Enhanced efficacy of combination therapy with adeno‑associated virus-delivered pigment epithelium-derived factor and cisplatin in a mouse model of Lewis lung carcinoma

Abstract

Pigment epithelium-derived factor (PEDF) is a potent inhibitor of angiogenesis, and the antitumor effect of adeno-associated virus (AAV)-mediated PEDF expression has been demonstrated in a range of animal models. The combined treatment of low-dose chemotherapy and gene therapy inhibits the growth of solid tumors more effectively than current traditional therapies or gene therapy alone. In the present study, the effect of treatment with an AAV2 vector harboring the human PEDF (hPEDF) gene in combination with low-dose cisplatin on the growth of Lewis lung carcinoma (LLC) in mice was assessed. LLC cells were infected with AAV-enhanced green fluorescent protein (EGFP) in the presence or absence of cisplatin, and then the effect of cisplatin on AAV-mediated gene expression was evaluated by image and flow cytometric analysis. Tumor growth, survival time, vascular endothelial growth factor (VEGF) expression, microvessel density (MVD) and apoptotic index were analyzed in C57BL/6 mice treated with AAV-hPEDF, cisplatin or cisplatin plus AAV-hPEDF. The results of the present study provide evidence that cisplatin treatment is able to enhance AAV-mediated gene expression in LLC cells. In addition, the combined treatment of cisplatin plus AAV‑hPEDF markedly prolonged the survival time of the mice and inhibited tumor growth, resulting in significant suppression of tumor angiogenesis and induction of tumor apoptosis in vivo, and also protected against cisplatin-related toxicity. These findings suggest that combination of AAV-hPEDF and cisplatin has potential as a novel therapeutic strategy for lung cancer.

Figure 1

Cisplatin enhanced adeno-associated virus (AAV)-enhanced green fluorescent protein (EGFP) expression and AAV-mediated human pigment epithelium-derived factor (PEDF) expression in the Lewis lung carcinoma (LLC) cell line. (A) The AAV-delivered EGFP expression in LLC cells in the (Aa) absence or (Ab) presence of 250 ng/ml cisplatin for 2 h (Ac) before and (Ad) after AAV-EGFP infection was analyzed by flow cytometry. (B) Western blot assay of LLC cells for human PEDF protein levels. (C) Inhibitory effect of cisplatin plus AAV-hPEDF on human umbilical vein endothelial cell tubule formation (magnification, ×100) and inhibition ratio of tube formation (E). (D) Fluorescence micrographs of LLC cells after the treatments described in A (magnification, ×400). ^*P<0.05 and ^**P<0.01 compared with NS or AAV-EGFP.

Figure 2

Efficacy of the combination of adeno-associated virus (AAV)-human pigment epithelium-derived factor (hPEDF) and cisplatin on survival, tumor growth and tumor weight. (A) Tumor growth curve. (B) Survival curve. (C) The average Lewis lung carcinoma (LLC) tumor weight of each group. (D) Changes in body weight of mice; there was a significant difference in body weight between the cisplatin group and the control groups. (E) Net body weight was calculated by subtracting the tumor weight from the total body weight.^*P<0.05 and ^**P<0.01 compared with normal saline (NS) or AAV-enhanced green fluorescent protein (EGFP), ^#P<0.05 compared with the combination group.

Figure 3

The combination of adeno-associated virus (AAV)-human pigment epithelium-derived factor (hPEDF) and cisplatin inhibited tumor angiogenesis in vivo. (A) hPEDF expression in vivo was analyzed by immunohistochemical staining (magnification ×400). (B) Immunohistochemical staining of vascular endothelial growth factor (VEGF) (magnification ×400) and the staining intensity of VEGF was quantified (D). (C) The combination of AAV-hPEDF and cisplatin greatly inhibited tumor angiogenesis in the LLC tumor (magnification ×400). (E) The average microvessel number in the tumors of the combined group was markedly decreased. ^*P<0.05 and ^**P<0.01 compared with normal saline (NS) or AAV-enhanced green fluorescent protein (EGFP).

Figure 4

The combination of adeno-associated virus (AAV)-human pigment epithelium-derived factor (hPEDF) and cisplatin induced tumor apoptosis in vivo. (A) Immunohistochemical staining of caspase-3 in Lewis lung carcinoma (LLC) tumors (×400). The average caspase-3 stained index was calculated for each group (D). (B) Apoptotic cells were identified by transferase-mediated dUTP nick end labelling assay in LLC tumor sections (×400). The mean apoptotic index was calculated (E). (D) Hematoxylin and eosin staining of LLC tumors in each group (×400). ^*P<0.05 and ^**P<0.01 compared with normal saline (NS) or AAV-enhanced green fluorescent protein (EGFP).