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Genetically engineered endostatin-lidamycin fusion proteins effectively inhibit tumor growth and metastasis.

Jiang WG, Lu XA, Shang BY, Fu Y, Zhang SH, Zhou D, Li L, Li Y, Luo Y, Zhen YS - BMC Cancer (2013)

Bottom Line: ES-LDP and ES show higher binding capability than LDP to lung carcinoma; in addition, ES-LDP and ES share similar binding capability.Furthermore, the enediyne-energized fusion protein ES-LDP-AE demonstrated significant efficacy against lung carcinoma xenograft in athymic mice.The ES-based fusion protein therapy provides some fundamental information for further drug development.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, P, R, China. yluo@tsinghua.edu.cn.

ABSTRACT

Background: Endostatin (ES) inhibits endothelial cell proliferation, migration, invasion, and tube formation. It also shows antiangiogenesis and antitumor activities in several animal models. Endostatin specifically targets tumor vasculature to block tumor growth. Lidamycin (LDM), which consists of an active enediyne chromophore (AE) and a non-covalently bound apo-protein (LDP), is a member of chromoprotein family of antitumor antibiotics with extremely potent cytotoxicity to cancer cells. Therefore, we reasoned that endostatin-lidamycin (ES-LDM) fusion proteins upon energizing with enediyne chromophore may obtain the combined capability targeting tumor vasculature and tumor cell by respective ES and LDM moiety.

Methods: In this study, we designed and obtained two new endostatin-based fusion proteins, endostatin-LDP (ES-LDP) and LDP-endostatin (LDP-ES). In vitro, the antiangiogenic effect of fusion proteins was determined by the wound healing assay and tube formation assay and the cytotoxicity of their enediyne-energized analogs was evaluated by CCK-8 assay. Tissue microarray was used to analyze the binding affinity of LDP, ES or ES-LDP with specimens of human lung tissue and lung tumor. The in vivo efficacy of the fusion proteins was evaluated with human lung carcinoma PG-BE1 xenograft and the experimental metastasis model of 4T1-luc breast cancer.

Results: ES-LDP and LDP-ES disrupted the formation of endothelial tube structures and inhibited endothelial cell migration. Evidently, ES-LDP accumulated in the tumor and suppressed tumor growth and metastasis. ES-LDP and ES show higher binding capability than LDP to lung carcinoma; in addition, ES-LDP and ES share similar binding capability. Furthermore, the enediyne-energized fusion protein ES-LDP-AE demonstrated significant efficacy against lung carcinoma xenograft in athymic mice.

Conclusions: The ES-based fusion protein therapy provides some fundamental information for further drug development. Targeting both tumor vasculature and tumor cells by endostatin-based fusion proteins and their enediyne-energized analogs probably provides a promising modality in cancer therapy.

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ES, ES-LDP or LDP-ES inhibited in vitro tubule formation. (A) ES or ES-based fusion proteins inhibited tubule formation of HMEC on Matrigel. Low (1 μM) and high (10 μM) concentrations were used. Bar, 200 μm. (B) Mean capillary tube number and (C) mean tube length was decreased by ES and ES-based fusion proteins after 12-h incubation. Three arbitral optical images were taken for each of the two independent experiments. Data are expressed as mean and standard deviation from six independent images, n = 6. *, P ≤ 0.05, **, P ≤ 0.001, compared with VEGF-control and #, P ≤ 0.05, compared with ES, in tube length and tube number, respectively. Cells were viewed with a microscope and pictures were taken at × 40.
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Figure 3: ES, ES-LDP or LDP-ES inhibited in vitro tubule formation. (A) ES or ES-based fusion proteins inhibited tubule formation of HMEC on Matrigel. Low (1 μM) and high (10 μM) concentrations were used. Bar, 200 μm. (B) Mean capillary tube number and (C) mean tube length was decreased by ES and ES-based fusion proteins after 12-h incubation. Three arbitral optical images were taken for each of the two independent experiments. Data are expressed as mean and standard deviation from six independent images, n = 6. *, P ≤ 0.05, **, P ≤ 0.001, compared with VEGF-control and #, P ≤ 0.05, compared with ES, in tube length and tube number, respectively. Cells were viewed with a microscope and pictures were taken at × 40.

Mentions: An endothelial tubule formation assay was used to further confirm the antiangiogenic activity of the fusion proteins. In this experiment, the use of Matrigel permits the growth and differentiation of endothelial cells into tubal structures that are reminiscent of blood vessels. Prominent tubal structures were observed in control cells (Figure 3A). ES or ES-based fusion proteins inhibited tube formation of HMEC in a concentration-dependent manner. At the low concentration (1 μM; Figure 3A, left column) ES or ES-based fusion proteins began to disrupt the formation of the tubes, as indicated by the arrows. At the high concentration (10 μM; Figure 3A, right column), ES or ES-based fusion proteins eliminated the tubal structures. As shown in Figure 3B and 3C, ES or ES-based fusion proteins reduced the number of closed capillary tubes as well as their length.


Genetically engineered endostatin-lidamycin fusion proteins effectively inhibit tumor growth and metastasis.

Jiang WG, Lu XA, Shang BY, Fu Y, Zhang SH, Zhou D, Li L, Li Y, Luo Y, Zhen YS - BMC Cancer (2013)

ES, ES-LDP or LDP-ES inhibited in vitro tubule formation. (A) ES or ES-based fusion proteins inhibited tubule formation of HMEC on Matrigel. Low (1 μM) and high (10 μM) concentrations were used. Bar, 200 μm. (B) Mean capillary tube number and (C) mean tube length was decreased by ES and ES-based fusion proteins after 12-h incubation. Three arbitral optical images were taken for each of the two independent experiments. Data are expressed as mean and standard deviation from six independent images, n = 6. *, P ≤ 0.05, **, P ≤ 0.001, compared with VEGF-control and #, P ≤ 0.05, compared with ES, in tube length and tube number, respectively. Cells were viewed with a microscope and pictures were taken at × 40.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4016579&req=5

Figure 3: ES, ES-LDP or LDP-ES inhibited in vitro tubule formation. (A) ES or ES-based fusion proteins inhibited tubule formation of HMEC on Matrigel. Low (1 μM) and high (10 μM) concentrations were used. Bar, 200 μm. (B) Mean capillary tube number and (C) mean tube length was decreased by ES and ES-based fusion proteins after 12-h incubation. Three arbitral optical images were taken for each of the two independent experiments. Data are expressed as mean and standard deviation from six independent images, n = 6. *, P ≤ 0.05, **, P ≤ 0.001, compared with VEGF-control and #, P ≤ 0.05, compared with ES, in tube length and tube number, respectively. Cells were viewed with a microscope and pictures were taken at × 40.
Mentions: An endothelial tubule formation assay was used to further confirm the antiangiogenic activity of the fusion proteins. In this experiment, the use of Matrigel permits the growth and differentiation of endothelial cells into tubal structures that are reminiscent of blood vessels. Prominent tubal structures were observed in control cells (Figure 3A). ES or ES-based fusion proteins inhibited tube formation of HMEC in a concentration-dependent manner. At the low concentration (1 μM; Figure 3A, left column) ES or ES-based fusion proteins began to disrupt the formation of the tubes, as indicated by the arrows. At the high concentration (10 μM; Figure 3A, right column), ES or ES-based fusion proteins eliminated the tubal structures. As shown in Figure 3B and 3C, ES or ES-based fusion proteins reduced the number of closed capillary tubes as well as their length.

Bottom Line: ES-LDP and ES show higher binding capability than LDP to lung carcinoma; in addition, ES-LDP and ES share similar binding capability.Furthermore, the enediyne-energized fusion protein ES-LDP-AE demonstrated significant efficacy against lung carcinoma xenograft in athymic mice.The ES-based fusion protein therapy provides some fundamental information for further drug development.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, P, R, China. yluo@tsinghua.edu.cn.

ABSTRACT

Background: Endostatin (ES) inhibits endothelial cell proliferation, migration, invasion, and tube formation. It also shows antiangiogenesis and antitumor activities in several animal models. Endostatin specifically targets tumor vasculature to block tumor growth. Lidamycin (LDM), which consists of an active enediyne chromophore (AE) and a non-covalently bound apo-protein (LDP), is a member of chromoprotein family of antitumor antibiotics with extremely potent cytotoxicity to cancer cells. Therefore, we reasoned that endostatin-lidamycin (ES-LDM) fusion proteins upon energizing with enediyne chromophore may obtain the combined capability targeting tumor vasculature and tumor cell by respective ES and LDM moiety.

Methods: In this study, we designed and obtained two new endostatin-based fusion proteins, endostatin-LDP (ES-LDP) and LDP-endostatin (LDP-ES). In vitro, the antiangiogenic effect of fusion proteins was determined by the wound healing assay and tube formation assay and the cytotoxicity of their enediyne-energized analogs was evaluated by CCK-8 assay. Tissue microarray was used to analyze the binding affinity of LDP, ES or ES-LDP with specimens of human lung tissue and lung tumor. The in vivo efficacy of the fusion proteins was evaluated with human lung carcinoma PG-BE1 xenograft and the experimental metastasis model of 4T1-luc breast cancer.

Results: ES-LDP and LDP-ES disrupted the formation of endothelial tube structures and inhibited endothelial cell migration. Evidently, ES-LDP accumulated in the tumor and suppressed tumor growth and metastasis. ES-LDP and ES show higher binding capability than LDP to lung carcinoma; in addition, ES-LDP and ES share similar binding capability. Furthermore, the enediyne-energized fusion protein ES-LDP-AE demonstrated significant efficacy against lung carcinoma xenograft in athymic mice.

Conclusions: The ES-based fusion protein therapy provides some fundamental information for further drug development. Targeting both tumor vasculature and tumor cells by endostatin-based fusion proteins and their enediyne-energized analogs probably provides a promising modality in cancer therapy.

Show MeSH
Related in: MedlinePlus