catenin and a silenced expression of E-cadherin, which may be induced by hypermethylation of the E-cadherin gene (CDH1) promoter and differed from conventional CRC cell lines. Therefore, CLY is an ideal cell model for further exploring the metastatic mechanisms of CRC and testing new therapeutic reagents for CRC with liver metastasis.

[Experimental study of effect of epidermal growth factor receptor tyrosine kinase inhibitor ZD1839 in combination with irinotecan].

OBJECTIVE: To assess the optimal regimen and its mechanism of ZD1839 in combination with SN38, the active metabolite of irinotecan (CPT-11), in the colon cancer cell lines HT-29 and LoVo. METHODS: Chou and Talalay method was used to analyze the combination effects of sequencing of ZD1839 and SN38. Western blotting and immunoprecipitation were used to determine the effects of ZD1839 and/or SN38 on their targeted enzymes and downstream markers. Apoptosis was assayed by analyzing histone-associated DNA fragment. RESULTS: Sequential SN38 followed by ZD1839 produced a synergistic effect. In contrast, SN38 following ZD1839 exhibited an antagonist effect. SN38 markedly inhibited topoisomerase I (Topo-I) activity. ZD1839 did not alter epidermal growth factor receptor (EGFR) expression, but resulted in a complete inhibition of EGFR phosphorylation. Sequential ZD1839 followed by SN38 did not show any enhanced inhibition effect on Topo-I activity, phosphorylation of EGFR and one of its downstream markers MAPK. However, simultaneous SN38 plus ZD1839, and sequential SN38 followed by ZD1839 administrations showed modest inhibition effect on EGFR's another downstream marker AKT. The combination schedules also showed prominent influence on cell cycle distribution. ZD1839 maintained SN38-induced DNA damage and apoptosis. CONCLUSION: Sequential SN38 followed by ZD1839 may be a favorable combination schedule.