Interferon-α enhances antitumor activities of oncolytic adenovirus-mediated IL-24 expression in hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) has a dismal 5-year-survival rate of 10%, so novel strategies are warranted. IL-24 mediates anti-tumor activity reducing STAT3 expression, which suggests that interferon (IFN) alpha may augment tumor cell lysis and reduce angiogenesis. We investigated the antitumor activity of treatment with IFN-α, with the oncolytic adenovirus SG600-IL-24, or the combination of both in HCC in vitro and in vivo. RESULTS: RT-PCR, ELISA assay and Western-blot confirmed that the exogenous IL-24 gene was highly expressed in HCC cells infected with SG600-IL-24. Treatment with combined IFN-α and SG600-IL-24 suppressed growth and promoted apoptosis of the HepG2, MHCC97L, and HCCLM3 cell lines compared with the normal cell line L02. The combined therapy increased STAT1 and SOCS1 and apoptosis, but decreased the expression of the metastatic and angiogenic proteins MMP-2, XIAP, OPN, and VEGF, which are regulated by STAT3 in HCC cells in vitro. To assess the effects in vivo, the HCC cell line HCCLM3 was transplanted subcutaneously into the right flanks of nude mice. Mice in the IFN-α group, the SG600-IL-24 group, or the combined therapy group had significantly suppressed growth of the HCC xenografted tumors compared to the PBS control group of mice. Among the mice treated with the combination of IFN-α and SG600-IL-24, three of those eight mice had long-term survival and no evidence of a tumor. These mice also had decreased expression of the metastatic and angiogenic proteins MMP-2, XIAP, OPN, and VEGF. CONCLUSIONS: The present study demonstrated for the first time the potential antitumor activity of IFN-α combined with the oncolytic adenovirus SG600-IL-24 in HCC both in vitro and in vivo, and suggests its further development as a potential candidate for HCC cancer gene therapy.

Transfection of IL-2 and/or IL-12 genes into spleen in treatment of rat liver cancer.

AIM: To test the efficacy of gene therapy in rat liver tumor. METHODS: A retroviral vector GCIL12EIL2PN encoding human IL-2 (hIL-2) and mouse IL-12 (mIL-12) fused gene and its packaging cell were constructed. The packaging cell lines contained of IL-2 and/or IL-12 genes were injected intrasplenically to transfect splenocyte at different time. The therapeutic effect, immune function and toxic effect were evaluated. RESULTS: The average survival times of the 4 groups using IL genes at days 1, 3, 5 and 7 after tumor implantation were 53.3+/-3.7, 49.3+/-4.2, 31.0+/-2.1 and 24.3+/-1.4 d respectively in IL-2/IL-12 fused gene group, 25.0+/-2.5, 23.5+/-2.0, 18.3+/-2.4 and 12.0+/-1.8 d respectively in IL-2 gene treatment group, and 39.0+/-4.8, 32.0+/-3.9, 23.0+/-2.5 and 19.4+/-2.1 d respectively in IL-12 gene treatment group (P<0.01, n=10). In the IL-12/IL-2 fused gene treatment group, 30% of rats treated at days 1 and 3 survived more than 60 d and serum mIL-12 and hIL-2 levels were still high at day 3 after treatment. Compared with IL alone, NK cell activity was strongly stimulated by IL-2/IL-12 gene. Microscopy showed that livers were infiltrated by a number of lymphocytes. CONCLUSION: IL-2 and/or IL-12 genes injected directly into spleen increase serum IL-2 and IL-12 levels and enhance the NK cell activity, which may inhibit the liver tumor growth. The therapy of fused gene IL-2/IL-12 is of low toxicity and relatively high NK cell activity. Our data suggest that IL-2/IL-12 fused gene may be a safe and efficient gene therapy for liver tumor. The gene therapy should be administrated as early as possible.

Relationship between the imaging features and pathologic alteration in hepatoma of rats.

AIM: The imaging features of MRI and DSA, using the models of implanted and induced hepatoma, were investigated in rats. METHODS: CBRH3 cancer cells were implanted for different liver site of rat liver and the diethylnitrosoamine was given orally to rats in order to induce liver cancer. Both experimental groups were detected by magnetic resonance imaging (MRI), digital subtraction angiography (DSA) and morphologic assay. RESULTS: Hypointensity on T1WI and homogenous high signal intensity on T2WI in MRI, and ring-like abnormal stain on DSA were found in implanted cancer. Induced cancers appeared as homogeneous or heterogeneous hypointensity on T1WI (10 cases), and equal or slight high intensity on T2WI (8 cases), but some as hypointensity on T2WI (2 cases). CONCLUSION: The imaging features of implanted cancers were similar to that of human liver metastases. Therefore, it could serve as an experimental model of human liver metastatic tumor. The imaging feature of induced cancers, whereas, were similar to that of human primary liver cancer. It could be use as an experimental model of human primary liver cancer.

[Treatment of hepatocellular carcinoma by transfecting interleukin-12 and interleukin-2 fusion gene intrasplenically, an experimental study].

OBJECTIVE: To study the inhibitory effect of retroviral packaging cells injected intrasplenically encoding mouse interleukin-12 (mIL-12) and human interleukin-2 (hIL-2) fusion gene on the growth of hepatocellular carcinoma. METHODS: The retroviral vectors encoding mIL-12 gene, hIL-2 gene, and mIL-12 and hIL-2 genes, GCIL12EXPN, GCXEIL2PN, and GCIL12EIL2PN were constructed and then transfected into the retroviral packaging cells PA317 to construct cells PA317-GCIL12EXPN, PA317-GCXEIL2PN, and PA317-GCIL12EIL2PN. Rat hepatocellular carcinoma cells CBRH3 were implanted into the livers of Wistar rats to establish hepatoma animal model. Then the rats were divided into 5 groups to be injected intrasplenically with normal saline one day after the implantation (0.8 ml/rat, group I, n = 10), blank vector PA317-GCXEXPN one day after the implantation (10(7) cells/rat, group II, n = 10), PA317-GCIL12EXPN containing IL-2 gene (1 x 10(7) cells/rat 1, 3, 5, or 7 days after the implantation, group III, n = 40), PA317-GCXEIL2PN containing mIL-12 gene (1 x 10(7) cells/rat 1, 3, 5, or 7 days after the implantation, group IV, n = 40), and PA317-GCIL12EIL2PN containing IL-12-IL-2 fusion gene (1 x 10(7) cells/rat 1, 3, 5, or 7 days after the implantation, group V, n = 40) respectively. The rats surviving longer than 2 months were re-injected with hepatocellular carcinoma cells. The therapeutic effect, immune function and toxic effect were evaluated. CT was conducted on the liver before and after the experiment. Laparotomy was performed 3 and 7 days after treatment to resect some of the carcinoma tissues to undergo pathological examination and OX8 immunohistostaining. Serum mIL-12 and hIL-2 were detected one day before and 3, 7, 30, and 60 days after treatment. RESULTS: The average survival times of the rats treated with IL-12-IL-2 fusion gene at the first, third, fifth and seventh day after tumor implantation were 53.3 +/- 3.7 days, 49.3 +/- 4.2 days, 31.0 +/- 2.1 days, and 24.3 +/- 1.4 days respectively, longer than those treated with IL-2 gene (25.0 +/- 2.5 days, 23.5 +/- 2.0 days, 18.3 +/- 2.4 days, and 12.0 +/- 1.8 days respectively, P < 0.001), and those treated with IL-12 gene (39.0 +/- 4.8 days, 32.0 +/- 3.9 days, 23.0 +/- 2.5 days, and 19.4 +/- 2.1 days respectively, P < 0.001). Long survival (>or= 60 days) rate in the rats treated with IL-12-IL-2 fusion gene on the first and third day was 30%. The serum mIL-12 and hIL-2 levels in these rats remained high on the 60th day after treatment. The pathological study showed that the number of infiltrating lymphocytes in liver tumor tissues was increased in the IL-12-IL-2 fusion gene treatment group. CONCLUSION: The retroviral packaging cell line injected intrasplenically encoding mIL-12 and hIL-2 fusion gene inhibits the growth of hepatocellular carcinoma significantly in rats. The therapeutical efficacy of early administration is superior to that of late one.

Cortactin expression confers a more malignant phenotype to gastric cancer SGC-7901 cells.

AIM: To study the effects of cortactin on the tumor biology of SGC-7901 cells and identify the mechanism involved in the process. METHODS: Cell lines in which cortactin was stably overexpressed or knocked down as well as the respective control cell lines were established by standard molecular methods. The effects of cortactin on the proliferation, migration and invasion capacity of SGC-7901 cells were assessed by the MTT assay, colony formation, flow cytometry, transwell migration and matrigel invasion. Nude mouse models were also used to assess the role of cortactin in the growth and metastasis of SGC-7901 cells in vivo. Western blotting analysis was performed to detect the expression of epidermal growth factor receptor (EGFR) and downstream molecules. RESULTS: Cell lines in which cortactin was stably overexpressed or knocked down as well as control cell lines were successfully established and designated as LV5-cortactin-SGC, LV5-SGC, LV3-shRNA-SGC and LV3-SGC. Cortactin overexpression promoted SGC-7901 cell migration (340.7 ±12.6 vs 229.1 ± 23.2, P < 0.01) and invasion (71.6 ± 5.2 vs 48.4 ± 3.6, P < 0.01). Cortactin downregulation impaired SGC-7901 cell migration (136.2 ± 19.8 vs 225 ± 17) and invasion (29.2 ± 5.2 vs 49.6 ± 3.8, P < 0.01). The results from the MTT and colony formation assays results indicated increased LV5-cortactin-SGC cell proliferation and decreased LV3-shRNA-SGC cell proliferation compared to the control cells. Flow cytometry analysis demonstrated that cortactin overexpression promoted the proliferation index of SGC-7901 cells, and the results were reversed when cortactin was downregulated. Mouse tumor models confirmed that cortactin expression increased SGC-7901 cell proliferation and metastasis in vivo. Western blotting analysis revealed that cortactin elevated EGFR expression and activated the downstream molecules. CONCLUSION: Cortactin expression promoted the migration, invasion and proliferation of SGC-7901 cells both in vivo and in vitro. The EGFR signaling pathway is mechanistically involved.

Inhibition of hepatocellular carcinoma by fulvestrant involves the estrogen receptor α and Wnt pathways in vitro and in patients.

The aim of the present study was to investigate the effect of anti-estrogen treatment (fulvestrant) on the biological activity of hepatocellular carcinoma (HCC), involving the estrogen receptor α (ERα) and Wnt pathways, and to evaluate whether ERα and Wnt inhibitory factor-1 (WIF1) could be biomarkers for anti-estrogen clinical therapy. H22 and HepG2 cells were treated with 0.04 to 625 nM fulvestrant and the WST-8 method was used to assess the inhibition rate after 72 h. Furthermore, prolactin (PRL) secretion by HepG2 cells was assessed at 24 h using an enzyme immunoassay. Quantitative polymerase chain reaction and western blot analysis were used to analyze the mRNA and protein expression levels of ERα, ß-catenin and WIF1, respectively, in HepG2 cells. For clinical patient analysis, the tumor volume was analyzed by magnetic resonance imaging methods, and PRL in the blood was detected by an enzyme immunoassay. In HepG2 cells, the mRNA and protein expression levels of ERα were downregulated (P<0.01), while ß-catenin expression remained unchanged and WIF1 expression was upregulated (P<0.01). Analysis of samples from clinical patients demonstrated that there was a positive correlation between PRL levels and tumor volume. In addition, as compared with non-cancerous tissues, the ERα mRNA levels in tumor tissue were upregulated (P<0.05), particularly in that of male patients, while WIF1 expression was significantly downregulated (P<0.01). In conclusion, fulvestrant inhibited the proliferation of HepG2 cells, involving the ERα and non-canonical Wnt pathways, and it may be a promising therapeutic for HCC.

ShRNA-mediated gene silencing of heat shock protein 70 inhibits human colon cancer growth.

Heat shock protein 70 (Hsp70), a chaperone involved in tumor progression, is overexpressed in various human tumors. However, its role in colon cancer progression is not completely understood. In the present study, two shRNA plasmid vectors against Hsp70 were constructed and stably transfected into the colon cancer cell line HT29 to determine the effect of Hsp70 on cell proliferation, cell cycle distribution and cell apoptosis in HT29 cells in vitro, and its effect on xenograft tumor growth and apoptosis in vivo. Cell proliferation was determined using MTT assay. The results revealed that Hsp70 silencing efficiently inhibited the growth of HT29 cells in culture, induced cell cycle arrest at the G1 phase, and significantly increased apoptosis. Moreover, stable clones from the Hsp70 shRNA-2 vector suppressed xenograft tumor growth and enhanced apoptosis in vivo compared with a mock and vector control group. In conclusion, specific Hsp70 shRNA silencing may inhibit colon cancer growth, indicating that Hsp70 silencing is a potential therapeutic strategy for the treatment of colon cancer.