Down-regulation of MFG-E8 by RNA interference combined with doxorubicin triggers melanoma destruction.

The pathogenic mechanism of malignant melanoma involves the dynamic interplay of transformed cell and normal host cell, but cancer treatments always target each partition separately. In the tumor microenvironment, milk fat globule epidermal growth factor-8 (MFG-E8) is a secreted glycoprotein highly expressed in the vertical growth phase of melanoma, leading to tumor progression through coordinated αvß3 and αvß5 integrin signaling in tumor cells and host cells. Doxorubicin (Dox) is one of the most widely used antitumor drugs against a lot of solid tumors, including melanoma. In this work, Dox was used to combine with down-regulation of MFG-E8 by RNA interference (RNAi) in order to determine the synergistic effect of the antitumor activity in vivo. And the possible mechanisms were investigated. Results showed that combination group (MFG-E8 RNAi plus Dox) could inhibit the growth of melanoma more effectively than monotherapy or control groups. We found that the combination treatment induced more tumor cell apoptosis and inhibited more neovascularization than other groups. Moreover, this combination treatment attenuated CD4(+) CD25(+) Foxp3(+) Treg cells in tumor-infiltrating lymphocytes compared with other groups. Our findings suggested that MFG-E8 down-regulation enhanced the antitumor function of chemotherapy through coordinated cell apoptosis and immune-mediated mechanisms, which might be a feasible way for cancer therapy.

[The molecular mechanism of extravasation in mouse melanoma lung metastasis model].

OBJECTIVE: To study molecular mechanisms underlying the extravasation of mice melanoma cells during lung metastasis. METHODS: B16-RED melanoma cell line was established which stably express the red fluorescent protein. B16-RED cells were compared with B16 cells in ability of proliferation and lung metastasis. A mouse lung metastasis model was established with B16-RED melanoma cells. FITC-dextran was injected i.v. and CD31 indirect immunoflourescence (IIF) staining was made to identify the location of the tumor cells and the time of tumor cell extravasation. Finally, at 48 hours post cell injection, the lung and a normal lung were removed and used for 32K mice microarray analysis. RESULTS: B16-RED was consistent with B16 in cell shape and ability of proliferation and lung metastasis. 52.7% of B16-RED melanoma cells completed the extravasation within 48 hours in mouse lung metastasis model. Many important signal pathways were involved during lung metastasis, including leukocyte transendothelial migration, MAPK signaling pathway, neuroactive ligand-receptor interaction, focal adhesion, cytokine-cytokine receptor interaction, regulation of actin cytoskeleton, axon guidance, calcium signaling pathway, tight junction, etc. CONCLUSION: The extravasation during metastasis is a complex and multiple-steps process, in which many important signal pathways in host tissues were involved.

Hyperthermia increases the therapeutic efficacy of survivinT34A in mouse tumor models.

The use of survivinT34A mutant targeted disruption of survivin, the strongest inhibitor of apoptosis protein overexpressed in tumors, has proved a promising strategy for advanced cancers. However, hyperthermia, as a cytotoxic enhancer, regularly activates the expression of survivin to counteract the heat-induced antitumor activity. Here, we investigated the combinational antitumor effect by using liposome-encapsulated mouse survivinT34A and hyperthermia in mouse models. We observed that the combination treatment of surivinT34A and hyperthermia significantly increased the growth inhibition and apoptosis of tumor cells in vitro compared with single treatment or other controls, which was similar to the effect of survivin silencing in combination with hyperthermia. Moreover, the inhibition of tumor growth in vivo was also remarkably enhanced by combination of surivinT34A and hyperthermia when compared with other treatments. Naturally, the tumor tissues in combination treatment presented the larger necrosis-like areas, more apoptotic cells and less microvessel density. Our findings suggest that the antitumor efficacy of survivin disruption can be enhanced by hyperthermia, which might be a new feasible approach for cancer therapy.

Enhanced antitumor efficacy by blocking activation of the phosphatidylinositol 3-kinase/Akt pathway during anti-angiogenesis therapy.

Anti-angiogenesis has been a promising strategy for cancer therapy. However, many signal pathways are activated during anti-angiogenic treatment to counteract the therapeutic efficacy. Among these pathways, evidence has directly pointed to the phosphatidylinositol 3-kinase/Akt (PI3K/Akt) pathway, whose activation resulted in tolerance to the absence of nutrients and oxygen when tumor angiogenesis has been inhibited. In the present study, we investigated the effects of blocking activation of the PI3K/Akt pathway on cell survival in vitro and tumor growth in vivo during anti-angiogenesis therapy. In modeled microenvironments in vitro, we observed that the phosphorylation of Akt in tumor cells was increased gradually in the absence of serum and oxygen in a time-dependent manner. The specific inhibitors of PI3K inhibited the proliferation of tumor cells in a dose-dependent manner in vitro. Moreover, inhibition was enhanced gradually with increased serum deprivation and/or hypoxia. In a mouse tumor model, we found the phosphorylation of Akt obviously increased following anti-angiogenic therapy using plasmids encoding soluble vascular endothelial growth factor receptor-2, but significantly reduced after treatment with LY294002. Consequently, the combinational treatment exhibited better antitumor effects compared with single treatments, presenting larger necrosis-like areas, more apoptotic cells, less microvessel density and less phosphorylated Akt in tumors. These results suggest that blocking activation of the PI3K/Akt pathway during anti-angiogenesis therapy could enhance antitumor efficacy. Thus, targeting the PI3K/Akt pathway might be a promising strategy to reverse tumor resistance to anti-angiogenesis therapy.

[Anti-tumor effects induced by the DNA vaccine coding human and mouse soluble VEGFR2].

OBJECTIVE: To develop a novel anti-angiogenesis strategy based on a DNA vaccine coding both human and mouse soluble VEGFR2. METHODS: The gene fragments coding human and mouse sVEGFR2 were amplified with PCR and cloned into pVITRO2 to generate pVITRO2-hm-sVEGFR2 recombinant. The in vitro VEGF blocking effect of the pVITRO2-hm-sVEGFR2 expression products on HUVEC cells were evaluated. The anti-tumor effect of pVITRO2-hm-sVEGFR2 was studied in mouse B16 model. The microvessels were stained by using CD31 antibody. RESULTS: The co-expressing vector pVITRO2-hm-sVEGFR2 was constructed successfully, confirmed by the restriction endonuclease digestion and sequencing. The expressing products of pVITRO2-hm-sVEGFR2 could obviously block the function of VEGF on promoting the proliferation of HUVEC in vitro. The tumor growth in mice was also significantly inhibited by pVITRO2-hm-sVEGFR2 expression. CD31 staining demonstrated that the microvessel density obviously decreased in tumor tissues treated with pVITRO2-hm-sVEGFR2. Both anti-tumor and anti-angiogenesis effects of pVITRO2-hm-sVEGFR2 were stronger than that of plasmids which coding only human or mouse sVEGFR2. CONCLUSION: pVITRO2-hm-sVEGFR2 could be a novel DNA vaccine for the anti-tumor therapy by inhibiting angiogenesis.

[IL-15 enhance the therapeutic effects of whole cell vaccine in mouse CT26 colon carcinoma model].

OBJECTIVE: To determine the enhancement effects of IL-15 to the tumor whole cell vaccine in tumor immunotherapy. METHODS: CT26 colon carcinoma model was established with BALB/c mice. Thirty two mice with CT26 colon carcinoma were divided randomly into four groups, which were subcutaneously injected at several spots with PBS (200 microL), CT26 whole cell vaccine (2 x 10(6) cells in 200 microL PBS), mIL-15 (20 microg, encapsulated with liposome in 200 microL 5% glucose solution) and CT26 whole cell vaccine (2 x 10(6) cells in 100 microL PBS) plus mIL-15 (20 microg, encapsulated with liposome in 100 microL 5% glucose solution) respectively every three days for six doses, the plasmid was injected beside the vaccine injecting spots. The size of tumors was measured every four days. All mice were sacrificed 30 days after tumor implantation. The pathologic observation and apoptotic analysis of tumors were preceded. RESULTS: CT26 whole cell vaccine combined with mIL-15 inhibited tumor growth by 45% compared with that of control group, the differences between CT26 + mIL-15 group and the other three groups were significant (P < 0. 05). The HE staining showed that the necrosis areas in tumors of the CT26 + mIL-15 group were larger than those of other three groups. The apoptotic index of tumors from the CT26 + mIL-15 group was (46.7 +/- 7.2)%, higher than that of the other three groups obviously (P < 0.05). CONCLUSION: IL-15 could enhance the therapeutic effects of CT26 whole cell vaccine in tumor immunotherapy.

An N-, C-terminally truncated basic fibroblast growth factor and LPD (liposome-polycation-DNA) complexes elicits a protective immune response against murine colon carcinoma.

Basic fibroblast growth factor (bFGF) is a mitogen for endothelial cells, which participates in tumor angiogenesis. Active immunity against bFGF could be a promising approach for the biotherapy of cancer. Because bFGF is abundant in normal and malignant tissues, it is presumably difficult for normal bFGF to induce immunity due to self-tolerance. In addition, previous studies have shown that a complex consisting of a cationic liposome and a non-coding plasmid DNA can be used to stimulate innate immunity. This stimulation initiates a potent cytokine response, which can inhibit tumor growth. To investigate the effects of immunity against bFGF on murine colon carcinomas, we employed an N-, C-terminally truncated basic fibroblast growth factor (tbFGF, of human origin) as an antigen and a liposome-DNA complex as an adjuvant. After six immunizations, a robust bFGF-specific immune response was elicited. Subsequently, inhibition of tumor growth and a significant reduction in tumor vasculature were observed. The antitumor effect was confirmed by adoptive therapy of activated spleen cells from the immunized mice. In vitro, a CTL assay revealed that bFGF-specific cytotoxic T lymphocytes (CTL) resulted in the lysis of mouse microvascular endothelial cells (MS1) rather than that of the CT26 colorectal cancer cells. These results suggest that anti-angiogenesis treatment induced by a bFGF-specific CTLs against microvascular endothelial cells may be a useful method for cancer therapy.