Steroids from the Deep-Sea-Derived Fungus Penicillium granulatum MCCC 3A00475 Induced Apoptosis via Retinoid X Receptor (RXR)-α Pathway.

Five new ergostanes, penicisteroids D-H (1-5), were isolated from the liquid culture of the deep-sea-derived fungus Penicillium granulatum MCCC 3A00475, along with 27 known compounds. The structures of the new steroids were established mainly on the basis of extensive analysis of 1D and 2D NMR as well as HRESIMS data. Moreover, the absolute configurations of 1 were confirmed unambiguously by the single-crystal X-ray crystallography. Compounds 2 and 4⁻7 showed moderate antiproliferative effects selectively against 12 different cancer cell lines with IC50 values of around 5 µM. Compounds 2 and 6, potent RXRα binders with Kd values of 13.8 and 12.9 µM, respectively, could induce apoptosis by a Retinoid X Receptor (RXR)-α-dependent mechanism by regulating RXRα transcriptional expression and promoting the poly-ADP-ribose polymerase (PARP) cleavage. Moreover, they could inhibit proliferation by cell cycle arrest at the G0/G1 phase.

Killing effect of Ad5/F35-APE1 siRNA recombinant adenovirus in combination with hematoporphrphyrin derivative-mediated photodynamic therapy on human nonsmall cell lung cancer.

The main goal of this work is to investigate the killing effects and molecular mechanism of photodynamic therapy (PDT) mediated by the Ad5/F35-APE1 siRNA recombinant adenovirus in combination with a hematoporphrphyrin derivative (HpD) in the A549 human lung adenocarcinoma cell line in vitro to provide a theoretical reference for treating lung cancer by HpD-PDT. By using the technologies of MTT, flow cytometry, ELISA, and western blot, we observed that the proliferation inhibition and apoptosis of the A549 cells were significantly higher than the control group (P < 0.05) after HpD-PDT was performed. The inhibitory efficiency is dependent on the HpD concentration and laser intensity dose. The inhibitory effect on the proliferation of A549 cells of Ad5/F35-APE1 siRNA is more significant after combining with PDT, as indicated by a significant elevation of the intracellular ROS level and the expression of inflammatory factors (P < 0.05). The HpD-PDT-induced expression of the APE1 protein reached the peak after 24 h in A549 cells. The inhibition of APE1 expression in A549 cells was most significant after 48 hours of infection by Ad5/F35-APE1 siRNA recombinant adenovirus (10 MOI). In conclusion, the Ad5/F35-APE1 siRNA recombinant adenovirus could efficiently inhibit the HpD-PDT-induced APE1 expression hence could significantly enhance the killing effect of HpD-PDT in lung cancer cells.

Human apurinic/apyrimidinic endonuclease 1 translocalizes to mitochondria after photodynamic therapy and protects cells from apoptosis.

Photodynamic therapy (PDT) is an effective therapeutic regime for lung cancer. Mitochondrial functional failure is considered to be one of the most important factors causing cell death after PDT. However, the detailed mechanisms that are involved are still unclear. We previously reported that apurinic/apyrimidinic endonuclease (APE1) plays a critical role in regulating sensitivity to PDT in the lung cancer A549 cell line. An important mitochondrial regulatory role for APE1 has recently been reported, so therefore we explored the role of APE1 in cell survival after PDT-induced oxidative stress through regulation of mitochondrial function. We first observed that photoirradiation induced the mitochondrial translocation of APE1. The ability of APE1 to regulate mitochondrial membrane potential and reactive oxygen species (ROS) production after photoirradiation was tested in APE1 knockdown A549 cells. APE1-deficient A549 cells were characterized as having a lower mitochondrial membrane potential and higher ROS production, which led to increased apoptosis through the mitochondrial pathway after PDT. Additionally, unexpected activity of APE1 was observed in mitochondria: the control of mitochondrial transcriptional activity by redox regulation of mitochondrial transcription factor A (TFAM). Furthermore, two dominant-negative mutants of APE1 were overexpressed to enhance their individual activities in mitochondria. The results suggest that both these APE1 activities play a role in the regulation of mitochondrial function but through different mechanisms. The present study not only provides possible mechanisms for APE1 in regulating survival after photoirradiation but also uncovers a new activity of APE1 in mitochondria.

Genetic polymorphism of DNA base-excision repair genes (APE1, OGG1 and XRCC1) and their correlation with risk of lung cancer in a Chinese population.

BACKGROUND AND AIMS: Reactive oxygen species (ROS) and numerous carcinogens may cause DNA damage including oxidative base lesions that contribute to the risk of lung cancer. The base excision repair (BER) pathway could effectively remove oxidative lesions in which 8-oxoguanine glycosylase-1 (OGG1), x-ray repair cross-complementing 1 (XRCC1), and apurinic/apyimidinic endonuclease 1 (APE1) play key roles. The aim of this study was to analyze the polymorphisms of DNA BER genes (OOG1, XRCC1 and APE1) and explore their associations, and the combined effects of these variants, with risk of lung cancer. METHODS: In a hospital-based, case-control study of 455 lung cancer cases and 443 cancer-free hospital controls, the SNPs of OGG1 (Ser326Cys), XRCC1 (Arg399Gln), APE1 (Asp148Glu and -141T/G) were genotyped and analyzed for their correlation with the risk of lung cancer in multivariate logistic regression models. RESULTS: Individuals homozygous for the variants APE1 -141GG showed a protective effect for lung cancer overall (OR=0.62; 95% CI: 0.42-0.91; p=0.02) and for lung adenocarcinoma (OR=0.65; 95% CI, 0.44-0.96; p=0.03). When analyzing the combined effects of variant alleles, 84 patients and controls were identified who were homozygous for two or three of the potential protective alleles (i.e., OGG1 326Cys, XRCC1 399Gln and APE1 -141G). ORs were significantly reduced when all patients were analyzed (OR=0.62; 95% CI: 0.38-0.99; p=0.05). CONCLUSIONS: The combined effects of polymorphisms within BER genes may contribute to the tumorigenesis of lung cancer.

[Identification of antigenic epitopes of human apurinic/apyrimidinic endonuclease monoclonal antibodies and their application].

AIM: To identify the antigenic epitopes of human apurinic/apyrimidinic endonuclease monoclonal antibodies (hAPE1 mAb) and to establish a one-step ELISA for quantitative measurement of hAPE1. METHODS: APE1-15 polypeptide array was used to identify the antigenic epitopes of hAPE1 mAb. Molsoft. ICM-Pro software was used to display the three dimensions of antigenic epitopes of hAPE1 mAbs. hAPE1 mAb was labeled with HRP by modified method of sodium periodate oxidization. A one-step ELISA was established by using two hAPE1 mAbs as a capture antibody and a detection antibody, respectively. RESULTS: The antigenic epitopes of 2-G1 mAb were localized within amino acids (aa) 76-90 and 109-123 in APE1 redox domain, belonging to the conformational epitope category. The antigenic epitope of 4-F6 mAb was within aa 109-147 in DNA repair endonuclease domain. In the range of 8.0 microg/L to 200 microg/L, the hAPE1 antigen showed a good linearity in the standard curve. The minimum detection threshold of this assay was 2.0 microg/L. The average of intra-assay precision and inter-assay precision were 8.67% and 12.45%, respectively. The average recovery rate was 105.47%. CONCLUSION: hAPE1 2-G1 mAb and 4-F6 mAb have different antigenic epitopes, and the one-step ELISA is established successfully to detect the serum hAPE1 level easily, quickly and accurately.

Microarray analysis of DNA damage repair gene expression profiles in cervical cancer cells radioresistant to 252Cf neutron and X-rays.

BACKGROUND: The aim of the study was to obtain stable radioresistant sub-lines from the human cervical cancer cell line HeLa by prolonged exposure to 252Cf neutron and X-rays. Radioresistance mechanisms were investigated in the resulting cells using microarray analysis of DNA damage repair genes. METHODS: HeLa cells were treated with fractionated 252Cf neutron and X-rays, with a cumulative dose of 75 Gy each, over 8 months, yielding the sub-lines HeLaNR and HeLaXR. Radioresistant characteristics were detected by clone formation assay, ultrastructural observations, cell doubling time, cell cycle distribution, and apoptosis assay. Gene expression patterns of the radioresistant sub-lines were studied through microarray analysis and verified by Western blotting and real-time PCR. RESULTS: The radioresistant sub-lines HeLaNR and HeLaXR were more radioresisitant to 252Cf neutron and X-rays than parental HeLa cells by detecting their radioresistant characteristics, respectively. Compared to HeLa cells, the expression of 24 genes was significantly altered by at least 2-fold in HeLaNR cells. Of these, 19 genes were up-regulated and 5 down-regulated. In HeLaXR cells, 41 genes were significantly altered by at least 2-fold; 38 genes were up-regulated and 3 down-regulated. CONCLUSIONS: Chronic exposure of cells to ionizing radiation induces adaptive responses that enhance tolerance of ionizing radiation and allow investigations of cellular radioresistance mechanisms. The insights gained into the molecular mechanisms activated by these "radioresistance" genes will lead to new therapeutic targets for cervical cancer.

Knock down of the dual functional protein apurinic /apyrimidinic endonuclease 1 enhances the killing effect of hematoporphrphyrin derivative-mediated photodynamic therapy on non-small cell lung cancer cells in vitro and in a xenograft model.

Photodynamic therapy (PDT) is considered to be effective treatment for many cancers including lung cancer, head and neck cancers, and prostate cancer. It uses the combination of nontoxic photosensitizers and harmless visible light to generate reactive oxygen species and kill cells. However, DNA repair and reactive oxygen species-induced signaling pathway activation play crucial roles in cellular response to PDT and may also result in therapeutic limitation of PDT. To improve the cancer therapeutic efficacy of PDT, we targeted apurinic/apyrimidinic endonuclease (APE1), which is essential for both DNA repair and redox regulation of gene transcription, as a potential candidate for PDT combined gene therapy. In our study, an adenovirus-mediated APE1 silencing strategy was introduced to test its therapeutic enhancement for the non-small cell lung cancer cell line A549 both in vitro and in vivo after hematoporphrphyrin derivative (HpD)-mediated PDT. The adenovirus vector Ad5/F35-shAPE1 was validated to significantly suppress the protein expression of APE1 in cultured A549 cell and in its xenograft of nude mice. Ad5/F35-shAPE1 effectively inhibited APE1 protein upregulation induced by PDT and resulted in an increase in A549 cell killing by photoirradiation compared with the hematoporphrphyrin derivative-PDT alone group. Ad5/F35-shAPE1 suppressed the DNA repair capacity for single-strand breaks and abolished the activation of some stress-related transcription factors such as hypoxia-induced factor (HIF)-1 that consequently lead to increased cell apoptosis after PDT. Additionally, knock down of APE1 enhanced the tumor suppression efficacy of PDT on the A549 xenograft. Our study indicated that APE1-targeted gene therapy combined with PDT is a promising strategy for enhancement of the efficacy of PDT in treatment of non-small cell lung cancer.

Rapid induction of PC3/BTG2 gene by hepatopoietin or partial hepatectomy and its mRNA expression in hepatocellular carcinoma.

BACKGROUND: The anti-proliferative gene, PC3 (pheoch-romocytoma cell 3)/BTG2 (B-cell translocation gene 2), is one of the early growth response genes and belongs to the BTG/Tob protein family. This study aimed to assess the effects of recombinant human hepatopoietin (HPO) and partial hepatectomy on rapidly induced expression of immediate-early genes and to investigate the expression of PC3/BTG2 mRNA in hepatocellular carcinoma (HCC) at different stages of progression. METHODS: After a rat model of partial hepatectomy was established, we investigated gene expression within 1 hour after 2/3 partial hepatectomy by representational difference analysis and in a primary cultured hepatocyte system. The expression levels of PC3/BTG2 from liver tissues of the rat model were assessed by RT-PCR and Northern blotting. Meanwhile, the expression of BTG2 mRNA in a tissue microarray of HCC was determined by in situ hybridization. RESULTS: The PC3/BTG2 gene was rapidly induced after 2/3 partial hepatectomy and its expression peaked within 1-2 hours after operation. HPO rapidly induced the expression of the genes c-fos, LRF-1, and PC3 in primary cultured rat hepatocytes, which might be one of the molecular mechanisms by which HPO stimulates hepatocyte proliferation. Positive BTG2 mRNA expression was detected in 71.19% (42/59) of the HCC samples and in 75% (3/4) of the normal liver tissue samples obtained from the region around the HCC tissues. PC3/BTG2 mRNA was located mainly in the cytoplasm of HCC cells and its expression was related to the degree of differentiation. CONCLUSIONS: Recombinant human HPO and partial hepatectomy rapidly induce the expression of the PC3/BTG2 gene. PC3/BTG2 mRNA is highly expressed in HCC cells and its expression is related to the degree of cell differentiation. The abnormal expression of PC3/BTG2 is closely related to the genesis and development of HCC, so PC3/BTG2 may play an important role in these processes.

[Preparation and characterization of rabbit anti-human apurinic/apyrimidinic endonuclease(APE1) polyclonal antibody].

AIM: To prepare a rabbit anti-human apurinic/apyrimidinic endonuclease polyclonal antibody and then have its characterization analyzed. METHODS: A rabbit polyclonal antibody was prepared through a modified rapid immune procedure and then it was purified using a specific avidity column. The efficacy of this antibody was detected by ELISA, Western blot and immunohistochemistry. RESULTS: The titer of the antiserum determined by ELISA was up to 1:128,000. The kaff value of the antibody was 8.96 x 10(-6) mol/L. Western blot analysis showed the antibody was of high specificity. The final purified antibody was highly specific to native form of APE1 protein in mice and rats. CONCLUSION: The rabbit anti-human APE1 polyclonal antibody with high titer and specificity has been successfully prepared. It can be used not only to elucidate the roles of APE1 protein in many important cellular procedures, but also to detect the expression of the APE1 protein in mice and rats.

[The expression of B-cell translocation gene 2 in diethylnitrosamine-induced primary hepatocellular carcinoma rat model.].

OBJECTIVE: To investigate the expression and role of B-cell translocation gene 2(BTG2) in the carcinogenesis of hepatocellular carcinoma (HCC). METHODS: Modified Diethylnitrosamine (DEN)-induced primary hepatocellular carcinoma rat model was established. The expression of BTG2, p53 and cyclinD1 was detected by RT-PCR, western blot and immunohistochemistry. RESULTS: The BTG2 protein was predominantly localized in the nucleus, with faint cytoplasmic staining in normal liver cells; however, it is mainly a cytoplasmic protein in HCC cells. BTG2 was over-expressed during the early stage after DEN treatment, the expression level peaked at 5 weeks and then it gradually decreased to the normal level after 16 weeks. The expression of cyclin D1 and cyclin E was increased gradually after DEN treatment, and peaked at 16 weeks and 5 weeks respectively. A significant increase in p53 was not observed until 5 weeks after DEN treatment, and it gradually decreased after 16 weeks. CONCLUSIONS: Decreased expression of BTG2 may be an important step in carcinogenesis of HCC. BTG2 may positively regulate p53 expression and negatively regulate cyclin D1 expression in the carcinogenesis of HCC.

[The expression of APE1 and its correlation with prognostic significance after 252Cf radiotherapy in cervical cancer].

OBJECTIVE: To investigate the expression feature of the apurinic/apyrimidinic endonuclease (APE1) and its correlation with clinicopathology and prognostic significance after 252Cf radiotherapy in cervical cancer. METHODS: The expression of APE1 was detected by immunohistochemistry technique in 89 cases of cervical cancer (treated by 252Cf), 15 cases cervical intraepithelial neoplasia (CIN) and 10 cases of normal cervical tissue, and its association with clinicopathological data as well as prognosis were analyzed. RESULTS: The expression of APE1 in cervical cancer is higher significantly than that in normal cervical tissue and CIN (P < 0.01). In normal cervical tissue and CIN, the APE1 express was located in the nucleus. In cervical cancer, the APE1 express was located in the nucleus (59), cytoplasm (8) or nucleus and cytoplasm (22), the location of APE1 was related with FIGO stage and pathological grade (P < 0.01), and not related with lymph node metastasis. The level of APE1 express related with FIGO stage, pathological grade and lymph node metastasis (P < 0.05), and not related with age and pathological type. The Kaplan-Meier survival analysis demonstrated that the survival time of the group of APE1 nucleus expression (median survival time is 70.9 months) and the group of APE1 low expression (median survival time is 75.8 months) is longer significantly than that of the group of APE1 cytoplasm expression (median survival time is 57.8 months) and the group of APE1 high expression (median survival time is 56.5 months) (P = 0.025, 0.001). CONCLUSION: The dystopic express of APE1 might play a pivotal role in carcinogenesis and progression of cervical cancer, and the express of APE1 might estimate the prognosis after 252Cf radiotherapy.

Targeting truncated APE1 in mitochondria enhances cell survival after oxidative stress.

The high steady-state level of mitochondrial DNA (mtDNA) oxidative lesions is assumed to be the result of high susceptibility to DNA damage attack and limited DNA repair capacity in mitochondria. As a key enzyme of base excision repair (BER), human apurinic/apyrimidinic endonuclease (APE1) is often scarce in mitochondria, and mitochondria-targeted APE1 with robust repair activity represents a promising therapeutic candidate. In this study, overexpression vectors of mitochondria-targeted truncated APE1 (mtAPE1) and that of full-length APE1 (flAPE1) were constructed and transfected to human umbilical vein endothelial cells to test their protective effects after hydrogen peroxide-induced oxidative stress. The overexpression of truncated APE1 was achieved at protein and enzyme activity levels in mitochondria of mtAPE1-transfected cells. In parallel, enhanced mtDNA repair capacity and increased cell survival were observed. MtAPE1 transfection also prevented apoptosis by blocking mitochondria-dependent pathways. In contrast, flAPE1 transfection rendered slight elevation of nuclear APE1 protein level and nuclear APE activity but no benefits for cell resistance to oxidative stress. The present results suggest that overexpression of the truncated APE1 in mitochondria appears to be a viable approach to protecting healthy cells from some deleterious effects of oxidative stress.

Vector-based Ape1 small interfering RNA enhances the sensitivity of human osteosarcoma cells to endostatin in vivo.

Osteosarcoma is a highly vascular and extremely destructive malignancy, and the survival of patients with osteosarcoma has not improved significantly in recent years. Antiangiogenic therapy currently holds great potential in conjunction with conventional treatment modalities for osteosarcoma. However, there are examples of gradual loss of response, and perhaps acquired resistance to antiangiogenic drugs. The acquired resistance of antiangiogenesis may be associated with a lot of hypoxia-response genes. The human apurinic/apyrimidinic endonuclease (Ape1) protein, a bifunctional redox factor and apurinic/apyrimidinic (AP) endonuclease, plays a crucial role in protecting against cell death due to hypoxia. We therefore hypothesized that Ape1 may contribute to the resistance of antiangiogenic therapy. To investigate the effect of Ape1 on the sensitivity of human osteosarcoma cells to endostatin, we constructed an Ape1 small interfering RNA expression vector, pSilenceApe1. Transfection of human osteosarcoma 9901 and HOS cells with pSilenceApe1 resulted in a dose-dependent loss of Ape1 protein. pSilenceApe1 also significantly suppressed the expression of vascular endothelial growth factor (VEGF) protein in the 9901 cells. Combined treatment with pSilenceApe1 and recombinant human endostatin (rhES) showed potent antiangiogenic effects in the transwell chamber invasion assay. Then, 20 nude mice bearing 9901 xenografts were divided into four groups: the phosphate-buffered saline treatment control group; the rhES treatment group (1.5 mg/kg, daily); the pSilenceApe1 treatment group (20 microg, once every 3 days); and the combination of rhES and pSilenceApe1 treatment group. pSilenceApe1 significantly suppressed the expression of Ape1 and VEGF protein in the 9901 xenografts. The tumor-inhibition rate of the pSilenceApe1, rhES, and combination of rhES and pSilenceApe1 treatment groups was 38.23, 35.29, and 62.18%, respectively. Furthermore, a significant decrease in microvessel density with an increase in apoptosis was observed following combined treatment with pSilenceApe1 and rhES, compared with control and either agent alone in 9901 xenografts. These results indicate that Ape1 small interfering RNA could enhance the sensitivity of osteosarcoma cells to endostatin.

[The expression of Tec and the level of its phosphorylation in primary hepatic carcinomas].

OBJECTIVES: To detect the expressions of Tec tyrosine kinase in hepatocellular carcinoma and the levels of phosphorylation of tyrosine kinase in liver cancer tissues, paracancerous tissues and normal liver tissues and to find the significance of their differences. METHODS: 200 specimens of tissues, including liver cancer tissues, surrounding liver tissues not more than 1.5 cm from the cancers, and normal liver tissues were investigated for Tec protein expression and Tec phosphorylation by tissue microarrays and immunohistochemistry (SP method). RESULTS: The positive immunohistochemical stainings of Tec in cancerous tissues and non-cancerous tissues showed no obvious differences, nevertheless, the immunostaining levels in liver cancer tissues were much higher than in non-cancerous tissues and they correlated with the grading of tumors (P < 0.05). The phosphorylation of Tec was significantly expressed in liver cancer tissues (73%) in comparison with other tissues (42%, 10% both P < 0.05), but it did not correlate with any clinicopathological characteristics. CONCLUSION: Overexpression of Tec is associated with the tumorigenesis and development of liver cancer; inhibiting Tec or degrading Tec phosphorylation directly might affect the progression of liver cancer.

[Effect of adenoviral N-methylpurine DNA glycosylase overexpression on chemosensitivity of human osteosarcoma cells].

OBJECTIVE: The overexpression of N-methylpurine DNA glycosylase (MPG) may imbalance the DNA base excision repair (BER) to sensitize tumor cells to current DNA damage chemotherapy. In an effort to improve the efficacy of cancer chemotherapy, we have constructed adenoviral vector of MPG, to study its ability to sensitize human osteosarcoma cell HOS to DNA damage agents. METHODS: The adenoviral infection and MPG expression, as well as enzyme activity were determined by flow cytometry, Western blot, and HEX labeled oligonucleotide-based assay respectively. The cell survival/proliferation was measured using MTS, SRB, and [(3)H] thymidine incorporation assay. Apoptosis cell death was assayed by flow cytometry after treatment using phycoerythin (PE)-conjugated Annexin V and 7-amino-actinomycin (7-AAD). RESULTS: A 10 MOI of recombinant nonreplicating adenovirus was found to infect more than 90% of HOS cells within 24 hours by EGFP fluorescence, in which the MPG overexpression and MPG enzyme activity were also detected. The MPG overexpression HOS cells were significantly more sensitive to the DNA damage agents, including MMS, MNNG, and TMZ, with changes in the IC50 of 6.0, 4.5, and 2.5 fold respectively. CONCLUSIONS: These data establish transient MPG overexpression as a potential therapeutic approach for increasing HOS cellular sensitivity to DNA damage agent chemotherapy.

[Characteristic and clinicopathologic significance of lymphangiogenesis in colorectal cancer].

OBJECTIVE: To investigate the distribution patterns and proliferative activity of lymphatic vessels in colorectal carcinomas (CRC) and their relationship with tumor metastasis and disease prognosis. METHODS: The microlymphatic density (MLD) and microvascular density in tumoral and non-tumoral areas of 96 cases of CRC were evaluated by immunohistochemistry, using monoclonal antibodies for podoplanin and CD34 respectively. The Ki-67 expression of the lymphatic and blood vessels was detected by double-labeling immunohistochemistry. The relationship between MLD and clinicopathologic features and prognosis was analyzed. RESULTS: The lymph vessels at central and superficia1 portions of CRC often had a reticular architecture with numerous tiny and ill-defined lumina, while those at the tumor borders had large and open lumina. The MLD at tumor borders (51.2 +/- 25.5) was significantly higher than that in normal colorectal mucosa (29.4 +/- 9.0) and other portions of CRC (P < 0.01). The Ki-67 labeling index of the lymphatic lining cells at tumor borders (0.23 +/- 0.17) was significantly higher than that in other portions of CRC (P < 0.05). The MLD significantly correlated with lymphatic involvement by tumor cells, regional lymph node metastasis and distant metastasis (P < 0.01). The 5-year survival rate was also significantly lower in patients with high MLD (P < 0.05). CONCLUSIONS: Neolymphatic vessels are commonly seen in CRC, especially at tumor borders. High MLD at tumor borders is associated with metastasis. The detection of MLD at tumor borders may thus be useful in predicting lymph node metastasis and prognosis in patients with CPC.

Effect of caffeic acid phenethyl ester on proliferation and apoptosis of colorectal cancer cells in vitro.

AIM: To study the effect of caffeic acid phenethyl ester (CAPE) on proliferation, cell cycle, apoptosis and expression of beta-catenin in cultured human colorectal cancer (CRC) cell line HCT116. METHODS: HCT116 cells were treated with CAPE at serial concentrations of 80, 40, 20, 10, 5, 2.5 mg/L. The proliferative status of HCT116 cells was measured by using methabenzthiazuron (MTT) assay. Cell cycle was analyzed by using flow cytometry (FCM) with propidium iodide (PI) labeling method. The rate of apoptosis was detected by using FCM with annexin V-FITC and PI double labeling method. beta-catenin levels were determined by Western blotting. beta-catenin localization in HCT116 was determined by indirect immunofluorescence. RESULTS: After HCT116 cells were exposed to CAPE (80, 40, 20, 10, 5, and 2.5 mg/L) for 24, 48, 72, 96 h, CAPE displayed a strong growth inhibitory effect in a dose- and time-dependent manner against HCT116 cells. FCM analysis showed that the ratio of G(0)/G(1) phase cells increased, S phase ratio decreased and apoptosis rate increased after HCT116 cells were exposed to CAPE (10, 5, and 2.5 mg/L) for 24 h. CAPE treatment was associated with decreased cytoplasmic beta-catenin, nuclear beta-catenin and a concurrent increase in beta-catenin protein expression at cell-cell junctions. CONCLUSION: CAPE could inhibit HCT116 cell proliferation and induce cell cycle arrest and apoptosis. Decreased beta-catenin protein expression may mediate the anti-proliferative effects of CAPE.

[Detecting protein expression of EphrinB2 ligand and its receptor EphB4 in astrocytoma using confocal laser scanning microscopy].

BACKGROUND & OBJECTIVE: EphrinB2 is a novel angiogenic factor. EphrinB2 and its receptor EphB4 express in several kinds of tumor cells,and correlate with tumorigenesis and neoangiogenesis. This study was designed to explore the characteristics of EphB4 and EphrinB2 protein expression in astrocytomas. METHODS: Double labelling immunofluorescence was used to detect co-expression of EphB4/EphrinB2 with glial fibrilary acid protein (GFAP)or CD34 protein in 35 fresh glioma specimens, and 2 kinds of human glioma cell lines (CHG-5,and SHG-44). RESULTS: EphB4/EphrinB2 and CD34 proteins co-expressed in some tumor stromal microvessels, and mainly localizing in endothelial cells. Co-expression of EphB4/EphrinB2 and GFAP proteins was also noticed in tumor cells,and 2 glioma cell lines. In poorly-differentiated SHG-44 cells, the average fluorescence intensity of EphB4 was 72.48+/-33.78,and that of EphrinB2 was 96.80+/-36.98, both higher than those in well-differentiated CHG-5 cells (56.7+/-21.7, and 53.6+/-18.8). But the green fluorescence intensity of GFAP in SHG-44 cells was 22.3+/-15.3, while in CHG-5 cells was 47.5+/-16.7. CONCLUSION: Expressions of EphB4 and EphrinB2 proteins may be related to differentiation degree of tumor cells.