Doxorubicin hydrochloride enhanced antitumour effect of CEA‐regulated oncolytic virotherapy in live cancer cells and a mouse model

Abstract Oncolytic adenovirus (OA) has attracted increasing attention due to their specific proliferation in tumour cells and resulting in lysis of tumour cells. To further improve the antitumour effect of OA, in this study, we combined CD55‐TRAIL‐IETD‐MnSOD (CD55‐TMn), a CEA‐controlled OA constructed previously, and chemotherapy to investigate their synergistic effect and possible mechanisms. MTT assay was performed to detect antitumour effects. Hoechst 33 342 and flow cytometric analysis were used to examine cell apoptosis. Western blotting was performed to examine cell pyroptosis and apoptosis mechanism. Animal experiment was used to detect antitumour effect of doxorubicin hydrochloride (Dox) combined with CD55‐TMn in vivo. We firstly found that Dox promotes gene expression mediated by CEA‐regulated OA and virus progeny replication by activating phosphorylation of Smad3, and Dox can enhance antitumour effect of CEA‐regulated CD55‐TMn by promoting cell apotopsis and cell pyroptosis. Thus, our results provide an experimental and theoretical basis on tumour therapy by combination treatment of the oncolytic virotherapy and chemotherapy and it is expected to become a novel strategy for liver cancer therapy.

by inserting an antitumour gene into an oncolytic viral vector (OA). 2,3 Besides, we have found that oncolytic viral vector carrying two different genes has stronger antitumour effect than oncolytic viral vector carrying any single gene, such as ZD55-TRAIL-IETD-Smac and CD55-TRAIL-IETD-MnSOD (CD55-TMn). 4,5 Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is considered as a potential effective antitumour agent by induction of tumour cell apoptosis. 6,7 Previous researches have reported that OV-mediated TRAIL has a good antitumour effect in hepatocellular carcinoma (HCC), 8 colorectal cancer 9 and lung adenocarcinoma. 10 Manganese superoxide dismutase (MnSOD) is a number of metal superoxide dismutase family. Our previous study showed complete suppression of tumour xenograft by combined OA-mediated MnSOD with TRAIL gene virotherapy via promoting tumour cell apoptosis. 11 Some reports suggest that MnSOD effectively inhibits tumour growth in HCC, 12 colorectal cancer 13 and pancreatic cancer. 14 Doxorubicin hydrochloride (Dox) is a kind of traditional chemotherapy drugs, and it has wide antitumour spectrum, which is used in a variety of cancer chemotherapy. 15 Dox belongs to the cycle non-specific drug that can suppress cell cycle of tumour cells.
However, high-dose Dox often causes cardiac toxicity. 16 Thus, how to improve the Dox sensitivity for chemotherapy and reduce its dose to achieve the best antitumour effect is the problem to be solved.
Previously, our study has showed that CD55-TMn has a strong antitumour effect in mouse tumour xenograft. 5 In this study, we further explored how to enhance the antitumour effect of CD55-TMn.
Thus, we found Dox could enhance antitumour effect of CD55-TMn in vitro and in vivo by a synergistic approach. Our results provide an experimental and theoretical basis on tumour therapy by combination treatment of the oncolytic virotherapy and chemotherapy.

| Cells and culture
The HEK293 (containing the E1A region of) was acquired from

| Cell viability assay
The 5000 cells were inoculated into 96-well plates, and after cultured 12 hours, they were treated with CD55-TMn, Dox (Beyotime, Nantong, China), or a combination of CD55-TMn and Dox. All cells were incubated at 37°C in a 5% CO2. At the indicated time after treatment, 15 µl solution containing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 0.5 mg/ml) was added to each well. After 4h in the incubator, the cell supernatants were completely removed and each well was added with 150 µl dimethyl sulphoxide. After mixing thoroughly, the 96-well plates were read by a microplate reader with absorbance for 490 nm (TECAN, Austria).

| Apoptotic cell staining
The 5 × 10 5 cells were inoculated into 6-well plates, and after one night, they were treated with CD55-TMn, Dox, or combination of CD55-TMn and Dox. All cells were incubated at 37°C in a 5% CO2.
After 48 hours, the cells were incubated with Hoechst 33 342 (Beyotime, Nantong, China) for 10min and then washed twice with phosphate buffered saline (PBS). Subsequently, the cells were observed under a fluorescence microscope.

| Western blot analysis
The 5 × 10 5 cells were inoculated into 6-well plates, and after one night, they were treated with CD55-TMn, Dox, or combination of CD55-TMn and Dox. All cells were incubated at 37°C in a 5% CO2.
After 48 hours, all cells were cracked and proteins were collected.
Concentration of protein collections was determined by Pierce BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA).

| Flow cytometric analysis
The 5 × 10 5 cells were inoculated into 6-well plates, and after one night, they were treated with CD55-TMn, Dox, or combination of CD55-TMn and Dox. All cells were incubated at 37°C in a 5% CO2. After 48 hours, the cells were trypsinized and harvested.
Aliquots of cell were resuspended with 500 µl binding buffer and stained with Annexin V FITC/PI (BD Biosciences, San Jose, CA, USA) based on the manufacturer's instructions. The cells were detected immediately by a fluorescence-activated cell sorting (BD Biosciences) assay.

| Histopathology, IHC and TUNEL assay
For analysis of histopathology, mice have been randomly selected from each group and were killed after 20 days of the first treatment in different groups. Tumour tissue, heart, liver, kidney and spleen were harvested and fixed in 5% paraformaldehyde, dehydrated with gradient increasing ethanol concentrations and embedded in paraffin wax, which were cut in 5-µm sections.
The sections were stained with haematoxylin and eosin for histological analysis. The sections were incubated with anti-TRAIL antibodies and then incubated with the avidin-biotin-peroxidase complex reagent (Vector Laboratories, Burlingame, CA, USA) for the IHC analysis. Haematoxylin was used as a counterstain.

In situ apoptosis detection kit (Sino-American Biotechnology
Co., Luoyang, China) was used to stain apoptotic cell tumour tissue sections on the base of the manufacture's instruction for the TUNEL assay. All sections were counterstained with haematoxylin.

| Statistical analysis
All experimental data were appeared as mean ± SD The differences were assessed by analysis of variance and Student's t test, and the data were considered statistically significant at P < 0.05.

| DOX enhanced virus progeny replication of CEA-regulating virus through activating phosphorylation of Smad3
Our research results first showed inhibition of TGF-β receptor type I/II and phosphorylation of Smad3 suppresses proliferation of CEAregulated oncolytic adenovirus CD55 in HCC (Fig. S1). In addition,  (Figure 2A and B). Furthermore, we also

| The combination of CD55-TMn and DOX induced pyroptosis through caspase-3 cleavage of GSDME
Pyroptosis is a form of cell death that is critical for immunity. In this study, the PLC/PRF/5 cells treated with the combination of CD55-TMn and Dox showed evident swelling with characteristic large bubbles from the plasma membrane ( Figure 3A). In addition, LDH assay showed the combination of CD55-TMn and Dox induced more releases of lactate dehydrogenase (LDH) compared to Dox alone or CD55-TMn alone ( Figure 3B). To verify that the combination of CD55-TMn and Dox induced pyroptosis through caspase-3 cleavage of GSDME, with difference dose Dox was detected by the Western blot analysis after 48 h, GAPDH was used as the internal control. All data are presented as the mean ± standard deviation. n = 3. *P < 0.05, **P < 0.01, ***P < 0.001. All CE is CD55-EGFP

F I G U R E 3
The combination of CD55-TMn and Dox induced pyroptosis through caspase-3 cleavage of GSDME. A, Phase-contrast imaging assay of pyroptosis. B, LDH release-based cell death. C, Caspase-3 cleavage of GSDME was detected by Western blotting, and GAPDH was used as the internal control. All data are presented as the mean ± standard deviation. n = 3. *P < 0.05, **P < 0.01, ***P < 0.001 the Western blotting was performed. The results showed cleaved caspase-3, GSDME-N, TNF-α and IL-1β were increased and GSDME were decreased in the combination of CD55-TMn and Dox compared to Dox or CD55-TMn ( Figure 3C), suggesting the combination of CD55-TMn and Dox induced pyroptosis through caspase-3 cleavage of GSDME.

| DOX significantly enhanced cell apoptosis in HCC infected with CD55-TMn
To further confirm whether Dox can promote cell apoptosis in- To explore the underlying mechanism in cell apoptosis, caspase signalling pathway was examined by Western blot analysis in the PLC/PRF/5 cells after 48 hours of various treatments. The result showed that CD55-TMn induced obvious activation of caspase signalling pathway, and Dox treatment largely enhanced the cleavage of procaspase-9, procaspase-8, procaspase-3, XIAP and PARP in PLC/ PRF/5 cells infected by CD55-TMn, revealing that the combination treatment can more effectively activate the caspase signalling pathway to induce the cell apoptosis that can enhance their antitumour effects ( Figure 4C). Therefore, Dox can enhance apoptosis inducement effect of CD55-TMn, and the combination treatment can more effectively kill the tumour cells via inducing cell apoptosis compared with each treatment alone.  Figure 5D). In clinicopathologic analysis of normal organ tissue by HE staining, combination therapy almost did not cause the cytopathic effects in the liver, kidney, spleen and heart, implying that the addition of Dox had no effect in the normal tissue treated with CD55-TMn and had a good security (Fig. S3). F I G U R E 5 Oncolytic virotherapy model demonstrates excellent antitumour effects in vivo. A, The tumour volume was measured every 5 days using the formula V (mm3)=1/2 × length × width 2. Data are presented as the mean ± standard deviation, n = 5. *P < 0.05, **P < 0.01, ***P < 0.001. B, The mouse survival rate in different treatment groups. C, The cellular necrotic areas in the tumours were detected using HE staining, and the apoptosis of tumour sections was assayed using TUNEL staining. Magnification, x200. D, The expression of E1A, MnSOD and TRAIL in vivo was detected by the IHC analysis. Magnification, x200. E, The toxicity to heart, liver, kidney and spleen tissues was detected by HE staining. Magnification, x200 and a tumour targeting oncolytic adenovirus can improve therapeutic outcomes in chemotherapy resistant metastatic human breast carcinoma. 21 In this study, we have found that the combination of Dox and CEA-regulated OA CD55-TMn exerted the synergic antitumour effect in HCC cells, compared to less than 10% inhibitory rate for Dox alone.  [34][35][36][37][38] In summary, we firstly found that Dox promotes gene expression mediated by CEA-regulated OA and virus progeny replication by activating phosphorylation of Smad3, and Dox can enhance antitumour effect of CEA-regulated CD55-TMn by promoting cell apotopsis and cell pyroptosis. Thus, our results provide an experimental and theoretical basis on tumour therapy by combination treatment of the oncolytic virotherapy and chemotherapy, and it is expected to become a novel strategy for liver cancer therapy.

CO N FLI C T O F I NTE R E S T
We have no conflicts of interest to declare.