Suppression of lung cancer in murine model: treated by combination of recombinant human endostsatin adenovirus with low-dose cisplatin

Abstract

Background: The sustained growth of tumors necessitates neovascularization. As one of the potent endogenous vascular inhibitors, endostatin has been widely used in antiangiogenesis therapy for tumor. Cisplatin is normally administered in chemotherapy for lung cancer but accompanied with serious side effects. In the current study, we investigated a novel chemo-antiangiogenesis therapeutic strategy to both improve toxic effects on lung cancer cells and reduce damages to normal cells in the anti-tumor therapy.

Methods: In vitro, we transduced LLC cells with Ad-hEndo and collected supernatants. Western blotting analysis of the supernatants revealed expression of endostatin. In vivo, to fully investigate the suppression effect on murine lung cancer of the combination therapy, we injected recombinant human endostatin adenovirus intratumorally plus a low dose of cisplatin intraperitoneally routinely. The tumor volume and survival time were observed. Angiogenesis was apparently inhibited within the tumor tissues and on the alginate beads. Assessment of apoptotic cells by the TUNEL assay was conducted in the tumor tissues.

Results: The combination treatment significantly suppressed the tumor growth and prolonged survival time of the murine LLC tumor model. This anti-tumor activity was associated with decreased microvessel density and increased apoptotic index of tumor cells.

Conclusion: According to the results in this study, recombinant human endostatin adenovirus in combination with a low dose of cisplatin demonstrated apparent synergistic anti-tumor activity without marked toxicity. Thus, these observations may provide a rational alternative for lung cancer treatment.

Figure 1

Expression of recombinant human endostatin. Recombinant human endostatin was expressed as a single band of appropriate 20 KD in Ad-hEndo transfected LLC cells(1), while no band was detected in Ad-null (2) transfected or untreated(3) tumor cells.

Figure 2

Tumor suppression and survival advantage in C57BL/6 mice bearing LLC. Tumor volume(A) and survival curves(B). Each group of Mice bearing LLC was s.c. injected intratumorally with corresponding treatment as described in "Methods". Treatment with combination of cisplatin and Ad-Endo resulted in the marked inhibition of tumor growth and longer life span(P < 0.05).

Figure 3

Inhibition of angiogenesis within tumor estimated by CD31 immunohistochemical analysis. (A) were representative sections from each group. a: Ad-hEndo+ cisplatin; b: Ad-hEndo; c: cisplatin; d: Ad-null; e: NS. (B) Vessel density was determined via counting the number of the microvessels per high-power field within hot spot area. Values were expressed as means ± SE (5 high power fields/slide). Tumors of the combination group showed smaller number of microvessel count than that of the other groups submitted to cisplatin or Ad-Endo alone, especially the NS (P < 0.05). a: Ad-hEndo+cisplatin; b: Ad-hEndo; c: cisplatin; d: Ad-null; e: NS.

Figure 4

Detection of apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining of tumor tissues. (A) Sections after treatment were stained with the TUNEL analysis to detect apoptotic cells. (B) Apoptotic index was determined by calculating the percentage of apoptotic cells among tumor cells (5 high power fields/slide). The combination group showed the highest apoptotic index. a: Ad-hEndo+ cisplatin; b: Ad-hEndo; c: cisplatin; d: Ad-null; e: NS.

Figure 5

Inhibition of antiangiogenesis assay by alginate bead in vivo. (A) Representative alginate beads from each group. (B) FITC-dextran uptake of beads from each group. The combination group showed a significant decrease in vascularization compared with the control groups(P < 0.05). The results were expressed as mean ± S.E. a: Ad-hEndo+ cisplatin; b: Ad-hEndo; c: cisplatin; d: Ad-null; e: NS.

Figure 6

Toxicity observation I. H & E staining of heart(a), liver(b), lung (c), spleen(d) and kidney(e) in recipient mice. No hemorrhage in organs appeared in the combination group and no differences were seen among groups. A: Ad-hEndo+ cisplatin; B: Ad-hEndo; C: cisplatin; D: Ad-null; E: NS.