A comparative study of the ability of recombinant oncolytic adenovirus, doxorubicin and tamoxifen to inhibit the proliferation of breast cancer cells

Abstract In this study, we compared the inhibitory effects of recombinant oncolytic adenovirus (Ad‐apoptin‐hTERTp‐E1a, Ad‐VT) with that of doxorubicin (DOX), a first‐line chemotherapy drug, and tamoxifen (TAM), an endocrine therapy drug, on the proliferation of breast cancer cells. We found that Ad‐VT could effectively inhibit the proliferation of breast cancer cells (p < 0.01); the inhibition rate of Ad‐VT on normal mammary epithelial MCF‐10A cells was less than 20%. DOX can effectively inhibit the proliferation of breast cancer cells and also has a strong inhibitory effect on MCF‐10A cells (p < 0.01). TAM also has a strong inhibitory effect on breast cancer cells, among which the oestrogen‐dependent MCF‐7 cell inhibition was stronger (p < 0.01), At higher concentrations, TAM also had a high rate of inhibition (>70%) on the proliferation of MCF‐10A cells. We also found that both recombinant adenovirus and both drugs could successfully induce tumour cell apoptosis. Further Western blot results showed that the recombinant adenovirus killed breast cancer cells through the endogenous apoptotic pathway. Analysis of the nude mouse subcutaneous breast cancer model showed that Ad‐VT significantly inhibited tumour growth (the luminescence rate of cancer cells was reduced by more than 90%) and improved the survival rate of tumour‐bearing mice (p < 0.01). Compared with DOX and TAM, Ad‐VT has a significant inhibitory effect on breast cancer cells, but almost no inhibitory effect on normal breast epithelial cells, and this inhibitory effect is mainly through the endogenous apoptotic pathway. These results indicate that Ad‐VT has significant potential as a drug for the treatment of breast cancer.


| INTRODUC TI ON
The incidence of breast cancer has increased rapidly worldwide over the last few years 1,2 and now ranks first as the most common form of cancer in women. 3 At present, the most common treatment for breast cancer is still the combination of surgical treatment and chemotherapy. 4,5 Doxorubicin is the main chemotherapy drug in clinical treatment of breast cancer. Adjuvant radiotherapy, chemotherapy, and endocrine and HER2-guided therapy are all clinical strategies that can significantly reduce the risk of disease recurrence and improve the overall survival rate of patients with breast cancer. However, the non-targeted toxicity of chemotherapy can seriously affect the quality of life of patients and approximately 40% of patients will relapse and die from metastasis. 3 In addition to chemotherapy and radiotherapy, the endocrine drug tamoxifen has become an indispensable clinical treatment for breast cancer. Although this drug has fewer side effects, it also has significant limitations. Therefore, there is an urgent need to find a drug with a strong inhibitory effect on the growth of various breast cancers.
The clinical concept of using a virus to treat cancer began in the mid-twentieth century. However, it was not until the 1990s that the emergence of recombinant DNA technology and viral genome engineering triggered a new wave of viral therapy. Viral therapy is a form of cancer treatment that uses viruses as vectors in which genetic engineering is used to modify the viruses themselves so that they are more inclined to target cancer cells; these viruses also alert the host's immune system to the presence of cancer. Viral therapy can be divided into two main types: (1) the use of non-replicating viruses as vectors for tumour gene therapy and (2) the use of replicating viruses as oncolytic agents. The emergence of oncolytic adenoviral therapy has led to a significant reduction in the side effects of chemotherapy. 4 There are many types of cell death, mainly divided into programmed cell death (apoptosis, necroptosis, pyroptosis, ferroptosis and autophagy) and cell unprogrammed necrosis. Of these, apoptosis is the most widely studied. Identifying ways to increase apoptosis and subsequent death in cancer cell death, thus inhibiting the progression of cancer, could provide a new concept for the treatment of cancer. Apoptin is a protein that is derived from the chicken anaemia virus and can selectively kill a variety of cancer cells. 6 In tumour cells, apoptin undergoes phosphorylation in the nucleus; however, in normal cells, this process occurs in the cytoplasm. 7 The transcription of human telomerase reverse transcriptase is a major step in the regulation of telomerase activity. Telomerase activity is essential for cancer cells to maintain immortality. By interfering with telomerase activity, it is possible to inhibit the growth of cancer cells.
The recombinant adenoviruses (Ad-VT, Ad-T, Ad-VP3 and Mock) constructed by our group are based on the RAPAd.I packaging system ( Figure 1). Ad-VT (Ad-Apoptin-hTERTp-E1A) includes a tumourspecific promoter (hTERTp, human telomerase reverse transcriptase) and the promoters of the E1A gene (necessary for viral replication), cytomegalovirus (CMV) and promoters that can activate the apoptin gene (Apoptin). Therefore, Ad-VT is a dual-specific oncolytic adenovirus that has the ability to specifically kill tumour cells and exerts tumour-specific replication capability. Ad-T (Ad-hTERTp-E1A), Ad-VP3 (Ad-CMV-Apoptin) and Ad-Mock are all viruses that can be used as controls. These four recombinant oncolytic adenoviruses have been confirmed in previous studies to have strong inhibitory effects on the growth of lung cancer, prostate cancer and liver cancer. 8,9 Bioluminescent imaging in vivo is a highly useful visualization technique that can be used to track cells and analyse tissue activity and gene behaviour in vivo. 10 Bioluminescence imaging in vivo is characterized by light scattering and is associated with a unique imaging advantage 11 ; since there is almost no endogenous luminescence in tissues and cells, the endogenous signal-to-noise ratio is low; therefore, background interference can be effectively eliminated, and the bioluminescence signal can be clearly observed in complex organisms.
The establishment of a luciferase-labelled animal tumour model would provide intuitive, real-time and continuous monitoring of the growth and metastasis of different tumour models with the aid of a live animal imaging system. This type of system could detect very slight changes over time, thus providing an ideal animal model for the more intuitive evaluation of the efficacy of anticancer drugs. In this study, we aimed to construct a luciferase-labelled human breast cancer cell line that could more intuitively and continuously detect       The media (G418) was refreshed every 2-3 days until monoclonal cells were detected. We selected monoclonal cells with good tolerance from the cell culture plates and continued to grow these cells in 24-well plates in G418 medium. When the degree of fusion was >90%, the cells were transferred into 96-well plates for culture and were then assayed for luciferase activity. Cell lines with the highest clonal luminescence value were inoculated on a 96-well plate, and different concentration gradients were generated. After 24 h, we added thee luciferase substrate (Promega Corporation, USA) and measured bioluminescence intensity with a small animal live imaging system (Merck KGaA, Darmstadt, Germany).

| Statistical processing
Statistical analyses were conducted using data from at least three independent experiments, and GraphPad Prism version 7 (GraphPad Software, USA) was used to process the data in the form of (x ± s).
Comparisons between the two groups were analysed by the t-test.
Comparisons were significant when p < 0.05 or p < 0.01.

| Growth inhibition of breast cancer cells and mammary epithelial cells by a recombinant oncolytic adenovirus and two drugs (doxorubicin and tamoxifen)
WST-1 was used to detect and analyse the inhibitory effect of re- In contrast, doxorubicin not only had a strong inhibitory effect on breast cancer cells but also inhibited the proliferation of normal breast epithelial cells. Tamoxifen only had a strong inhibitory effect on oestrogen-dependent breast cancer cells, had no obvious inhibitory effect on triple-negative breast cancer, but also had a strong inhibitory effect on normal breast epithelial cells.

| Recombinant oncolytic adenovirus and two drugs (doxorubicin and tamoxifen) increased apoptosis in breast cancer cells
Hoechst is a class of dye that can enter cells passively and bind to nucleic acids to exhibit blue fluorescence. As shown in Figure 3A,

| Construction of luciferase-labelled human breast cancer cells
As shown in Figure 5A Figure 5B shows results relating to cell cycle determination.
Before and after the construction of the two cell types, the proportion of cells in each phase was essentially the same (G1 phase, G2 phase and S phase) and there were no significant changes between the cell types with regard to the cell cycle (p > 0.05). Growth curves for the two cell types are shown in Figure 5C; these curves were similar before and after construction indicating that the growth characteristics of the two cell types were not significantly different  In summary, cell growth curves and cell cycle experiments showed that there were no significant changes between the two cell types in terms of their biological characteristics before and after construction. Luciferase activity assays, along with in vivo tumour formation experiments in nude mice, further confirmed that the two luciferase-labelled human breast cancer cell lines expressed luciferase in a stable manner. Collectively, these results indicated that the stable expression of luciferase did not affect the growth characteristics of cells.

| In vivo imaging of subcutaneous tumours in BALB/c nude mice and survival analysis
The bioluminescence intensity of tumours in nude mice was continuously detected with a small animal imaging system for 5 weeks from Week 0. The bioluminescence results are shown in Figure 6A During Weeks 4-5 weeks, the bioluminescence intensity of the tumour area in the Ad-VT treatment group was significantly lower than that in the control group (p < 0.01); the levels of luminescence from tumour cells followed a specific sequence: Ad-VT < Ad-T < Ad-VP3 < MOCK < Control. We used excess CO 2 to euthanize nude mice at the end of the experiment. The livers and kidneys of the non-model group (nude mice without subcutaneous tumour) and Ad-VT treatment groups were removed for HE staining. As shown in Figure 6D, the liver tissue structure of the nude mice in the Ad-VT treatment group was normal, and the hepatocytes were arranged in an orderly manner without obvious change; the kidney tissue structure of nude mice after intra-tumoral injection of Ad-VT was normal, the size of glomeruli was relatively uniform, the structure of renal tubules was normal, and there was no obvious degeneration of epithelial cells, intra-tumoral injection of Ad-VT treatment group and nude mice without subcutaneous tumour had no changes in tissue sections, and the morphology was similar. Ad-VT had no effect on the liver and kidney of nude After 48 h, we extracted total protein proteins from the cells and used Western blotting to detect the protein expression levels of PARP, caspase-3 and cytochrome C (D). All measurements were performed in triplicate; the mean ± standard deviation were then compared with the control group (*p < 0.05, **p < 0.01, ***p < 0.001) tumour loading; the mean survival time of mice in this group was significantly shorter than that in other groups ( Figure 6C). The mean survival rate of nude mice in the Ad-VT treatment group exceeded 50%; in addition, the total survival period was significantly prolonged in this group.
Nude mice treated with Ad-T and Ad-VT were still alive 50 days after the formation of tumours; the survival rate of mice in the Ad-VT treatment group was higher (p < 0.01) than that of mice in the Ad-T treatment group.

| DISCUSS ION
Cancer is one of the most significant and dangerous health issues in the world today and can seriously affect the health and lives of those affected. Over recent years, the global incidence of breast cancer has increased; this particular cancer is now the most common global cancer 12 ; however, the molecular mechanisms underlying breast cancer have yet to be elucidated. 13 There are many different treatment strategies for patients with breast cancer, including surgery, radiation, chemistry, endocrine therapy and HER2 molecular-targeted therapy. Surgery and che-  100 μl) were injected subcutaneously into the right hind limb of nude mice (10 mice in each group) to establish a xenograft model. An in vivo imaging luminescence system was used to continuously monitor changes in tumour bioluminescence intensity. (C) After successfully establishing the xenograft model in nude mice, we recorded the survival rates of mice daily for 5 weeks. The mean tumour inhibition rate in the 1 × 10 9 PFU/100 μl Ad-VT treatment group was significantly higher than that of the other groups. The survival rate of the 1 × 10 9 PFU/100 μl Ad-VT treatment group was also the highest of all groups; the mean survival rate of nude mice exceeded 50%. The survival time of mice in the 1 × 10 9 PFU/100 μl Ad-VT treatment group was significantly longer than those in the control group or the 1 × 10 9 PFU/100 μl MOCK treatment group. (D) After 5 weeks of continuous photography, the livers and kidneys of nude mice in the nonmodel group and the Ad-VT treatment group were taken for HE staining (three mice in each group). (*p < 0.05, **p < 0.01, ***p < 0.001) These findings provide a cytological foundation for the development of exciting new tumour models.
A previous study by Zhang et al. in 2020 showed that when compared with cisplatin, a new oncolytic adenovirus (Ad-TD-nsIL12), carrying human non-secretory IL-12, significantly inhibited tumour cell growth and tumour angiogenesis and improved the survival rate of experimental animals but without any notable side effects. 25 The recombinant adenovirus described in the present study can also kill breast cancer cells. WST-1 results showed that Ad-VP3, Ad-T and Ad-VT all caused significant mortality in breast cancer cells; the inhibitory rates produced by these viruses followed a specific sequence: Ad-VP3 < Ad-T < Ad-VT. Furthermore, these viruses had only minimal inhibitory effects on mammary epithelial cells. After  27 In the present study, we found that the mean luminescent intensity of tumours in the control group was significantly higher than that in any of the other treatment groups. During Weeks 3-5, the mean luminescent intensity of the Ad-VT treatment group was lower than that produced by the other treatment groups. We also found that the survival rate of mice in the recombinant oncolytic adenovirus treatment group was significantly prolonged (the mean survival rate of nude mice in the Ad-VT treatment group exceeded 50%). Collectively, these results indicate that Ad-VT and Ad-T significantly inhibited tumour growth in vivo and improved the survival rate of experimental mice.

CO N FLI C T O F I NTE R E S T
There is no conflict of interest in this article.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.