Oncolytic Vaccinia Virus Harboring Aphrocallistes vastus Lectin Inhibits the Growth of Hepatocellular Carcinoma Cells

Abstract

Oncolytic vaccinia virus has been developed as a novel cancer therapeutic drug in recent years. Our previous studies demonstrated that the antitumor effect of oncolytic vaccina virus harboring Aphrocallistes vastus lectin (oncoVV-AVL) was significantly enhanced in several cancer cells. In the present study, we investigated the underlying mechanisms of AVL that affect virus replication and promote the antitumor efficacy of oncolytic virus in hepatocellular carcinoma (HCC). Our results showed that oncoVV-AVL markedly exhibited antitumor effects in both hepatocellular carcinoma cell lines and a xenograft mouse model. Further investigation illustrated that oncoVV-AVL could activate tumor immunity by upregulating the expression of type I interferons and enhance virus replication by inhibiting ISRE mediated viral defense response. In addition, we inferred that AVL promoted the ability of virus replication by regulating the PI3K/Akt, MAPK/ERK, and Hippo/MST pathways through cross-talk Raf-1, as well as metabolism-related pathways. These findings provide a novel perspective for the exploitation of marine lectins in oncolytic therapy.

Keywords: Aphrocallistes vastus lectin; Huh7 cells; oncolytic vaccinia virus; viral replication.

Figure 1

OncoVV-AVL promotes cell apoptosis in HCC cells. (a) Cell viability was observed by MTT assay in Huh7, Hep-3B, and SK-Hep-1 cells infected with oncoVV or oncoVV-AVL (** p < 0.01). (b) Apoptosis rate of Huh7 cells was detected by flow cytometry. Cells were treated with oncoVV or oncoVV-AVL (MOI 5) for 36 h. (c) Statistical results of apoptosis rate (** p < 0.01). (d) Expression of apoptosis-related protein was verified by Western blot, and compared with control PBS, with GAPDH serving as reference protein.

Figure 2

Promotion by AVL of viral replication in Huh7 cells. (a) Replication of oncoVV-AVL and oncoVV in Huh7 cells was investigated by TCID50 assay (* p < 0.05). (b) Expressions of A27L and OASL were detected through Western blotting.

Figure 3

Upregulation of typeⅠinterferon transcription. (a) Evaluation of IFN-α/β transcription at 48 h post-infection with oncoVV or oncoVV-AVL in Huh7 cells. Comparative Ct (cycle threshold) value was used to investigate mRNA expression (* p < 0.05, ** p < 0.01). (b) Transcription activity of IRF-3 and AP-1. In dual-luciferase reporter gene assay, transfection efficiency was normalized by PRL-TK luciferase plasmid. Data are expressed as the mean ± SEM from at least three experimental repetitions (** p < 0.01). (c) Expressions of IRF-3 and p-IRF-3 determined by Western blotting.

Figure 4

Transcriptomic verification of oncoVV-AVL. (a) Heatmap of genes in Huh7 cells. Transcriptomic sequencing was performed at 36 h post-infection with oncoVV and oncoVV-AVL (MOI 5), separately. The key includes a histogram of the distribution of log2 fold variation values for specific genes. (b) ISRE transactivation determined using dual-luciferase reporter assay (** p < 0.01).

Figure 5

Virus yields in presence of activator or inhibitor. Huh7 cells were infected with oncoVV or oncoVV-AVL in presence of U0126 (a), SP600125 (b), KY12420 (c), XMU-MP-1 (d), EPI-001 (e), or capsaicin (f) (** p < 0.01).

Figure 6

OncoVV-AVL suppressed tumor in vivo. (a) Volume growth curve of Huh7 tumors in Balb/c nude mice that were intratumorally injected with control 0.9% normal saline, oncoVV, or oncoVV-AVL (** p < 0.01). (b) Photographs of tumors from sacrificed mice. (c) Immunohistochemistry results of A27L on Huh7 tumors. (d) Expression of A27L determined by Western blotting. (e) H&E-staining result of mouse tumor tissue.