Contributed equally
The aim of the present study was to establish a tamoxifen-resistant cell line (MCF-7/TAM-R) and to investigate the therapeutic effect of G47Δ on this cell line both
Breast cancer is the most common malignant neoplasm in women, and more than 1.6 million newly diagnosed cases were reported in 2012 worldwide (
Oncolytic herpes simplex virus (oHSV) is a promising agent for various types of cancers. It has been widely used in cancer research studies (
In the present study, we established the MCF-7 tamoxifen-resistant monoclonal subline and the MCF-7 wild-type monoclonal subline (
MCF-7 and Vero cells were provided by Dr Mu Sheng Zeng (Sun Yat-Sen University Cancer Center, Guangzhou, China). We cultured both types of cells in Dulbecco's modified Eagle's medium (DMEM; Gibco, Shanghai, China), which was supplemented with glucose (4.5 g/l) and 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA) at 37°C in an atmosphere containing 5% CO2.
Oncolytic HSV G47Δ was purchased from MediGene Inc. (San Diego, CA, USA), and it was reproduced in Vero cells. After achieving approximately 80% confluency, the Vero cells were infected with G47Δ at a multiplicity of infection (MOI) of 0.03 and incubated at 37°C in 5% CO2. All infected cells were harvested and centrifuged at 1,000 × g for 6 min, and then the liquid supernatant was discarded and 1 ml virus buffer (150 mM NaCl/20 mM Tris, pH 7.5) was added to the system. Thereafter, we subjected the cells to three freeze-thaw cycles; the resultant liquid supernatant was stored at −80°C for future analyses.
Virus titers were determined by plaque assays on the Vero cells. In brief, the Vero cells were seeded into 6-well plates with DMEM and 10% FBS at 37°C in 5% CO2 and infected with 700
We derived MCF-7/tamoxifen-resistant (MCF-7/TAM-R) and wild-type MCF-7 (MCF-7W) cells from the tamoxifen-sensitive MCF-7 cell line (
MCF-7/TAM-R and MCF-7W cells were separately seeded into a 96-well plate at a density of 5.0×103 cells/well and cultured in DMEM (5% FBS) at 37°C in 5% CO2. Cell proliferation was measured by Cell Counting Kit-8 assay (CCK-8; Dojindo, Japan) for 5 days. All experiments were triplicated.
MCF-7/TAM-R and MCF-7W cells were separately seeded overnight in a 96-well plate at a density of 5.0×103 cells/well. The cells were then treated with 4-OHT or vehicle (ethanol, control). In the treatment group, the doses of tamoxifen were exponentially increased (10−8, 10−7, 10−6, 10−5 and 10−4 mol/l). After 48 h, cell viability was evaluated using the CCK-8 assay. Prior to this measurement, we added 100
Cell survival rate = [(Average number of cells from virus-treated group)/(Average number of cells from control group)] × 100%
All of the experiments were performed in triplicate.
MCF-7/TAM-R and MCF-7W cells were seeded separately in 6-well plates (5.0×105 cells/well). Forty-eight hours after the intervention, we collected the virus-treated (MOI, 0.01), 4-OHT-treated (1
MCF-7/TAM-R and MCF-7W cells were separately seeded in a 6-well plate (5.0×105 cells/well) and harvested after 48 h. Each group of cells was washed twice with PBS. Then, 5.0×105 cells of each group were suspended in 500
MCF-7/TAM-R and MCF-7W cells were placed into 6-well plates. Then, the cells were treated with 4-OHT or vehicle (ethanol) for 48 h. After the intervention, the cells were washed twice with ice-cold PBS and lysed with a whole cell lysis kit (KeyGen). The protein density was measured using the BCA protein quantification kit (KeyGen). Then, all the samples were stored at −20°C before being subjected to electrophoresis. Briefly, the electrophoresis was carried out by separating 50
MCF-7W and MCF-7/TAM-R cells were either infected with G47Δ at different MOIs (MOI, 0.01, 0.1 or 1) or mock infected and incubated in DMEM (1% FBS) at 37°C in 5% CO2. After infection, the cells were stained with X-gal solution (Beyotime, Shanghai, China) and counted on a hemocytometer (Qiujing, Shanghai, China) for 5 days. The average number of cells from duplicate wells was recorded as the percentage of mock-infected cells.
All the protocols for the animal experiments were approved by the Ethics and Use Committee of the Research Institute at Sun Yat-Sen University, Guangdong, China. Female BALB/c nude mice (4 weeks of age, 14–16 g in weight) were purchased from Vital Rival Laboratories (Beijing, China). The mice were randomly divided into the following four groups (5 mice/group): MCF-7/TAM-R control group, MCF-7/TAM-R virus-treated group, MCF-7W control group, and MCF-7W virus-treated group. MCF-7/TAM-R and MCF-7W cells (1.0×107) were suspended in 100
The statistical analyses were carried out using SPSS 22.0 (IBM, Armonk, NY, USA). The reported values were presented as mean ± standard deviation (SD). The difference between the groups was evaluated using paired-sample t-test and one-way ANOVA. A P-value of <0.05 was considered to indicate a statistically significant result.
As shown in
After 48 h of treatment, 4-OHT (1
Annexin V-FITC/PI staining indicated that 4-OHT induced apoptosis in the MCF-7W cells (mock vs. 4-OHT, 0.74±0.89 vs. 5.89±0.88%; P=0.001). However, no significant difference was detected between the 4-OHT-treated MCF-7/TAM-R and the mock control cells (0.94±0.19 vs. 0.64±0.94%; P=0.71) (
Caspase-7 and BIK were assessed to determine whether 4-OHT induced the expression of apoptotic proteins in the MCF-7W and MCF-7/TAM-R cells. Western blot analyses showed that tamoxifen increased the expression of caspase-7 and BIK in the MCF-7W cell line; however, no significant difference was detected in the MCF-7/TAM-R cell line (
To determine the susceptibility of the tamoxifen-resistant MCF-7 cell line to G47Δ, MCF-7/TAM-R and MCF-7W cells were cultured in 6-well plates separately, and infected with G47Δ at MOI=0.01, 0.1 and 1. G47Δ exhibited a similar cytotoxic effect on the MCF-7/TAM-R and MCF-7W cell lines (
G47Δ significantly increased cell apoptosis in the MCF-7/TAM-R cells (control vs. virus: 2.43±0.97 vs. 14.88±0.85%; P=0.005) (
We successfully established subcutaneous tumor models of MCF-7/TAM-R and MCF-7W cells in 4-week-old female BALB/c nude mice. G47Δ significantly inhibited tumor growth in both models (for MCF-7/TAM-R: virus vs. control, 23.83±19.64 vs. 163.81±35.97 mm3; P=0.002; for MCF-7W: virus vs. control, 12.63±9.15 vs. 127.33±23.64 mm3; P=0.006). G47Δ was able to replicate in the MCF-7/TAM-R and MCF-7W models (
In the present study, we established MCF-7/TAM-R and MCF-7W monoclonal sublines to investigate the cytotoxic effect of G47Δ, a third generation oHSV, on tamoxifen-resistant breast cancer cells. The results indicated that G47Δ effectively targeted tamoxifen-resistant cells as well as wild-type MCF-7 cells. We further confirmed that when G47Δ was injected inside the tumor, it not only induced a significant slowdown in tumor growth but also prolonged the survival of both MCF-7/TAM-R and MCF-7W subcutaneous models.
Traditionally, tamoxifen is the most frequently used medication in endocrine therapy, since it effectively inhibits tumor growth and improves the survival rate of ER-positive patients. Nonetheless, the intrinsic or acquired therapeutic resistance towards tamoxifen limits its long-term application in breast cancer treatment. In the past decades, several scientists have tried to employ a virus vector in breast cancer treatment. G47Δ, a third generation HSV vector generated from G207, lacks both copies of neurovirulence γ34.5 gene and is inserted in an
Our data confirmed that tamoxifen arrested the cell cycle in the G0/G1 phase and reduced the proportion of MCF-7W cells in the S phase (
Tamoxifen inhibits breast cancer cell growth by blocking ER; therefore, the expression of ER-α is an important predictor in hormonal therapy. In ER-α-positive breast cancer cells, the expression of ER-β can increase the sensitivity towards tamoxifen as it downregulates HER2/HER3 signaling and increases the expression of PTEN (
In order to evaluate the impact of G47Δ on tamoxifen-resistant cells, MCF-7/TAM-R and MCF-7W cells were infected with G47Δ at various MOIs (0.01, 0.1 and 1). In both the MCF-7/TAM-R and MCF-7W cell lines, we observed extremely strong cytotoxic effects even at a very low MOI (0.01). Similarly, G47Δ significantly influenced cell cycle distribution, inducing cell cycle arrest at the G2/M phase in both the MCF-7W and MCF-7/TAM-R cells. Apoptosis analysis also confirmed that G47Δ introduced cell apoptosis in the MCF-7W and MCF-7/TAM-R cells. The above results indicate that G47Δ has great potential in reducing the replication and division of cancer cells. To further verify the antitumor effect of G47Δ on tamoxifen-resistant breast cancer
There are several limitations to our study. MCF-7 cells alone may not represent all the characteristics of ER-positive breast cancer cells. Additional ER-positive cell lines should be included in future studies. Furthermore, the underlying mechanism involved in the effective inhibition of MCF-7/TAM-R cell growth by G47Δ remains undetermined.
In conclusion, G47Δ, a third generation oHSV, is highly sensitive and safe for targeting tamoxifen-resistant breast cancer cells both
The present study was supported by the National Natural Science Foundation of China (grant nos. 81172523 and 81372815; principle investigator, R.B.L.).
Establishment of MCF-7/TAM-R and MCF-7W monoclonal sublines. At a density of 1.0×107 cells/dish, MCF-7 cells were seeded in a 10-cm dish. Then, they were cultured for 21 days in DMEM (5% FBS), containing 1
Identification of the tamoxifen-resistant characteristics of the MCF-7/TAM-R cells. (A) Cell proliferation experiments indicated that the MCF-7/TAM-R cell line had a higher proliferation rate than the MCF-7W cell line (P=0.018). (B) Tamoxifen cytotoxic tests showed that the ED50 of 4-OHT in the MCF-7W cells was lower than that in the MCF-7/TAM-R cells (P=0.007). (C–E) Cell cycle analyses and apoptosis assays confirmed the occurrence of 4-OHT-induced cell cycle arrest and apoptosis in the MCF-7W cells but not in the MCF-7/TAM-R cells. (F) Tamoxifen increased the expression of caspase-7 and BIK in the MCF-7W cells but not in the MCF-7/TAM-R cells. (G) The expression of ER-α and ER-β was significantly higher in the MCF-7/TAM-R cells.
Cytotoxicity of G47Δ in the MCF-7/TAM-R and MCF-7W cell lines. (A and B) X-gal staining of the MCF-7/TAM-R and MCF-7W cells (both magnification, ×100), which were infected with G47Δ. (C and D) Cell survival rate of the MCF-7/TAM-R and MCF-7W cells.
Apoptosis of G47Δ-treated MCF-7/TAM-R and MCF-7W cells. G47Δ significantly increased the apoptosis of the MCF-7/TAM-R (P=0.005) and MCF-7W cells (P=0.001).
Cell cycle analysis of G47Δ-treated MCF-7/TAM-R and MCF-7W cells. The virus infection induced cell cycle arrest at the G2/M phase in the MCF-7/TAM-R and MCF-7W cells (P=0.006 and P=0.004).
Gross specimens and X-gal staining (magnification, ×40) of implanted tumors. G47Δ significantly inhibited the tumor growth in both MCF-7/TAM-R and MCF-7W models.