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.

Keywords: CD55‐TMn; CEA; doxorubicin hydrochloride; liver cancer; oncolytic virotherapy.

FIGURE 1

Synergistic cytotoxic effect of the combination of CD55‐TMn and Dox in HCC cells. A, Cell viability of Hep 3B, Hep G2 and PLC/PRF/5 cell was examined via an MTT assay. B, The quantitated data of combination treatment were processed using CalcuSyn software, and the CI values of the treatment groups are represented by X‐marks. C, Cell viability of L‐02 cell was examined via an MTT assay. D, Cell viability of L‐02 cell and PLC/PRF/5 for 48 h was examined via an MTT assay. All data are presented as the mean ± standard deviation. n =3. **P < 0.01; ***P < 0.001. NS, not significant

FIGURE 2

Dox enhances gene expression and virus progeny replication of CD55‐TMn through activating phosphorylation of Smad3. (A) and (C) The bright‐field images and the EGFP fluorescence images were, respectively, captured by using a light microscope and a fluorescence microscope (0.2 mm fields; magnification, x200). (B) and (D) The E1A, p‐Smad3 and Smad3 with difference dose Dox were detected by the Western blot analysis after 48 h. GAPDH was used as the internal control. E, The virus yields with the treatment of Dox alone or Dox and SIS3 were detected by TCID50 analysis after 48 h. F, The expression of E1A, MnSOD, TRAIL, p‐Smad3 and Smad3 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

FIGURE 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

FIGURE 4

Dox significantly enhanced cell apoptosis in HCC infected with CD55‐TMn (A) After 48 h, nuclear fragmentation (arrows) was observed in PLC/PRF/5 cells (0.2 mm fields; magnification, x200) and L‐02 (0.2 mm fields; magnification, x100) cells in different treatment groups using Hoechst staining under an inverted fluorescence microscope. B, The percentage of apoptotic cells was detected using flow cytometric analysis. The data are presented as the mean ± standard deviation. n = 3. *P < 0.05, **P < 0.01, ***P < 0.001. C, Apoptosis‐associated protein expression was detected by Western blotting. GAPDH was used as the internal control

FIGURE 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