Recombinant oncolytic adenovirus combined with cyclophosphamide induces synergy in the treatment of breast cancer

doi:10.21203/rs.3.rs-1530123/v1

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Recombinant oncolytic adenovirus combined with cyclophosphamide induces synergy in the treatment of breast cancer

https://doi.org/10.21203/rs.3.rs-1530123/v1

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Purpose: Oncolytic virus therapy has gradually become an important approach in cancer treatment. We intend to explore the therapeutic effects of the combination of a dual cancer-selective anti-tumor recombinant adenovirus (Ad-Apoptin-hTERTp-E1a) and cyclophosphamide on breast cancer cells.

Methods: The inhibition of Ad-Apoptin-hTERTp-E1a (Ad-VT), cyclophosphamide, and Ad-VT + Cyclophosphamide on MCF-7 and MDA-MB-231 cells were investigated by the CCK-8 assay. According to the inhibition rate of different treatment combinations, the combination index (CI) was analyzed and calculated using the CalcuSyn software to determine the best combination. The CCK-8 assay and crystal violet staining were used to detect the cytotoxicity of the combination Ad-VT and cyclophosphamide in breast cancer cells and breast epithelial cells. Subsequently, hoechst staining, Annexin V flow cytometry and JC-1 staining were used to analyze the inhibitory pathway of the combination Ad-VT and cyclophosphamide on breast cancer cells. Cell migration and invasion of breast cancer cells were detected using cell-scratch and Transwell assays. The antitumor effect of different treatment groups on tumor-bearing nude mouse model were also analyzed.

Results: The results showed that the treatment combination of Ad-VT (40 MOI) and cyclophosphamide (400 µM) significantly inhibited MCF-7 and MDA-MB-231 cells and reduces the toxicity of cyclophosphamide in normal cells. Ad-VT mainly induces breast cancer cell apoptosis through the endogenous apoptotic pathway. This apoptotic effect is significantly increased after Ad-VT and cyclophosphamide combined treatment. This combination significantly inhibited the migration and invasion of MCF-7 and MDA-MB-231 cells. Then in vivo experiments showed that treatment combination of Ad-VT and cyclophosphamide significantly inhibited tumor growth and extended the survival time of mice.

Conclusions: The results showed that the combination of Ad-VT and cyclophosphamide had the effects of reducing toxicity and increasing efficiency in the treatment of breast cancer cells.

cyclophosphamide

Recombinant adenovirus

breast cancer

synergy

toxicity

According to the latest World Cancer Report, the number of newly diagnosed cancer cases is increasing year by year, and the number of cancer deaths has exceeded the million mark [1]. The report pointed out that the number of breast cancer (BC) patients exceeds the number of lung cancer patients, and breast cancer has become the highest incidence of cancer. Breast cancer still ranks first among women in terms of mortality.

At present, the treatment of breast cancer is mainly based on surgery combined with chemotherapy such as cyclophosphamide, adriamycin, raltitrexed, fluorouracil, capecitabine, and paclitaxel. Most breast cancer patients are sensitive to chemotherapeutic drugs, but there are still some patients who relapse after the first-line treatment is completely relieved. There are also many patients have severe reactions to chemotherapy. Therefore, alternative types of therapies are required.

The clinical concept of using virus to treat cancer began in the middle of 20th century, but it was not until the emergence of recombinant DNA technology and virus genome engineering in 1990s that a new wave of virus treatment was triggered. Viral therapy is a strategy of using live viruses as a treatment[2–4]. There are two kinds of oncolytic virus therapy: (1) tumor gene therapy with non-replicating virus as carrier; (2) Using replication virus as oncolytic agent[5–10]. Among them, the emergence of oncolytic adenovirus therapy greatly reduces the side effects of chemotherapy^[11]

Apoptin is a small protein consisting of 121 amino acids, which was first isolated and identified from chicken anemia virus (CAV) in Japan in 1974 and which is encoded by the VP3 gene of CAV [12–15]. Phosphorylated apoptin can be located in the nucleus of tumor cells and the cytoplasm of normal cells. Apoptin can only cause cell death when it is located in the nucleus[16]. Therefore, apoptin has the ability to selectively kill tumor cells [17–20].

Transcription of human telomerase reverse transcriptase is a major step in the regulation of telomerase activity. Telomerase is essential for cancer cells to maintain immortality. Interference with telomerase activity can inhibit the growth of cancer cells. [21, 22].

Ad-VT (Ad-Apoptin-hTERTp-E1A) includes a tumor-specific promoter (hTERTp, human telomerase reverse transcriptase) and apoptin gene, Therefore, Ad-VT is a dual specific oncolytic adenovirus with tumor-specific killer and tumor-specific replication ability [23]. This adenovirus replicates specifically in a large number of tumor cells, leading to the death of tumor cells mediated by effective apoptin [24–27]. In addition, we found in the previous study that Ad-VT combined with other chemotherapy drugs had a significant synergistic effect on lung cancer cells. In this study, we investigated the inhibitory pathway of Ad-VT combined with cyclophosphamide on breast cancer cells and its toxic effect on normal breast epithelial cells.

This study analyzed the optimal concentration of Ad-VT in combination with cyclophosphamide through a variety of assays with the aim of reducing the toxicity of cyclophosphamide and improving their therapeutic effects in BC cells.

Cells and viruses

The human breast cancer cell lines MCF-7 and MDA-MB-231 cells and human normal breast epithelial cell lines MCF-10A cells were cryopreserved by Changchun Veterinary Research Institute. The four recombinant adenoviruses (Ad-MOCK and Ad-VT) were constructed and preserved in our laboratory [23].

Female nude mice aged 4 to 5 weeks were purchased from Beijing Vital River Laboratory Animal Technology Co. Ltd. The animal experimental protocols were approved by the Institutional Animal Care and Use Committee of the Changchun University of Chinese Medicine.

Measurement of cytotoxic synergy

The BC cell were cultured in 96-well plates and respectively added with Ad-VT and cyclophosphamide (alone or combination). Before detection, the CCK-8 reagent was added to each well, and the plate incubated for 2 h in 5% CO₂ incubator at 37°C. The absorbance value of each well was measured at 450 nm using Infinite 200 multifunctional microplate reader (Tecan Trading AG, Switzerland).

CalcuSyn software analysis (Biosoft v2.0) was used to calculate the combination index (CI) value. CI = 1, > 1 and < 1 respectively indicate additive, antagonistic and synergistic effects. The best combination dose was selected by CI index and inhibition rate of each combination gradient.

Cell inhibition assays

The inhibition of the proliferation of MCF-7, MDA-MB-231, and MCF-10A cells was detected by crystal violet staining and CCK-8 assay. MCF-7, MDA-MB-231, and MCF-10A cells were cultured in 12-well plates. The Ad-VT (40 MOI) + cyclophosphamide (400 µM) combination group, Ad-VT (40 MOI) group, cyclophosphamide (400 µM) group, Ad-MOCK (40 MOI) group and blank control group were set up. After 48 h of treatment, a 0.4% crystal violet solution was added to each well for 5 min, then the dye solution was discarded, the wells washed three times with PBS and prepared for imaging. Subsequently, the CCK-8 assay was used for the analysis of the cell inhibition rate.

Measurement of apoptosis Levels in breast cancer cells

Hoechst staining and the annexin V method were used to detect the effect of the combination of Ad-VT and cyclophosphamide on apoptosis levels of BC cells. The BC cells were cultured in 12-well plates. The Ad-VT (40 MOI) + cyclophosphamide (400 µM) combination group, Ad-VT (40 MOI) group, cyclophosphamide (400 µM) group, Ad-MOCK (40 MOI) group and blank control group were set up. After 48 hours of treatment, the culture medium was discarded, Hoechst (Life Technologies, USA) staining solution (Hoechst staining solution diluted 1:1000) 1 ml / well was added and the cells were cultured in the dark of 5% CO₂ at 37°C for 8 minutes. Discard the working solution, wash it twice with PBS, and add culture medium. The cover slips was observed under fluorescence microscope and photographed.

For the Annexin V experiment, the BC cells were seeded into a 12-well plate. The above five groups were set up. After 48 hours of treatment, the cells were treated with FITC-coupled Annexin-V apoptotic kit (Cell Quest Pro, Becton Dickinson), and the apoptosis was detected by FACS flow cytometry.

JC-1 staining

The BC cells were seeded into a 12-well plate. The above five groups were set up. After 48 hours of treatment, the culture medium was discarded, JC-1 staining solution (1 ml / well) was added and the cells were cultured in the dark of 5% CO₂ at 37°C for 15 minutes. Discard the working solution, wash it twice with PBS, and add culture medium. The cover slips was observed under fluorescence microscope and photographed.

The BC cells were seeded into 96-well plates and the above five groups, were set up. After 48 hours of treatment, the medium was removed, a 100 µl of JC-1 solution was added to each well and the plate incubated in the dark for 15 minutes. The results were determined using a microplate reader. The absorbance value of each well was measured at 435 nm and 585 nm using a microplate reader.

Analysis of caspase and cytochrome c activities

The cells were seeded into a 12-well plate. After incubation, the above five e groups were set up. After 48 h, the cells were collected and lysed. Subsequently, the cellular total protein was extracted and caspases-3, 7, 9 and cytochrome c activities were measured using a Caspase activity assay kit.

Cell migration and invasion assays

Two methods were used for these experiments, the Biocat method and the scratch test. For the Biocat method, the cells were seeded into a 12-well plate and cultured for 24 h and the above five groups were set up. After 48 hours, the cells were collected and added to the upper chamber of the BioCoat and the plate was incubated for 24 h. After incubation, the cells were fixed with methanol, stained with crystal violet, observed under microscope and pictures were taken.

For the scratch test, the cells were seeded into a 6-well plate and cultured for 24 h. After incubation, the scratch was made on the cell layers with a sterile micropipette tip and the scratch width at 0 h was photographed under a microscope. The above five groups were set up. After 48 hours of treatment, the width of each scratch was measured. The cell migration ratio was calculated as follows: Cell migration ratio = (0 h scratch width – 24 / 48 h scratch width)/ 0 h scratch width.

Tumor inhibition assay of the tumor-bearing Model

MDA-MB-231-LUC cells (cell density 5 × 10⁶/100µL) were injected subcutaneously into the left hind limb of BALB/C nude mice. After the tumor-bearing model was established, the mice were randomly divided into 6 groups according to the type of tumor-bearing cells: 20 mg/kg and 50 mg/kg cyclophosphamide, Ad-VT (1×10⁹ PFU), 20 mg/kg cyclophosphamide + Ad-VT (1×10⁹ PFU), 50 mg/kg Cyclophosphamide + Ad-VT (1×10⁹ PFU). Before detecting the tumor size, nude mice were injected intraperitoneally with fluorescein (Promega Corporation, USA) 200µl /mouse (15mg/mL). After 5 minutes, each nude mouse was intraperitoneally injected with 1% sodium pentobarbital 120 µL for anesthesia. Photographs of tumor sites of nude mice were taken once a week for 6 weeks to observe changes in survival time and fluorescence intensity by small animal live imaging system (Merck KGaA, Darmstadt, Germany) [23, 28, 29].

Statistical analysis

All experiments were conducted at least three times independently, and the results were expressed as mean standard deviation (SD). The student's t-test is used to compare differences between the two groups. One-way analysis of variance (ANOVA) was used to determine the significance of differences between groups. Significance levels were defined as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Analysis of the synergistic effect of combination of Ad-VT and cyclophosphamide

The synergistic inhibiton of combination of Ad-VT and cyclophosphamide was detected by the CCK-8 assay (Fig. 1A-D). The purpose of this experiment was to use a combination of Ad-VT and cyclophosphamide to reduce the cytotoxicity of cyclophosphamide, while improving the therapeutic effect. To screen for an optimal concentration, different concentrations of Ad-VT and cyclophosphamide were used for the treatment of MCF-7 cells. Based on the calcusyn software calculation, we found that the combinations groups, 600 µM + 60 MOI, 600 µM + 40 MOI, 400 µM + 40 MOI, 800 µM + 20 MOI, and 600 µM + 20 MOI, were less than 1, indicating these combinations have synergistic inhibitory effect (Fig. 1A-B). Based on the above results, we further narrowed the concentration range of cyclophosphamide to 200 µM, 400 µM, and 600 µM and the Ad-VT to 20 MOI, 40 MOI, and 60MOI.

We also performed a combined experiment in MDA-MB-231 cells to select the optimal combined concentration of Ad-VT and cyclophosphamide. The analysis of the treated MDA-MB-231 cells showed that, the CI < 1 included the combinations, 400 µM + 40 MOI, 600 µM + 40 MOI, and 400 µM + 60 MOI. The combination group 400 µM + 40 MOI had the lowest CI value and a good synergistic effect, and therefore, was selected for subsequent in vitro assays (Fig. 1C-D).

The combination of Ad-VT and cyclophosphamide inhibits the proliferation of BC cells

Breast cancer cells and normal breast epithelial cells were treated with Ad-VT and cyclophosphamide. After 48 hours, the cells were stained with crystal violet. The results showed that Ad-VT inhibits MCF-7 and MDA-MB-231 cells but had no inhibitory effect on MCF-10A cells; however, cyclophosphamide showed some toxic effects on MCF-10A cells. When cyclophosphamide was combined with Ad-VT, the cytotoxicity of cyclophosphamide on MCF-10A cells was significantly lower than that on MCF-7 and MDA-MB-231 cells (Fig. 2A).

In the CCK-8 assay, we found that the use of Ad-VT alone could inhibit two kinds of breast cancer cells by about 40%, but had no toxic effect on MCF-10A cells (Fig. 2B). On the contrary, cyclophosphamide has obvious cytotoxicity on MCF-10A cells (p < 0.01). The combination of Ad-VT and cyclophosphamide significantly reduced the cytotoxicity that was caused by cyclophosphamide alone (p < 0.05). The inhibition rate of breast cancer cells following the combined treatment was higher than 60%, which was also significantly higher than that of the Ad-VT or Cyclophosphamide group (p < 0.05). Ad-MOCK had no significant killing effect on breast cancer cells (p > 0.05). The above results indicate that the combined use of Ad-VT and cyclophosphamide has a synergistic effect and significantly reduces cytotoxicity in normal breast epithelial cells.

The combination of Ad VT and cyclophosphamide increases apoptosis in BC cells

Hoechst is a type of dye that can enter cells at will and bind to nucleic acids to display blue fluorescence. The results showed the presence of a significant nuclear fragmentation and nuclear overstaining in the cells that were treated with a combination of Ad-VT and cyclophosphamide compared to that in the control and Ad-MOCK groups (Fig. 3A).

The quantitative experiment results of apoptosis showed that the combined treatment, Ad-VT, and cyclophosphamide induce cell apoptosis, but with different degrees. In MDA-MB-231 cells, the apoptosis rates of cyclophosphamide, Ad-VT, and the combined treatment groups were 15.62%, 37.59%, and 51.24%, respectively. In MCF-7 cells, the apoptotic rates of cyclophosphamide, Ad-VT, and combination therapy groups were 19.28%, 45.68%, and 62.76%, respectively. Among these, the apoptotic rate of BC cells in the combined treatment group was significantly higher than that of the other treatment groups (p < 0.05) (Fig. 3B-C).

Induction of the endogenous apoptosis pathway in BC Cells by the combination of Ad-VT and cyclophosphamide

The results of the JC-1 staining showed that the combination of Ad-VT and cyclophosphamide, Ad-VT and cyclophosphamide can cause mitochondrial damage and decrease MMP, but there is no obvious change in MMP of control group cells (Fig. 4A). After calculating the ratio of red fluorescence to green fluorescence, it was found that the red/green ratio in cells of combined treatment group was significantly lower than that of other groups (p < 0.01) (Fig. 4B).

From the results of the caspase and cytochrome c activity assays, it was found that the activities of caspase-3, -7, -9 and cytochrome c in the cells in the combination treatment and the Ad-VT groups are significantly increased, while in the cyclophosphamide group these levels did not significantly change (Fig. 4C). The caspase and cytochrome c activities of the combined group were higher than that of the single drug group (Fig. 4C). These results suggest that the combination of Ad-VT and cyclophosphamide induces apoptosis in BC cells by activating endogenous apoptosis pathway.

The combination of Ad-VT and cyclophosphamide increases the inhibition of BC Cell migration and invasion

The BioCat results showed that the ability of cell invasion in the combination of Ad-VT and cyclophosphamide, Ad-VT and cyclophosphamide groups was significantly lower than that in the control group (p < 0.001), indicating that all treatment groups can inhibit the invasion ability of BC cells. Among them, combination of Ad-VT and cyclophosphamide had the most significant effect (p < 0.05) (Fig. 5A-B).

The results of the scratch test showed that the combination of Ad-VT and cyclophosphamide, Ad-VT and cyclophosphamide significantly inhibits the healing ability of BC cells (p < 0.01). Among them, combination of Ad-VT and cyclophosphamide had the most significant effect (p < 0.01) (Fig. 5C-D). Above results indicated that the inhibiting tumor metastasis effect of the combination group was stronger than that of the single drug groups.

The anti-tumor effect of Ad-VT and cyclophosphamide treatment combination in vivo

During the 6-week monitoring of tumor bioluminescence intensity, it was found that the tumor bioluminescence intensity of control group increased rapidly all the time in the whole experimental period, while the growth rate of tumor bioluminescence intensity of other groups (20 mg/kg and 50 mg/kg cyclophosphamide, Ad-VT (1×10⁹ PFU), 20 mg/kg cyclophosphamide + Ad-VT (1×10⁹ PFU), 50 mg/kg Cyclophosphamide + Ad-VT (1×10⁹ PFU)) were slower. The difference in tumor volume between the treatment group and the control group was statistically significant (p < 0.001). The inhibition rates of each treatment group were 11.2%, 19.7%, 38.5%, 55.4%, and 64.2%, respectively (Fig. 6A-D).

After successful tumor bearing, the survival of nude mice in each group was recorded continuously. The survival rates in the 20 mg / kg cyclophosphamide + Ad-VT (1 × 10⁹ PFU), and the 50 mg/kg cyclophosphamide + Ad-VT (1 × 10⁹ PFU) groups were 70% and 80%, respectively, which were significantly higher than those of the control group (p < 0.001) (Fig. 6E). These results indicate that Ad-VT combined with cyclophosphamide could not only significantly inhibit the growth of tumor in vivo, but also significantly improve the survival rate of mice.

Cancer is the second leading cause of death in developing countries and its incidence and mortality are rapidly growing. According to the 2020 statistics, there were 19.29 million new cases and 9.96 million deaths worldwide [1]. Breast cancer is a common malignant tumor in women. At present, the main treatment for breast cancer is a combination of surgery and chemotherapy. However, these mainstream therapies have serious side effects, are ineffective for metastatic patients, and have a post-therapeutic recurrence risk. One of the most important causes of recurrence is the development of a chemoresistance. At present, the mechanisms of chemoresistance have not been clarified, and patients have serious toxic reactions during chemotherapy. Although molecular targeted therapy has made some achievements in BC treatment through immunotherapy and other precision therapies [30], most of these studies are still in the clinical second-line stage or above, due to small samples’ size and inconsistent results [31].

In recent years, with the development of gene therapy, many new therapeutic technologies have emerged, such as oncolytic virus therapy. Oncolytic viruses can specifically infect, replicate, and kill tumor cells. At present, newcastle disease virus (NDV), herpes simplex virus-1 (HSV-1), reovirus, oncolytic adenovirus and that like are modify into oncolytic viruses by oncophilic property. It specifically recognizes and infects tumor cells, eventually leading to cell swelling and destruction of tumor cells [32]. Among them, adenovirus is widely used in anti-tumor research due to its small size and easy transformation. Many studies have shown that modifing adenovirus to more efficient kill tumor is a very effective strategy.

In our previous studies, we construct constructed a recombinant adenovirus with tumor-specific replication and killing ability [33]. The recombinant adenovirus can replicate, proliferate, and express apoptin in tumor cells. As a pathogenic gene of chicken anemia virus, apoptin has the effect of specifically causing apoptosis in tumor cells. The hTERT promoter is a tumor-specific promoter that is only active in tumor cells. Using these two genes, this oncolytic adenovirus can effectively improve the anti-tumor effect of the virus.

At present, the combination of oncolytic adenoviruses and chemotherapy drugs, is one of the important directions in the treatment of cancer. This is mainly because the combination of oncolytic adenovirus and chemotherapy drugs can reduce the dosage of chemotherapy drugs, reduce the toxic effects of chemotherapy drugs, and improve the effect of killing tumors. By selecting the appropriate concentration for combined application, chemotherapy drugs can not interfere with the oncolytic effect of oncolytic adenovirus. Moreover, their combination can produce a synergistic effect, and the killing effect is significantly higher than that of chemotherapy alone or oncolytic adenovirus alone. Previous studies have shown that the anti-malignant melanoma drug, cyclophosphamide alone, cannot reduce the tumor growth in mouse models of melanoma, while a combination of low-dose cyclophosphamide and Ad5/3-Δ24-GM‐CSF leads to a complete tumor regression [34]. The mechanism may be related to the increase of angiogenesis inhibition and the decrease of regulatory T cells [35, 36].

In this study we analyzed the combined anti-breast cancer effect of Ad-VT and cyclophosphamide. Ad-VT and cyclophosphamide both have a significant inhibitory effect on BC cells. When Ad-VT is combined with cyclophosphamide, it has a more significant effect on BC cells, indicating that the combination of Ad-VT and cyclophosphamide has a significant synergistic effect. The cyclophosphamide has a significant toxic effect on MCF-10A cells, but the toxicity of cyclophosphamide on MCF-10A cells was significantly reduced after its combination with Ad-VT, indicating that the combination of two drugs can not only enhance the anti-tumor effect, but also reduce the toxicity of drugs to normal cells.

Other study have shown that Ad-shVEGF, an oncolytic adenovirus, can inhibit angiogenesis in vivo [37]. Another study showed that in the mouse tumor model established using renal cancer cells, TOS-3LN, the intra-tumoral injection of Axd Ad B-3 or the intra-peritoneal injection of gemcitabine (120mg/kg), can produce a cell killing effect on renal cell carcinoma cells. However, the combination of gemcitabine and Axd Ad B-3 has a more significant killing effect on tumor cells, thus significantly inhibiting the growth of tumor cells. In vivo anti-tumor experiment, the effect of Ad-VT combined with cyclophosphamide is significantly better than that of single drug group. These results are consistent with the experimental results in vitro.

The combination of Ad-VT and cyclophosphamide has a synergistic effect, which improves the inhibiton on BC cells and reduces the toxic effect of chemotherapy drugs on normal cells. In addition, we also found that this combination of Ad-VT and cyclophosphamide inhibits the metastatic ability of BC cells. These results indicate that Ad-VT has a great potential in the treatment of breast cancer when used alone or in combination with cyclophosphamide.

Ethics approval and consent to participate

The animal study was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the Changchun University of Chinese Medicine.

Consent for publication

Not applicable.

Informed consent

For this type of study, informed consent is not required

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests

The authors declare that they have no competing interests.

Funding

This work was supported by the Jilin Province Youth Scientific and Technological Talent Support Project (Grant No. QT202111).

Authors' contributions

Conceived and designed the experiments: Jing Wang, Xiao Li, Ningyi Jin and Yiquan Li. Performed the experiments: Jing Wang, Shuting Zuo, Yan Zhang, Shanzhi Li, Wei Yao, Ying Shi, Tonghua Du, and Ningyi Jin. Analyzed the data: Jing Wang, Xiao Li, Ningyi Jin and Yiquan Li. Contributed reagents/materials/analysis tools: Jing Wang, Shuting Zuo, Yan Zhang, Shanzhi Li, and Ying Shi. Wrote the paper: Jing Wang and Yiquan Li. All authors read and approved the final manuscript.

Acknowledgements

The authors would like to express their gratitude to EditSprings (https://www.editsprings.cn ) for the expert linguistic services provided.

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Recombinant oncolytic adenovirus combined with cyclophosphamide induces synergy in the treatment of breast cancer

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