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. 2024 Jun 7;14(1):13108.
doi: 10.1038/s41598-024-63325-z.

Oncolytic adenovirus encoding LHPP exerts potent antitumor effect in lung cancer

Yaru Zhao  1   2 , Huihui Liu  1 , Qi Zhan  1 , Hao Jin  1 , Yiqiang Wang  3 , Hui Wang  2 , Biao Huang  1 , Fang Huang  4 , Xiaoyuan Jia  5 , Yigang Wang  6 , Xiaoyan Wang  7
Affiliations

Oncolytic adenovirus encoding LHPP exerts potent antitumor effect in lung cancer

Yaru Zhao et al. Sci Rep. .

Abstract

LHPP has been shown to be a new tumor suppressor, and has a tendency to be under-expressed in a variety of cancers. Oncolytic virotheray is a promising therapeutics for lung cancer in recent decade years. Here we successfully constructed a new recombinant oncolytic adenovirus GD55-LHPP and investigated the effect of GD55-LHPP on the growth of lung cancer cells in vitro and in vivo. The results showed that LHPP had lower expression in either lung cancer cells or clinical lung cancer tissues compared with normal cells or tissues, and GD55-LHPP effectively mediated LHPP expression in lung cancer cells. GD55-LHPP could effectively inhibit the proliferation of lung cancer cell lines and rarely affected normal cell growth. Mechanically, the oncolytic adenovirus GD55-LHPP was able to induce stronger apoptosis of lung cancer cells compared with GD55 through the activation of caspase signal pathway. Notably, GD55-LHPP also activated autophagy-related signal pathway. Further, GD55-LHPP efficiently inhibited tumor growth in lung cancer xenograft in mice and prolonged animal survival rate compared with the control GD55 or PBS. In conclusion, the novel construct GD55-LHPP provides a valuable strategy for lung cancer-targeted therapy and develop the role of tumor suppress gene LHPP in lung cancer gene therapy.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Low expression of LHPP in lung cancer cells and tissues (a) The expression of LHPP mRNA in normal lung cell lines and different lung cancer cell lines by RT-qPCR . **P < 0.01, ***P < 0.001. (b) LHPP expression in five lung cancer tissue specimens and paracancer normal tissues (Orginal amplification: × 200). (c) The quantitative analysis of the LHPP expression staining images in five lung cancer tissue specimens and paracancer normal tissues. **P < 0.01, ***P < 0.001. (d) The expression of LHPP in lung adenocarcinoma derived from CPTAC samples.
Figure 2
Figure 2
Construction of recombination oncolytic virus GD55-LHPP and its characteristic. (a) The promoter activity of the GP73 promoter in normal lung cell lines and different lung cancer cell lines **P < 0.01, ***P < 0.001. (b) The expression of Coxsackie and Adenovirus Receptor (CAR) protein in normal lung cell lines and different lung cancer cell lines by Western blotting. **P < 0.01, ***P < 0.001. (c) Schematic diagram of the structure of the recombinant oncolytic adenovirus structure. ITR, inverted terminal repeat. (d) The expression of LHPP protein in four lung cancer cell lines (A549, H460, H1299, and H1975) and one human embryonic lung cell (MRC-5) infected with GD55-LHPP or GD55 at the 10 MOI for 48 h by Western blotting. (e) The progeny replication of the virus replicates in four lung cancer cell lines (A549, H460, H1299, and H1975) were infected with 10 MOI of GD55-LHPP or GD55 for 24, 48, 72, 96 h.
Figure 3
Figure 3
Killing effect of GD55-LHPP on lung cancer cell lines. (a) MTT assay was used to detect the anti-tumor effect of GD55 and GD55-LHPP in one human embryonic lung cell MRC-5 and lung cancer cells NCI-H460, A549, NCI-H1299, NCI-H1975. *P < 0.05, **P < 0.01, ***P < 0.001 (b) Crystal violet assay was employed to determine the cytopathic effect of GD55 and GD55-LHPP in one human embryonic lung cell (MRC-5) and four lung cancer cells (A549, H460, H1299, and H1975). MOI, the multiplicity of infection.
Figure 4
Figure 4
GD55-LHPP induced cell apoptosis and its mechanism in lung cancer cells. (a) After 48 h, nuclear fragmentation (arrows) was observed in MRC-5, A549, NCI-H460, NCI-H1299 cells (0.2 mm fields; original magnification, × 200) in different treatment groups using Hoechst staining under an inverted fluorescence microscope. (b) The percentage of apoptotic cells in NCI-H460 cells treated with PBS, GD55, or GD55-LHPP was detected using flow cytometric analysis. The data are presented as the mean ± standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001. (c) and (d) Expression of apoptosis-associated proteins expression was detected by Western blotting in NCI-H460 after treatment with PBS, GD55, or GD55-LHPP. GAPDH was used as the internal control. (e) Expression of TNF-α, and c-Myc mRNAs in NCI-H460 cells treated with PBS, GD55, or GD55-LHPP. *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 5
Figure 5
Antitumor effect of GD55-LHPP in mice. (a) A schematic diagram of building a tumor xenograft model and injecting an oncolytic virus. (b) The tumour volume was measured every 2 days using the formula V (mm3) = 0.52 × length × width2. Data are presented as the mean ± standard deviation, n = 5. *P < 0.05, **P < 0.01, ***P < 0.001. (c) The mouse survival rate in different treatment groups.
Figure 6
Figure 6
Histopathology assay. IHC and HE staining for detection of oncolytic adenovirus amplification and hepatorenal toxicity in different treatment groups. Magnification, × 200.
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