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, 11 (5), 1017-1026
eCollection

Target Therapy With Vaccinia Virus Harboring IL-24 For Human Breast Cancer

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Target Therapy With Vaccinia Virus Harboring IL-24 For Human Breast Cancer

Lili Deng et al. J Cancer.

Abstract

Background: Breast cancer is a heterogeneous disease with high aggression and novel targeted therapeutic strategies are required. Oncolytic vaccinia virus is an attractive candidate for cancer treatment due to its tumor cell-specific replication causing lysis of tumor cells as well as a delivery vector to overexpress therapeutic transgenes. Interleukin-24 (IL-24) is a novel tumor suppressor cytokine that selectively induces apoptosis in a wide variety of tumor types, including breast cancer. In this study, we used vaccinia virus as a delivery vector to express IL-24 gene and antitumor effects were evaluated both in vitro and in vivo. Methods: The vaccinia virus strain Guang9 armed with IL-24 gene (VG9-IL-24) was constructed via disruption of the viral thymidine kinase (TK) gene region. The cytotoxicity of VG9-IL-24 in various breast cancer cell lines was assessed by MTT and cell cycle progression and apoptosis were examined by flow cytometry. In vivo antitumor effects were further observed in MDA-MB-231 xenograft mouse model. Results: In vitro, VG9-IL-24 efficiently infected and selectively killed breast cancer cells with no strong cytotoxicity to normal cells. VG9-IL-24 induced increased number of apoptotic cells and blocked breast cancer cells in the G2/M phase of the cell cycle. Western blotting results indicated that VG9-IL-24-mediated apoptosis was related to PI3K/β-catenin signaling pathway. In vivo, VG9-IL-24 delayed tumor growth and improved survival. Conclusions: Our findings provided documentation that VG9-IL-24 was targeted in vitro and exhibited enhanced antitumor effects, and it may be an innovative therapy for breast cancer.

Keywords: apoptosis.; breast cancer; gene therapy; interleukin-24; oncolytic vaccinia virus.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Figure 1
Figure 1
Characterization of VG9-IL-24. (A) Schematic illustration of VG9-IL-24 construction. The IL-24 gene was inserted into TK locus of VG9 strain via homologous recombination. (B) Identification of VG9-IL-24 by PCR analysis. M, 1Kb Plus DNA Ladder (Invitrogen); Lane 1 (VG9-IL-24), P1/P2 primers amplify a 2020-bp fragment across the region of recombination, which confirm absence of TK. The TK positive fragment (534-bp) amplified of VG9 is shown in Lane 3 and negative control (H2O) is shown in Lane 5. Lane 2 (VG9-IL-24), P3/P4 primers amplify IL-24 to product a 546-bp band, which is absent in VG9 (Lane 4). Negative control (H2O) is shown in Lane 6. (C) Protein concentrations of IL-24 in breast malignant and normal cells. The concentrations of IL-24 protein from all breast cancer cells treated with VG9-IL-24 was higher than that in normal cells (all P<0.01). Each bar represents the mean ± SD (n=3). **P <0.01.
Figure 2
Figure 2
Oncolytic activity of VG9-IL-24. (A) Selective replication of VG9-IL-24 in breast cancer cells. MDA-MB-231 cells and MCF-10A cells in 12-well plates were infected with VG9-IL-24 at 0.1 MOI and samples were collected at indicated times. Virus titers in MDA-MB-231 cells were higher than those in MCF-10A cells at indicated times (all P<0.05). Each bar represents the mean ± SD (n=3). *P<0.05. (B) Breast cancer cell lines and normal cells were infected with VG9-IL-24 at various MOIs. After infection for 72 h, cell viability was measured by MTT assay. The cytotoxic effect was obvious on breast cancer cell lines, while little cytotoxicity on normal cells.
Figure 3
Figure 3
VG9-IL-24 induced apoptosis of breast cancer cells. (A) Cell apoptotic staining by Hoechst 33258. MDA-MB-231 cells and MCF-10A cells treated with PBS, VG9-IL-24 or VG9-EGFP were incubated with Hoechst 33258 for 30 min, and nuclear fragmentation and chromatin clumping were observed in virus-treated groups but not in normal cells. Bar: 20 μm. (B) The percentage of apoptotic cells was determined by flow cytometry. MDA-MB-231 cells treated with PBS, VG9-IL-24 or VG9-EGFP were harvested after 48 h and stained with FITC-labeled Annexin V and PI and immediately analyzed by flow cytometry. The apoptosis ratio of VG9-IL-24 group was significantly higher than that of VG9-EGFP or PBS group (P<0.01 compared with PBS group; P<0.05 compared with VG9-EGFP group). (C) Cell-cycle analysis by flow cytometry. MDA-MB-231 cells treated with PBS, VG9-IL-24 or VG9-EGFP were harvested after 48 h and stained with PI. Cell cycle distribution was analyzed by flow cytometry and the percentage of cell-cycle phases was analyzed. Higher G2/M proportion of the cell cycle was observed in VG9-IL-24 group compared with PBS or VG9-EGFP group (P<0.01 compared with PBS group; P<0.05 compared with VG9-EGFP group). Each bar represents the mean ± SD of three independent experiments. *P<0.05; **P <0.01.
Figure 4
Figure 4
VG9-IL-24 induced apoptosis in breast cancer cells via PI3K/β-catenin signaling pathway. (A) MDA-MB-231 cells treated with PBS, VG9-IL-24, VG9-EGFP for 48 h were harvested, lysed and prepared to be available for western blot analysis. Reduced expressions of PI3K, phosphorylation of Akt and GSK-3β, and β-catenin were observed in VG9-IL-24-treated cells. β-actin was used as a loading control. (B) Schematic illustration of VG9-IL-24 regulation of PI3K/β-catenin signaling pathway.
Figure 5
Figure 5
Antitumor effect of VG9-IL-24 in MDA-MB-231 xenograft mouse model. (A) Mean tumor volume in mice treated with PBS (Control), VG9-IL-24, or VG9-EGFP. Tumors developed rapidly in Control and VG9-EGFP groups, while tumor growth was significantly slow in VG9-IL-24 group. n=6 in each group. (B) Kaplan-Meier survival curves for tumor-bearing mice treated with PBS, VG9-IL-24, or VG9-EGFP. Higher survival rate was observed in VG9-IL-24 group compared with Control or VG9-EGFP group. n=6 in each group.
Figure 6
Figure 6
HE and immunohistochemical staining of the tumor tissue. Tumors from the mice treated with PBS (Control), VG9-IL-24, or VG9-EGFP were harvested, formalin fixed and paraffin embedded. Sections were subjected to HE staining and immunohistochemistry for Ki-67 and IL-24. Pyknosis events were obviously appeared in VG9-IL-24 group and decreased positive Ki-67 were also observed in VG9-IL-24 group. Expression of IL-24 was observed in tumors treated with VG9-IL-24. Original magnification, ×400. Bar: 20 μm.

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