Synergistic effect of therapeutic stem cells expressing cytosine deaminase and interferon-beta via apoptotic pathway in the metastatic mouse model of breast cancer

Abstract

As an approach to improve treatment of breast cancer metastasis to the brain, we employed genetically engineered stem cells (GESTECs, HB1.F3 cells) consisting of neural stem cells (NSCs) expressing cytosine deaminase and the interferon-beta genes, HB1.F3.CD and HB1.F3.CD.IFN-β. In this model, MDA-MB-231/Luc breast cancer cells were implanted in the right hemisphere of the mouse brain, while pre-stained GESTECs with redfluorescence were implanted in the contralateral brain. Two days after stem cells injection, 5-fluorocytosine (5-FC) was administrated via intraperitoneal injection. Histological analysis of extracted brain confirmed the therapeutic efficacy of GESTECs in the presence of 5-FC based on reductions in density and aggressive tendency of breast cancer cells, as well as pyknosis, karyorrhexis, and karyolysis relative to a negative control. Additionally, expression of PCNA decreased in the stem cells treated group. Treatment of breast cancer cells with 5-fluorouracil (5-FU) increased the expression of pro-apoptotic and anti-proliferative factor, BAX and p21 protein through phosphorylation of p53 and p38. Moreover, analysis of stem cell migratory ability revealed that MDA-MB-231 cells endogenously secreted VEGF, and stem cells expressed their receptor (VEGFR2). To confirm the role of VEGF/VEGFR2 signaling in tumor tropism of stem cells, samples were treated with the VEGFR2 inhibitor, KRN633. The number of migrated stem cells decreased significantly in response to KRN633 due to Erk1/2 activation and PI3K/Akt inhibition. Taken together, these results indicate that treatment with GESTECs, particularly HB1.F3.CD.IFN-β co-expressing CD.IFN-β, may be a useful strategy for treating breast cancer metastasis to the brain in the presence of a prodrug.

Keywords: 5-fluorocytosine; breast cancer; interferon-beta; metastasis; stem cell therapy.

Figure 1. Mouse model for breast cancer metastasis to brain and therapeutic effect of stem cells expressing therapeutic genes, cytosine deaminase (CD) and/or interferon-beta (IFN-β)

Luciferase labeled MDA-MB-231 breast cancer cells were implanted in the right hemisphere of anesthetized nude mice (AP + 1.0 mm, ML + 1.7 mm, DV −3.2 mm). Three stem cell lines, HB1.F3, HB1.F3.CD, and HB1.F3.CD. IFN-β cells, were transplanted to the opposite hemisphere of mice after two weeks. To activate the CD gene in stem cells, 5-FC (500 mg/kg/day) was administrated as a prodrug via intraperitoneal (i.p.) injection once a day 5 days per week for 2 weeks. (A) Schematic diagram of mouse models. (B) Volume of excised brain tumor. After the final 5-FC injection, brains were excised from several mice to measure tumor burden inside the brain (n = 8). (C) Bioluminescence imaging. During the experimental period, living images of all groups of mice were acquired at the indicated weeks. (D) Bioluminescence values (photons/sec/cm²/sr) (n = 8). (E) Survival rate of mouse models. Data shown are the mean ± SEM. *p < 0.05 vs. negative control (n = 8).

Figure 2. Hematoxylin and eosin (H & E) staining and PCNA expression level in metastatic breast cancer models

Excised brains were fixed in 4% normal formalin and embedded in paraffin. After being cut with a microtome, slides were deparaffined and rehydrated with xylene, ethanol, and tap water. (A) H & E staining. (B) Immunohistochemistry (IHC) staining for PCNA. To confirm the proliferation rate of tumor cells, brain sections were treated with the primary antibody, anti-mouse PCNA. Next, the slide was incubated with biotinylated anti-mouse secondary antibody and stained protein (brown color) was observed following DAB and hematoxylin staining. Dotted line: necrosis or apoptosis area of tumor cells in the brain section. Magnification ×100, ×200.

Figure 3. Effects of the vascular endothelial growth factor and their receptor 2 (VEGF/VEGFR2) pathway on stem cells migration

(A) Chemoattractant factor secreted by MDA-MB-231 breast cancer cells. Total RNA was obtained from MDA-MB-231 and investigated by real time PCR with SYBR green and ROX dye. (B) Transwell assay after treatment with the VEGFR2 inhibitor, KRN633. After treatment of stem cells in the culture dish with 100 μM KRN633, CM-DiI stained HB1.F3.CD cells were seeded in the upper chamber of the transwell for 1 day. The next day, non-migrated cells were removed from the upper chamber of the transwell and the number of migrated cells was counted by fluorescent microscopy. (C) The number of migrated and non-migrated cells following KRN633 treatment. Magnification ×100.

Figure 4. Alteration of VEGF/VEGFR2 signaling-related RNA and protein expression in stem cells following treatment with the vascular endothelial growth factor receptor 2 (VEGFR2) inhibitor, KRN633

(A) Expression of VEGFR2 gene in HB1.F3 cells. KRN633 at 50 μM or 100 μM was applied to HB1.F3 cells for up to 24 hours, during which time RNA was extracted at 3, 6, 9, and 24 h. To confirm the effects of KRN633, expression of the VEGFR2 gene was analyzed by RT-PCR. (B) Graph of the relative VEGFR2 mRNA level. (C) Expression of phospho-Erk1/2, phospho-Akt1/2/3, and c-fos protein in the HB1.F3.CD. Following KRN633 treatment (50 μM or 100 μM), protein was collected and separated by SDS-PAGE, then transferred to PVDF membrane and incubated with primary antibody, anti-p-Erk1/2, anti-p-Akt1/2/3, and anti-c-fos. (D) Graph of p-Erk1 related values. (E) Graph of p-Erk2 related values. (E) Graph of p-Akt1/2/3 related values. Each experiment was performed in triplicate and data shown are the mean ± SD. C: negative control (no treatment with KRN633) *p < 0.05 vs. negative control. ^#p < 0.05 vs. KRN633 50 μM treated cells.

Figure 5. Effect of 5-fluorouracil (5-FU) on expression or apoptosis associated marker in MDA-MB-231 cells

MDA-MB-231 cells were treated with 5-fluorocytosine (5-FC) at 5.0 μg/ml or 5-FU at 0.5, 1.0, and 5.0 μg/ml for 24 hours, after which BAX and Bcl-2 expression were determined by reverse transcriptase polymerase chain reaction (RT-PCR) analysis. GAPDH was used as a loading control and each sample was normalized to the GAPDH mRNA content. (A) BAX and Bcl-2 RNA expression levels. (B) Graph of BAX gene levels. (C) Graph of Bcl-2/BAX ratio. Data shown are the mean ± SD of three different experiments performed in triplicate. *p < 0.05 vs. negative control (no treatment with 5-FU).

Figure 6. Alteration of apoptosis and proliferation related protein expression in MDA-MB-231 cells after 5-fluorouracil (5-FU) treatment

To confirm the effects of 5-FU, serially diluted 5-FU (0.5, 1.0, and 5.0 μg/ml) and 5-fluorocytosine (5-FC) at 5.0 μg/ml were applied to MDA-MB-231 cells, which were then cultured in 6-well plates. Whole cell lysates were resolved by SDS-PAGE and immunoblotted with specific antibodies to BAX, c-fos, and p21 (Cip1/Waf1). (A) Expression of BAX and c-fos protein. (B) The relative value of BAX protein levels. (C) The relative value of c-fos protein levels. (D) The expression of p21 (Cip1/Waf1) protein. (E) Graph of p21 (Cip1/Waf1) protein levels. N: negative control (no treatment with 5-FU or 5-FC), C: 5-FC treatment (5.0 μg/ml), U: 5-FU treatment (0.5, 1.0, and 5.0 μg/ml, from the left). Data are presented as the mean ± SD of three different experiments each performed in triplicate. *p < 0.05 vs. negative control. #p < 0.05 vs. 5-FU 0.5 μg/ml treated cells. $p < 0.05 vs. 5-FU 5.0 μg/ml treated cells.

Figure 7. Regulation of transcriptional factor, p53 and pp38 in MDA-MB-231 cells

MDA-MB-231 cells were treated with 5-fluorouracil (5-FU) in a dose-dependent manner for one hour. Whole cell lysates were separated by SDS-PAGE and immunoblotted with primary antibodies to p53, phospho-p38, and GAPDH. Each sample was normalized to its GAPDH protein. (A) Expression of p53 and pp38 protein. (B) The value of p53 protein levels. (C) The value of pp38 protein levels. N: negative control (no treatment with 5-FU or 5-FC), C: 5-FC treatment (5.0 μg/ml), U: 5-FU treatment (0.5, 1.0, and 5.0 μg/ml, from the left). Data are presented as the mean ± SD of three different experiments each performed in triplicate. *p < 0.05 vs. negative control.

Figure 8. Synergistic effect of 5-fluorouracil (5-FU) and human interferon-beta (IFN-β) for treating MDA-MB-231 cells

MDA-MB-231 cells were co-treated with 5-FU 1.0 μg/ml and IFN-β 500 Unit/ml for different lengths of time. Cell lysates were used for immunoblotting analysis and incubated with primary antibodies, BAX, p21 (Cip1/Waf1), p53, and phospho-p38. (A) Expression of BAX protein. (B) The relative value of BAX protein. (C) Expression of p21 (Cip1/Waf1) protein. (D) The relative value of p21 (Cip1/Waf1) protein. (E) Expression of p53 and pp38 protein. (F) The relative value of p53 and pp38 protein. N: negative control (no treatment with 5-FU or 5-FC), C: 5-FC treatment (1.0 μg/ml), U: 5-FU treatment (1.0 μg/ml), B: IFN-β treatment (500 Unit/ml), U + B (5-FU 1.0 μg/ml and IFN-β 500 Unit/ml co-treatment). Data are presented as the mean ± SD of three different experiments each performed in triplicate. *p < 0.05 vs. negative control. #p < 0.05 vs. 5-FU 0.5 μg/ml treated cells. $p < 0.05 vs. 5-FU 5.0 μg/ml treated cells.