The combination of ANT2 shRNA and hNIS radioiodine gene therapy increases CTL cytotoxic activity through the phenotypic modulation of cancer cells: combination treatment with ANT2 shRNA and I-131

Abstract

Background: It is important to simultaneously induce strong cell death and antitumor immunity in cancer patients for successful cancer treatment. Here, we investigated the cytotoxic and phenotypic modulation effects of the combination of ANT2 shRNA and human sodium iodide symporter (hNIS) radioiodine gene therapy in vitro and in vivo and visualized the antitumor effects in an immunocompromised mouse colon cancer model.

Methods: A mouse colon cancer cell line co-expressing hNIS and the luciferase gene (CT26/hNIS-Fluc, named CT26/NF) was established. CT26/NF cells and tumor-bearing mice were treated with HBSS, scramble, ANT2 shRNA, I-131, and ANT2 shRNA + I-131. The apoptotic rates (%) and MHC class I and Fas gene expression levels were determined in treated CT26/NF cells using flow cytometry. Concurrently, the level of caspase-3 activation was determined in treated cells in vitro. For in vivo therapy, tumor-bearing mice were treated with scramble, ANT2 shRNA, I-131, and the combination therapy, and the anti-tumor effects were monitored using bioluminescence. The killing activity of cytotoxic T cells (CTLs) was measured with a lactate dehydrogenase (LDH) assay.

Results: For the in vitro experiments, the combination of ANT2 shRNA and I-131 resulted in a higher apoptotic cell death rate compared with ANT2 shRNA or I-131 alone, and the levels of MHC class I and Fas-expressing cancer cells were highest in the cells receiving combination treatment, while single treatment modestly increased the level of MHC class I and Fas gene expression. The combination of ANT2 shRNA and I-131 resulted in a higher caspase-3 activation than single treatments. Interestingly, in vivo combination treatment led to increased gene expression of MHC class I and Fas than the respective mono-therapies; furthermore, bioluminescence showed increased antitumor effects after combination treatment than monotherapies. The LDH assay revealed that the CTL killing activity against CT26/NF cells was most effective after combination therapy.

Conclusions: Increased cell death and phenotypic modulation of cancer cells in vitro and in vivo were achieved simultaneously after combination therapy with ANT2 shRNA and I-131, and this combination therapy induced remarkable antitumor outcomes through improvements in CTL immunity against CT26/NF. Our results suggest that combination therapy can be used as a new therapeutic strategy for cancer patients who show resistance to single therapy such as radiation or immunotherapy.

Figure 1

The cytotoxic effects of ANT shRNA or hNIS radioiodine gene therapy in CT26/NF cells. (A) and (C) Representative flow cytometry data for propidium iodide and Annexin V staining are shown. (B) and (D) The Y axis indicates the relative cell death (%), which is the sum of the early apoptotic portion (AV+PI-), the intermediate apoptotic portion (AV+PI+), and the late apoptotic portion (AV-PI-). The treated cells were stained with propidium iodide and FITC-conjugated Annexin V and analyzed using flow cytometry. The data shown are the mean of triplicate experiments; the bars represent the mean ± SD.

Figure 2

Enhanced cytotoxicity with ANT shRNA and hNIS radioiodine combination therapy in CT26/NF cells. (A) Representative flow cytometry data for propidium and Annexin V staining are shown. (B) The Y axis indicates the relative cell death (%), which is the sum the early apoptotic portion (AV+PI-), the intermediate apoptotic portion (AV+PI+), and the late apoptotic portion (AV-PI-). The treated cells were stained with propidium iodide and FITC-conjugated Annexin V and analyzed using flow cytometry. The data shown are the mean of triplicate experiments; the bars represent the mean ± SD.

Figure 3

The modulation of phenotypic markers in CT26/NF cells treated with ANT shRNA and hNIS radioiodine combination therapy. (A) and (C) Representative flow cytometry data for MHC class I and Fas are shown. (B) and (D) The Y axis indicates the relative increase in MHC class I and Fas expression levels in cancer cells. A total of 10,000 cells were analyzed, and the relative% depicts the increased percentage of surface marker gene expression of treated cells compared with control. The data shown are the mean of triplicate experiments; the bars represent the mean ± SD.

Figure 4

The change in phenotypic markers in the CT26/NF tumor model treated with ANT shRNA and hNIS radioiodine combination therapy. (A) and (C) Representative flow cytometry data for MHC class I and Fas are shown. (B) and (D) The Y axis indicates the relative increase of MHC class I and Fas expression in cancer cells. A total of 10,000 cells were analyzed, and the relative% depicts the increased percentage of surface marker gene expression of treated cells compared with control. The data shown are the mean of triplicate experiments; the bars represent the mean ± SD.

Figure 5

In vivo visualization of the antitumor effects of ANT2 shRNA and hNIS radioiodine combination therapy. (A) The in vivo tumor treatment schedule is shown. (B) The tumor growth inhibition effects were monitored in vivo using bioluminescent imaging. (C) The tumor growth quantification is shown. CT26/NF cells were transplanted s.c. into the right thighs of immunocompetent Balb/c mice. Fourteen days later, tumor growth was measured using bioluminescence. Then, the tumor-bearing mice were treated with scramble (supplemented with Lipofectamine 2000), ANT2 shRNA (supplemented with Lipofectamine 2000), I-131, and combination therapy according to a designated schedule through an intravenous or intratumoral route. The data shown are the mean of triplicate experiments; the bars represent the mean±SD (n = 7 mice/group).

Figure 6

The enhanced cytotoxic effects of CTLs on CT26/NF cancer cells by combination therapy. Specific lysis was observed in (A) CT26/NF and (B) B16F10 cells. The splenocytes of treated mice were prepared and stimulated with IL-2 and irradiated CT26/NF and B16F10 cells for 3 days. Irradiated CT26/NF and B16F10 cells (target cells) were then incubated with splenocytes (effectors) at a T:E ratio of 1:5, 1:15, and 1:30 for 4 h in 96-well plates. The experiments were performed in triplicate, and the bars represent the mean ±SD; *, P<0.01; n = 7 mice/group.