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. 2012 Sep 12;31(1):74.
doi: 10.1186/1756-9966-31-74.

Enforced effect of tk-MCP-1 fusion gene in ovarian cancer

Shuhui Hong  1 , Ping Zhang, Hui Zhang, Lin Jia, Xun Qu, Qifeng Yang, Fengnian Rong, Beihua Kong
Affiliations

Enforced effect of tk-MCP-1 fusion gene in ovarian cancer

Shuhui Hong et al. J Exp Clin Cancer Res. .

Abstract

Objective: The efficiency of HSV-tk/GCV system is not high because of insufficient gene transfer and incompletely initiative of host antineoplastic potency. The present study was designed to assess the antitumor efficacy of tk-MCP-1 on ovarian cancer in vitro and vivo.

Methods: A novel bicistronic expression system can help to improve the expression level of a gene in a stable manner. pLXSN/tk-MCP-1 co-expressing tk and MCP-1 genes was constructed using a pLXSN retroviral vector and an internal ribosome entry site sequence by restriction enzyme. Western blot was performed to determine tk and MCP-1 expression in the infected SKOV3. The GCV-sensitively tumoricidal activities of SKOV3/tk-MCP-1 with or without monocytes were compared to those of SKOV3 expressing HSV-tk or MCP-1. We investigated the growth of subcutaneous tumors in SCID mice immuno-reconstituted, and evaluated the antitumor effect of MCP-1 in conjunction with suicide gene.

Results: The significant GCV-sensitively tumoricidal activity of pLXSN/tk-MCP-1 was observed when compared with those of pLXSN/tk, pLXSN/MCP-1 and pLXSN/neo, especially when monocytes were added. The growth of subcutaneous tumors in SCID mice immuno-reconstituted was markedly suppressed by co-delivery of HSV-tk and MCP-1 genes, and the enhanced antitumor effect was associated with the recruitment of monocytes.

Conclusion: These results demonstrated pLXSN/tk-MCP-1 presented an enhanced antitumor effects on ovarian cancer by orchestration of immune responses.

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Figures

Figure 1
Figure 1
The plasmid characterization and confirmation of expression of tk and MCP-1 by RT-PCR and western blot. A. The construction of the bicistronic recombinant replication-defective retroviruses vector pLXSN/tk-MCP-1, pLXSN/tk and pLXSN/MCP-1. B. Restriction enzyme analysis of pLXSN/tk-MCP-1 showed that tk and/or MCP-1 gene fragment had insert in the proper orientation in the vector of pLXSN, pLXSN/tk, pLXSN/MCP-1 and pLXSN/neo. a: EcoR I + BamH I, b: Nco I + BamH I, c: Sal I + BamH I, d: EcoR I, M: Marker. C. The expression of tk and MCP-1 protein were detected by western blot 48 h after transfection. a: SKOV3/tk. b: SKOV3/MCP-1. c: SKOV3/neo. d: SKOV3/tk-MCP-1.
Figure 2
Figure 2
Antitumor effection. A: MTT assay of GCV on ovarian cancer cells. B: GCV at the density of 0.1 μg/ml, the beginning cytotoxic was 48 h and 40% kill rate at 96 h, however, the beginning cytotoxic was 48 h and 90% kill rate at 96 h when GCV at the density of 1.0 μg/ml. C: Lethal effect of mononuclear macrophage on SKOV3/MCP-1 and SKOV3/tk-MCP-1 was determined by MTT assay. D: There is a synergistic antitumor effect when cooperated tk-MCP-1 + GCV system with mononuclear macrophage.
Figure 3
Figure 3
CD25of SKOV3/tk (20.00 ± 2.04%) and SKOV3/tk-MCP-1 (38.82 ± 2.48%) was obviously higher than SKOV3/neo (8.73 ± 1.65%) (P < 0.05). CD25 of SKOV3/tk-MCP-1 was significantly higher than that of SKOV3/tk (P < 0.05). CD44v6 of SKOV3/tk (6.66 ± 2.01%) and SKOV3/tk-MCP-1 (6.51 ± 1.03%) was significantly lower than that of control group (40.74 ± 3.58%) (P < 0.01)
Figure 4
Figure 4
A–E. The ovarian tumors of the tk-MCP-1 group shrank significantly, followed by the tk or MCP-1 group. However, the tumor of the control group was still widespread in peritoneal cavity and cavitas pelvis. F. Kaplan-Meier survival analysis of mice intraperitoneally transplanted with diverse tumor cells. a. SKOV3/tk-MCP-1 b. SKOV3/MCP-1 c. SKOV3/tk d. SKOV3/neo.
Figure 5
Figure 5
Flow cytometry examination revealed that the number of macrophages (A) infiltrated the tumor tissues in the control group, tk or MCP-1 group and tk-MCP-1 group increased in order (P<0.05), so did TNF-α protein level from the activated microphages. There was no significant difference between tk and MCP-1 groups (P > 0.05) (B). a. SKOV3/neo b. SKOV3/tk c. SKOV3/MCP-1 d. SKOV3/tk-MCP-1.
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