Luteolin enhances the antitumor efficacy of oncolytic vaccinia virus that harbors IL‐24 gene in liver cancer cells

Abstract Background Interleukin 24 (IL‐24) is an IL‐10 family member and a secreted cytokine characterized by cancer‐targeted toxicity and can activate apoptosis by sensitizing cancer cells to chemotherapy. Cytotoxic effects of luteolin on different types of cancer cells suppress their growth by acting on the components of the apoptosis signaling cascade. Therefore, our study aimed to prove whether oncolytic vaccinia virus (VV) that harbors IL‐24 (VV‐IL‐24) combine with luteolin exerts a synergistic inhibitory effect in liver cancer cells. Methods Impacts on cell viability of VV‐IL‐24 and luteolin were assessed by MTT in various liver cancer cell lines. Then, liver cancer cell apoptosis was analyzed via flow cytometry and Western blotting. Besides, the MHCC97‐H xenograft mouse model was employed as a means of assessing in vivo antitumor efficacy. Results MTT assay confirmed that the combination treatment decreased liver cancer cells viability to a greater degree than treatment with VV‐IL‐24 or luteolin alone. Flow cytometry and Western blot assay proved that VV‐IL‐24 plus luteolin induced more liver cancer cells apoptosis than single treatment. Furthermore, in the MHCC97‐H xenograft model, 15 days of treatment with VV‐IL‐24 plus luteolin inhibited tumor growth significantly more than single treatment. Conclusion These data confirm that the synergistic mechanism of VV‐IL‐24 and luteolin elicits a stronger tumor growth inhibition than any single therapy. Thus, the combination of VV‐IL‐24 and luteolin could provide the basis for preclinical research in the treatment of liver cancer.

Vaccinia virus (VV) has a variety of mechanisms for antitumor efficacy, including direct oncolysis, suppression of tumor-induced immune response, and anti-angiogenesis. 3,4 Normal cells have generally low nucleotide concentrations, and thymidine kinase (TK) is linked with deoxyribonucleotide synthesis, which is necessary for DNA replication. However, cancer cells have high concentrations; therefore, TK is dispensable for cancer cells proliferation. 5,6 Thymidine kinase deleted VVs in many animal models including melanoma and breast carcinoma have shown that VVs replication selected in tumor. 7,8 A previous study has shown that vaccinia virus encoding IL-24 can target and kill lung cancer cells without impacting normal cells. 9 Furthermore, replication-competent oncolytic vaccine virus (VV) that selectively infect tumors are emerging as an attractive therapeutic target in liver cancer. 10 Thus, VV may be a good candidate for the systemic oncolytic virotherapy of human tumors.
Prior studies have assessed interleukin 24  in various cancer cells, 11 such as colon cancer, 12 prostate cancer 13 without exerting significant side effects on normal cells. The strong expression of IL-24 induces the promotion of tumor cell apoptosis which inhibits tumor growth, angiogenesis, or metastasis and even enhances immunoregulation. 14 The anticancer effect of IL-24 also includes the bystander effect killing nearby tumor cells. 15 To improve the therapeutic efficacy of IL-24, we searched for a synergistically acting antitumor drug that could be employed together with IL-24 in combination therapy.
Luteolin (3′,4′,5,7-tetrahydroxyflavone) can be obtained from various fruits and vegetables, 16 and is a flavonoid. Luteolin suppresses the growth of cell lines derived from different types of tumors, and this effect is mediated by the promotion of apoptosis and inhibition of proliferation. 17 The mechanism of the activation of liver cancer cells apoptosis by luteolin induces mitochondria translocation of Bax/Bak and the stimulation of JNK signaling. 18 Besides, luteolin enhances the expression of oncolytic adenovirus-mediated E1A and TRAIL proteins, which leads to augment antitumor effects by enhancing colorectal cancer cells apoptosis. 19 In this present study, the combination of VV-IL-24 and luteolin markedly decreased viability and significantly increased liver cancer cells apoptosis. The mechanism was explored to provide a novel strategy on the apply of VV-IL-24 and luteolin in the therapy of liver cancer.

| Cell lines and reagents
The human normal liver cells LO2, the human MHCC97-H, HepG2, PLC/PRF/5, Hep3B liver cancer cell lines, and HEK293 were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). All cells were grown in DMEM supplemented with 10% FBS (all from Gibco, MA, USA) in a humidified incubator at 5% CO 2 and 37°C Luteolin was purchased from Beyotime Institute of Biotechnology, Haimen, China, and stored at 4°C. Mycoplasma negativity was confirmed in all utilized cell lines.

| Construction and purification of VV-IL-24
An expression cassette under the control of the viral p-se/l and p-7.5 k promoters encoding the IL-24 and gpt genes was used to construct VV-IL-24 via insertion into the region of the viral TK gene, as previously described. 9 Viral amplification was conducted via the infection of HEK293 cells, with tissue culture infectious dose (TCID50) assay being used for tittering in HEK293 cells.

| MTT assay
Cells (5,000/well) were added to 96-well plates. The EC50 concentration of luteolin in normal liver cell LO2 for 48 h was 11.96 µg/ ml, which was detected by us. As a result, 5 μg/ml luteolin was considered as a safe dose and used in the following experiments.

| Western blotting
Cells (5 × 10 5 ) were added to 6-well plates per well. Then, liver cancer cells were subjected to treatment using VV-IL-24 (4 MOI) and luteolin (5 µg/ml) or both VV-IL-24 (4 MOI) and luteolin (5 µg/ml). After 48 h, the cells were washed and harvested in RIPA buffer (Beyotime Institute of Biotechnology, Shanghai, China) supplemented with protease inhibitors. A Pierce™ BCA Protein Assay Agent (Thermo Fisher Scientific, Inc) was then used to measure protein levels.
Samples corresponding to 20 µg protein were separated by 12% SDS-PAGE and transferred to PVDF membrane that was blocked using 5% non-fat dried milk for 1.5 h at room temperature. These membranes were probed with monoclonal antibodies either to IL-24 (cat. no. 60139-1-Ig) or to GAPDH (cat. no. 51332) overnight at 4°C and probed with appropriate secondary antibody (cat. no. 10230269) for 2 h. To detect apoptotic genes, 10% SDS-PAGE gels were instead used. The resultant PVDF membranes were probed using antibodies for cleaved poly-ADP-ribose polymerase (cat. no.

| Statistical analysis
All experiments were repeated three times, with data given as mean ± standard deviation (SD). Student's t test or one-way ANOVAs were used to analyze data with GraphPad Prism 6 (GraphPad Software, Inc, CA, USA). p < 0.05 was the significance threshold.

| Luteolin increases the inhibitory impact of VV-IL-24 on liver cancer cells viability
To determine whether VV-IL-24 (4 MOI) and luteolin (5 μg/ml) decreased cell viability more than VV-IL-24 (4 MOI) or luteolin (5 μg/ ml) alone, the liver cancer and control cells were infected for 24 h, The data suggested that the liver cancer cells viability of VV-IL-24 was obviously lower than that of luteolin, and the combination of VV-IL-24 with luteolin was significantly inhibited liver cancer cells viability than VV-IL-24 or luteolin alone and did not affect the proliferation of the normal liver cells LO2.

| VV-IL-24 and luteolin enhance the liver cancer cells apoptosis
To explore whether the antitumor properties of the combination treatment were due to an increase in cell death, flow cytometry measurements of apoptosis were performed. In MHCC97-H cells And in HepG2 cells ( Figure 3C), the test of cell death by flow cytometry displayed that compared with 11% for luteolin and 39% for VV-IL-24, treatment with VV-IL-24 and luteolin resulted in an obviously improvement percentage of apoptotic cells to 75%. Both

MHCC97-H and HepG2 cells treated with combined VV-IL-24 and
luteolin had a significantly higher percentage of apoptosis relative to either individual treatment (p < 0.001). Therefore, combination therapy induced more apoptosis compared with any single treatment in liver cancer cells.
We also examined the important caspase-dependent apoptotic signaling protein expression by Western blotting. Increased expression of cleaved PARP, cleaved caspase-3, cleaved caspase-8 and reduced procaspase-3 and procaspase-8, XIAP expression levels were significantly detected in combination-treated MHCC97-H ( Figure 3B) and HepG2 cells ( Figure 3D). We have quantified these blots of Figure 3B,D. The expression of cleaved PARP was the highest in the combination group.
These data indicated that combination treatment induced more cellular apoptosis than in VV-IL-24 or in luteolin treatment alone via activation of caspase-dependent apoptotic signaling pathway. F I G U R E 1 Cell viability of treated liver cancer and control cells. (A) The effect of VV-IL-24 (4 MOI) and luteolin (5 µg/ ml) was assessed via the MTT assay in MHCC97-H, HepG2, PLC/PRF/5, and Hep3B cells. (B) LO2 cells were subjected to treatment using luteolin (5 µg/ml) or both VV-IL-24 (4 MOI) and luteolin (5 µg/ml). Results are means ± standard deviation (n = 3). *p < 0.05; **p < 0.01; ***p < 0.001. NS, not significant; VV, vaccina virus; IL-24, interleukin 24; MOI, multiplicity of infection Hematoxylin-eosin (HE) staining showed that the combined VV-IL-24 and luteolin treatment resulted in a more severe cytopathic effect in tumor tissues than the treatment with VV-IL-24 or luteolin alone ( Figure 4B). In addition, the observation that the liver, kidney and spleen tissues of the combination group had no or little cell damage demonstrated that VV-IL-24 and luteolin had no or little toxic effects occurred on these tissues ( Figure 4C).

| D ISCUSS I ON
Many studies have demonstrated that luteolin is a key inhibitor of tumor cell proliferation that can induce apoptotic death, as shown in human myeloid leukemia 21 and melanoma cells. 22 Proliferation and chemo-resistance of the liver cancer cell can occur as a consequence of genetic and epigenetic changes, and all of these alterations can serve as potential therapeutic targets. [23][24][25] Optimal Recent studies have shown MDA-7/IL-24 to control many mi-croRNAs, such as miR-221, which is upregulated in many types of cancer. 33 MDA-7/IL-24 downregulated miR-221, which in turn induces Beclin-1, leading to autophagy. IL-24 has been shown to promote the populations of CD4 + and CD8 + T cells in diverse cancer models. 34,35 In our study, luteolin can increase VV-mediated IL-24 gene expression in liver cancer cells in vitro and in vivo. The ERK signaling pathway is necessary for the replication of vaccinia virus, 29 and whether luteolin promotes the expression of IL-24 through the ERK signaling pathway needs to be further explored. Furthermore, F I G U R E 4 VV-IL-24 and luteolin synergistically inhibit MHCC97-H cell growth in a mouse xenograft model. (A) Tumor dimensions were measured every 5 days and the volume was computed as (length × width 2 /2 [mm 3 ]). Results are means ± standard deviation (n = 6). ***p < 0.001. (B) The expression of IL-24, CD31, Ki67 and cleaved caspase-3 in tumor tissues was detected by IHC, and the areas of cellular necrosis within the tumors were examined by the HE staining. (C) Toxic effects in the liver, kidney and spleen tissues were examined by HE staining in each group. Enlargement, ×200. Bar: 50 µm. VV, vaccina virus; IL-24, interleukin 24; IHC, immunohistochemistry; HE, hematoxylin and eosin the combination therapy that stimulates liver cells autophagy and regulates the immune system should also be verified in the future.

CO N FLI C T O F I NTE R E S T
The authors have declared no competing interest for this work.

AUTH O R S ' CO NTR I B UTI O N S
Yigang Wang conceived and designed the experiments; Chunming Wang, Qiang Li, Boduan Xiao and Huiling Fang analyzed the data; Fang Huang performed the histological examination of animal experiment; Chunming Wang wrote the paper; Yigang Wang and Biao Huang revised the paper. All authors read and approved the final manuscript.

CO N S E NT FO R PU B LI C ATI O N
The authors have confirmed that all details can be published and prepared to provide copies upon reasonable request.

DATA AVA I L A B I L I T Y S TAT E M E N T
The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.