Efficient inhibition of lung cancer in murine model by plasmid-encoding VEGF short hairpin RNA in combination with low-dose DDP

Abstract

Background: VEGF is a well-validated target for antiangiogenic intervention in cancer. To date, RNAi technology has been proven to be a promising approach for targeted therapy. DDP is frequently used as a first-line drug in chemotherapy for lung cancer but usually causes severe toxicity. In this study, we investigated a novel strategy of administering and combining RNAi mediated VEGF-targeted therapy with DDP for treatment of lung cancer, with the aim of increasing efficacy and decreasing toxicity.

Methods: In this study, a plasmid encoding VEGF shRNA was constructed to knockdown VEGF both in vitro and in vivo. In vitro, specificity and potency of the targeting sequence were validated in A549 lung adenocarcinoma cells by RT-PCR and ELISA assays. In vivo, therapy experiments were conducted on nude mice bearing A549 xenograft tumors. The VEGF shRNA expressing plasmids were administered systemically in combination with low-dose DDP on a frequent basis. The tumor volume and weight were measured. MVD, the number of apoptotic cells and proliferation index in tumor tissues were assessed by CD31, TUNEL and PCNA immunostaining.

Results: The VEGF shRNA was highly effective in attenuating VEGF expression both in vitro and in vivo. The treatment with the VEGF shRNA alone reduced the mean tumor weight by 49.40% compared with the blank control (P < 0.05). The treatment with the VEGF shRNA plus DDP yielded maximal benefits by reducing the mean tumor weight by 83.13% compared with the blank control (P < 0.01). The enhanced antitumor efficacy was associated with decreased angiogenesis and increased induction of apoptosis.

Conclusions: Our study demonstrated synergistic antitumor activity of combined VEGF shRNA expressing plasmids and low-dose DDP with no overt toxicity, suggesting potential applications of the combined approach in the treatment of lung cancer.

Figure 1

Attenuation of VEGF expression in vitro. A) Photograph of agarose gel. Cultured A549 cells were transfected with pshVEGF or pshHK. Forty-eight hours after transfection, VEGF mRNA was semiquantified by RT-PCR. The β-actin gene was used as the internal control. B) Densitometric analysis of VEGF mRNA was done and the relative expression of each band was normalized to the blank control (5% GS). C) Forty-eight hours after transfection, supernatants were collected and assayed for secreted VEGF protein by ELISA. The results are presented as mean ± SD (n = 3). P < 0.05 versus pshHK.

Figure 2

Antitumor effect of VEGF silencing plus DDP on A549 cells in vivo. Tumor growth curves. Each point in the curves represents the mean ± SD (n = 5 tumors). The therapy started on day 7 when the tumors reached a volume of ~50 mm³. The combination of VEGF silencing plus DDP enhanced the inhibition of tumor growth, #P < 0.05 versus 5% GS, *P0.05 versus pshVEGF or DDP, **P < 0.01 versus 5% GS. B) Weight of the tumors. Each bar represents the mean ± SD (n = 5 tumors). *P < 0.05 versus pshVEGF or DDP. Mean weights of the tumors are 0.510 g, 0.490 g, 0.242 g, 0.228 g and 0.110 g, for the 5% GS group, the pshHK group, the pshVEGF group, the DDP group and the pshVEGF plus DDP group, respectively.

Figure 3

Knockdown of VEGF expression in vivo. A) Representative photographs of the tumor sections examined by immunohistochemical staining for VEGF (×400 magnification). The assessment of VEGF was based on a cytoplasmic staining pattern. B) The tumors were harvested and assayed for VEGF protein by ELISA. The results are presented as mean ± SD (n = 3 tumors). *P < 0.05 versus pshHK.

Figure 4

Inhibition of tumor angiogenesis, apoptosis and proliferation by VEGF silencing plus DDP in vivo. A) Representative photographs of the tumor sections examined by immunohistochemical staining for CD31 showing tumor vasculature (×400 magnification). Each bar represents the average vessel number for each group, expressed as mean ± SD. *P < 0.05 versus pshVEGF or DDP. B) Representative photographs of the tumor sections examined by TUNEL assay. TUNEL-positive cell nuclei (green) were observed under a fluorescence microscope (×400). Each bar represents the 'apoptosis index', expressed as mean ± SD.*P < 0.05 versus pshVEGF or DDP. C) Representative photographs of the tumor sections examined by immunohistochemical staining for PCNA (×400). The assessment of PCNA was based on a nuclear staining pattern. Each bar represents the ratio of PCNA positive cells to the total number of cells for each group, expressed as mean ± SD. *P < 0.05 versus pshVEGF or DDP.