Anti-tumor effect of a dual cancer-specific recombinant adenovirus on ovarian cancer cells

https://doi.org/10.1016/j.yexcr.2020.112185Get rights and content

Abstract

Background

Apoptin can specifically kill cancer cells but has no toxicity to normal cells. Human telomerase reverse transcriptase (hTERT) acts as a tumor-specific promoter, triggering certain genes to replicate or express only in tumor cells, conferring specific replication and killing abilities. This study aimed at investigating the anticancer potential of the recombinant adenovirus Ad-apoptin-hTERTp-E1a (Ad-VT) in ovarian cancer treatment.

Methods

Crystal Violet staining and WST-1 assays were used to analyze the inhibitory effect of Ad-VT on ovarian cancer SKOV3 and OVCAR-3Ā cells. Ad-VT-induced apoptosis of ovarian cancer cells, was detected using Hoechst, Annexin V-FITC/PI, JC-1 staining. Cell migration and invasion of ovarian cancer cells were detected using cell-scratch and Transwell assays. The pGL4.51 plasmid was used to transfect and to generate SKOV3-LUC cells, that stably express luciferase. The in vivo tumor inhibition effect of Ad-VT was subsequently confirmed using a tumor-bearing nude mouse model.

Results

Ad-VT had a strong apoptosis-inducing effect on SKOV3 and OVCAR-3Ā cells, that was mainly mediated through the mitochondrial apoptotic pathway. The Ad-VT could significantly increase the inhibition of ovarian cancer cell migration and invasion. The Ad-VT also can inhibit tumor growth and reduce toxicity in vivo.

Conclusions

The recombinant adenovirus, comprising the apoptin protein and the hTERTp promoter, was able to inhibit the growth of ovarian cancer cells and promote their apoptosis.

Introduction

Cancer is one of the most important public health problems in the world. With the changing disease structure and the aging trend of the population, the global burden of cancer has become increasingly prominent. Ovarian cancer, as a malignant tumor occurring in women's ovaries, ranks third among gynecological tumors, and its mortality rate ranks first among gynecological tumors [1]. Ovarian cancer is a common ovarian tumor, of which benign tumors account for about 50% of gynecological benign tumors, and malignant tumors account for 85%ā€“90% of ovarian malignant tumors and this tumor is more common in women over 50 years of age [2,3].

Currently, the most common treatment for ovarian cancer is surgery combined with radiotherapy and chemotherapy. However, most patients with ovarian cancer are susceptible to chemotherapy and relapse, leading to death [4]. Therefore, it is necessary to find new strategies to improve the treatment of ovarian cancer.

With the widespread use of genetic engineering in the biological and medical fields, gene-targeted therapies have become possible for the treatment of cancer. Gene therapy has shown an increasingly prominent advantage, especially treatment using adenovirus as a carrier to target cancer cells, which has received extensive research attention and has become a research hotspot. Adenovirus therapy is expected to replace traditional therapy for cancer treatment. However, how to accurately track and monitor the changes of genes and cells during treatment is critical to cancer treatment. Molecular imaging technology is an emerging technology that can effectively track and monitor genes and cells during treatment.

In vivo imaging tools will help to understand the molecular mechanisms that lead to cancer progression, metastasis and chemo resistance. It is very important to develop preclinical research tools that ensure faster and more accurate analyses of the molecular pathways, that are critical in improving diagnosis, the design and screening of new drugs, and cancer treatment. In vivo bioluminescence imaging is a visualization technique used to track cellular, tissue activity and genetic behavior in vivo [5,6]. In this study, we transfected cells with a plasmid containing the luciferase gene, screened them with selective antibiotics until a single resistant clone emerged, and finally the clones with the best luciferase activity and stability were selected. The labeled tumor cells were injected into the animal to establish a visualized tumor model.

Apoptin is a small apoptosis-inducing protein derived from the chicken anemia virus (CAV), which belongs to the genus Circoviridae and possesses a single-stranded minus-strand circular DNA [[7], [8], [9]]. It is a small 14Ā kDa protein that is rich in proline, serine, threonine and basic amino acids. Apoptin has the ability to selectively kill various human tumors or transformed cells, with little cytotoxic effect on normal cells [10]. It contains a two-core nuclear localization signal and a nuclear export signal that facilitates protein shuttle between the nucleus and the cytoplasm, and has several potential phosphorylation sites, including on threonine-108 (thrl-108). Apoptin is specifically phosphorylated on thr108 in tumor cells, and is not observed in normal cells [[11], [12], [13], [14]]. The tumor specific phosphorylation of Apoptin has generated interest in identifying cellular kinases with increased activity in tumor cells, and that might be responsible for Apoptin phosphorylation and its tumor-specific activation.

The length and viability of the human telomerase reverse transcriptase (hTERT) is related to cell senescence and immortalization. Telomerase is an RNA-dependent DNA polymerase that elongates 5ā€²-TTAGGG-3ā€² telomeric DNA [15]. Most normal human somatic cells lack telomerase activity due to the tight transcriptional suppression of the rate-limiting and catalytic component of the telomerase reverse transcriptase (hTERT) gene. However, hTERT expression and telomerase activation are observed in up to 90% of human malignances, giving them unlimited proliferation ability [16]. Studies have shown that the targeting of tumor cells and efficient expression of proteins of interest, is also dependent on the high efficiency and specificity of the hTERT promoter. This opens up new potential avenues for tumor therapy [17,18]. In previous studies, we took advantage of the characteristics of Apoptin and the hTERT promoter to construct an Apoptin expressing tumor-specific replication recombinant adenovirus (Ad-Apoptin-hTERTp-E1a, Ad-VT) [19]. This allowed the adenovirus to specifically replicate in large numbers, within tumor cells; therefore, expressing Apoptin and leading to Apoptin-mediated tumor cells death. Additionally, we have shown that the recombinant adenovirus has a significant killing effect on several other tumor cells [[20], [21], [22], [23]].

In the present study, we analyzed the inhibitory effect of Ad-VT on ovarian cancer cells, using various in vitro experiments and a BALB/c nude mouse subcutaneous tumor model. The findings of this study provide a theoretical basis for the treatment of ovarian cancer using oncolytic adenoviruses Ad-VT.

Section snippets

Cells, viruses and animals

Cryopreserved SKOV3 and OVCAR-3 human ovarian cancer cells were purchased from the Cell Bank of the Shanghai Institute for Biological Science (Shanghai, China), and maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 50 U/mL penicillin and 50 U/mL streptomycin, and incubated at 37Ā Ā°C with 5% CO2. All cell culture reagents were purchased from HyClone GE Healthcare Life Sciences (Logan, UT, USA).

The recombinant adenoviruses Ad-Apoptin-hTERTp-E1a (Ad-VT), Ad-hTERTp-E1a

Recombinant adenovirus Ad-VT can effectively inhibit the proliferation of ovarian cancer cells

In this study, we first examined the cytotoxic effects of recombinant adenovirus on ovarian cancer cells. Among them, Ad-VT contains the hTERT promoter, E1a gene and Apoptin gene; Ad-T contains the hTERT promoter and E1a gene; Ad-VP3 contains the Apoptin gene; and Ad-MOCK does not contain any other foreign genes (Fig. 1Aā€“D).

The crystal violet staining results showed that Ad-VT, Ad-T and Ad-VP3 can cause significant cytotoxicity in SKOV3 and OVCAR-3Ā cells (Fig. 1, Fig. 2C). From 24Ā h, the

Discussion

Cancer incidence and mortality are rapidly growing worldwide. It is the second leading cause of death in developing countries, and it is estimated that in 2018, there will be 18.1 million new cases and 9.6 million cancer deaths worldwide [[26], [27], [28]]. Ovarian cancer is one of the most common and deadly gynecological malignancies, with more than 295,000 new cases and more than 184,000 deaths in 2018 [26]. For patients with early ovarian cancer, surgery and chemotherapy are the main

CRediT authorship contribution statement

Yingli Cui: Conceptualization, Methodology, Data curation, Writing - original draft, Writing - review & editing. Yiquan Li: Software, Validation. Shanzhi Li: Visualization, Investigation. Wenjie Li: Visualization, Investigation. Yilong Zhu: Software, Validation. Jing Wang: Software, Validation. Xing Liu: Visualization, Investigation. Ying Yue: Conceptualization, Methodology, Writing - review & editing. Ningyi Jin: Conceptualization, Methodology, Supervision, Funding acquisition. Xiao Li:

Declaration of competing interest

The research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Acknowledgments

This work was supported by the National Science and Technology major Project (grant number 2018ZX10101003-005-003).

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