Expression characteristics of heparanase in colon carcinoma and its close relationship with cyclooxygenase-2 and angiogenesis.

BACKGROUND/AIMS: Heparanase (HPA) influences tumourigenesis and tumour progression by various mechanisms, including angiogenesis. Cyclooxygenase-2 (COX-2) was strongly correlated with microvessel density, and that COX-2 expression is up-regulated by HPA in esophageal cancer. In this study, we examined the relationship between HPA expression and that of COX-2 in colon carcinoma. The aim of this study was to determine whether the expression of HPA is related to the angiogenesis in colorectal cancer and whether it could be involved in clinical behaviour of colon carcinoma. METHODOLOGY: HPA and COX-2 was analyzed with Immunohistochemistry and Western blot. Microvessels in colon carcinoma were examined by using anti-CD34 antibody. Statistical analysis was applied to test for the prognostic and diagnostic associations. RESULTS: Immunohistochemistry revealed that HPA was expressed at low level in normal colonic mucosa (4/78, 5.1%), but at higher level in tumor tissues (63/78, 80.7%) and closely correlated with tumor lymph node metastasis (p < 0.05). This result was further confirmed by Western blot analysis. Furthermore, carcinomas with high HPA expression demonstrated high COX-2 expression and high MVD (microvesseldensity) labelled with CD34. In addition, mortality was higher in patients with HPA+ phenotype and HPA was an independent predictor of overall survival (p < 0.05). CONCLUSIONS: Our findings indicated that HPA might be an important biomarker for malignant transformation and be involved in promoting colon carcinoma metastasis by increasing angiogenesis.

Antiviral effects of Salvia miltiorrhiza (Danshen) against enterovirus 71.

In this study, the antiviral activities of seven different extracts of Salvia miltiorrhiza (danshen) were determined. The first two extracts, SA1 and SA2, isolated at room temperature by ethyl acetate and water extraction, respectively, neutralized the enterovirus 71-induced cytopathic effect in Vero, rhabdomyosarcoma and MRC-5 cells. The other five crude extracts, extracted with warm water (60-70 degrees C) or organic solvents, did not have any protective activity. The 50% inhibitory concentrations for neutralizing the enterovirus 71-induced cytopathic effect were 0.742 +/- 0.042 mg/ml for SA1 and 0.585 +/- 0.018 mg/ml for SA2 in Vero cells. No antiviral activity was observed in the other viruses tested. Antiviral activity was more efficient in cultures treated with SA1 or SA2 during viral infection compared to the cultures treated before or after infection, suggesting that these danshen extracts could interfere with viral entry. SA1 and SA2 were able to inhibit viral RNA synthesis in the infected cells and to abate the apoptotic process in enterovirus 71-infected Vero cells. We conclude that danshen extracts possess antiviral activity and have potential for the development as an anti-enterovirus 71 agent.

Study on the mechanism of epidermal growth factor-induced proliferation of hepatoma cells.

AIM: Many growth factors, such as epidermal growth factor (EGF), are associated with the carcinogenesis. EGF plays its role in the proliferation of hepatoma cells through binding with EGF receptor (EGFR) and a series of signal transduction. But the postreceptor pathway is still not clear. In the present experiment, we studied the effect of tyrosine kinase, protein kinase C, Na(+)/H(+) exchange, calmodulin and voltage-dependent Ca(2+) channel on EGF-induced hepatoma cell proliferation. METHODS: Hepatoma cell line SMMC7721 was cultured in RPMI1640 serum-free medium. In order to study the effect of thyrosine kinase, protein kinase C, Na(+)/H(+) exchange, calmodulin and voltage-dependent Ca(2+) channel on human heptoma cell proliferation induced by epidermal growth factor (EGF), DNA synthesis rate of hepatoma cells was measured by the method of (3)H-TdR incorporation. RESULTS: EGF (10(-9) M) stimulated the proliferation of heptoma cells significantly ((3)H-TdR incorporation was 1 880+/-281 cpm/well, P<0.05), and this effect was significantly inhibited by tyrosine kinase inhibitor genistein ((3)H-TdR incorporation was 808+/-209 cpm/well, P<0.001). Calmodulin inhibitor W-7, protein kinase C inhibitor H-7 and Na(+)/H(+) exchange inhibitor amiloride individually had significant inhibiting effect on EGF-induced proliferation of hepatoma cells ((3)H-TdR incorporation was 978+/-87.3 cpm/well, 1 241+/-147 cpm/well, 1 380+/-189 cpm/well, respectively, P<0.001, P<0.01, P<0.05), but they all had no effect on the basal level proliferation of cultured hepatoma cells ((3)H-TdR incorporation was 1 284+/-260 cpm/well, 1 179+/-150 cpm/well, 1 392+/-152 cpm/well, respectively, (3)H-TdR incorporation of the control was 1 353+/-175 cpm/well, P>0.05). Voltage-dependent Ca(2+) channel inhibitor verapamil had no inhibition on EGF-induced proliferation of hepatoma cells ((3)H-TdR incorporation was 1 637+/-133 cpm/well, P>0.05), it also had no effect on the basal level proliferation of cultured hepatoma cells ((3)H-TdR incorporation was 1 196+/-112 cpm/well, P>0.05). CONCLUSION: Our data suggest that tyrosine kinase, Ca(2+)-calmodulin-dependent pathway, protein kinase C and Na(+)/H(+) exchange play a critical role in EGF-induced proliferation of hepatoma cells and that the effect of EGF is independent of voltage-dependent Ca(2+) channel.

[Expression of human pituitary tumor transforming gene 1 in colorectal cancer detected with real-time fluorescent quantitative PCR and its clinical significance].

BACKGROUND & OBJECTIVE: Although human pituitary tumor transforming gene 1 (hPTTG1) is overexpressed in malignant tumors such as colorectal cancer, its correlation to clinicopathologic parameters and its value in diagnosis and prognosis prediction of colorectal cancer are still not clear. We investigated the expression of hPTTG1 in colorectal cancer tissues, and elucidated its correlation to some clinicopathologic parameters of colorectal cancer. METHODS: The expression of hPTTG1 in 60 specimens of colorectal cancer and corresponding noncancerous tissues were examined with real-time fluorescent quantitative polymerase chain reaction, and its correlation to seven clinicopathologic parameters were analyzed. RESULTS: The mRNA level of hPTTG1 was significantly higher in colorectal cancer tissues than in corresponding noncancerous tissues (0.42+/-0.07 vs. 0.03+/-0.01, P<0.001), significantly higher in colorectal cancer tissues with serum CEA level of > 5 ng/mL than in those with CEA of < 5 ng/mL (22.79+/-7.42 vs. 9.34+/-2.61, P<0.001), significantly higher in colorectal cancer tissues with diameter of > or = 3.5 cm than in those with diameter of < 3.5 cm (15.80+/-8.80 vs. 10.91+/-5.22, P<0.05), significantly lower in Dukes'A, B tumors than in Dukes' C, D tumors (9.03+/-0.35 and 9.58+/-2.93 vs. 15.88+/-8.09 and 25.69+/-7.67, P<0.001), and significantly higher in colorectal cancer tissues with lymph node metastasis (17.63+/-8.47), liver metastasis (31.07+/-4.10) and other organ metastasis (22.78+/-6.39) than in those without metastasis (11.15+/-6.65) (P<0.001). hPTTG1 expression had no relationship with patients' age, sex and histological type (P>0.05). CONCLUSIONS: hPTTG1 is overexpressed in colorectal cancer. It is closely related to the progression of colorectal cancer, and may be helpful for prognosis prediction of colorectal cancer.

Reticulon 3 binds the 2C protein of enterovirus 71 and is required for viral replication.

Enterovirus 71 is an enterovirus of the family Picornaviridae. The 2C protein of poliovirus, a relative of enterovirus 71, is essential for viral replication. The poliovirus 2C protein is associated with host membrane vesicles, which form viral replication complexes where viral RNA synthesis takes place. We have now identified a host-encoded 2C binding protein called reticulon 3, which we found to be associated with the replication complex through direct interaction with the enterovirus 71-encoded 2C protein. We observed that the N terminus of the 2C protein, which has both RNA- and membrane-binding activity, interacted with reticulon 3. This region of interaction was mapped to its reticulon homology domain, whereas that of 2C was encoded by the 25th amino acid, isoleucine. Reticulon 3 could also interact with the 2C proteins encoded by other enteroviruses, such as poliovirus and coxsackievirus A16, implying that it is a common factor for such viral replication. Reduced production of reticulon 3 by RNA interference markedly reduced the synthesis of enterovirus 71-encoded viral proteins and replicative double-stranded RNA, reducing plaque formation and apoptosis. Furthermore, reintroduction of nondegradable reticulon 3 into these knockdown cells rescued enterovirus 71 infectivity, and viral protein and double-stranded RNA synthesis. Thus, reticulon 3 is an important component of enterovirus 71 replication, through its potential role in modulation of the sequential interactions between enterovirus 71 viral RNA and the replication complex.

[Cloning, expression of human keratinocyte growth factor and its purification and identification].

To clone KGF-2 gene, get hKGF-2 protein and detemine its activity. The cNDA of human KGF-2 was isolated from fetal lung by RT-PCR and cloned into pBV220 plasmid. The recombinant pBV220-hKGF-2 plasmid was transformed into E. coli (BL21), induced at 42 degrees C for the expression of hKGF-2. Recombinant human KGF-2 was purified from the ultrasonic-treated BL21 by heparin-Sepharose CL-6B treated column chromatography and cation exchange column chromatography. MTT method was used for the determination of its biological activity. SDS-PAGE showed that rhKGF-2 was expressed in E. coli BL21 as soluble protein of approximately 20kD. The rhKGF-2 protein can stimulate the proliferation of NIH3T3 cells significantly from 1 ng/mL to 10 ng/mL. HKGF-2 cDNA wasclned and highly expressed in E. coli BL21 and the purified rhKGF-2 showed the mitogenic activity on NIH3T3 cells.