[Secretory expression vector V-pLNCX-s-hri inhibits the growth of mouse B16 melanoma].

BACKGROUND AND OBJECTIVE: Human ribonuclease inhibitor (hRI) extracted and purified from human placenta has been shown to remarkably inhibit some solid tumors in mice. This study was to construct V-pLNCX-s-hri, a secretory expression vector, and explore its inhibition effects on the growth of mouse B16 melanoma cells. METHODS: The hRI gene sequence conjugated with the synthesized signal peptide of mouse IgG was cloned into the retroviral vector V-pLNCX to construct V-pLNCX-s-hri. The PA317 cells were used for viral package and NIH3T3 cells were employed to determine the viral titer. The expression of hRI gene was detected by RT-PCR and Western blot. The content of RI was determined by enzyme-linked immunoabsorption assay (ELISA). The model of B16 melanoma-carrying mouse was established and received different treatments. The tumor weight and microvessle density (MVD) were assessed. Normal saline (NS), V-pLNCX, and V-pLNCX-hri were used as controls. RESULTS: The infection efficiency of V-pLNCX-s-hri on cultured B16 cells reached 38.5%. mRNA and protein levels of hRI were detected in B16 cells infected by V-pLNCX-s-hri. The hRI content in the supernatant of infected B16 cells reached 0.228 microg/mL. The hRI content in the peripheral blood of experimental mice was significantly higher in the V-pLNCX-s-hri group (0.249 microg/mL) than in the NS group (0.035 microg/mL), V-pLNCX group (0.028 microg/mL) and V-pLNCX-hri group (0.169 microg/mL) (P<0.01). The tumor weight and MVD were significantly lower in the V-pLNCX-s-hri group compared with those in the NS, V-pLNCX and V-pLNCX-hri groups (P>0.01). CONCLUSIONS: V-pLNCX-s-hri can effectively infect B16 cells and induce high expression of hRI. V-pLNCX-s-hri is superior to V-pLNCX-hri in inhibiting the growth of B16 cells.

Antitumor effects of human ribonuclease inhibitor gene transfected on B16 melanoma cells.

Human ribonuclease inhibitor (RI) is a cytoplasmic acidic protein. The experiment demonstrated that it might effectively inhibit tumor-induced angiogenesis and inhibit tumor growth. Ribonuclease inhibitor is constructed almost entirely of leucine-rich repeats, which might be involved in unknown biological effects besides inhibiting RNase A and angiogenin activities. The exact molecular mechanism of antitumor on ribonuclease inhibitor remains unclear so far. In order to further understand the function of ribonuclease inhibitor and investigate the relationship with tumor growth, our study established a transfection of human ribonuclease inhibitor cDNA into the murine B16 cells by the retroviral packaging cell line PA317. The cell line transfected with a stably high expression of ribonuclease inhibitor was identified. We found that the transfected ribonuclease inhibitor could obviously inhibit cell proliferation, regulate cell cycle and induce cell apoptosis in vitro. Mice that were injected with the B16 cells transfected RI cDNA showed a significant inhibition of the tumor growth with lighter tumor weight, lower density of microvessels, longer latent periods, and survival time than those in the other two control groups. In conclusion, the results reveal the novel mechanism that antitumor effect of ribonuclease inhibitor is also associated with inducing apoptosis, regulating cell cycle and inhibiting proliferation besides antiangiogenesis. These results suggest that ribonuclease inhibitor might be a candidate of tumor suppressor gene in some tissues. RI could become a target gene for gene therapy. Our study may be of biological and clinical importance.

[Preparation and purification of the antibody against ribonuclease inhibitor].

AIM: To prepare and purify the antibody against ribonuclease inhibitor(RI). METHODS: RI was extracted from human placenta and purified by affinity chromatography. Rabbits anti-RI antibody was obtained after immunization and then purified through rProtein A Sepharose Fast Flow chromatography column. The characters of the antibody was identified by SDS-PAGE, ELISA and Western blot. RESULTS: An anti-RI antibody was obtained and purified. SDS-PAGE analysis showed that the purified anti-RI antibody has high purity. The results of Western blot and ELISA indicated that anti-RI antibody had high specificity and good stability. CONCLUSION: The anti-RI antibody with high titer, high specificity and good stability has been acquired, which lays the foundation for further research on RI.

[Analysis of gene expression of ribonuclease inhibitor in human breast cancer tissue].

BACKGROUND & OBJECTIVE: Ribonuclease inhibitor (RI), which is rich in human placenta, is a multi-functional acidic glycoprotein. Our previous studies showed that the growth of some solid tumors (S180 sarcoma, Ca761 breast cancer, and H22 hepatoma) could be significantly inhibited by RI extracted and purified from human placenta. This study was designed to observe the change of RI gene expression in human breast cancer tissue. METHODS: The expression level of RI mRNA was determined by reverse transcription-polymerase chain reaction (RT-PCR) and capillary electrophoresis (CE). The RI content was determined by Western blot analysis. RESULTS: The electrophoretic stripes of the RT-PCR products of the breast cancer tissues were narrower and the plaques of Western blot of the breast cancer tissues were smaller compared with the control breast tissues. The peak altitude and width of capillary electrophoretic absorptive curve of the RT-PCR product of the breast cancer tissue were clearly smaller than that of the control breast tissue. The capillary electrophoresis integral values of the absorptive peak areas of RT-PCR product of the breast cancer tissue and the control breast tissue were (3.320+/-0.365)x10(6) and (4.385+/-0.880)x10(6), respectively. The comparison between two groups reveals a remarkably difference (P< 0.01). CONCLUSION: The gene expression of RI is clearly down-regulated in human breast cancer tissue that the RI mRNA level is remarkably lower and RI content also reduces significantly in human breast cancer tissue.

[Inhibitory effect of ginsenoside-Rg3 on lung metastasis of mouse melanoma transfected with ribonuclease inhibitor].

OBJECTIVE: To investigate the effect of ginsenoside-Rg3 on lung metastasis of ribonuclease inhibitor (RI) gene-transfected mouse B16 melanoma. METHODS: C57BL/6 mice were iv injected with parental or RI-transfected B16 melanoma cells. Lung metastasis was assessed by the number of surface tumor nodules. Mice were divided into 6 groups. Group I, II and III of mice were given parental, mock-transfected and RI-transfected B16 melanoma cells, respectively while in group IV, V and VI, Rg3 (1.5 mg/kg, iv q.o.d. x 10) was given to mice bearing parental, mock-transfected and RI-transfected B16 melanoma, respectively. Micovessel density (MVD) of the lung metastatic tumor was assessed by immunohistochemical staining of factor VIII-R expression. RESULTS: The number of tumor nodules was significantly decreased in mice injected with RI-transfected B16 melanoma (Gp III, compared to Gp I and II). Rg3 treatment per se could also decrease the number of lung tumor nodules but to a lesser extent (Gp IV and V compared to Gp III). However, Rg3 synergized with RI transfection resulting in most significant inhibition of lung metastasis (Gp VI). Mice in Gp I and II died within 26 days of the experiment, whereas all the mice in Gp VI were alive during the observation period of one and one half month. MVD was significantly decreased in the lung tumor nodules in mice injected with RI-transfected B16 melanoma. It was further decreased when additional Rg3 was given (Gp VI). CONCLUSION: Transfection of ribonuclease inhibitor gene significantly reduces the metastatic potential of B16 melanoma. Ginsenoside-Rg3 has a synergistic effect.