Ribonuclease inhibitor up-regulation inhibits the growth and induces apoptosis in murine melanoma cells through repression of angiogenin and ILK/PI3K/AKT signaling pathway.

Human ribonuclease inhibitor (RI), a cytoplasmic protein, is constructed almost entirely of leucine rich repeats. RI could suppress activities of ribonuclease and angiogenin (ANG) through closely combining with them. ANG is a potent inducer of blood vessel growth and has been implicated in the establishment, growth, and metastasis of tumors. ILK/PI3K/AKT signaling pathway also plays important roles in cell growth, cell-cycle progression, tumor angiogenesis, and cell apoptosis. Our previous experiments demonstrated that RI might effectively inhibit some tumor growth and metastasis. Our recent study showed that ILK siRNA inhibited the growth and induced apoptosis in bladder cancer cells as well as increased RI expression, which suggest a correlation between RI and ILK. However, the exact molecular mechanism of RI in anti-tumor and in the cross-talk of ANG and ILK signaling pathway remains largely unknown. Here we investigated the effects of up-regulating RI on the growth and apoptosis in murine melanoma cells through angiogenin and ILK/PI3K/AKT signaling pathway. We demonstrated that up-regulating RI obviously decreased ANG expression and activity. We also discovered that RI overexpression could remarkably inhibit cell proliferation, regulate cell cycle and induce apoptosis. Furthermore, up-regulation of RI inhibited phosphorylation of ILK downstream signaling targets protein kinase B/Akt, glycogen synthase kinase 3-beta (GSK-3ß), and reduced ß-catenin expression in vivo and vitro. More importantly, RI significant inhibited the tumor growth and angiogenesis of tumor bearing C57BL/6 mice. In conclusion, our findings, for the first time, suggest that angiogenin and ILK signaling pathway plays a pivotal role in mediating the inhibitory effects of RI on melanoma cells growth. This study identifies that RI may be a useful molecular target for melanoma therapy.

The expression of Bcl-XL, Bcl-XS and p27Kip1 in topotecan-induced apoptosis in hepatoblastoma HepG2 cell line.

BACKGROUND: To assess the efficacy of topotecan, a topoisomerase I specific inhibitor in S-phase, the reagent-induced apoptosis and cytotoxicity as well as related proteins expression, had been preliminarily investigated in human hepatoblastoma HepG2 cells. METHODS: Microculture tetrazolium assay (MTT), HE staining, transmission electron microscopy (TEM), flow cytometry (FCM), quantitative immunocytochemistry (QI), gene tranfection and RNAi technology were employed to carry out the exploration. RESULTS: Topotecan could potently kill HepG2 cells via inducing apoptosis and demonstrated strong cytotoxicity in a time, dose-dependent manner with IC50 of about 95 mu g/L. According to morphologic observation and FCM analyses, it was confirmed that the drug treatment, causing significant S-phase arrest, could trigger a typical interphase apoptosis, the main traits of which were identified as chromatin pycnosis and cytoplasm condensation. It was shown that the expression of Bcl-XL was simultaneously down-regulated with the up-regulation of Bcl-XS in cytoplasm, which was possibly a key downstream event following the topotecan-induced DNA damage in nucleus. The expression level of p27Kip1, a negative regulator in cell cycle at G1/S transient, was also elevated. Transfection of pcDNA 3.1-p27Kip1 into HepG2 cells could abrogate the cytotoxicity in a degree while silence of p27Kip1 with siRNA in drug treatments could significantly increased the chemosensitivity, strongly indicating that the up-regulation of p27Kip1 was not an apoptosis-promoting, but a self-rescue response against drug by moderate G0/G1 arrest. CONCLUSION: Topotecan had potent cytotoxicity against HepG2 cells by triggering an interphase apoptosis possibly mediated by increasing the ratio of Bcl-XS/Bcl-XL. Up-regulation of p27Kip1in TPT treatments could be a protective response for self-rescue and silence of the gene markedly augmented TPT cytotoxicity. Therefore, the experiment in vitro could provide a new idea for the clinical chemotherapy based on the combination of traditional drugs with the specific-siRNA targeted on the protective response gene.

[Preliminary study of apoptosis of human hepatocarcinoma cell line HepG2 induced by topotecan].

BACKGROUND & OBJECTIVE: Topotecan, a semisynthetic water-soluble derivative of camptothecin, is a potent inhibitor of DNA topoisomerase I and cell cycle specific antitumor agent; The incidence rate of hepatocarcinoma ranks the third in all types of malignant tumor in China. The clinical curative effect of the present treatments and drugs for hepatocarcinoma are not so satisfactory. The inducing effect of topotecan on apoptosis of hepatocarcinoma cell line HepG2 and its cytotoxicity on HepG2 were studied in this paper. METHODS: Microculture tetrazolium assay (MTT), HE staining, transmission electron microscope (TEM), flow cytometer (FCM) and immunohistochemistry were employed to determine and observe the apoptosis of HepG2 induced by topotecan and its lethal action on HepG2. RESULTS: Topotecan killed HepG2 cell by inducing apoptosis. Topotecan had strong anti-HepG2 activity in vitro, suggesting distinct dose- and time-dependent relationship. The drug IC50 was about 95 micrograms/L. Topotecan block HepG2 in S-phase, then further initiate apoptosis program. The morphologic characteristic of apoptosis showed that it is a typical interphase apoptosis process: the nuclei showed chromatin pycnosis, and clustered on the inner border of karyotheca, cytoplasm condensed, many vacuoles occurred in it. The expression of bcl-2 were not affected in the course of HepG2 apoptosis induced by topotecon (P > 0.05). CONCLUSION: Topotecan had significant cytotoxicity on HepG2 by inducing apoptosis.